Diversity of plasmid-mediated carbapenem-hydrolysing oxacillinases among carbapenem-resistant Acinetobacter baumannii isolates from Kingdom of Bahrain

Sir, Carbapenem resistance in Acinetobacter baumannii is now increasingly reported. The most prevalent mechanism of carbapenem resistance in A. baumannii corresponds to the production of acquired carbapenem-hydrolysing class D β-lactamases (CHDLs)1 encoded by blaOXA-23-like, blaOXA-40-like or blaOXA-58-like genes. Whereas the blaOXA-40 gene has been detected in Portugal, Spain and the USA, blaOXA-23 and blaOXA-58 genes have disseminated worldwide.1 The aim of our study was to identify the molecular mechanisms leading to carbapenem resistance among a panel of A. baumannii isolates exhibiting resistance or intermediate susceptibility to imipenem and collected from September 2007 to March 2008 in the Salmaniya Medical Complex, Kingdom of Bahrain, which is a 1000 bed tertiary care centre. A total of 454 A. baumannii isolates had been recovered during the studied period, among which 262 (58%) were resistant or intermediately susceptible to imipenem. Among the latter, eight randomly selected A. baumannii isolates collected from eight patients hospitalized in different units were further analysed. They were identified using the API20 NE system and 16S rRNA gene sequencing.2 All isolates had been recovered from deep tracheal aspirates. Although two isolates were considered as colonizers, six were involved in infections and, among those, four were responsible for septicaemia that led to the death of all these patients. Use of Etest strips (AB Biodisk, Solna, Sweden) combining imipenem and EDTA did not identify metallo-β-lactamase in the isolates. PCR experiments followed by sequencing as described previously3 led to the identification of CHDL genes. A blaOXA-23 gene was found in two isolates (1 and 2), preceded by insertion sequence ISAba1 as observed in transposon Tn2006;3 but no ISAba1 copy was identified downstream of blaOXA-23. Isolate 3 harboured a blaOXA-58 gene that was bracketed by two copies of ISAba3, as described previously.1 Five isolates possessed a blaOXA-40-like gene, and further sequencing revealed that this gene corresponded to blaOXA-72. The OXA-72 β-lactamase belongs to the OXA-40 subgroup together with OXA-25 and OXA-26 β-lactamases. The OXA-40-type β-lactamases differ by no more than five amino acids and possess weak carbapenemase properties. OXA-72 had been previously reported in a single Acinetobacter genomospecies 3 isolate in China,4 in a single Acinetobacter baylyi isolate in Korea5 and in several carbapenem-resistant A. baumannii isolates in Taiwan.6 Genotypic comparison was performed by PFGE, as described previously.7 The two OXA-23 producers (isolates 1 and 2) were clonally related (pulsotype I). The single blaOXA-58-positive isolate belonged to pulsotype II. The remaining blaOXA-72-positive isolates belonged to pulsotype III (isolates 4 and 5), IV (isolates 6 and 7) or V (isolate 8). All isolates were resistant to ticarcillin and piperacillin, and isolates carrying genes encoding CHDLs OXA-23 and OXA-72 were highly resistant to imipenem and meropenem, although the OXA-58 producer displayed lower MICs of imipenem and ceftazidime (Table 1). MICs (mg/L) of β-lactams for representative A. baumannii clinical isolates of the different pulsotypes, A. baumannii BM4547 harbouring pOXA-72, A. baumannii BM4547 RifR harbouring pOXA-23, A. baumannii BM4547 and BM4547 RifR reference strains, E. coli TOP10 harbouring recombinant plasmid pBK-OXA-72 and E. coli TOP10 reference strain CLA, clavulanic acid (4 mg/L); TZB, tazobactam (4 mg/L). MICs (mg/L) of β-lactams for representative A. baumannii clinical isolates of the different pulsotypes, A. baumannii BM4547 harbouring pOXA-72, A. baumannii BM4547 RifR harbouring pOXA-23, A. baumannii BM4547 and BM4547 RifR reference strains, E. coli TOP10 harbouring recombinant plasmid pBK-OXA-72 and E. coli TOP10 reference strain CLA, clavulanic acid (4 mg/L); TZB, tazobactam (4 mg/L). Mating-out experiments were performed as described previously,7 with OXA-23-, OXA-58- and OXA-72-positive isolates as donors and rifampicin-resistant A. baumannii BM4547 as the recipient strain. The transconjugants were selected on agar plates containing ticarcillin (50 mg/L) and rifampicin (50 mg/L). Transconjugants were obtained only with blaOXA-23-positive donors, and the transconjugants showed resistance to ticarcillin and decreased susceptibility to carbapenems (Table 1). The transconjugants harboured a 130 kb plasmid additionally conferring resistance to kanamycin and amikacin (data not shown). Plasmid extracts, obtained as described previously,3 with OXA-58- and OXA-72-positive donors were used for transformation experiments with A. baumannii BM4547 as the recipient strain. Electrotransformation products were selected on plates containing ticarcillin (50 mg/L). Electroporation of plasmid extracts gave rise to A. baumannii BM4547 (pOXA-72) transformants, whereas no transformant was obtained with the OXA-58-positive donor strain. The blaOXA-72 gene was located on a 10 kb plasmid in all OXA-72-positive isolates. The blaOXA-72-positive A. baumannii transformants showed resistance to ticarcillin and decreased susceptibility to carbapenems (Table 1), without any additional antibiotic resistance marker. In order to investigate the genetic structures surrounding the blaOXA-72 gene, cloning experiments were also performed. Total DNA of A. baumannii isolate 4 was digested with the SacI or HindIII restriction enzymes, ligated into corresponding sites of plasmid pBK-CMV and transferred by electroporation into Escherichia coli TOP10 as described previously.7E. coli strains harbouring recombinant plasmid pBK-OXA-72 were selected on agar plates containing ticarcillin (50 mg/L) and kanamycin (30 mg/L). Detailed sequence analysis of pBK-OXA-72 showed that a gene encoding a putative inner membrane protein was present 300 bp upstream of blaOXA-72, whereas downstream a gene encoding a putative replication protein was identified. No typical genetic element (such as an insertion sequence) that could explain the acquisition of blaOXA-72 was identified at each extremity. In the Middle East, clonal outbreaks of infections caused by carbapenem-resistant A. baumannii strains have been reported, having occurred in the United Arab Emirates, Qatar, Iran and Iraq.7–9 In the United Arab Emirates, resistance to carbapenems was associated with the production of OXA-23;6 the carbapenem resistance mechanisms were not investigated in the other studies. Here we identified heterogeneity of CHDL-encoding genes as the source of carbapenem resistance among A. baumannii isolates from the same hospital. This is, to the best of our knowledge, the first report of such diversity of carbapenem resistance determinants in a restricted geographical area. This diversity of A. baumannii isolates and of CHDL genes in Bahrain may be related to the fact that this region of the Middle East has a very mixed population originating from various parts of the world. This work was financed by a grant from the Ministère de la Recherche, Université Paris XI, Paris, France, and mostly by a grant from the European Community (DRESP2, LSHM-CT-2005-01705) and by the INSERM, France. None to declare.