Pfennigwerth Niels, Lange Felix, Belmar Campos Cristina, Hentschke Moritz, Gatermann Sören G, Kaase Martin
German National Reference Laboratory for Multidrug-Resistant Gram-negative Bacteria, Department of Medical Microbiology, Ruhr-University Bochum, Universitätsstraße 150, Bochum 44801, Germany.
Institute of Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany.
J Antimicrob Chemother. 2017 Apr 1;72(4):1068-1073. doi: 10.1093/jac/dkw554.
To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.
The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat .
P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem.
The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .
对一株多重耐药铜绿假单胞菌临床分离株中发现的新型B1类金属β-内酰胺酶（MBL）进行特性分析。
铜绿假单胞菌NRZ-03096于2012年从德国北部一名住院患者的肛门拭子中分离得到，对碳青霉烯类药物表现出高最低抑菌浓度（MIC）。通过多种表型试验分析MBL的产生情况。采用全基因组测序（WGS）对新型bla基因进行遗传特性分析和多位点序列分型（MLST）。将新型bla基因在大肠杆菌TOP10中表达，并对该酶进行生化特性分析以确定动力学参数Km和kcat。
铜绿假单胞菌NRZ-03096对所有测试的β-内酰胺类药物耐药，并表现出MBL表型。鸟枪法克隆实验得到一个克隆株，该克隆株产生一种新型B1类酶，与最相近的亲属KHM-1的同一性仅为74.3%。这种新型MBL被命名为HMB-1（代表汉堡MBL）。WGS数据分析表明，blaHMB-1基因位于染色体上，是一个名为Tn6345的Tn3家族转座子的一部分。blaHMB-1在大肠杆菌TOP10中的表达导致对β-内酰胺类药物的耐药性增加。Km和kcat的测定表明，HMB-1与KHM-1相比具有不同的水解特性，对头孢菌素的水解速率较低，对亚胺培南的水解速率较高。
HMB-1的鉴定进一步强调了碳青霉烯酶在革兰氏阴性人类病原体尤其是铜绿假单胞菌中的持续传播和多样化。
