Begum Yasmin A, Talukder K A, Azmi Ishrat J, Shahnaij Mohammad, Sheikh A, Sharmin Salma, Svennerholm A-M, Qadri Firdausi
International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh.
Molecular Microbiology and Microbial pathogenesis program, Division of Biology and Biomedical Sciences, Washington University in St. Louis, MO, United States of America.
PLoS One. 2016 Jul 18;11(7):e0157415. doi: 10.1371/journal.pone.0157415. eCollection 2016.
Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh.
A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE).
Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 μg/ml and 32μg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains.
Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.
产肠毒素大肠杆菌（ETEC）是导致婴幼儿以及前往ETEC流行国家的旅行者发生急性水样腹泻的常见细菌感染病因。环丙沙星是一种广谱抗菌剂，目前用于治疗腹泻。本研究旨在对从孟加拉国腹泻患者中分离出的耐环丙沙星ETEC菌株进行特征分析。
2005年至2009年期间，对达卡国际腹泻病研究中心医院腹泻患者的8580份粪便标本进行了ETEC筛查。采用聚合酶链反应（PCR）和神经节苷脂GM1-酶联免疫吸附测定（ELISA）检测ETEC的不耐热（LT）和耐热（ST）毒素。检测常用抗生素的药敏情况以及萘啶酸、环丙沙星和阿奇霉素的最低抑菌浓度（MIC）。对具有代表性的耐环丙沙星菌株进行DNA测序，以分析gyrA、gyrB、parC和parE喹诺酮耐药决定区的突变情况。采用PCR检测质粒介导的环丙沙星耐药基因qnr。通过脉冲场凝胶电泳（PFGE）确定耐环丙沙星（CipR）和环丙沙星敏感（CipS）菌株之间的克隆变异。
在鉴定出的1067株（12%）ETEC分离株中，42%产生LT/ST，仅28%产生ST，30%仅产生LT。通过斑点印迹免疫测定法确定，49%（n = 523）的ETEC菌株表达了13种检测的定植因子（CFs）中的一种或多种。ETEC菌株的抗生素耐药情况如下：氨苄西林66%、阿奇霉素27%、环丙沙星27%、头孢曲松13%、复方新诺明46%、强力霉素44%、红霉素96%、萘啶酸83%、诺氟沙星27%、链霉素48%和四环素42%。耐环丙沙星率从2005年的13%增至2009年的34%。所有菌株对美西林均不耐药。具有代表性的CipR菌株的萘啶酸和环丙沙星MIC分别为256μg/ml和32μg/ml。在耐萘啶酸的ETEC菌株中观察到gyrA的单个突变（Ser83-Leu）。相比之下，在耐环丙沙星菌株中发现了gyrA的双重突变（Ser83-Leu，Asp87-Asn）和parC的单个突变（Glu84-Ly）。在耐萘啶酸或耐环丙沙星菌株中均未发现gyrB突变。耐环丙沙星菌株中未发现qnr基因呈阳性。通过PFGE分析，在CF阳性和CF阴性ETEC菌株的所有耐环丙沙星菌株中均鉴定出不同的克隆。
耐环丙沙星ETEC菌株的出现给当前ETEC腹泻的治疗策略带来了重大挑战。
