Clonal diversity, virulence factors and antibiotic resistance genes of Enterococcus faecalis and Klebsiella pneumoniae from urban rats in Malaysia

Peridomestic rats serve as important carriers of zoonotic pathogens, including multidrug-resistant bacteria such as Enterococcus faecalis and Klebsiella pneumoniae . However, information on the clonal diversity, antibiotic resistance, and virulence of these pathogens in wild urban rats remains limited, particularly in tropical settings. This study investigated E. faecalis and K. pneumoniae in urban rats from wet markets across four Malaysian states. A total of 277 rats ( Rattus norvegicus , n = 177; Rattus rattus diardii , n = 100) were trapped, and lung tissues were processed for bacterial isolation. Isolates were identified by 16S rDNA sequencing and examined for antibiotic susceptibility, resistance genes, and virulence factors using PCR. Clonal relationships were determined through multilocus sequence typing (MLST). Nineteen E. faecalis and seven K. pneumoniae isolates were recovered. All E. faecalis isolates carried multiple virulence factors ( ace , asa , efaA , and gelE ), and four tetracycline-resistant isolates possessed tetL , tetM , and tetS . Five novel E. faecalis sequence types (ST730 - ST734) were identified. All K. pneumoniae isolates carried the SHV gene; two also harbored FOX, and two possessed aerobactin and kfu virulence factors. Two novel K. pneumoniae STs (ST2641 and ST2642) were detected. Phylogenetic analyses revealed that both species displayed diverse sequence types scattered across global lineages. These findings suggest that urban rats may act as potential carriers of virulent and antibiotic-resistant E. faecalis and K. pneumoniae . These findings support the inclusion of urban rats in future One Health surveillance programs investigating antimicrobial resistance and virulent bacterial pathogens.