Characterization of a newly isolated biosurfactant fengycin produced by Heyndrickxia coagulans strain.

PCR screening detected the presence of fen D gene in H. coagulans . The alignment of selected fenD genes of Bacillus species revealed an identity of 27.63–28.82% with fenD of H. coagulans , while the identity among Bacillus fenD genes ranged from 94.25 to 100.00%. Phylogenetic analysis showed that the fenD genes of the same species do not cluster together (Fig. S1). The UHPLC method with a DAD detector was used for purification of the biosurfactant produced by the strain H. coagulans 9FT27. The isolate of obtained lipopeptide was firstly dissolved in acetonitrile and then filtered through a 0.22 μm membrane filter. The standard of fengycin (Sigma Aldrich, Germany) was dissolved in acetonitrile at a concentration of 0.5 mg/mL. Optimization of the separation strategy was performed stepwise using a gradient elution focused on the mobile phase flow, ratio and gradient time (Englerová et al. 2021 ). The yield of partially purified lyophilised fengycin extract was 0.12%. The chromatograms of both fengycin standard and the test biosurfactant indicated a group of peaks between 2.6 and 7.7 min. (Fig.  1 ). The UHPLC chromatogram of the lipopeptides produced by H. coagulans 9FT27 showed peak similar to that in standard which could be assigned by comparing the retention times (Supplementary material, Table S2). These results were confirmed by the genetic and MALDI-TOF analyses mentioned in this article. Fig. 1 UHPLC chromatograms of biosurfactant isolated from H. coagulans 9FT27 ( A ) and fengycin standard ( B ) UHPLC chromatograms of biosurfactant isolated from H. coagulans 9FT27 ( A ) and fengycin standard ( B ) To clarify whether the biosurfactant produced by the strain H. coagulans 9FT27 was a lipopeptide, it was subjected to a NMR study. The chemical shifts were assigned using standard 1D and 2D NMR experiments. The 2D TOCSY study showed spin systems for protons of the saturated aliphatic chain in the range of 0.8–1.7 ppm, which were assigned to the fatty acid chain, one peak for fatty acid was also observed at 4.20 ppm as multiplet, and at 2.01 ppm as triplet assigned for protons on C-2 position of fatty acid which were assigned to the fatty acid chain (Fig.  2 A). The 1 H NMR spectrum also showed characteristic signals corresponding to the chemical groups in the aliphatic chain. The methyl (–CH 3 ) group was observed as a triplet at 0.89 ppm followed by methylene (–CH 2 ) functional groups overlapping at about 1.3 ppm. No signals for carbon–carbon double bonds were observed. Fig. 2 1 H NMR spectrum of the fengycin isolate of H. coagulans 9FT27, 2D TOCSY spectrum of the spin system belonging to the fatty acid chain ( A ) measured in DMSO- d 6 1 H NMR spectrum of the fengycin isolate of H. coagulans 9FT27, 2D TOCSY spectrum of the spin system belonging to the fatty acid chain ( A ) measured in DMSO- d 6 The 13 C NMR spectrum showed a series of signals for the aliphatic chain between 10.34 and 36.34 ppm. Other spin systems were observed between 2.5 and 4.5 ppm corresponding to the amino acid chain protons. Some cross-peaks overlapping into the aliphatic chain region were also observed. These signals were assigned to the aliphatic chains of the amino acids of the lipopeptide (Ala, Ile, Glu, etc.). Alpha hydrogens of amino acids were observed in the 4.0–5.0 ppm region as singlet signals. The NH protons of the peptide bonds were not well observed due to the use of deuterium oxide as a solvent as a result of proton exchange. The experiment in DMSO- d 6 (dimethyl sulfoxide) showed some NH protons, but HMBC spectrum did not show any connectivity to residue of amino acids, due to exchange of protons in DMSO- d 6 water traces observed at 3.33 ppm. Partial assignment of carbons and protons are listed in supplementary material (Table S3). Thus, comparing with the literature (Nam et al. 2015 ), it was assumed that the biosurfactant produced was lipopeptide fengycin, as proven further