Cell-Free Culture Supernatants of Lactobacilli Modify the Expression of Virulence Factors Genes in Staphylococcus aureus

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant microorganism and the predominant nosocomial pathogen all over the world. The potential benefits of probiotic lactobacilli against pathogenic bacteria have been shown in many studies.

Objectives: This study aimed to investigate the effect of cell-free culture supernatant (CFS) of probiotic Lactobacillus spp, including Lactobacillus reuteri, L. plantarum, and L. fermentum, on virulence factor gene expression of MRSA.

Methods: Lactobacilli were cultured in MRS and the cells were harvested by centrifuging at 10,000 x g for 10 min at 4°C. The pellet was discarded and 1/2 and 1/4 CFS concentrations from Lactobacillus spp were added to the medium (MHB) containing 107 CFU/mL of the MRSA strain. A quantitative polymerase chain reaction (qPCR) was performed to measure the expression of virulence factors at the transcriptional level.

Results: The results showed that lactobacilli CFS had no obvious inhibitoryeffectsonthe growth of S. aureus. The qPCR assay showed that the expression levels of sea, sae, agr A, tst, spa, and spi genes reduced at different levels, depending on the concentration of CFS and the species of lactobacilli so that the maximum significant down-regulation rate was observed in the sea and tst genes (up to 23.5 folds in the presence of 1/2 concentration of L. reuteri CFS after12 h incubation).

Conclusions: Cell-free culture supernatants of probiotic bacteria can down-regulate the virulence genes. Consequently, toxins and enzymes are less produced by S. aureus as a food-borne pathogen. Therefore, the presence of CFS in the food probably reduces diarrhea and vomiting caused by S. aureus.

Headnote

Received 2019 July31; Revised 2019 December 20; Accepted 2020 January 23.

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant microorganism and the predominant nosocomial pathogen all over the world. The potential benefits of probiotic lactobacilli against pathogenic bacteria have been shown in many studies.

Objectives: This study aimed to investigate the effect of cell-free culture supernatant (CFS) of probiotic Lactobacillus spp, including Lactobacillus reuteri, L. plantarum, and L. fermentum, on virulence factor gene expression of MRSA.

Methods: Lactobacilli were cultured in MRS and the cells were harvested by centrifuging at 10,000 x g for 10 min at 4°C. The pellet was discarded and 1/2 and 1/4 CFS concentrations from Lactobacillus spp were added to the medium (MHB) containing 107 CFU/mL of the MRSA strain. A quantitative polymerase chain reaction (qPCR) was performed to measure the expression of virulence factors at the transcriptional level.

Results: The results showed that lactobacilli CFS had no obvious inhibitoryeffectsonthe growth of S. aureus. The qPCR assay showed that the expression levels of sea, sae, agr A, tst, spa, and spi genes reduced at different levels, depending on the concentration of CFS and the species of lactobacilli so that the maximum significant down-regulation rate was observed in the sea and tst genes (up to 23.5 folds in the presence of 1/2 concentration of L. reuteri CFS after12 h incubation).

Conclusions: Cell-free culture supernatants of probiotic bacteria can down-regulate the virulence genes. Consequently, toxins and enzymes are less produced by S. aureus as a food-borne pathogen. Therefore, the presence of CFS in the food probably reduces diarrhea and vomiting caused by S. aureus.

Keywords: Lactobacillus, MRSA, Virulence Factors, Gene Expression

1.Background

Staphylococcus aureus is one of the most frequent foodborne pathogens causing various infections such as wound infection, septicemia, meningitis, toxic shock syndrome (TSS), and osteomyelitis in humans (1, 2). Staphylococcus aureus produces different toxins and enzymes including enterotoxins, DNase, coagulase, lipase, TSST-1 (tst), and hemolysin involved in the pathogenesis of the diseases (3). The enterotoxins (sea to see and seg to sej) are heat-resistant (4, 5). These toxins can cause food poisoning, staphylococcal gastroenteritis, and diverse autoimmune and allergic diseases (6, 7). Staphylococcus aureus also secretes protein A (spa) and second immunoglobulinbinding protein (sbi), virulence factors associated with staphylococcal pneumonia, and hypodermic infection (8, 9). Therefore, the sea, spa, tst, and sbi genes encode important virulence factors that are regulated by accessory gene regulators (agr) and two-component signal transduction system (sae) in response to environmental stimulating factors (10-12).

