{"paper_id":"26d821cd-5e41-4933-83cd-437ade7dfb71","body_text":"Non-peroxide antibacterial activity of Meliponula (Axestotrigona) ferruginea honey from \nTanzania \nChristopher Alphonce Mduda | ORCID: 0000-0003-4720-2163 \nAffiliation: Department of Crop Science and Beekeeping Technology , College of \nAgriculture and Food Technology, University of Dar es Salaam, Dar es Salaam, Tanzania. \nEmail address: mduda@udsm.ac.tz \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nAbstract \nHoney serves as a medicinal food that is utilized for both preventative care and the \ntreatment of various ailments. Amidst the contemporary challenge of antibiotic resistance, \nhoney emerges as a promising natural antimicrobial solution. The effica cy of honey in \ntherapy hinges on its mechanisms of antimicrobial action. Thus, this study investigated \nthe non-peroxide antibacterial properties of honey sources from a stingless bee species, \nMeliponula (Axestotrigona) ferruginea, that is commonly managed in Tanzania. The \nfindings reveal that honey from stingless bees exhibits remarkable antibacterial efficacy \nagainst both resistant and susceptible bacterial strains. Notably, the studied honey \nsamples retained a substantial portion of their antibacterial po tency (89.9 - 98.7%) even \nafter the removal of hydrogen peroxide. Interestingly, the antibacterial activity of honey \ndid not correlate with its total phenolic and flavonoid content, suggesting the influence of \nspecific bioactive compounds rather than overa ll phytochemical content. Stingless bee \nhoney was most effective against gram -positive bacterial strains, particularly \nStaphylococcus aureus. These results underscore the therapeutic potential of stingless \nbee honey for the management of pathogenic bacteria. Future investigations should focus \non elucidating the specific bioactive compounds present in stingless bee honey to bolster \nits clinical applications. \nKeywords: antimicrobial activity, non -peroxide activity, stingless bee honey, resistant \nbacteria, apitherapy, polyphenols \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n1.0 Introduction \nBacterial pathogens constantly evolve, acquiring resistance to existing antibiotics \n(Christaki et al. 2020). This evolutionary arms race complicates treatment strategies,  as \nonce-effective antibiotics lose their efficacy against resistant strains (Jalalifar et al. 2024). \nThe overuse and misuse of antibiotics in both clinical and agricultural settings exacerbate \nthis problem, fostering the emergence and spread of drug -resistant bacteria (Endale et \nal. 2023). Resistant bacterial strains such as Methicillin Resistant Staphylococcus aureus \n(MRSA) have become cosmopolitan due to their capacity to spr ead rapidly (Nishio et al. \n2015). Furthermore, the development of new antibiotic s lags behind the emergence of \nresistance, with few novel drugs reaching the market in recent decades. The discovery of \nnew antibiotics has slowed down over the past years due to high costs of drug research, \nresulting into few effective antimicrobials which are often associated with high costs and \nmultiple side effects for patients (Cardozo et al. 2013, Zainol et al. 2013). For this reason, \nthe search for novel antimicrobial compounds sourced from natural products is \nparamount. \nHoney is a sweet substance produced by bees from the nectar collected from plants. It \ncontains sugars as the major component, alongside water, organic acids, enzymes, \nvitamins, proteins, amino acids, minerals, polyphenols and volatile compounds ( da Silva \net al. 2016). There is a broad array of honey varieties with dinstinct flavor, color, and odor, \noriginating from various floral sources and bee species. Honey has been used as a food \nand an integral part of traditional medicine since ancient times (Kuropatnicki et al. 2018). \nThe medicinal uses of honey persisted to the modern era, giving rise to an alternative \nmedicine