Short communication
Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp.

https://doi.org/10.1016/j.ijantimicag.2006.01.004Get rights and content

Abstract

Carbapenem resistance in Acinetobacter baumannii is a growing public health concern and is most often mediated by OXA carbapenemases. We describe a novel multiplex polymerase chain reaction (PCR) assay able to detect and distinguish alleles encoding three subgroups of acquired OXA carbapenemases (OXA-23-like, OXA-24-like and OXA-58-like) that are scattered in Acinetobacter spp., and a fourth subgroup, OXA-51-like, which appears to be intrinsic to Acinetobacter baumannii. Isolates belonging to two prevalent UK A. baumannii ‘OXA’ clones (OXA-23 clones 1 and 2) had alleles encoding both an intrinsic OXA-51-like and an acquired OXA-23 enzyme, whereas isolates of the ‘SE clone’ had only an intrinsic blaOXA-51-like allele. Genes encoding OXA-58 were detected (with blaOXA-51-like) in a cluster of related isolates from a single hospital. This simple assay will assist in monitoring the mechanisms responsible for carbapenem resistance in Acinetobacter spp.

Introduction

The emergence of carbapenem resistance in Acinetobacter spp. is a significant public health concern, leaving few therapeutic options remaining [1]. Carbapenem-hydrolysing β-lactamases (carbapenemases) belonging to molecular class D (OXA enzymes) have emerged globally as the main mechanism responsible for this resistance, although metallo-enzymes are locally prevalent, especially in East Asia. The OXA carbapenemases of Acinetobacter spp. are divided into four phylogenetic subgroups: OXA-23-like; OXA-24-like; OXA-51-like; and OXA-58 [2]. Recently it has been suggested that enzymes belonging to the OXA-51-like subgroup are intrinsic to Acinetobacter baumannii [3], occurring in most or all strains, although they are very variably expressed.

We have previously identified three prevalent, multi-resistant clones of A. baumannii in the UK, based on their pulsed-field gel electrophoretic banding patterns [1], [4], [5]. Two of these clones show carbapenem resistance, produce OXA-23 enzyme and have been designated OXA-23 clone 1 and OXA-23 clone 2 [1], [5]. Isolates belonging to the third group, the SE clone, show variable carbapenem resistance [4], contingent on the activation or not of blaOXA-51-like by insertion sequences [6].

We sought to develop a multiplex polymerase chain reaction (PCR) assay to differentiate alleles encoding the four subgroups of OXA carbapenemases found in Acinetobacter and to investigate their distribution among a collection of isolates, including members of the three prevalent UK clones of A. baumannii.

Section snippets

Bacterial isolates and susceptibility testing

The blaOXA alleles encoding known carbapenemases were sought in 250 clinical isolates of Acinetobacter spp. from the UK, none of which produced metallocarbapenemases. These included 168 isolates representing three prevalent UK A. baumannii clones (as defined by pulsed-field gel electrophoresis (PFGE) of ApaI-digested genomic DNA) [1], [4], [5], 65 isolates from a survey of Acinetobacter spp. conducted in 2000 in the UK [7] and 17 recently-referred carbapenem-resistant clinical isolates not

Results and discussion

The multiplex PCR assay (using eight primers) amplified fragments of blaOXA alleles encoding each of the three subgroups of acquired OXA carbapenemases in Acinetobacter spp. and the blaOXA-51-like alleles intrinsic to A. baumannii. The subgroups assigned by this assay to the control isolates were consistent with the blaOXA alleles previously characterised by sequencing, and amplicons matched their predicted sizes (Fig. 1).

All 154 PFGE-defined isolates belonging to A. baumannii OXA-23 clone 1

Acknowledgments

We are grateful to AstraZeneca for supporting the work of J.M.C. on carbapenem-resistant Acinetobacter. The work of N.W., D.M.L. and M.E.W. on emerging β-lactamases is supported, in part, by the EU/FP6-funded COBRA project (6-PCRD LSHM-CT-2003-503-335). The work of S.B. and S.G.B.A. was supported by the Chief Scientist Office of the Scottish Executive Health Department (grant number CZB/4/42). This work was presented in December 2005 at the 45th ICAAC, Washington, USA; Poster D-1706.

References (10)

There are more references available in the full text version of this article.

Cited by (919)

View all citing articles on Scopus
View full text