by the MALDI-TOF method. The lyophilised extract of BS from H. coagulans 9FT27 was subjected to MALDI-TOF/MS analysis. All peaks from the fengycin standard were observed in BS 9FT27 sample, except for 1530 and 1544 (Fig.  3 ). In supplementary material (Table S4) are listed m/z values. Fig. 3 MALDI-TOF/MS spectra of fengycin isolated from  H. coagulans 9FT27 and commercial fengycin standard MALDI-TOF/MS spectra of fengycin isolated from  H. coagulans 9FT27 and commercial fengycin standard The effects of BS at different concentrations on the antibiofilm activity against S. aureus CCM 4223 and MRSA are presented in Table  1 . The inhibition of biofilm formation by BS 9FT27 was compared with the control by absorption analysis. It was found that BS at concentrations of 15 mg/mL to 0.2 mg/mL significantly ( P  < 0.0001) inhibited the biofilm formation of both pathogenic strains. The concentration of BS from 15 to 0.9 mg/mL inhibited the biofilm formation of S. aureus CCM 4223 in the range of 92–97% and of MRSA in the range of 60–75%. At a concentration of BS 0.1 mg/mL, the biofilm of S. aureus CCM 4223 ( P  = 0.0043) and MRSA ( P  = 0.0019) was significantly inhibited. Table 1 Antibiofilm activity of BS´ 9FT27 different concentrations on S. aureus CCM 4223 and MRSA BS 9FT27 concentration (mg/mL) S. aureus CCM 4223 MRSA Mean % inhibition ± SD P value Mean % inhibition ± SD P value 15 92.35 ± 2.89  < 0.0001 59.80 ± 5.49  < 0.0001 7.5 95.59 ± 1.69  < 0.0001 73.63 ± 3.66  < 0.0001 3.8 97.16 ± 0.46  < 0.0001 74.69 ± 3.19  < 0.0001 1.9 96.25 ± 0.49  < 0.0001 74.53 ± 3.46  < 0.0001 0.9 91.66 ± 1.11  < 0.0001 74.04 ± 1.34  < 0.0001 0.5 57.97 ± 2.88  < 0.0001 61.92 ± 6.14  < 0.0001 0.2 31.11 ± 5.52  < 0.0001 53.13 ± 6.57  < 0.0001 0.1 11.86 ± 4.66 0.0043 14.31 ± 8.98 0.0019 Antibiofilm activity of BS´ 9FT27 different concentrations on S. aureus CCM 4223 and MRSA The relative expression of the ica A gene was significantly downregulated in all concentrations of both MRSA and S. aureus CCM 4223 compared to the controls at different significance levels (Fig.  4 ). Fig. 4 Relative expression of the ica A gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the ica A gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) The relative expression of the ica B gene was significantly downregulated in all concentrations used in both MRSA and S. aureus CCM 4223 compared to controls (Fig.  5 ). Fig. 5 Relative expression of the ica B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the ica B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative gene expression of the ica D gene was significantly downregulated in all concentrations used compared to the MRSA control (Fig.  6 ). Fig. 6 Relative expression of the ica D gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the ica D gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) The relative expression of the srt A gene was significantly upregulated in both controls compared to the tested concentrations (Fig.  7 ) . Fig. 7 Relative expression of the srt A gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the srt A gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) The relative gene expression of gyrase B was markedly downregulated at different significance levels in all concentrations used compared to S. aureus CCM 4223 and the MRSA control (Fig.  8 ). Fig. 8 Relative expression of the gyr B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the gyr B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) The relative expression of clf B was upregulated in both controls compared to all concentrations used, with different levels of significance (Fig.  9 ). Fig. 9 Relative expression of the clf B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL) Relative expression of the clf B gene in MRSA and S. aureus CCM 4223 samples ( n  = 3), at each concentration (mg/mL)

