结核分枝杆菌对氨基糖苷类药物耐药相关基因突变特征分析
车洋, 杨天池, 平国华, 林律
宁波市疾病预防控制中心,宁波 315010

作者简介:Email: chey@nbcdc.org.cn

摘要

目的 分析宁波地区耐多药结核分枝杆菌(Multiple drug-resistant tuberculosis, MDR-TB)不同基因型构成及氨基糖苷类药物耐药相关基因突变特征,为本地区耐多药结核病防控提供科学依据。方法 宁波市疾病预防控制中心2014-2016年耐药监测组共收集106例MDR-TB临床分离株,对其进行3种氨基糖苷类药物卡那霉素(Km)、阿米卡星(Am)、卷曲霉素(Cm)耐药检测。应用RD105缺失基因检测法鉴定北京基因型及非北京基因型菌株。采用DNA直接测序法对所有MDR-TB菌株氨基糖苷类药物耐药相关基因 rrs tlyA eis gidB进行测序分析。结果 106株MDR-TB菌株中,北京基因型83株78.3%(83/106),非北京基因型23株(21.7%,23/106)。耐氨基糖苷类药物的菌株中北京基因型10株12.0%(10/83),非北京基因型0株0.0%(0/23)。10例耐氨基糖苷类药物的耐多药菌株中有4株发生耐药基因突变,突变率为40.0%(4/10),96例对氨基糖苷类药物全敏感的耐多药菌株中有24株发生耐药基因突变,突变率为26.7%(24/96),差异无统计学意义(χ2=1.048, P>0.05)。结论 宁波地区MDR-TB中北京基因型与氨基糖苷类药物耐药相关,目前已知的耐药相关基因突变不能完全解释结核分枝杆菌对氨基糖苷类药物耐药的原因。

关键词: 结核分枝杆菌; 抗药性; 多种; 细菌; 基因型
中图分类号:R378 文献标志码:A 文章编号:1002-2694(2018)02-0144-06
Second-line injectable drugs resistance-associated mutation among multi-drug resistant tuberculosis strains in Ningbo, China
CHE Yang, YANG Tian-chi, PING Guo-hua, LIN Lv
Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010, China
Abstract

We identified the Beijing family strains of multiple drug-resistant tuberculosis and find out the distribution of second-line injectable drugs resistance-associated nucleotide alteration among the MDR strains in Ningbo. The 106 MDR isolates were selected from the first drug resistant survey in Ningbo during 2014 and 2016. The conventional drug susceptibility testing was used to detect the drug-resistant profiles against 3 second-line injectable drugs (kanamycin, amikacin, capreomycin). The RD105 deletion-targeted multiplex PCR method was used to distinguish the genotype among 106 MDR strains. The gene mutations of second-line injectable drugs resistance-associated among MDR-TB strains were detected by direct DNA sequencing. Results showed that out of the 106 MDR isolates, 83(78.3%, 83/106) belonged to Beijing genotype, while the other 23(21.7%, 23/106) were non-Beijing genotype. There were 10 strains with second-line injectable drugs resistance in 83 Beijing genotype MDR strains and there were no strains with second-line injectable drugs resistance in 23 non-Beijing genotype MDR strains. The Beijing MDR strains had significantly higher proportions of second-line injectable drugs resistance than non-Beijing strains. There were 4 with mutations in 10 MDR-TB with second-line injectable drugs resistance and there were 24 with mutations in 96 MDR-TB without second-line injectable drugs resistance (χ2=1.048, P>0.05). Beijing genotype MDR strains revealed a significant association with second-line injectable drugs resistance. The mechanism of second-line injectable drugs resistance in MDR-TB is mainly in no connection with the mutation of the genes.

Key words: Mycobacterium tuberculosis; drug resistance; multiple; bacterial; genotypet

中国是仅次于印度和印尼的结核病高负担国家, 耐多药肺结核疫情较为严峻[1]。卡那霉素、阿米卡星及卷曲霉素3种氨基糖苷类药物是治疗耐多药肺结核的核心药物, 作用机制都是与结核分枝杆菌核糖体结合, 阻碍细菌蛋白质合成。有研究显示, 结核菌的rrstlyAeisgidB基因突变与氨基糖苷类药物耐药相关[2]。国内耐多药结核分枝杆菌的主要基因型是北京基因型, 但是北京基因型是否与耐药相关一直存在争议[3, 4, 5]。本研究通过RD105缺失基因检测法[6]鉴定宁波地区耐多药结核分枝杆菌临床分离株基因型, 并对氨基糖苷类药物耐药相关基因进行突变特征分析, 为本地区耐多药肺结核的有效防控提供科学依据。

