Acinetobacter baumannii is an opportunistic pathogen that is an important source of nosocomial infections, mostly pneumonia . The treatment of infections due to this microorganism is becoming a serious clinical concern because A. baumannii is frequently resistant to multiple antibiotics . Resistance to carbapenems in A. baumannii is mostly related to the production of carbapenem-hydrolysing class D β-lactamases and to a lesser extent to metallo-β-lactamases .
Recent reports showed that the blaNDM-1 gene encoding New Delhi metallo-β-lactamase 1 (NDM-1) is spreading worldwide among gram-negative bacteria . The blaNDM-1 gene was initially identified in Klebsiella pneumoniae and Escherichia coli isolates but has been additionally reported in many other Gram-negative rods [3,4]. In particular, the blaNDM-1 and blaNDM-2 genes have been recently identified in A. baumannii [5–9] and in other Acinetobacter species [10,11]. Notably, both genes have been identified as located on the same composite transposon named Tn125, being 10 099-bp long and comprising two copies of an identical insertion sequence ISAba125 bracketing the blaNDM-1/-2 genes [12,13].
Our study aimed to study the clonal relationship of NDM-1-producing A. baumannii and the genetic context of blaNDM-1 responsible for dissemination of this resistance trait, by analysing a collection of blaNDM-1-positive A. baumannii isolates recovered from different European countries. Isolate 161/07 was recovered in Germany in 2007 with a Balkan origin , isolate Slo was from a respiratory sample taken in Slovenia in 2008, isolate JH was collected in Switzerland in 2010 with a Balkan origin , and the two remaining isolates were isolated in France in 2011, one of those two being imported in France from Algeria (Table 1) .
Table 1. Features of NDM-1 producing Acinetobacter baumannii
| A. baumannii isolates||Country of isolation||Year of isolation||Sample||Carbapenemase genotyping||MICsc||ECa//STb||Pulsotype||Support||Genetic vehicle|
|JH||Switzerland||2010||Rectal swab||NDM-1, OXA-23, OXA-69||>256||>256||>32||>32||>32||I/1||A||Chromosomal||Tn125|
|Slo||Slovenia||2008||Respiratory sample||NDM-1, OXA-64||>256||>256||>32||>32||>32||nt/25||B||Chromosomal||Tn125|
|161/07||Germany||2007||Skin, respiratory sample||NDM-1, OXA-64||>256||>256||>32||>32||>32||nt/25||B||Chromosomal||Tn125|
|Ora-1||France||2011||Rectal swab||NDM-1, OXA-94||>256||>256||>32||>32||>32||nt/85||C||Chromosomal||ΔTn125|
|StN||France||2011||Rectal swab||NDM-1, OXA-94||>256||>256||>32||>32||>32||nt/85||C||Chromosomal||ΔTn125|
The isolates were identified by 16S rRNA gene sequencing . Susceptibility testing was performed by disc-diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France) and interpreted according to updated CLSI guidelines . The MICs of β-lactams including imipenem, meropenem and doripenem were determined by Etest (AB bioMérieux; Solna, Sweden) as described previously . All isolates were resistant to β-lactams including carbapenems (Table 1). The production of metallo-β-lactamases was assessed using a combined disc-test, based on the inhibition of the metallo-β-lactamase activity by EDTA as described elsewhere . All isolates were positive for the production of metallo-β-lactamases. The PCR experiments performed to detect carbapenemase genes as described previously , followed by sequence analysis, led to the identification of the blaNDM-1 gene in the five isolates in addition to naturally occurring blaOXA-51-like genes (respectively encoding OXA-64, OXA-69 or OXA-94) (Table 1). In addition, the blaOXA-23 gene, coding for the unrelated carbapenemase OXA-23, was identified in isolate JH, as found previously in one Indian A. baumannii isolate .
To determine the genetic location of the blaNDM-1 gene, the plasmid DNA of the different A. baumannii isolates was extracted by using the Kieser technique  and Southern hybridization was performed by using internal PCR-obtained amplicons as probe for the blaNDM gene. DNA hybridization gave negative results, suggesting that the blaNDM-1 gene was chromosomally located in all isolates (data not shown). Transfer of the ticarcillin-resistance marker into A. baumannii BM4547 was attempted by liquid mating-out assays at 37°C and by electroporation of a plasmid DNA suspension extracted from A. baumannii clinical isolate into A. baumannii BM4547. It gave negative results, reinforcing the hypothesis of a chromosomal location of the blaNDM-1 gene in all isolates.
