16 February 2012
NDM-1 producing Acinetobacter baumannii isolated from a patient repatriated to the Czech Republic from Egypt, July 2011
We describe the isolation of an NDM-1-producing Acinetobacter baumannii in a Czech patient repatriated in July 2011 from Egypt. The infection spread to another patient on the same ward. Both isolates showed the same resistance pattern and were susceptible only to colistin. They had an identical PFGE pattern and belonged to the same sequence type ST 1. Sequencing of the blaNDM gene identified the NDM-1 variant of the carbapenemase, surrounded by two copies of insertion sequence ISAba125.
Here we describe the isolation of a New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Acinetobacter baumannii
in a Czech citizen repatriated from Egypt in July 2011. The patient was hospitalised in Egypt, and then transferred to a hospital in the Czech Republic. The patient developed ventilator-associated pneumonia caused by A. baumannii
in addition to a primary neurological diagnosis. A carbapenem-resistant A. baumannii
strain (V509) was isolated from bronchoalveolar lavage and an oral cavity swab. He was initially treated by meropenem and metronidazole. Due to progression of the primary disease, the patient was transferred to a long-term intensive care unit. Although the antibiotic regimen was not changed, the patient recovered according to the biochemical markers of inflammation within seven days and the antibiotic therapy was then stopped. The available data are not conclusive as to whether this patient was infected or colonised. However, the resistant isolate has been detected in low quantity in oral swab and bronchoalveolar lavage until the transfer to the long-term intensive care unit. The intensive care centre was informed about the epidemiological risk associated with this patient so that they could prepare for appropriate measures upon transfer.
A second A. baumannii
isolate (V566) with the same resistance pattern was recovered six days later from the airways of another ventilated patient sharing the same room. The patient was treated with amoxicillin/clavulanic acid, chloramphenicol and ciprofloxacin. He died due to respiratory failure four days after the first isolation of NDM-1-producing A. baumannii
The isolates from both patients were identified as A. baumannii by biochemical test API ID32 GN (bioMérieux, France) and by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Germany). The minimum inhibitory concentrations (MICs) to 14 antibiotics were tested and the results were interpreted according to the EUCAST recommendation . The isolates from both patients were resistant to all beta-lactams tested including carbapenems and other antibiotics (Table).
Table. Antimicrobial susceptibility of the NDM-1-producing Acinetobacter baumannii isolates, Czech Republic, July 2011 (n=2)
Typing performed by pulsed-field gel electrophoresis (PFGE)  showed that the isolates had indistinguishable macrorestriction patterns. Carbapenemase production was confirmed by MALDI-TOF mass spectrometry . Production of metallo-beta-lactamase (MBL) activity was verified by ethylenediaminetetraacetic acid (EDTA) double-disk synergy test .
The blaNDM gene of both isolates was amplified and sequenced as described previously , and revealed the NDM-1 variant of the enzyme. The blaNDM-1 together with other genes was located between two copies of the insertion sequence ISAba125 in the same orientation as found by Pfeifer et al. . Because plasmid preparations from the two isolates did not yield any plasmids visible after electrophoretic separation, and no transformants were obtained after transformation experiments performed as previously described , it can be hypothesised that blaNDM-1 is located on the bacterial chromosome.
Multi-locus sequence typing (MLST) was performed  and the MLST database available at the website of the Pasteur Institute was used to assign the sequence type (ST). Both isolates belonged to sequence type (ST) 1 (allelic profile 1-1-1-1-5-1-1) which represents the epidemiologically successful European clone I .
Discussion and conclusion
Reports describing NDM-type carbapenemase producers isolated from patients previously hospitalised in high-prevalence countries have been increasing. Pfeifer et al. detected NDM-1 in A. baumannii isolated from a patient repatriated to Germany from Serbia in 2007 . Importation of NDM-1-producing A. baumannii strain from Serbia has also been described by Poirel et al. . Other A. baumannii isolates expressing NDM-1 MBL have been isolated in China and India [11,12]. It is remarkable that blaNDM-1 was also found on a plasmid in A. lwoffii in China . The new NDM-2 variant was first detected in A. baumannii from a patient transferred from Egypt to Germany . Recently, clonal spread of NDM-2-producing A. baumannii strains have been described in a rehabilitation ward in Israel and in the United Arab Emirates [14,15].
Until this report, no NDM-1 producing bacterium had been described in the Czech Republic, a country with a low prevalence of carbapenemase-producing bacteria [16-18]. Although routine procedures were in place in the hospital department, the strain quickly spread within one ward to another patient. After the death of the second patient and the transfer of the first patient to the long-term intensive care unit centre, the department was closed for two weeks and general cleaning including decontamination of all equipment was undertaken. No NDM-1-producing strain has been detected after the cleaning. Due to the importance of international travel in the spread of bacterial resistance, fast detection and active surveillance of bacteria producing acquired carbapenemases is needed [5-7,10,16,18,19].
We also tested the new MALDI-TOF mass spectrometry approach  for the detection of carbapenemase activity in the isolates. Although phenotypical detection of carbapenem-hydrolyzing enzymes in A. baumannii seems to be difficult by conventional methods , we were able to see a clear carbapenemase activity by this assay. Further validation, however, is necessary.
This work was supported by the research project grants NT11032-6/2010 and NS9717-4/2008 from the Ministry of Health of the Czech Republic and MSM0021620819 from the Ministry of Education of the Czech Republic.
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