Probiotic and postbiotic strategies against foulbrood in honeybees: in vitro and in vivo insights

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Due to the lack of safe and effective treatments, interest is growing in the use of beneficial bacterial supplements as a promising alternative to antibiotics. This study evaluated the probiotic and postbiotic potential of selected bacterial strains against foulbrood pathogens. An initial screening of 25 strains for anti-foulbrood activity led to the selection of the most active candidates for further investigation. The inhibitory effect of their cell-free supernatants (CFS) was assessed and their mode of action was investigated. The probiotic and postbiotic properties were further evaluated using P. larvae -infected larvae reared under laboratory conditions. Five lactic acid bacteria exhibited strong antagonistic activity against one or both pathogens, as their CFS displayed inhibitory effects. Notably, the CFS of Lactobacillus crispatus and Lactiplantibacillus plantarum completely inhibited P. larvae at a dose of 12.5% (v/v). Further characterisation of these CFS, suggested a bacteriostatic effect, mainly attributed to organic acids. In vivo assays demonstrated a significant increase in larval survival when supplemented with live L. plantarum , whereas CFS treatments failed to rescue infected larvae. These findings highlight the potential of probiotic and postbiotic-based strategies as sustainable alternatives for managing foulbrood in beekeeping. Antagonistic activity Lactic Acid Bacteria Apis mellifera Paenibacillus larvae Melissococcus plutonius Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Managed honeybees are among the most important pollinators, contributing approximately 70% of pollination services to the main crops cultivated for human consumption and wild plants [ 1 – 3 ]. However, over the past decade, declines in pollinator populations have been reported, as exemplified by the Colony Collapse Disorder (CCD). First reported in the USA in 2006, CCD is characterised by the sudden collapse of the colony and the loss of adult workers. Only the queen and some bees remains, as well as abundant brood and food reserves [ 4 , 5 ]. Multiple factors were identified as possible drivers for this global decline. While abiotic factors such as pesticide exposure, climate change, and habitat loss contribute to this decline, biotic stressors, particularly pathogens, are recognized as major threats exerting strong pressure on colony health. The two main bacterial pathogens affecting honeybees are Paenibacillus larvae and Melissococcus plutonius , the etiological agents of American and European Foulbrood (AFB and EFB), respectively [ 6 , 7 ]. Both are Gram-positive bacteria. P. larvae is rod-shaped and forms highly resistant spores that can remain infectious for several years [ 8 ], whereas M. plutonius is a lanceolate coccus. These brood diseases, first described in the 20th century, are now distributed worldwide [ 9 ]. Infection begins when larvae ingest contaminated food brought by nurse bees, allowing the bacteria to proliferate extensively in the larval midgut. P. larvae produces various virulence factors that contribute to the infection success by degrading the peritrophic matrix and enabling invasion of the haemocoel [ 10 – 12 ]. In contrast, M. plutonius generally proliferates in the gut lumen, although its pathogenicity varies between strains and the pathomechanisms remains elusive, notably for the “typical” strain [ 13 , 14 ]. Ultimately, both pathogens cause septicaemia and the death of the infected larvae, resulting in highly infectious cadavers. During cleaning duties, nurses spread the infection both within and between colonies [ 15 – 17 ]. The lack of population renewal weakens the colony, and without treatment, the disease can lead to colony collapse [ 18 – 20 ]. Despite extensive research, effective treatments for foulbrood remain limited. Oxytetracycline (OTC) is among the most widely used antibiotics in beekeeping, but its use raises several concerns. The emergence of antibiotic-resistant bacterial strains has been reported [ 21 , 22 ] and OTC is ineffective against the highly resistant endospores of P. larvae . Moreover, antibiotic residues have been detected in hive matrices, including honey, potentially compromising both its quality and safety for human consumption [ 23 ]. Honeybees are also impacted by antibiotics, reducing their lifespan and disturbing gut microbiota homeostasis [ 24 – 26 ]. These issues have led to increasing controversy over the use of antibiotics in beekeeping, prompting stricter regulations. Within the European Union, no Maximum Residue Limits (MRLs) have been established for antibiotics in beekeeping, and their use is highly restricted for both preventive and curative purposes [ 27 , 28 ]. Given these limitations, alternative control strategies have been adopted, such as the shook swarm technique or the incineration of infected hives [ 27 , 29 , 30 ]. However, these measures are preventive rather than curative [ 31 ]. Currently, no effective treatment exists for clinically infected colonies, generating important economic losses for beekeepers [ 32 ]. Due to their impact on epizootiology and beekeeping sustainability, these diseases are now included in the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH), requiring member states to report any outbreaks [ 33 ]. To address these challenges, many studies are underway to develop alternative strategies to antibiotics that ensure the safety of both consumers and honeybees. Natural products, which are seen as safer and more environmentally friendly than synthetic chemicals, are gaining global interest and support a sustainable One Health approach. Among these, plant extracts and essential oils have emerged as promising candidates and have been extensively studied in recent years [ 34 , 35 ]. Other innovant solutions include phage therapy [ 36 , 37 ], trans-generational immune priming through oral administration of inactivated P. larvae cells [ 38 , 39 ], selective breeding of colonies with hygienic behaviour [ 40 , 41 ], and the development of specific molecules such as indole analogues and lipid-like carbohydrate derivatives [ 42 , 43 ]. Among these strategies, the use of beneficial microorganisms has received particular attention. An increasing number of studies are underscoring the pivotal function of the intestinal microbiota in honeybee physiology, influencing development, behaviour, nutrition and detoxification processes [ 44 ]. Studies have suggested that the evolutionary relationship between gut bacteria and honeybees dates back over 80 million years [ 45 ]. The core microbiota in honeybees is specialized, highly conserved and is orally transmitted upon emergence through social interactions or contact with hive components [ 46 , 47 ]. This microbial community contributes to host protection against pathogens through various mechanisms, including the production of antimicrobial compounds, induction of the innate immune response, modulation of intestinal pH, promotion of beneficial bacteria growth and competitive exclusion of pathogens [ 48 ]. Among these microbes, symbiotic lactic acid bacteria (LAB), mostly located in the crop (honey stomach), are thought to play a key role in antimicrobial properties of honey [ 49 , 50 ], which contribute to colony-level social immunity [ 51 ]. Probiotic research in beekeeping has expanded in recent years, focusing on restoring the gut microbiota after antibiotic treatment and exploring new management strategies for disease control [ 52 – 54 ]. Probiotics are defined as “live micro-organisms that, when administered in adequate amounts, confer a health benefit to the host” [ 55 ] and are often included in the GRAS (Generally Recognized As Safe) and QPS (Qualified Presumption of Safety) lists, which attests to their safety. However, challenges such as ensuring probiotic viability, stability, effective colonization, and effective delivered dose can limit their efficacy [ 56 , 57 ]. As many probiotics effects can rely on secreted metabolites, an emerging alternative is the use of postbiotics. These are defined as non-living microorganisms and their components that also confer health benefits to the host [ 58 ]. Postbiotics comprise a range of bioactive compounds, including exopolysaccharides (EPS), enzymes, short-chain fatty acids (SCFA), bacterial lysates, cell wall fragments, and cell-free supernatants (CFS), all of which have potential to modulate the gut microbiota and inhibit pathogen [ 59 , 60 ]. However, the precise mechanisms underlying the benefits of postbiotics in honeybees remain insufficiently understood and need further investigation. The purpose of the present study was to assess the probiotic and postbiotic potential of bacterial candidates against the two foulbrood agents, P. larvae and M. plutonius . We first screened the anti-foulbrood activity of a range of commercial bacterial strains and selected the most active ones for further investigation. Particular attention was given to the characterisation of their cell-free supernatants (CFS) to identify bioactive compounds and elucidate their antagonistic mechanisms. Finally, the probiotic and postbiotic effects of two selected strains, L. plantarum and L. crispatus , were evaluated in P. larvae -infected and laboratory-reared larvae. The specific properties of these strains were analysed and discussed. Overall, our study provides novel insights into the development of probiotic and postbiotic-based products as a sustainable alternative for controlling AFB and EFB in beekeeping. 2. Materials and Methods Bacterial strains and growth conditions In this study, a total of 25 bacterial isolates comprising lactic acid bacteria (LAB), Bacillus and Paenibacillus strains, from the private collection of GREENCELL (GREENTECH Group) were used. These strains included: Lactobacillus acidophilus (n = 2), Lactobacillus crispatus (n = 1), Lactobacillus johnsonii (n = 2), Lactiplantibacillus plantarum (n = 2), Lacticaseibacillus rhamnosus (n = 3), Weissella cibaria (n = 1), Bacillus amyloliquefaciens (n = 5), Bacillus licheniformis (n = 1), Priestia megaterium (formerly Bacillus megaterium ) (n = 1), Bacillus subtilis (n = 2), Paenibacillus polymyxa (n = 5). Strains were selected according to previous studies reporting evidence of inhibitor activity against P. larvae and M. plutonius and their registration in QPS and GRAS safety lists. P. larvae B641 and M. plutonius B642 were acquired from the Pasteur institute strain collection (CIP 104618T and CIP 104052T). These strains were previously assigned to the ERIC 1 genotype [ 61 ] and to the phylogenetic group sequence type 1, Clonal Complex 13 [ 62 ] respectively. Unless otherwise stated, LAB strains were routinely cultured under anaerobic conditions using DeMan, Rogosa et Sharpe (MRS) broth [ 63 ] or agar (OXOID). AnaeroGen™ Atmosphere Generation Systems (Thermo Fisher Scientific) were used to create anaerobic conditions for agar media. Bacillus and Paenibacillus strains were grown on Nutrient Broth (NB) or Nutrient Agar (NA), composed of 5 g/L peptone (Gibco) and 3 g/L meat extract (Millipore). P. larvae was cultured in Brain Heart Infusion (BHI) medium (OXOID), while M. plutonius was grown anaerobically in KS-BHI medium according to Arai et al. , (2012) [ 64 ], containing 37 g/L BHI, 20.4 g/L KH 2 PO 4 (Sigma Aldrich), and 10 g/L Soluble starch (BD Difco). To obtain P. larvae spores, vegetative cells were cultured on BHI agar plates for 7 days at 37°C. After incubation, the plates were flooded with 5 mL sterile distilled water, and the surface was scrapped with a sterile spreader to collect the spore suspension. This procedure was repeated twice and the resulting suspensions were pooled. The combined aliquot was then heated at 65°C for 20 min to eliminate any remaining vegetative cells, followed by centrifugation at 4000 x g for 20 min. The pellet was washed twice and finally resuspended in sterile distilled water. Spore concentration was assessed using a Malassez counting chamber (Glaswarenfabrik Karl Hecht) under a phase-contrast light microscope (Olympus CX41, 400x). To determine viability, spore suspensions were plated onto MYPGP agar medium, as described by Dingman and Stahly (1983) [ 65 ]. The medium consisted of 10 g/L Mueller-Hinton broth (Millipore), 15 g/L yeast extract (Sigma Aldrich), 3 g/L KH 2 PO 4 , 1 g/L sodium pyruvate (Sigma Aldrich), and 2 g/L dextrose (OXOID). Plates were incubated at 37°C for 3 days. Spore suspensions were stored at 4°C and heat-activated at 65°C for 20 min before experiments [ 66 ]. Antagonistic assays of beneficial strains Agar slab method To assess the inhibitory activity of the LAB strains, inhibition assays were carried out using a modified agar slab method based on Leska et al. , (2022) [ 67 ]. Briefly, each LAB strain was cultured overnight and adjusted to the density of 1 x 10 8 CFU/mL, then 500 µL of the suspension were spread on MRS agar plates. Petri dishes were incubated anaerobically at 37°C for 48h. Agar disks (8mm diameter) were then cut in triplicate using sterile, reversed tips and placed on freshly inoculated plates containing P. larvae or M. plutonius , which were spread to a final density of 1 x 10 8 CFU/mL. The medium used was a modified MYT agar [ 68 ], containing 21 g/L Mueller-Hinton broth, 15 g/L yeast extract, and 0.1 mg/L thiamine hydrochloride (Sigma Aldrich). Plates were incubated for 48 h and the inhibition diameters were measured and defined as zones of inhibition (ZOI). The diameter of the agar disc was subtracted from inhibition diameters. Tetracycline (Conda Pronadisa) at 30 µg/mL was used as a positive control and MRS broth was used as a negative control. Perpendicular streak method To evaluate the inhibitory activity of Bacillus and Paenibacillus strains, the perpendicular streak method was adapted to account for the extensive and diffuse growth of these bacteria on agar plates, following a modification of the protocol by Alippi and Reynaldi, (2006) [ 69 ]. The 14 tested strains were cultured overnight and adjusted to a density of 1 x 10 8 CFU/mL and streaked (2 cm) with a 10 µL oese on the side of a 3% agar (Millipore) MYT petri dish. The plates were incubated at 30 or 37°C for 48h, depending on the optimal growth temperature for each strain. On the same time, P. larvae and M. plutonius were grown overnight and adjusted at 1 x 10 8 CFU/mL. Using a 1 µL oese, five perpendicular streaks were made on the plate, ensuring they did not contact the initial streak. The edge of the streaks was marked, and the plates were incubated for a further 48 h at 37°C. Inhibition zones (ZOI) were then measured starting from the marked edges. Tetracycline at 30 µg/mL and BHI broth was used as positive and negative control respectively. Antibacterial activity of LAB metabolites CFS production We selected the five most active LAB strains and five Bacillus and Paenibacillus strains to investigate the antagonistic activity of their metabolites. To obtain cell-free supernatants, the strains were cultured for 48h at 37°C and under agitation at 110 rpm. LAB strains were grown anaerobically in MRS medium, while Bacillus and Paenibacillus species were cultured aerobically in MYT medium. After incubation, cultures were centrifuged (4500 x g for 10 min) and the supernatants were collected. The supernatants were then filtered-sterilized using a 0.22 µm filter and stored at -18°C. CFS antagonistic activity assays To determine the minimal inhibitory dose of the CFS from LAB and Bacillus and Paenibacillus species, the two foulbrood pathogens were exposed to different concentrations of CFS using the two-fold dilution method in a 96-well flat-bottom plate. The broth microdilution method was adapted from the CA-SFM / EUCAST guidelines for antimicrobial susceptibility testing [ 70 ]. The CFS was serially diluted in MYT broth, to reach final concentrations ranging from 25% (v/v) to 0.195% (v/v). Overnight cultures of honeybee pathogens were grown, adjusted to a final concentration of 10 4 CFU/mL and distributed in the wells. The plate was incubated at 37°C, under agitation at 110 rpm for 48h. After incubation, the optical density (OD) was measured at 600 nm with a Multiskan SkyHigh microplate reader. To corroborate the OD measurements, a resazurin reduction-based assay was conducted to assess the metabolic activity of the pathogens, providing an additional information of their response to the CFS. Twenty-five microliters of filter-sterilized 0.1% resazurin was added to each well and the plate was incubated for an additional 2 h. The fluorescence was measured using a Fluoroskan Ascent FL microplate reader with excitation and emission wavelengths of 544 nm and 590 nm, respectively. The experiments were conducted in biological triplicates, and each value was normalized to its corresponding blank value (CFS optical density value). Bacterial growth inhibition (%) was calculated according to the following Eq. (1) : (1) Growth inhibition (%) = \(\:100-\frac{{A}_{t}}{{A}_{c}}\times\:100\) A t was the OD value of the test condition after 48 h and A c was the OD value of the negative control (pathogen growth in normal conditions) after 48 h. Characterisation of antibacterial compounds of CFS After selecting the two CFS with the strongest inhibitory activity, the nature of this activity was further investigated. CFS were treated with proteolytic enzymes, heated and modified by varying pH values. Heat treatments were done at 40°C, 80°C and 100°C for 1h, and at 121°C (110 kPa) for 20 min. The pH of the CFS was adjusted to values of pH 3, 5, 7 and 10. Additionally, the CFS were exposed to proteases. The CFS was first adjusted to pH 8 and pH 3.5 and incubated at 37°C under agitation at 110 rpm for 1h, in the presence of proteinase K and trypsin at 10 mg/mL, respectively. This was followed by a heat treatment at 80°C for 10 min to inactivate the enzymes. Afterwards, the pH was readjusted to the natural pH of the CFS. Finally, the antagonistic activity was determined using the broth microdilution method, as previously done. The experiments were conducted in technical duplicates and biological quadruplicates, and growth inhibition percentages were calculated as previously mentioned. The CFS vehicle (MRS broth) was used as a negative control. CFS effect on honeybee pathogens growth To evaluate the impact of the CFS on the growth of the two pathogens, growth kinetics were performed. Overnight cultures of the pathogens were adjusted to a concentration of 10 8 CFU/mL. Then, 27 mL aliquots of MYT were inoculated to a final concentration of 10 6 CFU/mL and incubated at 37°C under agitation at 110 rpm. At two-hour intervals, the OD 600nm was measured with the Ultrospec 2000 Spectrophotometer (Pharmacia Biotech), and cultures were plated on BHI agar to determine viable cell counts. The plates were incubated at 37°C for 48h. Once the cultures reached the late-exponential phase (OD of 0.2), the CFS was added to achieve a final concentration of 12.5% (v/v), corresponding to the previously determined minimal inhibitory dose. In the negative control, the CFS vehicle (MRS broth) was added at the same dose. The kinetic growth was monitored for 24 h. In vivo antagonistic assay To evaluate the antagonistic potential of LAB and their CFS, an in vivo infection assay was performed using laboratory-reared larvae. Frames containing first-instar larvae from three different colonies of the same apiary (UMR 6023, Clermont Auvergne University, Clermont-Ferrand, France) were removed and brought to the laboratory. Subsequently, larvae were grafted using a Chinese grafting tool and placed in plastic queen cups. They were equally distributed into a 48-well plate to minimize colony bias, with groups of n = 30 larvae per condition. The larvae were checked under a binocular magnifying glass and those showing signs of graft injury were identified and were excluded from the experiment if dead after 12 h, resulting in 19–25 larvae per condition. The experimental groups were then established as follows: the uninfected group was fed 10 µL of worker jelly, while the infected group received worker jelly inoculated with P. larvae at a concentration of 10 4 spores/mL. The CFS-treated groups received worker jelly supplemented with CFS at 6,25% (v/v) and inoculated with P. larvae . Finally, the last group was treated with live LAB. LAB strains were grown overnight, adjusted to 10 8 CFU/mL in PBS, and 2 µL of this suspension was provided to the larvae. After 4 h, the larvae were then fed worker jelly inoculated with 10 4 sp/mL of P. larvae . The worker jelly diet consisted of 50% organic royal jelly (Naturapi) and 50% aqueous solution of D-glucose (Thermo Fisher Scientific), D-fructose (Thermo Fisher Scientific) and yeast extract, in varying amounts depending on the larvae’s developmental stage, and was fed for a period of six days [ 71 ]. To prepare the supplemented diet, the CFS were first mixed with a small quantity of distilled water, which was then added to the sugars and yeast extract. The water content was adjusted to a final volume of 5 mL, before adding the royal jelly in a 1:1 (w/w) ratio. All diets were prepared beforehand and stored in aliquots at -18°C, with P. larvae spores being added just before infection. The next days, larvae were fed with uninfected food, with or without CFS supplementation, according to the experimental groups. The amount and composition of the diets are provided in Supplementary Table S1 . Before feedings, diets were pre-warmed to 34.5°C in an incubator. Larvae were reared in desiccators with a relative humidity (R.H.) of 94%, and kept in an incubator at 34.5°C. Larvae survival was monitored daily under a stereomicroscope. Individuals were considered dead when they remained motionless, with no contractile movements of their spiracles, showing colour changes from pearly white to brown and lost body elasticity. Dead larvae were removed and observed under light microscope to confirm infection status. Between 6 and 7 days post-infection, when the larvae had consumed all of the provided diet, they were gently transferred to a new 48-well plate, with wells lined with a piece of Kimwipe at the bottom. The plate was then incubated in desiccators with 75% relative humidity, and incubated at 34.5°C. Pupae were monitored daily and considered dead when their growth stopped and exhibited a developmental stage that is incongruent with the other individuals. After a few days of observations, death was confirmed, and dead pupae were removed. Statistical Analysis Statistical analyses were performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, California, USA). To evaluate statistical differences between groups, non-parametric data were analysed using a Mann-Whitney U test for pairwise comparisons, and a Kruskal-Wallis test for comparisons involving more than two groups, followed by a Dunn’s post-hoc test. A significance threshold of p < 0.05 was applied. These analyses were performed to compare antagonistic activities observed in agar diffusion assays and broth microdilution experiments. To analyse the effect of treatments on P. larvae growth, two statistical approaches were conducted. Differences in growth between groups at each time point were analysed using the Kruskal-Wallis test, followed by Dunn’s post-hoc test. The Friedman test was employed to analyse growth over time within one group, followed by Dunn's post-hoc test. Additionally, linear regression models were performed with R Statistical Software (v4.1.2; R Core Team 2023) and the “growthrate” R package [ 72 , 73 ]. The generation time (g) for each replicate was calculated based on the maximum specific growth rate (µmax or k), according to the following Eq. (2): (2) Generation time (g) = \(\:\frac{Ln\left(2\right)}{k}\) To assess the survival rate of honeybee larvae in response to treatments, the Kaplan–Meier method was used to generate survival curves, followed by the Log-rank (Mantel-Cox) test to evaluate differences between groups. 