Methicillin-resistant S. aureus (MRSA) like other multidrug-resistant bacteria is now resistant to many antimicrobial agents and cause serious infections in inpatients and outpatients (6,13). These isolates from human sources and dairy products are related to foodborne diseases (e.g., diarrhea) in humans who consume contaminated dairy products. Therefore, there is an increasing need to study alternative agents with maximum efficacy and minimum side effects (14,15).

Probiotic bacteria like Lactobacillus species (Lactobacillus reuteri, L. fermentum, and L. plantarum), as a beneficial group of microbiota, play a considerable role in hu- man health (16,17). Lactic acid bacteria (LAB) demonstrate the inhibitory and therapeutic effects on some intestinal pathogenic infections, antibiotic-associated gastrointestinal complications, diarrhea, allergies, cancers, and certain inflammatory and immunomodulatory disorders (18-20). The supernatant of the LAB culture includes aroma components, fatty acids, organic acids, low-molecular-mass compounds, and hydrogen peroxide that can kill pathogens (21). In recent studies, the effect of the supernatant of LAB has been probed on the inhibition of the growth of pathogenic bacteria such as S. aureus and less attention has been put on the effect of the supernatant on the expression of virulence factors.

2. Objectives

In this study, we evaluated the effect of the CFS of three probiotic LAB (L. reuteri, L. fermentum, and L. plantarum) on the gene expression regulator system (sae and agr A) and other genes (sea, tst, spa, and sbi) at the transcriptional level.

3. Methods

3.1. Bacterial Strains and Growth Conditions

The Methicillin-resistant S. aureus (MRSA) strain (ATCC 33591), L. reuteri (ATCC 23272), L.plantarum (ATCC 8014), and L. fermentum (ATCC 9338) were obtained from the Persian type culture collection (PTCC). Methicillin-resistant S. aureus was cultured in Muller Hinton Broth (MHB) at 37°C until it reached the exponential phase of growth. Lactobacilli were cultured in MRS (De Man, Rogosa, and Sharpe) broth at 37°C until they reached the stationary phase of growth. After a 24-h period, the lactobacilli cells were harvested by centrifuging at 10,000 x g for 10 minat 4°C. The pellet was discarded and the cell-free supernatants (CFS) were then adjusted to natural pH (pH = 7) with NaOH (1 N) and sterilized through a 0.22-^m filter. These supernatants were stored at -20°C for later use.

3.2. Microbial Interactions

The density and the number (CFU/mL) of MRSA in cultures were measured through the use of spectrophotometry at OD600. The 1/2 and 1/4 x CFS of Lactobacilli spp. were added to the medium (MHB) containing 107 CFU/mL of MRSA bacteria. After 6 and 12 h of incubation, the bacterial density was quantifiedatOD600,and then centrifuged at 12,000 x rpm/10 min at 4°C. The supernatants were removed and the pellet was used for RNA extraction. A lactobacilli-free culture of MRSA was used as control at the logarithmic phase of growth.

3.3. RNA Extraction and cDNA Synthesis

The RNA was extracted using a Rapid Bacterial RNA Isolation Kit (Bio Basic, Inc., Canada) according to the manufacturer's instructions. The total RNA was solved in 30 ^l of RNase-free water. To remove all DNA, the purified RNA was treated with DNase I, RNase-free (Cinna Gene, Iran) after extraction. The purity of RNA was determined by calculating A260/A230 and A260/A280 ratios (Denovix, NanoDrop, USA) and the integrity of total RNA was assessed with 1% agarose gel (data not shown). The cDNA was synthesized using a cDNA reverse transcription kit (Pars Tous, Iran).

3.4. Quantitative PCR

The quantitative polymerase chain reaction (qPCR) was performed using primers described previously (22-24). Briefly, all PCR assays were accomplished in a total volume of 20 ^L, containing 10 pL of Sybr Green Mix (Takara Bio®, Japan), 0.1 pM of primers, 5 ng of cDNA, and deionized water. The RT-qPCR amplification was carried out by the Applied Biosystems 7500 and the Step One Plus Realtime PCR system (ABI, France). The cycling parameters for qPCR included an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. We used LinRegPCRsoftware to calculate the amplification efficiency of each sample (25). We entered the raw dataset of RT-qPCR outputs into LinRegPCR version 2017.1 software (https: //LinRegPCR.HFRC.nl). The performance and efficacy of all primers was evaluated separately on the qPCR data of samples. The housekeeping gene 16S rRNA was applied as an internal control. The expression ratio (fold) and the up-regulation and down-regulation of genes were calculated using the ДДИ formula. The data were represented as mean ± standard deviation (SD). All experiments were accomplished independently at least three times. Statistical analysis was performed using GraphPad Prism version 5.00 software (GraphPad software, Inc., CA, USA).

