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.

中文翻译：

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vanM型粪肠球菌中的万古霉素异抗性。

尽管万古霉素异抗性存在于肠球菌中，但使用常规方法不易检测到，并且机制尚不清楚。在这项研究中，我们表征了临床vanM型粪肠球菌菌株中万古霉素异抗性的分子机制。最初的粪肠球菌分离株hVREm7通过肉汤微稀释和Etest对万古霉素敏感。但是，在Etest抑制区内存在耐万古霉素的亚克隆。对hVREm7进行了三次传代，以消除污染的可能性。选择了hVREm7和三个抗性变异体以研究异抗性机制。序列分析表明，所有四个菌株均含有未改变的vanM簇。Southern印迹分析表明，vanM在三种变体中都存在于染色体外和染色体外，而仅存在于hVREm7中。的大小vanM在三个变种荷瘤外DNA片段明显高于hVREm7较大，表明的变化vanM基因扩增在变体。一致地，变异株VREm7-1中vanM拷贝数和表达水平增加。这些结果表明，通过尚未确定的机制部分或完全扩增/转移了vanM基因簇，导致vanM的拷贝数增加和表达增强。基因，该基因向万古霉素heteroresistant转化屎肠球菌到高级别万古霉素抗性变体。