discipline known as Apitherapy, which utilizes honey and other bee products for \nhealth treatment (Mandal and Mandal 2011). The medicinal potential of honey is \nacknowledged for its antimicrobial, antioxidant, anti -inflammatory, anti -cancer, \nantidiabetic and immunomodulatory properties (Meo et al. 2017). Due its local availability, \naffordability, and minimal risks of toxicity and microbial resistance, honey stands out as a \nvaluable alternative for treating bacterial pathogens (Mduda et al. 2023e). Currently, \nseveral types of honey are marketed as medical -grade honeys with standardized levels \nof antibacterial activity.  The best known is the Manuka honey which is pro duced from \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nLeptospermum species and is reported to be effective against more than 60 species of \nbacteria (Mandal and Mandal 2011, Nolan 2020).  \nThe diverse components and physical properties of honey collectively contribute to its \nantimicrobial potency. In an undiluted state, the antimicrobial activity of honey is largely \nattributed to its high osmolarity and low pH (Zainol et al. 2013). High sugar concentration \nin honey exerts osmotic pressure on bacterial cells, leading to dehydration and cell \nshrinkage (Albaridi 2019). Additionally, pH of honey (3.2 – 4.5) is far below the optimal \npH for the growth of most bacteria which ranges from 6.5 to 7.5 (Almasaudi 2021). Dilution \nof honey activate the enzyme glucose oxidase which catalyzes the conversion of glucose \nto gluconic acid and hydrogen peroxide (Zainol et al. 2013).  Hydrogen peroxide is a \nstrong disinfectant which contributes to the antimicrobial efficacy of honey. The maximum \nlevel of hydrogen peroxide is achieved when honey is diluted by 30 to 50% (Alm asaudi \n2021). However, hydrogen peroxide is susceptible to degradation by catalase enzyme in \nliving tissues mak ing it less effective during therapy (Ewnetu et al. 2013, Mduda et al. \n2023e). The antibacterial activity of honey can decrease by up to 100 -fold following the \nremoval of hydrogen peroxide (Mandal and Mandal 2011). Nonetheless, certain varieties \nof honey possess non -peroxide activity which allows them to  maintain antibacterial \npotency even after the removal of hydrogen peroxide. The non-peroxide activity results \nfrom various elements found in honey, such as phenolic compounds, flavonoids, \nantibacterial peptides, methylglyoxal, methyl syringate, and other trace components \n(Zainol et al. 2013). \nThe use of honey in traditional medicine is widespread in  eastern Africa (Kiprono et al. \n2022, Mduda et al. 2023d, Héger et al. 2023). To date, various studies have been \nconducted to investigate the antimicrobial properties of honeys from this region (Ewnetu \net al. 2023, Mokaya et al. 2020, Mduda et al. 2023e, R ikohe et al. 2023, Mduda et al. \n2024). Findings from Ethiopia and Tanzania revealed that stingless bee honey was more \neffective against both gram -positive and gram -negative bacteria in comparison to Apis \nmellifera honey (Ewnetu et al. 2013, Mduda et al. 20 24). However, little is still known \nabout the mechanisms that underlie the antimicrobial potency of stingless bee honey. The \ncurrent study investigated for the first time the non-peroxide antibacterial activity of honey \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nsamples produced by Meliponula (Axestotrigona) ferruginea, a commonly managed \nstingless bee species in Tanzania (Mduda et al. 2023d, Mduda et al. 2023b). Specifically, \nhoney samples from Siha and Kibiti districts were tested against resistant and susceptible \nstrains of common pathogenic bacteria. The findings of this study will offer valuable \ninsights into the effectiveness of stingless bee honey as a powerful antimicrobial agent \nagainst prevalent pathogenic bacteria, potentially expanding its utility in clinical therapy.  \n \n2.0 Materials and Methods \n2.1 Honey samples \nHoney samples were gathered from Meliponula (Axestotrigona) ferruginea hives from two \ndistricts, namely  Siha and Kibiti . Siha district, situated in the northern highlands of \nTanzania, featured sampling locations at the western foothills of the Mount Kilimanjaro. \nThis area boasts Afromontane vegetation, known for its multi -layered, evergreen flora, \nspanning elevations between 1,200 to 2,500 meters with remarkable plant diversity (Foley \net al. 2014). Conversely, Kibiti district lies along the eastern coast region, where honey \nsamples were collected from the Rufiji Delta. This delta is renowned for hosting the largest \nconcentration of mangroves on the eastern coast of Africa, representing six distinct \nfamilies: Avicenniaceae, Combretaceae, Meliaceae, Rhizophoraceae, Sonneratiaceae, \nand Sterculiaceae (Monga et al., 2018).  At both locations, stingless bee hives were \nmanaged within semi-natural settings, characterized by the natural vegetation . Sample \ncollection was done in September 2023, with seven hives sampled from each district, \nresulting in a total of fourteen honey samples. The h oney was harvested using the pot -\npuncture technique that is outlined in Mduda et al. (2023d). Subsequently, a ll honey \nsamples were filtered using a clean food -grade filter cloth, then transferred into amber \nplastic containers, and stored at 4°C pending laboratory analyses. \n2.2 Test microorganisms \nAll test microorganisms used in this study were of standard reference strains from the \nAmerican Type Culture Collection (ATCC, US) . The microbes comprised  three Gram-\npositive bacteria; Methicillin R esistant Staphylococcus aureus  (MRSA) ATCC 33592, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nStaphylococcus aureus ATCC 6538P and Bacillus subtilis ATCC 6633, and two Gram -\nnegative bacteria; Escherichia coli  ATCC 11229 and Salmonella typhimurium  ATCC \n14028. \n2.3 Chemicals \nNutrient broth and Mueller Hinton agar were supplied from Himedia Laboratories Private \nLimited (India). Folin –Ciocalteu phenol reagent, gallic acid (99%), quercetin (98%), \naluminium chloride, sodium nitrite, sodium chloride, barium chloride, sodium hydroxide, \nhydrogen peroxide and methanol were supplied from Glentham Life Sciences (England). \nCatalase (C100) was supplied from Sigma Aldrich (Germany). \n2.4 Instrumentation \nA class II biosafety cabinet (BSC-1300IIA2-X, BIOBASE) was used to provide controlled \nenvironment microbial handling and other sanitary procedures . An inc ubator (LFZ-TSI-\n200D, LABFREEZ Instruments) and a UV/Vis spectrophotometer (Cary 60 UV –Vis \nSpectrophotometer, Agilent Technologies) were also used in this study. \n2.5 Assessment of the antimicrobial activity of honey \n2.5.1 Preparation of inoculum and culture media \nPreparation of inoculum and culture media employed the methods outlined in Mduda et \nal. (2023e). Mueller Hinton agar medium (38 g of Mueller Hinton agar in 1000 mL of \ndistilled water) was prepared and sterilized in an autoclave at 121°C for 15 minutes. The \nresulting suspension was poured into sterile petri dishes and allowed to solidify at room \ntemperature. Meanwhile, the test microorganisms were inoculated into nutrient broth \nmedia (8 g of nutrient broth in 1000 mL of distilled water) in test tubes and then incubated \nat 37°C for 24 hours. A 0.5 McFarland standard solution was prepared by mixing 0.5 mL \nof 1.175% (w/v) barium chloride with 99.5 mL of 1% v/v sulfuric acid, and then distributed \ninto screw-capped test tubes. Subsequently, 100 microliters  of the inoculated microbe \nsample from the nutrient broth medium was added to 5 mL of saline, and the \nconcentration was adjusted to 1.5 × 10 8 colony-forming units (CFU) per milliliter by \ncomparison with the prepared McFarland standard. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \n2.5.2 Agar-well diffusion assay \nThe agar-well diffusion assay was carried out according to the procedures outlined in \nEwnetu et al. (2013). The bacterial strains were inoculated by streaking the surface of an \nagar plate with a sterile swab until complete coverage of the agar surface was achieved. \nWells were created on the agar plates using a sterile cork borer (6 mm). For the \ndetermination of total antibacterial activity, 100µL of 50% (w/v) honey sample in deionized \ndistilled water was added into the agar wells. For non -peroxide activity, 100µL of 50% \n(w/v) honey sample in catalase solution (10 mg/mL) was used instead (Zainol et al. 2013). \nThe culture plates were then incubated at 37°C for 24 hours.   The diameter of inhibition \nzone was determined by measuring the clear area  surrounding the  agar wells . \nMeasurements were conducted in both horizontal and vertical directions using a Vernier \ncaliper and recorded in millimeters.  Deionized distilled water and Ciprofloxacin (10 µg) \nwere used as negative and positive controls, respectively. \n2.5.3 Catalase effectiveness test \nConfirmation of catalase removal in honey samples was done following the methods \noutlined in Zainol et al. (2013). Two honey samples were selected for the test against S. \naureus ATCC 6538P. Six tubes of test solution were prepared and labeled as follows: \ntube 1 (50% (w/v) honey solution, 45 mmol/L hydrogen peroxide, and 10 mg/mL catalase \nsolution); tube 2 (50% (w/v) honey solution and 10 mg/mL catalase solution); tube 3 (45 \nmmol/L hydrogen peroxide and 10 mg/m L catalase solution); tube 4 (50% (w/v) honey \nsolution and 45 mmol/L hydrogen peroxide; tube 5 (50% (w/v) honey solution); and tube \n6 (45 mmol/L hydrogen peroxide). These solutions were then tested in the same manner \nas the agar well diffusion assays on the same plate. \n2.6 Determination of phytochemical content in honey \n2.6.1 Total phenolic content \nDetermination of phenolic content in honey was conducted following the Folin-Ciocalteau \nmethod described by Singleton et al. (1999). Initially, three grams of honey sample were \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nmixed with 30 mL of methanol and subjected to sonication for 15 minutes. Subsequently, \nthe mixture was centrifuged at 9000 rpm, after which the supernatant was carefully \ndecanted and stored at 20°C. Following this step, 2.5 mL of diluted Fo lin-Ciocalteau \nphenol reagent and 2 mL of 7.5% sodium carbonate solution were added to 0.5 mL of the \nextracted sample in a separate tube. The mixture was thoroughly mixed and allowed to \nstand at room temperature for 2 hours. Absorbance was measured at 765 nm against the \nblank using a UV –Vis spectrophotometer. A standard calibration curve using gallic acid \n(0.02 to 0.20 mg/mL) was generated, and the total phenolic content (TPC) was expressed \nas milligrams of gallic acid equivalent per 100 g of honey (mg GAE/100 g). \n2.6.2 Total flavonoid content \nDetermination of total flavonoid content in honey followed the methods of Zhishen  et al. \n(1999). One mL of the sample solution (5 g of honey in 20 mL of distilled water) was mixed \nwith 4 mL of distilled water and 0.3 mL of 5% sodium nitrite. After a five -minute interval, \n0.3 mL of 10% aluminum chloride was introduced into the mixture a nd allowed to stand \nfor 1 minute. Following this, 2 mL of 1 M sodium hydroxide was added, followed by 2.4 \nmL of distilled water. The absorbance of the resulting mixture was measured against the \nblank at 510 nm using a UV/Vis spectrophotometer. A standard c alibration curve (0.01–\n0.10 mg/mL) was established using quercetin, and the total flavonoid content (TFC) was \nexpressed as milligrams of quercetin equivalent per 100 g of honey (mg QE/100 g). \n2.7 Data analysis \nOne-way analysis of variance (ANOVA) and a Tukey post-hoc test were employed for the \ncomparison of the total and non -peroxide antibacterial activities of honey samples from \nthe two locations. Non-metric multidimensional scaling (NMDS) ordination plot was \ncreated using Euclidean