Open AccessArticle Integrative Phenotypic and Genomic Analysis Reveals Antimicrobial and Stress-Resistance Mechanisms of Lacticaseibacillus rhamnosus MG0718 as a Promising Probiotic Candidate for Food Applications 1 Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530004, China 2 Key Laboratory of China (Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning 530001, China 3 Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China 4 Department of Engineering, Virginia Tech, Blacksburg, VA 24061, USA * Authors to whom correspondence should be addressed. † These authors contributed equally to this work. Microorganisms 2026, 14(6), 1290; https://doi.org/10.3390/microorganisms14061290 (registering DOI) Submission received: 29 April 2026 / Revised: 2 June 2026 / Accepted: 2 June 2026 / Published: 7 June 2026 Abstract Lactobacilli species have emerged as a focal point in food microbiology due to their core probiotic properties, including the regulation of intestinal homeostasis and the enhancement of immunity. This study focuses on Lacticaseibacillus rhamnosus MG0718 (hereinafter referred to as MG0718), employing a combined approach of phenotypic evaluation and whole-genome sequencing to assess its probiotic potential and analyze the correlation between its phenotype and genotype. In vitro experiments demonstrated that MG0718 possesses broad-spectrum antibacterial activity against pathogenic bacteria. In vitro experiments showed that MG0718 had broad-spectrum antibacterial activity against pathogenic bacteria such as Escherichia coli ( E. coli), with an inhibition zone diameter of up to 13.67 ± 1.56 mm. It survived pH 2.5 for 6 h with only a 1.72 log10 reduction, and showed 0.78 and 1.11 log10 CFU/mL reductions in artificial gastric and intestinal fluids after 2 h. DPPH scavenging was 56.7% and total reducing power was 91.1%. In vivo, 7-day preventive administration maintained 100% survival against S. Typhimurium infection and alleviated weight loss. Bacterial loads in spleen, liver, and cecum dropped from 4.5, 4.5, and 4.2 to 3.6, 1.8, and 2.5 lg CFU/g, respectively. Whole-genome sequencing analysis indicated that the complete genome of MG0718 is 2,574,565 bp in length, containing 2813 CDS. Among these genomic components, 203 stress-related protein genes elucidate its superior environmental tolerance; one bacteriocin gene cluster, one EPS gene cluster and two secondary metabolite gene clusters provide the genetic basis for its antibacterial activity. Notably, no virulence factors were detected, ensuring the safety of the strain for application. In summary, the functional phenotypes of MG0718 are highly consistent with its genetic characteristics, identifying it as a probiotic candidate of significant developmental value. Future research should focus on clinical trials to further verify its practical benefits for human intestinal health and immunomodulation, thereby providing a robust scientific basis for its application in functional foods. 1. Introduction Probiotics are defined as viable microorganisms that confer health benefits to the host [ 1]. As microorganisms with specific functional capabilities, they have demonstrated immense potential in food preservation, quality enhancement, and the protection of consumer health. Over the past two decades, families such as Lactobacillaceae and Bifidobacteriaceae, along with certain probiotic yeasts, have become the focus of research in food microbiology. A substantial body of evidence from both in vitro and in vivo models demonstrates that these strains can enhance food safety and consumer health by optimizing the gut microbiota, accelerating the repair of intestinal epithelial injury, and modulating host immunity [ 2]. Among these, Lacticaseibacillus rhamnosus ( Lbs. rhamnosus) [ 3] has emerged as one of the most commercially promising strains due to its tolerance to gastric acid and bile, as well as its ability to colonize the upper small intestine [ 3, 4, 5, 6]. Its surface exopolysaccharides and pili structures significantly enhance adhesion efficiency to intestinal epithelial cells, laying the foundation for long-term colonization and sustained metabolic activity [ 7, 8, 9]. Since the completion of the first whole-genome sequence (WGS) of Lactococcus lactis in 2001, whole-genome sequencing has superseded traditional identification methods, becoming a core tool for elucidating the genetic background and functional potential of probiotics [ 10]. Hundreds of complete maps for lactobacilli strains are now available in public databases. Through pan-genome analysis, comparative genomics, and functional annotation, researchers can rapidly identify key genes associated with bacteriocin synthesis, acid and bile salt tolerance, and immunomodulation [ 11, 12]. Currently, the integration of WGS and bioinformatics allows for a comprehensive exploration of strain genetic and biological characteristics with higher resolution and sensitivity. This approach offers valuable insights into genetic information, evolutionary relationships, physiological traits, probiotic potential, and safety profiles [ 13]. However, it must be acknowledged that phenotypic analysis remains crucial for the discovery of probiotics, as sequencing processes may contain errors, and the consistency between genomic sequences and existing databases is finite [ 14]. For example, Lbs. rhamnosus KF7 isolated from kefir was evaluated through whole-genome sequencing, phenotypic safety assays, and a Caenorhabditis elegans in vivo model. This confirmed the absence of virulence factors and