Heyndrickxia coagulans 9FT27 was collected from healthy horse skin and identified as previously described in Styková et al. ( 2022 ). The strain was deposited in the Czech Culture Collection (Masaryk University, Brno, Czech Republic) as Bacillus coagulans Biocenol™ 9FT27 CCM 9014 and preserved in Microbank™ (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at − 70 °C. The determination of extracellular BS was performed according to Morikawa et al. ( 2000 ) and the determination of cell-bound BS was performed according to Ghasemi et al. ( 2019 ) as described in Styková et al. ( 2022 ). Genomic DNA was isolated using DNAzol Direct (Molecular Research Center Inc., Cincinnati, USA) according to the manufacturer’s instructions. In brief, a pure bacterial colony, approximately the size of an inoculation loop, was added to 50 μL of DNAzol Direct and heated at 95 °C for 15 min. PCR was then performed as described by Styková et al. ( 2022 ). The sequences of the fengycin fen D gene of the strain were sent to GenBank and accepted under the accession number MN197533 for H. coagulans 9FT27. For comparison with other fenD genes, the fenD sequences of Bacillus amyloliquefaciens , Bacillus subtilis and Bacillus velezensis were downloaded from GenBank, and a phylogenetic tree was constructed using Geneious Prime v2025.1.2 (Biomatters). Biosurfactants were extracted according to the method of Plaza et al. ( 2015 ) with modifications as described in Englerová et al. ( 2021 ). The preparation of isolated lipopeptide and a subsequent UHPLC analysis was performed as described in Englerová et al. ( 2021 ). NMR spectra were obtained at room temperature in deuterium oxide (D 2 O) and hexadeuteriodimethyl sulfoxide (DMSO- d 6 ) using a Varian Mercury Plus NMR spectrometer operated at 600 MHz for 1 H and 150 MHz for 13 C. Tetramethylsilane was used as an internal standard for both 1 H and 13 C nuclei ( δ TMS  = 0.00 ppm for both). Heteronuclear HSQC and HMBC 2D experiments were optimised on 145 Hz (one-bond) and 8 Hz (long-range) for J H,C couplings. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI TOF/MS) was applied to check the purity and molecular weight of isolated surfactant (9FT27 fenD) in comparison with standard of fengycin (Sigma Aldrich, Germany). Briefly, the purified surfactant was desalted by the ZipTip (Merck Millipore) as per the manufacturer’s instructions. Surfactant attached in the ZipTip matrix was eluted with 1 µL of the α-Cyano-4-hydroxycinnamic acid matrix (HCCA matrix; Bruker Daltonics, Germany) dissolved in TA50 (50:50 [v/v] acetonitrile: 0.1% trifluoroacetic acid, TFA). Two microliters of the eluate were spotted on AnchorChip (Bruker Daltonics) and air-dried. The spectra were obtained with MALDI-TOF Microflex™-LRF mass-spectrometer (Bruker-Daltonics). Calibration standard II (Bruker-Daltonics) was used for calibrating the instrument. The acquisition was performed in flexControl v3.4 (Burker Daltonics) in reflectron-negative mode at a laser frequency of 35 Hz (100 shots) (Mlynarcik et al. 2012 ). Mass was analysed in flexAnalysis v3.4 software (Bruker Daltonics). The pathogenic strain  S. aureus  CCM 4223, isolated from a wound, was sourced from the Czech Collection of Microorganisms (Brno, Czech Republic). Our clinical MRSA isolate was recovered from the infected fetlock joint of a horse during arthroscopic surgery. Both strains harbor the biofilm-associated operon genes ica ADCB, agr A, srt A and ica R, which are involved in biofilm formation (Englerová et al. 2021 ; Styková et al. 2022 ). The MRSA isolate was identified at LABOKLIN (Laboratory for Clinical Diagnostics GmbH & Co., KG, Bad Kissingen, Germany), while the presence of the mec A gene was confirmed at Microsynth (Vienna, Austria). The MRSA strain was deposited in the GenBank database under accession number MN822720 (Styková et al. 2022 ). The wells of a polystyrene microtitre plate (Greiner ELISA 8 Well Strips, 350 μL, Flat Bottom, Medium Binding; Cruinn Diagnostics Ltd., Dublin, Ireland) were filled with 100 μL of BHI medium (mBHI; Brain Heart Infusion Broth; HIMEDIA) enriched with 1% glucose and 2% NaCl. This BHI medium also contained BS 9FT27 at