similarity index  to highlight the similarities in the total and non -\nperoxide antibacterial activities. Data were log transformed before generating the NMDS \nplot to fit them in the same scale. ANOVA was also used to compare susceptibility of the \nbacterial strains to the studied h oney samples. Two sample t -test was conducted to \ncompare the phytochemical content of honey samples from the two locations. Pearson’s \ncorrelation coefficient was performed to evaluate association between diameter s of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\ninhibition zones and phytochemical cont ent of honey . Data analysis was done using \nPAleontological STatistics (PAST)  Software V.  4.03 and graph s were plotted by  \nGraphPad Prism V. 9.5.1. \n \n3.0 Results and Discussion \n3.1 Total versus non-peroxide antibacterial activity of stingless bee honey \nResults of the total antibacterial activity of stingless bee honey against the test microbes \nare presented in Table 1 . Honey samples exhibited antibacterial activity against all \nbacterial strains except for two samples from Kibiti (KB02 and KB03) which failed to inhibit \nthe growth of E. coli ATCC 11229. The highest total antibacterial activity was observed in \nsample KB05 from Kibiti against S. aureus ATCC 6538P (Table 1).  \nTable 1 Diameters of inhibition zones (mm) produced by honey samples before treatment \nwith catalase against five bacterial strains. \nTest \nmicrobes \nMRSA \nATCC 33592 \nS. aureus \nATCC \n6538P \nB. subtilis \nATCC 6633 \nE. coli ATCC \n11229 \nS. typhimurium \nATCC 14028 \nMean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD \nSH01 13.0 ± 1.0 16.0 ± 0.0 14.9 ± 0.3 11.7 ± 0.6 11.3 ± 1.2 \nSH02 15.0 ± 0.5 15.7 ± 0.6 12.5 ± 1.1 11.0 ± 1.0 13.3 ± 1.2 \nSH03 16.0 ± 0.5 14.3 ± 0.6 12.3 ± 0.6 10.5 ± 0.3 12.0 ± 0.0 \nSH04 13.0 ± 1.0 13.7 ± 1.2 14.0 ± 0.3 10.5 ± 0.5 11.7 ± 0.6 \nSH05 14.0 ± 1.0 14.3 ± 0.6 13.8 ± 0.3 12.5 ± 0.5 13.0 ± 0.0 \nSH06 13.5 ± 0.5 13.5 ± 1.2 12.5 ± 0.5 11.0 ± 0.6 16.0 ± 1.5 \nSH07 13.5 ± 0.3 14.8 ± 0.3 14.4 ± 0.6 12.0 ± 0.0 13.3 ± 1.5 \nKB01 12.0 ± 0.0 13.3 ± 1.5 13.0 ± 1.1 10.0 ± 0.0 11.7 ± 0.6 \nKB02 11.0 ± 0.0 14.2 ± 0.6 11.2 ± 0.3 *6.0 ± 0.0 12.3 ± 0.6 \nKB03 12.5 ± 1.5 13.3 ± 0.6 11.0 ± -.3 *6.0 ± 0.0 10.5 ± 0.5 \nKB04 13.5 ± 0.5 13.7 ± 1.2 12.5 ± 0.6 14.5 ± 0.5 12.8 ± 0.3 \nKB05 11.8 ± 0.3 16.3 ± 0.6 12.3 ± 0.6 11.0 ± 0.0 13.3 ± 0.6 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nKB06 12.0 ± 0.0 13.7 ± 0.6 13.5 ± 0.3 10.0 ± 0.0 12.0 ± 1.0 \nKB07 12.5 ± 0.5 13.7 ± 0.6 12.3 ± 0.6 10.2 ± 0.8 12.0 ± 0.0 \nSamples with codes SH and KB originated from Siha and Kibiti districts, respectively. Size \nof the agar well is 6.00 mm. Values with * sign indicate no microbial inhibition. \nHoney exhibits potent antimicrobial activity due to various attributes such as its low pH, \nhigh osmolarity and the presence of hydrogen peroxide and non -peroxide components \n(Mandal and Mandal 2011). Previous studies have reported pH of M. ferruginea honey \nranging between 3.8 and 4.9 (Mokaya et al 2022, Mduda et al. 2023a) which is low \nenough to be inhibitory to bacteria. Honey acidity is influenced by the presence of organic \nacids, particularly gluconic acid which is the dominant acid in honey (Dardón et al. 2013). \nHowever, pH is raised when honey is diluted making it less effective as an antimicrobial \nfactor (Mduda et al. 2013e). Additionally, M. ferruginea honey has lower sugar content \n(70.3 – 73.9 °Brix) and higher water content (26.1 - 28.8%) compared to  A. mellifera \nhoney (Mokaya et al. 2022, Mduda et al. 2023a), resulting to low osmolarity.  \nHoney generates hydrogen peroxide via the enzyme glucose oxidase which oxidizes \nglucose to gluconic acid and hydrogen peroxide (Mandal and Mandal 2011). Antimicrobial \npotency due to hydrogen peroxide (peroxide activity) is the most common in many types \nof honey , with maximum activity when  the honey is diluted (Almasaudi 2021). The \ndownside of the peroxide activity is that hydrogen peroxide can be easily destroyed by \nheat or in the presence of catalase enzyme ( Mduda et al. 2023e ). In that regard, the \neffectiveness of hydrogen peroxide as an antimicrobial agent is limited when honey is \nmixed in bodily fluids (Almasaudi 2021, Mduda et al. 2024). \nNon-peroxide antibacterial activity was assayed after treating the studied honey samples \nwith catalase enzyme and the results are presented in Table 2.  Catalase effectiveness \ntest showed that the enzyme was effective in removing all hydrogen peroxide molecules \nfrom the honey samples  and its activity was not affected by other components  \n(Supplementary table). The highest non -peroxide antibacterial activity was observed in \nsample SH05 against S. aureus ATCC 6538P. Similar to the results of total antibacterial \nactivity, two honey samples from Kibiti failed to inhibit the growth of E. coli ATCC 11229. \nNo significant difference was observed between  the total and non-peroxide antibacterial \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nactivity against all bacterial strains for honey samples from Siha and Kibiti (Fig. 1). Honey \nsamples from both locations retained the majority of antibacterial activity after treatment \nwith catalase (Fig. 2, Table 3). Similarly, the NMDS plot (Fig. 3) shows minimal differences \nbetween the total and non-peroxide antibacterial activity based on the extent to which the \nconvex hulls overlap. \nTable 2 Diameters of inhibition zones (mm) produced by catalase-treated honey samples \nagainst five bacterial strains. \nTest \nmicrobes \nMRSA \nATCC 33592 \nS. aureus \nATCC 6538P \nB. subtilis \nATCC 6633 \nE. coli ATCC \n11229 \nS. typhimurium \nATCC 14028 \nMean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD \nSH01 12.3 ± 0.3 16.0 ± 0.0 14.2 ± 0.3 10.3 ± 0.6 9.8 ± 0.3 \nSH02 13.8 ± 0.8 15.0 ± 0.5 12.3 ± 0.6 10.3 ± 1.2 12.5 ± 0.5 \nSH03 14.5 ± 0.3 14.0 ± 1.0 11.7 ± 1.1 8.7 ± 0.6 11.8 ± 0.3 \nSH04 12.0 ± 0.0 13.0 ± 1.0 13.3 ± 0.6 9.7 ± 0.6 11.0 ± 0.0 \nSH05 13.0 ± 0.5 16.0 ± 0.0 13.3 ± 0.6 12.0 ± 1.0 12.5 ± 0.5 \nSH06 12.8 ± 0.8 12.5 ± 0.5 11.8 ± 1.1 10.3 ± 0.6 15.5 ± 0.5 \nSH07 12.5 ± 0.5 14.5 ± 0.5 13.0 ± 0.6 11.3 ± 0.6 10.5 ± 0.8 \nKB01 10.5 ± 0.5 12.5 ± 0.5 12.3 ± 0.6 9.7 ± 0.6 10.5 ± 0.5 \nKB02 10.0 ± 0.0 13.0 ± 1.0 10.7 ± 1.1 *6.0 ± 0.0 11.0 ± 1.0 \nKB03 11.0 ± 0.5 12.8 ± 0.3 10.3 ± 0.6 *6.0 ± 0.0 9.3 ± 0.6 \nKB04 11.8 ± 0.3 13.0 ± 1.0 11.7 ± 0.6 13.7 ± 0.6 10.5 ± 0.5 \nKB05 11.5 ± 0.5 11.5 ± 0.5 11.7 ± 0.3 9.7 ± 0.6 12.5 ± 0.5 \nKB06 11.3 ± 0.3 13.8 ± 0.3 12.8 ± 0.3 9.0 ± 1.0 11.3 ± 0.3 \nKB07 12.3 ± 0.8 12.3 ± 0.8 11.7 ± 0.6 10.0 ± 0.0 11.0 ± 0.0 \nSamples with codes SH and KB originated from Siha and Kibiti districts, respectively. Size \nof the agar well is 6.00 mm. Values with * sign indicate no microbial inhibition. \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \n \nFig. 1 Grouped bar-graphs showing mean diameters of inhibition zones of untreated (total \nactivity) and catalase-treated (non-peroxide activity) honey samples against the bacterial \nstrains. Ciprofloxacin (10 µg) was used as a positive control. Superscripts with different \nletters within the same group indicate significant differences in diameters of inhibition \nzones.  \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \nFig. 2 A representative agar plate showing zones of inhibition produced by stingless bee \nhoney. Wells in Tr1 and Tr2 received catalase-treated honey (Non-peroxide activity) while \nTr3 received untreated honey (Total activity). \nTable 3: Antibacterial activity retained after treatment of honey samples with catalase \nenzyme. \nHoney \nsource \n MRSA \nATCC \n33592 \nS. aureus \nATCC \n6538P \nB. subtilis \nATCC \n6633 \nE. coli \nATCC \n11229 \nS. typhimurium \nATCC 14028 \nSiha \nMean total activity \n(mm) 14.0 14.6 13.5 11.3 13.0 \nMean non-peroxide \nactivity (mm) 13.0 14.4 12.8 10.4 11.9 \nRetained activity 92.60% 98.70% 95.10% 91.80% 92.10% \nKibiti \nMean total activity \n(mm) 12.2 14.0 12.2 9.7 12.1 \nMean non-peroxide \nactivity (mm) 11.2 12.7 11.6 9.1 10.9 \nRetained activity 91.80% 90.40% 94.80% 94.70% 89.90% \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \nFig. 3 Non-metric multidimensional scaling (NMDS) ordination plot showing honey \ntreatments exhibiting total and non-peroxide antibacterial activity. Overlapping of convex \nhulls indicate the degree of similarity between sample treatments.  