Multiplex PCR for detection of bacterial uropathogens in nosocomial samples from Northeastern Mexico: one health-relevant urinary tract pathogens

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, accounting for approximately 150 million cases annually and an estimated 3.8 million cases each year in Mexico. The principal uropathogens are Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, all of which are opportunistic zoonotic bacteria associated with humans, companion animals, livestock, and wildlife. Within the One Health framework, these pathogens and their antimicrobial resistance (AMR) determinants may circulate across healthcare settings, communities, animals, and the environment, highlighting the need for integrated surveillance. Although conventional urine culture remains the diagnostic gold standard, rapid molecular methods can facilitate timely diagnosis and support antimicrobial stewardship. This study analyzed 300 urine samples collected from three hospitals representing different levels of healthcare complexity in Reynosa, Tamaulipas, Mexico. Conventional urine culture and species-specific PCR assays targeting E. coli, K. pneumoniae, and P. mirabilis were performed. Fifty-six samples yielded positive urine cultures, with E. coli identified as the predominant uropathogen (85.7%, 48/56), followed by K. pneumoniae (10.7%, 6/56) and P. mirabilis (3.6%, 2/56). Positive cases were more frequent among females (70%). In addition, a multiplex PCR assay was developed and validated for the simultaneous detection of the three pathogens. The assay was also successfully applied directly to unprocessed urine samples, provided that urine represented no more than 20% of the total PCR reaction volume. The multiplex PCR demonstrated 100% concordance with conventional culture results while substantially reducing diagnostic turnaround time. To our knowledge, this is the first study conducted in Reynosa, Tamaulipas, to integrate conventional microbiological and molecular diagnostics for UTIs within a One Health framework. These findings establish a methodological baseline for regional surveillance of uropathogens at the human–healthcare–environment interface, supporting future cross-border One Health surveillance initiatives.