4.Results

While the growth of MRSA in the presence of CFS of lactobacilli was not inhibited over the six and 12-h periods of post-treatment, the level of gene expression was significantly reduced (Table 1) depending on the species of Lactobacillus, the supernatant concentration, and the incubation time.

4.1. Gene Expression at 6-h Post-Treatment

The attenuation of virulence gene expression of MRSA was observed at 6-h post-treatment (Figure 1). At this primary time-point, no significant change was observed in the expression of any of the study genes (Figure 1A, E, F, B, and C). By increasing the CFS concentration to 1/2 x CFS, the ratio of gene expression decreased significantly from 2.18 to 7.98 folds compared to that of the control in the spa, sea, sae, tst, and agr A genes in the presence of the CFS of all the three Lactobacilli studied (P< 0.05). The maximum reduction belonged to the sea gene (7.89 folds) (Figure 1C). However, by increasing the concentration of L. reuteri and L. fermentum CFS, there was no significant decrease in the expression of the sbi and spa genes (Figure 1E and D).

4.2. Gene Expression at 12-h Post-Treatment

The expression levels of the studied genes also decreased at 12-h post-treatment. However, they were different from the expression levels at 6-h post-treatment (Figure 2). The expression ratios of the agr A, tst and sea genes in all types of CFS markedly decreased 12-h post-treatment in comparison with the control group at a concentration of 1/2 x CFS from 14.3 to 26.6 folds (Figure 2A-C). The significant reductions in the expression of the sae gene were also observed in the presence of L. plantarum and L. reuteri CFS (Figure 2F). However, by increasing the incubation time, L. fermentum CFS (1/2 and 1/4) could not significantly affect the expression ratios of the sae, sbi, and spa genes (Figure 2F and E). The maximum reduction, up to 23.5 folds, was observed in the tst and sea genes (Figure 2B and C).

5.Discussion

Despite various studies conducted on the virulence genes of S. aureus aiming to reduce its adverse effects, this human pathogen is still considered an extremely prevalent pathogen worldwide (26, 27). The objective of this study was to compare the potential effects of CFS from three different probiotic lactobacilli including L. reuteri, L. fermentum, and L. plantarum on the expression of some virulence factor encoding genes of MRSA at different incubation times and concentrations. Lactobacilli spp. produce a class of molecules with antimicrobial capacity (28). For example, bacteriocins produced by L. fermentum can reduce adhesion and cytotoxicity properties of MRSA (29). The CFS of L. salivarius and L. fermentum can inhibit the biofilm formation of S. aureus (30). The complete inhibition of Salmonella growth has been shown in the presence of the CFS from L.johnsonii, L. rhamnosus, L. casei, and L.plantarum (31,32). Lactobacillus reuteri, L. plantarum, and L. fermentum have been extensively investigated for their probiotic effects on pathogenic bacteria (27,33-35).

Vahedi-Shahandashti et al. (36) stated that some probiotic lactobacilli culture supernatants had an inhibitory effect on Serratia marcescens swarming and antibiotic resistanc. Hiawy et al. in Iraq showed that the CFS of L. fermentum has inhibitory effects on the growth rate and expression reduction of the gene responsible for biofilm formation in MRSA (21). Our results indicated that L. reuteri, L. fermentum, and L. plantarum reduced the expression of the tst, sae, and sea genes, as well as the agr quorum-sensing system, in MRSA without any tangible effects on its growth. The agr A system is an important global regulatory system that controls the expression of many genes associated with secreting proteins and virulence determinants including protease and collagenase in S. aureus (37, 38). Based on our results and in parallel with the Li et al.'s (39) investigations, it is proven that the reduction in the expression of virulence factors is related to the reduction in the agr system. In fact, the agr A gene is down-regulated at the transcriptional level by CFS. Consequently, it reduces the expression of the sae, tst, sea, sbi, and spa genes. In parallel with other similar studies, it seems that the tst gene is regulated by the sae and agr systems (40).