1 材料与方法
1.1 材料

1.1.1 菌株来源 用于检测的106例耐多药结核分枝杆菌临床分离株来源于宁波地区11个县(市)、区2014-2016年耐药监测期间收集的痰培养阳性菌株, 经菌种鉴定及一线抗结核药物耐药检测确定为同时耐异烟肼和利福平的耐多药临床分离株。结核分枝杆菌H37RV标准株来源于中国疾病预防控制中心。

1.1.2 试剂来源 分离培养、菌种鉴定及药敏试验培养基均购自珠海贝索生物技术有限公司。结核分枝杆菌DNA提取、PCR扩增及基因测序相关试剂及技术服务购自上海桑尼生物科技有限公司。

1.2 方法

1.2.1 菌株的分离培养、菌种鉴定 按照《结核病诊断实验室检验规程》[7]进行。药物敏感性检测采用WHO/IUATLD《耐药监测指南》推荐的1%比例法对耐多药结核分枝杆菌菌株进行卡那霉素(Km)、阿米卡星(Am)、卷曲霉素(Cm)3种氨基糖苷类药物的耐药性检测, 含药培养基中药物终浓度分别为Km 30.0 μ g/mL、Am 30.0 μ g/mL、Cm 40.0 μ g/mL。耐药百分比=(含药培养基上生长的菌落数/对照培养基上生长的菌落数)× 100%, 以耐药百分比大于1%定义为耐药。

1.2.2 结核分枝杆菌基因组DNA制备 取一接种环L-J培养基上生长状态良好菌落溶于400 μ L TE(PH 8.0)中, 震荡混匀后沸水浴15 min, 12 000 g/min离心3 min取上清。

1.2.3 北京基因型的鉴定 根据北京基因型菌株RD105片段缺失特点, 参照Chen[6]建立的检测方法进行检测。

1.2.4 氨基糖苷类药物耐药相关基因PCR扩增和测序 根据结核分枝杆菌H37RV标准株rrs(GeneID2700429)、tlyA(GeneID885396)、eis(GeneID885903)、gidB(GeneID886243)基因通过Primer Premier 5.0设计引物, 见表1。采用20 μ L PCR反应体系:模板1 μ L(50ng), 2× Taq PCR MasterMix 10 μ L(TIANGEN), Forward Primer (20mmol/L) 1 μ L, Reverse Primer (20mmol/L) 1 μ L, ddH2O 7 μ L。反应条件:95 ℃ 5 min, 95 ℃ 30s, 57 ℃ 30s, 72 ℃ 40 s, 30次循环, 72 ℃ 3 min。

1.2.5 统计学分析 应用Seqman pro 7.1(version 7.1, DNA star Lasergene, Inc. USA)进行基因测序数据分析。参比序列为H37RV标准株DNA序列。采用SPSS16.0对北京基因型和非北京基因型菌株的氨基糖苷类药物耐药率、人群分布特征及耐药基因突变情况进行χ 2检验, 以P< 0.05为差异有统计学意义。

表1 耐药基因引物序列及其扩增片段 Tab.1 Primer sequence of genes with drug resistance
2 结 果
2.1 不同基因型耐多药菌株耐药情况及人群特征分布

106株耐多药结核分枝杆菌中, 有83株78.3%(83/106)属于北京基因型, 有23株21.7%(23/106)属于非北京基因型。83株北京基因型菌株中, 有10株耐氨基糖苷类药物12.0%(10/83); 23株非北京基因型菌株对氨基糖苷类药物全部敏感, 见表2

表2 不同基因型耐多药结核分枝杆菌氨基糖苷类药物耐药情况 Tab.2 Distribution of Beijing genotype with second-line injectable drugs resistance among MDR-TB

治疗分类在不同基因型耐多药菌株中差异有统计学意义(χ 2=4.318, P< 0.05), 不同基因型耐多药菌株人群分布特征, 见表3

表3 不同基因型耐多药结核分枝杆菌人群分布特征 Tab.3 Characteristics of population among MDR-TB
2.2 氨基糖苷类药物耐药相关基因突变特征

