Although vancomycin heteroresistance exists in enterococci, it is not easily detected using routine methods and the mechanism is not clearly understood. In this study, we characterized the molecular mechanism underlying vancomycin heteroresistance in a clinical vanM-type Enterococcus faecium strain. The original E. faecium isolate, hVREm7, was susceptible to vancomycin by broth microdilution and Etest. However, vancomycin-resistant subcolonies were present within the Etest zone of inhibition. Three passages of hVREm7 were carried out to eliminate the possibility of contamination. hVREm7 and three resistant variants were selected to study the heteroresistance mechanism. Sequence analysis revealed that all four strains contained an unaltered vanM cluster. Southern blot analysis showed that vanM was present both chromosomally and extrachromosomally in the three variants, but only extrachromosomally in hVREm7. The size of the vanM-bearing extrachromosomal DNA fragments in the three variants was larger than that in hVREm7, indicating a variation of vanM gene amplification in the variants. Consistently, vanM copy number and expression level were increased in variant strain VREm7-1. These results suggest that partial or complete amplification/transfer of the vanM gene cluster by an as-yet-unidentified mechanism leads to increased copy number and augmented expression of the vanM gene, which contribute to the transformation of vancomycin-heteroresistant E. faecium into high-level vancomycin-resistant variants.
Keywords: Enterococcus faecium; copy number; heteroresistance; vanM; vancomycin.