A Feline Milk-Drived Pediococcus acidilactici M22 Alleviates Escherichia coli O157:H7 Infection Through Anti-Adhesion, Anti-Inflammation, and Microbiota Modulation

TL;DR

A probiotic from feline milk, Pediococcus acidilactici M22, reduces E. coli O157:H7 infection in mice by blocking adhesion, reducing inflammation, and improving gut health.

Contribution

This study identifies a novel feline milk-derived probiotic effective against E. coli O157:H7 through multiple protective mechanisms.

Findings

M22 reduced body weight loss and disease activity in infected mice.

M22 preserved colon length and reduced inflammation and oxidative stress.

M22 restored gut microbiota diversity and suppressed harmful bacteria.

Abstract

Escherichia coli O157:H7 is a pathogenic bacterium that causes severe intestinal infections characterized by inflammation and disruption of the intestinal barrier. Probiotic lactic acid bacteria (LAB) from milk can support intestinal health and combat enteric pathogens; however, the potential of feline milk-derived LAB against E. coli O157:H7 infection remains unclear. In this study, Pediococcus acidilactici (P. acidilactici) M22, isolated from feline milk, was evaluated for probiotic activity in vitro and in vivo in a C57BL/6 mouse model of Escherichia coli O157:H7 infection. In vitro assays demonstrated that M22 significantly inhibited the adhesion of Escherichia coli O157:H7 to intestinal epithelial cells. For in vivo assessment, C57BL/6 mice were orally administered M22 prior to infection with E. coli O157:H7. Protective effects were evaluated by monitoring body weight loss, colon length, disease activity index (DAI), myeloperoxidase (MPO) activity, cytokine levels, tight junction protein expression, oxidative stress markers, and gut microbiota composition. M22-treated mice exhibited significantly less body weight loss and lower DAI scores than infected controls. M22 also prevented colon shortening, indicating reduced colonic damage. Probiotic treatment attenuated neutrophil infiltration and mucosal inflammation, as evidenced by decreased colonic MPO activity, reduced levels of pro-inflammatory cytokines, and elevated anti-inflammatory IL-10. Additionally, M22 preserved intestinal barrier function by upregulating tight junction proteins and mitigating infection-induced histopathological changes. M22 supplementation enhanced antioxidant defenses in colonic tissue (lower malondialdehyde, higher superoxide dismutase and glutathione), indicating reduced oxidative stress. Furthermore, gut microbiota analysis (16S rRNA sequencing) revealed that M22 counteracted infection-induced dysbiosis, restoring microbial diversity and a healthy composition (enrichment of beneficial commensals and suppression of harmful bacteria). By safeguarding intestinal integrity and homeostasis, M22 emerges as a promising next-generation probiotic for improving intestinal health in companion animals.

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1. Introduction

Gastrointestinal disorders caused by enteric pathogens are a major health concern in companion animals, especially in puppies and kittens during early life [1]. Among these pathogens, enterohemorrhagic Escherichia coli O157:H7 stands out as a notorious foodborne bacterium that produces Shiga toxins, which can damage the intestinal lining [2]. Infection with E. coli O157:H7 can induce severe diarrhea and systemic illness in both humans and animals [3]. During the early stage of socialization, young cats and dogs exhibit strong curiosity and exploratory behaviors toward their surroundings, which markedly increase their exposure to and risk of infection by pathogenic microorganisms [4]. At the same time, their intestinal barrier function and immune systems are not yet fully developed, rendering them particularly susceptible to enteric infections [5]. Severe infections during early life can result in dehydration, growth retardation, and long-term disruption of the gut microbiota, thereby exerting lasting adverse effects on overall health and development [6]. With mounting restrictions on antibiotic use in animal health, there is an urgent demand for alternative strategies to prevent and treat enteric infections in pets [7].

Probiotics, especially lactic acid bacteria (LAB), have emerged as promising candidates for this role [8]. Various Lactobacillus and Bifidobacterium strains are known to enhance mucosal immunity, modulate gut microbiota, and inhibit pathogen colonization [9]. For instance, certain probiotic formulations (e.g., combining Saccharomyces boulardii and P. acidilactici) have been shown to promote intestinal health in cats [10]. Probiotics protect the host through multiple mechanisms. They compete with pathogens for adhesion sites and nutrients, produce antimicrobial compounds (such as organic acids and bacteriocins), and strengthen the intestinal epithelial barrier. By these means, probiotics can effectively prevent pathogenic bacteria from colonizing the gut and causing disease [11].