The results showed that the significant downregulation of gene expressions was in the preference of the 1/2 x CFS concentration while none of the genes was significantly altered at the low concentration of CFS (1/4 x CFS). It has been proven that the sub-lethal dosage of these antimicrobial compounds can reduce the expression of virulence-associated genes in S. aureus and Vibrio cholera, without affecting their growth (7,39,41-43). These results reveal that increasing the incubation time causes a significant reduction in the gene expression so that a significant reduction in the sbi and spa gene expressions achieved by increasing the incubation time. In addition, the CFS of these species of LAB was effective on the level of gene expression. Our findings showed that the CFS of L. reuteri and L. plantarum had a more powerful effect than the CFS of L. fermentum on gene expression. Previous researches suggested that human probiotic isolates, such as L. plantarum, inhibited bacterial virulence, inhibited bacterial virulence, representing a promising alternative to antibiotic prophylaxis of staphylococcal menstrual TSS and potentially of other S .aureus-mediated diseases (40, 44). Though the actual physiological function of supernatant compounds is unclear, these compounds may serve signaling molecules involved in host-bacterial interactions, considering their biological effects in humans (39,45).

It is necessary to mention that L. fermentum is not recognized as a well-known probiotic bacterium. It had an inhibitory effect on virulence gene expression, depending on concentration (1/2 more effective than 1/4 CFS concen- tration) and incubation time. It seems that a reduction in gene expression is associated with the pathogenesis and consequently their supernatant component is due to the down-regulation of virulence genes. Therefore, the compounds found in CFS can influence the agr, sae, sea, tst, sbi, and spa genes to reduce expression at the transcription level.

5.1. Conclusions

Probiotic bacteria and their products in food can inhibit the growth and down-regulate virulence gene expression. Consequently, toxins (enterotoxins) and enzymes are less produced by S. aureus as a bacterium causing vomiting. Therefore, the presence of CFS in the food probably reduces diarrhea and vomiting caused by bacteria.

Acknowledgments

This project was funded by a grant (no.: 9.3692) from the Vice Chancellery for Research and Technology, Rafsanjan University of Medical Sciences (RUMS). The authors thank the RUMS Molecular Medicine Research Center for kindly providing the required equipment.

Footnotes

Authors' Contribution: Study concept and design: Mohammad Zare-Bidaki and Vahid Bagheri. Analysis and interpretation of data: Mahnaz Ramezani. Drafting of the manuscript: Mahnaz Ramezani. Critical revision of the manuscript for important intellectual content: Nahid Zainodini, Hamid Hakimi, Ebrahim Rezazadehzarandi, and Reza Bahramabadi. Statistical analysis: Mahnaz Ramezani.

Conflict of Interests: There is no conflict of interest declared.

Ethical Approval: The ethical approval was 9.3692.

Funding/Support: Rafsanjan University of Medical Sciences (RUMS) funded the study.

References

References

1. Zanger P, Holzer J, Schleucher R, Scherbaum H, Schittek B, Gabrysch S. Severity of Staphylococcus aureus infection of the skin is associatedwith inducibilityofhuman beta-defensin 3 but not human betadefensin 2. Infect immun. 2010;78(7):3112-7. doi: 10.1128/IAI.00078-10. [PubMed: 20404083]. [PubMed Central: PMC28 9 7370].

2. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: An overview of basic and clinical research. Nat Rev Microbiol. 2019;17(4):203-18. doi: 10.1038/s41579-018-0147-4. [PubMed: 30737488]. [PubMed Central: PMC6939889].

3. Spaulding AR, Salgado-Pabon W, Kohler PL, Horswill AR, Leung DY, Schlievert PM. Staphylococcal and streptococcal superantigen exotoxins. ClinMicrobiolRev. 2013;26(3):422-47. doi: 10.1128/CMR.00104-12. [PubMed: 23824366]. [PubMed Central: PMC3719495].

4. Babic M, Pajic M, Nikolic A, Teodorovic V, Mirilovic M, Milojevic L. Expression of toxic shock syndrome toxin-1 gene of Staphylococcus aureus in milk: Proof of concept. Mljekarstvo. 2018;68(1). doi: 10.15567/mljekarstvo.2018.0102.