antibiotic resistance genes and demonstrated colonization potential [ 15]. Similarly, strain Lbs. rhamnosus SAL2 was characterized through hybrid Illumina/PacBio sequencing and multidimensional phenotypic benchmarking against the reference strain Lbs. rhamnosus GG. The results revealed comparable safety and superior antioxidant performance [ 16]. Other studies, such as those on dairy-derived Lbs. rhamnosus 044AE [ 17], also combined in vitro probiotic assays with genomic annotation to identify stress-response genes and bacteriocin clusters. Together, these studies show that phenotype-genotype integration is a powerful tool for probiotic screening. However, several key gaps remain. Most screening studies are still limited to in vitro assays. They lack in vivo pathogen challenge validation to confirm protective efficacy under real host infection conditions. Although phenotypic and genomic data are often presented together, the mechanistic link between specific genetic loci and observed functional advantages is frequently unclear. This limits the ability to predict phenotype from genotype. Moreover, probiotic traits are highly strain-specific. The functional mechanisms of newly isolated strains require independent characterization, rather than inference from model strains such as Lbs. rhamnosus GG. To address this, the present study focuses on Lbs. rhamnosus MG0718 (hereinafter referred to as MG0718), a strain isolated from Sour Bamboo Shoot that exhibits favorable antibacterial activity, hereinafter designated as MG0718. We first systematically determined key probiotic phenotypes, including acid and bile salt tolerance, cell adhesion, and antimicrobial spectrum. Subsequently, we utilized Illumina NovaSeq to complete whole-genome sequencing, mining for potential bacteriocin and stress tolerance genes through comparative genomics and functional annotation. On this basis, we established a mouse model of S. Typhimurium infection to evaluate the protective effects of MG0718 on intestinal colonization, pathological injury, inflammatory cytokine levels, and survival rates. By correlating the genome, phenotype, and in vivo protective efficacy, we aim to provide theoretical and experimental evidence for the development of Lbs. rhamnosus preparations that function as both preservatives and intestinal protectants. 2. Materials and Methods 2.1. Strains and Culture Methods S. Typhimurium SM022 was generously provided by Professor Alejandro Aballay of Duke University, USA. Escherichia coli ATCC 25922, Staphylococcus aureus subsp. aureus ATCC 6583 ( S. aureus ATCC 6583), and MG0718 (isolated from traditional Sour Bamboo Shoot in the Guangxi region) were obtained from the Guangxi Veterinary Research Institute (Nanning, China). MG0718 was inoculated into MRS medium (Haibo, Shanghai, China) and incubated at 37 °C for 18 h. Cell-free supernatant (CFS) was collected after centrifugation (8000× g, 4 °C, 10 min). To obtain bacterial suspensions (BS) (Haibo, Shanghai), strains’ cells were washed three times with phosphate-buffered saline (PBS) and adjusted to 1 × 10 9 CFU/mL. Pathogenic strains’cells were inoculated into LB medium (Haibo, Shanghai), incubated at 37 °C for 18 h, centrifuged (8000× g, 4 °C, 10 min), washed three times with PBS, and adjusted to 1 × 10 9 CFU/mL. 2.2. In Vitro Antibacterial Experiment of MG0718 2.2.1. The Oxford Cup Assay Modified from He et al. [ 19], the pathogenic strains were used as indicator bacteria to observe the inhibitory capability against common pathogens. Pathogenic bacteria were cultured to the logarithmic growth phase. A 100 μL aliquot of a bacterial suspension containing 1.0 × 10 6–10 7 CFU/mL was spread onto an LB agar plate. Oxford cups were placed on LB plates spread with pathogenic bacteria BS. Subsequently, 200 µL of the test bacterial solution or supernatant was added to the Oxford cups and incubated at 37 °C for 18 h. The diameter of the inhibition zone was observed, with three replicates set for each strain. 2.2.2. Characterization of MG0718 Antimicrobial Substances Following the method of Scillato M et al. [ 20]. The CFS was treated as follows: (1) pH Neutralization: The pH of the CFS was adjusted to 6.5 using 1 mol/L sterile NaOH. MRS medium adjusted to pH 6.5 with lactic acid and acetic acid served as blank controls. (2) The CFS was adjusted to pH 6.5, and catalase (final concentration 5 mg/L) was added, followed by a water bath at 37 °C for 2 h. MRS medium adjusted to pH 6.5 with catalase added served as a blank control. (3) Proteolytic Enzyme Treatment: To determine the proteinaceous nature of the antimicrobial substances, trypsin, papain, proteinase K (Haibo, Shanghai), and pepsin (final concentration 1 mg/mL) were added to the CFS separately and incubated in a water bath at 37 °C for 2 h. (4) Untreated fermentation supernatant served as the control. 200 µL of the treated CFS was added to Oxford cups, incubated at 37 °C for 18 h, and the diameter of the inhibition zone was measured in triplicate. 2.3. Prophylactic Protection of MG0718 Against S. Typhimurium Infection in C57BL/6 Mice 2.3.1. Strain Preparation S. Typhimurium SM022 and MG0718 were grown to the logarithmic phase in LB broth and MRS broth (Haibo, Shanghai), respectively. Bacterial cells were collected by centrifugation at 894× g for 10 min, washed twice with PBS, and adjusted to a concentration of 1 × 10 8 CFU/mL prior to gavage. 