P. acidilactici is a Gram-positive, homofermentative LAB widely recognized for its probiotic potential due to its robust acid/bile tolerance and ability to survive gastrointestinal transit. Notably, P. acidilactici M22—a strain we originating from feline milk—exhibits strong adhesion and resilience under GI conditions. Based on this context, P. acidilactici M22 emerges as a promising next-generation probiotic strain for enhancing resistance to enteric pathogen infections during early life in cats and dogs. By supporting intestinal barrier integrity and immune maturation at a critical developmental window, M22 may help reduce infection susceptibility and mitigate the adverse consequences of early-life pathogenic challenges, thereby contributing to healthier growth and long-term gut homeostasis in companion animals [12].

In the present study, we evaluated the protective role of P. acidilactici M22 against E. coli O157:H7-induced intestinal infection through both in mice. Specifically, we investigated whether pre-treatment with M22 could inhibit E. coli O157 adhesion to intestinal epithelial cells, attenuate infection-induced inflammation, and restore epithelial barrier function. Our findings provide novel insights into the mechanisms by which this feline-derived probiotic exerts its protective effects and represent a viable probiotic strategy for promoting gut health and suggesting potential relevance for the protection of companion animals against enteric pathogens.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

P. acidilactici M22 was previously isolated from feline milk and preserved in our laboratory culture collection. The strain was maintained in MRS broth (Haibo, Qingdao, China) and incubated at 37 °C under anaerobic conditions [12]. Escherichia coli O157:H7 (ATCC 43895) was obtained from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China) and cultured in LB broth at 37 °C with constant shaking (200 rpm) [13]. For experimental use, bacterial suspensions were collected at the mid-logarithmic growth phase, washed twice with sterile phosphate-buffered saline (PBS, pH 7.4), and adjusted to approximately 1 × 10^8^ CFU/mL according to optical density at 600 nm (OD_600_ ≈ 0.8) [9].

2.2. In Vitro Pathogen Inhibition Assay

The adhesion inhibition of P. acidilactici M22 against E. coli O157:H7 was assessed using differentiated Caco-2 cells (ATCC HTB-37, Manassas, VA, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO_2_ atmosphere [14]. With minor modifications, freshly cultured bacterial suspensions were harvested by centrifugation at 4 °C and 4000 rpm for 5 min and washed three times with sterile phosphate-buffered saline (PBS). After the final wash, the bacterial pellets were resuspended in fluorescein isothiocyanate (FITC) solution that had been equilibrated to room temperature for 30 min and incubated at 37 °C for 2 h in the dark with gentle shaking. Following incubation, the bacteria were centrifuged (4 °C, 4000 rpm) and washed three times with PBS to remove unbound FITC. The labeled bacteria were then resuspended in Dulbecco’s modified Eagle’s medium (DMEM), and the bacterial concentration was adjusted to 2 × 10^8^ CFU/mL. The relative fluorescence intensity (RFU) of the labeled bacteria was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm and recorded as the relative fluorescence intensity before adhesion (R_0_).

2.2.1. Fluorescent Labeling of Pathogenic Bacteria

Escherichia coli O157:H7 (ATCC 43895) was fluorescently labeled according to the same procedure described in Section 2.2 for labeling probiotic candidate strains. The labeled bacteria were used for subsequent adhesion inhibition assays under light-protected conditions.

2.2.2. Preparation of Probiotic Suspensions

P. acidilactici M22 were subcultured three consecutive times in MRS broth to ensure viability. Cells were collected by centrifugation at 4 °C (4000 rpm for 10 min) and subsequently washed three times with sterile phosphate-buffered saline (PBS, pH 7.4), and resuspended in DMEM medium to a final concentration of 2 × 10^8^ CFU/mL.

2.2.3. Competition Assay

Each experimental well contained 0.3 mL of probiotic suspension and 0.3 mL of fluorescently labeled E. coli O157:H7 suspension. The control wells contained 0.3 mL of DMEM medium and 0.3 mL of labeled E. coli O157:H7. All assays were performed in triplicate. The plates were incubated for 2 h at 37 °C in a 5% CO_2_ humidified incubator under dark conditions.

