Staphylococcus aureus, as a foodborne pathogen causing significant harm worldwide, was studied to assess the effectiveness of probiotic strains  Lactobacillus casei, Lactobacillus plantarum, and Bifidobacterium bifidum individually and collectively (as consortia) in controlling its growth. The growth patterns of S. aureus were observed when co-cultured with each probiotic strain and a consortium of all three strains over 72 hours. Additionally, the antimicrobial activity of probiotic cell-free supernatants (CFS) against S. aureus was tested using the agar well diffusion method. This study underscores the potential of L. plantarum and a consortium of L. casei, L. plantarum, and B. bifidum in controlling S. aureus growth.

Keywords: Foodborne pathogen, Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium bifidum, Staphylococcus aureus, probiotic

Staphylococcus aureus is a foodborne, disease-causing bacteria which is a major cause of mortality and morbidity in the world.¹ It is gram positive, non-spore forming bacteria that can contaminate food product during food processing and food preparation.² Besides being commonly found in environmental sites such as marine and freshwater, soil surfaces, plant surfaces, dust and air, they commonly colonize the mucous membrane, skin and skin glands of the human body.³ It can also form complex 3 dimensional structures called biofilms on food contact surfaces in food processing facilities, further increasing the likelihood of their contamination in food,⁴ the reason for their occurrence in all categories of food (the raw, semi and the processed).⁵ S. aureus produces a large variety of enterotoxins which when consumed along with food can cause intoxication. S. aureus produces enterotoxins, releasing them in the medium (food) when it reaches a quorum, which is to the concentration of 10000-100000 cfu/gm of food.⁶ About 20 types of enterotoxins are produced by S. aureus that is pyrogenic exotoxins.⁷ The concentration of the staphylococcal enterotoxin has to reach a concentration of 10-20 ng to be able to cause foodborne intoxication.⁶ It is evident therefore that containing the growth of S. aureus in food is vital to reducing the incidence of staphylococcal intoxication and concurring concerns of food intoxication. Lactic Acid Bacteria (LAB) have been known to have inhibitory and antagonistic behavior against foodborne pathogens, including S. aureus.⁸ Also, given the emerging concerns of antimicrobial resistance and increasing demand for an alternative solution to control pathogens,⁹ it is imperative that possible bio-control agents are explored to serve this purpose.¹⁰ Probiotic strains of Lactic Acid Bacteria can be beneficial and worthy of application in this context.

Lactic Acid Bacteria are living organisms that have several health benefits.^11,12 In the food industry, reducing the growth of foodborne bacteria can be brought about LAB. Among LAB, Lactobacilli and Bifidobacteria are the most common probiotic strains.¹³ Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium bifidum have been studied for their capacity to competitively inhibit or exclude pathogens.^14,15 The objective of this study was to evaluate the efficacy of Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium bifidum in controlling Staphylococcus aureus.

Bacterial strains and media

Lactobacillus casei (ATCC 12116) and Staphylococcus aureus (ATCC 5345) were procured from National Collection of Industrial Microorganisms (NCIM) Pune, India and Bifidobacterium bifidum (NRRL /ATCC 29521) And Lactobacillus plantarum (NRRL /ATCC 8014) were procured from Northern Regional Research Laboratory (NRRL), Agriculture Research Services (ARS) United State Department of Agriculture (USDA), USA. Mannitol Salt Agar (Himedia M118-500G), Lactobacillus MRS Agar Media (Himedia M614-500G), Soyabean Casein Digestive Medium (SCDM) (Himedia M011- 500G), and Muller Hinton Agar (Himedia M173-100G) were used in this study.

Microbial analysis of S. aureus with LAB

Inhibition of S. aureus [SA] in co-culture conditions with Lactobacillus casei [LC], Lactobacillus plantarum [LP] and Bifidobacterium bifidum [BB] in broth were studied. S. aureus as pure culture was used as control. The cell concentration of S. aureus was estimated at specific time intervals 0, 24, 48 and 72 hours, 0 hours being the initial concentration of the culture at the start of the experiment. 1 ml of S. aureus culture was inoculated into 100ml of 5 Soyabean Casein Digestive Medium broths each, labelled as SA, SA+LC, SA+LP, SA+BB, SA+ Consortium (Table 1). Except for the pure culture broths labelled as SA, the rest were inoculated with the 1ml of respective cultures of LC, LP and BB accordingly. In the last broth, besides SA all other cultures (LC, LP & BB) were inoculated 1ml each. All 5 culture broths were incubated at 37°C. The concentration of SA was estimated at the start of the experiment (0hrs) and thereafter at specified time intervals (24, 48 and 72hrs) from each of the culture conditions (SA, SA+LC, SA+LP and SA+BB) by standard plating technique.

