Antimicrobial Susceptibility Studies
Multiplex PCR for detection of acquired carbapenemase genes

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Abstract

A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: blaIMP, blaVIM, blaNDM, blaSPM, blaAIM, blaDIM, blaGIM, blaSIM blaKPC, blaBIC, and blaOXA-48. Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes.

Introduction

The current emergence of carbapenemase-producing Gram-negatives is of concern because it is often associated to the occurrence of multidrug-resistant isolates for which very few (if any) antibiotic options remain available (Poirel et al., 2007). This is the reason why detection and early recognition of carbapenemase producers are important. This is not always easy taking into account that those resistance determinants may sometimes confer only a slight increase of MIC values for carbapenems, and this is the reason why using molecular approaches and not only phenotypic tests are sometimes very helpful (Cornaglia et al., 2007). Among the current threats related to carbapenemase spread, metallo-β-lactamases (MBLs) of the IMP and VIM types are relevant because they have been identified in distantly related geographical areas among many different enterobacterial species and also often among Pseudomonas spp. (Walsh et al., 2005). The MBL SPM-1 is widespread in Brazil and has been identified so far only in Pseudomonas aeruginosa (Sader et al., 2005, Toleman et al., 2002, Poirel et al., 2004a, Picão et al., 2009). The last but not less worrying threat corresponds to the recent emergence of the MBL NDM-1 among different enterobacterial species (Yong et al., 2009) and also in Acinetobacter baumannii (Karthikeyan et al., 2010), not only in UK but also in India and Pakistan; those latter countries currently considered to be its main reservoir (Kumarasamy et al., 2010).

In addition, we have witnessed the emergence of the Ambler class A KPC β-lactamase during the recent years, mostly in Klebsiella pneumoniae (but also in P. aeruginosa, Escherichia coli, and A. baumannii) identified mostly in the United States, in Israel, and in Greece (Nordmann et al., 2009, Poirel et al., 2010a). The carbapenem-hydrolyzing class D β-lactamase (CHDL) OXA-48 is currently disseminating in Europe, being mostly identified in K. pneumoniae, with Turkey being the main identified reservoir (Carrër et al., 2010, Poirel et al., 2004b). However, recent studies showed the emergence of OXA-48 producers in several European countries (Poirel et al., 2010a), but also the emergence of some other carbapenem-hydrolyzing blaOXA-48-like encoding genes, such as blaOXA-163 in Argentina (Poirel et al., 2010b) and blaOXA-181 in India (Poirel et al., 2011). Besides those frequently identified resistance determinants, scattered reports of several unrelated acquired carbapenemases are known, such as the Ambler class A BIC-1 (from Pseudomonas fluorescens) (Girlich et al., 2010), the Ambler class B AIM-1 (from P. aeruginosa) (Yong et al., 2007), DIM-1 (from Pseudomonas stutzeri) (Poirel et al., 2010c), GIM-1 (from P. aeruginosa) (Castanheira et al., 2004), and SIM-1 (from A. baumannii) (Lee et al., 2005).

The purpose of this work was to develop a PCR-based technique allowing easy and efficient recognition of those clinically relevant carbapenemase genes. The previously published multiplex PCR schemes designed by Ellington et al. (2007) and by Mendes et al. (2007) were interesting but did not include the emerging carbapenemase genes encoding the class A KPC, the class B NDM-1, and the class D OXA-48, neither the recently identified genes encoding DIM-1, BIC-1, or AIM-1 enzymes.

Section snippets

Bacterial isolates

For optimization of the multiplex PCR, previously well-characterized strains that had been recovered from different parts of the world were used as positive controls. Five P. aeruginosa isolates carrying, respectively, blaSPM-1, blaVIM-2, blaGIM-1, blaAIM, and blaBIC-1, were used; 3 K. pneumoniae isolates carrying, respectively, blaKPC-2, blaIMP-1, and blaNDM-1; 1 A. baumannii isolate carrying blaSIM-1; 1 P. stutzeri isolate carrying blaDIM-1; and 1 Enterobacter cloacae isolate carrying bla

Results and discussion

Positive controls (used separately or mixed) yielded expected bands and confirmed the specificity of the PCR primers used (Fig. 1). The primer pairs were tested in simplex PCR (only one gene screened) and with a multiplex approach. Although the specificity was good using the simplex approach (data not shown), with amplification of all the expected fragments, the multiplex approach gave also good results (Fig. 1). We did not observe any lack of specificity when using the multiplex approach.

Acknowledgments

This work was funded by grants from the INSERM (U914), the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and by a grant from the European Community (TEMPOtest-QC, HEALTH-2009-241742). We are grateful to Dr Chong who kindly provided us with a SIM-1–producing A. baumannii isolate.

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