2.2.4. Exclusion Assay

In the exclusion test, 0.6 mL of probiotic suspension was added to each experimental well, whereas control wells received 0.6 mL of DMEM medium. After incubation for 1 h at 37 °C under 5% CO_2_, cells were washed three times with PBS to remove non-adherent bacteria. A fluorescently labeled E. coli O157:H7 suspension (0.6 mL) was introduced into each well, followed by incubation for 1 h in the absence of light.

2.2.5. Displacement Assay

For the displacement assay, 0.6 mL of fluorescently labeled E. coli O157:H7 suspension was first added to each well and incubated for 1 h at 37 °C under 5% CO_2_. After washing with PBS to remove unbound cells, 0.6 mL of probiotic suspension (or DMEM medium for the control) was added and incubated for another 1 h under the same conditions.

2.2.6. Detection and Calculation of Adhesion Inhibition Rate

After incubation, wells were washed three times with PBS and treated with 0.3 mL of trypsin for 5 min to detach adherent cells. The digestion was stopped by adding 0.5 mL of DMEM medium. The relative fluorescence intensity (RFU) of the cell suspension was measured using a microplate reader at the same excitation and emission wavelengths. The inhibition rate of pathogen adhesion (%) was calculated using the following equation:

$$[eqn]$$

where R represents the relative fluorescence intensity of the experimental group and R_0_ represents that of the C group. All assays were conducted in triplicate under dark conditions.

2.3. Animal Experimental Design

30 male C57BL/6 mice (6 weeks old, 18–20 g) were purchased from Jinan Pengyue Laboratory Animal Co., Ltd. (Jinan, China). All procedures were approved by the Institutional Animal Care and Use Committee of Liaocheng University. After a 7-day acclimation, mice were randomly divided into five groups (n = 6 per group): Control (C), model group (MG), and M22 low (L), medium (M), and H groups (10^7^, 10^8^, 10^9^ CFU/mL). Mice received M22 by oral gavage once daily for 14 days and were subsequently challenged with 200 µL of E. coli O157 (1 × 10^8^ CFU/mL) on day 15. M22 was administered by oral gavage at a volume of 200 µL per mouse per day. Bacterial concentrations were standardized based on the optical density (OD_600_) values determined from the established growth curve of P. acidilactici M22 prior to administration. Fresh bacterial suspensions were prepared daily to ensure bacterial viability and consistency. Mice were euthanized 48 h post-infection based on established acute E. coli O157 infection models to capture peak inflammatory responses.

2.4. Sample Collection

At 48 h post-infection, mice were anesthetized, and blood samples were collected from the retro-orbital venous plexus using sterile capillary tubes. Serum was separated by centrifugation at 3000 rpm for 10 min and stored at −80 °C for further analysis.

After euthanasia, the colon tissues were excised, gently flushed with cold PBS to remove luminal contents, and divided into portions for histological examination (fixed in 4% paraformaldehyde), immunofluorescence analysis, and biochemical assays.

2.5. Disease Activity Index (DAI)

The DAI score was calculated daily based on three parameters: body weight loss, stool consistency, and presence of blood in feces, as previously described. Each score ranged from 0 to 4, and the average was used to represent disease severity [9].

2.6. Serum Cytokine Measurement

Serum concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) were determined using commercial ELISA kits (Solarbio, Beijing, China) according to the manufacturer’s protocols [15].

2.7. Myeloperoxidase (MPO) Activity

MPO activity in colon tissue homogenates was measured using a colorimetric MPO activity assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Results were expressed as units per gram of tissue [16].

2.8. H&E Histological Staining

After fixation in 4% paraformaldehyde, colon tissues were paraffin-embedded, cut into 5 µm sections, and subjected to hematoxylin and eosin staining [17]. Histological changes, including epithelial integrity, crypt structure, and inflammatory infiltration, were evaluated under a light microscope (Olympus, Tokyo, Japan).

2.9. Immunofluorescence Analysis

Paraffin-embedded colon sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Sections were incubated overnight at 4 °C with primary antibodies against ZO-1, occludin, and MUC2 (Proteintech, Wuhan, China), followed by fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI, and images were captured using a fluorescence microscope (Leica, Wetzlar, Germany).

2.10. Oxidative Stress Analysis

Colon homogenates were analyzed for malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) using commercial kits (Nanjing Jiancheng Bioengineering Institute, China) in accordance with the manufacturer’s instructions [18].