3. Results Screening of antagonistic activity of bacterial strains against P. larvae and M. plutonius A first screening of the antimicrobial activity of eleven LAB strains and fourteen Bacillus and Paenibacillus strains against P. larvae and M. plutonius was performed using the agar slab and perpendicular streak methods. A significant difference in sensitivity was observed between the two foulbrood bacteria (Mann-Whitney test, p < 0.0001), with P. larvae showing greater susceptibility to probiotic antagonism compared to M. plutonius . As shown in Fig. 1 A, all tested LAB strains exhibited antagonistic activities against P. larvae , with a mean inhibition zone (ZOI) of 8.2 ± 3.0 mm ranging from 4.0 ± 4.0 mm to 14.3 ± 3.1 mm. Only eight LAB strains showed inhibitory activity against M. plutonius , with a mean inhibition zone of 2.4 ± 1.8 mm, varying from 0 ± 0 mm to 4.7 ± 1.5 mm. The strains that demonstrated the greatest antimicrobial activity against P. larvae (ZOI > 10 mm) were Lactobacillus johnsonii B275.B, L. acidophilus B274.A and L. crispatus B629, whereas the most active strains against M. plutonius (ZOI > 3.5 mm) included L. plantarum B276, L. rhamnosus G113 and L. johnsonii B275.A. All fourteen Bacillus and Paenibacillus strains tested showed inhibitory activity against P. larvae , with a mean inhibition (ZOI) of 13.6 ± 6.1 mm, ranging from 7.7 ± 3.8 mm to 27.1 ± 6.3 mm (Fig. 1 B). In contrast, their activity against M. plutonius was significantly lower, with a mean inhibition zone of 0.5 ± 1.3 mm. Only two strains, Paenibacillus polymyxa B435.B and Bacillus amyloliquefaciens B450, showed measurable activity against M. plutonius , with ZOI of 2.7 ± 2.5 mm and 4.3 ± 7.5 mm, respectively. The strongest inhibitory effect against P. larvae (ZOI > 17 mm) was observed with P. polymyxa B435.A, B. amyloliquefaciens B24, B. subtilis B115 and P. megaterium G124. Antibacterial activity of CFS The previous approaches highlighted the activity of secreted metabolites that could diffuse through the agar and inhibit pathogens without direct cell contact, which led us to explore the antimicrobial potential of bacterial cell-free supernatants (CFS). Thus, after 48h culture, CFS of the most active LAB strains (n = 5) along with five Bacillus and Paenibacillus strains were collected and assessed for their antimicrobial activity using the broth microdilution method. The CFS from all five LAB strains completely inhibited P. larvae growth at a dose of 25% after 48h of exposure (Fig. 2 A). However, when the dose was reduced to 12.5%, the CFS from L. acidophilus B274.A lost its inhibitory effect, followed by those from L. johnsonii B275.A and B. In contrast, the CFS from L. crispatus B629 and L. plantarum B276 maintained antimicrobial activity down to 6.25%, though with only moderate inhibition, averaging 22% and 34%, respectively. Consistent with the latest results, the tested CFS demonstrated a weaker activity against M. plutonius (Fig. 2 B). As observed in the agar diffusion assay, the CFS from L. acidophilus showed no inhibitory effect at any tested doses. The other CFS remained active at 25%, but all lost their activity at 12.5%. Among the five LAB strains, L. crispatus B629 and L. plantarum B276 CFS demonstrated the strongest antimicrobial activity. Surprisingly, the antibacterial activity of Bacillus and Paenibacillus species could not be confirmed through their CFS, as either no effect was measured (data not shown). The exception was B. amyloliquefaciens B24, whose CFS exhibited inhibitory activity against P. larvae at a 25% concentration. We therefore decided to elucidate the mode of action of the LAB-derived CFS, specifically those from L. crispatus B629 and L. plantarum B276, which displayed the highest antimicrobial activity. Characterisation of CFS antibacterial activity To unravel the antagonistic mode of action of the CFS from L. crispatus B629 and L. plantarum B276, we evaluated their sensitivity to various treatments, including exposure to proteases, heat and pH variations. These treatments aimed to provide insights into the chemical nature of the antimicrobial compounds involved, as well as to evaluate the stability of the CFS, an essential factor for potential industrial-scale applications. The treated CFS were then subjected to the broth microdilution method to determine their residual activity. The assays were performed at the dose of 12.5% (v/v), previously identified as the minimum effective dose against P. larvae . After one hour of exposure to various temperatures ranging from 40°C to 121°C, conditions that mimic certain industrial processing treatments, the CFS from both L. crispatus B629 and L. plantarum B276 retained their antimicrobial activity, with no significant decrease compared to the untreated control (Fig. 3 A). Similarly, treatment with proteolytic enzymes had no impact on the antimicrobial activity of both CFS. In contrast, pH adjustment to neutral (pH 7) and alkaline (pH 10) conditions abolished their inhibitory effects. Specifically, the percentage of growth inhibition significantly decreased to 15.56 ± 28.37% and 3.68 ± 31.69% for L. crispatus B629, and to 5.95 ± 33.19% and 0.63 ± 26.41% for L. plantarum B276, at pH 7 and 10 respectively. These results suggest that the antimicrobial activity of the LAB-derived CFS is closely linked to their acidic nature. Indeed, the physiological pH values of the CFS from L. crispatus B629 and L. plantarum B276 were measured at 3.49 and 3.58, respectively. To further investigate the role of pH in the observed inhibitory effects, we exposed P. larvae to the CFS vehicle (MRS broth) adjusted to pH 3 and compared the results to those obtained with the full CFS under the same conditions (Fig. 3 B). While both CFSs maintained their antimicrobial activity at pH 3 at doses of 25% (data not shown) and 12.5%, the CFS vehicle alone at pH 3 showed a clear reduction in activity at 12.5%, with only 41.24 ± 21.56% inhibition. In contrast, the CFS from L. crispatus and L. plantarum demonstrated significantly higher inhibition rates of 99.76 ± 2.70% and 97.22 ± 25.44%, respectively (Kruskal-Wallis test, p-value = 0.028 and p-value = 0.048, respectively). Growth kinetics of P. larvae and M. plutonius exposed to CFS To further characterise the mode of action and to assess the impact of the CFS on pathogen growth, 24 h growth kinetics were carried out. The CFS were added at a concentration of 12.5% (v/v) during the mid-to-late exponential phase. This same dose was used for M. plutonius to compare the response of the two pathogens. The growth of P. larvae was significantly affected following treatment with the L. crispatus CFS, as evidenced by both a reduction in optical density and a decrease in CFU counts (Fig. 4 ) , indicating a clear slowdown in bacterial proliferation. We observed a significant increase in P. larvae growth at 24h (14h after addition of the CFS vehicle), as OD 600nm was 4.7 times higher (p-value = 0.0322). In contrast, the addition of L. crispatus B629 CFS suppressed bacterial growth, resulting in only a 1.4-fold increase in OD 600nm over the same period (p-value = 0.0869). Indeed, at the end of the kinetics, treatment with L. crispatus B629 CFS led to a 1-log10 reduction in viable P. larvae cells (p-value = 0.0417), along with a 2.73-fold decrease in OD 600nm (p-value = 0.0219) compared to the vehicle-treated group. Although weaker, L. plantarum B276 CFS also seems to exhibit antagonistic activity. Fourteen hours after CFS addition, P. larvae growth increased 2.9-fold (p-value = 0.006). At the end of the kinetic, this treatment resulted in a 0.5-log10 reduction in CFU (p-value = 0.4008) and a 1.51-fold reduction in OD 600nm (p-value = 0.5391) compared to the control. To confirm the CFS inhibitory effect, generation times were calculated for each condition (Fig. 5 ) . The mean generation time of the vehicle-exposed group was 3.674 ± 0.627 h. P. larvae exposure to L. plantarum B276 CFS extended its generation time by 1.6-fold (6.009 ± 1.040 h; p-value = 0.5391), whereas L. crispatus B629 CFS caused a significant 3.8-fold increase, with a mean generation time of 13.84 ± 2.479 h (p-value = 0.0219). Although CFS at 25% was previously identified as the minimal inhibitory dose, exposure to a 12.5% dose still resulted in a reduction of M. plutonius growth ( data not shown ). Due to variability in growth among replicates, statistical analysis could not be performed, although a similar trend in response to treatment was observed. Over a 14 h period following treatment, OD 600nm values increased 3.5-fold in the vehicle control group, compared to 2.36-fold and 1.84-fold increases in the L. plantarum B276 C and L. crispatus B629 CFS-treated groups, respectively. At the 24 h time point, growth in the L. crispatus B629 CFS group was reduced by 2.47-fold relative to the control. As observed against P. larvae , L. plantarum CFS showed a lower inhibitory effect on M. plutonius , resulting in a 1.96-fold reduction. Generation time analysis supported these observations: L. plantarum CFS-treated cultures showed a 1.7-fold increase in generation time (7.306 ± 2.279 h) compared to the control group (4.263 ± 0.7353; p-value = 0.3032), while L. crispatus CFS-treated group extended the generation time by 2.4-fold (10.41 ± 3.635 h; p value = 0.0512) (Fig. 5 ). Although these differences did not reach statistical significance, they suggest a clear tendency toward growth inhibition of M. plutonius in response to both CFS treatments. Evaluation of postbiotic and probiotic effects on P. larvae -infected laboratory-reared honeybee larvae To assess the antagonistic potential of L. plantarum B276 and L. crispatus B629, along with their CFS, an in vivo infection assay was conducted using laboratory-reared honeybee larvae. The survival of larvae was monitored until emergence with P. larvae ERIC I genotype exhibiting a slower killing phenotype, with LT100 occurring around day 12. As a preliminary experiment revealed that infected larvae fed daily with worker jelly supplemented with CFS at 12.5% exhibited slight toxicity (data not shown), we decided to use a lower concentration (6.25%) for the current study. In another previous experiment, we found that daily oral supplementation of live LAB through worker jelly had no effect on the survival rate of infected larvae. Furthermore, plating of the worker jelly supplemented with LAB revealed no survival of the LAB after 24h (data not shown). In the subsequent experiment, the survival rate of uninfected larvae was 85.2% (Fig. 6 ), corresponding to 14.8% mortality, which is within the accepted mortality threshold of 20–25% as reported by Crailsheim et al ., (2013) [ 74 ]. In contrast, the survival rate of P. larvae -infected larvae was significantly lower, with only 16.4% surviving by day 17 (p-value < 0.0001). Daily supplementation with 6.25% L. plantarum B276 or L. crispatus B629 CFS did not improve the survival of infected larvae, with survival rates of 7.14% and 15%, respectively. However, when LAB supplementation occurred 4 h before infection, a significant increase in survival was observed. Supplementation with L. crispatus B629 resulted in a 38.1% survival rate (Log rank (Mantel-Cox), df = 1, χ2 = 0.16, p-value = 0.6895), while L. plantarum B276 supplementation led to a significant increase in survival, reaching 58.9% compared to the infected group (Log rank (Mantel-Cox), df = 1, χ2 = 5.13, p-value = 0.0235). From day 6, L. plantarum B276 fed larvae displayed improved survival during pupation, whereas the infected and untreated group exhibited a persistent decline in survival. 4. Discussion Colony collapse disorder is gaining attention as researchers seek to unravel its underlying causes. Pathogens and parasites are among the key contributors to colony losses, with foulbrood diseases posing a severe and highly contagious threat to honeybees. Although various in vitro and in vivo treatments have shown promise, achieving colony-level efficacy remains challenging, and prophylactic use is not widespread. The honeybee gut microbiome plays a crucial role in development, nutrition, and defence against pathogens [ 44 ]. Disruptions in these microbial communities may predispose colonies to American and European foulbrood (AFB and EFB) [ 75 ]. In this context, bacterial supplementation emerges as a sustainable strategy to maintain gut balance and enhance disease resistance or tolerance. Our study therefore explores postbiotic and probiotic approaches against P. larvae and M. plutonius , focusing on strains capable of producing antimicrobial metabolites, their modes of action, and their potential against foulbrood in laboratory-reared larvae. The lactic acid bacteria (LAB) strains used were selected from industrial collections rather than directly isolated from honeybees, but were previously detected in honeybee-related environments (e.g., gut, honey) [ 76 – 78 ]. These strains are classified as “Generally Recognized as Safe" (GRAS) by the FDA and/or hold the "Qualified Presumption of Safety" (QPS) status from the EFSA, ensuring their suitability for large-scale production and potential application in beekeeping practices. Using agar diffusion assays, we assessed the antagonistic activity of LAB, Bacillus , and Paenibacillus species against foulbrood pathogens. LAB demonstrated strong inhibition effects on both P. larvae and M. plutonius , according to previous research [ 67 , 79 , 80 ]. Notably, Lactobacillus crispatus , a relatively unstudied species, exhibited significant inhibition of P. larvae , supporting findings on its presence in the honeybee gut [ 78 , 81 ]. We also showed inhibitory activity of several Bacillus species against P. larvae , aligning with previous studies identifying P. megaterium and B. licheniformis as effective inhibitors [ 69 ]. However, the antagonistic potential of Bacillus species against M. plutonius remains largely unexplored. Honeybee protection using probiotic strains involves the production and release of antimicrobial metabolites by bacteria. LAB are known to produce a wide range of such compounds, including organic acids, bacteriocins, and bacteriocin-like inhibitory substances (BLIS) [ 82 ]. Investigating the antimicrobial potential of LAB-derived cell-free supernatants (CFS) offers a promising alternative to live probiotics, avoiding challenges associated with maintaining bacterial viability. However, few studies have examined LAB CFS effects on foulbrood pathogens. In our study, CFS from L. plantarum and L. crispatus showed the strongest inhibition, achieving complete inhibition of P. larvae at a 12.5% dose and M. plutonius at 25% respectively. These findings align with Leska et al ., who reported L. plantarum strain 21/1 as highly effective against P. larvae ATCC 255367 (ERIC IV) [ 67 ]. Similar inhibitory patterns were observed for L. plantarum against both pathogens, while L. acidophilus was the least effective [ 80 , 83 ]. The antagonistic activity of L. crispatus against P. larvae CCM 4483 (ERIC I) was first reported by Kačániová et al ., demonstrating a zone of inhibition (ZOI) of approximately 20 mm on agar plates, one of the highest among LAB strains [ 78 , 81 ]. The same study also found that Bacillus species ( B. licheniformis , P. megaterium , B. subtilis ) exhibited weaker antagonistic effects compared to LAB. In this study, Bacillus CFS did not demonstrate antimicrobial activity, unlike in agar diffusion assays, possibly due to suboptimal conditions for metabolite production during incubation. We further analysed L. crispatus and L. plantarum CFS to identify active antimicrobial compounds and suggest their mode of action. Our results suggest that organic acids are the primary antimicrobial agents, as neutralizing the pH significantly reduced CFS activity. However, acidity alone does not fully explain the inhibitory effect, since the CFS vehicle adjusted to pH 3 at a 12.5% concentration showed only weak activity compared to LAB-derived CFS under the same conditions. Moreover, while other LAB CFS demonstrated acidic pH levels (ranging from 3.51 to 3.98), they did not display strong antimicrobial effects. Prior studies have attributed CFS antimicrobial effects to both organic acids [ 67 , 83 ] and proteinaceous compounds [ 84 – 87 ]. The observed activity likely results from a synergistic interaction of various organic acids [ 88 ], including lactic, phenyl-lactic, acetic, propionic, and butyric acids, which disrupt pathogens through multiple mechanisms. LAB are also known to produce other antimicrobial compounds, such as diacetyl, exopolysaccharides (EPS), phenyl-lactic acid (PLA), and hydrogen peroxide (H₂O₂) [ 89 ], though their presence in this study was not characterised. Despite the presence of bacteriocin-producing genes (data not shown), we have no evidence of the activity of such compounds, as CFS retained their antimicrobial activities even after protease treatment (Fig. 3 ). The twenty-four hour growth kinetics of P. larvae and M. plutonius revealed that treatment with 12.5% CFS slowed bacterial growth, as evidenced by extended generation times. Lactobacillus crispatus CFS was the most effective, though M. plutonius exhibited greater resistance. Organic acids can exert bacteriostatic or bactericidal effects, depending on factors such as the physiological state of the target organism and environmental conditions [ 90 ]. In acidic environments, weak acids lower external pH. When pH drops below their pKa, the undissociated acid form diffuses into cells, where it dissociates, releasing protons and anions. This disrupts intracellular pH, depletes ATP by interfering with the proton-motive force, and induces bacteriostasis. Additionally, acid-sensitive macromolecules, such as DNA and proteins, may denature, ultimately leading to cell death [ 90 – 92 ]. Lactic acid at a concentration of 29 mg/mL, a dose produced by LAB strains, has demonstrated antibacterial activity against Gallibacterium anatis , an opportunistic poultry pathogen. Transmission electron microscopy (TEM) analysis showed no morphological changes but revealed membrane disruption over time, including intracellular leakage and condensation after one hour of exposure. Scanning electron microscopy (SEM) further confirmed membrane rupture and pore formation [ 92 ]. In our study, TEM observations of P. larvae treated with CFS showed only minimal structural alterations, though prolonged exposure may yield stronger effects ( Supplementary Figure S1 ). Further investigation is needed to confirm intracellular condensation and alterations in cell length [ 93 ]. These results suggest that at concentrations of 12.5% for P. larvae and 25% for M. plutonius , CFS exerts a primarily bacteriostatic effect driven by organic acids. This is supported by SEM observations indicating a reduction in the number of dividing P. larvae cells following CFS treatment ( Supplementary Figure S2 ). The in vivo antagonistic activity of L. crispatus , L. plantarum , and their respective CFS was assessed on laboratory-reared honeybee larvae infected with P. larvae . CFS at doses of 12.5% and 6.25% had no significant effect on larval survival, while the highest concentration tested showed slight toxicity (data not shown). Our data showed no significant effect of CFS on the survival of infected larvae, while a previous study found that daily administration of CFS from a mixture of 13 honeybee-specific LAB strains significantly reduced larval mortality caused by P. larvae (ERIC I and II) [ 94 ]. Notably, CFS derived from multiple LAB strains showed stronger in vitro inhibitory activity than those extracted from a single strain [ 79 , 80 , 83 ]. To date, few studies have examined the use of CFS or purified metabolites against AFB or EFB at the individual level, and even fewer at the colony level. The midgut, the largest part of the honeybee larval digestive tract, serves as the primary site for P. larvae colonization [ 12 , 95 ]. Its pH ranges between 5 and 6.8 [ 18 , 96 , 97 ], similar to that of adult bees with a conventional gut microbiota [ 98 ]. In contrast, germ-free bees exhibit a more alkaline midgut environment and lower concentrations of short-chain fatty acids (SCFAs), highlighting the critical role of the microbiota in maintaining gut pH and overall physiological balance [ 98 ]. P. larvae grows at pH levels above 5 in MYPGP medium, while spore germination is significantly reduced under pH 5 [ 99 , 100 ]. Therefore, fluctuations in midgut pH may affect the dissociation dynamics of organic acid (e.g., lactic acid, pKa = 3.86), limiting the diffusion of their undissociated forms across pathogen cell membranes [ 90 ]. Additionally, during digestion, CFS components may be metabolised or absorbed by the host, reducing their effective concentration [ 101 ]. Other host-related factors may also affect infection outcomes and CFS efficiency, including immune responses, the antimicrobial properties of larval diets [ 98 , 102 , 103 ], gut microbiota defences, and general intestinal health [ 44 ]. Dietary components such as proteins and minerals may also buffer the antimicrobial action of CFS [ 104 ]. Thus, all these factors can influence the antimicrobial efficacy of CFS and, consequently, P. larvae infection potential, as it could be the case in our study. Future research should focus on isolating antimicrobial compounds and/or enhancing CFS stability, concentration, and synergies through encapsulation or stabilization [ 105 , 106 ]. Nevertheless, our experiment found that a 4 h pre-treatment with L. plantarum probiotic cells suspended in PBS significantly increased larval survival following P. larvae infection. For L. crispatus , we observed a trend toward improved larval survival although this effect was not significant. The use of probiotics in honeybees has yielded mixed results. Some studies reported no significant effect from LAB supplementation via sugar syrup at the colony level [ 107 , 108 ], while other research conducted under laboratory conditions demonstrated protective effects against AFB [ 94 ]. Daisley et al . later confirmed the efficacy of a three-strain LAB formulation delivered through nutrient patties at the colony level [ 79 ]. These discrepancies are often attributed to differences in methodology and honeybee social behaviour. Sucrose syrup, commonly used in colony-scale studies, can induce osmotic stress, leading to probiotic cell lysis. Moreover, royal jelly, a key component of larval diets in laboratory studies, contains antimicrobial compounds that may reduce probiotic efficacy [ 86 , 109 , 110 ]. Thus, assessing probiotic resistance to osmotic stress is essential when using sucrose solutions as a delivery method. Alternative approaches, such as pollen patties or direct application of LAB suspensions onto hive frames, may improve probiotic effectiveness [ 111 ]. L. plantarum is a well-characterised probiotic with strong adaptability, thriving in diverse environments [ 80 , 91 , 112 , 113 ]. Its resilience may allow it to withstand adverse factors like royal jelly and successfully colonize the larval gut, enabling its anti-AFB activity. L. plantarum has been successfully established in the honeybee gut following probiotic application [ 114 ]. The 4 h pre-treatment period used in our study may have allowed sufficient time for early colonization. This strain has demonstrated both in vitro and in vivo antagonistic activity against foulbrood pathogens [ 67 , 79 , 112 , 115 ]. L. plantarum is also known to produce exopolysaccharides that facilitate biofilm formation and exert antimicrobial effects [ 77 , 116 , 117 ]. Biofilms limit pathogen virulence and spread through niche and nutrient competition [ 118 , 119 ]. L. plantarum can also harbours tyrosine decarboxylase, which may metabolize tyrosine, a germinant for P. larvae spores [ 79 ]. Additionally, ingested L. plantarum may enhance innate immune priming in larvae by upregulating genes involved in antimicrobial peptide (AMP) production and the peritrophic matrix, increasing resistance to AFB [ 79 , 120 ]. Evidence suggests that probiotics reduce AFB through multiple mechanisms: direct inhibition of pathogens via antimicrobial compounds (e.g., organic acids), gut colonization coupled with competitive exclusion, and indirect modulation of the host immune system through AMP upregulation [ 75 ]. While not fully understood in honeybees, postbiotics also offer health benefits, including immunomodulatory effects, enhanced resistance to infections, regulation of lipid metabolism, and antioxidant activity, presenting a promising therapeutic alternative [ 121 ]. SCFAs, fermentation by-products accumulating in the bee hindgut, play key roles in microbial cross-feeding. In mammals, they are also involved in gut-brain axis communication [ 122 , 123 ]. Such mechanisms may act synergistically, potentially enhancing treatment efficacy. Postbiotics have also been shown to improve probiotic properties like auto-aggregation, hydrophobicity, and co-aggregation [ 124 ]. Additionally, exopolysaccharides produced by L. plantarum may stimulate the growth of other beneficial bacteria like B. infantis and L. acidophilus [ 125 ]. Combining highly adaptable strains like L. plantarum with honeybee-associated species like L. kunkeei may potentiate synergistic effects [ 79 ]. Thus, blends of probiotics and postbiotics may offer interesting strategies to explore in the context of honeybee health. Different strategies may be needed to control AFB and EFB. Our study found that the M. plutonius type strain was more resistant to inhibition by both live LAB and their CFS. Similarly, Leska et al. reported that only 55 out of 103 LAB strains inhibited M. plutonius , while all strains were effective against P. larvae [ 67 ]. Previous studies have shown that Bifidobacteria species slightly reduced M. plutonius growth, though not significantly [ 126 ], and Bombella apis failed to rescue infected larvae [ 127 ]. In contrast, anti- M. plutonius activity has been demonstrated for specific peptide-based antimicrobials such as kunkecin A, nisin A, and Bacillus -derived metabolites [ 85 – 87 ]. To date, only Mojgani et al . have reported in vitro inhibition of M. plutonius by LAB-derived organic acids, though their effects were less effective than those of bacteriocin- or BLIS-producing strains over a 96 h period [ 83 ]. This suggests that M. plutonius may be more susceptible to peptide-based antimicrobials than organic acids, potentially due to acid resistance mechanisms. Despite being identified over a century ago, M. plutonius remains challenging to study due to its fastidious growth and variable virulence, likely linked to genetic factors or microbial interactions [ 64 , 128 ]. Further research is needed to elucidate its acid tolerance and resistance mechanisms, which could inform the development of safe and effective treatment strategies against foulbrood diseases. 