Antimicrobial activity assay

The Antimicrobial activity assay was performed based on agar well diffusion by Kirby-Bauer method as discussed in this section. Culture conditions were similar to the earlier study (SA, SA+LC, SA+LP, SA+BB and SA+ Consortium, for convenience hereafter referred to as T1, T2, T3 & T4). Pure culture and co-cultures were cultured in SCDM broth and incubated at 37^oC for 24 hrs. Post the incubation, the cultures were centrifuged at 5000 rpm for 20 minutes to sediment bacterial cells and cell free supernatant (CFS) was collected. The volume of CFS was 10ml.  CFS was filtered with syringe filter (0.45µm in pore size). The CFS filtrate was lyophilized¹⁶ and the lyophilized CFS was further used for antimicrobial activity assay.The lyophilized CFS was rehydrated to a volume of 2ml. Antimicrobial activity of the lyophilized CFS of LAB culture against SA was determined by agar-well diffusion method on Mueller-Hinton Agar media (M173; HiMedia). The volume of rehydrated CFS (rCFS) and the standard antibiotics (as control) used for the assay were 200μl per well. The standard antibiotics used were Penicillin G, Oxacillin, Cephalothin, Amoxycycline, Clindamycin and Erythromycin (HiMedia HX001-1PK).  After 24 hours, the plates were observed for zone of inhibition (mm) around the well.

Growth pattern of S. aureus with L. casei

Log 10 cell concentrations of S. aureus in control (SA) and co-culture test (SA+ LC) at fixed time interval is as shown in Figure-1.0. While the cell concentration of SA at the start of the experiment (0 hrs) was 5.46, the cell concentration after 24hrs of incubation was estimated as 7.7 in Control and 7.32 in co-culture condition after 24 hours of incubation. Similar growth pattern of SA was observed at 48 and 72 hours, when grown with L. casei. The cell concentrations of SA were enumerated to be 7.57, 6.7 in control, and 7.01, 6.38 in co-cultured test at 48 and 72hrs respectively (Figure 1).

Figure 1 Growth Pattern of S. aureus as control and in the presence of L .casei.

Growth pattern of S. aureus with L. plantarum

The growth pattern of S. aureus in control (SA) and co-culture test with L. casei (SA+ LC) at fixed time interval is as shown in Figure-2.0. While the cell concentration (Log10) of SA was 7.05 at the start of the experiment (0 hrs), the cell concentration of SA as pure culture (control) was estimated as 8.98 (SA) and 7.07 in co-culture with LP (SA+LP) after 24 hours of incubation. Similar change in Log 10 cell concentration of SA was observed at 48 and 72 hours when grown with L. plantarum. The cell concentrations of SA was estimated to be  8.2 and 7.2  in control, and 7.18 and 5.86  in co-cultured test at 48 and 72 hours respectively (Figure 2).

Figure 2 Growth Pattern of S. aureus as control and in the presence of L. plantarum.

Growth pattern of S. aureus with B. bifidum

The growth pattern of S. aureus in control (SA) and co-culture test (SA+ BB) at fixed time interval is shown in Figure 3. While the cell concentration of SA at 0hrs was estimated as 9.93 (log 10). The cell concentration of SA was estimated as 8.08 (Control) and 7.85(co-culture test) after 24 hours of incubation. The log 10 cell concentration of SA was observed at 48 and 72hrs when grown with B. bifidum The cell concentration of SA was estimated 7.56 and 7.17 in control (SA),  7.17 and 6.82 in co-cultured (SA+BB) at 48 and 72 hours respectively (Figure 4).

Figure 3 Growth Pattern of S. aureus as control and in the presence of B.bifidum.

Figure 4 Growth Pattern of S. aureus as control and in the presence of Consortia of cultures.

Growth pattern of S. aureus with consortium

Growth pattern of S. aureus in control (SA) and co-culture test with a consortium of LC+ LP+BB was observed. The log10 concentration of SA at 0 hrs was 7.28. The culture concentration of SA as pure culture was 8.18, 7.32 and 7.20, while the concentration of SA in the presence of consortium of cultures were 7.30, 6.0and 5.8 at 24, 48 and 72 hours respectively (Figure 5).