2.11. 16S rRNA Sequencing of Intestinal Microbiota

Cecal samples were aseptically obtained and processed for 16S rRNA gene sequencing. Bacterial genomic DNA was isolated using the E.Z.N.A.^®^ Soil DNA Kit (Omega, Norcross, GA, USA), and the V3–V4 hypervariable region of the 16S rRNA gene was PCR-amplified with primers 341F and 806R [19]. Sequencing was performed on the Illumina NovaSeq 6000 platform (Majorbio, Shanghai, China). Microbial diversity was analyzed using QIIME2 software (version 2024.8). α-Diversity indices (Chao1, Shannon) and β-diversity (principal coordinate analysis, PCoA) were calculated, and differential taxa were identified using LEfSe [20,21].

2.12. Statistical Analysis

All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. A value of p < 0.05 was considered statistically significant. For body weight and DAI, statistical comparisons were performed at individual time points using one-way ANOVA. In vitro experiments were performed with three independent biological replicates.

3. Results

3.1. M22 Significantly Restrains the Adhesion of E. coli O157 In Vitro

The adhesion inhibition ability of P. acidilactici M22 against Escherichia coli O157 was evaluated using three adhesion models: competition, exclusion, and displacement assays. As shown in Table 1, M22 exhibited distinct inhibition efficiencies under different experimental conditions. In the competition assay, where M22 and E. coli O157 were co-incubated simultaneously, the inhibition rate reached 36.04 ± 0.15%, indicating a strong competitive ability to occupy epithelial binding sites. In the exclusion assay, when epithelial cells were pretreated with M22 before pathogen exposure, the inhibition rate was 25.94 ± 0.23%, suggesting that pre-adhesion of M22 partially prevented pathogen attachment. In the displacement assay, in which M22 was added after the pathogen had already adhered, a 23.37% ± 0.41% reduction in adhesion was observed, demonstrating that M22 could moderately displace previously attached E. coli O157 cells.

3.2. M22 Pretreatment Alleviates Clinical Symptoms in E. coli O157-Infected Mice

Compared with the C group, the MG group showed a pronounced reduction in body weight (Figure 1A, p < 0.01). In contrast, M22 pretreatment attenuated body weight loss in a dose-dependent manner, with a significant effect observed in the H group (p < 0.05). Consistently, the DAI score was markedly increased in the MG group (p < 0.0001), indicating severe disease symptoms (Figure 1B). M22 pretreatment significantly reduced DAI scores, particularly in the M and H groups. As shown in Figure 1C,D, E. coli O157 infection caused a significant shortening of the colon (p < 0.001), while M22 pretreatment effectively preserved colon length, with the H group showing a significant recovery (p < 0.05). In addition, colonic MPO activity was significantly elevated in the MG group (p < 0.01), whereas M22 pretreatment significantly reduced MPO levels (Figure 1E), indicating attenuation of intestinal inflammation. Overall, these results demonstrate that M22 pretreatment effectively alleviates clinical symptoms induced by E. coli O157 infection in mice.

3.3. M22 Pretreatment Modulates Inflammatory Responses and Oxidative Stress in E. coli O157-Infected Mice

As shown in Figure 1F–H, E. coli O157 infection significantly increased the levels of TNF-α, IL-6, and IL-1β in the M group compared with the C group (p < 0.05–0.0001). M22 pretreatment markedly reduced the concentrations of these cytokines in a dose-dependent manner, with the M and H groups showing significant decreases compared with the M group. In contrast, the anti-inflammatory cytokine IL-10 was significantly reduced in the M group (p < 0.05), whereas M22 pretreatment significantly increased IL-10 levels, with the highest levels observed in the H group (Figure 1I). Oxidative stress analysis showed that MDA levels were significantly elevated in the M group (p < 0.001), while M22 pretreatment markedly reduced MDA concentrations (Figure 1J). Meanwhile, the activities of antioxidant enzymes SOD and GSH were significantly decreased following E. coli O157 infection but were effectively restored by M22 pretreatment, particularly in the H group (Figure 1K,L).

3.4. M22 Preserves Intestinal Morphology and Epithelial Integrity

Histopathological scoring revealed severe intestinal damage in the MG group, as evidenced by extensive inflammatory cell infiltration, mucosal destruction, and crypt loss, which are indicated by red arrows in Figure 2A. In contrast, probiotic-treated groups showed dose-dependent attenuation of intestinal injury, with the H group exhibiting near-normal mucosal architecture and minimal histological alterations (Figure 2B).

