Antimicrobial activity of postbiotics from Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™)

Antimicrobial activity of postbiotics from Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™) | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 October 2025 V1 Latest version Share on Antimicrobial activity of postbiotics from Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™) Authors : Maryam Ghaffari , Azin Aghajani Amiri , Amir Reza Rad , Faraz Fotouhi , Mohammad Hasan Neghabi , Kianoush Forouhar Majd , Hamed Azad 0009-0007-8870-9784 , and Arian Ghasemi 0009-0001-2665-6863 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175999901.16120771/v1 174 views 114 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The increasing resistance of pathogenic bacteria to conventional antibiotics has necessitated the search for alternative antimicrobial agents. This study investigated the antimicrobial activity of postbiotic metabolites produced by Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™ ). The broth microdilution method determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the postbiotics. Additionally, the disk diffusion and well diffusion methods were employed to evaluate the antimicrobial activity. The results demonstrated that the postbiotic metabolites exhibited significant antimicrobial effects, with an MIC of 31.25 μg/mL and an MBC of 62.5 μg/mL. Inhibition zones of 8.33±0.58 a mm, 10.00±1.00 a mm, and 11.33±1.53 b mm were observed in the disk diffusion method at concentrations of 105, 125, and 145 μg/mL, respectively. In the well diffusion method, inhibition zones of 22.00±1.00 a mm, 26.00±1.00 b mm, 26.67±0.58 b mm, and 29.33±0.58 c mm were observed at concentrations of 125, 135, 145, and 155 μg/mL, respectively. These findings suggest that postbiotic metabolites from Pediococcus acidilactici have potential as alternative antimicrobial agents against Staphylococcus aureus . Antimicrobial activity of postbiotics from Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™ ) Title: Antimicrobial activity of postbiotics from Pediococcus acidilactici against Escherichia coli (ATCC 25922™ ) Authors: Maryam Ghaffari 1 ., Azin Aghajani Amiri 2 ., Amir Reza Rad 3 , Faraz Fotouhi 4 , Mohammad Hasan Neghabi 5 , Kianoush Forouhar Majd 6 , Hamed Azad 2 , Arian Ghasemi 2 Affiliations: 1 Department of Veterinary Medicine, Zabol University, Zabol, Iran 2 Department of Veterinary Medicine, Islamic Azad University, Babol, Iran 3 Department of Medical Laboratory Science, Gonabad Medical University,gonabad, Iran 4 Graduated Student of the Faculty of Veterinary Medicine, Urmia University, Urmia, Iran. 5 Graduated Student of the Faculty of veterinary Medicine, Islamic Azad University, shabestar, Iran. 6 Graduated Student of the Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran. Keywords: Antimicrobial activity, Postbiotic, Probiotic, Staphylococcus aureus , Pediococcus acidilactici Correspondence: Arian Ghasemi E-mail: [email protected] ABSTRACT The increasing resistance of pathogenic bacteria to conventional antibiotics has necessitated the search for alternative antimicrobial agents. This study investigated the antimicrobial activity of postbiotic metabolites produced by Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™ ). The broth microdilution method determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the postbiotics. Additionally, the disk diffusion and well diffusion methods were employed to evaluate the antimicrobial activity. The results demonstrated that the postbiotic metabolites exhibited significant antimicrobial effects, with an MIC of 31.25 μg/mL and an MBC of 62.5 μg/mL. Inhibition zones of 8.33±0.58 a mm, 10.00±1.00 a mm, and 11.33±1.53 b mm were observed in the disk diffusion method at concentrations of 105, 125, and 145 μg/mL, respectively. In the well diffusion method, inhibition zones of 22.00±1.00 a mm, 26.00±1.00 b mm, 26.67±0.58 b mm, and 29.33±0.58 c mm were observed at concentrations of 125, 135, 145, and 155 μg/mL, respectively. These findings suggest that postbiotic metabolites from Pediococcus acidilactici have potential as alternative antimicrobial agents against Staphylococcus aureus . INTRODUCTION Pediococcus acidilactici is a gram-positive bacterium belonging to the group of homofermentative lactic acid bacteria, which is considered one of the most important known members of probiotic bacteria and can prevent the growth of dangerous pathogenic microbes [1]. These bacteria have antimicrobial properties and can also reduce the growth and proliferation of pathogenic and spoilage microorganisms and neutralize the metabolites and toxins [2]. The antimicrobial activity of these bacteria is caused by the production of inhibitory substances such as organic acids (lactic acid and acetic acid), oxygen compounds like hydrogen peroxide, protein compounds such as bacteriocins, low molecular weight peptides, and antifungal proteins [3]. Postbiotics