concentrations of 0.1–15 mg/mL. The medium was inoculated with the indicator strain S. aureus CCM 4223 (McFarland 1; 15 μL per well). The negative control was mBHI with physiological solution, or mBHI with corresponding concentrations of BS 9FT27 served as a positive control. The plates were incubated at 37 °C for 24 h. A modified crystal violet assay (O´Toole et al. 1999 ) was used to evaluate biofilm formation with modifications as described in Styková et al. ( 2022 ). A volume of 500 µL of TRI-reagent (Sigma-Aldrich, St. Louis, USA) was added to each tube containing the controls ( S. aureus CCM 4223 or MRSA) and also to these pathogens with different concentrations of BS. The contents of each tube were pipetted to extract total RNA. The RNAeasy mini kit (Qiagen, Hilden, Germany) was used to purify total RNA according to the manufacturer’s instructions. The concentration and purity of total RNA were assessed spectrophotometrically using a NanoPhotometer P-Class P 300 (Implen, Munich, Germany) in the ratios 260/280 and 260/230. For all isolated RNA samples, the 260/280 ratio was approximately 2.0 and the 260/230 ratio was around 1.8 to 2.2, confirming minimal contamination. Reverse transcription was subsequently carried out using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, California, USA), as described in Karaffová et al. ( 2017 ). The relative gene expression of selected biofilm-related genes in S. aureus biofilms ( ica A, ica B, ica D, srt A, gyr B, clf B) was determined. In addition, the relative mRNA expression of the reference gene encoding 16 s rRNA was evaluated. Changes in transcription and relative expression of selected genes were measured by real-time PCR performed on a Lightcycler 480 II Instrument (Roche, Basel, Switzerland) using SsoAdvanced TM universal SYBR green supermix (Bio-rad Laboratories, Hercules, California) and specific primers. The primer sequences for qRT-PCR were used according to previous studies (Frank et al. 2008 ; Iqbal et al. 2016 ; Nourbakhsh and Namvar 2016 ; Pereyra et al. 2016 ; Farnsworth et al. 2017 ; Englerová et al. 2021 ) and listed in Table S1. The DNA amplification efficiency was between 94 and 100%. The reaction mixture contained 10 μL of master mix, 4 μL water, 1 μL of each primer and 4 μL of the cDNA sample. The qRT-PCR reaction was initiated by denaturation at 95 °C for 30 s and followed by amplification: UDG activation at 50 °C for 2 min, initial denaturation at 95 °C for 2 min, subsequent 39 cycles: denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min and extension step at 72 °C for 2 min. For each RT-qPCR plate, a melting curve from 65 °C to 95 °C with readings at 0.5 °C each was obtained. Analysis was performed after each run to ensure a single amplified product for each reaction. Each real-time PCR reaction was performed in duplicate, and the mean values of the duplicate were used for further analysis. Amplification efficiency of each gene (including HPRT) was confirmed between 94 and 100% in the exponential phase of the reaction, where the quantification cycle (Cq) was calculated. The Cq values of the studied genes were normalised to the average Cq value of the reference gene (ΔCq), and the relative expression of each gene was mathematically calculated as 2 −ΔΔCq . The data were analysed using GraphPad Prism v10.4.0 software (GraphPad Software Inc., San Diego, CA, USA). The results were assessed using two-way ANOVA followed by Dunnett’s test for multiple comparisons. Significance was set at P  < 0.05. The Kolmogorov–Smirnov test for normality was used for statistical analyses of quantitative real-time PCR to assess the distribution of the data. The significance of the differences between the concentrations in both groups (MRSA and S. aureus CCM 4223) and the control was determined using an unpaired T-test. The level of statistical significance was expressed as a P value ( P  < 0.05, P  < 0.01, P  < 0.001, P  < 0.001). The values in the figures are given as mean values with standard deviations (± SD).

