碳青霉烯酶表型检测方法

A simple phenotypic method for the differentiation of metallo-b -lactamases and class A KPC carbapenemases in

Enterobacteriaceae clinical isolates

Athanassios Tsakris 1*,Aggeliki Poulou 1,2,Spyros Pournaras 3,Evangelia Voulgari 1,Georgia Vrioni 1,

Katerina Themeli-Digalaki 4,Dimitra Petropoulou 5and Danai Sofianou 6

1

Department of Microbiology,Medical School,University of Athens,Athens,Greece;2Department of Microbiology,Serres General Hospital,Serres,Greece;3Department of Microbiology,Medical School,University of Thessaly,Larissa,Greece;4Department of Microbiology,Tzaneion General Hospital,Piraeus,Greece;5Department of Microbiology,Saint Panteleimon Hospital,Nicea,Greece;

6

Department of Microbiology,Hippokration University Hospital,Thessaloniki,Greece

*Corresponding author.Tel:+30-210-7462011;Fax:+30-210-7462210;E-mail:atsakris@med.uoa.gr

Received 12March 2010;returned 15April 2010;revised 27April 2010;accepted 11May 2010

Background:The increasing frequency of class A KPC enzymes and class B metallo-b -lactamases (MBLs)among Enterobacteriaceae as well as their possible co-production makes their early differentiation urgent.

Methods:A simple phenotypic algorithm employing three combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA),EDTA,or both PBA and EDTA was designed for the differentiation of KPC and MBL enzymes.Augmentation of the zone of inhibition by ≥5mm was considered a positive combined-disc test result.A total of 141genotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates (63KPC producers,47MBL producers,and 31KPC and MBL producers)with various carbapenem MICs were exam-ined.For comparison,84genotypically confirmed carbapenemase-negative Enterobacteriaceae clinical isolates [39extended-spectrum b -lactamase (ESBL)producers,22AmpC producers,and 23ESBL and AmpC producers]were also tested.

Results:The phenotypic algorithm was able to differentiate MBL from KPC producers as well as to detect the possible co-production of both carbapenemases (positive result only with the combined-disc test using mero-penem alone and with both PBA and EDTA).The method detected all KPC or MBL producers (sensitivity 100%)as well as 30of the KPC and MBL producers (sensitivity 96.8%).All three combined-disc tests were negative for non-carbapenemase-producing isolates,except two ESBL and AmpC producers that gave positive combined-disc tests using meropenem alone and with PBA and both PBA and EDTA (specificity for KPC detection 98.8%).Conclusions:This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes,especially in regions where KPC-and MBL-pos-sessing Enterobacteriaceae are highly prevalent.

Keywords:class A carbapenemases,MBLs,extended-spectrum b -lactamases,plasmid-mediated AmpC,combined-disc test,EDTA,boronic acid

Introduction

Carbapenems are often considered last resort antibiotics in the treatment of infections due to multidrug-resistant Enterobacter-iaceae clinical isolates,since they are stable even in response to extended-spectrum b -lactamases (ESBLs)and AmpC enzymes.However,during the last decade carbapenem resistance has been increasingly reported among Enterobacteriaceae and is largely attributed to the production of Ambler class B acquired metallo-b -lactamases (MBLs).1These enzymes efficiently

hydrolyse all b -lactams except monobactams.They have shown a worldwide dissemination although they are more fre-quently reported in southern Europe and the Asiatic-Pacific region.1,2More recently,a new type of class A b -lactamase,the Klebsiella pneumoniae carbapenemase (KPC),has also spread among K.pneumoniae isolates and other Enterobacteria-ceae.3These enzymes confer various levels of resistance to all b -lactams including carbapenems,even though cephamycins and ceftazidime are only weakly hydrolysed.3,4They have become widespread in several regions of North and South

#The Author 2010.Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.All rights reserved.For Permissions,please e-mail:journals.permissions@

J Antimicrob Chemother 2010;65:1664–1671

doi:10.1093/jac/dkq210Advance Access publication 11June 2010

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