Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-β-lactamase found in a Pseudomonas aeruginosa clinical isolate

TY - JOUR

T1 - Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-β-lactamase found in a Pseudomonas aeruginosa clinical isolate

AU - Pfennigwerth, Niels

AU - Lange, Felix

AU - Belmar Campos, Cristina

AU - Hentschke, Moritz

AU - Gatermann, Sören G

AU - Kaase, Martin

PY - 2017/4/1

Y1 - 2017/4/1

N2 - Objectives: To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.Methods: 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 .Results: 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.Conclusions: The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

AB - Objectives: To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.Methods: 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 .Results: 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.Conclusions: The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

KW - Aged

KW - Anti-Bacterial Agents

KW - Carbapenems

KW - Drug Resistance, Multiple, Bacterial

KW - Escherichia coli

KW - Feces

KW - Female

KW - Gene Expression

KW - Genome, Bacterial

KW - Germany

KW - Humans

KW - Microbial Sensitivity Tests

KW - Multilocus Sequence Typing

KW - Pseudomonas Infections

KW - Pseudomonas aeruginosa

KW - Recombinant Proteins

KW - Sequence Analysis, DNA

KW - beta-Lactamases

KW - Case Reports

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1093/jac/dkw554

DO - 10.1093/jac/dkw554

M3 - SCORING: Journal article

C2 - 28065891

VL - 72

SP - 1068

EP - 1073

JO - J ANTIMICROB CHEMOTH

JF - J ANTIMICROB CHEMOTH

SN - 0305-7453

IS - 4

ER -