3.5. M22 Enhances Tight Junction Protein Expression in the Colon

As shown in Figure 3A, the fluorescence intensities of ZO-1 and occludin were markedly reduced in the MG group compared with the C group, indicating compromised tight junction integrity following Escherichia coli O157 infection. In contrast, M22 pretreatment significantly restored the expression and distribution of ZO-1 and occludin in a dose-dependent manner, with more continuous and intense signals observed in the M and H groups. Consistent with these observations, quantitative analysis demonstrated that the fluorescence intensities of ZO-1 and occludin were significantly decreased in the M group but were significantly increased after M22 pretreatment, particularly in the H group (Figure 3C). In addition, immunofluorescence staining of MUC2 showed a marked reduction in mucin expression in the M group compared with the C group. M22 pretreatment significantly enhanced MUC2 expression, indicating improved mucosal barrier function (Figure 3B,C).

3.6. M22 Modulates the Intestinal Microbiota Composition

3.6.1. Alpha Diversity

To evaluate the impact of M22 on the gut microbial diversity of mice challenged with Escherichia coli O157, α-diversity indices including Chao1, ACE, Shannon, and Simpson were analyzed (Figure 4A–D). Infection with E. coli O157 markedly reduced microbial richness, as reflected by significantly lower Chao1 and ACE indices, was observed in the MG group. Significant reductions in the Chao1 and ACE indices were observed after infection, reflecting diminished bacterial richness. Similarly, the Shannon index decreased while the Simpson index also dropped, reflecting a reduction in microbial evenness and overall community diversity.

Pretreatment with P. acidilactici M22 effectively reversed these infection-induced alterations. The M and H M22 groups displayed significantly higher Chao1, ACE, and Shannon indices and an increased Simpson index compared with the O157 group (p < 0.05). These findings suggest that M22 mitigates the loss of microbial richness and diversity induced by E. coli O157 infection, thereby supporting a more stable and balanced intestinal microbiota.

As shown in Figure 4E, all Shannon rarefaction curves gradually reached a plateau, indicating that the sequencing depth was sufficient to cover most of the bacterial diversity in each group. The E. coli O157-infected group (MG) showed a lower Shannon index than the control (C), while pretreatment with P. acidilactici M22 (L, M, H) increased microbial diversity in a dose-dependent manner.

3.6.2. Beta Diversity

Principal coordinate analysis (PCoA) based on Bray–Curtis distance at the phylum level revealed distinct clustering of microbial communities among groups (Figure 4E,F). The E. coli O157-infected group (MG) separated clearly from the C group (C) along the first principal coordinate (PC1, 61.05%), indicating that infection markedly altered the gut microbiota composition. Phylum-level Bray–Curtis PCoA was used to visualize global community shifts, while genus-level analyses were used for biological interpretation. In contrast, the M22-treated groups (L, M, and H) clustered closer to the C group, particularly the H group (H), suggesting that P. acidilactici M22 pretreatment partially restored the microbial structure toward a healthy profile. The ANOSIM test (R = 0.373, p < 0.001) further confirmed significant differences in β-diversity among groups.

3.6.3. Cecal Microbiota Composition Following

As shown in Figure 4G, the gut microbial composition varied markedly among groups. The E. coli O157-infected group (MG) showed a decreased abundance of beneficial genera such as Lactobacillus and Bifidobacterium, while potentially pathogenic taxa including Escherichia–Shigella were enriched. Pretreatment with P. acidilactici M22, especially at medium and high doses, restored the relative abundance of beneficial bacteria and reduced harmful genera, leading to a restoration of the microbial profile toward that observed in the C group.

Phylum-Level Composition Analysis

Across all groups, the gut microbial community was dominated by Firmicutes, Bacteroidota, Actinobacteriota, and Proteobacteria (Figure 4H). E. coli O157 infection (MG group) caused a noticeable shift in microbial structure, characterized by an increased relative abundance of Proteobacteria and a reduction in Firmicutes and Bacteroidota compared with the C group (C). Pretreatment with P. acidilactici M22 restored the microbial balance, especially in the M and H groups (M and H), where the proportions of Firmicutes and Bacteroidota increased while Proteobacteria decreased, resembling the C group pattern.

