广州地区艰难梭菌耐药检测与耐药机制分析

摘要

目的：了解广州地区腹泻患者中分离的艰难梭菌（ CD）耐药情况及耐药发生的可能机制。方法收集广州3 家医院的腹泻患者粪便标本467 份。PCR 方法直接检测粪便中CD 种特异性tpi 基因，对tpi 基因阳性的粪便标本富集芽孢后，采用厌氧培养法用CCFA 培养基进行CD 的分离培养，然后PCR 鉴定可疑菌株的CD 种特异性tpi 基因；采用Etest 法测定CD 对常用抗菌药物的MIC 值并判定菌株对这些药物的敏感性；对耐药菌株，通过PCR 和基因测序方法检测相关的耐药基因如克林霉素耐药相关基因ermB 及喹诺酮类耐药主要相关DNA 旋转酶基因gyrA 和gyrB 的突变情况。结果 467 份粪便标本共检测出tpi 阳性标本29 份，从中共培养出22 株CD。药敏结果显示，所有菌株对甲硝唑、万古霉素、阿莫西林／克拉维酸、哌拉西林、哌拉西林／他唑巴坦均敏感，未检测到对上述5 种抗菌药物耐药和中介的菌株。测试菌对克林霉素、青霉素、莫西沙星的耐药率分别是90畅9％（20／22）、27畅3％（6／22）和13畅6％（3／22）。所有耐药菌株中，有2 株同时对克林霉素、莫西沙星、青霉素耐药。20 株对克林霉素耐药的CD 中，有40％（8／20）的菌株ermB 基因阳性，3 株莫西沙星耐药菌株中均检出gyrA、gyrB 基因发生突变。结论 广州地区CD 对临床常用抗菌药物耐药率不高，但对克林霉素有较高的耐药率，ermB 基因的存在是CD 对克林霉素耐药的原因之一，而gyrA、gyrB 基因的突变与CD 对喹诺酮类耐药有关。另外，已出现同时耐3种抗菌药物的多重耐药CD 菌株。%Objective To investigate the drug resistance and the possible mechanisms of Clostridium difficile ( CD) on patients with diarrhea in Guangzhou.Methods A total of 467 stool samples were collected from patients with diarrhea from 3 hospitals in Guangzhou.PCR methods was used to detect the CD species-specific tpi gene, so as the CD spores were enriched from the stool specimens with positive gene tpi, innoculated on the CCFA medium, and incubated in anaerobic environment.Suspicious colonies were subcultured and identified by PCR methods.MIC values of isolated CD to common drugs were tested by the Etest method.To the drug-resistance strains, drug-resistance-associated genes, in-cluding clindamycin-resistance-associated gene ermB and quinolone resistance-associated DNA gyrase gene gyrA and gyrB were detected by PCR and sequenced.Results Gene tpi was obtained from 29 of 467 stool specimens, from which 22 CD strains were isolated.Drugs susceptibility test showed that all the 22 strains were sensitive to metronidazole, vanco-mycin, amoxicillin/clavulanic acid, piperacillin and piperacillin/tazobactam, while none of 22 CD strains was resistant or intermediary to the 5 drugs.The drug-resistance rates to clindamycin, penicillin and moxifloxacin were 90.9% ( 20/22), 27.3%(6/22) and 13.6%(3/22), respectively;among which 2 CD strains were resistant to clindamycin, moxi-floxacin and penicillin, presenting as multidrug resistance CD strains.Gene ermB was obtained from 8 of 20 clindamycin-resistance CD strains, with the positive rate of 40% (8/20).The mutations of gene gyrA and gyrB were observed in 3 moxifloxacin-resistance strains.Conclusion There are not prominent drug -resistance of CD to antimicrobial agents commonly used in hospitals in Guangzhou, though there is a high drug-resistance rate to clindamycin.The gene ermB is one of the reasons of drug-resistance to clindamycin, while the mutations of the gene gyrA and gyrB are correlated with quinolone resistance in CD strains.Multidrug resistance CD strains have been found.