produced by Pediococcus acidilactici include a variety of bioactive compounds, such as bacteriocins (e.g., pediocin), organic acids (e.g., lactic acid and acetic acid), and other metabolites that exhibit antimicrobial, immunomodulatory, and anti-inflammatory properties. These postbiotics have shown promising results in inhibiting the growth of foodborne pathogens and spoilage microorganisms, making them potential candidates for use in food preservation and therapeutic applications [4]. Pediococcus species produce small, heat-resistant, non-lanthionine bacteriocins that contain class 2 peptides. These bacteriocins have demonstrated antimicrobial activity against food-borne pathogens such as Listeria monocytogenes , Clostridium perfringens , Bacillus cereus , and Staphylococcus aureus [5]. Staphylococcus aureus is a major opportunistic pathogen and one of the most common causes of both community-acquired and healthcare-associated bacterial infections in humans. This bacterium is a leading cause of skin and soft tissue infections and also causes life-threatening conditions such as bacteremia, endocarditis, and osteomyelitis [6]. The treatment of infections caused by Staphylococcus aureus has been profoundly complicated by the emergence of antimicrobial resistance. The global spread of methicillin-resistant Staphylococcus aureus (MRSA) is a major public health crisis, driven largely by the horizontal transfer of resistance genes located on mobile genetic elements like staphylococcal cassette chromosomes (SCC mec ) [7]. The efficacy of antibacterial drugs has significantly decreased, largely due to the rise in bacterial resistance, which is currently one of the major public health concerns [8]. Most infections caused by antibiotic-resistant microorganisms do not respond to conventional treatments, and even last-resort antibiotics have lost their effectiveness. The increase in multidrug-resistant bacteria over recent decades has led to serious problems [9]. Postbiotics refer to the non-living functional components of probiotic cells, typically produced by living bacteria during the fermentation process or through various physical and chemical methods in a laboratory setting. Postbiotics can include both bacterial and fungal species [10]. MATERIALS AND METHODS Bacterial Strains and Culture Conditions The study utilized Staphylococcus aureus (ATCC 13813™ ) as the target pathogen. The strain was obtained from the Iranian Biological Resource Center (IBRC) located in Tehran, Iran. The strain was cultured on Mueller-Hinton agar (MHA) and maintained at 37 °C for 24 hours. For experimental purposes, a single colony was inoculated into Mueller-Hinton broth (MHB) and incubated at 37 °C for 18-24 hours to achieve a turbidity equivalent to 0.5 McFarland standard (approximately 1.5 × 10 8 CFU/mL). Preparation of Postbiotic Metabolites The postbiotic metabolites were obtained from Pediococcus acidilactici , a probiotic strain cultured in De Man, Rogosa, and Sharpe (MRS) broth at 37 °C for 48 hours under anaerobic conditions. After incubation, the culture was centrifuged at 10,000 × g for 15 minutes to separate the bacterial cells from the supernatant. The supernatant, containing the postbiotic metabolites, was filtered through a 0.22 μm membrane filter to ensure sterility. The concentration of the postbiotic metabolites was determined using spectrophotometry at a wavelength of 600 nm, and the final concentration was adjusted to 500 mg/mL for further experiments. Extraction of Metabolites The contents of the Erlenmeyer flasks, containing approximately 100 mL of MHB culture medium, were divided equally into 10 centrifuge tubes. The tubes were centrifuged three times at 3000 rpm for 10 minutes each. After centrifugation, the supernatant was carefully extracted using a Landa 1000 sampler, excluding the bottom 1 mL containing bacterial cells. The extracted supernatant, containing the bacterial metabolites, was transferred to separate Erlenmeyer flasks for further use [11]. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The MIC and MBC of the postbiotic metabolites against Staphylococcus aureus (ATCC 13813™ ) were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, two-fold serial dilutions of the postbiotic metabolites were prepared in MHB, ranging from 500 μg/mL to 7.8 μg/mL. Each well was inoculated with 100 μL of bacterial suspension (1.5 × 10 6 CFU/mL) and incubated at 37 °C for 24 hours. The MIC was defined as the lowest concentration of the postbiotic that completely inhibited visible bacterial growth. For MBC determination, 10 μL from each well showing no visible growth was subcultured on MHA plates and incubated at 37 °C for 24 hours. The MBC was defined as the lowest concentration that resulted in no bacterial growth on the agar plates. Disk Diffusion and Well Diffusion Assays The antimicrobial activity of the postbiotic metabolites was also evaluated using the disk diffusion and well diffusion