2.3.2. Experimental Grouping Thirty-two 4-week-old female C57BL/6 mice were housed under standardized conditions (22–26 °C, 20% humidity, 12 h light/dark cycle) with ad libitum access to standard diet and water. After one week of acclimatization, mice were randomly divided into four groups ( n = 8 per group). All animal experiments described in this paper were conducted in strict accordance with the National Standard of the People’s Republic of China, Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892-2018), and the Guidance on the Humane Treatment of Laboratory Animals. The experimental protocol was reviewed and approved by the Laboratory Animal Ethics Committee of the Guangxi Veterinary Research Institute (Approval No.: GXV-2026-005). Throughout the experiments, all animals were treated humanely and in accordance with the ‘3Rs’ principle, minimising both animal suffering and the number of animals used. Negative Control (CON): No treatment; MG0718 Control (LR): Received MG0718; MG0718 Prevention (LR + Sty): Received MG0718 followed by S. Typhimurium SM022; Positive Control (Sty): Received S. Typhimurium SM022; Detailed grouping is shown in Figure 1. Mice were euthanized and analyzed 14 days after S. Typhimurium SM022 gavage. 2.3.3. Detection of Body Weight Changes and Survival Rate Mouse weight and quantity were recorded before feeding with S. Typhimurium SM022. Following infection, the number of surviving mice was recorded daily for 7 days to generate a survival curve. Mice were weighed prior to euthanasia to plot body weight changes. 2.3.4. Detection of S. Typhimurium Load in Colon, Liver, and Spleen Adapted from the article by Ou et al. [ 21]. The liver, spleen, and cecum of each mouse were weighed, thoroughly ground in PBS, and serially diluted (10 0 to 10 −5). 100 µL of each dilution was spread evenly on bismuth sulfite agar plates using sterile glass beads. After incubation at 37 °C for 18 h, Salmonella load was calculated based on colony counts in each tissue. 2.3.5. Detection of Liver Lesions Adapted from the article by Yin et al. [ 22]. On the 5th day challenge, three mice from each group were randomly selected for euthanasia. Livers were aseptically removed and immediately fixed in 4% formaldehyde solution for 48 h. Fixed tissue samples were transported to the Guangxi University of Chinese Medicine for histological processing and analysis. 2.3.6. Measurement of Inflammatory Factors Adapted from the article by Li et al. [ 23]. Total RNA was extracted from liver tissue using the Cwbio kit (CW0581S) (Shanghai, Shenggong, China). cDNA synthesis was performed using the Cwbio kit (CW2020M) (Shanghai Shenggong). Quantitative analysis was conducted using Sangon Biotech SYBR Green qPCR reagent (B690016-0005) (Shanghai Shenggong). Gene expression differences between groups were calculated using the 2 −ΔΔCt method, normalized to β-actin. 2.5. Bacterial Adhesion Ability Detection 2.5.1. Auto-Aggregation Detection Adapted from the article by Chaudhari et al. [ 26]. After 18 h of culture, activated bacteria were centrifuged (8000× g, 5 min), washed twice with PBS, and resuspended. The initial absorbance (OD 600 recorded as D 0) was measured. The suspension was vortexed for 30 s and incubated statically at 30 °C. Subsequent absorbance values (D t) were recorded at 1, 2, 3, 4, and 24 h. The auto-aggregation rate (A, %) was calculated as: A = 1 − D t D 0 ୍ଠ 100 % 2.5.2. Hydrophobicity Determination Adapted from the article by Apostolakos et al. [ 27]. 2 mL of bacterial suspension was mixed with an equal volume of hexadecane, vortexed for 1 min, and allowed to separate at room temperature for 30 min. The OD 600 of the aqueous phase (D 1) was measured. Hydrophobicity (H, %) was calculated as: H = 1 − D 1 D 0 ୍ଠ 100 % 2.5.3. Determination of Antioxidant Activity DPPH Radical Scavenging Activity Adapted from the article by Yin et al. [ 28]. 1 mL of CFS was mixed with 2 mL of DPPH (Solarbio, Beijing, China) ethanol solution (0.2 mmol/L), vortexed, and incubated in the dark at room temperature for 30 min. After centrifugation (3578× g, 10 min), absorbance was measured at 517 nm. Scavenging capacity was calculated as: DPPH scavenging ( % ) = ( 1 − A 1 − A 2 A 0 ) ୍ଠ 100 A 0: Absorbance value measured by the same treatment with an equal volume of anhydrous ethanol instead of the sample to be tested. A 1: absorbance value of the experimental group. A 2: equal volume of anhydrous ethanol instead of DPPH solution; the absorbance value measured by the same treatment. Superoxide Anion Radical Scavenging Activity Adapted from the article by Yin et al. [ 28]. 3.4 mL of Tris-HCl (50 mmol/L, pH 8.2) (Solarbio, Beijing, China) was mixed with 0.5 mL pyrogallol (50 mmol/L) (Solarbio, Beijing) and 1 mL of sample, then incubated at 25 °C for 4 min. The reaction was terminated with 0.1 mL of 8 mol/L HCl, and absorbance was measured at 325 nm. Scavenging rate was calculated as: O 2 − scavenging rate ( % ) = A 3 − A 2 A 1 − A 0 ୍ଠ 100 A 0: Equal volumes of water were used to replace the samples to be tested and o-triacontanol, respectively, and the absorbance values were measured by the same treatment. A 1: equal volume of water instead of the sample to be tested, the same treatment of the measured absorbance values. A 2: equal volume of water instead of o-o-benzenetriol, the same treatment of the measured absorbance values. A 3: absorbance value of the experimental group. Determination of Total Reducing Power (TP) Adapted from the article by Yin et al. [ 28]. A mixture of 0.5 mL potassium permanganate (1%) (Solarbio, Beijing), 0.5 mL PBS, and 0.5 mL sample was incubated at 50 °C for 20 min. Then, 0.5 mL trichloroacetic acid (10%) was added, followed by centrifugation (224× g, 10 min). The supernatant was mixed with an equal volume of FeCl 3 (0.1%), and absorbance was measured at 700 nm. TP was calculated as: TP ( % ) = A 1 − A 0 A 1 ୍ଠ 100 A 0: Absorbance value measured by the same treatment with PBS instead of the sample to be tested. A 1: absorbance value of the experimental group. 