Competitive interactions between canine-derived lactic acid bacteria and Klebsiella michiganensis: Implications for probiotic potential in canine cystitis.

The Klebsiella oxytoca complex comprises several closely related bacterial species, including Klebsiella michiganensis, for which reports in veterinary medicine remain scarce, particularly in the context of urinary tract infections. This prospective in vitro study aimed to (i) molecularly characterize, using next-generation sequencing (NGS), a K. michiganensis strain isolated from canine cystitis, including its plasmid content and antimicrobial resistance gene repertoire; and (ii) identify, among six host-specific lactic acid bacteria (LAB) recovered from the urogenital tract of healthy dogs, candidate strains exhibiting in vitro antagonism against this pathogen and a probiotic phenotype compatible with future ex vivo and in vivo validation. The in vitro antagonism was performed through five bacterial competition assays: spot-on-the-lawn (SOTL), radial streak (RS), modified cross-streak (MCS), agar well diffusion (AWD), and liquid coculture (LCA) assays. The probiotic properties of LAB strains were assessed through resistance to artificial gastric juice, bile salt tolerance, hydrophobicity, growth capacity, hydrogen peroxide production, antimicrobial susceptibility, complemented by class 1 integron genes screening and KEGG functional profile characterization. NGS analysis of K. michiganensis revealed the presence of IncF and IncR plasmids and 26 resistance-associated genes (6 AMR and 20 stress-response genes). KEGG orthology analysis of the LAB strains revealed strain-specific metabolic KOs in four out of the six isolates. Among the tested strains, Enterococcus faecalis showed greater inhibition in the SOTL assays (p<0.0001), whereas Lacticaseibacillus casei exhibited significantly greater inhibitory activity in the RS (p < 0.05). Although some E. faecalis strains showed limited inhibition in the AWD and MCS assays, both species effectively inhibited the pathogen in the LCA. All LAB strains demonstrated tolerance to gastrointestinal conditions, moderate to high hydrophobicity, and hydrogen peroxide production, with no class 1 integron genes detected. Overall, K. michiganensis was identified for the first time as a cause of canine cystitis, and LAB strains, particularly L. casei, displayed key probiotic traits, supporting its potential for therapeutic application.