Stress value of the \nNMDS plot is 0.159. \nPrevious studies have highlighted the prevalence of non-peroxide antibacterial activity in \nstingless bee honey ( Temaru et al. 2007, Zainol et al. 2013, Jibril et al. 2020). Honey \nsamples from 14 stingless bee species displayed remarkable no n-peroxide antibacterial \nactivity against gram positive and gram -negative bacterial strains (Temaru et al. 2007). \nFurther, Jibril et al. (2020) reported that stingless bee honey retained 98.9% of the \nantibacterial activity after treatment with catalase (Ji bril et al. 2020). Contrary, non -\nperoxide activity is uncommon in Apis mellifera honey except for special honey types \nsuch as Manuka honey (Johnston et al. 2018). For example, honey samples from various \norigins in UK and Denmark  had no detectable non-peroxide activity despite exhibiting \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nbroad-spectrum total antibacterial activity  (Sulaiman et al. 2012 , Matzen et al. 2018 ). \nAdditionally, the removal of hydrogen peroxide by catalase resulted in substantial \ndecrease in the antibacterial activity of A. mellifera honeys from Western Australia \n(Roshan et al. 2017). \nSeveral non-peroxide components have been reported to contribute to the antibacterial \nactivity of honey. The most extensively studied are methylglyoxal and methyl syringate \nwhich are found predominantly in Manuka honey (Johnston et al. 2018, El-Senduny et al. \n2021, Hossain et al. 2023 ). Honey also contains a diverse array of phytochemical s \nincluding polyphenols which are derived from the nectar of flowers (Mduda et al. 2023c). \nPolyphenols such as phenolic acids and flavonoids can exhibit antibacterial potency by \ninterfering with the bacterial cell functioning, disrupting cell growth and effecting cell lysis \n(Shehu et al. 2016, Kumar Singh et  al. 2019). Additionally, honey may co ntain bee \nderived proteins such as the antimicrobial peptides  which ma y also contribute to its \nantimicrobial potency (Almasaudi 2021). \n3.2 Phytochemical content of stingless bee honey \nStingless bee honey exhibited remarkable levels of total phenolic (197.0  – 263.1 mg \nGAE/100 g) and flavonoid content (118.5 – 156.7 mg QE/100 g) (Fig. 4). Polyphenols can \nplay a critical role in the non-peroxide antibacterial activity of honey (Tuksitha et al. 2018). \nPrevious studies reported that antimicrobial activity of honey strongly correlated with both \nTPC and TFC (Sousa et al. 2016, Mduda et al. 2023e). In contrast, it was observed in this \nstudy that both TPC and TFC lacked significant correlation with the diameters of inhibition \nzones against any of the bacterial strain s (Fig. 5 ). When comparing the two locations, \nhoney samples from Kibiti produced smaller mean diameters of inhibition zones (Fig. 1), \ndespite having  significantly higher amounts of total phenolic and flavonoid content.  \nTuksitha et al. (2018) also observed that honey samples with the highest TPC failed to  \ninhibit the growth of gram negative bacteria. In addition, honey samples from Brazil and \nScotland showed no correlation between TPC and antimicrobial activity against a variety \nof bacterial strains including Shigella dysentery , Salmonella typhimurium , \nStaphylococcus aureus and Bacillus cereus (Bueno-Costa et al. 2016, Fyfe et al. 2017).  \nThese results indicate that the antimicrobial activity displayed by the studied honey \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\nsamples may be attributed to specific polyphenolic compounds present in honey rather \nthan the total content.  