Biosurfactants are secondary metabolites that are synthesised non-ribosomally by actively growing and/or resting microbial cells (Ron and Rosenberg 2001 ). Due to their amphiphilic nature, their antimicrobial activity lies in their ability to directly disrupt the integrity of membranes in a detergent-like manner. Therefore, they are less likely to develop resistance compared to conventional antibiotics (Ndlovu et al. 2017 ). BSs are very attractive molecules due to their specificity, low toxicity, high biodegradability, broad applicability and efficacy at extreme pH, temperature and salinity (Muthusamy et al. 2008 ; Ben Ayed et al. 2014 ). The study of Lin et al. ( 2020 ) demonstrated that fengycins produced by Bacillus amyloliquefaciens JFL21 maintained stable anti- Listeria activity across a wide range of pH and temperatures, and remained effective following exposure to ultraviolet sterilization, various chemical agents, and proteolytic enzymes. Fengycins offer a potential biological control strategy against multidrug-resistant foodborne pathogens. The oral administration of biosurfactants, recently also referred to as postbiotics, is said to provide several health benefits to the consumer (Hiramoto et al. 2023 ). Peptides produced by members of the genus Bacillus have been shown to exhibit broad-spectrum antimicrobial activity against pathogenic microbes (Sumi et al. 2015 ). Most studies have used oral probiotics, but studies investigating topical probiotics are also becoming increasingly common (Yu et al. 2020 ). The extracellular metabolite of H. coagulans MTCC 5856 LactoSporin® was used in the treatment of mild-to-moderate acne lesions and seborrhoeic conditions. It also showed anti-inflammatory and dose-dependent anti-collagenase activity, suggesting that it may maintain skin firmness and prevent wrinkle formation (Majeed et al. 2020 ). Partially purified lactosporin extracted from H. coagulans has also been used as an effective alternative for the application as wound dressings due to its strong antibacterial properties (Ekrami et al. 2024 ). The production of fengycin was reported e.g. in B. subtilis (Hu et al. 2019 ), B. thuringiensis (Roy et al. 2013 ), B. circulans (Niekerk et al. 2023 ), B. megaterium (Wang et al. 2020 ) and B. amyloliquefaciens (Englerová et al. 2021 ), but as far as we know, not in H. coagulans . The aim of the present study was to extract and characterise lipopeptide fengycin ( fen D) produced by H. coagulans 9FT27. The results obtained by UHPLC-DAD, NMR and MALDI-TOF/MS analyses confirmed the production of the lipopeptide biosurfactant fengycin by H. coagulans 9FT27. These methods are useful tools for the identification of a variety of substances in biological samples using different protocols (Kim et al. 2010 ; Biniarz et al. 2017 ). MALDI-TOF is a suitable method for the identification of peptides in the 1–5 kDa range (Stein 2008 ). The fengycin class of biosurfactant lipopeptides has been studied in detail by various authors. For example, Sarangi et al. ( 2009 ) identified fengycin mass peaks produced by different Bacillus species, which are in good agreement with our results. A variety of HPLC methods for the identification of lipopeptides have been proposed. Our method for identifying fengycin was developed specifically for our conditions. The identification of fengycin isoforms by the method developed was rapid and efficient. The antibiofilm activity of BSs is associated with their ability to affect bacterial adhesion due to changes in surface tension and their influence on biofilm-related gene expression (Shokouhfard et al. 2022 ). Surfactin effectively inhibits adhesion and disrupts biofilms of S. aureus (including MRSA) on surface. Mechanistically, surfactin reduces production of alkali-soluble exopolysaccharides, downregulates expression of ica A and ica D needed for PIA-mediated biofilm, and interferes with the AI 2 quorum-sensing system (Liu et al. 2019 ). In MRSA clinical isolates, surfactin significantly prevents biofilm formation at sub-inhibitory concentrations, while