Genus-Level Composition Analysis

At the genus level (Figure 4I), following E. coli O157 infection, the relative abundance of potentially pathogenic taxa, including Shigella, was significantly increased, accompanied by a decrease in beneficial genera such as Lactobacillus, Muribaculaceae, and Akkermansia. In contrast, M22 pretreatment effectively reversed these alterations. The M and H groups exhibited higher relative abundances of Lactobacillus and Bifidobacterium, along with a marked reduction in Escherichia–Shigella, indicating that P. acidilactici M22 modulated the intestinal microbial composition and promoted the recovery of a healthy gut flora structure.

3.7. LEfSe Analysis and Predicted Functional Profiling of the Gut Microbiome

LEfSe analysis identified distinct bacterial taxa that were significantly enriched in each group based on an LDA score > 2.0 (Figure 5A). The E. coli O157-infected group (MG) was characterized by a higher abundance of Bacteroidota, Muribaculaceae, and Burkholderiales, whereas the C group (C) was dominated by Firmicutes, particularly members of the Lactobacillaceae family such as Lactobacillus and Ligilactobacillus. Pretreatment with P. acidilactici M22 altered the microbial composition in a dose-dependent manner, enriching beneficial taxa including Bacilli, Lactobacillales, and Patescibacteria, which are associated with gut health and anti-inflammatory effects. These results indicate that M22 modulated the intestinal microbiota structure by promoting the growth of probiotic-related taxa and suppressing infection-related bacteria.

Functional prediction of the gut microbiome using PICRUSt2 revealed that the dominant functional categories were amino acid transport and metabolism (E), carbohydrate transport and metabolism (G), energy production and conversion (C), and transcription (K) (Figure 5B). The O157-infected group showed reduced functional abundance in carbohydrate and energy metabolism pathways, whereas M22 pretreatment restored these functions toward control levels. These findings suggest that P. acidilactici M22 not only reshaped the microbial community composition but also enhanced microbial metabolic capacity, supporting intestinal homeostasis after E. coli O157 infection.

4. Discussion

Escherichia coli O157:H7 (EHEC) is a major foodborne pathogen that can adhere tightly to intestinal epithelial cells, disrupt epithelial integrity, and trigger severe enteritis in both humans and animals [22,23]. Adhesion to the intestinal mucosa represents a critical initial step for successful colonization and infection, making it an important target for preventive strategies [24].

To our knowledge, this study is the first to demonstrate that the feline milk-derived probiotic P. acidilactici M22 effectively inhibited E. coli O157 adhesion and alleviated infection-induced intestinal injury. The protective effects of M22 were achieved through multiple coordinated mechanisms, including competitive, exclusion, and displacement inhibition of pathogen adhesion, enhancement of epithelial barrier integrity, modulation of inflammatory responses, and restoration of gut microbiota balance. These findings highlight the potential of P. acidilactici M22 as a promising probiotic candidate for preventing E. coli O157 infection by targeting the early stage of pathogen–host interaction.

The Caco-2 cell model is widely used not only to evaluate the adhesion capacity of probiotic strains but also to rapidly screen for strains capable of preventing or attenuating intestinal infections caused by enteric pathogens such as Escherichia coli and Salmonella Typhimurium [25]. Conventional treatments for enteric infections mainly rely on antibiotics; however, excessive antibiotic use can disrupt the intestinal microbiota and promote the emergence of antibiotic-resistant bacteria. Therefore, identifying probiotic strains with the ability to inhibit pathogen adhesion has become an attractive strategy for developing antibiotic alternatives in the prevention of intestinal diseases [26].

In this study, the inhibitory effect of P. acidilactici M22 on E. coli O157 adhesion to epithelial cells was evaluated through three adhesion models: competition, exclusion, and displacement assays [27]. As shown in Table 1, M22 exerted significant adhesion-inhibitory effects in all three models, with the competition assay showing the highest inhibition rate, followed by displacement and exclusion. This suggests that M22 can effectively compete with E. coli O157 for epithelial binding sites, thereby limiting pathogen attachment at the early stage of infection. Similarly, Wang et al. [28] reported that Lactiplantibacillus plantarum L15 exhibited the strongest inhibitory effect in the competition assay, followed by exclusion and displacement, which is consistent with the findings of the present study. These results indicate that P. acidilactici M22 primarily exerts its anti-adhesion activity through competitive exclusion, occupying epithelial receptors or surface glycoconjugates that would otherwise serve as binding sites for pathogenic bacteria.