5. Conclusions The honey bee gut microbiome has become a central concern to assess host health, contributing to digestion, development, immunity and protection against pathogens. The enhancement or restoration of the gut microbiota through the introduction of beneficial microorganisms has emerged as a promising management strategy in the fight against the deadly foulbrood diseases. The present findings corroborate earlier research and offer further insights into the mode of action of beneficial lactic acid bacteria, both in vitro and in vivo . The efficacy of CFS of L. crispatus and L. plantarum against P. larvae and M. plutonius was demonstrated in vitro at varying levels of intensity and indicated a bacteriostatic effect that is predominantly attributable to organic acids. However, under our experimental conditions, only live L. plantarum rescued AFB-infected larvae. FFurther research is necessary to enhance the in vivo efficacy of both probiotics and postbiotics, with a focus on the development of innovative formulations and delivery methods. In order to ascertain the beneficial effect of probiotic and postbiotic substances, future studies should also take into account interaction with the microbiota and effect on host health. In addition, it is imperative to assess the in-hive efficiency of the treatment and its long-lasting effect in order to validate these novel alternatives for controlling AFB and EFB. Declarations The authors declare no conflict of interest. Author contributions statement Conceptualization, CM, ADZ, HEA methodology, investigation, CM, LF, JG, CP, MD, PG, CT, HEA validation, formal analysis; CM, LF, ADZ, HEA writing-original draft preparation CM writing- review and editing, CM, FD, EP, CT, MD, JYB, PG, ADZ, HEA supervision; ADZ, HEA project administration ; ADZ, HEA funding acquisition, JYB, ADZ, PG, HEA All authors have read and agreed to the published version of the manuscript. funding acquisition, JYB, ADZ, PG, HEA All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank all the members of the research team for their involvement in time-consuming bee experiments and also Christelle Blavignac and Claire Szczepaniak from the CICS platform for their help in TEM and SEM experiments. References Gallai N, Salles J-M, Settele J, Vaissière BE (2009) Economic valuation of the vulnerability of world agriculture confronted with pollinator decline. Ecol Econ 68:810–821. https://doi.org/10.1016/j.ecolecon.2008.06.014 Khalifa SAM, Elshafiey EH, Shetaia AA, El-Wahed AAA, Algethami AF, Musharraf SG, AlAjmi MF, Zhao C, Masry SHD, Abdel-Daim MM, Halabi MF, Kai G, Al Naggar Y, Bishr M, Diab MAM, El-Seedi HR (2021) Overview of Bee Pollination and Its Economic Value for Crop Production. Insects 12:688. https://doi.org/10.3390/insects12080688 Klein A-M, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc Biol Sci 274:303–313. https://doi.org/10.1098/rspb.2006.3721 Oldroyd BP (2007) What’s Killing American Honey Bees? PLOS Biol 5:e168. https://doi.org/10.1371/journal.pbio.0050168 Stokstad E (2007) Puzzling Decline of U.S. Bees Linked to Virus From Australia. Science 317:1304–1305. https://doi.org/10.1126/science.317.5843.1304 Bailey L (1983) Melissococcus pluton , the cause of European foulbrood of honey bees ( Apis spp). J Appl Bacteriol 55:65–69. https://doi.org/10.1111/j.1365-2672.1983.tb02648.x Genersch E, Forsgren E, Pentikäinen J, Ashiralieva A, Rauch S, Kilwinski J, Fries I (2006) Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. IJSEM 56:501–511. https://doi.org/10.1099/ijs.0.63928-0 Hasemann L (1961) How long can spores of American foulbrood live? Am. bee J 101:298–299 Matheson A (1993) World Bee Health Report. Bee World 74:176–212. https://doi.org/10.1080/0005772X.1993.11099183 Garcia-Gonzalez E, Genersch E (2013) Honey bee larval peritrophic matrix degradation during infection with Paenibacillus larvae , the aetiological agent of American foulbrood of honey bees, is a key step in pathogenesis. Environ Microbiol 15:2894–2901. https://doi.org/10.1111/1462-2920.12167 Poppinga L, Genersch E (2015) Molecular pathogenesis of American Foulbrood: how Paenibacillus larvae kills honey bee larvae. Curr Opin Insect Sci 10:29–36. https://doi.org/10.1016/j.cois.2015.04.013 Yue D, Nordhoff M, Wieler LH, Genersch E (2008) Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae , the causative agent of American foulbrood of honeybees ( Apis mellifera ). Environ Microbiol 10:1612–1620. https://doi.org/10.1111/j.1462-2920.2008.01579.x Grossar D, Kilchenmann V, Forsgren E, Charrière J-D, Gauthier L, Chapuisat M, Dietemann V (2020) Putative determinants of virulence in Melissococcus plutonius , the bacterial agent causing European foulbrood in honey bees. Virulence 11:554–567. https://doi.org/10.1080/21505594.2020.1768338 Nakamura K, Okumura K, Harada M, Okamoto M, Okura M, Takamatsu D (2021) Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain. Sci Rep 11:87–98. https://doi.org/10.1038/s41598-021-88302-8 Belloy L, Imdorf A, Fries I, Forsgren E, Renseigné N, Kuhn R, Charrière J-D (2007) Spatial distribution of Melissococcus plutonius in adult honey bees collected from apiaries and colonies with and without symptoms of European foulbrood. Apidologie 38:136–140. https://doi.org/10.1051/apido:2006069 Lindström A, Korpela S, Fries I (2008) The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee ( Apis mellifera ) colonies. J Invertebr Pathol 99:82–86. https://doi.org/10.1016/j.jip.2008.06.010 McKee BA, Djordjevic SP, Goodman RD, Hornitzky MA (2003) The detection of Melissococcus pluton in honey bees ( Apis mellifera ) and their products using a hemi-nested PCR. Apidologie 34:19–27. https://doi.org/10.1051/apido:2002047 Bailey L, Ball BV (1991) Honey Bee Pathology. Elsevier Hansen H, Brødsgaard CJ (1999) American foulbrood: a review of its biology, diagnosis and control. Bee World 80:5–23. https://doi.org/10.1080/0005772X.1999.11099415 Hansen H, Brødsgaard CJ (1995) Field trials with induced infection of Bacillus larvae. In: Proceedings of the XXIV International Apiculture Congress of Apimondia, Lausanne. pp 166–171 Masood F, Thebeau JM, Cloet A, Kozii IV, Zabrodski MW, Biganski S, Liang J, Marta Guarna M, Simko E, Ruzzini A, Wood SC (2022) Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees. Sci Rep 12:5906. https://doi.org/10.1038/s41598-022-09796-4 Miyagi T, Peng CYS, Chuang RY, Mussen EC, Spivak MS, Doi RH (2000) Verification of Oxytetracycline-Resistant American Foulbrood Pathogen Paenibacillus larvae in the United States. J Invertebr Pathol 75:95–96. https://doi.org/10.1006/jipa.1999.4888 Martel A-C, Zeggane S, Drajnudel P, Faucon J-P, Aubert M (2006) Tetracycline residues in honey after hive treatment. Food Addit Contam 23:265–273. https://doi.org/10.1080/02652030500469048 Jia S, Wu Y, Chen G, Wang S, Hu F, Zheng H (2022) The Pass-on Effect of Tetracycline-Induced Honey Bee ( Apis mellifera ) Gut Community Dysbiosis. Front Microbiol 12:781746. https://doi.org/10.3389/fmicb.2021.781746 Peng Y-SC, Mussen E, Fong A, Montague MA, Tyler T (1992) Effects of chlortetracycline of honey bee worker larvae reared in vitro . J Invertebr Pathol 60:127–133. https://doi.org/10.1016/0022-2011(92)90085-I Raymann K, Shaffer Z, Moran NA (2017) Antibiotic exposure perturbs the gut microbiota and elevates mortality in honeybees. PLOS Biol 15:e2001861. https://doi.org/10.1371/journal.pbio.2001861 FAO, IZSLT (2021) Responsible use of antimicrobials in beekeeping. FAO Animal Production and Health Guidelines No. 26. Rome, FAO. https://doi.org/10.4060/cb6918en European Commission (2010) Commission Regulation (EU) 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Official J Eur Union L15:1–72 Matović K, Žarković A, Debeljak Z, Vidanović D, Vasković N, Tešović B, Ćirić J (2023) American Foulbrood—Old and Always New Challenge. Vet Sci 10:180. https://doi.org/10.3390/vetsci10030180 Somerville D, Laffan J (2015) Healthy Bees: Managing pests, diseases and other disorders of the honey bee: AgGuide - A Practical Guide. NSW Agriculture Mosca M, Gyorffy A, Pietropaoli M, Giannetti L, Cersini A, Fortugno L, Formato G (2024) IPM Strategy to Control EFB in Apis mellifera : Oxytetracycline Treatment Combined with Partial Shook Swarm and Queen Caging. Vet Sci 11:28. https://doi.org/10.3390/vetsci11010028 Laate E, Emunu J, Duering A, Ovinge L (2020) Potential Economic Impact of European and American Foulbrood on Alberta’s Beekeeping Industry. Agriculture and Forestry, Government of Alberta World Organization for Animal Health (WOAH) (2024) Terrestrial Animal Health Code (32nd ed., Vol. 2, Ch. 9.2–9.3). Available at: https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&L=1&htmfile=titre_1.9.htm (Accessed March 03, 2025) Alonso-Salces RM, Cugnata NM, Guaspari E, Pellegrini MC, Aubone I, De Piano FG, Antunez K, Fuselli SR (2017) Natural strategies for the control of Paenibacillus larvae , the causative agent of American foulbrood in honey bees: a review. Apidologie 48:387–400. https://doi.org/10.1007/s13592-016-0483-1 Bava R, Castagna F, Ruga S, Nucera S, Caminiti R, Serra M, Bulotta RM, Lupia C, Marrelli M, Conforti F, Statti G, Domenico B, Palma E (2023) Plants and Their Derivatives as Promising Therapeutics for Sustainable Control of Honeybee ( Apis mellifera ) Pathogens. Pathogens 12:1260. https://doi.org/10.3390/pathogens12101260 Brady TS, Merrill BD, Hilton JA, Payne AM, Stephenson MB, Hope S (2017) Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of Paenibacillus larvae in honeybee hives. J Invertebr Pathol 150:94–100. https://doi.org/10.1016/j.jip.2017.09.010 Brady TS, Roll CR, Walker JK, Fajardo CP, Breakwell DP, Eggett DL, Hope S (2021) Phages Bind to Vegetative and Spore Forms of Paenibacillus larvae and to Vegetative Brevibacillus laterosporus . Front Microbiol 12:588035. https://doi.org/10.3389/fmicb.2021.588035 Dickel F, Bos NMP, Hughes H, Martín-Hernández R, Higes M, Kleiser A, Freitak D (2022) The oral vaccination with Paenibacillus larvae bacterin can decrease susceptibility to American Foulbrood infection in honey bees—A safety and efficacy study. Front Vet Sci 9:946237. https://doi.org/10.3389/fvets.2022.946237 Kim YH, Kim BY, Choi YS, Lee KS, Jin BR (2023) Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin-hypopharyngeal gland axis in honeybees. Dev Comp Immunol 144:104709. https://doi.org/10.1016/j.dci.2023.104709 Spivak M, Masterman R, Ross R, Mesce KA (2003) Hygienic behavior in the honey bee ( Apis mellifera L.) and the modulatory role of octopamine. J Neurobiol 55:341–354. https://doi.org/10.1002/neu.10219 Spivak M, Reuter GS (2001) Resistance to American foulbrood disease by honey bee colonies Apis mellifera bred for hygienic behavior. Apidologie 32:555–565. https://doi.org/10.1051/apido:2001103 Alvarado I, Margotta JW, Aoki MM, Flores F, Agudelo F, Michel G, Elekonich MM, Abel-Santos E (2017) Inhibitory effect of indole analogs against Paenibacillus larvae , the causal agent of American foulbrood disease. J Insect Sci 17:104. https://doi.org/10.1093/jisesa/iex080 Šamšulová V, Šedivá M, Kóňa J, Klaudiny J, Poláková M (2023) A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae . Molecules 28:2516. https://doi.org/10.3390/molecules28062516 Motta EVS, Moran NA (2023) The honeybee microbiota and its impact on health and disease. Nat Rev Microbiol 22:122–137. https://doi.org/10.1038/s41579-023-00990-3 Vásquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, Szekely L, Olofsson TC (2012) Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. PLoS ONE 7:e33188. https://doi.org/10.1371/journal.pone.0033188 Kwong WK, Moran NA (2016) Gut Microbial Communities of Social Bees. Nat Rev Microbiol 14:374–384. https://doi.org/10.1038/nrmicro.2016.43 Powell JE, Martinson VG, Urban-Mead K, Moran NA (2014) Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera . AEM 80:7378–7387. https://doi.org/10.1128/AEM.01861-14 Hamdi C, Balloi A, Essanaa J, Crotti E, Gonella E, Raddadi N, Ricci I, Boudabous A, Borin S, Manino A, Bandi C, Alma A, Daffonchio D, Cherif A (2011) Gut microbiome dysbiosis and honeybee health: Gut microbiome dysbiosis and honeybee health. J Appl Entomol 135:524–533. https://doi.org/10.1111/j.1439-0418.2010.01609.x Olofsson TC, Butler È, Markowicz P, Lindholm C, Larsson L, Vásquez A (2016) Lactic acid bacterial symbionts in honeybees – an unknown key to honey’s antimicrobial and therapeutic activities. Int Wound J 13:668–679. https://doi.org/10.1111/iwj.12345 Raymann K, Moran NA (2018) The role of the gut microbiome in health and disease of adult honey bee workers. Curr Opin Insect Sci 26:97–104. https://doi.org/10.1016/j.cois.2018.02.012 Erler S, Denner A, Bobiş O, Forsgren E, Moritz RFA (2014) Diversity of honey stores and their impact on pathogenic bacteria of the honeybee, Apis mellifera . Ecol Evol 4:3960–3967. https://doi.org/10.1002/ece3.1252 Daisley BA, Chmiel JA, Pitek AP, Thompson GJ, Reid G (2020) Missing Microbes in Bees: How Systematic Depletion of Key Symbionts Erodes Immunity. Trends Microbiol 28:1010–1021. https://doi.org/10.1016/j.tim.2020.06.006 Daisley BA, Pitek AP, Chmiel JA, Gibbons S, Chernyshova AM, Al KF, Faragalla KM, Burton JP, Thompson GJ, Reid G (2020) Lactobacillus spp. attenuate antibiotic-induced immune and microbiota dysregulation in honey bees. Commun Biol 3:1–13. https://doi.org/10.1038/s42003-020-01259-8 Sabaté DC, Cruz MS, Benítez-Ahrendts MR, Audisio MC (2012) Beneficial Effects of Bacillus subtilis subsp. subtilis Mori2, a Honey-Associated Strain, on Honeybee Colony Performance. Probiotics Antimicro Prot 4:39–46. https://doi.org/10.1007/s12602-011-9089-0 Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME (2014) The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11:506–514. https://doi.org/10.1038/nrgastro.2014.66 Motta EVS, Powell JE, Leonard SP, Moran NA (2022) Prospects for probiotics in social bees. Phil Trans R Soc B 377:20210156. https://doi.org/10.1098/rstb.2021.0156 Sbaghdi T, Garneau JR, Yersin S, Chaucheyras-Durand F, Bocquet M, Moné A, El Alaoui H, Bulet P, Blot N, Delbac F (2024) The Response of the Honey Bee Gut Microbiota to Nosema ceranae Is Modulated by the Probiotic Pediococcus acidilactici and the Neonicotinoid Thiamethoxam. Microorganisms 12:192. https://doi.org/10.3390/microorganisms12010192 Salminen S, Collado MC, Endo A, Hill C, Lebeer S, Quigley EMM, Sanders ME, Shamir R, Swann JR, Szajewska H, Vinderola G (2021) The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat Rev Gastroenterol Hepatol 18:649–667. https://doi.org/10.1038/s41575-021-00440-6 Thorakkattu P, Khanashyam AC, Shah K, Babu KS, Mundanat AS, Deliephan A, Deokar GS, Santivarangkna C, Nirmal NP (2022) Postbiotics: Current Trends in Food and Pharmaceutical Industry. Foods 11:3094. https://doi.org/10.3390/foods11193094 Wegh CAM, Geerlings SY, Knol J, Roeselers G, Belzer C (2019) Postbiotics and Their Potential Applications in Early Life Nutrition and Beyond. Int J Mol Sci 20:4673. https://doi.org/10.3390/ijms20194673 Genersch E (2008) Paenibacillus larvae and American Foulbrood – long since known and still surprising. J Verbr Lebensm 3:429–434. https://doi.org/10.1007/s00003-008-0379-8 Haynes E, Helgason T, Young JPW, Thwaites R, Budge GE (2013) A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants. Environ Microbiol Rep 5:525–529. https://doi.org/10.1111/1758-2229.12057 Holdeman LV, Moore WEC, Cato EP (1977) Anaerobe Laboratory Manual, 4th edn. Anaerobe Laboratory, Blacksburg Arai R, Tominaga K, Wu M, Okura M, Ito K, Okamura N, Onishi H, Osaki M, Sugimura Y, Yoshiyama M, Takamatsu D (2012) Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates. PLoS ONE 7:e33708. https://doi.org/10.1371/journal.pone.0033708 Dingman DW, Stahly DP (1983) Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components. Appl Environ Microbiol 46:860–869 Mahdi OS, Fisher NA (2018) Sporulation and Germination of Paenibacillus larvae Cells. Curr Protoc Microbiol 48 9E.2.1–9E.2.10. https://doi.org/10.1002/cpmc.46 Leska A, Nowak A, Szulc J, Motyl I, Czarnecka-Chrebelska KH (2022) Antagonistic Activity of Potentially Probiotic Lactic Acid Bacteria against Honeybee ( Apis mellifera L.) Pathogens. Pathogens 11:1367. https://doi.org/10.3390/pathogens11111367 Gende LB, Eguaras MJ, Fritz R (2008) Evaluation of culture media for Paenibacillus larvae applied to studies of antimicrobial activity. Rev argent microbiol 40:147–150 Alippi AM, Reynaldi FJ (2006) Inhibition of the growth of Paenibacillus larvae , the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources. J Invertebr Pathol 91:141–146. https://doi.org/10.1016/j.jip.2005.12.002 Société Française de Microbiologie (2019) Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM) (Recommandations 2019 V.2.0 Mai) [French]. Available at: https://www.sfm-microbiologie.org/2019/05/06/casfm-eucast-2019-v2/ (Accessed September, 2023) Aupinel P, Fortini D, Dufour H, Tasei J-N, Michaud B, Odoux F, Pham-Delègue M-H (2005) Improvement of artificial feeding in a standard in vitro method for rearing Apis mellifera larvae. Bull Insectology 58:107–111 Hall BG, Acar H, Nandipati A, Barlow M (2014) Growth Rates Made Easy. Mol Biol Evol 31:232–238. https://doi.org/10.1093/molbev/mst187 Petzoldt T (2016) Estimation of growth rates with package growthrates. https://github.com/tpetzoldt/growthrates/blob/master/doc/Introduction.pdf Crailsheim K, Brodschneider R, Aupinel P, Behrens D, Genersch E, Vollmann J, Riessberger-Gallé U (2013) Standard methods for artificial rearing of Apis mellifera larvae. J Apic Res 52:1–16. https://doi.org/10.3896/IBRA.1.52.1.05 Daisley BA, Pitek AP, Mallory E, Chernyshova AM, Allen-Vercoe E, Reid G, Thompson GJ (2022) Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection. Trends Microbiol 31:521–534. https://doi.org/10.1016/j.tim.2022.11.012 Brudzynski K (2021) Honey as an Ecological Reservoir of Antibacterial Compounds Produced by Antagonistic Microbial Interactions in Plant Nectars, Honey and Honey Bee. Antibiotics 10:551. https://doi.org/10.3390/antibiotics10050551 Iorizzo M, Testa B, Lombardi SJ, Ganassi S, Ianiro M, Letizia F, Succi M, Tremonte P, Vergalito F, Cozzolino A, Sorrentino E, Coppola R, Petrarca S, Mancini M, De Cristofaro A (2020) Antimicrobial Activity against Paenibacillus larvae and Functional Properties of Lactiplantibacillus plantarum Strains: Potential Benefits for Honeybee Health. Antibiotics 9:442. https://doi.org/10.3390/antibiotics9080442 Kačániová M, Terentjeva M, Žiarovská J, Kowalczewski PŁ (2020) Vitro Antagonistic Effect of Gut Bacteriota Isolated from Indigenous Honey Bees and Essential Oils against Paenibacillus larvae . Int J Mol Sci 21:6736. https://doi.org/10.3390/ijms21186736 . In Daisley BA, Pitek AP, Chmiel JA, Al KF, Chernyshova AM, Faragalla KM, Burton JP, Thompson GJ, Reid G (2020) Novel probiotic approach to counter Paenibacillus larvae infection in honey bees. ISME J 14:476–491. https://doi.org/10.1038/s41396-019-0541-6 Urcan AC, Criste AD, Bobiș O, Cornea-Cipcigan M, Giurgiu A-I, Dezmirean DS (2024) Evaluation of Functional Properties of Some Lactic Acid Bacteria Strains for Probiotic Applications in Apiculture. Microorganisms 12:1249. https://doi.org/10.3390/microorganisms12061249 Kačániová M (2019) Antagonistic effect of gut microbiota of honeybee ( Apis mellifera ) against causative agent of american foulbrood Paenibacillus larvae . JMBFS 9:478–481. https://doi.org/10.15414/jmbfs.2019.9.special.478-481 Mallory E, Freeze G, Daisley BA, Allen-Vercoe E (2024) Revisiting the role of pathogen diversity and microbial interactions in honeybee susceptibility and treatment of Melissococcus plutonius infection. Front Vet Sci 11:1495010. https://doi.org/10.3389/fvets.2024.1495010 Mojgani N, Bagheri M, Moharrami M, Toutiaee S, Rousta HM (2023) Bioactive metabolites from autochthonous lactic acid bacteria inhibit the growth of Melissococcus plutonius , causal agent of European foulbrood disease in honey bees. Entomol Exp Appl 171:386–394. https://doi.org/10.1111/eea.13295 Benitez LB, Velho RV, de Souza da Motta A, Segalin J, Brandelli A (2012) Antimicrobial factor from Bacillus amyloliquefaciens inhibits Paenibacillus larvae , the causative agent of American foulbrood. Arch Microbiol 194:177–185. https://doi.org/10.1007/s00203-011-0743-4 Endo A, Salminen S (2013) Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Syst Appl Microbiol 36:444–448. https://doi.org/10.1016/j.syapm.2013.06.002 Wu M, Sugimura Y, Iwata K, Takaya N, Takamatsu D, Kobayashi M, Taylor D, Kimura K, Yoshiyama M (2014) Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica , against Melissococcus plutonius , the causal agent of European foulbrood disease. J Insect Sci 14:129. https://doi.org/10.1093/jis/14.1.129 Zendo T, Ohashi C, Maeno S, Piao X, Salminen S, Sonomoto K, Endo A (2020) Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, Apilactobacillus kunkeei FF30-6 Isolated From Honey Bees. Front Microbiol 11:571903. https://doi.org/10.3389/fmicb.2020.571903 Ji Q-Y, Wang W, Yan H, Qu H, Liu Y, Qian Y, Gu R (2023) The Effect of Different Organic Acids and Their Combination on the Cell Barrier and Biofilm of Escherichia coli . Foods 12:3011. https://doi.org/10.3390/foods12163011 Mani-López E, Arrioja‐Bretón D, López‐Malo A (2022) The impacts of antimicrobial and antifungal activity of cell‐free supernatants from lactic acid bacteria in vitro and foods. Comp Rev Food Sci Food Safe 21:604–641. https://doi.org/10.1111/1541-4337.12872 Ricke S (2003) Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult Sci 82:632–639. https://doi.org/10.1093/ps/82.4.632 Rocchetti MT, Russo P, Capozzi V, Drider D, Spano G, Fiocco D (2021) Bioprospecting Antimicrobials from Lactiplantibacillus plantarum : Key Factors Underlying Its Probiotic Action. Int J Mol Sci 22:12076. https://doi.org/10.3390/ijms222112076 Zhang H, HuangFu H, Wang X, Zhao S, Liu Y, Lv H, Qin G, Tan Z (2021) Antibacterial Activity of Lactic Acid Producing Leuconostoc mesenteroides QZ1178 Against Pathogenic Gallibacterium anatis . Front Vet Sci 8:630294. https://doi.org/10.3389/fvets.2021.630294 Thompson JL, Hinton M (1996) Effect of short-chain fatty acids on the size of enteric bacteria. Lett Appl Microbiol 22:408–412. https://doi.org/10.1111/j.1472-765X.1996.tb01191.x Lamei S, Stephan JG, Riesbeck K, Vasquez A, Olofsson T, Nilson B, de Miranda JR, Forsgren E (2019) The secretome of honey bee-specific lactic acid bacteria inhibits Paenibacillus larvae growth. J Apic Res 58:405–412. https://doi.org/10.1080/00218839.2019.1572096 Gonçalves WG, Fernandes KM, Santana WC, Martins GF, Zanuncio JC, Serrão JE (2017) Post-embryonic changes in the hindgut of honeybee Apis mellifera workers: Morphology, cuticle deposition, apoptosis, and cell proliferation. Dev Biol 431:194–204. https://doi.org/10.1016/j.ydbio.2017.09.020 Chauvin R (1962) Nutrition De L’abeille. Annales de la nutrition et de l’alimentation 16:A41–A60. https://www.jstor.org/stable/45124670 Wardell G (1983) Honeybee nutrition and European foulbrood. Quebec, Canada Zheng H, Powell JE, Steele MI, Dietrich C, Moran NA (2017) Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. PNAS 114:4775–4780. https://doi.org/10.1073/pnas.1701819114 Alvarado I, Phui A, Elekonich MM, Abel-Santos E (2013) Requirements for In vitro Germination of Paenibacillus larvae Spores. J Bacteriol 195:1005–1011. https://doi.org/10.1128/jb.01958-12 Šedivá M, Laho M, Kohútová L, Mojzisova A, Majtan J, Klaudiny J (2018) 10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae . Molecules 23:32–36. https://doi.org/10.3390/molecules23123236 Hume ME, Corrier DE, Ivie GW, Deloach JR (1993) Metabolism of [14 C] Propionic Acid In Broiler Chicks. Poult Sci 72:786–793. https://doi.org/10.3382/ps.0720786 Evans JD, Aronstein K, Chen YP, Hetru C (2006) Immune pathways and defence mechanisms in honey bees Apis mellifera . Insect Mol Biol 15:645–656. https://doi.org/10.1111/j.1365-2583.2006.00682.x Fontana R, Mendes MA, de Souza BM, Konno K, César LMM, Malaspina O, Palma MS (2004) Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees ( Apis mellifera ). Peptides 25:919–928. https://doi.org/10.1016/j.peptides.2004.03.016 Dibner JJ, Buttin P (2002) Use of Organic Acids as a Model to Study the Impact of Gut Microflora on Nutrition and Metabolism. J Appl Poult Res 11:453–463. https://doi.org/10.1093/japr/11.4.453 Lopes LQS, Santos CG, De Almeida Vaucher R, Gende L, Raffin RP, Santos RCV (2016) Evaluation of antimicrobial activity of glycerol monolaurate nanocapsules against American foulbrood disease agent and toxicity on bees. Microb Pathog 97:183–188. https://doi.org/10.1016/j.micpath.2016.05.014 Mitsuwan W, Saengsawang P, Jeenkeawpieam J, Nissapatorn V, Pereira M, de Kitpipit L, Thomrongsuwannakij W, Poothong T, Vimon S S (2023) Development of a microencapsulated probiotic containing Pediococcus acidilactici WU222001 against avian pathogenic Escherichia coli . Vet World 16:1131–1140. https://doi.org/10.14202/vetworld.2023.1131-1140 Lamei S, Stephan JG, Nilson B, Sieuwerts S, Riesbeck K, de Miranda JR, Forsgren E (2020) Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or Paenibacillus larvae Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community. Microb Ecol 79:743–755. https://doi.org/10.1007/s00248-019-01434-3 Stephan JG, Lamei S, Pettis JS, Riesbeck K, de Miranda JR, Forsgren E (2019) Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies. Appl Environ Microbiol 85:e00606–e00619. https://doi.org/10.1128/AEM.00606-19 Forsgren E, Olofsson TC, Vásquez A, Fries I (2010) Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie 41:99–108. https://doi.org/10.1051/apido/2009065 Ptaszyńska AA, Borsuk G, Zdybicka-Barabas A, Cytryńska M, Małek W (2016) Are commercial probiotics and prebiotics effective in the treatment and prevention of honeybee nosemosis C? Parasitol Res 115:397–406. https://doi.org/10.1007/s00436-015-4761-z Daisley BA, Pitek AP, Torres C, Lowery R, Adair BA, Al KF, Niño B, Burton JP, Allen-Vercoe E, Thompson GJ, Reid G, Niño E (2023) Delivery mechanism can enhance probiotic activity against honey bee pathogens. ISME J 17:1382–1395. https://doi.org/10.1038/s41396-023-01422-z Leska A, Nowak A, Czarnecka-Chrebelska KH (2022) Adhesion and Anti-Adhesion Abilities of Potentially Probiotic Lactic Acid Bacteria and Biofilm Eradication of Honeybee ( Apis mellifera L.) Pathogens. Molecules 27:8945. https://doi.org/10.3390/molecules27248945 Zhang Z, Niu H, Qu Q, Guo D, Wan X, Yang Q, Mo Z, Tan S, Xiang Q, Tian X, Yang H, Liu Z (2025) Advancements in Lactiplantibacillus plantarum : probiotic characteristics, gene editing technologies and applications. Crit Rev Food Sci Nutr 0:1–22. https://doi.org/10.1080/10408398.2024.2448562 Javorský P, Fecskeová LK, Hrehová L, Sabo R, Legáth J, Pristas P (2017) Establishment of Lactobacillus plantarum strain in honey bee digestive tract monitored using gfp fluorescence. BM 8:291–298. https://doi.org/10.3920/BM2016.0022 Pietropaoli M, Carpana E, Milito M, Palazzetti M, Guarducci M, Croppi S, Formato G (2022) Use of Lactobacillus plantarum in Preventing Clinical Cases of American and European Foulbrood in Central Italy. Appl Sci 12:1388. https://doi.org/10.3390/app12031388 Iorizzo M, Ganassi S, Albanese G, Letizia F, Testa B, Tedino C, Petrarca S, Mutinelli F, Mazzeo A, De Cristofaro A (2022) Antimicrobial Activity from Putative Probiotic Lactic Acid Bacteria for the Biological Control of American and European Foulbrood Diseases. Vet Sci 9:236. https://doi.org/10.3390/vetsci9050236 Silva LA, Lopes Neto JHP, Cardarelli HR (2019) Exopolysaccharides produced by Lactobacillus plantarum : technological properties, biological activity, and potential application in the food industry. Ann Microbiol 69:321–328. https://doi.org/10.1007/s13213-019-01456-9 Berríos P, Fuentes JA, Salas D, Carreño A, Aldea P, Fernández F, Trombert AN (2018) Inhibitory effect of biofilm-forming Lactobacillus kunkeei strains against virulent Pseudomonas aeruginosa in vitro and in honeycomb moth ( Galleria mellonella ) infection model. BM 9:257–268. https://doi.org/10.3920/BM2017.0048 Salas-Jara MJ, Ilabaca A, Vega M, García A (2016) Biofilm Forming Lactobacillus : New Challenges for the Development of Probiotics. Microorganisms 4:35. https://doi.org/10.3390/microorganisms4030035 Yoshiyama M, Wu M, Sugimura Y, Takaya N, Kimoto-Nira H, Suzuki C (2013) Inhibition of Paenibacillus larvae by lactic acid bacteria isolated from fermented materials. J Invertebr Pathol 112:62–67. https://doi.org/10.1016/j.jip.2012.09.002 Żółkiewicz J, Marzec A, Ruszczyński M, Feleszko W (2020) Postbiotics—A Step Beyond Pre- and Probiotics. Nutrients 12:2189. https://doi.org/10.3390/nu12082189 Cabirol A, Moriano-Gutierrez S, Engel P (2023) Neuroactive metabolites modulated by the gut microbiota in honey bees. Mol Microbiol 122:284–293. https://doi.org/10.1111/mmi.15167 Kešnerová L, Mars RAT, Ellegaard KM, Troilo M, Sauer U, Engel P (2017) Disentangling metabolic functions of bacteria in the honey bee gut. PLOS Biol 15:e2003467. https://doi.org/10.1371/journal.pbio.2003467 Khojah E, Gomaa MS, Elsherbiny EG, Elawady A, Darwish MS (2022) The In vitro Analysis of Postbiotics in Functional Labneh to Be Used as Powerful Tool to Improve Cell Surfaces Properties and Adherence Potential of Probiotic Strains. Fermentation 8:122. https://doi.org/10.3390/fermentation8030122 Das D, Baruah R, Goyal A (2014) A food additive with prebiotic properties of an α-d-glucan from Lactobacillus plantarum DM5. Int J Biol Macromol 69:20–26. https://doi.org/10.1016/j.ijbiomac.2014.05.029 Wu M, Sugimura Y, Takaya N, Takamatsu D, Kobayashi M, Taylor D, Yoshiyama M (2013) Characterization of Bifidobacteria in the digestive tract of the Japanese honeybee, Apis cerana japonica . J Invertebr Pathol 112:88–93. https://doi.org/10.1016/j.jip.2012.09.005 Floyd AS, Mott BM, Maes P, Copeland DC, McFrederick QS, Anderson KE (2020) Microbial Ecology of European Foul Brood Disease in the Honey Bee ( Apis mellifera ): Towards a Microbiome Understanding of Disease Susceptibility. Insects 11:555. https://doi.org/10.3390/insects11090555 Giersch T, Barchia I, Hornitzky M (2010) Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood? Apidologie 41:151–159. https://doi.org/10.1051/apido/2009066 Additional Declarations No competing interests reported. 