2.6. Genomic DNA Extraction, Library Construction, and Sequencing Genomic DNA was extracted and purified using the Qiagen DNA extraction kit (Qiagen, Düsseldorf, Germany) following the manufacturer’s instructions. Sequencing library construction and paired-end sequencing were performed by OE Biotech Co., Ltd. (Shanghai, China). Briefly, 2.5 μg of genomic DNA was fragmented, end-repaired, and A-tailed for library preparation. Paired-end sequencing (2 × 250 bp) was subsequently conducted on the Illumina platform. Raw sequencing data were quality-controlled using FastQC and assembled using Unicycler. 2.7. Virulence Factor Analysis Protein coding sequences were BLASTed against the Virulence Factor Database (VFDB) with thresholds of E-value 1 × 10 −5, identity > 60%, coverage > 70%, and gap length 87%), while hydrophobicity was low (16.7%) ( Figure 6f). Antioxidant assays revealed a DPPH scavenging capacity of 56.7% and a total reducing power of 91.1%, highlighting its potential to mitigate oxidative damage ( Figure 6g). 3.3. Basic Features of MG0718 Genome The MG0718 genome consists of one circular chromosome and four circular plasmids ( Figure 7). The chromosome spans 2,574,565 bp with an average GC content of 47%. It contains 2813 predicted coding sequences (CDS) and 59 tRNA genes ( Table 4). 3.3.1. Functional Annotation of MG0718 3.3.2. Virulence No virulence factors were detected. 3.3.3. Mining of Secondary Metabolite Gene Clusters in MG0718 In this study, antiSMASH 5.0 was employed to mine the genome of strain MG0718 for secondary metabolite biosynthetic gene clusters (BGCs). As shown in Figure 9, two putative BGCs were identified. The first was annotated as a Type III polyketide synthase (T3PKS) cluster, harboring the core gene mvaS, which encodes 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase. The second cluster was predicted as a ribosomally synthesized and post-translationally modified peptide (RiPP)-like cluster, carrying the core gene lagD. This gene encodes an ATP-binding cassette (ABC) transporter accessory protein responsible for the processing and secretion of bacteriocins (e.g., lactococcin G), constituting the key genetic basis for the strain’s antimicrobial peptide production. 3.3.4. Antimicrobial Activity and Bacteriocin Production The bacteriocin detection tool BAGELv.4.0 identified a putative bacteriocin gene cluster within the MG0718 genome ( Figure 10), in which all four core proteins were classified as Class IIb bacteriocins. Their sequence identities were determined to be 43.182% (E-value = 6.89 × 10 −9), 46.154% (E-value = 1.07 × 10 −13), and 32.727% (E-value = 5.03 × 10 −21), respectively. Notably, one of the bacteriocin gene clusters exhibited no matching reference cluster, indicating the potential presence of a novel Class IIb bacteriocin, which warrants further investigation. 3.3.5. Annotation of the Extracellular Polysaccharide Gene Cluster of MG0718 In LAB, genes responsible for EPS synthesis often exist as single genes or gene clusters; this structure confers efficiency and precision to the EPS synthesis pathway. Currently, genes involved in LAB EPS biosynthesis generally include those encoding nucleoside synthases, polymerases, invertases, and the CpsB/CpsD proteins involved in the regulation of EPS biosynthesis. Based on gene annotation and BLAST alignment, the gene clusters and sequence information of MG0718 are shown in Figure 11. The chromosome of MG0718 contains a single EPS gene cluster with a length of 20,346 bp and a modular structure. The EPS gene cluster is flanked by the regulatory protein genes wzb, brpA, and wze. At the center of the cluster are four glycosylate synthase genes ( rmlB, rmlC, rfbA, and rmlA), one initiator glycosyltransferase gene ( epsL), three glycosyltransferase genes ( mshA, wbbI, and epsG), two genes encoding transmembrane proteins ( wzx and wzy), one gene encoding UDP-furan galactose isomerase ( glf), one gene encoding a transposase (IS element), and one domain protein of unknown function. Based on the above, the gene cluster of MG0718 EPS is consistent with known gene cluster structures and contains genes related to EPS synthesis. 3.3.6. Antibacterial Gene As shown in Table 5, functional screening was performed on 15 genes of MG0718, resulting in the identification of 5 genes encoding bacteriocins and 7 genes encoding lysozymes. Therefore, it is speculated that MG0718 possesses specific antibacterial efficacy at the genetic level. 3.3.7. Antistress Gene Analysis of MG0718 A comprehensive analysis of the whole genome of strain MG0718 identified a total of 198 genes associated with stress resistance ( Table 6). Among these, genes encoding antioxidant activity were the most prevalent, numbering 45, followed by 24 genes encoding proteins related to osmotic balance. 