KGs are widely recognized for their probiotic properties and potential health benefits, particularly their antimicrobial activity [27]. The presence of lactic acid bacteria and acetic acid bacteria in KGs produces bioactive compounds for instance organic acids, bacteriocins, and other metabolites that exert these antimicrobial effects [28]. These antimicrobial compounds protect against gastrointestinal infections and diseases caused by pathogenic bacteria and fungi [29] and may serve as a potent alternative to antibiotics [30].
Studies from USA-origin KGs manifested compositional variation across different studies. According to Mohammadi et al., four distinct isolates were identified including Lactococcus lactis, Lactobacillus paracasei, Pediococcus pentosaceus, and Bacillus species. Lactococcus lactis, Lactobacillus paracasei, Pediococcus pentosaceus, and various Bacillus species are known for their notable antimicrobial activities. Lactococcus lactis produces nisin, a well-studied bacteriocin, which exhibits strong antibacterial properties against a broad variety of Gram-positive bacteria, including foodborne pathogens like Listeria monocytogenes. Lactobacillus paracasei is effective against both Gram-positive and Gram-negative bacteria owing to its production of organic acids, hydrogen peroxide, and bacteriocins such as paracasein. Pediococcus pentosaceus secretes pediocin, a bacteriocin that targets Listeria and other spoilage microorganisms, thus playing a crucial role in food preservation. Bacillus species, particularly Bacillus subtilis and Bacillus licheniformis, are known for synthesizing a wide array of antimicrobial compounds, including subtilin, bacitracin, and surfactin. These compounds exhibit activity against various pathogens, including Staphylococcus aureus and Escherichia coli, contributing to their application in both food safety and as probiotics. Collectively, these microorganisms and their antimicrobial products are pivotal in maintaining microbial balance, enhancing food safety, and offer therapeutic potential. Notably, Lactococcus lactis subsp. lactis CAB701 demonstrates resilience in acidic, bile, and pancreatin conditions [31], and exhibits a remarkable propensity for adhesion to enterocytes, suggesting its potential for establishing favorable interactions with the intestinal epithelium. P. pentosaceus and Lactobacillus paracasei manifested a remarkable adherence to human colon cancer cells (P<0.01), leading to a notable decrease in cell growth (P<0.05) [32]. According to another study, researchers highlighted the abundant presence of Actinobacteria and Saccharomyces spp in USA-oriented KGs, which exhibited 67% antibacterial activity against E. coli, S. aureus, B. subtilis, S. Typhimurium, Curvularia spp., F. exquisite, and C. gloeosporioides [33]. Furthermore, another study reported the presence of Lactobacillus kefiri from USA-oriented KGs and exhibited bactericidal effect against Enterococcus faecalis, Staphylococcus aureus, Shigella flexneri, Pseudomonas aeruginosa, enterohemorrhagic Escherichia coli, and Listeria monocytogenes [34].
In our analysis, USA-origin KGs exhibited a remarkable presence of Lactococcus species and one Bacillus cereus. Lactococcus species, particularly Lactococcus lactis, are well-known for their advantageous roles in food fermentation, especially in dairy products like cheese and yogurt. These bacteria produce various antimicrobial substances, including nisin, which is a well-characterized bacteriocin that inhibits the growth of several pathogenic and spoilage bacteria. Nisin's mode of action involves binding to the bacterial cell membrane, leading to pore formation and cell death, making it a valuable natural preservative in the food industry. Additionally, Lactococcus species contribute to the improvement of gut health by producing lactic acid, which lowers the pH of the gut environment, inhibiting the growth of harmful bacteria and promoting a balanced microbiota. Their probiotic properties also extend to immunomodulation, where they help in the enhancement of the immune response and reduction of inflammation. Overall, the beneficial effects of Lactococcus species encompass antimicrobial activity, gut health improvement, and immunomodulatory properties, making them valuable in both food production and health applications [35]. In addition, B. cereus produces bacteriocin known as cerein, which inhibits the growth of other S. aureus, P. fluorescens, and Bacillus spp., in particular, B. thuringiensis and gram-negative bacteria like E. coli by inhibiting the sporulation which causes food-borne infections [36]. Furthermore, it also releases some proteolytic enzymes i.e. protease K, which helps to prevent L. monocytogenes by compromising the integrity of bacterial cell walls and reduces GIT infections such as listeriosis, S.aureus food intoxication and B. thuringiensis diarrheal infection [37].
