Shariati A, Azimi T, Ardebili A, Chirani A S, Bahramian A, Pormohammad A, Sadredinamin M, Erfanimanesh S, Bostanghadiri N, Shams S, Hashemi A
Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
New Microbes New Infect. 2017 Dec 1;21:75-80. doi: 10.1016/j.nmni.2017.10.013. eCollection 2018 Jan.
In this study, we report the insertion sequence IS21 in the D porin gene of carbapenem-resistant isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of and efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS-PAGE. The genetic relationship between the isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of harboured the IS21 insertion element in the D gene. The extended-spectrum β-lactamase-encoding gene in IS21-carrying isolates was . PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The A, A, B and A genes were positive in all IS21 harbouring isolates. The relative expression levels of the X, B, T and D genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain PAO1. Evaluation of the outer membrane protein by SDS-PAGE suggested that D was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing IS21 were shown to be clonally related. The present study describes a novel molecular mechanism, IS21 insertion of the D gene, associated with carbapenem resistance in clinical isolates.
在本研究中，我们报告了在伊朗德黑兰烧伤患者的耐碳青霉烯分离株的D孔蛋白基因中发现插入序列IS21。对分离株进行了抗生素敏感性试验。评估了金属β-内酰胺酶（MBL）和碳青霉烯酶的产生情况，并通过PCR和测序方法研究了β-内酰胺酶编码基因和氨基糖苷修饰酶基因。通过实时PCR评估了外排泵基因的mRNA转录水平。通过SDS-PAGE确定外膜蛋白谱。通过随机扩增多态性DNA PCR评估分离株之间的遗传关系。总体而言，10.52%（10/95）的临床分离株在D基因中携带IS21插入元件。携带IS21的分离株中编码超广谱β-内酰胺酶的基因是。针对MBL和碳青霉烯酶编码基因的PCR检测在所有十个分离株中也均为阴性。在所有携带IS21的分离株中，A、A、B和A基因均为阳性。十个分离株中X、B、T和D基因的相对表达水平分别为0.1至1.4倍、1.1至3.68倍、0.3至8.22倍和1.7至35.17倍。十个分离株中该基因的相对表达水平为0.57至35.01倍，远高于对照菌株PAO1。通过SDS-PAGE对外膜蛋白的评估表明，所有分离株产生的D水平都非常低。使用随机扩增多态性DNA PCR基因分型，十个含有IS21的分离株中有八个显示出克隆相关性。本研究描述了一种与临床分离株耐碳青霉烯相关的新分子机制，即D基因的IS21插入。