Figure 5 Effect of individual probiotic cultures and as consortia.

Result of antimicrobial activity assay

The antimicrobial activity of CFS of LAB was evaluated against S. aureus using the agar well diffusion method. The selected species of LAB namely L.casei, L.plantarum and B.bifidum were each inoculated individually and as consortium to grow in SCDM broth along with S. aureus. The objective was to screen for expression of antibiosis by the strains of LAB against S.aureus and experiments based on Kirby-bauer method as discussed in section 2.3 were performed. As discussed the CFS collected from each of the tests (1 to 4) were screened for antibiosis against S. aureus.  As control, antibiotics recommended sensitive for Gram positive bacteria, namely penicillin G, oxacillin, cephalothin and amoxycycline are inhibitors of cell wall synthesis¹⁷ and other clindamycin and erythromycin are inhibitors of protein synthesis¹⁸ were used. While the result showed that the strain of S.aureus was sensitive to all the antibiotics used as control in the form of Zone of inhibition or no growth around the wells carrying the control antibiotics, no inhibition was observed around the wells with Cell Free supernatants from any of tests 1 to 4.

Staphylococcal food poisoning is among the most prevalent foodborne intoxication in world.¹⁹ Control and prevention of S. aureus therefore is a crucial public health concern.²⁰ Probiotics as live microorganisms that confer benefits to human health have also been studied for their potency to control pathogens through competition or inhibition via antibiosis.²¹ The objective of the study was to screen for possible control of S. aureus using probiotic species namely L. casei, L. plantarum and B. bifidum by understanding their effect on the growth and proliferation of S.aureus.

It was observed that among the tests (1 to 4) wherein, S.aureus was incubated individually with each of the selected probiotic strains (L. casei[LC], L. plantarum[LP] and B. bifidum[BB]) and together as consortium, the growth of S.aureus was maximum affected when grown with L. plantarum. Although individually the effect of L.casei and B. bifidum on the growth of S. aureus was marginal, the effect of L. casei, L. plantarum and B. bifidum as consortia had a significant effect on the growth of S. aureus.  It is also noteworthy that the growth pattern of S. aureus both with L. plantarum individually and with consortia did not show log scale rise as observed in the control. From 0hrs to 24hrs in the presence of L. plantarum and consortia the concentration of S. aureus (in Log10 value) remained close to constant. This gives us to infer that the growth rate of S. aureus, equated their numbers to remain at close to constant as concentration was contained by L. plantarum individually and also by the consortia of all the 03 probiotic strains. The variation in concentration as Log10 values/ml of S .aureus in control to test was -1.91 at 24hrs, -1.02 at 48hrs and -1.34 at 72hrs, in the presence of L. plantarum. It has been reported earlier that L. plantarum strains can produce variety of antimicrobial compounds like diacetyl, hydrogen peroxide, organic acid and also bacteriocins and antimicrobial peptides²² and these probably could affect the growth S. aureus. Also, according to other previous studies, antagonistic behaviour via expression of phenyllactic acid and lactic acid that show antimicrobial activity have been reported.^23,24 These organic acids have been shown to have adverse effect on microorganisms sharing the same niche. It is also possible that the containing effect of the probiotic strain namely L. plantarum in this case could be due to competition between LAB and pathogens for limited resources and space as reported in earlier studies.²⁵

Similarly, the consortia of all the 03 strains collectively contained the growth of S. aureus and the variation in concentration as Log10 values of CFU/ml from control to test was -0.88 at 24hrs, -1.32 at 48hrs and -1.2 at 72hrs. The probable mechanism of inhibition of S. aureus could be attributed to the reasons as cited above. At the same time, it is also be noted that when in the same niche as consortia the probiotic strains could be competing and or exhibiting antibiosis against each other as well. Nevertheless, the possibility of cooperation among the denizens of a niche is also a possibility which is beyond the scope of this study. Our observations that L. casei did not show significant effect on the growth of S. aureus is contrary to the previous reports. According to previous reports L. casei showed strong antimicrobial activity and was inferred to be probably due to production of bacteriocin, competition for resources or production of organic acid such as lactic acid which decreases the pH levels.²⁴ The variation in concentration as Log10 values of CFU/ml from control to test was -0.4, -0.87 and -0.63 at 24, 48 and 72 hrs respectively.

