Abstract
BackgroundCarbapenemase producing Enterobacteriaceae are becoming a major public health concern globally, however, relatively little is known about the molecular and clinical epidemiology of these organisms in many parts of the world.MethodsAs part of a laboratory surveillance programme, 96 carbapenem non-susceptible Enterobacteriaceae isolates from clinical samples from patients in seven hospitals were referred for investigation for carbapenemases. Using polymerase chain reaction (PCR) to screen for a collection of genes encoding carbapenemases, 33 of 96 (34.5%) isolates were confirmed as carbapenemase producers. NDM-1 producers were the most prevalent at 64% (21/33) whilst OXA-181 was the second most common carbapenemase constituting 24.5% (8/33) of the carbapenemase producing isolates. Seven of these eight OXA-181 positive isolates underwent further characterisation with screening for other transmissible antimicrobial resistance determinants using PCR. Clonal relatedness was explored using Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Plasmid characterisation was performed including restriction analysis and transfer by conjugation or transformation.ResultsIn addition to the OXA-181 gene, all contained other transmissible resistance determinants including extended spectrum β-lactamases, oxacillinases or 16S rRNA methylase genes, but none contained metallo-β-lactamases or serine carbapenemases. All isolates had a multidrug resistant phenotype with two isolates being resistant to every antibiotic tested including colistin. Multilocus sequence typing confirmed five isolates belonged to ST17 and two to ST14, with those belonging to the same sequence type having identical PFGE profiles. The OXA-181 gene was typically carried on large plasmids which were mostly non-conjugative.ConclusionsOXA-181 carbapenemase appears to be an important and probably under-recognised cause of carbapenem resistance in Enterobacteriaceae in Singapore. Further coordinated research into clinical and molecular epidemiology of carbapenemases is urgently required in Singapore and throughout Asia.
Highlights
Carbapenemase producing Enterobacteriaceae are becoming a major public health concern globally, relatively little is known about the molecular and clinical epidemiology of these organisms in many parts of the world
These carbapenem hydrolysing Class D βlactamases are unusual among carbapenemases in their weak hydrolysis of cephalosporins and resistance to inhibitors such as clavulanate, making them difficult to identify in the laboratory [2]
Antimicrobial susceptibility testing was performed with VITEK-2 instrument and carbapenem Minimum inhibitory concentration (MIC) confirmed with Etest with susceptibility defined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints
Summary
All isolates were confirmed as K. pneumoniae using matrix assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-ToF-MS, Bruker Daltonics GmHB, Bremen, Germany). Detection of blaOXA was based on a multiplex PCR assay developed by Woodford et al [12] with addition of blaOXA-48-like primers (OXA48L 50- GTGGGATGGACAGACGCG-30 and OXA-48 LL 50-CCACACATTATCATCAAGTTC-30) using National Collection of Type Cultures (NTCT) 13442 K. pneumoniae as positive control for blaOXA-48. Smaller plasmids were extracted using the QIAprep spin miniprep kit (Qiagen GmbH, Hilden, Germany). Transformation studies were performed with plasmid DNA extracted using QIAprep Spin Miniprep kit introduced by electroporation into E. coli DH5α cells using Gene Pulser Xcell (Bio-Rad, Hercules, CA) with transformants selected on Luria-Bertani (LB) agar supplemented with imipenem (1 mg/L). To analyse the immediate genetic environment of the blaOXA-181 gene in our isolates, primers (IRF 50-CCTAGATTCTACGTCAGTAC-30 and IRR2 50CTCTCTAGTCGGACAACACC-30) based on GenBank reference sequence NC_019160.1, designed to walk in from the left and right inverted long repeats (IRL) in the direction of blaOXA-181 were used
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