methods. For the disk diffusion assay, sterile paper discs (6 mm diameter) were impregnated with 10 μL of the postbiotic metabolites at concentrations of 105, 125, and 145 μg/mL. The discs were placed on MHA plates inoculated with Staphylococcus aureus (0.5 McFarland standard) and incubated at 37 °C for 24 hours. The zones of inhibition were measured in millimeters. For the well diffusion assay, wells (6 mm diameter) were punched into MHA plates inoculated with Staphylococcus aureus . Each well was filled with 100 μL of the postbiotic metabolites at concentrations of 125, 135, 145, and 155 μg/mL. The plates were incubated at 37 °C for 24 hours, and the zones of inhibition were measured. Statistical Analysis All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism, version 9.0. The significance of differences between groups was assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value < 0.05 was deemed statistically significant. RESULTS The outcome of identifying the minimum inhibitory concentration of metabolites on Staphylococcus aureus (ATCC 13813™) . In this study, the minimum concentration that inhibits the growth of Staphylococcus aureus (ATCC 13813™) in the presence of the metabolite obtained from Pediococcus acidilactici was determined to be 31.25 μg/mL. The test results for the minimum bactericidal concentration of Pediococcus acidilactici metabolites against Staphylococcus aureus (ATCC 13813™) . In this study, the minimum bactericidal concentration of Staphylococcus aureus (ATCC 13813™) near the metabolite obtained from Pediococcus acidilactici was 62.5 μg/mL. The results of the disk method test for Staphylococcus aureus ’s non-growth halo. The non-growth halo test results indicated that the metabolite of Pediococcus acidilactici at concentrations of 105, 125, and 145 μg/mL produced halos with diameters of 8.33±0.58 a , 10.00±1.00 a , and 11.33±1.53 b mm,respectively. The effect of extract concentrations of 105, 125, and 145 μg/mL on the growth inhibition of Staphylococcus aureus bacteria was evaluated using the disk diffusion method. An ANOVA test was conducted to assess the differences in average growth inhibition zones among the various extract volumes . The statistical analysis revealed a significant difference (p=0.043) between the concentrations used in the antibacterial disk diffusion test. The 145 μg/mL concentration showed a significant increase in the inhibition zone compared to 105 μg/mL and 125 μg/mL, but the 125 μg/mL concentration did not show a significant difference in the inhibition zone when compared to 105 μg/mL. The results of the test to determine the non-growth halo of Staphylococcus aureus using the well method. The results of the bacteria growth halo test indicated that the wells with metabolite concentrations of 125, 135, 145, and 155 μg/mL produced growth halos measuring 22.00±1.00 a , 26.00±1.00 b , 26.67±0.58 b , and 29.33±0.58 c mm in diameter, respectively. The effect of extract concentrations of 125, 135, 145, and 155 μg/mL on the inhibition of Staphylococcus aureus growth was evaluated using the well method (see Table 2). An ANOVA statistical test was employed to assess the differences in the mean sizes of the non-growth halos. The statistical analysis revealed a significant difference between the concentrations used in the antibacterial well diffusion test for the extract (p=0.001). Post hoc analysis showed that the 125 μg/mL concentration produced the smallest inhibition zone, the zones of inhibition at concentrations of 135 μg/mL and 145 μg/mL of the extract did not show a significant difference from each other. However, they showed a significantly larger zone of inhibition compared to the concentration of 125 μg/mL, and a smaller zone of inhibition compared to the concentration of 155 μg/mL. On the other hand, the concentration of 155 μg/mL of the extract showed the largest zone of inhibition compared to the other three concentrations, and this increase was also statistically significant. 4. DISCUSSION The antimicrobial activity of Pediococcus acidilactici metabolites is attributed to a variety of bioactive compounds produced during fermentation. These metabolites include organic acids, bacteriocins, hydrogen peroxide, and other antimicrobial peptides, which collectively contribute to their inhibitory effects against pathogenic bacteria such as Staphylococcus aureus . Pediococcus acidilactici produces significant amounts of lactic acid and acetic acid during fermentation. These organic acids lower the pH of the environment, creating unfavorable conditions for bacterial growth. The undissociated forms of these acids can penetrate bacterial cell membranes, disrupting intracellular pH homeostasis and leading to cell death [13]. One of the most well-known bacteriocins produced by Pediococcus acidilactici is pediocin, a class IIa bacteriocin. Pediocin exhibits strong antimicrobial activity against gram-positive bacteria. Pediocin disrupts bacterial