5. Clarisse T, Michele S, Olivier T, Valerie E, Vincent LM, Jacques-Antoine H, et al. Detection and quantification of staphylococcal enterotoxin A in foods with specific and sensitive polyclonal antibodies. Food Control. 2013;32(1):255-61. doi: 10.1016/j.foodcont.2012.11.021.

6. Le Loir Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. GenetMolRes. 2003;2(1):63-76. [PubMed: 12917803].

7. Smith-Palmer A, Stewart J, Fyfe L. Influence of subinhibitory concentrations of plant essential oils on the production of enterotoxins A and B and alpha-toxin by Staphylococcus aureus. J Med Microbiol. 2004;53(Pt10):1023-7. doi: 10.1099/jmm.0.45567-0. [PubMed: 15358826].

8. Le KY, Otto M. Quorum-sensing regulation in staphylococci-an overview. Front Microbiol. 2015;6:1174. doi: 10.3389/fmicb.2015.01174. [PubMed: 26579084]. [PubMed Central: PMC4621875].

9. Upadhyay A, Burman JD, Clark EA, Leung E, Isenman DE, van den Elsen JM, et al. Structure-function analysis of the C3 binding region of Staphylococcus aureus immune subversion protein Sbi. J Biol Chem. 2008;283(32):22113-20. doi: 10.1074/jbc.M802636200. [PubMed: 18550524]. [PubMed Central: PMC2494919].

10. Bibalan MH, Shakeri F, Javid N, Ghaemi A, Ghaemi EA. Accessory gene regulator types of Staphylococcus aureus isolated in Gorgan, North of Iran. J Clin Diagn Res. 2014;8(4):DC07-9. doi: 10.7860/JCDR/2014/6971.4219. [PubMed: 24959440]. [PubMed Central: PMC4064853].

11. Thompson TA, Brown PD. Association between the agr locus and the presence of virulence genes and pathogenesis in Staphylococcus aureus using a Caenorhabditis elegans model. Int J Infect Dis. 2017;54:726. doi: 10.1016/j.ijid.2016.11.411. [PubMed: 27915107].

12. Liu Q, Yeo WS, Bae T. The SaeRS two-component system of Staphylococcus aureus. Genes (Basel). 2016;7(10). doi: 10.3390/genes7100081. [PubMed: 27706107]. [PubMed Central: PMC5083920].

13. Madahi H, Rostami F, Rahimi E, Safarpoor Dehkordi F. Prevalence of Enterotoxigenic Staphylococcus aureus Isolated From Chicken Nugget in Iran. Jundishapur J Microbiol. 2014;7(8). e10237. doi: 10.5812/jjm.10237. [PubMed: 25485044]. [PubMed Central: PMC4255206].

14. Hasanpour Dehkordi A, Khaji L, Sakhaei Shahreza MH, Mashak Z, Safarpoor Dehkordi F, Safaee Y, et al. One-year prevalence of antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus recovered from raw meat. Trop Biomed. 2017;34(2):396-404.

15. Safarpoor Dehkordi F, Gandomi H, Basti AA, Misaghi A, Rahimi E. Phenotypic and genotypic characterization of antibiotic resistance of methicillin-resistant Staphylococcus aureus isolated from hospital food. AntimicrobResistInfect Control. 2017;6:104. doi: 10.1186/s13756-0170257-1. [PubMed: 29034091]. [PubMed Central: PMC5628482].

16. Prince T, McBain AJ, O'Neill CA. Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion. Appl Environ Microbiol. 2012;78(15):5119-26. doi: 10.1128/AEM.00595-12. [PubMed: 22582077]. [PubMed Central: PMC3416415].

17. Sarika AR, Lipton AP, Aishwarya MS. Bacteriocin production bya new isolate of Lactobacillus rhamnosus GP1 under different culture conditions. AdvJFood Sci Technol. 2010;2(5):291-7.

18. Al-Ghazzewi FH, Tester RF. Effect of konjac glucomannan hydrolysates and probiotics on the growth of the skin bacterium Propionibacterium acnes in vitro. Int J Cosmet Sci. 2010;32(2):139-42. doi: 10.1111/j.1468-2494.2009.00555.x. [PubMed: 19818083].