MPO activity is a widely used and reliable marker of neutrophil infiltration and acute intestinal inflammation [29]. In the present study, pretreatment with P. acidilactici M22 markedly alleviated E. coli O157-induced clinical symptoms in mice. Infection resulted in significant body weight loss, colon shortening, and elevated MPO activity, all of which are indicative of acute intestinal inflammation. These pathological alterations were substantially mitigated by M22 supplementation, especially at higher doses [30]. The restoration of colon length and reduction in MPO activity suggest that M22 effectively attenuates neutrophil infiltration and local inflammatory responses in the colon. Similar findings have been reported for other lactic acid bacteria, such as Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus, which protect intestinal tissues by preserving mucosal integrity and reducing inflammatory cell recruitment. These results demonstrate that M22 exerts a potent anti-inflammatory effect and contributes to the maintenance of intestinal structural integrity during O157 infection.

Pro- and anti-inflammatory cytokines are central mediators of host immune responses and play a critical role in the initiation, amplification, and resolution of intestinal inflammation [31]. Inflammatory cytokine profiling further confirmed the immunomodulatory effects of P. acidilactici M22. E. coli O157 infection significantly increased serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, while markedly decreasing the anti-inflammatory cytokine IL-10. M22 pretreatment reversed these trends in a dose-dependent manner—suppressing pro-inflammatory cytokines while restoring IL-10 levels to near-control values [32]. These findings indicate that M22 helps rebalance the host immune response by downregulating excessive inflammation and promoting anti-inflammatory pathways. This modulation may be mediated through inhibition of NF-κB activation or enhancement of regulatory T-cell responses, as previously reported for other Pediococcus and Lactobacillus strains [33]. Together, these results suggest that P. acidilactici M22 alleviates E. coli O157-induced intestinal inflammation via both inhibition of inflammatory cytokine overproduction and activation of host anti-inflammatory defenses.

Oxidative stress is one of the major pathological mechanisms underlying intestinal injury caused by pathogenic infection. Excessive accumulation of reactive oxygen species (ROS) leads to lipid peroxidation, disruption of tight junctions, and impaired intestinal barrier function. In this study, E. coli O157 infection caused a significant increase in MDA levels, accompanied by a reduction in antioxidant molecules such as SOD and GSH, indicating severe oxidative imbalance [34]. Pretreatment with P. acidilactici M22 effectively reversed these changes, suggesting its strong antioxidative potential. The observed antioxidant protection may be attributed to several mechanisms: (1) P. acidilactici produces metabolites such as exopolysaccharides and short-chain fatty acids that can scavenge free radicals; (2) M22 may upregulate host antioxidant enzyme systems through the activation of the Nrf2–Keap1 signaling pathway; and (3) by modulating the gut microbiota, M22 reduces oxidative metabolism from pathogenic bacteria, indirectly lowering ROS generation [35]. These findings are consistent with reports that other lactic acid bacteria, such as Lactiplantibacillus plantarum and Limosilactobacillus fermentum, exert similar antioxidative effects in infection and inflammation models. Collectively, these data indicate that P. acidilactici M22 protects the host from E. coli O157-induced oxidative injury by strengthening antioxidant enzyme systems and maintaining redox homeostasis [36].

In fact, our previous study demonstrated that M22 effectively protected mice against Salmonella Typhimurium infection by alleviating intestinal inflammation and oxidative stress [37]. The intestinal barrier is a critical defense against enteric pathogen invasion, relying on the coordinated integrity of epithelial morphology, tight junction structures, and the mucus layer [22]. In the present study, E. coli O157 infection induced pronounced epithelial injury, as evidenced by severe mucosal destruction, crypt loss, and inflammatory cell infiltration, highlighting substantial disruption of barrier integrity. M22 pretreatment markedly alleviated these histopathological alterations in a dose-dependent manner, with high-dose administration nearly restoring normal mucosal architecture, indicating a robust protective effect on epithelial structure. Consistent with these morphological improvements, M22 significantly preserved the expression and continuity of key tight junction proteins ZO-1 and occludin, whose disruption is a hallmark of increased intestinal permeability during pathogenic infection. Furthermore, the restoration of MUC2 expression suggests that M22 not only reinforces intercellular junctions but also enhances the mucus barrier, which serves as the first line of defense against luminal pathogens [38]. Collectively, these findings demonstrate that M22 effectively maintains intestinal barrier integrity by simultaneously protecting epithelial morphology, strengthening tight junction complexes, and promoting mucin production, thereby limiting barrier breakdown and contributing to its preventive efficacy against E. coli O157-induced intestinal injury.