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CNRS","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Fourel","suffix":""},{"id":448595122,"identity":"f3eb5910-3774-40af-9b63-b8dca7f006c2","order_by":2,"name":"Juliette Gilbert","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Juliette","middleName":"","lastName":"Gilbert","suffix":""},{"id":448595123,"identity":"f42b222a-445b-40ee-bc12-e439701ab551","order_by":3,"name":"Christophe Portelli","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Portelli","suffix":""},{"id":448595124,"identity":"8c1aa52c-66d0-41df-aa3a-6b5d296726c6","order_by":4,"name":"Marie Diogon","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Diogon","suffix":""},{"id":448595125,"identity":"7850c153-1b76-43ea-99a5-7bc4e393827b","order_by":5,"name":"Frédéric Delbac","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Delbac","suffix":""},{"id":448595126,"identity":"542167e6-be0f-466d-918f-59028c4e49b5","order_by":6,"name":"Catherine Texier","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Texier","suffix":""},{"id":448595127,"identity":"de41b5db-b45c-490f-89fa-e7b2ffaade50","order_by":7,"name":"Eric Peyretaillade","email":"","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Peyretaillade","suffix":""},{"id":448595128,"identity":"45dc2e13-d347-4507-bb7b-cc8d5d500ab7","order_by":8,"name":"Pascale Goupil","email":"","orcid":"","institution":"Université Clermont Auvergne, INRAE, PIAF","correspondingAuthor":false,"prefix":"","firstName":"Pascale","middleName":"","lastName":"Goupil","suffix":""},{"id":448595129,"identity":"4dc76ef7-266f-4b0d-bef5-9558c44b0838","order_by":9,"name":"Jean Yves Berthon","email":"","orcid":"","institution":"Greentech","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Yves","lastName":"Berthon","suffix":""},{"id":448595130,"identity":"348a6274-7cda-4834-b418-c731c1cc6de3","order_by":10,"name":"Assia Dreux-Zigha","email":"","orcid":"","institution":"Greencell","correspondingAuthor":false,"prefix":"","firstName":"Assia","middleName":"","lastName":"Dreux-Zigha","suffix":""},{"id":448595131,"identity":"293a864c-c888-4caf-9fc1-ba53e4ac522b","order_by":11,"name":"Hicham El Alaoui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3PsQrCMBCA4SuBTJGuKSK+QkVIHQp5lYDQKZtLJ+1UJ3d9kc5CQJeCaydRhG5CQRAdClqVUgfT1SH/EnLwcQmAyfSHuQpwdc5csGIQoeL2a+63E+EC2kCRKuJE1T3QkHVNcGCtYkWekxayRfnhLitCGOo8yTAbHwsQ+5+EK+wNFglMa8KyYEhBTDR/IZh2ksYWlkmg1k1oiVO+iH1FZfWwpUQ3EHrS/driUompnmDW7SX0TUgaEJrmbCR0ZKdy55z4Mx4R70JCn9vz8SkrNOQTBegfGvdWYDKZTCZtD3HUTdAVFOkQAAAAAElFTkSuQmCC","orcid":"","institution":"Université Clermont Auvergne, CNRS","correspondingAuthor":true,"prefix":"","firstName":"Hicham","middleName":"El","lastName":"Alaoui","suffix":""}],"badges":[],"createdAt":"2025-04-23 08:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6510472/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6510472/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-025-10739-4","type":"published","date":"2025-09-11T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81532885,"identity":"a6f2627d-6a82-4fd7-a776-7071426c3727","added_by":"auto","created_at":"2025-04-28 09:47:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat map of the antagonistic activity of lactic acid bacteria (A) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePaenibacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species (B) against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. larvae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. plutonius\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Antagonistic activities of LAB were evaluated by the agar slab method and represented as zones of inhibition (ZOI) mean in mm (diameter of ZOI without the agar disc size). Antagonistic activities of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003especies foulbroods was evaluated by the perpendicular streak method and represented as ZOI mean in mm (ZOI correspond to the distance between each streaks). MRS, NB and BHI broth medium served as negative controls, while Tetracycline (30 µg/mL) served as positive control. Results are means of three independent experiments with three (A) and five (B) technical replicates.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/4ce379546cb12d57e4c8ef96.jpg"},{"id":81532888,"identity":"96e8ac95-0be4-4d9a-8622-5d339f5e24c1","added_by":"auto","created_at":"2025-04-28 09:47:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth inhibition (%) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. larvae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (A) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. plutonius\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(B) by cell-free supernatant of LAB species determined by the broth microdilution method.\u003c/strong\u003e Results are presented as mean ± standard deviation (SD) of growth inhibition (%) of three independent experiments. Data were obtained by OD 600 nm measurement.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/c7189a8d88d9407261b245a8.jpg"},{"id":81532883,"identity":"76d6e0b8-d44a-4127-914b-f24a6f4c3c11","added_by":"auto","created_at":"2025-04-28 09:47:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth inhibition of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. larvae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. crispatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. plantarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etreated cell-free supernatant evaluated by the broth microdilution method.\u003c/strong\u003e(A) Growth inhibition (%) of \u003cem\u003eP. larvae\u003c/em\u003e by LABs treated CFS (heat, pH and proteases) at 12,5% (v/v). (B) Growth inhibition (%) of \u003cem\u003eP. larvae \u003c/em\u003eby LABs CFS and CFS vehicle (corresponding to MRS broth) adjusted at pH 3 at 12,5% (v/v). Results are presented as box and whiskers of growth inhibition (%) of four independent experiments. Statistical differences in inhibition compared to the positive control untreated CFS are represented as * at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/f20e74ea2cf41dc60fb074ef.jpg"},{"id":81532904,"identity":"add86acd-15f4-427f-b021-5bd5d14bbf3b","added_by":"auto","created_at":"2025-04-28 09:47:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. plantarum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eB276 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. crispatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e B629 cell-free supernatant addition at 12.5% (v/v) on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. larvae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egrowth\u003c/strong\u003e. Turbidity (coloured circles) and cell viability (open circles) were monitored for 24 hours. The arrow indicates the time of CFS addition. Points are mean ± standard deviation (SD) of three replicates represented on a log-10 scale. Statistical differences are represented as * at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/2228d0842c287912935d5f8e.jpg"},{"id":81532889,"identity":"73127f46-0062-4efb-a8fc-5c479a918224","added_by":"auto","created_at":"2025-04-28 09:47:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration time of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. larvae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. plutonius\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, exposed to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. plantarum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eB276 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. crispatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eB629 cell-free supernatant at 12.5% (v/v). \u003c/strong\u003eResults are presented as scattered plot with mean ± standard deviation (SD) of three replicates. Statistical differences are represented as * at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/40effb406b577fd3735f7565.jpg"},{"id":81534640,"identity":"983a7110-4503-4568-bbe5-24e58620c6c0","added_by":"auto","created_at":"2025-04-28 10:03:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival curves of laboratory-reared first instar honey bee larvae infected and treated with cell-free supernatant or live LAB. \u003c/strong\u003eIndividuals were infected with \u003cem\u003eP. larvae \u003c/em\u003espores at 10\u003csup\u003e4\u003c/sup\u003e spores/mL and were either daily supplemented with 6.25% \u003cem\u003eL. plantarum \u003c/em\u003eB276 or \u003cem\u003eL. crispatus \u003c/em\u003eB629 CFS, or were supplemented with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of individual LAB 4-hours post-infection. Results are presented as percent survival of n= 19 – 25 larvae per condition, calculated with Kaplan-Meier method. Statistical comparison were assessed with the Mantel-Cox test and groups compared to the infected group are represented as * at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/b5078b9c26a7c4225d26318e.jpg"},{"id":91359013,"identity":"0131ddff-afb4-4be7-8c4a-aa6f8b0df6d2","added_by":"auto","created_at":"2025-09-15 16:04:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1932664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/aebe9827-c651-4b81-82e2-60f24387d02f.pdf"},{"id":81533951,"identity":"043cb5cf-c8b4-4a81-8a87-f2339c510ccc","added_by":"auto","created_at":"2025-04-28 09:55:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":213596,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/3db1654568a0070da6197cb6.pdf"},{"id":81532884,"identity":"be32d62d-d551-4514-a8b0-80d333263608","added_by":"auto","created_at":"2025-04-28 09:47:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20981,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/2ad4e48d84876bc73c37c9fc.pdf"},{"id":81534641,"identity":"b0a3dcd9-b358-4559-8cdc-2ab4c3b5a6fb","added_by":"auto","created_at":"2025-04-28 10:03:03","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":106829,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/12495b886757f41dbb29ac8f.pdf"},{"id":81532886,"identity":"98fc6c24-93c5-4e3d-924a-a3f9a15040e3","added_by":"auto","created_at":"2025-04-28 09:47:02","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16048,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6510472/v1/5fc745d874d85b8e257d191c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Probiotic and postbiotic strategies against foulbrood in honeybees: in vitro and in vivo insights","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eManaged honeybees are among the most important pollinators, contributing approximately 70% of pollination services to the main crops cultivated for human consumption and wild plants [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, over the past decade, declines in pollinator populations have been reported, as exemplified by the Colony Collapse Disorder (CCD). First reported in the USA in 2006, CCD is characterised by the sudden collapse of the colony and the loss of adult workers. Only the queen and some bees remains, as well as abundant brood and food reserves [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Multiple factors were identified as possible drivers for this global decline. While abiotic factors such as pesticide exposure, climate change, and habitat loss contribute to this decline, biotic stressors, particularly pathogens, are recognized as major threats exerting strong pressure on colony health.\u003c/p\u003e \u003cp\u003eThe two main bacterial pathogens affecting honeybees are \u003cem\u003ePaenibacillus larvae\u003c/em\u003e and \u003cem\u003eMelissococcus plutonius\u003c/em\u003e, the etiological agents of American and European Foulbrood (AFB and EFB), respectively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Both are Gram-positive bacteria. \u003cem\u003eP. larvae\u003c/em\u003e is rod-shaped and forms highly resistant spores that can remain infectious for several years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], whereas \u003cem\u003eM. plutonius\u003c/em\u003e is a lanceolate coccus. These brood diseases, first described in the 20th century, are now distributed worldwide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Infection begins when larvae ingest contaminated food brought by nurse bees, allowing the bacteria to proliferate extensively in the larval midgut. \u003cem\u003eP. larvae\u003c/em\u003e produces various virulence factors that contribute to the infection success by degrading the peritrophic matrix and enabling invasion of the haemocoel [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, \u003cem\u003eM. plutonius\u003c/em\u003e generally proliferates in the gut lumen, although its pathogenicity varies between strains and the pathomechanisms remains elusive, notably for the \u0026ldquo;typical\u0026rdquo; strain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Ultimately, both pathogens cause septicaemia and the death of the infected larvae, resulting in highly infectious cadavers. During cleaning duties, nurses spread the infection both within and between colonies [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The lack of population renewal weakens the colony, and without treatment, the disease can lead to colony collapse [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite extensive research, effective treatments for foulbrood remain limited. Oxytetracycline (OTC) is among the most widely used antibiotics in beekeeping, but its use raises several concerns. The emergence of antibiotic-resistant bacterial strains has been reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and OTC is ineffective against the highly resistant endospores of \u003cem\u003eP. larvae\u003c/em\u003e. Moreover, antibiotic residues have been detected in hive matrices, including honey, potentially compromising both its quality and safety for human consumption [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Honeybees are also impacted by antibiotics, reducing their lifespan and disturbing gut microbiota homeostasis [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These issues have led to increasing controversy over the use of antibiotics in beekeeping, prompting stricter regulations. Within the European Union, no Maximum Residue Limits (MRLs) have been established for antibiotics in beekeeping, and their use is highly restricted for both preventive and curative purposes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Given these limitations, alternative control strategies have been adopted, such as the shook swarm technique or the incineration of infected hives [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, these measures are preventive rather than curative [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Currently, no effective treatment exists for clinically infected colonies, generating important economic losses for beekeepers [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Due to their impact on epizootiology and beekeeping sustainability, these diseases are now included in the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH), requiring member states to report any outbreaks [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo address these challenges, many studies are underway to develop alternative strategies to antibiotics that ensure the safety of both consumers and honeybees. Natural products, which are seen as safer and more environmentally friendly than synthetic chemicals, are gaining global interest and support a sustainable One Health approach. Among these, plant extracts and essential oils have emerged as promising candidates and have been extensively studied in recent years [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Other innovant solutions include phage therapy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], trans-generational immune priming through oral administration of inactivated \u003cem\u003eP. larvae\u003c/em\u003e cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], selective breeding of colonies with hygienic behaviour [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and the development of specific molecules such as indole analogues and lipid-like carbohydrate derivatives [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Among these strategies, the use of beneficial microorganisms has received particular attention. An increasing number of studies are underscoring the pivotal function of the intestinal microbiota in honeybee physiology, influencing development, behaviour, nutrition and detoxification processes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Studies have suggested that the evolutionary relationship between gut bacteria and honeybees dates back over 80\u0026nbsp;million years [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The core microbiota in honeybees is specialized, highly conserved and is orally transmitted upon emergence through social interactions or contact with hive components [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This microbial community contributes to host protection against pathogens through various mechanisms, including the production of antimicrobial compounds, induction of the innate immune response, modulation of intestinal pH, promotion of beneficial bacteria growth and competitive exclusion of pathogens [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Among these microbes, symbiotic lactic acid bacteria (LAB), mostly located in the crop (honey stomach), are thought to play a key role in antimicrobial properties of honey [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], which contribute to colony-level social immunity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Probiotic research in beekeeping has expanded in recent years, focusing on restoring the gut microbiota after antibiotic treatment and exploring new management strategies for disease control [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Probiotics are defined as \u0026ldquo;live micro-organisms that, when administered in adequate amounts, confer a health benefit to the host\u0026rdquo; [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and are often included in the GRAS (Generally Recognized As Safe) and QPS (Qualified Presumption of Safety) lists, which attests to their safety. However, challenges such as ensuring probiotic viability, stability, effective colonization, and effective delivered dose can limit their efficacy [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. As many probiotics effects can rely on secreted metabolites, an emerging alternative is the use of postbiotics. These are defined as non-living microorganisms and their components that also confer health benefits to the host [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Postbiotics comprise a range of bioactive compounds, including exopolysaccharides (EPS), enzymes, short-chain fatty acids (SCFA), bacterial lysates, cell wall fragments, and cell-free supernatants (CFS), all of which have potential to modulate the gut microbiota and inhibit pathogen [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, the precise mechanisms underlying the benefits of postbiotics in honeybees remain insufficiently understood and need further investigation.\u003c/p\u003e \u003cp\u003eThe purpose of the present study was to assess the probiotic and postbiotic potential of bacterial candidates against the two foulbrood agents, \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e. We first screened the anti-foulbrood activity of a range of commercial bacterial strains and selected the most active ones for further investigation. Particular attention was given to the characterisation of their cell-free supernatants (CFS) to identify bioactive compounds and elucidate their antagonistic mechanisms. Finally, the probiotic and postbiotic effects of two selected strains, \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. crispatus\u003c/em\u003e, were evaluated in \u003cem\u003eP. larvae\u003c/em\u003e-infected and laboratory-reared larvae. The specific properties of these strains were analysed and discussed. Overall, our study provides novel insights into the development of probiotic and postbiotic-based products as a sustainable alternative for controlling AFB and EFB in beekeeping.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cb\u003eBacterial strains and growth conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, a total of 25 bacterial isolates comprising lactic acid bacteria (LAB), \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains, from the private collection of GREENCELL (GREENTECH Group) were used. These strains included: \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eLactobacillus crispatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eWeissella cibaria\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5), \u003cem\u003eBacillus licheniformis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003ePriestia megaterium\u003c/em\u003e (formerly \u003cem\u003eBacillus megaterium\u003c/em\u003e) (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eBacillus subtilis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5). Strains were selected according to previous studies reporting evidence of inhibitor activity against \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e and their registration in QPS and GRAS safety lists. \u003cem\u003eP. larvae\u003c/em\u003e B641 and \u003cem\u003eM. plutonius\u003c/em\u003e B642 were acquired from the Pasteur institute strain collection (CIP 104618T and CIP 104052T). These strains were previously assigned to the ERIC 1 genotype [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and to the phylogenetic group sequence type 1, Clonal Complex 13 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] respectively. Unless otherwise stated, LAB strains were routinely cultured under anaerobic conditions using DeMan, Rogosa et Sharpe (MRS) broth [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] or agar (OXOID). AnaeroGen\u0026trade; Atmosphere Generation Systems (Thermo Fisher Scientific) were used to create anaerobic conditions for agar media. \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains were grown on Nutrient Broth (NB) or Nutrient Agar (NA), composed of 5 g/L peptone (Gibco) and 3 g/L meat extract (Millipore). \u003cem\u003eP. larvae\u003c/em\u003e was cultured in Brain Heart Infusion (BHI) medium (OXOID), while \u003cem\u003eM. plutonius\u003c/em\u003e was grown anaerobically in KS-BHI medium according to Arai \u003cem\u003eet al.\u003c/em\u003e, (2012) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], containing 37 g/L BHI, 20.4 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (Sigma Aldrich), and 10 g/L Soluble starch (BD Difco).\u003c/p\u003e \u003cp\u003eTo obtain \u003cem\u003eP. larvae\u003c/em\u003e spores, vegetative cells were cultured on BHI agar plates for 7 days at 37\u0026deg;C. After incubation, the plates were flooded with 5 mL sterile distilled water, and the surface was scrapped with a sterile spreader to collect the spore suspension. This procedure was repeated twice and the resulting suspensions were pooled. The combined aliquot was then heated at 65\u0026deg;C for 20 min to eliminate any remaining vegetative cells, followed by centrifugation at 4000 x g for 20 min. The pellet was washed twice and finally resuspended in sterile distilled water. Spore concentration was assessed using a Malassez counting chamber (Glaswarenfabrik Karl Hecht) under a phase-contrast light microscope (Olympus CX41, 400x). To determine viability, spore suspensions were plated onto MYPGP agar medium, as described by Dingman and Stahly (1983) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The medium consisted of 10 g/L Mueller-Hinton broth (Millipore), 15 g/L yeast extract (Sigma Aldrich), 3 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1 g/L sodium pyruvate (Sigma Aldrich), and 2 g/L dextrose (OXOID). Plates were incubated at 37\u0026deg;C for 3 days. Spore suspensions were stored at 4\u0026deg;C and heat-activated at 65\u0026deg;C for 20 min before experiments [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntagonistic assays of beneficial strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAgar slab method\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo assess the inhibitory activity of the LAB strains, inhibition assays were carried out using a modified agar slab method based on Leska \u003cem\u003eet al.