Isolation of Tn1546-like elements in vancomycin-resistant Enterococcus faecium isolated from wood frogs: an emerging risk for zoonotic bacterial infections to humans

Aim: Isolation and characterization of vancomycin-resistant enterococci (VRE), mainly Enterococcus faecium, from the faecal pellet of wood frogs (Rana sylvatica). Methods and Results: The frog VRE isolates were tested for their susceptibility to various antibiotics and were found resistant to ampicillin (Am), chloramphenicol (Cm), erythromycin (Em), gentamicin (Gm), tetracycline (Tc), teicoplanin (Tp) and vancomycin (Vn). The linkage of multiple antibiotic resistances to Em, Tc, Tp and Vn was observed in 84% of resistant Ent. faecium. Inducible antibiotic resistance (MIC >= 512 mu g ml-1) to Vn was also detected in these isolates. PCR analysis revealed the presence of vanA in all strains, and none of the strains were positive for vanB, indicating the existence of vanA phenotype. Furthermore, the PCR-RFLP analysis of the frog vanA amplicon with PstI, BamHI and SphI generated identical restriction patterns similar to Tn1546-like elements found in human VRE isolates. DNA homoduplex analysis also confirmed that vanA from the frog VRE has DNA sequence homology with the vanA of Tn1546-like elements of human and animal isolates. Blastx analysis of frog vanA sequence showed similarities with protein sequences generated from protein database of Vn-resistant Ent. faecium, Baccilus circulans, Paenibacillus apiarius and Oerskovia turbata isolates. Horizontal transfer of Vn resistance was not detected in frog isolates as revealed by filter mating conjugal experiment. Conclusions: In summary, our results demonstrated that wood frogs carry Vn-resistant bacteria, and resistance genes (vanA) are located on Tn1546-like elements. Significance and Impact of the Study: This study highlights a previously less recognized role of amphibians as sentinels for multidrug-resistant bacteria and alerts the public health workers for an emerging risk of zoonotic bacterial infections to humans.