These antimicrobial properties are attributed to the presence of various organic acids, such as lactic acid, which lowers the pH and creates an inhospitable environment for harmful pathogens such as E. coli and S. aureus by interfering with bacterial cell membrane integrity, causing leakage of cellular contents and compromising cell function. Additionally, these organic acids can act as energy sources for certain bacteria but inhibit the growth of others by disrupting cell glucose metabolism [38]. Moreover, exopolysaccharide (EPS) of LAB can interfere with quorum sensing, a communication system used by pathogens to coordinate virulence and biofilm formation, thus disrupting their ability to establish infections. Additionally, some EPS possess antimicrobial properties directly targeting pathogens, either by disrupting their cell membranes or inhibiting essential metabolic processes [39].
In our analysis, KMS obtained from kefir at concentrations of 50%, 60%, and 70% effectively suppressed the growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhi, which are known causative agents of GIT illnesses. Findings from various outbreak studies indicate that dairy products are the primary reservoir of S. aureus-related illnesses (30.3%), followed by grains and legumes (12.9%), meat (10.5%), and other sources (7.0%). S. aureus has been implicated in cases of antibiotic-associated diarrhea, exacerbated by diminished gastric acidity, facilitating bacterial proliferation within the intestines, leading to enteritis and subsequent diarrhea in 59% of people annually [40].
P. aeruginosa, the most frequently associated strain with human infections, is primarily transmitted through food. Analysis of samples revealed prevalence rates of multidrug-resistant P. aeruginosa in bovine meat (47.8%), fresh fish (33.1%), and smoked fish (20.0%). This pathogen can cause infections in various anatomical sites, including the bloodstream and lungs (resulting in pneumonia), particularly post-surgery. It can lead to conditions such as gastroenteritis, characterized by symptoms like diarrhea, abdominal pain, and fever. In more severe cases, particularly in hospitalized or immunocompromised patients, it can cause infections like peritonitis or typhlitis in about 8% to 25% of cases, which are serious and potentially life-threatening [41].
Salmonella contamination is mainly associated with the consumption of foods like eggs, milk, and poultry meat that have been contaminated. Globally, around 20% of poultry yields are affected by Salmonella and this bacterium can endure for long periods in both animal and human settings due to its ability to form biofilms. In Europe, there were approximately 94,625 reported cases of Salmonella infections [42]. Salmonella enterica (S. enterica) serotypes typhi and paratyphi are responsible for causing typhoid fever in approximately 1.4 million people worldwide each year. This disease is marked by systemic infection, fever, and gastrointestinal symptoms such as diarrhea. In addition, S. enterica serotype Typhimurium (S. Typhimurium) generally leads to enterocolitis in humans [43].
In this study, K. pneumoniae, E. coli, V. cholera, H. pylori, and B. subtilis were resistant at all three KMS concentrations as well as with SILB. Consistent with these findings, an analysis by Kolakowski et al. elucidated the emergence of E. coli, H. pylori, and V. cholerae resistance toward KMS [44]. This is attributed to resistance through mechanisms such as efflux pumps that actively pump out the antibacterial compounds from bacterial cells, by quorum sensing in which bacteria communicate, and alterations in cell membrane permeability reduce the entry of these agents. Additionally, biofilm formation can create a defending atmosphere that shields the bacteria from the antimicrobial effects of kefir components [45,46]. B. subtilis' catalase-positive trait endows it with the capacity to effectively counter oxidative stress induced by L. lactis [47].
Understanding why SILB did not show inhibition requires further investigation into its specific characteristics, genetic makeup, and interactions with antimicrobial agents like KMS under various conditions [48]. One possible explanation is that inhibition may require the synergistic activity of the entire microbial consortia. In isolation, SILB may lack the necessary interactions or metabolic pathways that contribute to the overall inhibitory activity observed when the consortia are present. However, further research is needed to explore these interactions and identify the specific factors contributing to the observed inhibition in the consortia.
To the best of our understanding, this study represents the pioneering effort in Pakistan's context. It significantly contributes to our comprehension of the intricate microbial composition of USA-based kefir and underscores the significance of kefir as an alternative to antibiotics. However, present research incurs certain limitations, including a deficiency in understanding the comprehensive microbial profile of these KGs, and an incomplete grasp of the dynamics governing these antimicrobial attributes. Future studies are warranted to fully elucidate these intricacies of KGs and to optimize doses for the effective treatment of gastrointestinal illnesses.