\u003c/em\u003e, (2022) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Briefly, each LAB strain was cultured overnight and adjusted to the density of 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, then 500 \u0026micro;L of the suspension were spread on MRS agar plates. Petri dishes were incubated anaerobically at 37\u0026deg;C for 48h. Agar disks (8mm diameter) were then cut in triplicate using sterile, reversed tips and placed on freshly inoculated plates containing \u003cem\u003eP. larvae\u003c/em\u003e or \u003cem\u003eM. plutonius\u003c/em\u003e, which were spread to a final density of 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. The medium used was a modified MYT agar [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], containing 21 g/L Mueller-Hinton broth, 15 g/L yeast extract, and 0.1 mg/L thiamine hydrochloride (Sigma Aldrich). Plates were incubated for 48 h and the inhibition diameters were measured and defined as zones of inhibition (ZOI). The diameter of the agar disc was subtracted from inhibition diameters. Tetracycline (Conda Pronadisa) at 30 \u0026micro;g/mL was used as a positive control and MRS broth was used as a negative control.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePerpendicular streak method\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the inhibitory activity of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains, the perpendicular streak method was adapted to account for the extensive and diffuse growth of these bacteria on agar plates, following a modification of the protocol by Alippi and Reynaldi, (2006) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The 14 tested strains were cultured overnight and adjusted to a density of 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and streaked (2 cm) with a 10 \u0026micro;L oese on the side of a 3% agar (Millipore) MYT petri dish. The plates were incubated at 30 or 37\u0026deg;C for 48h, depending on the optimal growth temperature for each strain. On the same time, \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e were grown overnight and adjusted at 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Using a 1 \u0026micro;L oese, five perpendicular streaks were made on the plate, ensuring they did not contact the initial streak. The edge of the streaks was marked, and the plates were incubated for a further 48 h at 37\u0026deg;C. Inhibition zones (ZOI) were then measured starting from the marked edges. Tetracycline at 30 \u0026micro;g/mL and BHI broth was used as positive and negative control respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial activity of LAB metabolites\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCFS production\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe selected the five most active LAB strains and five \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains to investigate the antagonistic activity of their metabolites. To obtain cell-free supernatants, the strains were cultured for 48h at 37\u0026deg;C and under agitation at 110 rpm. LAB strains were grown anaerobically in MRS medium, while \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e species were cultured aerobically in MYT medium. After incubation, cultures were centrifuged (4500 x g for 10 min) and the supernatants were collected. The supernatants were then filtered-sterilized using a 0.22 \u0026micro;m filter and stored at -18\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCFS antagonistic activity assays\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo determine the minimal inhibitory dose of the CFS from LAB and \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e species, the two foulbrood pathogens were exposed to different concentrations of CFS using the two-fold dilution method in a 96-well flat-bottom plate. The broth microdilution method was adapted from the CA-SFM / EUCAST guidelines for antimicrobial susceptibility testing [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The CFS was serially diluted in MYT broth, to reach final concentrations ranging from 25% (v/v) to 0.195% (v/v). Overnight cultures of honeybee pathogens were grown, adjusted to a final concentration of 10\u003csup\u003e4\u003c/sup\u003e CFU/mL and distributed in the wells. The plate was incubated at 37\u0026deg;C, under agitation at 110 rpm for 48h. After incubation, the optical density (OD) was measured at 600 nm with a Multiskan SkyHigh microplate reader. To corroborate the OD measurements, a resazurin reduction-based assay was conducted to assess the metabolic activity of the pathogens, providing an additional information of their response to the CFS. Twenty-five microliters of filter-sterilized 0.1% resazurin was added to each well and the plate was incubated for an additional 2 h. The fluorescence was measured using a Fluoroskan Ascent FL microplate reader with excitation and emission wavelengths of 544 nm and 590 nm, respectively. The experiments were conducted in biological triplicates, and each value was normalized to its corresponding blank value (CFS optical density value). Bacterial growth inhibition (%) was calculated according to the following Eq.\u0026nbsp;(1) :\u003c/p\u003e \u003cp\u003e(1) Growth inhibition (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:100-\\frac{{A}_{t}}{{A}_{c}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eA\u003csub\u003et\u003c/sub\u003e was the OD value of the test condition after 48 h and A\u003csub\u003ec\u003c/sub\u003e was the OD value of the negative control (pathogen growth in normal conditions) after 48 h.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCharacterisation of antibacterial compounds of CFS\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAfter selecting the two CFS with the strongest inhibitory activity, the nature of this activity was further investigated. CFS were treated with proteolytic enzymes, heated and modified by varying pH values. Heat treatments were done at 40\u0026deg;C, 80\u0026deg;C and 100\u0026deg;C for 1h, and at 121\u0026deg;C (110 kPa) for 20 min. The pH of the CFS was adjusted to values of pH 3, 5, 7 and 10. Additionally, the CFS were exposed to proteases. The CFS was first adjusted to pH 8 and pH 3.5 and incubated at 37\u0026deg;C under agitation at 110 rpm for 1h, in the presence of proteinase K and trypsin at 10 mg/mL, respectively. This was followed by a heat treatment at 80\u0026deg;C for 10 min to inactivate the enzymes. Afterwards, the pH was readjusted to the natural pH of the CFS. Finally, the antagonistic activity was determined using the broth microdilution method, as previously done. The experiments were conducted in technical duplicates and biological quadruplicates, and growth inhibition percentages were calculated as previously mentioned. The CFS vehicle (MRS broth) was used as a negative control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCFS effect on honeybee pathogens growth\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the impact of the CFS on the growth of the two pathogens, growth kinetics were performed. Overnight cultures of the pathogens were adjusted to a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Then, 27 mL aliquots of MYT were inoculated to a final concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU/mL and incubated at 37\u0026deg;C under agitation at 110 rpm. At two-hour intervals, the OD\u003csub\u003e600nm\u003c/sub\u003e was measured with the Ultrospec 2000 Spectrophotometer (Pharmacia Biotech), and cultures were plated on BHI agar to determine viable cell counts. The plates were incubated at 37\u0026deg;C for 48h. Once the cultures reached the late-exponential phase (OD of 0.2), the CFS was added to achieve a final concentration of 12.5% (v/v), corresponding to the previously determined minimal inhibitory dose. In the negative control, the CFS vehicle (MRS broth) was added at the same dose. The kinetic growth was monitored for 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eantagonistic assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the antagonistic potential of LAB and their CFS, an \u003cem\u003ein vivo\u003c/em\u003e infection assay was performed using laboratory-reared larvae. Frames containing first-instar larvae from three different colonies of the same apiary (UMR 6023, Clermont Auvergne University, Clermont-Ferrand, France) were removed and brought to the laboratory. Subsequently, larvae were grafted using a Chinese grafting tool and placed in plastic queen cups. They were equally distributed into a 48-well plate to minimize colony bias, with groups of n\u0026thinsp;=\u0026thinsp;30 larvae per condition. The larvae were checked under a binocular magnifying glass and those showing signs of graft injury were identified and were excluded from the experiment if dead after 12 h, resulting in 19\u0026ndash;25 larvae per condition. The experimental groups were then established as follows: the uninfected group was fed 10 \u0026micro;L of worker jelly, while the infected group received worker jelly inoculated with \u003cem\u003eP. larvae\u003c/em\u003e at a concentration of 10\u003csup\u003e4\u003c/sup\u003e spores/mL. The CFS-treated groups received worker jelly supplemented with CFS at 6,25% (v/v) and inoculated with \u003cem\u003eP. larvae\u003c/em\u003e. Finally, the last group was treated with live LAB. LAB strains were grown overnight, adjusted to 10\u003csup\u003e8\u003c/sup\u003e CFU/mL in PBS, and 2 \u0026micro;L of this suspension was provided to the larvae. After 4 h, the larvae were then fed worker jelly inoculated with 10\u003csup\u003e4\u003c/sup\u003e sp/mL of \u003cem\u003eP. larvae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe worker jelly diet consisted of 50% organic royal jelly (Naturapi) and 50% aqueous solution of D-glucose (Thermo Fisher Scientific), D-fructose (Thermo Fisher Scientific) and yeast extract, in varying amounts depending on the larvae\u0026rsquo;s developmental stage, and was fed for a period of six days [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. To prepare the supplemented diet, the CFS were first mixed with a small quantity of distilled water, which was then added to the sugars and yeast extract. The water content was adjusted to a final volume of 5 mL, before adding the royal jelly in a 1:1 (w/w) ratio. All diets were prepared beforehand and stored in aliquots at -18\u0026deg;C, with \u003cem\u003eP. larvae\u003c/em\u003e spores being added just before infection.\u003c/p\u003e \u003cp\u003eThe next days, larvae were fed with uninfected food, with or without CFS supplementation, according to the experimental groups. The amount and composition of the diets are provided in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Before feedings, diets were pre-warmed to 34.5\u0026deg;C in an incubator. Larvae were reared in desiccators with a relative humidity (R.H.) of 94%, and kept in an incubator at 34.5\u0026deg;C. Larvae survival was monitored daily under a stereomicroscope. Individuals were considered dead when they remained motionless, with no contractile movements of their spiracles, showing colour changes from pearly white to brown and lost body elasticity. Dead larvae were removed and observed under light microscope to confirm infection status. Between 6 and 7 days post-infection, when the larvae had consumed all of the provided diet, they were gently transferred to a new 48-well plate, with wells lined with a piece of Kimwipe at the bottom. The plate was then incubated in desiccators with 75% relative humidity, and incubated at 34.5\u0026deg;C. Pupae were monitored daily and considered dead when their growth stopped and exhibited a developmental stage that is incongruent with the other individuals. After a few days of observations, death was confirmed, and dead pupae were removed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, California, USA). To evaluate statistical differences between groups, non-parametric data were analysed using a Mann-Whitney U test for pairwise comparisons, and a Kruskal-Wallis test for comparisons involving more than two groups, followed by a Dunn\u0026rsquo;s post-hoc test. A significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied. These analyses were performed to compare antagonistic activities observed in agar diffusion assays and broth microdilution experiments. To analyse the effect of treatments on \u003cem\u003eP. larvae\u003c/em\u003e growth, two statistical approaches were conducted. Differences in growth between groups at each time point were analysed using the Kruskal-Wallis test, followed by Dunn\u0026rsquo;s post-hoc test. The Friedman test was employed to analyse growth over time within one group, followed by Dunn's post-hoc test. Additionally, linear regression models were performed with R Statistical Software (v4.1.2; R Core Team 2023) and the \u0026ldquo;growthrate\u0026rdquo; R package [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The generation time (g) for each replicate was calculated based on the maximum specific growth rate (\u0026micro;max or k), according to the following Eq.\u0026nbsp;(2):\u003c/p\u003e \u003cp\u003e(2) Generation time (g) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Ln\\left(2\\right)}{k}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTo assess the survival rate of honeybee larvae in response to treatments, the Kaplan\u0026ndash;Meier method was used to generate survival curves, followed by the Log-rank (Mantel-Cox) test to evaluate differences between groups.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eScreening of antagonistic activity of bacterial strains against\u003c/b\u003e \u003cb\u003eP. larvae\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eM. plutonius\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA first screening of the antimicrobial activity of eleven LAB strains and fourteen \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains against \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e was performed using the agar slab and perpendicular streak methods. A significant difference in sensitivity was observed between the two foulbrood bacteria (Mann-Whitney test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with \u003cem\u003eP. larvae\u003c/em\u003e showing greater susceptibility to probiotic antagonism compared to \u003cem\u003eM. plutonius\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, all tested LAB strains exhibited antagonistic activities against \u003cem\u003eP. larvae\u003c/em\u003e, with a mean inhibition zone (ZOI) of 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 mm ranging from 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 mm to 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 mm. Only eight LAB strains showed inhibitory activity against \u003cem\u003eM. plutonius\u003c/em\u003e, with a mean inhibition zone of 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mm, varying from 0\u0026thinsp;\u0026plusmn;\u0026thinsp;0 mm to 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mm. The strains that demonstrated the greatest antimicrobial activity against \u003cem\u003eP. larvae\u003c/em\u003e (ZOI\u0026thinsp;\u0026gt;\u0026thinsp;10 mm) were \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e B275.B, \u003cem\u003eL. acidophilus\u003c/em\u003e B274.A and \u003cem\u003eL. crispatus\u003c/em\u003e B629, whereas the most active strains against \u003cem\u003eM. plutonius\u003c/em\u003e (ZOI\u0026thinsp;\u0026gt;\u0026thinsp;3.5 mm) included \u003cem\u003eL. plantarum\u003c/em\u003e B276, \u003cem\u003eL. rhamnosus\u003c/em\u003e G113 and \u003cem\u003eL. johnsonii\u003c/em\u003e B275.A. All fourteen \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains tested showed inhibitory activity against \u003cem\u003eP. larvae\u003c/em\u003e, with a mean inhibition (ZOI) of 13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 mm, ranging from 7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 mm to 27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, their activity against \u003cem\u003eM. plutonius\u003c/em\u003e was significantly lower, with a mean inhibition zone of 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 mm. Only two strains, \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e B435.B and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B450, showed measurable activity against \u003cem\u003eM. plutonius\u003c/em\u003e, with ZOI of 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 mm and 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5 mm, respectively. The strongest inhibitory effect against \u003cem\u003eP. larvae\u003c/em\u003e (ZOI\u0026thinsp;\u0026gt;\u0026thinsp;17 mm) was observed with \u003cem\u003eP. polymyxa\u003c/em\u003e B435.A, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e B24, \u003cem\u003eB. subtilis\u003c/em\u003e B115 and \u003cem\u003eP. megaterium\u003c/em\u003e G124.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial activity of CFS\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe previous approaches highlighted the activity of secreted metabolites that could diffuse through the agar and inhibit pathogens without direct cell contact, which led us to explore the antimicrobial potential of bacterial cell-free supernatants (CFS). Thus, after 48h culture, CFS of the most active LAB strains (n\u0026thinsp;=\u0026thinsp;5) along with five \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e strains were collected and assessed for their antimicrobial activity using the broth microdilution method. The CFS from all five LAB strains completely inhibited \u003cem\u003eP. larvae\u003c/em\u003e growth at a dose of 25% after 48h of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, when the dose was reduced to 12.5%, the CFS from \u003cem\u003eL. acidophilus\u003c/em\u003e B274.A lost its inhibitory effect, followed by those from \u003cem\u003eL. johnsonii\u003c/em\u003e B275.A and B. In contrast, the CFS from \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276 maintained antimicrobial activity down to 6.25%, though with only moderate inhibition, averaging 22% and 34%, respectively. Consistent with the latest results, the tested CFS demonstrated a weaker activity against \u003cem\u003eM. plutonius\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As observed in the agar diffusion assay, the CFS from \u003cem\u003eL. acidophilus\u003c/em\u003e showed no inhibitory effect at any tested doses. The other CFS remained active at 25%, but all lost their activity at 12.5%. Among the five LAB strains, \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276 CFS demonstrated the strongest antimicrobial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSurprisingly, the antibacterial activity of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePaenibacillus\u003c/em\u003e species could not be confirmed through their CFS, as either no effect was measured (data not shown). The exception was \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e B24, whose CFS exhibited inhibitory activity against \u003cem\u003eP. larvae\u003c/em\u003e at a 25% concentration. We therefore decided to elucidate the mode of action of the LAB-derived CFS, specifically those from \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276, which displayed the highest antimicrobial activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterisation of CFS antibacterial activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo unravel the antagonistic mode of action of the CFS from \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276, we evaluated their sensitivity to various treatments, including exposure to proteases, heat and pH variations. These treatments aimed to provide insights into the chemical nature of the antimicrobial compounds involved, as well as to evaluate the stability of the CFS, an essential factor for potential industrial-scale applications. The treated CFS were then subjected to the broth microdilution method to determine their residual activity. The assays were performed at the dose of 12.5% (v/v), previously identified as the minimum effective dose against \u003cem\u003eP. larvae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAfter one hour of exposure to various temperatures ranging from 40\u0026deg;C to 121\u0026deg;C, conditions that mimic certain industrial processing treatments, the CFS from both \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276 retained their antimicrobial activity, with no significant decrease compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, treatment with proteolytic enzymes had no impact on the antimicrobial activity of both CFS. In contrast, pH adjustment to neutral (pH 7) and alkaline (pH 10) conditions abolished their inhibitory effects. Specifically, the percentage of growth inhibition significantly decreased to 15.56\u0026thinsp;\u0026plusmn;\u0026thinsp;28.37% and 3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;31.69% for \u003cem\u003eL. crispatus\u003c/em\u003e B629, and to 5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;33.19% and 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;26.41% for \u003cem\u003eL. plantarum\u003c/em\u003e B276, at pH 7 and 10 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results suggest that the antimicrobial activity of the LAB-derived CFS is closely linked to their acidic nature. Indeed, the physiological pH values of the CFS from \u003cem\u003eL. crispatus\u003c/em\u003e B629 and \u003cem\u003eL. plantarum\u003c/em\u003e B276 were measured at 3.49 and 3.58, respectively.\u003c/p\u003e \u003cp\u003eTo further investigate the role of pH in the observed inhibitory effects, we exposed \u003cem\u003eP. larvae\u003c/em\u003e to the CFS vehicle (MRS broth) adjusted to pH 3 and compared the results to those obtained with the full CFS under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). While both CFSs maintained their antimicrobial activity at pH 3 at doses of 25% (data not shown) and 12.5%, the CFS vehicle alone at pH 3 showed a clear reduction in activity at 12.5%, with only 41.24\u0026thinsp;\u0026plusmn;\u0026thinsp;21.56% inhibition. In contrast, the CFS from \u003cem\u003eL. crispatus\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e demonstrated significantly higher inhibition rates of 99.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.70% and 97.22\u0026thinsp;\u0026plusmn;\u0026thinsp;25.44%, respectively (Kruskal-Wallis test, p-value\u0026thinsp;=\u0026thinsp;0.028 and p-value\u0026thinsp;=\u0026thinsp;0.048, respectively).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth kinetics of\u003c/b\u003e \u003cb\u003eP. larvae\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eM. plutonius\u003c/b\u003e \u003cb\u003eexposed to CFS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further characterise the mode of action and to assess the impact of the CFS on pathogen growth, 24 h growth kinetics were carried out. The CFS were added at a concentration of 12.5% (v/v) during the mid-to-late exponential phase. This same dose was used for \u003cem\u003eM. plutonius\u003c/em\u003e to compare the response of the two pathogens. The growth of \u003cem\u003eP. larvae\u003c/em\u003e was significantly affected following treatment with the \u003cem\u003eL. crispatus\u003c/em\u003e CFS, as evidenced by both a reduction in optical density and a decrease in CFU counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, indicating a clear slowdown in bacterial proliferation. We observed a significant increase in \u003cem\u003eP. larvae\u003c/em\u003e growth at 24h (14h after addition of the CFS vehicle), as OD\u003csub\u003e600nm\u003c/sub\u003e was 4.7 times higher (p-value\u0026thinsp;=\u0026thinsp;0.0322). In contrast, the addition of \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS suppressed bacterial growth, resulting in only a 1.4-fold increase in OD\u003csub\u003e600nm\u003c/sub\u003e over the same period (p-value\u0026thinsp;=\u0026thinsp;0.0869). Indeed, at the end of the kinetics, treatment with \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS led to a 1-log10 reduction in viable \u003cem\u003eP. larvae\u003c/em\u003e cells (p-value\u0026thinsp;=\u0026thinsp;0.0417), along with a 2.73-fold decrease in OD\u003csub\u003e600nm\u003c/sub\u003e (p-value\u0026thinsp;=\u0026thinsp;0.0219) compared to the vehicle-treated group. Although weaker, \u003cem\u003eL. plantarum\u003c/em\u003e B276 CFS also seems to exhibit antagonistic activity. Fourteen hours after CFS addition, \u003cem\u003eP. larvae\u003c/em\u003e growth increased 2.9-fold (p-value\u0026thinsp;=\u0026thinsp;0.006). At the end of the kinetic, this treatment resulted in a 0.5-log10 reduction in CFU (p-value\u0026thinsp;=\u0026thinsp;0.4008) and a 1.51-fold reduction in OD\u003csub\u003e600nm\u003c/sub\u003e (p-value\u0026thinsp;=\u0026thinsp;0.5391) compared to the control. To confirm the CFS inhibitory effect, generation times were calculated for each condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The mean generation time of the vehicle-exposed group was 3.674\u0026thinsp;\u0026plusmn;\u0026thinsp;0.627 h. \u003cem\u003eP. larvae\u003c/em\u003e exposure to \u003cem\u003eL. plantarum\u003c/em\u003e B276 CFS extended its generation time by 1.6-fold (6.009\u0026thinsp;\u0026plusmn;\u0026thinsp;1.040 h; p-value\u0026thinsp;=\u0026thinsp;0.5391), whereas \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS caused a significant 3.8-fold increase, with a mean generation time of 13.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.479 h (p-value\u0026thinsp;=\u0026thinsp;0.0219).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough CFS at 25% was previously identified as the minimal inhibitory dose, exposure to a 12.5% dose still resulted in a reduction of \u003cem\u003eM. plutonius\u003c/em\u003e growth (\u003cb\u003edata not shown\u003c/b\u003e). Due to variability in growth among replicates, statistical analysis could not be performed, although a similar trend in response to treatment was observed. Over a 14 h period following treatment, OD\u003csub\u003e600nm\u003c/sub\u003e values increased 3.5-fold in the vehicle control group, compared to 2.36-fold and 1.84-fold increases in the \u003cem\u003eL. plantarum\u003c/em\u003e B276 C and \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS-treated groups, respectively. At the 24 h time point, growth in the \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS group was reduced by 2.47-fold relative to the control. As observed against \u003cem\u003eP. larvae\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e CFS showed a lower inhibitory effect on \u003cem\u003eM. plutonius\u003c/em\u003e, resulting in a 1.96-fold reduction. Generation time analysis supported these observations: \u003cem\u003eL. plantarum\u003c/em\u003e CFS-treated cultures showed a 1.7-fold increase in generation time (7.306\u0026thinsp;\u0026plusmn;\u0026thinsp;2.279 h) compared to the control group (4.263\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7353; p-value\u0026thinsp;=\u0026thinsp;0.3032), while \u003cem\u003eL. crispatus\u003c/em\u003e CFS-treated group extended the generation time by 2.4-fold (10.