Similarly no significant effect on S. aureus growth was observed with B. bifidum in our study. However, earlier reports have indicated that B. bifidum could inhibit the growth of S .aureus and that B. bifidum produced bacteriocins, besides organic acids and short chain fatty acids that create unfavorable condition for pathogens.^26,27 Also, possible mechanism of competition for nutrients and space by B. bifidum that is inhibitory to other species in co-culture has been reported,^28,29 further emphasizing the inhibitory effect of B. bifidum on co-habitants. But results from our experiments indicated that as compared to L. casei and L. plantarum, B. bifidum was less inhibitory to S. aureus. The variation in concentration as Log10 values of CFU/ml from control to test was -0.23, -0.39 and -0.32 at 24, 48 and 72 hrs respectively.

Given the results that clearly indicated inhibition of S. aureus by the selected strains of probiotic, significant inhibition in case of L. plantarum and the consortia of L. casei, L. plantarum and B. bifidum, it was compelling to probe for possible antibiosis by the strains against S. aureus. Experiments based on the Kirby-bauer method using the CFS from each of the culture conditions (LP, LC, BB and Consortia) that were concentrated by lyophilization and thereafter rehydrated, were performed. The results however did not show any marked zone of inhibition of the S.aureus growth, which was otherwise explicit with antibiotics (Pencillin G, Oxacillin, Cephalothin, Amoxycycline, Clindamycin and Erthyromycin) used as reference control.

Based on the results of the study and previous reports on the subject it could be inferred that inhibition of S. aureus by Lactic Acid Bacteria could be strain dependent, and also that the inhibition could be more by competition for space and nutrients, and less by expression of antibiosis. Nevertheless further probing into possible expression of Quorum Quenching molecules and or bacteriocin types of molecules cannot be negated.

S. aureus, owing to its rapid and progressive evolution to multi-drug resistant forms, has emerged to be of major concern. LAB that have been reported to competitively exclude, and exhibit antimicrobial properties are optimal candidates for containing S. aureus. The inhibitory action of lactic acid bacteria against other microorganisms cannot be attributed to the production of various metabolites and antimicrobial compounds. It is important to note that the inhibitory action of LAB can vary depending on the specific strain of bacteria, type of food matrix and the environmental conditions. Further study is needed to understand the mode of action of LAB, especially to evaluate the potential use of LAB in biocontrol of foodborne pathogens and as a substitute in bio-preservation.

The authors extend their heartfelt appreciation to the Instrumentation facility, Indian Institute of Technology, Delhi for their valuable support. We would like to express our gratitude to Priyanka Chaturvedi and the Department of Agriculture Sciences, Dayalbagh Educational Institute for constant support and encouragement.

The author declares no conflicts of interest.

