Calf Gut Bacteria Show Strain-Specific Power Against Diarrhea-Causing Pathogens

Neonatal calf diarrhea remains one of the most damaging problems in modern dairy farming, driving illness and death in young calves and imposing heavy economic losses on cattle operations worldwide. A new laboratory study has now mapped, in unusual detail, how twenty-two lactic acid bacterial strains originally isolated from the feces of healthy newborn calves stand up against five enteric pathogens in vitro, and has used hierarchical clustering to sort those strains into four groups based on their inhibitory fingerprints. The work, published in Dairy Science and Management, identifies a cluster of fourteen strains with the broadest and strongest antimicrobial activity and singles out several Ligilactobacillus salivarius isolates as the most promising candidates for a multi-strain probiotic adjunct aimed at preventing the disease.

The research team, led by Sachin Kumar and colleagues, tested strains belonging to three species: Ligilactobacillus salivarius, Limosilactobacillus reuteri, and Enterococcus faecalis. All twenty-two had been isolated and characterized in earlier studies from fecal samples of healthy newborn Sahiwal cattle and Murrah buffalo calves, and each had already been identified by 16S rDNA gene sequencing, with sequences deposited in the NCBI database. The indicator pathogens in the assays were reference strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 14028, Salmonella arizonae ATCC 13314, and Bacillus cereus ATCC 14569. These organisms represent the kinds of bacterial pathobionts associated with neonatal calf diarrhea, a condition the authors note can contribute to calf mortality of up to 20 percent within the first three months of age and can diminish net herd profitability by as much as 40 percent.

Two complementary laboratory methods formed the core of the study. In the agar spot assay, each live LAB strain was spotted onto brain-heart infusion agar seeded with roughly one million pathogen cells per milliliter, and the diameter of any clear halo of inhibited pathogen growth around the spot was measured after 48 hours of aerobic incubation at 37 degrees Celsius. In the agar well diffusion assay, the researchers instead used cell-free supernatant, the liquid broth in which each LAB strain had grown for 24 hours, filtered through a 0.22-micrometer membrane to remove the bacteria themselves. Two hundred microliters of this supernatant went into seven-millimeter wells punched in pathogen-seeded agar, and inhibition zones were measured after another day of incubation. All experiments were run in triplicate, and differences between groups were assessed with one-way ANOVA, with p < 0.005 treated as statistically significant.

The agar spot results revealed inhibition zones ranging from zero to 35 millimeters, with dramatic variation from strain to strain and pathogen to pathogen. The strongest effects came from L. salivarius RBL68 against S. typhi, from L. salivarius RBL62 and RBL12 against S. arizonae, and from L. salivarius RBL68 against E. coli. Quantitatively, RBL62 produced halos of 32.83 ± 0.67 millimeters against S. arizonae, RBL12 reached 31.83 ± 1.17 millimeters against the same organism, and RBL68 delivered 31.33 ± 0.67 millimeters against E. coli. Against S. typhi, RBL22 and RBL12 produced halos of 31.83 ± 0.67 and 30.83 ± 0.88 millimeters respectively. The selectivity was just as striking: L. reuteri E7 showed no detectable inhibition of S. arizonae, S. typhi, S. aureus, or B. cereus, while L. salivarius RBL26 failed to inhibit S. arizonae, S. aureus, and E. coli, and RBL38 showed no activity against S. arizonae and S. aureus.

The well diffusion assay, which probes the activity of secreted compounds rather than living cells, told a consistent story. L. salivarius RBL62 produced clear zones of 14.33 ± 0.33 millimeters against S. arizonae, 17.00 ± 0.58 against S. typhi, 14.33 ± 0.88 against S. aureus, 17.67 ± 0.67 against E. coli, and 11.00 ± 0.58 against B. cereus. L. salivarius RBL50 and L. reuteri SW27 also inhibited all five pathogens, with SW27 reaching 17.33 ± 0.88 millimeters against E. coli. Across all strains, RBL68 showed the strongest activity against E. coli at 18.33 millimeters, followed by RBL12 at 18.00 and RBL62 at 17.67, while robust inhibition of S. typhi came from L. salivarius S26 and RBL37, both at 17.33 millimeters. Some strains showed no inhibition at all in this format, including L. salivarius J7 against S. aureus and RBL40 against S. arizonae and E. coli. The authors report that the agreement between the two assays suggests both cellular activity and secreted metabolites contribute to the observed effects.