also showing synergistic antibacterial effects with antibiotics like ampicillin, oxacillin, and tetracycline (Li et al. 2023 ). Iturin W (especially the C₁₄ variant) exhibits strong antibacterial activity against S. aureus ATCC 25923 and MRSA strains (Ji and Wu 2024 ). However, its biofilm-inhibitory properties against S. aureus or MRSA biofilms are not well-documented. Fengycin alone has limited antibacterial and antibiofilm efficacy against S. aureus and MRSA compared to surfactin. It predominantly acts against filamentous fungi (Wu et al. 2025 ). However, the study by Piewngam et al. ( 2018 ) showed fengycin alone can inhibit S. aureus quorum sensing via competitive binding to AgrC, the sensor kinase in the Agr regulatory system. This disruption leads to reduced virulence and limits colonization by both wild-type and MRSA strains in murine models (Chung et al. 2019 ). The results showed that co-incubation of fengycin from  H. coagulans 9FT27 with  S. aureus  CCM 4223 and MRSA significantly inhibited biofilm formation. The percentage of inhibition of biofilm formation at concentrations from 15 to 0.1 mg/mL ranged from 97 to 12% for S. aureus  CCM 4223 and 75–14% for MRSA. The formation of a biofilm is one of the key virulence factors in many pathogens. It consists of an extracellular matrix made up of surface proteins, exopolysaccharides and extracellular DNA. The regulatory mechanisms underlying biofilm formation are complex and depend on the co-expression of several genes (Peng et al. 2022 ). In S. aureus , the production of the polysaccharide intercellular adhesin (PIA), which is considered one of the main exopolysaccharide components of the staphylococcal biofilm, is regulated by the ica operon comprising icaA , icaB , icaC and icaD genes. More importantly, the expression of this operon can be influenced by various factors such as the use of antibiotics, extreme temperatures, the availability of oxygen and other stress conditions. It has also been shown that the genes of the ica ADBC operon were noticeably upregulated during the maturation stages of biofilm formation, particularly at the 24 h, 32 h and 48 h with a 7-, 12- and 15-fold increase, respectively (Oknin et al. 2023 ). Previous reports indicated that while curcumin at a concentration of 2 mg/mL exerted antibiofilm and antimicrobial activity that downregulated the expression of icaA , icaB and icaD , the relative expression of icaA was increased 9- and 20-fold in the presence of 4% and 6% NaCl, respectively (Kot et al. 2018 ; Lee et al. 2014 ). In the present study, the expression of the genes icaA , icaB and icaD was significantly inhibited by the different concentrations of fengycin. The test BS at all the concentrations used also downregulated srt A encoding sortase A, an enzymatic membrane protein that assists in anchoring certain surface proteins to the cell wall and its inactivation reduced virulence of the pathogen by reducing its adhesion capacity (Schneewind et al. 2019 ; Zong et al. 2004 ). In the study, Wang et al. ( 2021 ) reported that the flavonoid compound taxifolin inhibits the activity of sortase A, and suggested the potential of SrtA inhibitors for their possible application in the treatment of various Gram-positive bacterial infections. Clumping factor B, encoded by the gene clfB, is another key virulence factor of S. aureus , a protein associated with its ability to colonise epithelial cells (Ganesh et al. 2011 ). It was reported that clfB gene is involved in infection, pathogenicity and cell adhesion, and is increasingly expressed during biofilm formation (Kang et al. 2022 ). Even deletion of this gene in S. aureus strains virtually eliminated their ability to form a biofilm (Abraham and Jefferson 2012 ). In this study, all concentrations of BS significantly inhibited the expression of clfB in both S. aureus CCM 4223 and MRSA. In addition to the genes that are usually analysed during the application of antimicrobial agents, the expression of gyrB gene, which encodes a subunit of DNA gyrase, was also determined. DNA gyrase is an important