Short communicationMultiplex PCR for genes encoding prevalent OXA carbapenemases 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.
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