Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae

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Abstract

Background

Multidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern detection, followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates.

Methods

A panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and validation of results was achieved by genotypic analysis.

Results

The most accurate identification of resistance determinants was obtained with the combined disc test (Rosco Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection of AmpC enzymes. However, in general, phenotypic tests failed to characterize isolates harboring multiple carbapenem resistance determinants, which were successfully assessed only by PCR-based analysis.

Conclusions

To detect and control the spread of pathogens with complicated resistance patterns, both optimized phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and genotypic assays are recommended in the routine diagnostic of clinical laboratories.

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Most cited references 27

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Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe.

R Canton, M. Akova, Y Carmeli … (2012)

Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most β-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum β-lactamase (mainly CTX-M) producers in all European countries. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

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Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR.

F. Javier Pérez-Pérez, Nancy Hanson (2002)

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC beta-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC beta-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.

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Identification and screening of carbapenemase-producing Enterobacteriaceae.

P. Nordmann, M Gniadkowski, C.G. Giske … (2012)

Carbapenem-hydrolysing β-lactamases are the most powerful β-lactamases, being able to hydrolyse almost all β-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

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Author and article information

Contributors

Andrea Bartolini

Ilaria Frasson

Antonietta Cavallaro

Sara N Richter

Giorgio Palù

Journal

Journal ID (nlm-ta): Gut Pathog

Journal ID (iso-abbrev): Gut Pathog

Title: Gut Pathogens

Publisher: BioMed Central

ISSN (Electronic): 1757-4749

Publication date Collection: 2014

Publication date (Electronic): 19 May 2014

Volume: 6

Page: 13

Affiliations

[1 ]Padua Hospital, Microbiology and Virology Unit, via Giustiniani 2, Padua 35121, Italy

[2 ]Department of Molecular Medicine, University of Padua, via Gabelli 63, Padua 35121, Italy

Article

Publisher ID: 1757-4749-6-13

DOI: 10.1186/1757-4749-6-13

PMC ID: 4032584

PubMed ID: 24860620

SO-VID: 1745ec45-83e9-4288-b3aa-5e22f7970f8e

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae

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Karger: Gastroenterology

Most cited references 27

Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe.

Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR.

Identification and screening of carbapenemase-producing Enterobacteriaceae.

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