cell membranes by forming pores, leading to leakage of intracellular contents and cell death [12]. Pediococcus acidilactici also produces hydrogen peroxide as a byproduct of aerobic metabolism. Hydrogen peroxide induces oxidative stress in bacterial cells, damaging DNA, proteins, and lipids, which ultimately results in cell death [14]. In addition to the above, Pediococcus acidilactici produces other bioactive compounds, such as diacetyl, reuterin, and exopolysaccharides, which contribute to its antimicrobial and immunomodulatory properties. These compounds can inhibit bacterial adhesion, biofilm formation, and quorum sensing, further enhancing their antimicrobial efficacy [15]. The results of our study demonstrated that the secondary metabolites of Pediococcus acidilactici exhibit significant antimicrobial activity against Staphylococcus aureus . The minimum inhibitory concentration (MIC) was determined to be 31.25 μg/mL, while the minimum bactericidal concentration (MBC) was 62.5 μg/mL. In the disk diffusion assay, the metabolite produced inhibition zones of 8.33±0.58 mm, 10.00±1.00 mm, and 11.33±1.53 mm at concentrations of 105, 125, and 145 μg/mL, respectively. Similarly, in the well diffusion method, concentrations of 125, 135, 145, and 155 μg/mL resulted in inhibition zones measuring 22.00±1.00^a^mm, 26.00±1.00 mm, 26.67±0.58 mm, and 29.33±0.58 mm, respectively. The antimicrobial action of Pediococcus acidilactici metabolites against Staphylococcus aureus is likely mediated through multiple synergistic mechanisms. Organic acids lower the intracellular pH and disrupt membrane integrity, bacteriocins form pores in the bacterial cell membrane, and hydrogen peroxide induces oxidative stress. These combined effects lead to substantial growth inhibition, as evidenced by the MIC and MBC values [12,14]. The increasing prevalence of antibiotic-resistant Staphylococcus aureus , particularly the global spread of methicillin-resistant Staphylococcus aureus (MRSA), is a major public health crisis, driven largely by the horizontal transfer of resistance genes, which underscores the urgent need for alternative antimicrobial agents. Probiotic-derived compounds, such as those from Pediococcus acidilactici , represent promising candidates due to their efficacy and safety profile [15]. The findings of this study highlight the potential of these metabolites as a viable strategy to combat resistant bacterial strains. In a seminal multinational study published in 2017 by Giske et al., which analyzed invasive isolates from numerous countries, Staphylococcus aureus was a leading cause of bloodstream infections. The prevalence of methicillin-resistant S. aureus (MRSA) varied significantly by region, exceeding 25% in many parts of the world. Resistance to oxacillin (which indicates methicillin resistance) among S. aureus isolates in this global collection was a primary driver of therapeutic failure. As a result, the high prevalence of MRSA continues to complicate treatment regimens, intensifying the need to develop and preserve the efficacy of last-line antimicrobials like vancomycin [16]. In a 2019 study on skin and soft tissue infections by Esposito et al., Staphylococcus aureus was identified as the most prevalent pathogen, isolated in over 50% of culture-positive cases. Methicillin resistance was detected in a significant proportion of these S. aureus isolates. Resistance to clindamycin among community-acquired MRSA isolates was found to be over 30% in some patient groups. Consequently, the high resistance rates to common oral agents limit therapeutic options for outpatient management, underscoring the critical need for new antimicrobial strategies [17]. Several studies have investigated the antimicrobial potential of probiotic metabolites, and our findings are consistent with and expand upon these previous works. For instance, a study by Arena et al. (2018) demonstrated that Pediococcus acidilactici strains produce bacteriocins with strong inhibitory activity against Listeria monocytogenes and Staphylococcus aureus . In another study, Garsa et al. (2014) reported that Pediococcus acidilactici metabolites, particularly pediocin, exhibited significant antimicrobial activity against foodborne pathogens such as Salmonella and Clostridium . Their findings align with our results, as we observed similar inhibitory effects against Staphylococcus aureus . However, our study provides additional insights by quantifying the MIC (31.25 μg/mL) and MBC (62.5 μg/mL) values, which are crucial for understanding the therapeutic potential of these metabolites against Staphylococcus aureus strains. This quantitative approach offers a clearer framework for future clinical or industrial applications of postbiotics in combating gram-positive pathogens. The antimicrobial properties of Pediococcus acidilactici metabolites make them a promising candidate for the treatment of infections caused by Staphylococcus aureus , particularly