10株耐氨基糖苷类药物的耐多药菌株中有4株发生耐药相关基因突变, 突变率为40.0%, 96株对氨基糖苷类药物敏感的耐多药菌株中有24株发生耐药相关基因突变, 突变率为26.7%, 差异无统计学意义(χ 2=1.048, P> 0.05), 见表4。96株对氨基糖苷类药物敏感的耐多药菌株中有5株发生rrs基因突变, 突变类型为1株A1401G及4株A514C; 有12株发生tlyA基因突变, 突变类型均为T708G; 有2株发生eis基因突变, 突变类型为1株发生A10G、C12T、T14A、G15C、T37C多位点联合突变, 1株发生A10T、C12T联合突变; 有5株发生gidB基因突变, 突变类型为4株发生115位缺失C碱基, 1株发生A208C突变。

表4 氨基糖苷类药物耐药相关基因突变特征 Tab.4 Characteristics of gene mutations with second-line injectable drugs resistance among MDR-TB
3 讨 论

北京基因型结核分枝杆菌是全球广泛分布及流行的优势菌群, 且是我国耐多药结核分枝杆菌主要基因型[3, 4, 5]目前针对北京基因型的流行与耐药结核病传播之间关系的研究存在争议, 有的研究认为[8, 9], 北京基因型菌株易对抗结核药物产生耐药, 耐药因素间接促进了该基因型的流行。但部分研究显示[10, 11, 12, 13], 北京基因型与非北京基因型菌株在耐药性方面无差异。本次研究表明纳入研究的耐氨基糖苷类药物的耐多药结核分枝杆菌均为北京基因型, 提示北京基因型这一因素可能与本地区耐多药菌株对氨基糖苷类药物耐药有关。

有研究显示, 编码16SrRNA的rrs基因突变与氨基糖苷类药物的耐药有关, 且rrs基因1401位A-G的突变是氨基糖苷类药物3种药物之间交叉耐药的分子机制之一[14, 15]。本次研究表明, 同时耐卡那霉素与阿米卡星的5株耐多药菌株中有4株发生了1401位A-G的突变80.0%(4/5), 在氨基糖苷类药物敏感的耐多药菌株中发生了5株rrs基因突变5.2%(5/96), 差异有统计学意义(χ 2=32.752, P< 0.05), 与现有研究相符[16, 17]

gidB基因广泛存在于细菌基因组中, 其编码蛋白是一种rRNA甲基转移酶, 其靶点16S rRNA的G527正常甲基化对于氨基糖苷类药物与16S rRNA的稳定结合起到至关重要的作用。Okamoto[18]研究发现gidB基因突变会导致16S rRNA的530环不能得到修饰而引起氨基糖苷类药物耐药。gidB基因突变类型较为多样, 存在点突变, 缺失, 插入等[19, 20]。本研究显示, 耐氨基糖苷类药物的耐多药结核分枝杆菌未发生gidB基因突变, 相反在对氨基糖苷类药物敏感的菌株中存在gidB基因缺失和点突变情况。因此, gidB基因突变与氨基糖苷类药物的耐药关系值得商榷。同时由于gidB基因突变与低水平的链霉素耐药有关[21], gidB基因突变的这部分菌株可能存在低水平的链霉素耐药。

结核分枝杆菌增强胞内存活基因(enhanced invracellular survival, eis)及其编码蛋白EIS(乙酰转移酶家族成员)可增强结核分枝杆菌在巨噬细胞内的存活能力。有研究显示, eis基因启动子区域突变与氨基糖苷类药物低水平耐药有关[2]。本次研究结果显示, 耐氨基糖苷类药物的耐多药结核分枝杆菌中未发生eis基因启动子区域突变, 相反在对氨基糖苷类药物敏感的耐多药菌株中有发生突变, 提示eis基因启动子区域突变与氨基糖苷类药物耐药之间关系尚不明确。

tlyA基因的开放阅读框(ORF)内包含一个具有甲基转移酶活性的功能基团, 在结核分枝杆菌rRNA甲基化过程中起到促进rRNA成熟, 促进药物转运的作用。有研究显示, 该基因发生断裂、缺失或突变, 则细菌会对药物产生耐药性, 且结核分枝杆菌该基因突变主要为点突变[22, 23]。本研究表明, 耐氨基糖苷类药物的菌株中仅有一株发生tlyA基因突变, 且是和rrs基因的联合突变, 在对氨基糖苷类药物敏感的菌株中却有12株发生了tlyA基因突变, 提示tlyA基因突变与氨基糖苷类药物的耐药性关系无特异性。

综上所述, 宁波地区MDR-TB中北京基因型与氨基糖苷类药物耐药相关, 目前已知的耐药相关基因突变不能完全解释结核分枝杆菌对氨基糖苷类药物耐药的原因, 有关分子机制还需进一步研究。

编辑:李友松

The authors have declared that no competing interests exist.

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