Characterization of a multidrug-resistant Acinetobacter baumannii strain carrying the blaNDM-1 and blaOXA-23 carbapenemase genes from the Czech Republic.

Sir, Since its discovery in 2008, the metallo-b-lactamase (MBL) NDM-1 has been identified in different Enterobacteriaceae species and recently also in a number of other bacterial species isolated from water supplies in India, such as Vibrio cholerae and Pseudomonas spp. Genes encoding NDM b-lactamases have been additionally identified in multidrugresistant Acinetobacter spp. Here, we report on the identification and characterization of an Acinetobacter baumannii strain carrying the blaNDM-1 gene that was isolated in the Czech Republic in 2011. A. baumannii strain ANC 4097 was isolated in mid-2011 during a prospective, nationwide study in the Czech Republic focused on the Acinetobacter population structure and resistance mechanisms. It was recovered from the sputum of an elderly patient hospitalized in an intensive care unit (ICU) of a hospital located in the north-western part of the Czech Republic. The patient, who had a malignant tumour, died of complications of the underlying disease several days after the isolation of the strain. The MICs of b-lactams for ANC 4097 determined using Etest (bioMérieux, Solna, Sweden) showed high-level resistance to all b-lactams, including carbapenems (MICs of both imipenem and meropenem .32 mg/L) and broad-spectrum cephalosporins (MICs of both ceftazidime and cefepime .256 mg/L), as well as penicillins in combination with inhibitors (MICs of ampicillin/sulbactam and piperacillin/tazobactam .256 mg/L). The strain was also resistant to fluoroquinolones (MIC of ciprofloxacin .32 mg/L), aminoglycosides (MIC of amikacin .64 mg/L and MIC of gentamicin 32 mg/L) and tetracycline (MIC 128 mg/L). An MBL Etest (bioMérieux) revealed a 24-fold reduction of imipenem MIC when combined with EDTA, which suggested production of an MBL. Isolate ANC 4097 remained susceptible only to tigecycline (MIC 2 mg/L), doxycycline (MIC 2 mg/L), netilmicin (MIC 4 mg/L), tobramycin (MIC 0.5 mg/L) and colistin (MIC 0.25 mg/L). Strain ANC 4097 was genotyped by multilocus sequence typing (MLST) using the Pasteur Institute scheme (http://www.pasteur.fr/ recherche/genopole/PF8/mlst/). It belongs to sequence type (ST) 1 (allelic profile 1-1-1-1-5-1-1), which is typical of the European clone I epidemic lineage. PCR screening followed by sequencing of resistance determinants revealed that the strain harboured genes encoding five b-lactamases: NDM-1, class D carbapenemases OXA-23 (acquired) and OXA-69 (intrinsic), acquired narrowspectrum class A b-lactamase TEM-1 and intrinsic AmpC-type b-lactamase ADC. This strain co-harboured the tetA(A) tetracycline resistance gene and the aacC1, aphA1, aphA6 and aadA1 aminoglycoside resistance genes, but no armA 16S RNA methylase gene was identified. Insertion sequence ISAba1 was detected upstream of each of blaOXA-23, blaOXA-69 and blaADC, thus providing strong promoter sequences likely to enhance the expression of these genes in ANC 4097. In addition, the comM gene (encoding a polypeptide containing an ATPase domain) was found to be disrupted, which, together with the presence of the blaTEM-1, aacC1, aphA1, aadA1 and tetA(A) genes, suggested the presence of an AbaR3-like genomic resistance island typical of the European clone I lineage. Genetic structures associated with the blaOXA-23 and blaNDM-1 genes were analysed using PCR mapping and sequencing as described elsewhere. The blaNDM-1 gene was located in the previously characterized composite transposon Tn125 (two copies of ISAba125 bracketing a 7925 bp region), while the blaOXA-23 gene was found to be part of transposon Tn2008. In order to determine the genetic location of the blaOXA-23 and blaNDM-1 genes, plasmid DNA of ANC 4097 was isolated as described previously. Transfer of the ticarcillin resistance marker was attempted by both electroporation of the ANC 4097 plasmid suspension into A. baumannii BM4547 and liquid mating-out assays of the ANC 4097 and BM4547 strains at 378C. Selection was performed on agar plates supplemented with ticarcillin (100 mg/L). Transformants or transconjugants harbouring the blaNDM-1 and blaOXA-23 genes were not obtained, suggesting a chromosomal location of both genes, as described previously. This study reports on the first blaNDM-1-positive A. baumannii strain in the Czech Republic and adds to the body of evidence of the current spread of multidrug-resistant Acinetobacter harbouring this MBL in Europe.