This study was approved by the Ethics Committee of the Inner Mongolia Agricultural University (NND2022046), dated 2 March 2022. All experimental procedures adhered to the guidelines for the care and use of experimental animals.

The genomic investigation of Lactobacillus salivarius Y20 began with the isolation and cultivation of the strain. Single colonies were obtained by streak-inoculating Lactobacillus salivarius Y20 onto LB agar plates, followed by incubation at 37°C for 12–18 h. These colonies were then transferred to LB liquid medium and cultured overnight at 37°C with shaking at 110 rpm. Genomic DNA extraction was performed using the Biomed Gene Genomic DNA Extraction Kit (Model DL111-01), adhering to the manufacturer’s protocol to ensure high-quality DNA for downstream sequencing applications (Liang, 2021).

A hybrid sequencing approach was employed to comprehensively characterize the Lactobacillus salivarius Y20 genome, utilizing both second-generation (Illumina MiSeq) and third-generation (PacBio) sequencing technologies. The Whole Genome Shotgun (WGS) strategy was used for library construction, ensuring broad genomic coverage.

The Illumina sequencing data underwent rigorous quality control using Fast QC software. Parameters evaluated included GC content distribution, base quality distribution, sequence base quality, and base content distribution to ensure data integrity and suitability for downstream assembly and analysis (Liang, 2021).

Given the extended read lengths provided by third-generation sequencing, the Falcon assembler was selected for de novo assembly of the PacBio reads. This approach simplified the assembly process and enhanced genome continuity.

Functional annotation of the coding proteins within the Lactobacillus salivarius Y20 genome was conducted using a curated set of databases, including Nr, SwissProt, KEGG, GO, COG, Pfam Scan, and CAZy.

To contextualize the genomic features of Lactobacillus salivarius Y20 within its broader taxonomic framework, comparative genomics analyses were undertaken.

To further elucidate the evolutionary relationships of Lactobacillus salivarius Y20, population evolutionary analyses were conducted.

The statistical analysis of the data was conducted using MEGA version 6.06 and SPSS version 23.0. For comparing means across multiple groups, a one-way analysis of variance (ANOVA) was employed, followed by Tukey’s post hoc test to identify specific pairwise differences. Visual representations of the data were generated using GraphPad Prism version 8 (Zhang et al., 2025).

Through a systematic approach, this part of the results describes the isolation, screening, and identification of an optimal vaginal Lactobacillus strain (Y20) from Mongolian mares. This was achieved by evaluating acid production, resistance, growth kinetics, and morphological traits (Figures 1–3).

Initially, 675 acid-producing colonies were isolated through dilution-plating on a bromocresol purple-containing medium. Color changes from purple to yellow (Figure 1A) served as an indicator of successful lactic acid production. Among these colonies, 80 strains (Y1-Y80) were selected using a litmus milk color test. In this test, a pink curd-like morphology (highlighted in the green-lined regions of Figure 1B) was found to correlate with proteolytic activity and acidification potential.

Subsequent assays were conducted to assess the strains’ growth under acidic stress (pH 2–4). The results showed that Y42 and Y47 exhibited superior growth at pH 2, with OD₆₀₀ values of 0.25–0.23 (Figure 2A). Meanwhile, Y66 demonstrated excellent growth at pH 3, reaching an OD₆₀₀ of 0.8 (Figure 2B). At pH 4, Y11, Y18, and Y20 achieved comparable growth, with OD₆₀₀ values around 1.7 (Figure 2C), while Y25 had a slightly lower OD₆₀₀ of 1.5. In the blank medium, Y20, Y25, and Y66 entered the logarithmic phase by 6 h, with an OD₆₀₀ of 1.8 at 32 h (Figure 2D), outperforming the other strains. Notably, Y20 acidified the medium most rapidly, reaching a pH of 3.7 within 12 h, with a ΔpH of 2.0 (Figure 2E). It also maintained lower pH values (≤4.1 at 16 h) compared to all its competitors.

