Molecular and Genomic Investigation Unveils Pseudomonas putida as an Emerging Multidrug-Resistant Pathogen Linked to Bovine Clinical Mastitis.

Pseudomonas putida is one of the emerging pathogens responsible causing mastitis in lactating animals. This study investigated the prevalence, antimicrobial resistance (AMR), genetic diversity and virulence factor genes (VFGs) to highlight the pathogenic potentials of P. putida strains isolated from milk, feces and farm soil of bovine clinical mastitis (CM). A total of 110 samples were collected and analyzed, revealing an overall prevalence of P. putida in dairy farms at 40.90%, with specific prevalence rates of 42.22% in milk, 26.67% in feces, and 31.11% in farm soil. In vitro antimicrobial assays demonstrated that 76.0% P. putida isolates exhibited multidrug resistance (MDR, resistance to ≥ 3 antibiotics), particularly showing high resistance to oxacillin, ampicillin, nalidixic acid, and aztreonam. Conversely, P. putida isolates showed the highest susceptibility against imipenem. The genome analysis of three MDR P. putida strains 11CM-M1 (milk), 11CM-F1 (feces) and 11CM-S1 (farm soil), showed a close evolutionary relationship with different strains of Pseudomonas spp. isolated from bovine mastitis milk and feces samples, human stool, and the hospital environment. The assembled genomes of three P. putida strains encoded nine antibiotic resistance genes (ARGs), 36 VFGs, and 367 metabolic subsystems, highlighting a complex functional profile and potential for pathogenicity. The detailed analysis of these ARGs and VFGs demonstrated that P. putida strains employ distinct mechanisms of resistance (e.g., efflux pumps), biofilm formation, and virulence factors, including adhesins, secreted toxins, and lipopolysaccharides, which contribute to their pathogenic potential. Given the lack of reports linking P. putida strains to bovine mastitis in Bangladesh, the increasing trend of AMR, along with the presence of significant ARGs and VFGs in the studied strains, underscores the need for more intensive research, including animal model experiment, to better elucidate the pathogenesis and inform treatment decisions for mastitis in dairy animals.