4株利奈唑胺不敏感粪肠球菌的23S rRNA基因突变分析

doi:10.3969/j.issn.1673-8640.2018.05.018

检验医学 ›› 2018, Vol. 33 ›› Issue (5): 452-456.DOI: 10.3969/j.issn.1673-8640.2018.05.018

4株利奈唑胺不敏感粪肠球菌的23S rRNA基因突变分析

林贺¹, 薛苗², 汪怀周², 黄晓春², 秦阳华²

1.上海交通大学医学院附属瑞金医院北院检验科,上海 201801
2.第二军医大学附属长海医院实验诊断科,上海 200433

收稿日期:2017-05-05 出版日期:2018-05-20 发布日期:2018-05-30
作者简介:null
作者简介：林贺,女,1992年生,学士,主要从事临床检验工作。

通信作者：秦阳华,联系电话：021-31162076。
基金资助:
科技部863计划基金资助项目（2011AA02A119）

Study of 23S rRNA gene mutation in 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis

LIN He¹, XUE Miao², WANG Huaizhou², HUANG Xiaochun², QIN Yanghua²

1. Department of Clinical Laboratory,North Campus,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 201801,China
2. Department of Clinical Laboratory,Changhai Hospital,the Second Military Medical University,Shanghai 200433,China

Received:2017-05-05 Online:2018-05-20 Published:2018-05-30

摘要/Abstract

摘要：

目的研究临床分离的4株利奈唑胺不敏感粪肠球菌的耐药机制。方法采用Vitek 2 Compact全自动微生物分析系统对临床分离的4株粪肠球菌进行鉴定和体外药物敏感性试验,利奈唑胺的最低抑菌浓度（MIC）采用E-test法进行复核。采用聚合酶链反应（PCR）扩增其氯霉素-氟甲砜霉素耐药（cfr）基因、23S rRNA V区和基因组23S rRNA 4个拷贝基因,经测序确定突变情况。结果 4株利奈唑胺不敏感粪肠球菌均未扩增出cfr基因, 23S rRNA V区扩增片段连接T载体随机挑选6克隆测序,未发现利奈唑胺耐药最常见的G2576U突变。进一步分别扩增23S rRNA 4个拷贝基因并测序,仅1株菌存在1拷贝G2576U突变,4株菌均存在U2826C突变,另外还分别存在G2389A、U2363C和A2881G突变。U2826C位于23S rRNA可变区边缘,进一步扩增同期临床分离的利奈唑胺敏感粪肠球菌,测序结果显示均存在U2826C突变。结论临床分离的4株利奈唑胺不敏感粪肠球菌的23S rRNA存在散发突变,是其耐药的主要分子机制。

关键词: 利奈唑胺, 粪肠球菌, 23S rRNA, 耐药突变

Abstract:

Objective To study the drug resistance mechanism of 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis. Methods Phenotype identification and in vitro antimicrobial susceptibility test of the 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis were performed by Vitek 2 Compact microbial analysis system,and the minimum inhibitory concentration （MIC） of linezolid was rechecked by E-test strips. Chloramphenicol-florfenicol resistance （cfr） gene,23S rRNA V region and 4 copies of 23S rRNA were amplified and sequenced to find mutations. Results No cfr gene was found in the 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis,and sequencing 23S rRNA V region did not find G2576U mutation. After further amplification and sequencing of 4 copies of 23S rRNA,only G2576U mutation was found in 1 isolate,and all 4 isolates contained U2826C mutation. G2389A,U2363C and A2881G mutations were also determined. Since U2826C was located at the edge of variable region of 23S rRNA,the sequcening results showed that linezolid non sensitive Enterococcus faecalis harbored U2826C mutation. Conclusions For the 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis 23S rRNA,random mutation might be the main mechanism of drug resistance to linezolid.

Key words: Linezolid, Enterococcus faecalis, 23S rRNA, Drug resistance mutation

中图分类号:

R446.5

林贺, 薛苗, 汪怀周, 黄晓春, 秦阳华. 4株利奈唑胺不敏感粪肠球菌的23S rRNA基因突变分析[J]. 检验医学, 2018, 33(5): 452-456.

LIN He, XUE Miao, WANG Huaizhou, HUANG Xiaochun, QIN Yanghua. Study of 23S rRNA gene mutation in 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis[J]. Laboratory Medicine, 2018, 33(5): 452-456.

图/表 6

参考文献 15

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菌株号	样本类型	MIC（μg/mL）	结果解释
1507490	引流液	4	中介
1512104	引流液	32	耐药
1512160	中段尿	32	耐药
1512163	中段尿	16	耐药

菌株号	样本类型	MIC（μg/mL）	结果解释
1507490	引流液	4	中介
1512104	引流液	32	耐药
1512160	中段尿	32	耐药
1512163	中段尿	16	耐药

基因	前向引物	逆向引物	片段大小（bp）
23S V	GCAGAAGGGAGCTTGACTGCGAG	ACCCAGCAATGCCCTTGGCAG	429
cfr	TGAAGTATAAAGCAGGTTGGGAGTCA	ACCATATAATTGACCACAAGCAGC	746
23S rRNA1	GGCGCTGGTGGGATACTA	GGACGGTTATGAGCCGTC	1 898
23S rRNA2	GGCGCTGGTGGGATACTA	GCGATCTCCTGCGTGAC	1 415
23S rRNA3	GGCGCTGGTGGGATACTA	CCCTTCTTCAAGCTTATC	1 111
23S rRNA4	GGCGCTGGTGGGATACTA	CCACAGTGATTTTGCCCA	1 952

基因	前向引物	逆向引物	片段大小（bp）
23S V	GCAGAAGGGAGCTTGACTGCGAG	ACCCAGCAATGCCCTTGGCAG	429
cfr	TGAAGTATAAAGCAGGTTGGGAGTCA	ACCATATAATTGACCACAAGCAGC	746
23S rRNA1	GGCGCTGGTGGGATACTA	GGACGGTTATGAGCCGTC	1 898
23S rRNA2	GGCGCTGGTGGGATACTA	GCGATCTCCTGCGTGAC	1 415
23S rRNA3	GGCGCTGGTGGGATACTA	CCCTTCTTCAAGCTTATC	1 111
23S rRNA4	GGCGCTGGTGGGATACTA	CCACAGTGATTTTGCCCA	1 952

4株利奈唑胺不敏感粪肠球菌的23S rRNA基因突变分析

Study of 23S rRNA gene mutation in 4 clinical isolates of linezolid non-sensitive Enterococcus faecalis

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