Morphological analysis (Figure 3) classified Y8, Y18, Y42, Y47, and Y66 as round or oval in shape (Figures 3A,C,F–H), while Y11, Y20, and Y25 were short-rod-shaped (Figures 3B,D,E). This morphological classification was consistent with their growth and acidification profiles. Given Y20’s exceptional acid production capabilities, its resilience to pH values ranging from 2 to 4, and its rapid growth, it was prioritized for advanced investigation into equine vaginal microbiome dynamics and probiotic applications. This highlights its critical role in maintaining vaginal health and contributing to the overall well-being of the host.

The identification results of the vaginal Lactobacillus strain Y20 from Mongolian mares are as follows (Figure 4 and Tables 1–3).

In the physiological and biochemical tests, strain Y20 was capable of fermenting salicin, sorbitol, sucrose, raffinose, inulin, and lactose, and it might also ferment cellobiose and mannitol. It did not produce indole, gelatinase, or H₂S, nor could it hydrolyze starch (Table 1). Based on these results and in conjunction with the data from Bergey’s Manual of Determinative Bacteriology (9th edition), strain Y20 was preliminarily identified as belonging to the genus Lactobacillus.

In the Biolog microwell plate bacterial identification, after the Biolog microwell plate reaction, there were 38 positive reactions, 47 negative reactions, and 11 intermediate-value reactions (Supplementary Figure 1). Positive reactions were observed for substrates such as raffinose, maltose, trehalose, and sucrose, while negative reactions were observed for substrates like arabitol and inositol. Intermediate-value reactions were noted for substrates such as cellobiose and turanose. After integrating all the reactions and using the Biolog bacterial identification system, the highest similarity index (SIM) was 0.682 (>0.5), indicating reliable data. The strain with the highest SIM value was identified as Lactobacillus salivarius. Specifically, the top four strains with the highest SIM values were Lactobacillus salivarius, Lactobacillus salivarius ss araffinosus, Leuconostoc mesenteroides ss dextranicum, and Lactobacillus brevis (Table 2).

For the 16S rRNA gene identification, during the 16S rRNA gene PCR electrophoresis detection, analysis of the amplified products via 1.2% agarose gel electrophoresis revealed a distinct and singular target band of approximately 1,408 bp for strain Y20, aligning with the anticipated size (Figure 4A); subsequently, in the 16S rRNA gene PCR product sequencing and homology comparison, the PCR products were dispatched to BGI Genomics for sequencing, and BLAST software was employed to assess the homology of the 16S rRNA gene sequence of strain Y20 (Table 3), with the construction of a phylogenetic tree using MEGA6.06 indicating that strain Y20 exhibited the closest phylogenetic relationship to Lactobacillus salivarius, sharing a homology exceeding 99% (Figure 4B).

A meticulous and multifaceted evaluation of the biological attributes of Lactobacillus salivarius Y20, isolated from the vaginal microenvironment of Mongolian mares, was undertaken utilizing a series of rigorously designed and executed assays (Figures 5–9 and Tables 4, 5).

During the heat-resistance assessment, the heat-survival ratio of strain Y20 was precisely determined, with 37°C serving as the reference survival rate (Figure 5). A striking and statistically robust decline (p < 0.001) in the survival rate was observed as the temperature was elevated to 55°C, 65°C, and 75°C. At the extreme temperature of 75°C, the survival rate of Y20 experienced a precipitous drop to less than 1%, clearly indicating its relatively limited thermotolerance.

Shifting focus to the antibacterial testing, strain Y20 demonstrated a broad-spectrum antimicrobial activity against six well-established and thoroughly characterized quality-control strains (Table 4 and Figure 6). Among these, the most pronounced inhibitory effect was exerted against Staphylococcus aureus, as evidenced by an exceptionally large inhibition zone diameter of 20.42 mm. A hierarchical order of inhibitory efficacy was established, with Escherichia coli exhibiting the second-highest level of inhibition, succeeded by Salmonella typhi, Streptococcus equi subsp. zooepidemicus, Gardnerella vaginalis, and finally Streptococcus hemolyticus group B, which displayed the least inhibitory response among the tested strains.