19. Cinque B, La Torre C, Melchiorre E, Marchesani G, Zoccali G, Palumbo P. Use of probiotics for dermal applications. Probiotics. Germany: Springer; 20U. p. 221-41. doi:10.1007/978-3-642-20838-6.

20. Di Marzio L, Centi C, Cinque B, Masci S, Giuliani M, Arcieri A, et al. Effect of the lactic acid bacterium Streptococcus thermophilus on stratum corneum ceramide levels and signs and symptoms ofatopic dermatitis patients. Exp Dermatol. 2003;12(5):615-20. doi: 10.1034/j.16000625.2003.00051.x. [PubMed: 14705802].

21. Allayeh A, Dardeer E, Kotb N. Effects of cell-free supernatants of yogurts metabolites on Coxsackie B 3 virus in vitro and in vivo. Middle EastJAppl Sci. 2015;5:353-8.

22. Long DR, Mead J, Hendricks JM, Hardy ME, Voyich JM. 18betaglycyrrhetinic acid inhibits methicillin-resistant Staphylococcus aureus survival and attenuates virulence gene expression. Antimicrob Agents Chemother. 2013;57(1):241-7. doi:10.1128/AAC.01023-12. [PubMed: 23114775]. [PubMed Central: PMC3535912].

23. Subrt N, Mesak LR, Davies J. Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus.JAntimicrob Chemother. 2011;66(5):979-84. doi: 10.1093/jac/dkr043. [PubMed: 21393149].

24. Lee JH, Cho HS, Kim Y, Kim JA, Banskota S, Cho MH, et al. Indole and 7-benzyloxyindole attenuate the virulence of Staphylococcus aureus. ApplMicrobiolBiotechnol. 2013;97(10):4543-52. doi: 10.1007/s00253-0124674-z. [PubMed: 23318836].

25. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, et al. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37(6). e45. doi: 10.1093/nar/gkp045. [PubMed: 19237396]. [PubMed Central: PMC2665230].

26. Even S, Charlier C, Nouaille S, Ben Zakour NL, Cretenet M, Cousin FJ, et al. Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures. Appl Environ Microbiol. 2009;75(13):4459-72. doi: 10.1128/AEM.02388-08. [PubMed: 19429556]. [PubMed Central: PMC2704850].

27. Laughton JM, Devillard E, Heinrichs DE, Reid G, McCormick JK. Inhibition of expression of a staphylococcal superantigen-like protein by a soluble factor from Lactobacillus reuteri. Microbiology. 2006;l52(Pt 4):1155-67. doi: 10.1099/mic.0.28654-0. [PubMed:16549678].

28. Gopal PK, Prasad J, Smart J, Gill HS. In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli. Int J Food Microbiol. 2001;67(3):207-16. doi: 10.1016/s0168-1605(01)00440-8. [PubMed: 11518430].

29. Jayashree S, Karthikeyan R, Nithyalakshmi S, Ranjani J, Gunasekaran P, Rajendhran J. Anti-adhesion property of the potential probiotic strain Lactobacillus fermentum 8711 against methicillin-resistant Staphylococcus aureus (MRSA). Front Microbiol. 2018;9:411. doi: 10.3389/fmicb.2018.00411. [PubMed: 29568290]. [PubMed Central: PMC5852077].

30. Kang MS, Lim HS, Oh JS, Lim YJ, Wuertz-Kozak K, Harro JM, et al. Antimicrobial activity of Lactobacillus salivarius and Lactobacillus fermentum against Staphylococcus aureus. Pathog Dis. 2017;75(2). doi: 10.1093/femspd/ftx009. [PubMed: 28158586].

31. Botes M, Loos B, van Reenen CA, Dicks LM. Adhesion of the probiotic strains Enterococcus mundtii ST4SAand Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments. Arch Microbiol. 2008;190(5):573-84. doi: 10.1007/s00203-008-0408-0. [PubMed: 18641972].

32. Fayol-Messaoudi D, Berger CN, Coconnier-Polter MH, Lievin-Le Moal V, Servin AL. pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium. Appl Environ Microbiol. 2005;71(10):6008-13. doi: 10.1128/AEM.71.10.6008-6013.2005. [PubMed: 16204515]. [PubMed Central: PMC1266002].