in hospital-acquired infections. They could also be used in the food industry as natural preservatives to inhibit the growth of pathogenic bacteria in food products. Given their potential safety and efficacy, these metabolites could be developed into novel therapeutic agents or incorporated into probiotic formulations to enhance gut health and prevent infections. While our study provides valuable insights into the antimicrobial potential of Pediococcus acidilactici metabolites, it has certain limitations. For instance, the study was conducted in vitro, and further research is needed to evaluate the efficacy and safety of these metabolites in vivo. Additionally, the specific bioactive compounds responsible for the observed effects need to be isolated and characterized. Future studies should also explore the synergistic effects of these metabolites with existing antibiotics, as well as their potential applications in clinical and industrial settings. In conclusion, the secondary metabolites of Pediococcus acidilactici exhibit significant antimicrobial activity against Staphylococcus aureus , making them a promising candidate for the development of novel therapeutic agents. Their natural origin, combined with their potent antimicrobial effects, positions them as a viable alternative to traditional antibiotics in the fight against antibiotic-resistant infections. Further research is warranted to fully explore their potential and translate these findings into practical applications. ACKNOWLEDGMENTS This research was conducted in the laboratory of the Faculty of Veterinary Medicine at Islamic Azad University in Babol. CONFLICT OF INTEREST The authors declare that they have no potential conflicts of interest. DAS: The data supporting this study’s findings are available from the corresponding author, [Arian Ghasemi], upon reasonable request. REFERENCES 1. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014 Aug;11(8):506-14. 2. Gaggìa, F., Mattarelli, P., & Biavati, B. 2010. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology , 141, S15-S28.https://doi.org/10.1016/j.ijfoodmicro.2010.02.031 3. Żółkiewicz, J., Marzec, A., Ruszczyński, M., & Feleszko, W. 2020. Postbiotics: A step beyond pre- and probiotics. Nutrients , 12(8),https://doi.org/10.3390/nu12082189 4. Aguilar-Toalá, J. E., Garcia-Varela, R., Garcia, H. 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Antibiotic resistance of Escherichia coli, Listeria and Salmonella isolates from retail meat tables in Ibadan municipal abattoir, Nigeria. African Journal of Biotechnology, 10(30), 5795-5799. Table 1 . Zones of inhibition of metabolite of Pediococcus acidilactici at different concentrations using disk-diffusion method 0.043 11.33±1.53 b 10.00±1.00 a 8.33±0.58 a Zone of Inhibition (mm) ± SD Table 2 . Zones of inhibition of metabolite of Pediococcus acidilactici at different concentrations using well-diffusion method <0.001 29.33±0.58 c 26.67±0.58 b 26.00±1.00 b 22.00±1.00 a Zone of Inhibition (mm) ± SD List of Abbreviations • ANOVA: Analysis of Variance • ATCC: American Type Culture Collection • CFU: Colony Forming Units • CLSI: Clinical and Laboratory Standards Institute • IBRC: Iranian Biological Resource Center • MHA: Mueller-Hinton Agar • MHB: Mueller-Hinton Broth • MIC: Minimum Inhibitory Concentration • MBC: Minimum Bactericidal Concentration • MRS: de Man, Rogosa and Sharpe • MRSA: Methicillin-Resistant Staphylococcus aureus • rpm: Revolutions Per Minute • SD: Standard Deviation Information & Authors Information Version history V1 Version 1 09 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords pediococcus acidilactici staphylococcus aureus antimicrobial activity postbiotic probiotic Authors Affiliations Maryam Ghaffari University of Zabol View all articles by this author Azin Aghajani Amiri Islamic Azad University Babol Branch View all articles by this author Amir Reza Rad Gonabad University of Medical Sciences View all articles by this author Faraz Fotouhi Urmia University Faculty of Veterinary Medicine View all articles by this author Mohammad Hasan Neghabi Islamic Azad University Shabestar Branch View all articles by this author Kianoush Forouhar Majd Shahrekord University Faculty of Veterinary Medicine View all articles by this author Hamed Azad 0009-0007-8870-9784 Islamic Azad University Babol Branch View all articles by this author Arian Ghasemi 0009-0001-2665-6863 [email protected] Islamic Azad University Babol Branch View all articles by this author Metrics & Citations Metrics Article Usage 174 views 114 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Maryam Ghaffari, Azin Aghajani Amiri, Amir Reza Rad, et al. Antimicrobial activity of postbiotics from Pediococcus acidilactici against Staphylococcus aureus (ATCC 13813™). Authorea . 09 October 2025. DOI: https://doi.org/10.22541/au.175999901.16120771/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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