Biavasco F, Miele A, Vignaroli C, Manso E, Lupidi R, Varaldo P E
Institute of Microbiology, University of Ancona Medical School, Italy.
Microb Drug Resist. 1996 Summer;2(2):231-7. doi: 10.1089/mdr.1996.2.231.
Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.
尽管作为医院病原体的耐万古霉素肠球菌（VRE）越来越受到关注，尤其是在美国，但在意大利，对于大多数诊断实验室来说，VRE仍然是一种不常见且偶尔才会遇到的情况。我们报告了意大利首次报道的医院内VRE暴发的基因型特征。进行了一些实验，包括质粒分析和脉冲场凝胶电泳（PFGE）检测，旨在研究VRE分离株的遗传相关性。其他基于杂交和聚合酶链反应（PCR）检测的实验旨在鉴定万古霉素耐药决定因素。在6个月的时间里，在一个神经外科重症监护病房中，从8名患者（均曾早期接受糖肽类抗生素治疗）身上分离出21株VRE，所有菌株均被鉴定为粪肠球菌。所有分离株具有相同的生化特征和抗生素敏感性模式，包括对氨基糖苷类高水平耐药，对万古霉素和替考拉宁的最低抑菌浓度（MIC）分别为256和128微克/毫升。在21株VRE分离株中，除最后一株外，其余所有菌株均检测到三种质粒，其中一种与vanA探针强烈杂交。该簇的最后一株分离株缺少三种质粒中最小的一种。用几种核酸内切酶消化质粒DNA后获得了相似的限制性图谱，仅在第一株和最后一株分离株中发现了微小差异。通过PFGE分析基因组DNA限制性片段模式证实，所报告的VRE分离株簇是由单一的医院内粪肠球菌菌株引起的，尽管在暴发开始和结束时质粒DNA有一些变化。同期从同一重症监护病房住院患者中分离出的对万古霉素敏感的粪肠球菌菌株产生了完全不同的PFGE图谱。用vanA和vanS - vanH探针进行的杂交实验以及使用14对针对vanA簇基因（vanR、vanS、vanH、vanA和vanY）、orf1、orf2、vanB和vanC的PCR引物对进行的DNA扩增检测表明，所有流行的VRE分离株中耐药决定因素的组织方式相同。这种组织方式似乎与VanA原型菌株屎肠球菌BM4147中描述的Tn1546相同。