4. Discussion A key characteristic of potential probiotic strains, particularly within the field of food microbiology, is their antimicrobial activity [ 30]. This property is crucial as it enables these strains to inhibit the growth of pathogenic and spoilage microorganisms, thereby enhancing food safety and quality [ 31]. During growth and metabolism, lactic acid bacteria (LAB) produce and secrete a variety of bioactive metabolites—including short-chain fatty acids, organic acids, glyoxylic acid, peptides, hydrogen peroxide, exopolysaccharides, and bacteriocins [ 32]—which mediate cell signaling and confer probiotic effects upon the host [ 33]. In the present study, MG0718 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria in vitro. Furthermore, mice pre-treated with MG0718 achieved a 100% survival rate following infection with S. Typhimurium. Weight loss induced by S. Typhimurium infection was significantly alleviated, and liver damage was mitigated; concurrently, tissue bacterial load decreased, and the inflammatory response was reduced. These findings suggest that MG0718 possesses potent in vivo antimicrobial activity, consistent with the demonstrated anti-infective capabilities of numerous LAB strains [ 23, 34, 35]. This broad-spectrum activity aligns with the findings of other researchers [ 36, 37]. Although LAB typically demonstrate higher activity against Gram-positive pathogens [ 38, 39], some studies suggest there is no correlation between the antagonistic activity of lactobacilli and the Gram status of the pathogen [ 40]. Although we demonstrated MG0718’s protective efficacy against S. Typhimurium infection in vivo, its effect on the overall intestinal microbiome was not assessed. Current evidence suggests that Lbs. rhamnosus strains generally exert minimal effects on the global gut microbial community, although specific taxa may be affected [ 41, 42]. Whether MG0718 modulates particular taxa (e.g., enriching beneficial genera or suppressing pathogens) requires direct evaluation through 16S rRNA or metagenomic sequencing in future studies. The lagD gene in the RiPP cluster encodes a protein responsible for bacteriocin processing and transport. This genetic architecture is typically co-localized with immunity genes, allowing the strain to secrete antimicrobial peptides without self-harm [ 45, 46]. This mechanism likely provides MG0718 with a competitive advantage in complex microbial communities and supports its potential use as a biopreservative strain. Additionally, BAGEL analysis detected a class IIb bacteriocin cluster with low sequence homology to known references, suggesting that this strain may harbor novel bacteriocins. Functional bacterial gene clusters responsible for producing extracellular cationic peptides (bacteriocins) are typically organized as operons, containing genes involved in bacteriocin synthesis, immunity, ABC transport, and accessory proteins [ 47]. Therefore, AOI_01 is likely involved in bacteriocin biosynthesis and contributes to the antimicrobial activity of MG0718. However, further investigation revealed that neutralizing the supernatant pH to 7.0 completely eliminated its antimicrobial activity, indicating that organic acids are the primary mediators of the inhibitory effect exerted by MG0718. Consistent with previous findings [ 48, 49], hydrogen peroxide was not the source of antimicrobial activity in MG0718, a result that aligns with the findings of Reuben et al. [ 50]. Phenotypic studies indicate that organic acids are the primary contributors to the antimicrobial activity of LAB. Apart from using BAGEL to detect bacteriocin clusters, we also conducted a functional screen across the entire genome. Five bacteriocin-encoding genes and seven lysozyme-encoding genes were found. We hypothesize that these genes form part of the genetic foundation for the antimicrobial effects of MG0718. However, machine learning models predicted the presence of Class II bacteriocins and lysozymes in this strain. Secondary metabolite gene clusters in lactic acid bacteria are highly diverse and strain-specific, and many remain uncharacterized. Even when bacteriocin gene clusters are predicted, their expression may not be detectable due to strain specificity or experimental conditions. Therefore, integrating metabolomic and transcriptomic analyses with machine learning models is crucial for guiding the discovery of functional bacteriocins [ 44]. In addition to bacteriocin clusters, the MG0718 genome also contains an EPS gene cluster. Exopolysaccharides can enhance cell surface hydrophobicity and adhesion. They also regulate the intestinal microenvironment and inhibit pathogen adhesion. In addition, their spatial structures are closely related to antioxidant and immunomodulatory activities [ 51]. The MG0718 genome contains one EPS gene cluster. It is 20,346 bp long and encodes 17 open reading frames. This cluster provides a complete pathway for polysaccharide synthesis. It includes three regulatory genes ( wzb, brpA, wze) and four sugar nucleotide synthesis genes ( rmlB, rmlC, rfbA, rmlA). These genes control EPS synthesis and supply dTDP-rhamnose precursors. This structure is similar to the EPS clusters of other Lbs. rhamnosus strains. Such clusters usually span 18–20 kb and encode 16–17 ORFs [ 52, 53]. However, MG0718 contains only three glycosyltransferase genes ( mshA, wbbI, epsG). This number is lower than that of Lbs. rhamnosus GG (six) [ 53] and Lbs. rhamnosus ATCC 9595 (five) [ 52]. Nevertheless, MG0718 retains the minimum functional unit needed for heteropolysaccharide synthesis. Notably, an IS element is inserted upstream of the rmlA gene in MG0718. Such insertions are common in Lbs. rhamnosus EPS clusters. They often cause genomic plasticity and EPS diversity among strains [ 54]. In Lbs. rhamnosus GG, an IS element also interrupts rmlA. However, Lbs. rhamnosus GG carries a complete rmlABCD operon at another locus as compensation [ 53]. Whether MG0718 has a similar compensatory mechanism remains to be verified. The