First identification of NDM-5 associated with OXA-181 in Escherichia coli from Egypt

Emerging Microbes and Infections (2016) 5, e30; doi:10.1038/emi.2016.24; published online 6 April 2016

to amikacin, tigecycline and colistin was retained ( Table 1). The PCR and sequence analysis for carbapenemases, extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC cephalosporinaseencoding genes, plasmid-mediated quinolone resistance genes, aminoglycoside-modifying enzymes and methyltransferases [5][6][7] identified the presence of bla NDM-5 , bla CTX-M-15 , bla OXA-181 , bla CMY-2 , aac(3)-IIa and aac(6´)-Ib-cr. PCR-based plasmid replicon typing detected the presence of two replicons, FIA and FIB. 6 Multilocus sequence typing (MLST), performed according to the Genotyping of Pathogens and Public Health (Institute Pasteur, Paris, France; http://www.pateur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html), identified the isolate as ST410, which is different from those in previously reported cases of NDM-5, which were ST648 and ST2659 and was infrequently encountered in North Africa. 8 Whereas an association between ST410 and NDM-1 has been reported in Norway, the United Kingdom, Switzerland, France and the United States, more recently, it was found in Poland in a patient who had previously received care in Tunisia after a terrorist attack. 8,9 To the best of our knowledge, this is the first report of ST410 in Egypt. The genetic environment of bla NDM-5 , assessed by PCR mapping as previously described, 10 showed 99% similarity to that of the plasmid pHC105  3 Conjugation experiments using the azide-resistant E. coli J53 as the recipient strain at two different temperatures and with plates containing meropenem at 0.5 μg/mL failed. Therefore, plasmid extraction using a QIAGEN Midi Kit (Qiagen, Hilden, Germany) followed by electrotransformation experiments into E. coli Top10 was performed.
The resistance phenotype and the gene content of the transformants were assessed and compared with those of the parental cells (Table 1).
Plasmid analysis by an S1 nuclease digestion of the whole genomic DNA followed by pulsed-field gel electrophoresis (S1-PFGE) showed that the isolate had three plasmids of sizes approximately 48.5, 88 and 100 kb. Southern blot hybridization of the S1-PFGE plasmid DNA was performed using a DIG DNA Labeling and Detection Kit (Roche, Mannheim, Germany) with DIG-labeled probes for bla NDM-5 , bla OXA-181 , bla CTX-M-15 , FIA and FIB showing that the bla NDM-5 and bla CTX-M-15 were all located on the same plasmid (an ≈100 kb plasmid), in both the parental and the transformed cells, which were also collocated with FIA and FIB, indicating the presence of a multireplicon plasmid, whereas bla OXA-181 was found on another plasmid of size ≈48.5 kb only in the parental cell.
In Egypt, NDM-1 was first identified in 2013 in one Klebsiella pneumoniae isolate, and then more cases were found in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. 11 By contrast, NDM-2 was previously reported in A. baumannii in 2011. 12 Compared with NDM-1, NDM-5 has two amino-acid substitutions (Val 88 → Leu) and (Met 154 → Leu), which confer enhanced hydrolytic activity against carbapenems.
Additionally, OXA-181, a variant of OXA-48, is associated with other carbapenemase genes, such as bla NDM-1 and bla VIM-5 . 13 The coproduction of OXA-181 with NDM-5 has been recently reported in K. pneumoniae; 14 however, the emergence of this co-existence in E. coli is alarming as it is believed that the worldwide spread of this enzyme is a mirror image to that of NDM-1. 13 We hereby report the first case of NDM-5 in Egypt, confirming the pervasiveness of the NDM enzymes in North Africa and the urgent need for public health concern towards the evolution and spread of these enzymes.