bacterial enzyme that consists of two subunits, gyrA and gyrB, and is involved in the cleavage of DNA duplexes by double-stranded cleavage (Kato et al. 2023 ). While gyrB i s used as a reference gene (housekeeping gene) in many studies, it is also one of the factors involved in biofilm formation in S. aureus (Gurram and Azam 2021 ). This study confirmed that all tested BS concentrations significantly reduced gyrB expression. The inhibition of DNA gyrase and the associated antibacterial activity were also demonstrated in the study by Alt et al. ( 2011 ), in which various aminocoumarin compounds were used as inhibitors of S. aureus DNA gyrase. In conclusion, our results suggested that the BS produced by H. coagulans ( B. coagulans ) Biocenol™ 9FT27 CCM 9014 showed promise as a potential staphylococcal biofilm disruption agent. Although downregulation of biofilm-associated genes has been demonstrated, the exact mechanism of action of fengycin remains unknown. Further studies are required to elucidate this mechanism and support technological advances to optimise and scale up its production.

Heyndrickxia coagulans  was first described by Narsing Rao et al. ( 2023 ) and was formerly recognised as Bacillus coagulans and Weizmannia coagulans (Gupta et al. 2020 ). The strain B. coagulans Hammer (ATCC 7050; DSM1) was first described as the type strain of  B. coagulans  in 1915 by Hammer, who isolated this organism from spoiled canned milk (De Clerck et al. 2004 ). The growing concern about the development of pathogenic bacteria and their increasing resistance to antibiotics has prompted the search for alternative therapeutic methods. In recent decades, very few novel antimicrobial agents and therapies have been made available for clinical use, although the need is increasing (Wiman et al. 2023 ). Bacteriotherapy with  H. coagulans is a promising approach for the prevention and treatment of dysbiosis in various parts of the body such as the respiratory tract, skin and ears (Maresca et al. 2024 ). Antimicrobial peptides from Bacillus species are ideal therapeutic tools due to their broad specificity and their specific and rapid killing activity against various pathogens (Sumi et al. 2015 ). Biosurfactants (BSs) are postbiotics defined as „non-living preparations of microorganisms and/or their components that confer a health benefit on their host” (Salminen et al. 2021 ). They are known to have surface-active and antibiotic properties and have the potential to be used in bioremediation and microbially enhanced oil recovery (Huszcza and Burczyk 2003 ; Das et al. 2009 ).  H. coagulans produces compounds with antimicrobial activity against Gram‐positive and Gram‐negative bacteria (Le Marrec et al. 2000 ; Kim et al.  2021 ) and is used in various commercial food products (Konuray et al. 2021 ). Protein components with strong antibacterial and anti-aging properties have been isolated from H. coagulans  (Zhang et al. 2025 ). LactoSporin is an extracellular metabolite preparation extracted from the probiotic strain  H. coagulans MTCC 5856 (LactoSpore®) (Majeed et al. 2020 ). In addition, H. coagulans  has not been shown to cause skin/eye irritation or sensitization issues on the skin (Endres et al. 2009 ). The aim of the present study was to isolate BS from H. coagulans 9FT27 and test its antibiofilm activity against the biofilm-producing reference strain Staphylococcus aureus CCM 4223 and the clinical isolate MRSA (methicillin-resistant Staphylococcus aureus ) under in vitro conditions. The isolated and partially purified lipopeptide fengycin was identified and characterised using molecular tools and by UHPLC-DAD (ultrahigh-performance liquid chromatography with diode array detector), MALDI-TOF/MS (matrix-assisted laser desorption/ionization time-of-flight/molecular mass) and NMR (nuclear magnetic resonance spectroscopy). The expression of biofilm-related genes was also determined.

Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 Supplementary Material 5 Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 Supplementary Material 5