Purpose

To investigate the occurrence of vancomycin-variable enterococci (VVE) in a hospital in central Italy.

Methods

vanA positive but vancomycin-susceptible Enterococcus faecium isolates (VVE-S) were characterized by antibiotic susceptibility tests, molecular typing (PFGE and MLST), and WGS approach. The reversion of VVE-S to a resistant phenotype was assessed by exposure to increasing vancomycin concentrations, and the revertant isolates were used in filter mating experiments. qPCR was used to analyze the plasmid copy number.

Results

Eleven putative VVE-S were selected. WGS revealed two categories of vanA cluster plasmid located: the first type showed the lack of vanR, the deletion of vanS, and an intact vanH/vanA/vanX cluster; the second type was devoid of both vanR and vanS and showed a deletion of 544-bp at the 5′-end of the vanH. Strains (n = 7) carrying the first type of vanA cluster were considered VVE-S and were able to regain a resistance phenotype (VVE-R) in the presence of vancomycin, due to a 44-bp deletion in the promoter region of vanH/vanA/vanX, causing its constitutive expression. VVE-R strains were not able to transfer resistance by conjugation, and the resistance phenotype was unstable: after 11 days of growth without selective pressure, the revertants were still resistant but showed a lower vancomycin MIC. A higher plasmid copy number in the revertant strains was probably related to the resistance phenotype.

Conclusion

We highlight the importance of VVE transition to VRE under vancomycin therapy resulting in a potential failure treatment. We also report the first-time identification of VVE-S isolates pstS-null belonging to ST1478.

中文翻译：

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意大利万古霉素变异屎肠球菌临床分离株的遗传分析

目的

调查意大利中部一家医院万古霉素变异肠球菌 (VVE) 的发生情况。

方法

通过抗生素敏感性试验、分子分型（PFGE 和 MLST）和全基因组测序方法对vanA 阳性但万古霉素敏感的屎肠球菌分离株 (VVE-S) 进行了表征。通过暴露于增加的万古霉素浓度来评估VVE-S向抗性表型的回复，并将回复分离株用于过滤交配实验。 qPCR用于分析质粒拷贝数。

结果

选择了 11 个假定的 VVE-S。 WGS显示两类vanA簇质粒定位：第一类显示vanR的缺失、 vanS的缺失以及完整的vanH / vanA / vanX簇；第二种类型缺乏vanR和vanS ，并且在vanH的 5' 端显示 544-bp 的缺失。 携带第一种vanA簇的菌株 ( n = 7) 被认为是 VVE-S，并且由于vanH启动子区域的 44 bp 缺失，能够在万古霉素存在下重新获得抗性表型 (VVE-R) /vanA/vanX，导致其组成型表达。 VVE-R菌株不能通过接合转移耐药性，并且耐药表型不稳定：在没有选择压力的情况下生长11天后，回复体仍然具有耐药性，但显示出较低的万古霉素MIC。回复体菌株中较高的质粒拷贝数可能与抗性表型有关。

结论

我们强调万古霉素治疗下 VVE 过渡到 VRE 的重要性，这可能导致治疗失败。我们还首次鉴定了属于 ST1478的 VVE-S 分离株pstS -null。