small intestine contains 0.03–0.3% bile salts, which can inhibit microbial growth and metabolism. Therefore, bile salt tolerance is a key factor for the survival of LAB in the intestinal environment [ 59]. In our phenotypic assays, strain MG0718 exhibited tolerance to 0.3% bile salts, although tolerance decreased as the concentration increased. Bile salt tolerance in the genus lactobacilli is primarily mediated by bile salt hydrolases [ 60] and bile salt transporters [ 61]. While genes related to bile salt hydrolase were not detected in strain MG0718, a rich set of transporter genes was identified. These abundant transporter genes may facilitate the expulsion of bile salts via bile efflux systems, thereby alleviating osmotic stress and enabling the strain to tolerate low concentrations of bile salts. Tolerance to gastrointestinal fluids is another critical characteristic of probiotics. MG0718 demonstrated robust tolerance in simulated gastric and intestinal fluids, with a substantial population surviving after 2 h of exposure. Acid tolerance tests showed that the strain could maintain viability in a solution at pH 2.5 for 6 h. Its acid tolerance is consistent with the presence of acid-resistance-related genes in its genome, including genes encoding dTDP-glucose 4,6-dehydratase [ 62], 4-hydroxy-tetrahydrodipicolinate synthase, ATP-dependent Clp endopeptidase proteolytic subunit ClpP [ 63], and F0F1-type ATPase subunits [ 64]. These genes likely play a pivotal role in adaptation to low pH environments. In phenotypic assays, strain MG0718 exhibited robust tolerance to both low and high temperatures. After treatment at temperatures ranging from −20 °C to 10 °C for 48 h, its survival rate remained stable, demonstrating remarkable cryotolerance. Conversely, heat treatment reduced its survival rate: viable counts were 6 log10 CFU/mL at 60 °C, and viable bacteria persisted even after treatment at 70 °C. Compared to other well-studied strains, MG0718 demonstrated superior temperature tolerance. For instance, the widely recognized probiotic strain Lbs. rhamnosus GG showed significantly reduced viability at 60 °C and completely lost viability at 70 °C [ 65]. Similarly, Lbs. rhamnosus OF44 exhibited lower activity at 60 °C, indicating weaker heat tolerance [ 66]. This temperature tolerance is likely attributed to genes regulating thermal adaptation in strain MG0718. Cold shock DNA-binding domain proteins (CSD) and cold shock proteins maintain genome stability and efficient protein synthesis under low-temperature conditions by binding to nucleic acids and preventing the formation of secondary RNA structures [ 67]. Under heat stress conditions, small heat shock proteins (sHsps) (such as HslO) and transcriptional regulators (such as HrcA) are induced, thereby protecting proteins from heat damage and regulating the expression of stress response genes in MG0718 [ 68]. Collectively, these mechanisms enhance the strain’s tolerance to dual cold and heat stresses. These traits provide MG0718 with a significant advantage in food and probiotic applications, particularly in environments requiring tolerance to extreme temperatures. The ability of potential probiotic strains to adhere to intestinal cells is a fundamental criterion for probiotic screening [ 69]. It has been suggested that auto-aggregation—which supports bacterial adhesion to host gastrointestinal epithelial cells and prevents pathogen colonization—should exceed 40% in potential probiotic strains [ 69, 70]. Through whole-genome analysis, this study revealed that the adhesion capability of strain MG0718 relies on various surface proteins capable of binding to host cell receptors or extracellular matrix components. Phenotypic experiments indicated that the auto-aggregation rate of MG0718 reached 87%, which is significantly higher than other common LAB such as L. acidophilus (21.4%), Lbs. rhamnosus GG (13.1%), and Bifidobacterium species, which are typically below 20% [ 71], This suggests a potential advantage for MG0718 in gastrointestinal colonization. However, its cell surface hydrophobicity was relatively low (16.7% when mixed with xylene). This is consistent with previous studies indicating that many lactobacilli strains, including Lbs. rhamnosus, typically exhibit low to moderate hydrophobicity [ 72]. This low hydrophobicity may be attributed to the presence of surface proteins and exopolysaccharides (EPS), which mask hydrophobic sites on the cell surface [ 72]. Despite this, the high auto-aggregation capability of MG0718 and the presence of specific surface proteins may compensate for its lower hydrophobicity, thereby enabling effective adhesion to intestinal cells [ 72]. Notably, low hydrophobicity may also affect the intestinal residence time of MG0718. Previous studies have shown that probiotic colonization is generally transient. In murine models, Lbs. rhamnosus GG was eliminated from feces within approximately 7 days after a single gavage, and only in vivo evolution extended its persistence to 14–21 days [ 73]. Thus, daily administration is likely required to maintain effective intestinal concentrations for wild-type strains. The exact dosing frequency and residence time of MG0718, however, remain to be determined in future studies. Lactic acid bacteria are widely used in the food and health sectors, playing vital roles in maintaining cell viability, enhancing host health, and adapting to complex environments. Their antioxidant mechanisms allow them to survive in oxidative stress environments, such as the gut, and protect cells from damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) [ 74]. In this study, we conducted a comprehensive analysis of the oxidative stress resistance genes of strain MG0718 and evaluated its in vitro antioxidant efficacy. The superoxide anion scavenging ability of the strain was 16.5%, which is relatively low compared to most LAB. However, its DPPH radical scavenging ability reached 56.7%, significantly surpassing that of other strains. Previous studies have shown that the DPPH radical scavenging activity of parsley cheese ranges from 31% to 48% [ 75]. Afify et al. [ 76] reported that the DPPH scavenging rate of L. plantarum C88 was 53.1%, slightly lower than that of strain MG0718. Ferricyanide reduction experiments showed that the total reducing power of strain MG0718 reached 91.1%, which may be associated with the high number of genes encoding oxidoreductases and antioxidant proteins. These antioxidant genes cover multiple aspects, including sulfur metabolism, oxidoreductases, antioxidant proteins, and transcriptional regulators, collectively constituting a complex defense network. Key components include cysteine desulfurase and thioredoxin involved in sulfur metabolism [ 77, 78]. SDR family oxidoreductases and NADP-dependent oxidoreductases maintain redox balance [ 79, 80], while peroxiredoxins and glutathione peroxidase serve to mitigate oxidative damage [ 81, 82]. Transcriptional regulators (such as the DeoR/GlpR and Crp/Fnr families) regulate the expression of antioxidant genes according to the cellular redox state [ 83, 84, 85]. The synergistic action of these genes enhances the strain’s tolerance to oxidative stress, ensuring its survival in complex environments. Future research could further investigate the expression patterns and regulatory mechanisms of these stress-resistance genes under different environmental conditions, as well as their interactions. This will deepen our understanding of the stress resistance of strain MG0718 and provide a solid theoretical foundation for its application in food and pharmaceutical fields. The genomic and phenotypic traits of MG0718 reflect strong niche adaptation to its isolation source. This strain was isolated from traditional sour bamboo shoots in Guangxi, a naturally fermented vegetable product. Unlike intestinal strains, MG0718 evolved under open, highly acidic, and microbially competitive fermentation conditions. This ecological pressure likely selected for its unique genotype. Phenotypically, its robust acid tolerance, broad-spectrum antibacterial activity, and thermal stability meet the functional demands of fermentation survival and food processing. These findings emphasize that probiotic characteristics are profoundly habitat-imprinted. Therefore, strain-specific evaluation must consider the original ecological niche rather than extrapolating from human-derived model strains. 5. Conclusions MG0718 exhibited broad-spectrum inhibitory effects against pathogenic bacteria such as E. coli in vitro (inhibition zone: 13.67 ± 1.56 mm). Prophylactic administration for 7 days achieved 100% survival in mice infected with S. Typhimurium (vs. 0% in the infection group), significantly reduced tissue bacterial loads, and alleviated infection-induced weight loss and liver damage. The strain possesses superior biological traits, including potent antioxidant capacity (DPPH scavenging: 56.7%; total reducing power: 91.1%), robust gastrointestinal tolerance (only 1.72, 0.78, and 1.11 log10 reductions after exposure to pH 2.5, artificial gastric fluid, and intestinal fluid, respectively), and strong auto-aggregation (>87%). Whole-genome analysis (2,574,565 bp; 2813 CDS) identified 203 stress-related protein genes, one bacteriocin gene cluster, one EPS gene cluster, and two secondary metabolite clusters, providing a genetic basis for its probiotic features. Collectively, these findings designate MG0718 as a highly promising probiotic candidate. Its exceptional adaptability to food processing conditions and the digestive environment, combined with its safety profile, bridges the gap between food microbial resources and human health promotion. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms14061290/s1, Figure S1. Antipathogenic activity; a: E. coli; b: S. aureus; c: S. Typhimurium. Figure S2. Characterization of MG0718 antimicrobial substances a: E. coli; b: S. aureus; c: S. Typhimurium; A: Acid excretion test: 1: MG0718 CFS, 2: MG0718 CFS-pH6.5, 3: MRS + lactic acid, 4: MRS + acetic acid; B: Hydrogen peroxide excretion test: 1: MG0718 CFS, 2: MG0718 CFS + H 2O 2. 3: MRS + H 2O 2; C: Protein removal test: 1: MG0718 CFS, 2: MG0718 CFS + pepsin, 3: MG0718 CFS + proteinase K, 4: MG0718 CFS + trypsin, 5: MG0718 CFS + chymotrypsin. Author Contributions Conceptualization, Y.Y.; Methodology, Y.Y.; Validation, Y.H.; Formal analysis, Y.H. and C.L. (Chunling Li); Resources, Y.Y. and Z.C.; Data curation, C.L. (Changting Li); Writing—original draft, Z.P. and H.B.; Writing—review and editing, C.L. (Changting Li); Visualization, C.M.; Supervision, C.M. and J.L.; Project administration, H.C. and H.P.; Funding acquisition, H.C. and H.P. All authors have read and agreed to the published version of the manuscript. Funding This research was supported by the Guangxi key research and development Program (AB241484045, FN2600640468), the Nanning Key Research and Development Program (NNKJ202408), the Fangchenggang District Key Research and Development Program (Fangke AB24002021), the Guangxi Academy of Agricultural Sciences stable funding research team project (2021YT109), the