利奈唑胺不敏感肠球菌的耐药机制及分子分型

The resistance mechanism and molecular typing of linezolid-insensitive Enterococcus

目的了解利奈唑胺不敏感肠球菌(linezolid-insensitive Enterococcus,LISE)检出率及分布特点,分析LISE的主要耐药机制和分子分型,为LISE医院感染精准预防与控制提供理论依据。方法回顾性分析2018—2019年福建医科大学附属泉州第一医院临床分离肠球菌属细菌药敏试验数据,对经仪器法检测出的LISE采用肉汤微量稀释法和纸片法进行确认,采用多位点序列分型(MLST)分析LISE菌株的同源性。采用PCR法检测23S rRNA、rplC、rplD、rplV、optrA、poxtA、cfr、cfr(B)利奈唑胺耐药相关基因。结果2018—2019年共分离鉴定肠球菌522株,检测出LISE共44株(均为粪肠球菌),检出率为8.4%(44/522),其中利奈唑胺中介粪肠球菌(linezolid-intermediate Enterococcus faecalis,LIEf)占3.1%(16/522);利奈唑胺耐药粪肠球菌(linezolid-resistant Enterococcus faecalis,LREf)占5.4%(28/522)。利奈唑胺对28株LRE的MIC值为8~16 mg/L。44株LISE分布的科室主要为泌尿外科(43.2%,19/44)、肿瘤科(9.1%,4/44)、老年病科和肝胆外科(均为6.8%,3/44);标本来源主要为尿液52.3%(23/44)、抽出液18.2%(8/44)、分泌物13.6%(6/44)、引流液4.5%(2/44)。44株LISE分为32个不同的ST型,其中ST16是主要型别,占20.5%(9/44)。发现耐药基因阳性的LISE有33株,可分单耐药基因和双耐药基因两类。单耐药基因:optrA阳性为7株,rplD发生突变(C348T、A144G)的有10株。双耐药基因:同时携带optrA、cfr和同时携带optrA、poxtA的LRE各有1株;optrA阳性合并23S rRNA点突变(G2601C)的1株;optrA阳性合并rplC突变(C291T、C369T、T107A)的4株;optrA阳性合并rplD突变的9株。未发现任何耐药基因的LISE有11株。结论LRE菌株的主要耐药机制是携带optrA,该基因可用作耐药性筛选的标志。cfr、poxtA和23S rRNA突变参与耐药,但并不是主要耐药机制。

Objective To investigate the prevalence and distribution of linezolid-insensitive Enterococcus(LISE),and analyze the resistance mechanism and molecular types of LISE for precise prevention and control of LISE hospital infections.Methods The antibiotic resistance patterns of Enterococcus strains were retrospectively reviewed in the period from 2018 to 2019 in Quanzhou Frist Hospital.Kirby-Bauer method and broth microdilution method were used to confirm the identification of the LISE identified by instrument.Multilocus sequence typing(MLST)was used to analyze the homology of LISE strains.The linezolid-related resistance genes including 23S rRNA,rplC,rplD,rplV,optrA,poxtA,cfr,and cfr(B),were detected by polymerase chain reaction(PCR)and DNA sequencing.Results A total of 522 Enterococcus strains were isolated and identified in Quanzhou Frist Hospital in the period from 2018 to 2019,of which 44(8.4%)were identified as LISE(all Enterococcus faecalis).The prevalence of linezolid-intermediate Enterococcus faecalis(LIEfs)and linezolid-resistant Enterococcus faecalis(LREfs)was 3.0%(16/522)and 5.4%(28/522),respectively.The minimum inhibitory concentration(MIC)of linezolid was 8–16 mg/L against the 28 LRE strains.About 43%(19/44)of the LISE strains were isolated from Departments of Urology,followed by Department of Oncology(9%,4/44),Geriatrics(7%,3/44),and Hepatobiliary Surgery(7%,3/44).The 44 LISE strains were isolated from urine(52%,23/44),aspirate(18%,8/44),secretions(14%,6/44),and drainage(5%,2/44).The 44 LISE strains were classified into 32 different ST types by MLST,primarily ST16 type(20.5%,9/44).Relevant single or double drug-resistant genes were detected in 33 LISE strains.Specifically,optrA in 7 strains and rplD mutations(C348T,A144G)in 10 strains;optrA+cfr in one LRE strain,optrA+poxtA in one LRE strain;optrA and 23S rRNA point mutation(G2601C)in another strain;optrA and rplC mutations(C291T,C369T,T107A)in 4 strains,optrA and rplD mutation in 9 strains.None of the relevant genes was detected in the remaining 11 LISE strains.Conclusions The optrA gene is the main mechanism of linezolid resistance in Enterococcus strains,which can be used as a marker for resistance screening.In addition,Cfr gene,poxtA gene,and 23S rRNA gene mutations may also contribute to linezolid resistance.