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.635 h; p value\u0026thinsp;=\u0026thinsp;0.0512) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although these differences did not reach statistical significance, they suggest a clear tendency toward growth inhibition of \u003cem\u003eM. plutonius\u003c/em\u003e in response to both CFS treatments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of postbiotic and probiotic effects on\u003c/b\u003e \u003cb\u003eP. larvae\u003c/b\u003e\u003cb\u003e-infected laboratory-reared honeybee larvae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the antagonistic potential of \u003cem\u003eL. plantarum\u003c/em\u003e B276 and \u003cem\u003eL. crispatus\u003c/em\u003e B629, along with their CFS, an \u003cem\u003ein vivo\u003c/em\u003e infection assay was conducted using laboratory-reared honeybee larvae. The survival of larvae was monitored until emergence with \u003cem\u003eP. larvae\u003c/em\u003e ERIC I genotype exhibiting a slower killing phenotype, with LT100 occurring around day 12. As a preliminary experiment revealed that infected larvae fed daily with worker jelly supplemented with CFS at 12.5% exhibited slight toxicity (data not shown), we decided to use a lower concentration (6.25%) for the current study. In another previous experiment, we found that daily oral supplementation of live LAB through worker jelly had no effect on the survival rate of infected larvae. Furthermore, plating of the worker jelly supplemented with LAB revealed no survival of the LAB after 24h (data not shown). In the subsequent experiment, the survival rate of uninfected larvae was 85.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), corresponding to 14.8% mortality, which is within the accepted mortality threshold of 20\u0026ndash;25% as reported by Crailsheim \u003cem\u003eet al\u003c/em\u003e., (2013) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In contrast, the survival rate of \u003cem\u003eP. larvae\u003c/em\u003e-infected larvae was significantly lower, with only 16.4% surviving by day 17 (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Daily supplementation with 6.25% \u003cem\u003eL. plantarum\u003c/em\u003e B276 or \u003cem\u003eL. crispatus\u003c/em\u003e B629 CFS did not improve the survival of infected larvae, with survival rates of 7.14% and 15%, respectively. However, when LAB supplementation occurred 4 h before infection, a significant increase in survival was observed. Supplementation with \u003cem\u003eL. crispatus\u003c/em\u003e B629 resulted in a 38.1% survival rate (Log rank (Mantel-Cox), df\u0026thinsp;=\u0026thinsp;1, χ2\u0026thinsp;=\u0026thinsp;0.16, p-value\u0026thinsp;=\u0026thinsp;0.6895), while \u003cem\u003eL. plantarum\u003c/em\u003e B276 supplementation led to a significant increase in survival, reaching 58.9% compared to the infected group (Log rank (Mantel-Cox), df\u0026thinsp;=\u0026thinsp;1, χ2\u0026thinsp;=\u0026thinsp;5.13, p-value\u0026thinsp;=\u0026thinsp;0.0235). From day 6, \u003cem\u003eL. plantarum\u003c/em\u003e B276 fed larvae displayed improved survival during pupation, whereas the infected and untreated group exhibited a persistent decline in survival.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eColony collapse disorder is gaining attention as researchers seek to unravel its underlying causes. Pathogens and parasites are among the key contributors to colony losses, with foulbrood diseases posing a severe and highly contagious threat to honeybees. Although various \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e treatments have shown promise, achieving colony-level efficacy remains challenging, and prophylactic use is not widespread. The honeybee gut microbiome plays a crucial role in development, nutrition, and defence against pathogens [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Disruptions in these microbial communities may predispose colonies to American and European foulbrood (AFB and EFB) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In this context, bacterial supplementation emerges as a sustainable strategy to maintain gut balance and enhance disease resistance or tolerance. Our study therefore explores postbiotic and probiotic approaches against \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e, focusing on strains capable of producing antimicrobial metabolites, their modes of action, and their potential against foulbrood in laboratory-reared larvae. The lactic acid bacteria (LAB) strains used were selected from industrial collections rather than directly isolated from honeybees, but were previously detected in honeybee-related environments (e.g., gut, honey) [\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. These strains are classified as \u0026ldquo;Generally Recognized as Safe\" (GRAS) by the FDA and/or hold the \"Qualified Presumption of Safety\" (QPS) status from the EFSA, ensuring their suitability for large-scale production and potential application in beekeeping practices.\u003c/p\u003e \u003cp\u003eUsing agar diffusion assays, we assessed the antagonistic activity of LAB, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003ePaenibacillus\u003c/em\u003e species against foulbrood pathogens. LAB demonstrated strong inhibition effects on both \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e, according to previous research [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Notably, \u003cem\u003eLactobacillus crispatus\u003c/em\u003e, a relatively unstudied species, exhibited significant inhibition of \u003cem\u003eP. larvae\u003c/em\u003e, supporting findings on its presence in the honeybee gut [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. We also showed inhibitory activity of several \u003cem\u003eBacillus\u003c/em\u003e species against \u003cem\u003eP. larvae\u003c/em\u003e, aligning with previous studies identifying \u003cem\u003eP. megaterium\u003c/em\u003e and \u003cem\u003eB. licheniformis\u003c/em\u003e as effective inhibitors [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. However, the antagonistic potential of \u003cem\u003eBacillus\u003c/em\u003e species against \u003cem\u003eM. plutonius\u003c/em\u003e remains largely unexplored.\u003c/p\u003e \u003cp\u003eHoneybee protection using probiotic strains involves the production and release of antimicrobial metabolites by bacteria. LAB are known to produce a wide range of such compounds, including organic acids, bacteriocins, and bacteriocin-like inhibitory substances (BLIS) [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Investigating the antimicrobial potential of LAB-derived cell-free supernatants (CFS) offers a promising alternative to live probiotics, avoiding challenges associated with maintaining bacterial viability. However, few studies have examined LAB CFS effects on foulbrood pathogens. In our study, CFS from \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. crispatus\u003c/em\u003e showed the strongest inhibition, achieving complete inhibition of \u003cem\u003eP. larvae\u003c/em\u003e at a 12.5% dose and \u003cem\u003eM. plutonius\u003c/em\u003e at 25% respectively. These findings align with Leska \u003cem\u003eet al\u003c/em\u003e., who reported \u003cem\u003eL. plantarum\u003c/em\u003e strain 21/1 as highly effective against \u003cem\u003eP. larvae\u003c/em\u003e ATCC 255367 (ERIC IV) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Similar inhibitory patterns were observed for \u003cem\u003eL. plantarum\u003c/em\u003e against both pathogens, while \u003cem\u003eL. acidophilus\u003c/em\u003e was the least effective [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The antagonistic activity of \u003cem\u003eL. crispatus\u003c/em\u003e against \u003cem\u003eP. larvae\u003c/em\u003e CCM 4483 (ERIC I) was first reported by Kač\u0026aacute;niov\u0026aacute; \u003cem\u003eet al\u003c/em\u003e., demonstrating a zone of inhibition (ZOI) of approximately 20 mm on agar plates, one of the highest among LAB strains [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The same study also found that \u003cem\u003eBacillus\u003c/em\u003e species (\u003cem\u003eB. licheniformis\u003c/em\u003e, \u003cem\u003eP. megaterium\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e) exhibited weaker antagonistic effects compared to LAB. In this study, \u003cem\u003eBacillus\u003c/em\u003e CFS did not demonstrate antimicrobial activity, unlike in agar diffusion assays, possibly due to suboptimal conditions for metabolite production during incubation.\u003c/p\u003e \u003cp\u003eWe further analysed \u003cem\u003eL. crispatus\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e CFS to identify active antimicrobial compounds and suggest their mode of action. Our results suggest that organic acids are the primary antimicrobial agents, as neutralizing the pH significantly reduced CFS activity. However, acidity alone does not fully explain the inhibitory effect, since the CFS vehicle adjusted to pH 3 at a 12.5% concentration showed only weak activity compared to LAB-derived CFS under the same conditions. Moreover, while other LAB CFS demonstrated acidic pH levels (ranging from 3.51 to 3.98), they did not display strong antimicrobial effects. Prior studies have attributed CFS antimicrobial effects to both organic acids [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] and proteinaceous compounds [\u003cspan additionalcitationids=\"CR85 CR86\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. The observed activity likely results from a synergistic interaction of various organic acids [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e], including lactic, phenyl-lactic, acetic, propionic, and butyric acids, which disrupt pathogens through multiple mechanisms. LAB are also known to produce other antimicrobial compounds, such as diacetyl, exopolysaccharides (EPS), phenyl-lactic acid (PLA), and hydrogen peroxide (H₂O₂) [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e], though their presence in this study was not characterised. Despite the presence of bacteriocin-producing genes (data not shown), we have no evidence of the activity of such compounds, as CFS retained their antimicrobial activities even after protease treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe twenty-four hour growth kinetics of \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e revealed that treatment with 12.5% CFS slowed bacterial growth, as evidenced by extended generation times. \u003cem\u003eLactobacillus crispatus\u003c/em\u003e CFS was the most effective, though \u003cem\u003eM. plutonius\u003c/em\u003e exhibited greater resistance. Organic acids can exert bacteriostatic or bactericidal effects, depending on factors such as the physiological state of the target organism and environmental conditions [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. In acidic environments, weak acids lower external pH. When pH drops below their pKa, the undissociated acid form diffuses into cells, where it dissociates, releasing protons and anions. This disrupts intracellular pH, depletes ATP by interfering with the proton-motive force, and induces bacteriostasis. Additionally, acid-sensitive macromolecules, such as DNA and proteins, may denature, ultimately leading to cell death [\u003cspan additionalcitationids=\"CR91\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Lactic acid at a concentration of 29 mg/mL, a dose produced by LAB strains, has demonstrated antibacterial activity against \u003cem\u003eGallibacterium anatis\u003c/em\u003e, an opportunistic poultry pathogen. Transmission electron microscopy (TEM) analysis showed no morphological changes but revealed membrane disruption over time, including intracellular leakage and condensation after one hour of exposure. Scanning electron microscopy (SEM) further confirmed membrane rupture and pore formation [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. In our study, TEM observations of \u003cem\u003eP. larvae\u003c/em\u003e treated with CFS showed only minimal structural alterations, though prolonged exposure may yield stronger effects (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Further investigation is needed to confirm intracellular condensation and alterations in cell length [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. These results suggest that at concentrations of 12.5% for \u003cem\u003eP. larvae\u003c/em\u003e and 25% for \u003cem\u003eM. plutonius\u003c/em\u003e, CFS exerts a primarily bacteriostatic effect driven by organic acids. This is supported by SEM observations indicating a reduction in the number of dividing \u003cem\u003eP. larvae\u003c/em\u003e cells following CFS treatment (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e antagonistic activity of \u003cem\u003eL. crispatus\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e, and their respective CFS was assessed on laboratory-reared honeybee larvae infected with \u003cem\u003eP. larvae\u003c/em\u003e. CFS at doses of 12.5% and 6.25% had no significant effect on larval survival, while the highest concentration tested showed slight toxicity (data not shown). Our data showed no significant effect of CFS on the survival of infected larvae, while a previous study found that daily administration of CFS from a mixture of 13 honeybee-specific LAB strains significantly reduced larval mortality caused by \u003cem\u003eP. larvae\u003c/em\u003e (ERIC I and II) [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Notably, CFS derived from multiple LAB strains showed stronger \u003cem\u003ein vitro\u003c/em\u003e inhibitory activity than those extracted from a single strain [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. To date, few studies have examined the use of CFS or purified metabolites against AFB or EFB at the individual level, and even fewer at the colony level. The midgut, the largest part of the honeybee larval digestive tract, serves as the primary site for \u003cem\u003eP. larvae\u003c/em\u003e colonization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Its pH ranges between 5 and 6.8 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e], similar to that of adult bees with a conventional gut microbiota [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. In contrast, germ-free bees exhibit a more alkaline midgut environment and lower concentrations of short-chain fatty acids (SCFAs), highlighting the critical role of the microbiota in maintaining gut pH and overall physiological balance [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. \u003cem\u003eP. larvae\u003c/em\u003e grows at pH levels above 5 in MYPGP medium, while spore germination is significantly reduced under pH 5 [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. Therefore, fluctuations in midgut pH may affect the dissociation dynamics of organic acid (e.g., lactic acid, pKa\u0026thinsp;=\u0026thinsp;3.86), limiting the diffusion of their undissociated forms across pathogen cell membranes [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Additionally, during digestion, CFS components may be metabolised or absorbed by the host, reducing their effective concentration [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. Other host-related factors may also affect infection outcomes and CFS efficiency, including immune responses, the antimicrobial properties of larval diets [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e, \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e], gut microbiota defences, and general intestinal health [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Dietary components such as proteins and minerals may also buffer the antimicrobial action of CFS [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. Thus, all these factors can influence the antimicrobial efficacy of CFS and, consequently, \u003cem\u003eP. larvae\u003c/em\u003e infection potential, as it could be the case in our study. Future research should focus on isolating antimicrobial compounds and/or enhancing CFS stability, concentration, and synergies through encapsulation or stabilization [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, our experiment found that a 4 h pre-treatment with \u003cem\u003eL. plantarum\u003c/em\u003e probiotic cells suspended in PBS significantly increased larval survival following \u003cem\u003eP. larvae\u003c/em\u003e infection. For \u003cem\u003eL. crispatus\u003c/em\u003e, we observed a trend toward improved larval survival although this effect was not significant. The use of probiotics in honeybees has yielded mixed results. Some studies reported no significant effect from LAB supplementation via sugar syrup at the colony level [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e, \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e], while other research conducted under laboratory conditions demonstrated protective effects against AFB [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Daisley \u003cem\u003eet al\u003c/em\u003e. later confirmed the efficacy of a three-strain LAB formulation delivered through nutrient patties at the colony level [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. These discrepancies are often attributed to differences in methodology and honeybee social behaviour. Sucrose syrup, commonly used in colony-scale studies, can induce osmotic stress, leading to probiotic cell lysis. Moreover, royal jelly, a key component of larval diets in laboratory studies, contains antimicrobial compounds that may reduce probiotic efficacy [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e, \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. Thus, assessing probiotic resistance to osmotic stress is essential when using sucrose solutions as a delivery method. Alternative approaches, such as pollen patties or direct application of LAB suspensions onto hive frames, may improve probiotic effectiveness [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e is a well-characterised probiotic with strong adaptability, thriving in diverse environments [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e, \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e]. Its resilience may allow it to withstand adverse factors like royal jelly and successfully colonize the larval gut, enabling its anti-AFB activity. \u003cem\u003eL. plantarum\u003c/em\u003e has been successfully established in the honeybee gut following probiotic application [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. The 4 h pre-treatment period used in our study may have allowed sufficient time for early colonization. This strain has demonstrated both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antagonistic activity against foulbrood pathogens [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e, \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e is also known to produce exopolysaccharides that facilitate biofilm formation and exert antimicrobial effects [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e]. Biofilms limit pathogen virulence and spread through niche and nutrient competition [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e, \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e can also harbours tyrosine decarboxylase, which may metabolize tyrosine, a germinant for \u003cem\u003eP. larvae\u003c/em\u003e spores [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Additionally, ingested \u003cem\u003eL. plantarum\u003c/em\u003e may enhance innate immune priming in larvae by upregulating genes involved in antimicrobial peptide (AMP) production and the peritrophic matrix, increasing resistance to AFB [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvidence suggests that probiotics reduce AFB through multiple mechanisms: direct inhibition of pathogens via antimicrobial compounds (e.g., organic acids), gut colonization coupled with competitive exclusion, and indirect modulation of the host immune system through AMP upregulation [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. While not fully understood in honeybees, postbiotics also offer health benefits, including immunomodulatory effects, enhanced resistance to infections, regulation of lipid metabolism, and antioxidant activity, presenting a promising therapeutic alternative [\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e]. SCFAs, fermentation by-products accumulating in the bee hindgut, play key roles in microbial cross-feeding. In mammals, they are also involved in gut-brain axis communication [\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e]. Such mechanisms may act synergistically, potentially enhancing treatment efficacy. Postbiotics have also been shown to improve probiotic properties like auto-aggregation, hydrophobicity, and co-aggregation [\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e]. Additionally, exopolysaccharides produced by \u003cem\u003eL. plantarum\u003c/em\u003e may stimulate the growth of other beneficial bacteria like \u003cem\u003eB. infantis\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e [\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e]. Combining highly adaptable strains like \u003cem\u003eL. plantarum\u003c/em\u003e with honeybee-associated species like \u003cem\u003eL. kunkeei\u003c/em\u003e may potentiate synergistic effects [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Thus, blends of probiotics and postbiotics may offer interesting strategies to explore in the context of honeybee health.\u003c/p\u003e \u003cp\u003eDifferent strategies may be needed to control AFB and EFB. Our study found that the \u003cem\u003eM. plutonius\u003c/em\u003e type strain was more resistant to inhibition by both live LAB and their CFS. Similarly, Leska \u003cem\u003eet al.\u003c/em\u003e reported that only 55 out of 103 LAB strains inhibited \u003cem\u003eM. plutonius\u003c/em\u003e, while all strains were effective against \u003cem\u003eP. larvae\u003c/em\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Previous studies have shown that Bifidobacteria species slightly reduced \u003cem\u003eM. plutonius\u003c/em\u003e growth, though not significantly [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e], and \u003cem\u003eBombella apis\u003c/em\u003e failed to rescue infected larvae [\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e]. In contrast, anti-\u003cem\u003eM. plutonius\u003c/em\u003e activity has been demonstrated for specific peptide-based antimicrobials such as kunkecin A, nisin A, and \u003cem\u003eBacillus\u003c/em\u003e-derived metabolites [\u003cspan additionalcitationids=\"CR86\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. To date, only Mojgani \u003cem\u003eet al\u003c/em\u003e. have reported \u003cem\u003ein vitro\u003c/em\u003e inhibition of \u003cem\u003eM. plutonius\u003c/em\u003e by LAB-derived organic acids, though their effects were less effective than those of bacteriocin- or BLIS-producing strains over a 96 h period [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. This suggests that \u003cem\u003eM. plutonius\u003c/em\u003e may be more susceptible to peptide-based antimicrobials than organic acids, potentially due to acid resistance mechanisms. Despite being identified over a century ago, \u003cem\u003eM. plutonius\u003c/em\u003e remains challenging to study due to its fastidious growth and variable virulence, likely linked to genetic factors or microbial interactions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e]. Further research is needed to elucidate its acid tolerance and resistance mechanisms, which could inform the development of safe and effective treatment strategies against foulbrood diseases.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe honey bee gut microbiome has become a central concern to assess host health, contributing to digestion, development, immunity and protection against pathogens. The enhancement or restoration of the gut microbiota through the introduction of beneficial microorganisms has emerged as a promising management strategy in the fight against the deadly foulbrood diseases. The present findings corroborate earlier research and offer further insights into the mode of action of beneficial lactic acid bacteria, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The efficacy of CFS of \u003cem\u003eL. crispatus\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e against \u003cem\u003eP. larvae\u003c/em\u003e and \u003cem\u003eM. plutonius\u003c/em\u003e was demonstrated \u003cem\u003ein vitro\u003c/em\u003e at varying levels of intensity and indicated a bacteriostatic effect that is predominantly attributable to organic acids. However, under our experimental conditions, only live \u003cem\u003eL. plantarum\u003c/em\u003e rescued AFB-infected larvae. FFurther research is necessary to enhance the \u003cem\u003ein vivo\u003c/em\u003e efficacy of both probiotics and postbiotics, with a focus on the development of innovative formulations and delivery methods. In order to ascertain the beneficial effect of probiotic and postbiotic substances, future studies should also take into account interaction with the microbiota and effect on host health. In addition, it is imperative to assess the in-hive efficiency of the treatment and its long-lasting effect in order to validate these novel alternatives for controlling AFB and EFB.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, CM, ADZ, HEA\u003c/p\u003e\n\u003cp\u003emethodology, investigation, CM, LF, JG, CP, MD, PG, CT, HEA\u003c/p\u003e\n\u003cp\u003evalidation, formal analysis; CM, LF, ADZ, HEA\u003c/p\u003e\n\u003cp\u003ewriting-original draft preparation CM\u003c/p\u003e\n\u003cp\u003ewriting- review and editing, CM, FD, EP, CT, MD, JYB, PG, ADZ, HEA\u003c/p\u003e\n\u003cp\u003esupervision; ADZ, HEA\u003c/p\u003e\n\u003cp\u003eproject administration ; ADZ, HEA\u003c/p\u003e\n\u003cp\u003efunding acquisition, JYB, ADZ, PG, HEA\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003efunding\u003c/h2\u003e \u003cp\u003eacquisition, JYB, ADZ, PG, HEA\u003c/p\u003e \u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank all the members of the research team for their involvement in time-consuming bee experiments and also Christelle Blavignac and Claire Szczepaniak from the CICS platform for their help in TEM and SEM experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGallai N, Salles J-M, Settele J, Vaissi\u0026egrave;re BE (2009) Economic valuation of the vulnerability of world agriculture confronted with pollinator decline. Ecol Econ 68:810\u0026ndash;821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecolecon.2008.06.014\u003c/span\u003e\u003cspan address=\"10.1016/j.ecolecon.2008.06.