Guangxi Scientific Research Project (22-5, XKJ2325, XKJ2335), the Special Project for the Construction of National beef cattle Industry Technical system (GZCYTX-03) and Qiankehe Platform Talent-YQK [2023]020, the Guike Special (25-04, 25-05), the Guangxi Forestry Science and Technology Projects (2025GXZCLK 09, 2025GXZCLK11, 2025GXZCLK 60). Institutional Review Board Statement The experimental protocol was reviewed and approved by the Laboratory Animal Ethics Committee of the Guangxi Veterinary Research Institute (Approval No.: GXV-2026-005, approved date: 10 January 2026). Throughout the experiments, all animals were treated humanely and in accordance with the ‘3Rs’ principle, minimising both animal suffering and the number of animals used. Informed Consent Statement Not applicable. Data Availability Statement The datasets used and/or analysed during the current study are available in the NCBl Sequence ReacArchive repository [CP196043]. The GenBan accession number for accessing the Lacticaseibacillus rhamnosus MG0718 genome sequence is [CP196043]. Acknowledgments Many thanks to Yanwen Zhang, Xian Li, and Yinzheng Wu for their support and help with the entire paper. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this article. Abbreviations bp base pairs BS bacterial suspension CDSs protein-coding genes CSD Cold stress DNA-binding domain proteins CFS Cell-free supernatant DPPH 1,1-Diphenyl-2-picrylhydrazyl CAZy Carbohydrate-Active enZymes GIT gastrointestinal tract GO Gene Ontology GHs glycoside hydrolases COG Clusters of Orthologous Groups of proteins GTs glycosyltransferases KEGG Kyoto Encyclopedia of Genes and Genomes Hsp Heat shock proteins Lbs. rhamnosus Lacticaseibacillus rhamnosus LAB lactic acid bacteria MRS de Man, Rogosa and Sharpe LB Luria–Bertani PBS phosphate-buffered saline NS Normal Saline Solution SM secondary metabolite PLs polysaccharide lyases ROS Reactive Oxygen Species O 2−Superoxide Anion Radical T3PKS type III polyketide synthase RNS Reactive Nitrogen Species WGS Whole Genome Shotgun TP Total Reducing Power uspA universal stress protein References Mercer, S.D.; Doherty, C.; Singh, G.; Willmott, T.; Cheesapcharoen, T.; Teanpaisan, R.; O’Neill, C.; Ledder, R.G.; McBain, A.J. Lactobacillus lysates protect oral epithelial cells from pathogen-associated damage, increase secretion of pro-inflammatory cytokines and enhance barrier integrity. Sci. Rep. 2025, 15, 5894. [ Google Scholar] [ CrossRef] Filidou, E.; Kandilogiannakis, L.; Shrewsbury, A.; Kolios, G.; Kotzampassi, K. Probiotics: Shaping the gut immunological responses. World J. Gastroenterol. 2024, 30, 2096–2108. [ Google Scholar] [ CrossRef] [ PubMed] Todorov, S.D.; Baretto Penna, A.L.; Venema, K.; Holzapfel, W.H.; Chikindas, M.L. Recommendations for the use of standardised abbreviations for the former Lactobacillus genera, reclassified in the year 2020. Benef. Microbes 2023, 15, 1–4. [ Google Scholar] [ CrossRef] [ PubMed] Leser, T.; Baker, A. Molecular Mechanisms of Lacticaseibacillus rhamnosus, LGG ପ୍ପ Probiotic Function. Microorganisms 2024, 12, 794. [ Google Scholar] [ CrossRef] [ PubMed] Petrova, M.I.; Reid, G.; Ter Haar, J.A. Lacticaseibacillus rhamnosus GR-1, a.k.a. Lactobacillus rhamnosus GR-1: Past and Future Perspectives. Trends Microbiol. 2021, 29, 747–761. [ Google Scholar] [ CrossRef] Verdenelli, M.C.; Ghelfi, F.; Silvi, S.; Orpianesi, C.; Cecchini, C.; Cresci, A. Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces. Eur. J. Nutr. 2009, 48, 355–363. [ Google Scholar] [ CrossRef] Kant, R.; Rintahaka, J.; Yu, X.; Sigvart-Mattila, P.; Paulin, L.; Mecklin, J.P.; Saarela, M.; Palva, A.; von Ossowski, I. A comparative pan-genome perspective of niche-adaptable cell-surface protein phenotypes in Lactobacillus rhamnosus. PLoS ONE 2014, 9, e102762. [ Google Scholar] [ CrossRef] von Ossowski, I.; Reunanen, J.; Satokari, R.; Vesterlund, S.; Kankainen, M.; Huhtinen, H.; Tynkkynen, S.; Salminen, S.; de Vos, W.M.; Palva, A. Mucosal adhesion properties of the probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED pilin subunits. Appl. Environ. Microbiol. 2010, 76, 2049–2057. [ Google Scholar] [ CrossRef] Zhou, B.; Jin, G.; Pang, X.; Mo, Q.; Bao, J.; Liu, T.; Wu, J.; Xie, R.; Liu, X.; Liu, J.; et al. Lactobacillus rhamnosus GG colonization in early life regulates gut-brain axis and relieves anxiety-like behavior in adulthood. Pharmacol. Res. 2022, 177, 106090. [ Google Scholar] [ CrossRef] Huang, Y.-Y.; Liu, D.-M.; Jia, X.-Z.; Liang, M.-H.; Lu, Y.; Liu, J. Whole genome sequencing of Lactobacillus plantarum DMDL 9010 and its effect on growth phenotype under nitrite stress. LWT 2021, 149, 111778. [ Google Scholar] [ CrossRef] Panthee, S.; Paudel, A.; Blom, J.; Hamamoto, H.; Sekimizu, K. Complete Genome Sequence of Weissella hellenica 0916-4-2 and Its Comparative Genomic Analysis. Front. Microbiol. 2019, 10, 1619. [ Google Scholar] [ CrossRef] Qureshi, N.; Gu, Q.; Li, P. Whole genome sequence analysis and in vitro probiotic characteristics of a Lactobacillus strain Lactobacillus paracasei ZFM54. J. Appl. Microbiol. 2020, 129, 422–433. [ Google Scholar] [ CrossRef] Zhao, M.; Liu, K.; Zhang, Y.; Li, Y.; Zhou, N.; Li, G. Probiotic characteristics and whole-genome sequence analysis of Pediococcus acidilactici isolated from the feces of adult beagles. Front. Microbiol. 2023, 14, 1179953. [ Google Scholar] [ PubMed] Zhang, X.; R
Integrative Phenotypic and Genomic Analysis Reveals Antimicrobial and Stress-Resistance Mechanisms of Lacticaseibacillus rhamnosus MG0718 as a Promising Probiotic Candidate for Food Applications
