Polycystic ovary syndrome (PCOS) is a complex disorder that impacts both the endocrine and metabolic systems, often resulting in infertility, obesity, insulin resistance, and cardiovascular complications. The aim of this study is to investigate the role of intestinal flora and its metabolites, particularly short-chain fatty acids (SCFAs), in the development of PCOS, and to assess the effects of metformin therapy on these components. SCFA levels in fecal and blood samples from women with PCOS ($n=69$) and healthy controls ($n=18$) were analyzed using Gas Chromatography–Mass Spectrometry (GC/MS) for precise measurement. Fecal microbiota were quantitatively detected by real-time polymerase chain reaction (PCR). To assess the efficacy of six months of metformin treatment, changes in the microbiota and SCFAs in the PCOS group ($n=69$) were also evaluated. The results revealed that women with PCOS exhibited a significant reduction in beneficial bacteria (namely, the C. leptum group and Prevotella spp.) alongside a notable overgrowth of opportunistic microorganisms (C. perfringens, C. difficile, Staphylococcus spp., and Streptococcus spp.). An overproduction of acetic acid (AA, $\mathrm{FC}=0.47$, $p<0.05$) and valeric acid (VA, $\mathrm{FC}=0.54$, $p<0.05$) suggests a link between elevated SCFAs and the development of obesity and PCOS. Interestingly, AA in the bloodstream might offer a protective effect against PCOS by ameliorating key symptoms such as high body mass index ($r=-0.33$, $p=0.02$), insulin resistance ($r=-0.39$, $p=0.02$), and chronic inflammation. Although serum SCFA levels showed non-significant changes following metformin treatment ($p>0.05$), the normalization of AA in the gut underscores that metformin exerts a more pronounced effect locally within the gastrointestinal tract. Furthermore, the study identified the most effective model for predicting the success of metformin therapy, based on serum concentrations of butyric acid (BA) and VA, achieving a 91% accuracy rate, 100% sensitivity, and 80% specificity. These promising findings highlight the potential for developing targeted interventions and personalized treatments, ultimately improving clinical outcomes for women with PCOS.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor—intestinal alkaline phosphatase (IAP)—in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman’s intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0–1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

Background/Objectives: Campylobacter jejuni (CJ) is the etiological agent of the world’s most common intestinal infectious food-borne disease, ranging from mild symptoms to fatal outcomes. The development of innovative synbiotics that inhibit the adhesion and reproduction of multidrug-resistant (MDR) CJ in animals and humans, thereby preserving intestinal homeostasis, is relevant. We have created a synbiotic based on the consortium of Lactobacillus crispatus 2029 (LC2029), Ligilactobacillus salivarius 7247 (LS7247), and a mannan-rich prebiotic (Actigen^®). The purpose of this work was to study the in vitro anti-adhesive and antagonistic activities of the created synbiotic against MDR CJ strains, along with its role in preventing intestinal barrier dysfunction, which disrupts intestinal homeostasis. Methods: A complex of microbiological, immunological, and molecular biological methods was used. The ability of the LC2029 and LS7247 consortium to promote intestinal homeostasis in vitro was assessed by the effectiveness of controlling CJ-induced TLR4 activation, secretion of pro-inflammatory cytokines, development of intestinal barrier dysfunction, and production of intestinal alkaline phosphatase (IAP). Results: All MDR CJ strains showed marked adhesion to human Caco-2, pig IPEC-J2, chicken CPCE, and bovine BPCE enterocytes. For the first time, we found that the prebiotic and cell-free culture supernatant (CFS) from the consortium of LC2029 and LS7247 strains exhibit an additive effect in inhibiting the adhesion of MDR strains of CJ to human and animal enterocytes. CFS from the LC2029 and LS7247 consortium increased the permeability of the outer and inner membranes of CJ cells, which led to extracellular leakage of ATP and provided access to the peptidoglycan of the pathogen for the peptidoglycan-degrading bacteriocins nisin and enterolysin A produced by LS7247. The LC2029 and LS7247 consortium showed a bactericidal effect on CJ strains. Co-cultivation of the consortium with CJ strains resulted in a decrease in the viability of the pathogen by 6 log. CFS from the LC2029 and LS7247 consortium prevented the growth of CJ-induced TLR4 mRNA expression in enterocytes. The LC2029 and LS7247 consortium inhibited a CJ-induced increase in IL-8 and TNF-α production in enterocytes, prevented CJ-induced intestinal barrier dysfunction, maintained the transepithelial electrical resistance of the enterocyte monolayers, and prevented an increase in intestinal paracellular permeability and zonulin secretion. CFS from the consortium stimulated IAP mRNA expression in enterocytes. The LC2029 and LS7247 consortium and the prebiotic Actigen represent a new synergistic synbiotic with anti-CJ properties that prevents intestinal barrier dysfunction and preserves intestinal homeostasis. Conclusions: These data highlight the potential of using a synergistic synbiotic as a preventive strategy for creating feed additives and functional nutrition products based on it to combat the prevalence of campylobacteriosis caused by MDR strains in animals and humans.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human β-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.