To bring order to this patchwork of activity, the team subjected the halo diameters from the spot assay to hierarchical clustering using Euclidean distance as the similarity measure and average linkage as the linkage criterion. The analysis resolved the twenty-two strains into four distinct groups. Group A contained a single member, L. reuteri E7, which was effective only against E. coli. Group B held two strains, L. salivarius RBL38 and RBL26, whose activity was selective, targeting mainly S. typhi and E. coli. Group C comprised five strains, L. reuteri S27, L. salivarius J7, L. reuteri S26, L. salivarius RBL27, and L. reuteri S23, which showed broad-spectrum activity against all five pathogens but at lower inhibition levels than the top cluster. Group D, the largest, contained fourteen strains, including L. salivarius RBL62, RBL50, RBL22, RBL12, RBL68, RBL37, and Enterococcus faecalis SW28, and this cluster inhibited every tested pathogen significantly more than strains in the other three groups.

Why do these bacteria inhibit pathogens at all? The authors attribute the effects primarily to the metabolic arsenal of lactic acid bacteria. Fermentation of simple dietary carbohydrates produces lactic acid, dropping the pH of the surrounding medium, and LAB also generate hydrogen peroxide, diacetyl, bacteriocins, reuterin, and a range of organic acids including acetic, formic, succinic, malic, and propionic acids. In their undissociated form, organic acids can penetrate bacterial membranes, causing intracellular acidification and disrupting the transmembrane proton motive force, a mechanism that is pH-dependent because the undissociated fraction increases when the pH falls below the acid’s pKa. Lactate itself can permeabilize the outer membrane of Gram-negative bacteria, altering the structure of its phospholipid component. Consistent with this, the study’s own findings point to the bioactive compounds in cell-free supernatant, rather than the bacterial cells alone, as the major driver of inhibition in the well diffusion format.

Strain identity matters as much as mechanism. The discussion highlights prior work showing that specific L. salivarius strains owe their anti-staphylococcal activity to low pH plus particular proteins, including a LysM-containing peptidoglycan-binding protein and a peptidase M23B protein, and that L. salivarius strains can inhibit carbapenem-resistant E. coli and Klebsiella pneumoniae. L. reuteri strains produce organic acids, ethanol, and reuterin, and the authors infer that their own L. reuteri isolate is a reuterin producer given its significant effects against both Gram-positive and Gram-negative pathogens. Notably, Enterococcus faecalis SW28 inhibited both Gram-positive and Gram-negative bacteria, an effect the authors attribute to bacteriocins, the ribosomally synthesized antimicrobial peptides many enterococci produce. The paper also acknowledges the limitations of bacteriocin-based approaches: these peptides are vulnerable to proteolytic degradation in the digestive system, and problems of poor solubility, adsorption, and uneven dispersion can reduce their effectiveness, whereas organic acids resist proteases, act broadly against Gram-negative bacteria, and dissolve more readily.

The authors are careful about what these results do and do not establish. Every finding reported here comes from in vitro assays on agar plates and in broth, against reference pathogen strains under controlled laboratory conditions. No calves were treated, no gut colonization was measured, and no clinical outcomes were observed. The paper explicitly states that further in vivo research is needed to assess the strains’ efficacy and their potential as supportive treatments for gastrointestinal disorders in young dairy calves. What the study does deliver is a systematic, quantified screening and a clustering framework that sorts candidate probiotics by their antimicrobial profiles, allowing researchers to select strains with complementary or overlapping activity when designing multi-strain formulations. Within that framework, Group D stands out as the most promising cluster, and strains such as L. salivarius RBL68, RBL62, and RBL12 emerge as the leading candidates.

The broader context gives the work its urgency. Sub-therapeutic antibiotics have long been a routine feature of calf nutrition, used to address enteric infections and promote growth, but the escalating emergence of antimicrobial resistance has made alternatives a pressing need for both human and animal health. Probiotics, defined by the FAO and WHO as live microorganisms that confer health benefits when administered in adequate amounts, offer one such alternative, working through competitive exclusion and the synthesis of antimicrobial compounds to curb the colonization and proliferation of enteric pathogens. By grounding strain selection in measured inhibition data and a reproducible clustering method, this study provides a concrete starting point for building targeted multi-strain probiotic formulations, even as the decisive test, whether these calf-derived bacteria can actually prevent diarrhea in living animals, remains to be run.

Subject of Research: In vitro antimicrobial screening and clustering of calf-derived lactic acid bacteria against calf diarrhea pathogens

Article Title: Calf-derived lactic acid bacteria as antimicrobial agents against enteric pathogens: a clustering approach

Article References: Kumar, S., Chauhan, N., Chauhan, T., Tyagi, N., Rizwan, M., & Samanta, A. K. (2025). Calf-derived lactic acid bacteria as antimicrobial agents against enteric pathogens: a clustering approach. Dairy Science and Management, 2(1), Article 13. https://doi.org/10.1186/s44363-025-00014-w

Image Credits: AI Generated

DOI: 10.1186/s44363-025-00014-w

Keywords: lactic acid bacteria, neonatal calf diarrhea, probiotics, Ligilactobacillus salivarius, antimicrobial activity, cluster analysis, enteric pathogens, cell-free supernatant, bacteriocins, organic acids, dairy calves, antimicrobial resistance