The genetic context of blaNDM-1 gene was determined by PCR mappings or by shotgun clonings using HindIII-restricted genomic DNA and the HindIII-restricted pBK-CMV plasmid as described previously . Recombinant plasmids were selected onto Trypticase soy agar plates containing ticarcillin (100 mg/mL) and kanamycin (30 mg/mL). Sequencing of the recombinant plasmids showed that the blaNDM-1 gene was located in a transposon Tn125 . In isolate JH, transposon Tn125 was inserted into a chromosomal gene encoding a putative protein of A. baumannii and surrounded by a 3-bp duplication (TTG) being the signature of the transposition process. In isolates SLO and 161/07, Tn125 was inserted in a gene encoding a major facilitator superfamily transporter protein (CTG duplication). In the two remaining isolates (StN and Ora-1), Tn125 was truncated at its righthand extremity by insertion sequence ISAba14 located upstream of the dct gene, giving rise to a truncated Tn125 (ΔTn125) (Table 1; Fig. 1). Further PCR assays did not identify a second copy of ISAba125, suggesting that Tn125 was no more functional in that latter structure.
Figure 1. Schematic representation of transposon Tn125 carrying the blaNDM-1 gene. Genes and their transcription orientations are indicated by arrows. The lengths of the target genes and the exact location of the target site are not to scale. The oriIS of ISCR21 is indicated by a circle. Gene names are abbreviated according to their corresponding proteins: iso for phosphoribosylanthranilate isomerase; tat for twin-arginine translocation pathway signal sequence protein; dvt for divalent cation tolerance protein; Δpac for truncated phospholipid acetyltransferase. IRL and IRR are for inverted repeat left and right, respectively. The Tn125 complete was found in isolates JH, Slo and 161/07 and the truncated isoform of Tn125 (ΔTn125) was found in isolates Ora-1 and StN.
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Genotypic comparison was performed by multilocus sequence typing as described by Diancourt et al. , by sequence-type multiplex PCR , by DiversiLab following the manufacturer’s instructions (bioMérieux, La Balme-les-Grottes, France), and by pulsed-field gel electrophoresis as described . The pulsed-field gel electrophoresis analysis showed that the five isolates were grouped into three distinct clones named A to C (Table 1), with strains Ora-1 and StN (from France) sharing identical patterns, strains Slo (from Slovenia) and 161/07 (from Germany) being clonally related according to the Tenover’s criteria (two bands of difference) , and strain JH (from Switzerland) belonging to a different clone. Analysis by the Diversilab technique resulted in the same interpretation (Fig. 2). Further analysis with sequence-type multiplex PCR showed that isolate JH belonged to European clone I although other isolates did not correspond to any defined European clone (Table 1). Multilocus sequence typing analysis showed that isolate JH belonged to ST1, isolates Slo and 161/07 belonged to ST25, and the two remaining isolates belonged to ST85 (Table 1). ST1-type isolates are widely distributed throughout the world whereas ST25 and ST85 strains have been rarely reported (http://www.pasteur.fr/cgi-bin/genopole/PF8/mlstdbnet.pl?file=acin_isolates.xml).
In conclusion, this report highlights scattered diffusion of NDM-1 producing A. baumannii in Europe from the west to the east. This dissemination was neither due to a single clone nor to any plasmid diffusion but rather to different clones carrying the transposon Tn125 or Tn125-derivatives which are truncated. While this study was in progress, two NDM-1-producing A. baumannii were isolated in Belgium and Czech Republic, [25,26]. The clinical isolate from the Czech Republic belonged to ST1, similar to isolate JH from Switzerland; whereas the clinical isolate from Belgium belonged to European clone II (whichcorresponds to ST2 in the multilocus sequence typing Pasteur Institute scheme) [25,26]. These two reports reinforce the fact that the spread of the blaNDM-1 gene in A. baumannii is not linked to a clonal spread but to the spread of a genetic structure. The spread of transposon Tn125 in Acinetobacter species harbouring blaNDM genes mirrors what has been observed with the blaKPC carbapenemase gene, which is associated with transposon Tn4401 . Interestingly, we report here the first occurrence of an NDM-1 producer in Slovenia and therefore further confirm that Balkan countries constitute a significant reservoir for NDM-1-producing bacteria.