In the hydrogen peroxide production assay, all colonies of Y20 exhibited a characteristic blue reaction (Figure 7), unequivocally indicating a positive result and underscoring its capacity to synthesize hydrogen peroxide, a crucial antimicrobial metabolite with significant implications for microbial competition and host defense. The antioxidant capacity analysis revealed that strain Y20 possesses remarkable antioxidant properties. It achieved a DPPH radical-scavenging rate of 73.77%, a total antioxidant capacity (T-AOC) of 67.49 U/mL, and a total superoxide dismutase activity (T-SOD) of 59.66 U/mL. These findings suggest its potential to effectively counteract oxidative stress within the vaginal environment, which is a critical factor in maintaining vaginal health and preventing oxidative-damage-related pathologies.

The hemolysis test provided compelling evidence of the safety profile of Lactobacillus salivarius Y20. In contrast to Staphylococcus aureus, which produced a distinct and well-defined hemolysis ring, strain Y20 did not generate any such ring (Figure 8). This observation unequivocally indicates its non-hemolytic nature, thereby offering a high level of assurance regarding its safety for potential therapeutic or probiotic applications.

In the drug-sensitivity analysis, a panel of 10 antibiotics, including penicillin, carbenicillin, piperacillin, cefazolin, cefradine, doxycycline, minocycline, midecamycin, ofloxacin, and clindamycin, exhibited inhibitory effects on strain Y20. The strain demonstrated a high degree of susceptibility to carbenicillin, piperacillin, cefradine, and doxycycline, while displaying moderate susceptibility to midecamycin, ofloxacin, and clindamycin. Conversely, it exhibited resistance to penicillin, cefazolin, and minocycline, and showed no susceptibility to the remaining 20 antibiotics included in the test panel (Table 5 and Figure 9).

With regard to the auto-aggregation and biofilm-formation capacity assessments, strain Y20 displayed an outstanding auto-aggregation rate (A%) of 86.8127%. In the biofilm-formation evaluation, the OD595 value of strain Y20 was measured at 1.2834, with an ODC of 0.4244. Given the criterion of 2 ODc < OD ≤ 4 ODc, this indicates a moderate (++) biofilm-formation ability. These attributes are of paramount significance for Lactobacilli, as they facilitate robust adhesion to vaginal epithelial cells, enhance their adaptability and survival within the complex vaginal ecosystem, and enable them to effectively compete with pathogenic bacteria, thereby playing a pivotal role in maintaining vaginal homeostasis and preventing infections.

The equine industry, while experiencing significant growth, concurrently confronts numerous reproductive health challenges, with bacterial vaginosis (BV) in mares emerging as a critical disease that severely compromises their reproductive health. This condition markedly reduces conception and fertility rates, imposing substantial economic losses on production and breeding efficiency within the equine sector and thus posing a significant bottleneck to its further industrial transformation, upgrading, and sustainable development (Coudray and Madhivanan, 2020; Marnach et al., 2022).