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalifa SAM, Elshafiey EH, Shetaia AA, El-Wahed AAA, Algethami AF, Musharraf SG, AlAjmi MF, Zhao C, Masry SHD, Abdel-Daim MM, Halabi MF, Kai G, Al Naggar Y, Bishr M, Diab MAM, El-Seedi HR (2021) Overview of Bee Pollination and Its Economic Value for Crop Production. Insects 12:688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects12080688\u003c/span\u003e\u003cspan address=\"10.3390/insects12080688\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlein A-M, Vaissi\u0026egrave;re BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc Biol Sci 274:303\u0026ndash;313. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2006.3721\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2006.3721\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOldroyd BP (2007) What\u0026rsquo;s Killing American Honey Bees? PLOS Biol 5:e168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.0050168\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.0050168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStokstad E (2007) Puzzling Decline of U.S. Bees Linked to Virus From Australia. Science 317:1304\u0026ndash;1305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.317.5843.1304\u003c/span\u003e\u003cspan address=\"10.1126/science.317.5843.1304\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey L (1983) \u003cem\u003eMelissococcus pluton\u003c/em\u003e, the cause of European foulbrood of honey bees (\u003cem\u003eApis\u003c/em\u003e spp). J Appl Bacteriol 55:65\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2672.1983.tb02648.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.1983.tb02648.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenersch E, Forsgren E, Pentik\u0026auml;inen J, Ashiralieva A, Rauch S, Kilwinski J, Fries I (2006) Reclassification of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e subsp. \u003cem\u003epulvifaciens\u003c/em\u003e and \u003cem\u003ePaenibacillus larvae\u003c/em\u003e subsp. \u003cem\u003elarvae\u003c/em\u003e as \u003cem\u003ePaenibacillus larvae\u003c/em\u003e without subspecies differentiation. IJSEM 56:501\u0026ndash;511. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijs.0.63928-0\u003c/span\u003e\u003cspan address=\"10.1099/ijs.0.63928-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasemann L (1961) How long can spores of American foulbrood live? Am. bee J 101:298\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatheson A (1993) World Bee Health Report. Bee World 74:176\u0026ndash;212. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/0005772X.1993.11099183\u003c/span\u003e\u003cspan address=\"10.1080/0005772X.1993.11099183\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia-Gonzalez E, Genersch E (2013) Honey bee larval peritrophic matrix degradation during infection with \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the aetiological agent of American foulbrood of honey bees, is a key step in pathogenesis. Environ Microbiol 15:2894\u0026ndash;2901. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.12167\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.12167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoppinga L, Genersch E (2015) Molecular pathogenesis of American Foulbrood: how \u003cem\u003ePaenibacillus larvae\u003c/em\u003e kills honey bee larvae. Curr Opin Insect Sci 10:29\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cois.2015.04.013\u003c/span\u003e\u003cspan address=\"10.1016/j.cois.2015.04.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYue D, Nordhoff M, Wieler LH, Genersch E (2008) Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) analysis of the interactions between honeybee larvae and \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the causative agent of American foulbrood of honeybees (\u003cem\u003eApis mellifera\u003c/em\u003e). Environ Microbiol 10:1612\u0026ndash;1620. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1462-2920.2008.01579.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-2920.2008.01579.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrossar D, Kilchenmann V, Forsgren E, Charri\u0026egrave;re J-D, Gauthier L, Chapuisat M, Dietemann V (2020) Putative determinants of virulence in \u003cem\u003eMelissococcus plutonius\u003c/em\u003e, the bacterial agent causing European foulbrood in honey bees. Virulence 11:554\u0026ndash;567. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21505594.2020.1768338\u003c/span\u003e\u003cspan address=\"10.1080/21505594.2020.1768338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura K, Okumura K, Harada M, Okamoto M, Okura M, Takamatsu D (2021) Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent \u003cem\u003eMelissococcus plutonius\u003c/em\u003e strain. Sci Rep 11:87\u0026ndash;98. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-88302-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-88302-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelloy L, Imdorf A, Fries I, Forsgren E, Renseign\u0026eacute; N, Kuhn R, Charri\u0026egrave;re J-D (2007) Spatial distribution of \u003cem\u003eMelissococcus plutonius\u003c/em\u003e in adult honey bees collected from apiaries and colonies with and without symptoms of European foulbrood. Apidologie 38:136\u0026ndash;140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido:2006069\u003c/span\u003e\u003cspan address=\"10.1051/apido:2006069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindstr\u0026ouml;m A, Korpela S, Fries I (2008) The distribution of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e) colonies. J Invertebr Pathol 99:82\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2008.06.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2008.06.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKee BA, Djordjevic SP, Goodman RD, Hornitzky MA (2003) The detection of \u003cem\u003eMelissococcus pluton\u003c/em\u003e in honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e) and their products using a hemi-nested PCR. Apidologie 34:19\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido:2002047\u003c/span\u003e\u003cspan address=\"10.1051/apido:2002047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey L, Ball BV (1991) Honey Bee Pathology. Elsevier\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansen H, Br\u0026oslash;dsgaard CJ (1999) American foulbrood: a review of its biology, diagnosis and control. Bee World 80:5\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/0005772X.1999.11099415\u003c/span\u003e\u003cspan address=\"10.1080/0005772X.1999.11099415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansen H, Br\u0026oslash;dsgaard CJ (1995) Field trials with induced infection of \u003cem\u003eBacillus larvae.\u003c/em\u003e In: Proceedings of the XXIV International Apiculture Congress of Apimondia, Lausanne. pp 166\u0026ndash;171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasood F, Thebeau JM, Cloet A, Kozii IV, Zabrodski MW, Biganski S, Liang J, Marta Guarna M, Simko E, Ruzzini A, Wood SC (2022) Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees. Sci Rep 12:5906. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-09796-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-09796-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyagi T, Peng CYS, Chuang RY, Mussen EC, Spivak MS, Doi RH (2000) Verification of Oxytetracycline-Resistant American Foulbrood Pathogen \u003cem\u003ePaenibacillus larvae\u003c/em\u003e in the United States. J Invertebr Pathol 75:95\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/jipa.1999.4888\u003c/span\u003e\u003cspan address=\"10.1006/jipa.1999.4888\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartel A-C, Zeggane S, Drajnudel P, Faucon J-P, Aubert M (2006) Tetracycline residues in honey after hive treatment. Food Addit Contam 23:265\u0026ndash;273. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/02652030500469048\u003c/span\u003e\u003cspan address=\"10.1080/02652030500469048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia S, Wu Y, Chen G, Wang S, Hu F, Zheng H (2022) The Pass-on Effect of Tetracycline-Induced Honey Bee (\u003cem\u003eApis mellifera\u003c/em\u003e) Gut Community Dysbiosis. Front Microbiol 12:781746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2021.781746\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.781746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng Y-SC, Mussen E, Fong A, Montague MA, Tyler T (1992) Effects of chlortetracycline of honey bee worker larvae reared \u003cem\u003ein vitro\u003c/em\u003e. J Invertebr Pathol 60:127\u0026ndash;133. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-2011(92)90085-I\u003c/span\u003e\u003cspan address=\"10.1016/0022-2011(92)90085-I\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaymann K, Shaffer Z, Moran NA (2017) Antibiotic exposure perturbs the gut microbiota and elevates mortality in honeybees. PLOS Biol 15:e2001861. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.2001861\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.2001861\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFAO, IZSLT (2021) Responsible use of antimicrobials in beekeeping. FAO Animal Production and Health Guidelines No. 26. Rome, FAO. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4060/cb6918en\u003c/span\u003e\u003cspan address=\"10.4060/cb6918en\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Commission (2010) Commission Regulation (EU) 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Official J Eur Union L15:1\u0026ndash;72\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatović K, Žarković A, Debeljak Z, Vidanović D, Vasković N, Tešović B, Ćirić J (2023) American Foulbrood\u0026mdash;Old and Always New Challenge. Vet Sci 10:180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vetsci10030180\u003c/span\u003e\u003cspan address=\"10.3390/vetsci10030180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomerville D, Laffan J (2015) Healthy Bees: Managing pests, diseases and other disorders of the honey bee: AgGuide - A Practical Guide. NSW Agriculture\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosca M, Gyorffy A, Pietropaoli M, Giannetti L, Cersini A, Fortugno L, Formato G (2024) IPM Strategy to Control EFB in \u003cem\u003eApis mellifera\u003c/em\u003e: Oxytetracycline Treatment Combined with Partial Shook Swarm and Queen Caging. Vet Sci 11:28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vetsci11010028\u003c/span\u003e\u003cspan address=\"10.3390/vetsci11010028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaate E, Emunu J, Duering A, Ovinge L (2020) Potential Economic Impact of European and American Foulbrood on Alberta\u0026rsquo;s Beekeeping Industry. Agriculture and Forestry, Government of Alberta\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Organization for Animal Health (WOAH) (2024) Terrestrial Animal Health Code (32nd ed., Vol. 2, Ch. 9.2\u0026ndash;9.3). Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169\u0026amp;L=1\u0026amp;htmfile=titre_1.9.htm\u003c/span\u003e\u003cspan address=\"https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169\u0026amp;L=1\u0026amp;htmfile=titre_1.9.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Accessed March 03, 2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlonso-Salces RM, Cugnata NM, Guaspari E, Pellegrini MC, Aubone I, De Piano FG, Antunez K, Fuselli SR (2017) Natural strategies for the control of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the causative agent of American foulbrood in honey bees: a review. Apidologie 48:387\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13592-016-0483-1\u003c/span\u003e\u003cspan address=\"10.1007/s13592-016-0483-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBava R, Castagna F, Ruga S, Nucera S, Caminiti R, Serra M, Bulotta RM, Lupia C, Marrelli M, Conforti F, Statti G, Domenico B, Palma E (2023) Plants and Their Derivatives as Promising Therapeutics for Sustainable Control of Honeybee (\u003cem\u003eApis mellifera\u003c/em\u003e) Pathogens. Pathogens 12:1260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens12101260\u003c/span\u003e\u003cspan address=\"10.3390/pathogens12101260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrady TS, Merrill BD, Hilton JA, Payne AM, Stephenson MB, Hope S (2017) Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e in honeybee hives. J Invertebr Pathol 150:94\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2017.09.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2017.09.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrady TS, Roll CR, Walker JK, Fajardo CP, Breakwell DP, Eggett DL, Hope S (2021) Phages Bind to Vegetative and Spore Forms of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e and to Vegetative \u003cem\u003eBrevibacillus laterosporus\u003c/em\u003e. Front Microbiol 12:588035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2021.588035\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.588035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDickel F, Bos NMP, Hughes H, Mart\u0026iacute;n-Hern\u0026aacute;ndez R, Higes M, Kleiser A, Freitak D (2022) The oral vaccination with \u003cem\u003ePaenibacillus larvae\u003c/em\u003e bacterin can decrease susceptibility to American Foulbrood infection in honey bees\u0026mdash;A safety and efficacy study. Front Vet Sci 9:946237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2022.946237\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2022.946237\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim YH, Kim BY, Choi YS, Lee KS, Jin BR (2023) Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin-hypopharyngeal gland axis in honeybees. Dev Comp Immunol 144:104709. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dci.2023.104709\u003c/span\u003e\u003cspan address=\"10.1016/j.dci.2023.104709\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpivak M, Masterman R, Ross R, Mesce KA (2003) Hygienic behavior in the honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e L.) and the modulatory role of octopamine. J Neurobiol 55:341\u0026ndash;354. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/neu.10219\u003c/span\u003e\u003cspan address=\"10.1002/neu.10219\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpivak M, Reuter GS (2001) Resistance to American foulbrood disease by honey bee colonies \u003cem\u003eApis mellifera\u003c/em\u003e bred for hygienic behavior. Apidologie 32:555\u0026ndash;565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido:2001103\u003c/span\u003e\u003cspan address=\"10.1051/apido:2001103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarado I, Margotta JW, Aoki MM, Flores F, Agudelo F, Michel G, Elekonich MM, Abel-Santos E (2017) Inhibitory effect of indole analogs against \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the causal agent of American foulbrood disease. J Insect Sci 17:104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jisesa/iex080\u003c/span\u003e\u003cspan address=\"10.1093/jisesa/iex080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŠamšulov\u0026aacute; V, Šediv\u0026aacute; M, K\u0026oacute;ňa J, Klaudiny J, Pol\u0026aacute;kov\u0026aacute; M (2023) A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against \u003cem\u003ePaenibacillus larvae\u003c/em\u003e. Molecules 28:2516. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28062516\u003c/span\u003e\u003cspan address=\"10.3390/molecules28062516\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotta EVS, Moran NA (2023) The honeybee microbiota and its impact on health and disease. Nat Rev Microbiol 22:122\u0026ndash;137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-023-00990-3\u003c/span\u003e\u003cspan address=\"10.1038/s41579-023-00990-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026aacute;squez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, Szekely L, Olofsson TC (2012) Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. PLoS ONE 7:e33188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0033188\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0033188\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwong WK, Moran NA (2016) Gut Microbial Communities of Social Bees. Nat Rev Microbiol 14:374\u0026ndash;384. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro.2016.43\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2016.43\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell JE, Martinson VG, Urban-Mead K, Moran NA (2014) Routes of Acquisition of the Gut Microbiota of the Honey Bee \u003cem\u003eApis mellifera\u003c/em\u003e. AEM 80:7378\u0026ndash;7387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.01861-14\u003c/span\u003e\u003cspan address=\"10.1128/AEM.01861-14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamdi C, Balloi A, Essanaa J, Crotti E, Gonella E, Raddadi N, Ricci I, Boudabous A, Borin S, Manino A, Bandi C, Alma A, Daffonchio D, Cherif A (2011) Gut microbiome dysbiosis and honeybee health: Gut microbiome dysbiosis and honeybee health. J Appl Entomol 135:524\u0026ndash;533. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1439-0418.2010.01609.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0418.2010.01609.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlofsson TC, Butler \u0026Egrave;, Markowicz P, Lindholm C, Larsson L, V\u0026aacute;squez A (2016) Lactic acid bacterial symbionts in honeybees \u0026ndash; an unknown key to honey\u0026rsquo;s antimicrobial and therapeutic activities. Int Wound J 13:668\u0026ndash;679. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/iwj.12345\u003c/span\u003e\u003cspan address=\"10.1111/iwj.12345\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaymann K, Moran NA (2018) The role of the gut microbiome in health and disease of adult honey bee workers. Curr Opin Insect Sci 26:97\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cois.2018.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.cois.2018.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErler S, Denner A, Bobiş O, Forsgren E, Moritz RFA (2014) Diversity of honey stores and their impact on pathogenic bacteria of the honeybee, \u003cem\u003eApis mellifera\u003c/em\u003e. Ecol Evol 4:3960\u0026ndash;3967. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ece3.1252\u003c/span\u003e\u003cspan address=\"10.1002/ece3.1252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaisley BA, Chmiel JA, Pitek AP, Thompson GJ, Reid G (2020) Missing Microbes in Bees: How Systematic Depletion of Key Symbionts Erodes Immunity. Trends Microbiol 28:1010\u0026ndash;1021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tim.2020.06.006\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2020.06.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaisley BA, Pitek AP, Chmiel JA, Gibbons S, Chernyshova AM, Al KF, Faragalla KM, Burton JP, Thompson GJ, Reid G (2020) \u003cem\u003eLactobacillus\u003c/em\u003e spp. attenuate antibiotic-induced immune and microbiota dysregulation in honey bees. Commun Biol 3:1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-020-01259-8\u003c/span\u003e\u003cspan address=\"10.1038/s42003-020-01259-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabat\u0026eacute; DC, Cruz MS, Ben\u0026iacute;tez-Ahrendts MR, Audisio MC (2012) Beneficial Effects of \u003cem\u003eBacillus subtilis\u003c/em\u003e subsp. \u003cem\u003esubtilis\u003c/em\u003e Mori2, a Honey-Associated Strain, on Honeybee Colony Performance. Probiotics Antimicro Prot 4:39\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12602-011-9089-0\u003c/span\u003e\u003cspan address=\"10.1007/s12602-011-9089-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME (2014) The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11:506\u0026ndash;514. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrgastro.2014.66\u003c/span\u003e\u003cspan address=\"10.1038/nrgastro.2014.66\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotta EVS, Powell JE, Leonard SP, Moran NA (2022) Prospects for probiotics in social bees. Phil Trans R Soc B 377:20210156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rstb.2021.0156\u003c/span\u003e\u003cspan address=\"10.1098/rstb.2021.0156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSbaghdi T, Garneau JR, Yersin S, Chaucheyras-Durand F, Bocquet M, Mon\u0026eacute; A, El Alaoui H, Bulet P, Blot N, Delbac F (2024) The Response of the Honey Bee Gut Microbiota to \u003cem\u003eNosema ceranae\u003c/em\u003e Is Modulated by the Probiotic \u003cem\u003ePediococcus acidilactici\u003c/em\u003e and the Neonicotinoid Thiamethoxam. Microorganisms 12:192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms12010192\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms12010192\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalminen S, Collado MC, Endo A, Hill C, Lebeer S, Quigley EMM, Sanders ME, Shamir R, Swann JR, Szajewska H, Vinderola G (2021) The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat Rev Gastroenterol Hepatol 18:649\u0026ndash;667. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41575-021-00440-6\u003c/span\u003e\u003cspan address=\"10.1038/s41575-021-00440-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThorakkattu P, Khanashyam AC, Shah K, Babu KS, Mundanat AS, Deliephan A, Deokar GS, Santivarangkna C, Nirmal NP (2022) Postbiotics: Current Trends in Food and Pharmaceutical Industry. Foods 11:3094. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods11193094\u003c/span\u003e\u003cspan address=\"10.3390/foods11193094\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWegh CAM, Geerlings SY, Knol J, Roeselers G, Belzer C (2019) Postbiotics and Their Potential Applications in Early Life Nutrition and Beyond. Int J Mol Sci 20:4673. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms20194673\u003c/span\u003e\u003cspan address=\"10.3390/ijms20194673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenersch E (2008) \u003cem\u003ePaenibacillus larvae\u003c/em\u003e and American Foulbrood \u0026ndash; long since known and still surprising. J Verbr Lebensm 3:429\u0026ndash;434. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00003-008-0379-8\u003c/span\u003e\u003cspan address=\"10.1007/s00003-008-0379-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaynes E, Helgason T, Young JPW, Thwaites R, Budge GE (2013) A typing scheme for the honeybee pathogen \u003cem\u003eMelissococcus plutonius\u003c/em\u003e allows detection of disease transmission events and a study of the distribution of variants. Environ Microbiol Rep 5:525\u0026ndash;529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1758-2229.12057\u003c/span\u003e\u003cspan address=\"10.1111/1758-2229.12057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoldeman LV, Moore WEC, Cato EP (1977) Anaerobe Laboratory Manual, 4th edn. Anaerobe Laboratory, Blacksburg\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArai R, Tominaga K, Wu M, Okura M, Ito K, Okamura N, Onishi H, Osaki M, Sugimura Y, Yoshiyama M, Takamatsu D (2012) Diversity of \u003cem\u003eMelissococcus plutonius\u003c/em\u003e from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates. PLoS ONE 7:e33708. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0033708\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0033708\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDingman DW, Stahly DP (1983) Medium Promoting Sporulation of \u003cem\u003eBacillus larvae\u003c/em\u003e and Metabolism of Medium Components. Appl Environ Microbiol 46:860\u0026ndash;869\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahdi OS, Fisher NA (2018) Sporulation and Germination of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e Cells. Curr Protoc Microbiol 48 9E.2.1\u0026ndash;9E.2.10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cpmc.46\u003c/span\u003e\u003cspan address=\"10.1002/cpmc.46\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeska A, Nowak A, Szulc J, Motyl I, Czarnecka-Chrebelska KH (2022) Antagonistic Activity of Potentially Probiotic Lactic Acid Bacteria against Honeybee (\u003cem\u003eApis mellifera\u003c/em\u003e L.) Pathogens. Pathogens 11:1367. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens11111367\u003c/span\u003e\u003cspan address=\"10.3390/pathogens11111367\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGende LB, Eguaras MJ, Fritz R (2008) Evaluation of culture media for \u003cem\u003ePaenibacillus larvae\u003c/em\u003e applied to studies of antimicrobial activity. Rev argent microbiol 40:147\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlippi AM, Reynaldi FJ (2006) Inhibition of the growth of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources. J Invertebr Pathol 91:141\u0026ndash;146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2005.12.002\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2005.12.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoci\u0026eacute;t\u0026eacute; Fran\u0026ccedil;aise de Microbiologie (2019) Comit\u0026eacute; de l\u0026rsquo;Antibiogramme de la Soci\u0026eacute;t\u0026eacute; Fran\u0026ccedil;aise de Microbiologie (CA-SFM) (Recommandations 2019 V.2.0 Mai) [French]. Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sfm-microbiologie.org/2019/05/06/casfm-eucast-2019-v2/\u003c/span\u003e\u003cspan address=\"https://www.sfm-microbiologie.org/2019/05/06/casfm-eucast-2019-v2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Accessed September, 2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAupinel P, Fortini D, Dufour H, Tasei J-N, Michaud B, Odoux F, Pham-Del\u0026egrave;gue M-H (2005) Improvement of artificial feeding in a standard \u003cem\u003ein vitro\u003c/em\u003e method for rearing \u003cem\u003eApis mellifera\u003c/em\u003e larvae. Bull Insectology 58:107\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall BG, Acar H, Nandipati A, Barlow M (2014) Growth Rates Made Easy. Mol Biol Evol 31:232\u0026ndash;238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/mst187\u003c/span\u003e\u003cspan address=\"10.1093/molbev/mst187\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetzoldt T (2016) Estimation of growth rates with package growthrates. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tpetzoldt/growthrates/blob/master/doc/Introduction.pdf\u003c/span\u003e\u003cspan address=\"https://github.com/tpetzoldt/growthrates/blob/master/doc/Introduction.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrailsheim K, Brodschneider R, Aupinel P, Behrens D, Genersch E, Vollmann J, Riessberger-Gall\u0026eacute; U (2013) Standard methods for artificial rearing of \u003cem\u003eApis mellifera\u003c/em\u003e larvae. J Apic Res 52:1\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3896/IBRA.1.52.1.05\u003c/span\u003e\u003cspan address=\"10.3896/IBRA.1.52.1.05\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaisley BA, Pitek AP, Mallory E, Chernyshova AM, Allen-Vercoe E, Reid G, Thompson GJ (2022) Disentangling the microbial ecological factors impacting honey bee susceptibility to \u003cem\u003ePaenibacillus larvae\u003c/em\u003e infection. Trends Microbiol 31:521\u0026ndash;534. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tim.2022.11.012\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2022.11.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrudzynski K (2021) Honey as an Ecological Reservoir of Antibacterial Compounds Produced by Antagonistic Microbial Interactions in Plant Nectars, Honey and Honey Bee. Antibiotics 10:551. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antibiotics10050551\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics10050551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIorizzo M, Testa B, Lombardi SJ, Ganassi S, Ianiro M, Letizia F, Succi M, Tremonte P, Vergalito F, Cozzolino A, Sorrentino E, Coppola R, Petrarca S, Mancini M, De Cristofaro A (2020) Antimicrobial Activity against \u003cem\u003ePaenibacillus larvae\u003c/em\u003e and Functional Properties of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e Strains: Potential Benefits for Honeybee Health. Antibiotics 9:442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antibiotics9080442\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics9080442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKač\u0026aacute;niov\u0026aacute; M, Terentjeva M, Žiarovsk\u0026aacute; J, Kowalczewski PŁ (2020) \u003cem\u003eVitro\u003c/em\u003e Antagonistic Effect of Gut Bacteriota Isolated from Indigenous Honey Bees and Essential Oils against \u003cem\u003ePaenibacillus larvae\u003c/em\u003e. Int J Mol Sci 21:6736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms21186736\u003c/span\u003e\u003cspan address=\"10.3390/ijms21186736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cem\u003eIn\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaisley BA, Pitek AP, Chmiel JA, Al KF, Chernyshova AM, Faragalla KM, Burton JP, Thompson GJ, Reid G (2020) Novel probiotic approach to counter \u003cem\u003ePaenibacillus larvae\u003c/em\u003e infection in honey bees. ISME J 14:476\u0026ndash;491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-019-0541-6\u003c/span\u003e\u003cspan address=\"10.1038/s41396-019-0541-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrcan AC, Criste AD, Bobiș O, Cornea-Cipcigan M, Giurgiu A-I, Dezmirean DS (2024) Evaluation of Functional Properties of Some Lactic Acid Bacteria Strains for Probiotic Applications in Apiculture. Microorganisms 12:1249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms12061249\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms12061249\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKač\u0026aacute;niov\u0026aacute; M (2019) Antagonistic effect of gut microbiota of honeybee (\u003cem\u003eApis mellifera\u003c/em\u003e) against causative agent of american foulbrood \u003cem\u003ePaenibacillus larvae\u003c/em\u003e. JMBFS 9:478\u0026ndash;481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15414/jmbfs.2019.9.special.478-481\u003c/span\u003e\u003cspan address=\"10.15414/jmbfs.2019.9.special.478-481\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMallory E, Freeze G, Daisley BA, Allen-Vercoe E (2024) Revisiting the role of pathogen diversity and microbial interactions in honeybee susceptibility and treatment of \u003cem\u003eMelissococcus plutonius\u003c/em\u003e infection. Front Vet Sci 11:1495010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2024.1495010\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2024.1495010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMojgani N, Bagheri M, Moharrami M, Toutiaee S, Rousta HM (2023) Bioactive metabolites from autochthonous lactic acid bacteria inhibit the growth of \u003cem\u003eMelissococcus plutonius\u003c/em\u003e, causal agent of European foulbrood disease in honey bees. Entomol Exp Appl 171:386\u0026ndash;394. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/eea.13295\u003c/span\u003e\u003cspan address=\"10.1111/eea.13295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenitez LB, Velho RV, de Souza da Motta A, Segalin J, Brandelli A (2012) Antimicrobial factor from Bacillus amyloliquefaciens inhibits \u003cem\u003ePaenibacillus larvae\u003c/em\u003e, the causative agent of American foulbrood. Arch Microbiol 194:177\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00203-011-0743-4\u003c/span\u003e\u003cspan address=\"10.1007/s00203-011-0743-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEndo A, Salminen S (2013) Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Syst Appl Microbiol 36:444\u0026ndash;448. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.syapm.2013.06.002\u003c/span\u003e\u003cspan address=\"10.1016/j.syapm.2013.06.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Sugimura Y, Iwata K, Takaya N, Takamatsu D, Kobayashi M, Taylor D, Kimura K, Yoshiyama M (2014) Inhibitory effect of gut bacteria from the Japanese honey bee, \u003cem\u003eApis cerana japonica\u003c/em\u003e, against \u003cem\u003eMelissococcus plutonius\u003c/em\u003e, the causal agent of European foulbrood disease. J Insect Sci 14:129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jis/14.1.129\u003c/span\u003e\u003cspan address=\"10.1093/jis/14.1.129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZendo T, Ohashi C, Maeno S, Piao X, Salminen S, Sonomoto K, Endo A (2020) Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, \u003cem\u003eApilactobacillus kunkeei\u003c/em\u003e FF30-6 Isolated From Honey Bees. Front Microbiol 11:571903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2020.571903\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.571903\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi Q-Y, Wang W, Yan H, Qu H, Liu Y, Qian Y, Gu R (2023) The Effect of Different Organic Acids and Their Combination on the Cell Barrier and Biofilm of \u003cem\u003eEscherichia coli\u003c/em\u003e. Foods 12:3011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods12163011\u003c/span\u003e\u003cspan address=\"10.3390/foods12163011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMani-L\u0026oacute;pez E, Arrioja‐Bret\u0026oacute;n D, L\u0026oacute;pez‐Malo A (2022) The impacts of antimicrobial and antifungal activity of cell‐free supernatants from lactic acid bacteria \u003cem\u003ein vitro\u003c/em\u003e and foods. Comp Rev Food Sci Food Safe 21:604\u0026ndash;641. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1541-4337.12872\u003c/span\u003e\u003cspan address=\"10.1111/1541-4337.12872\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicke S (2003) Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult Sci 82:632\u0026ndash;639. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ps/82.4.632\u003c/span\u003e\u003cspan address=\"10.1093/ps/82.4.632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRocchetti MT, Russo P, Capozzi V, Drider D, Spano G, Fiocco D (2021) Bioprospecting Antimicrobials from \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e: Key Factors Underlying Its Probiotic Action. Int J Mol Sci 22:12076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms222112076\u003c/span\u003e\u003cspan address=\"10.3390/ijms222112076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, HuangFu H, Wang X, Zhao S, Liu Y, Lv H, Qin G, Tan Z (2021) Antibacterial Activity of Lactic Acid Producing \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e QZ1178 Against \u003cem\u003ePathogenic Gallibacterium anatis\u003c/em\u003e. Front Vet Sci 8:630294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2021.630294\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2021.630294\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson JL, Hinton M (1996) Effect of short-chain fatty acids on the size of enteric bacteria. Lett Appl Microbiol 22:408\u0026ndash;412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1472-765X.1996.tb01191.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1472-765X.1996.tb01191.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamei S, Stephan JG, Riesbeck K, Vasquez A, Olofsson T, Nilson B, de Miranda JR, Forsgren E (2019) The secretome of honey bee-specific lactic acid bacteria inhibits \u003cem\u003ePaenibacillus larvae\u003c/em\u003e growth. J Apic Res 58:405\u0026ndash;412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/00218839.2019.1572096\u003c/span\u003e\u003cspan address=\"10.1080/00218839.2019.1572096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves WG, Fernandes KM, Santana WC, Martins GF, Zanuncio JC, Serr\u0026atilde;o JE (2017) Post-embryonic changes in the hindgut of honeybee \u003cem\u003eApis mellifera\u003c/em\u003e workers: Morphology, cuticle deposition, apoptosis, and cell proliferation. Dev Biol 431:194\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ydbio.2017.09.020\u003c/span\u003e\u003cspan address=\"10.1016/j.ydbio.2017.09.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauvin R (1962) Nutrition De L\u0026rsquo;abeille. Annales de la nutrition et de l\u0026rsquo;alimentation 16:A41\u0026ndash;A60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jstor.org/stable/45124670\u003c/span\u003e\u003cspan address=\"https://www.jstor.org/stable/45124670\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWardell G (1983) Honeybee nutrition and European foulbrood. Quebec, Canada\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng H, Powell JE, Steele MI, Dietrich C, Moran NA (2017) Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. PNAS 114:4775\u0026ndash;4780. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1701819114\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1701819114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarado I, Phui A, Elekonich MM, Abel-Santos E (2013) Requirements for \u003cem\u003eIn vitro\u003c/em\u003e Germination of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e Spores. J Bacteriol 195:1005\u0026ndash;1011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jb.01958-12\u003c/span\u003e\u003cspan address=\"10.1128/jb.01958-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŠediv\u0026aacute; M, Laho M, Koh\u0026uacute;tov\u0026aacute; L, Mojzisova A, Majtan J, Klaudiny J (2018) 10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e. Molecules 23:32\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules23123236\u003c/span\u003e\u003cspan address=\"10.3390/molecules23123236\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHume ME, Corrier DE, Ivie GW, Deloach JR (1993) Metabolism of [14 C] Propionic Acid In Broiler Chicks. Poult Sci 72:786\u0026ndash;793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3382/ps.0720786\u003c/span\u003e\u003cspan address=\"10.3382/ps.0720786\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans JD, Aronstein K, Chen YP, Hetru C (2006) Immune pathways and defence mechanisms in honey bees \u003cem\u003eApis mellifera\u003c/em\u003e. Insect Mol Biol 15:645\u0026ndash;656. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2583.2006.00682.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2583.2006.00682.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontana R, Mendes MA, de Souza BM, Konno K, C\u0026eacute;sar LMM, Malaspina O, Palma MS (2004) Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (\u003cem\u003eApis mellifera\u003c/em\u003e). Peptides 25:919\u0026ndash;928. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.peptides.2004.03.016\u003c/span\u003e\u003cspan address=\"10.1016/j.peptides.2004.03.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDibner JJ, Buttin P (2002) Use of Organic Acids as a Model to Study the Impact of Gut Microflora on Nutrition and Metabolism. J Appl Poult Res 11:453\u0026ndash;463. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/japr/11.4.453\u003c/span\u003e\u003cspan address=\"10.1093/japr/11.4.453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopes LQS, Santos CG, De Almeida Vaucher R, Gende L, Raffin RP, Santos RCV (2016) Evaluation of antimicrobial activity of glycerol monolaurate nanocapsules against American foulbrood disease agent and toxicity on bees. Microb Pathog 97:183\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2016.05.014\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2016.05.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitsuwan W, Saengsawang P, Jeenkeawpieam J, Nissapatorn V, Pereira M, de Kitpipit L, Thomrongsuwannakij W, Poothong T, Vimon S S (2023) Development of a microencapsulated probiotic containing \u003cem\u003ePediococcus acidilactici\u003c/em\u003e WU222001 against avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Vet World 16:1131\u0026ndash;1140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14202/vetworld.2023.1131-1140\u003c/span\u003e\u003cspan address=\"10.14202/vetworld.2023.1131-1140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamei S, Stephan JG, Nilson B, Sieuwerts S, Riesbeck K, de Miranda JR, Forsgren E (2020) Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or \u003cem\u003ePaenibacillus larvae\u003c/em\u003e Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community. Microb Ecol 79:743\u0026ndash;755. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-019-01434-3\u003c/span\u003e\u003cspan address=\"10.1007/s00248-019-01434-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStephan JG, Lamei S, Pettis JS, Riesbeck K, de Miranda JR, Forsgren E (2019) Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies. Appl Environ Microbiol 85:e00606\u0026ndash;e00619. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00606-19\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00606-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForsgren E, Olofsson TC, V\u0026aacute;squez A, Fries I (2010) Novel lactic acid bacteria inhibiting \u003cem\u003ePaenibacillus larvae\u003c/em\u003e in honey bee larvae. Apidologie 41:99\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido/2009065\u003c/span\u003e\u003cspan address=\"10.1051/apido/2009065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePtaszyńska AA, Borsuk G, Zdybicka-Barabas A, Cytryńska M, Małek W (2016) Are commercial probiotics and prebiotics effective in the treatment and prevention of honeybee nosemosis C? Parasitol Res 115:397\u0026ndash;406. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00436-015-4761-z\u003c/span\u003e\u003cspan address=\"10.1007/s00436-015-4761-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaisley BA, Pitek AP, Torres C, Lowery R, Adair BA, Al KF, Ni\u0026ntilde;o B, Burton JP, Allen-Vercoe E, Thompson GJ, Reid G, Ni\u0026ntilde;o E (2023) Delivery mechanism can enhance probiotic activity against honey bee pathogens. ISME J 17:1382\u0026ndash;1395. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-023-01422-z\u003c/span\u003e\u003cspan address=\"10.1038/s41396-023-01422-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeska A, Nowak A, Czarnecka-Chrebelska KH (2022) Adhesion and Anti-Adhesion Abilities of Potentially Probiotic Lactic Acid Bacteria and Biofilm Eradication of Honeybee (\u003cem\u003eApis mellifera\u003c/em\u003e L.) Pathogens. Molecules 27:8945. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27248945\u003c/span\u003e\u003cspan address=\"10.3390/molecules27248945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Niu H, Qu Q, Guo D, Wan X, Yang Q, Mo Z, Tan S, Xiang Q, Tian X, Yang H, Liu Z (2025) Advancements in \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e: probiotic characteristics, gene editing technologies and applications. Crit Rev Food Sci Nutr 0:1\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10408398.2024.2448562\u003c/span\u003e\u003cspan address=\"10.1080/10408398.2024.2448562\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJavorsk\u0026yacute; P, Fecskeov\u0026aacute; LK, Hrehov\u0026aacute; L, Sabo R, Leg\u0026aacute;th J, Pristas P (2017) Establishment of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e strain in honey bee digestive tract monitored using gfp fluorescence. BM 8:291\u0026ndash;298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3920/BM2016.0022\u003c/span\u003e\u003cspan address=\"10.3920/BM2016.0022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePietropaoli M, Carpana E, Milito M, Palazzetti M, Guarducci M, Croppi S, Formato G (2022) Use of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e in Preventing Clinical Cases of American and European Foulbrood in Central Italy. Appl Sci 12:1388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app12031388\u003c/span\u003e\u003cspan address=\"10.3390/app12031388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIorizzo M, Ganassi S, Albanese G, Letizia F, Testa B, Tedino C, Petrarca S, Mutinelli F, Mazzeo A, De Cristofaro A (2022) Antimicrobial Activity from Putative Probiotic Lactic Acid Bacteria for the Biological Control of American and European Foulbrood Diseases. Vet Sci 9:236. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vetsci9050236\u003c/span\u003e\u003cspan address=\"10.3390/vetsci9050236\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva LA, Lopes Neto JHP, Cardarelli HR (2019) Exopolysaccharides produced by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e: technological properties, biological activity, and potential application in the food industry. Ann Microbiol 69:321\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13213-019-01456-9\u003c/span\u003e\u003cspan address=\"10.1007/s13213-019-01456-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerr\u0026iacute;os P, Fuentes JA, Salas D, Carre\u0026ntilde;o A, Aldea P, Fern\u0026aacute;ndez F, Trombert AN (2018) Inhibitory effect of biofilm-forming \u003cem\u003eLactobacillus kunkeei\u003c/em\u003e strains against virulent \u003cem\u003ePseudomonas aeruginosa in vitro\u003c/em\u003e and in honeycomb moth (\u003cem\u003eGalleria mellonella\u003c/em\u003e) infection model. BM 9:257\u0026ndash;268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3920/BM2017.0048\u003c/span\u003e\u003cspan address=\"10.3920/BM2017.0048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalas-Jara MJ, Ilabaca A, Vega M, Garc\u0026iacute;a A (2016) Biofilm Forming \u003cem\u003eLactobacillus\u003c/em\u003e: New Challenges for the Development of Probiotics. Microorganisms 4:35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms4030035\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms4030035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshiyama M, Wu M, Sugimura Y, Takaya N, Kimoto-Nira H, Suzuki C (2013) Inhibition of \u003cem\u003ePaenibacillus larvae\u003c/em\u003e by lactic acid bacteria isolated from fermented materials. J Invertebr Pathol 112:62\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2012.09.002\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2012.09.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŻ\u0026oacute;łkiewicz J, Marzec A, Ruszczyński M, Feleszko W (2020) Postbiotics\u0026mdash;A Step Beyond Pre- and Probiotics. Nutrients 12:2189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu12082189\u003c/span\u003e\u003cspan address=\"10.3390/nu12082189\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabirol A, Moriano-Gutierrez S, Engel P (2023) Neuroactive metabolites modulated by the gut microbiota in honey bees. Mol Microbiol 122:284\u0026ndash;293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mmi.15167\u003c/span\u003e\u003cspan address=\"10.1111/mmi.15167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKešnerov\u0026aacute; L, Mars RAT, Ellegaard KM, Troilo M, Sauer U, Engel P (2017) Disentangling metabolic functions of bacteria in the honey bee gut. PLOS Biol 15:e2003467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.2003467\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.2003467\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhojah E, Gomaa MS, Elsherbiny EG, Elawady A, Darwish MS (2022) The \u003cem\u003eIn vitro\u003c/em\u003e Analysis of Postbiotics in Functional Labneh to Be Used as Powerful Tool to Improve Cell Surfaces Properties and Adherence Potential of Probiotic Strains. Fermentation 8:122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/fermentation8030122\u003c/span\u003e\u003cspan address=\"10.3390/fermentation8030122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas D, Baruah R, Goyal A (2014) A food additive with prebiotic properties of an α-d-glucan from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e DM5. Int J Biol Macromol 69:20\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2014.05.029\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2014.05.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Sugimura Y, Takaya N, Takamatsu D, Kobayashi M, Taylor D, Yoshiyama M (2013) Characterization of Bifidobacteria in the digestive tract of the Japanese honeybee, \u003cem\u003eApis cerana japonica\u003c/em\u003e. J Invertebr Pathol 112:88\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2012.09.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2012.09.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFloyd AS, Mott BM, Maes P, Copeland DC, McFrederick QS, Anderson KE (2020) Microbial Ecology of European Foul Brood Disease in the Honey Bee (\u003cem\u003eApis mellifera\u003c/em\u003e): Towards a Microbiome Understanding of Disease Susceptibility. Insects 11:555. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects11090555\u003c/span\u003e\u003cspan address=\"10.3390/insects11090555\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiersch T, Barchia I, Hornitzky M (2010) Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood? Apidologie 41:151\u0026ndash;159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido/2009066\u003c/span\u003e\u003cspan address=\"10.1051/apido/2009066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antagonistic activity, Lactic Acid Bacteria, Apis mellifera, Paenibacillus larvae, Melissococcus plutonius","lastPublishedDoi":"10.21203/rs.3.rs-6510472/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6510472/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmong the most severe honeybee diseases, American and European foulbroods caused by \u003cem\u003ePaenibacillus larvae\u003c/em\u003e and \u003cem\u003eMelissococcus plutonius\u003c/em\u003e, respectively pose a significant threat to larval health and colony survival. Due to the lack of safe and effective treatments, interest is growing in the use of beneficial bacterial supplements as a promising alternative to antibiotics. This study evaluated the probiotic and postbiotic potential of selected bacterial strains against foulbrood pathogens. An initial screening of 25 strains for anti-foulbrood activity led to the selection of the most active candidates for further investigation. The inhibitory effect of their cell-free supernatants (CFS) was assessed and their mode of action was investigated. The probiotic and postbiotic properties were further evaluated using \u003cem\u003eP. larvae\u003c/em\u003e-infected larvae reared under laboratory conditions. Five lactic acid bacteria exhibited strong antagonistic activity against one or both pathogens, as their CFS displayed inhibitory effects. Notably, the CFS of \u003cem\u003eLactobacillus crispatus\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e completely inhibited \u003cem\u003eP. larvae\u003c/em\u003e at a dose of 12.5% (v/v). Further characterisation of these CFS, suggested a bacteriostatic effect, mainly attributed to organic acids. \u003cem\u003eIn vivo\u003c/em\u003e assays demonstrated a significant increase in larval survival when supplemented with live \u003cem\u003eL. plantarum\u003c/em\u003e, whereas CFS treatments failed to rescue infected larvae. These findings highlight the potential of probiotic and postbiotic-based strategies as sustainable alternatives for managing foulbrood in beekeeping.\u003c/p\u003e","manuscriptTitle":"Probiotic and postbiotic strategies against foulbrood in honeybees: in vitro and in vivo insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 09:46:58","doi":"10.21203/rs.3.rs-6510472/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-25T12:48:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T16:06:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-05T11:10:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322496361529445689108730815176622625923","date":"2025-04-29T18:51:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274032127183416562923084335941271257871","date":"2025-04-27T11:17:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170724780942098250270233793786373035814","date":"2025-04-24T15:31:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324553514752470361577553709259804722053","date":"2025-04-24T13:35:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-24T13:26:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-24T03:05:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T03:05:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-04-23T08:12:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"404a3a77-a450-4153-b69e-4a032a779bde","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T16:00:22+00:00","versionOfRecord":{"articleIdentity":"rs-6510472","link":"https://doi.org/10.1007/s12602-025-10739-4","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2025-09-11 15:57:28","publishedOnDateReadable":"September 11th, 2025"},"versionCreatedAt":"2025-04-28 09:46:58","video":"","vorDoi":"10.1007/s12602-025-10739-4","vorDoiUrl":"https://doi.org/10.1007/s12602-025-10739-4","workflowStages":[]},"version":"v1","identity":"rs-6510472","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6510472","identity":"rs-6510472","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}