1. Kirk MD, Pires SM, Black RE, et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. PLoS Med. 2015;12(12):e1001921.
2. Yan P, Chelliah R, Jo KH, et al. Stability and antibiofilm efficiency of slightly acidic electrolyzed water against mixed-species of Listeria monocytogenes and Staphylococcus aureus. Front Microbiol. 2022;13:865918.
3. Huang D, Chen H, Shen M, et al. Recent advances on the transport of microplastics/nanoplastics in abiotic and biotic compartments. J Hazard Mater. 2022;438:129515.
4. Schönborn S, Krömker V. Detection of the biofilm component polysaccharide intercellular adhesin in Staphylococcus aureus infected cow udders. Vet Microbiol. 2016;196:126–128.
5. Pal M, Kerorsa GB, Marami LM. Epidemiology, pathogenicity, animal infections, antibiotic resistance, public health significance, and economic impact of staphylococcus aureus: a comprehensive review. Am J Public Health Res. 2020;8(1):14–21.
6. Zhao Y, Zhu A, Tang J, et al. Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning. Lett Appl Microbiol. 2017;65(1):27–34.
7. Elahi S, Fujikawa H. Comprehensive study of the boundaries of enterotoxin A production and growth of Staphylococcus aureus at various temperatures and salt concentrations. J Food Sci. 2019;84(1):121–126.
8. Lani MN, Ismail A, Hasim NN, et al. antagonistic activity and surface decontaminant potential of lactic acid bacteria from fermented Oreochromis niloticus. 2022;17(3).
9. Singh N, Rai V, Tripathi CKM. Purification and chemical characterization of antimicrobial compounds from a new soil isolate Streptomyces rimosus MTCC 10792. Prikl Biokhim Microbiol. 2013;49(5):467–475.
10. Singh N, Rai V, Tripathi CKM. Production and optimization of oxytetracycline by a new isolate Streptomyces rimosus using response surface methodology. Med Chem Res. 2012;21:3140–3145.
11. Arqués JL, Rodríguez E, Nuñez M, et al. Combined effect of reuterin and lactic acid bacteria bacteriocins on the inactivation of food-borne pathogens in milk. Food Control. 2011;22(3–4):457–461.
12. Tsai YT, Cheng PC, Pan TM. The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits. Appl Microbiol Biotechnol. 2012;96(4):853–862.
13. Holzapfel WH, Haberer P, Geisen R, et al. Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr. 2001;73(2):365S–373S.
14. Kim JH, Lee ES, Song KJ, et al. Development of desiccation-tolerant probiotic biofilms inhibitory for growth of foodborne pathogens on stainless steel surfaces. Foods. 2022;11(6):831.
15. Sharma H, Fidan H, Özogul F, et al. Recent development in the preservation effect of lactic acid bacteria and essential oils on chicken and seafood products. Front Microbiol. 2022;13:1092248.
16. Georgieva R, Yocheva L, Tserovska L, et al. Antimicrobial activity and antibiotic susceptibility of Lactobacillus and Bifidobacterium spp. intended for use as starter and probiotic cultures. Biotechnol Biotechnol Equip. 2015;29(1):84–91.
17. Olaimat AN, Al‐Holy MA, Shahbaz HM, et al. Emergence of antibiotic resistance in Listeria monocytogenes isolated from food products: a comprehensive review. Compr Rev Food Sci Food Saf. 2018;17(5):1277–1292.
18. Mahfouz AA, Said HS, Elfeky SM, et al. Inhibition of erythromycin and erythromycin-induced resistance among Staphylococcus aureus clinical isolates. Antibiotics (Basel). 2023;12(3):503.
19. Zeaki N, Johler S, Skandamis PN, et al. The role of regulatory mechanisms and environmental parameters in staphylococcal food poisoning and resulting challenges to risk assessment. Front Microbiol. 2019;10:1307.
20. Sedarat, Z, Taylor-Robinson AW. Biofilm formation by pathogenic bacteria: applying a staphylococcus aureus model to appraise potential targets for therapeutic intervention. Pathogens. 2022;11(4):388.
21. Yousaf S, Nouman HM, Ahmed I, et al. A review of probiotic applications in poultry: improving immunity and having beneficial effects on production and health. Postępy Mikrobiologii Adv Microbiol. 2022;61(3):115–123.
22. Kos B, Beganović J, Jurašić L, et al. Coculture-inducible bacteriocin biosynthesis of different probiotic strains by dairy starter culture Lactococcus lactis. Mljekarstvo/Dairy. 2011;61(4).
23. Nataraj BH, Mallappa RH. Antibiotic resistance crisis: an update on antagonistic interactions between probiotics and methicillin-resistant Staphylococcus aureus (MRSA). Curr Microbiol. 2021;78(6):2194–2211.
24. Tharmaraj N, Shah NP. Antimicrobial effects of probiotics against selected pathogenic and spoilage bacteria in cheese-based dips. Int Food Res J. 2009;16(1):261–276.
25. Knipe H, Temperton B, Lange A, et al. Probiotics and competitive exclusion of pathogens in shrimp aquaculture. Rev Aquacult. 2020;13(1):324–352.
26. Asadpoor M, Ithakisiou GN, Henricks PA, et al. Non-digestible oligosaccharides and short chain fatty acids as therapeutic targets against enterotoxin-producing bacteria and their toxins. Toxins. 2021;13(3):175.
27. Rauf A, Khalil AA, Rahman UU, et al. Recent advances in the therapeutic application of short-chain fatty acids (SCFAs): An updated review. Crit Rev Food Sci Nutr. 2022;62(22):6034–6054.
28. Pan D, Yu Z. Intestinal microbiome of poultry and its interaction with host and diet. Gut Microbes. 2014;5(1):108–119.
29. Fernández Juliá, PJ, Commane D, van Sinderen, D, et al. Cross-feeding interactions between human gut commensals belonging to the Bacteroides and Bifidobacterium genera when grown on dietary glycans. Microbiome Res Rep. 2022;1:12.