A balanced intestinal microbiota is integral to maintaining host physiological homeostasis and limiting pathogen invasion. Infection with Escherichia coli O157 is known to disrupt this delicate microbial balance, leading to reduced diversity, expansion of pathogenic taxa, and suppression of beneficial bacteria. In the present study, E. coli O157 infection significantly decreased α-diversity indices (Chao1, ACE, Shannon, and Simpson), indicating a loss of microbial richness and evenness. These alterations are consistent with previous findings that pathogenic infection induces dysbiosis characterized by decreased microbial diversity and dominance of opportunistic bacteria [39,40]. Notably, pretreatment with P. acidilactici M22 markedly restored microbial richness and diversity, particularly in the M and H groups, suggesting that M22 helps maintain microbial homeostasis and prevent infection-induced dysbiosis. Similar protective effects on microbial diversity have been observed for other lactic acid bacteria such as Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus, which can stabilize gut ecology under pathogenic or inflammatory stress [41].

β-Diversity analysis further revealed distinct clustering between the E. coli O157-infected and C groups, whereas M22-treated groups, especially the H group, clustered closely with the C group. This pattern suggests that M22 restructured the microbial community toward a healthy profile. At the phylum level, infection increased the relative abundance of Proteobacteria—a phylum often associated with intestinal inflammation—while reducing Firmicutes and Bacteroidota. Pretreatment with M22 reversed these changes by restoring Firmicutes and Bacteroidota proportions and decreasing Proteobacteria, indicating improved microbial stability. At the genus level, E. coli O157 infection enriched pathogenic Escherichia–Shigella and decreased beneficial genera such as Lactobacillus, Muribaculaceae, and Akkermansia. M22 supplementation, particularly at higher doses, significantly increased Lactobacillus and Bifidobacterium abundance while suppressing Escherichia–Shigella, thereby promoting the reestablishment of a balanced and health-associated microbial structure. These findings suggest that P. acidilactici M22 not only protects against infection but also acts as a microbiota-modulating probiotic that favors beneficial taxa and reduces pathogen overgrowth.

Consistent with taxonomic changes, LEfSe analysis identified several bacterial taxa enriched in different groups. In the C group, the predominant taxa included Lactobacillus, Ligilactobacillus, and Bacilli, whereas Bacteroidota and Muribaculaceae were predominant in the infected group [42]. In contrast, M22-treated mice showed enrichment of beneficial taxa such as Lactobacillales and Patescibacteria, which are linked to short-chain fatty acid (SCFA) production and anti-inflammatory properties. These compositional shifts indicate that M22 fosters a beneficial microbial environment conducive to intestinal health and epithelial protection. Functional prediction by PICRUSt2 further revealed that infection impaired carbohydrate and energy metabolism pathways, while M22 pretreatment restored these metabolic functions [43]. Enhanced microbial functions related to amino acid metabolism, carbohydrate utilization, and transcriptional activity may contribute to improved nutrient absorption and intestinal energy balance, further supporting the host’s resistance to infection.

Taken together, these findings demonstrate that P. acidilactici M22 alleviates E. coli O157-induced intestinal dysbiosis by restoring microbial richness and diversity, reshaping the taxonomic structure, enriching beneficial bacteria, and enhancing microbial metabolic potential. The capacity of M22 to stabilize the gut microbiota and strengthen metabolic resilience may represent a key mechanism underlying its probiotic protective effects against enteric infections.

5. Conclusions

In summary, this study demonstrates that the feline milk-derived P. acidilactici M22 provides effective prophylactic protection against Escherichia coli O157:H7-induced intestinal infection in a C57BL/6 mouse model. M22 pretreatment markedly alleviated clinical symptoms, reduced intestinal inflammation and oxidative stress, preserved epithelial morphology and barrier integrity, and mitigated infection-associated gut microbiota dysbiosis. These protective effects are mediated through coordinated inhibition of pathogen adhesion, modulation of host immune responses, reinforcement of the intestinal barrier, and restoration of microbial homeostasis. Collectively, our findings identify P. acidilactici M22 as a promising next-generation probiotic and provide a solid scientific basis for its development as an antibiotic-alternative strategy to promote intestinal health and prevent enteric infections, particularly in companion animals.