In traditional livestock production practices, antibiotic therapy remains the predominant approach for the treatment of bacterial vaginosis in mares. However, the prolonged and widespread use of antibiotics has triggered a series of severe problems. Antimicrobial resistance (AMR) has escalated dramatically, with many pathogens, such as Staphylococcus aureus, progressively developing resistance to multiple antibiotic agents and evolving into concerning “superbugs” (Liu et al., 2021; Mlynarczyk-Bonikowska et al., 2022). S. aureus colonizes not only the host’s gastrointestinal tract, skin, and perineal regions but also secretes diverse toxins, including alpha-hemolysin (Hla), enterotoxins, and leukocidins, which collectively endanger host health. The emergence of resistance undermines the efficacy of conventional antibiotic treatments, thereby increasing the complexity and cost of disease management (references omitted for brevity). Beyond vaginal infections, Escherichia coli is capable of cause severe systemic illnesses, such as diarrhea, sepsis, hemolytic uremic syndrome, and urinary tract infections, posing threats to both animal and human health (Ahmad-Mansour et al., 2021). Salmonella spp., as zoonotic pathogens, present substantial risks in livestock production and public health, with outbreaks potentially leading to large-scale infections in animals and humans, severely impacting the economic viability of breeding operations and public health safety (Takaya et al., 2020). Streptococcus pyogenes can induce severe invasive diseases, damaging local tissues and causing conditions such as scarlet fever and erysipelas, with significant implications for animal welfare (Yu et al., 2023). Although primarily a commensal bacterium in horses, Streptococcus equi subsp. zooepidemicus (SEZ) is a common pathogen in bacterial endometritis in mares and can also infect dogs, pigs, and other animals, particularly in high-value breeding sectors like horse racing, resulting in substantial economic losses (Ikhuoso et al., 2020; Boyle, 2023). Gardnerella vaginalis is widely implicated in bacterial vaginosis and associated with adverse reproductive health outcomes, adversely affecting mare reproductive function (Joseph et al., 2024). The AMR, drug residues, and complex pathogenicity mechanisms induced by these pathogens have emerged as critical global health threats, necessitating urgent exploration of safe, efficient, and sustainable alternative therapeutic strategies (McDonald and Linde, 2002).

Probiotics, as potential alternatives to antibiotics, have garnered considerable research attention due to their safety, controllability, eco-friendliness, and sustainability (Mnisi et al., 2025). Probiotics exhibit immense potential in modulating microecological balance, enhancing host immunity, and inhibiting pathogenic growth, offering novel solutions to combat antibiotic misuse (Suez et al., 2019; Ahire et al., 2023). Through a rigorous screening strategy, this study successfully isolated Lactobacillus salivarius Y20, a strain characterized by robust growth performance, strong acid tolerance, and high acid production efficiency. In preliminary screening, Y20 demonstrated rapid growth kinetics and achieved high cell density. Subsequent acid tolerance and acid production assays confirmed its ability to thrive under acidic conditions while efficiently producing organic acids. Critically, Y20 exhibited potent inhibitory effects against multiple pathogens, including S. aureus, E. coli, Salmonella enterica serovar Typhi, G. vaginalis, SEZ, and S. pyogenes. Theoretically, Y20’s superior growth traits enable rapid colonization and proliferation within the mare’s vaginal microenvironment, establishing dominant microbiota. Its acid tolerance ensures survival and sustained functionality in the acidic vaginal milieu, while its high acid production capacity further lowers vaginal pH, reinforcing pathogen suppression. Collectively, these attributes underscore the potential of Y20 as a probiotic for preventing and treating bacterial vaginosis in mares, offering a viable alternative to antibiotic therapy and mitigating the risks associated with AMR and drug residues. Future studies should focus on elucidating Y20’s colonization dynamics in the mare’s vagina, its long-term impact on vaginal microecology, practical therapeutic efficacy, and safety profiles. Such investigations will provide a robust theoretical foundation and empirical support for its clinical application in the equine industry, advancing the sector toward healthier, more sustainable practices.