The amount and types of polyphenols present in honey vary \ndepending on the botanical source, geographical location as well as the bee species \norigin (Mduda et al. 2023e).  Specific phenolic acids such as Syringic acid, Chlorogenic \nacid, Caffeic acid and Hydroxyc innamic acid s, and flavonoids such as Naringenin , \nApigenin, Quercetin and Myricetin, have been identified to exhibit broad spectrum \nantibacterial activity (Jibril et al. 2019). Hence, future research should focus on identifying \nthe specific polyphenols and other bioactive compounds responsible for the antimicrobial \npotency observed in Tanzanian stingless bee honeys. \n \nFig. 4 Grouped bar-graph showing total phenolic and flavonoid content of stingless bee \nhoney from the studied locations. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \nFig. 5  Correlation matrix showing pairwise Pearson’s coefficients among variables. \nCrossed boxes (×) indicate correlations which are not statistically significant ( p > 0.05). \nMRSA = Methicillin resistant S. aureus ATCC 33592, SA = S. aureus ATCC 6538P, BS \n= B. subtilis ATCC 6633, EC = E. coli ATCC 11229, ST = S. typhimurium ATCC 14028, \nTPC total phenolic content, TFC = total flavonoid content. \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \n3.3 Microbial susceptibility to stingless bee honey \nThe bacterial strains displayed different levels of susceptibility to both untreated and \ncatalase treated honey samples  (Fig. 4). Stingless bee honey was more effective in \ninhibiting the growth of gram-positive bacteria compared to gram-negative bacteria. The \nlargest and smallest diameters of inhibition zones were observed against S. aureus ATCC \n6538P and E. coli  ATCC 11229, respectively. Several authors have also reported \nstingless bee honey to be more effective against gram -positive bacteria compared to \ngram-negative bacteria (Zainol et al. 2013, Domingos et al. 2021 , Mduda et al. 2023e). \nMalaysian stingless bee honey displayed remarkably high total and non-peroxide activity \nagainst S. aureus  than the standard me dicinal Manuka honey (Zainol et al. 2023).  \nSimilarly, honey samples from two Scaptotrigona species effectively inhibited the growth \nof two MRSA strains, while being least effective against strains of E. coli (Nishio et al. \n2016). In contrast, Ng. et al (20 20) reported Malaysian stingless bee honey to be highly \neffective against E. coli. Variation in microb ial susceptibility to honey may  result from \ndifferences in growth rate and cell -wall permeability to antimicrial components (Dżugan \net al. 2020).  Additionally, honey samples from different origins may comprise bioactive \ncompounds with different effects on bacterial cells.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint \n\n \nFig. 6 Grouped bar-graph showing differences in microbial susceptibility to the untreated \n(total activity) and catalase-treated (non-peroxide activity) honey samples. \n \n4.0 Conclusion \nThe studied honey  samples demonstrated broad-spectrum antibacterial activity against \ncommon pathogens. Primarily, the antibacterial efficacy of M. ferruginea stemmed from \nits non -peroxide component, indicating substantial therapeutic value. Its notable \neffectiveness against Methicillin Resistant S. aureus  advocates it as  a potent natural \nsolution for the  treatment of wound infections and dr ug-resistant pathogens.  Further \ninvestigations are needed to  elucidate the mechanisms and bioactive compounds \nunderlying the observed activity in order to ascertain its clinical potential for treating \nbacterial infections including resistant strains. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is made \nThe copyright holder for this preprintthis version posted March 20, 2024. ; https://doi.org/10.1101/2024.03.20.585900doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}