33. Das JK, Mishra D, Ray P, Tripathy P, Beuria TK, Singh N, et al. In vitro evaluation of anti-infective activity of a Lactobacillus plantarum strain against Salmonella enterica serovar Enteritidis. Gut Pathog. 2013;5(1):11. doi: 10.1186/1757-4749-5-11. [PubMed: 23668384]. [PubMed Central: PMC3662602].

34. Gan BS, Kim J, Reid G, Cadieux P, Howard JC. Lactobacillus fermentum RC-14 inhibits Staphylococcus aureus infection of surgical implants in rats. J Infect Dis. 2002;185(9):13 69-72. doi: 10.1086/340126. [PubMed: 12001060].

35. Haileselassie Y, Navis M, Vu N, Qazi KR, Rethi B, Sverremark-Ekstrom E. Lactobacillus reuteri and Staphylococcus aureus differentially influence the generation of monocyte-derived dendritic cells and subsequent autologous T cell responses. Immun Inflamm Dis. 2016;4(3):31526. doi: 10.1002/iid3.115. [PubMed: 27621814]. [PubMed Central: PMC5004286].

36. Vahedi-Shahandashti R, Kasra-Kermanshahi R, Shokouhfard M, Ghadam P, Feizabadi MM, Teimourian S. Antagonistic activities of some probiotic lactobacilli culture supernatant on Serratia marcescens swarming motility and antibiotic resistance. Iran J Microbiol. 2017;9(6):348-55. [PubMed: 29487733]. [PubMed Central: PMC5825935].

37. Arvidson S, Tegmark K. Regulation of virulence determinants in Staphylococcus aureus. Int J Med Microbiol. 2001;291(2):159-70. doi: 10.1078/1438-4221-00112. [PubMed: 11437338].

38. Bronner S, Monteil H, Prevost G. Regulation of virulence determinants in Staphylococcus aureus: Complexity and applications. FEMS MicrobiolRev. 2004;28(2):183-200. doi: 10.1016/j.femsre.2003.09.003. [PubMed: 15109784].

39. Li J, Wang W, Xu SX, Magarvey NA, McCormick JK. Lactobacillus reuteri-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin-1 in staphylococci. Proc Natl Acad Sci U S A. 2011;108(8):3360-5. doi: 10.1073/pnas.1017431108. [PubMed: 21282650]. [PubMed Central: PMC3044419].

40. Jeong DW, Cho H, Jones MB, Shatzkes K, Sun F, Ji Q, et al. The auxiliary protein complex SaePQ activates the phosphatase activity of sensor kinase SaeS in the SaeRS two-component system of Staphylococcus aureus. Mol Microbiol. 2012;86(2):331-48. doi: 10.1111/j.1365-2958.2012.08198.x. [PubMed: 22882143]. [PubMed Central: PMC3468659].

41. Yamasaki S, AsakuraM, Neogi SB, HinenoyaA, Iwaoka E, Aoki S. Inhibition of virulence potential of Vibrio cholerae by natural compounds. IndianJMedRes. 2011;133:232-9. [PubMed: 21415500]. [PubMed Central: PMC3089057].

42. Qiu J, Zhang X, Luo M, Li H, Dong J, Wang J, et al. Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus. PLoS One. 2011;6(1). e16160. doi: 10.1371/journal.pone.0016160. [PubMed: 21283822]. [PubMed Central: PMC3023776].

43. Chatterjee S, Asakura M, Chowdhury N, Neogi SB, Sugimoto N, Haldar S, et al. Capsaicin, a potential inhibitor of cholera toxin production in Vibrio cholerae. FEMS Microbiol Lett. 2010;306(1):54-60. doi: 10.1111/j.1574-6968.2010.01931.x. [PubMed: 20337712].

44. Nagarajan V, Peng M, Tabashsum Z, Salaheen S, Padilla J, Biswas D. Antimicrobial effect and probiotic potential of phage resistant Lactobacillus plantarum and its interactions with zoonotic bacterial pathogens. Foods. 2019;8(6). doi: 10.3390/foods8060194. [PubMed: 31195676]. [PubMed Central: PMC6616511].

45. Pu S, Wang F, Ge B. Characterization of toxin genes and antimicrobial susceptibility of Staphylococcus aureus isolates from Louisiana retail meats. Foodborne Pathog Dis. 2011;8(2):299-306. doi: 10.1089/fpd.2010.0679. [PubMed: 21034265].