Whole-genome high-throughput sequencing technology has been widely employed in microbiology-related research. For the genomic study of Lactobacillus salivarius, the first published genome of Lactobacillus salivarius was that of Lactobacillus salivarius UCC118 (Raftis et al., 2014). The genome of Lactobacillus salivarius UCC118 comprises a 1.83 Mb chromosome, a large plasmid of 242 kb (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44) (Fang et al., 2008). With the advancement of sequencing technology, related research has continued to increase. For instance, Ayala et al. (2017) isolated Lactobacillus salivarius L28 from ground beef. The genome size of L28 is approximately 2,028,405 bp, with an average GC content of 32.7%. A total of 1982 coding sequences, 64 tRNAs, and 31 rRNAs were predicted. Kergourlay et al. (2012) isolated Lactobacillus salivarius SMXD51 from chickens. Using the RAST server, they achieved functional annotation of predicted genes. SMXD51 contains 1795 coding sequences, 103 RNAs, and 78 tRNA genes. Raftis et al. (2014) isolated Lactobacillus salivarius JCM1046 from pigs, and its genome comprises a 1.83 Mb chromosome and four plasmids. Mackenzie et al. (2014) isolated Lactobacillus salivarius cp400 from pigs and performed automated gene prediction using GeneMark and Glimmer3. Cho et al. (2011) isolated Lactobacillus salivarius GJ-24 from human feces. By utilizing the RAST server and comparing with the KEGG and COG databases, they achieved functional annotation of predicted genes. Sun et al. (2015) isolated Lactobacillus salivarius LsR from the feces of a centenarian. Its genome contains a 1,751,565 bp circular chromosome and two plasmids. This study also identified several important genes that enable LsR to survive in the gastrointestinal tract and adhere to the intestine. Additionally, it was found that LsR can inhibit the occurrence of oral cancer and colorectal cancer caused by some factors. Jiménez et al. (2010) determined the complete genome sequence of Lactobacillus salivarius CECT 5713 isolated from a mother-infant pair (breast milk and infant feces) using whole-genome shotgun sequencing. Its genome consists of a 1,828,169 bp circular chromosome and two plasmids. They observed that CECT 5713 has anti-inflammatory, immunomodulatory, and anti-infective properties. In this study, the circular genome size of Lactobacillus salivarius Y20 is approximately 1.74 Mb, which is smaller than that of Lactobacillus salivarius IBB3154 (from hens), JCM1046 (from pigs), LPM01 (from humans), 2D (from horses), and S01 (from fish). Lee et al. (2017) found that the genome size of human-derived Lactobacillus salivarius strains is smaller than that of pig-derived strains. This could be attributed to the internal environment of humans is more attractive to strains than that of pigs and chickens, and humans are exposed to harsh environments (such as diseases, toxins, etc.) less frequently than livestock. Symbiotic bacteria accompany the evolution of the host, and in non-harsh environments, symbiotic bacteria lose more genes than in harsh environments. The genome size of Lactobacillus salivarius Y20 derived from the horse vagina is much smaller than that of many other sources of Lactobacillus salivarius. It is hypothesized that the vaginal environment of Mongolian horses is more attractive to bacteria, and the strains are exposed to fewer harsh conditions in the Mongolian horse vaginal environment.

In the analysis of the GO annotation of the Y20 genome, several antioxidant-related genes were found, such as thioredoxin, which, together with nicotinamide adenine dinucleotide phosphate and thioredoxin reductase, plays a positive role in stabilizing the redox state of organisms (Ball et al., 2022; Bradford et al., 2024). In the KEGG annotation, immune-related pathways (NOD-like receptor signaling pathway) and the TrxA gene for regulating redox were identified. Comparative genomics analysis revealed the unique genes of Lactobacillus salivarius Y20. One immune-related unique gene, LRS64_05055 (RAMP protein Csm5), possesses an immune defense function. Three immune-related genes are shared among six strains, namely LRS64_01800 (thioredoxin TrxA), LRS64_03595 (thioredoxin TrxA), and LRS64_05635 (GTPase). The aforementioned results collectively indicate that Y20 possesses a robust foundation for immunity and antioxidant properties.

Based on the findings of this study, several avenues for future research emerge. First, in vivo studies are warranted to evaluate the efficacy of Y20 in preventing or treating equine reproductive disorders. These studies should investigate the optimal dosage, duration of treatment, and potential interactions with other vaginal microorganisms. Second, further exploration of the molecular mechanisms underlying the probiotic effects of Y20, such as the production of antimicrobial substances and modulation of the host immune response, would provide deeper insights into its therapeutic potential. Additionally, comparative studies with other Lactobacillus salivarius strains from different hosts could identify strain-specific traits that contribute to their ecological success in specific niches. Finally, long-term safety studies are essential to ensure the absence of any adverse effects associated with the prolonged use of Y20 as a probiotic.