Identification of Enterobacteriaceae isolates with OXA-48 and coproduction of OXA-181 and NDM-1 in Norway

Sir, The global emergence and dissemination of acquired carbapenemases among Gram-negative bacteria are considered a major public health problem.1 The dominant carbapenemases include the class A serine β-lactamases (e.g. KPC), the class B metallo-β-lactamases (e.g. NDM, VIM and IMP) and the class D OXA group.1 The carbapenemase-encoding genes are often located on plasmids along with other resistance genes, resulting in multidrug-resistant, extremely drug-resistant or pan-drug-resistant bacteria.1 Among the class D β-lactamases, OXA-48-like carbapenemases have increasingly been identified in Enterobacteriaceae with a wide geographical distribution.2 After the first identification in Turkey, the dissemination of OXA-48-like carbapenemases was mainly identified in North African and Middle Eastern countries.2 However, a growing number of reports show a global dissemination of OXA-48-like carbapenemases and, in some European countries, OXA-48-like has become the predominant carbapenemase.3 The OXA-48-like enzymes have limited activity against extended-spectrum cephalosporins, in contrast to other carbapenemases, and the MIC values of carbapenems are often below the clinical breakpoints.4 This has implications for detection procedures in the laboratory.4 In the Nordic countries, the emergence of carbapenemase producers has mainly been associated with patient transfer from other countries.3 In this study, we characterized the first three OXA-48-like producing Enterobacteriaceae from Norway, of which one isolate coproduced NDM-1. The bacterial isolates, two Klebsiella pneumoniae and one Escherichia coli, were submitted by three different laboratories to the National Reference Centre for Detection of Antimicrobial Resistance in Norway, based on reduced susceptibility to carbapenems (Table 1). All three isolates were isolated from patients who had recently been hospitalized or in contact with a hospital abroad in three separate countries (Romania, Morocco and Thailand) (Table 1). National guidelines in Norway recommend that patients who have been hospitalized in countries with a known high prevalence of multidrug-resistant Gram-negative bacteria are screened; two of the isolates were obtained from rectal screening samples. The third isolate was associated with a urinary tract infection. Reasons for hospitalization abroad included a traffic accident or heart problems. Relevant characteristics of OXA-48-like isolates and respective transconjugants MEM, meropenem; IPM, imipenem; ETP, ertapenem; CAZ, ceftazidime; CTX, cefotaxime; TZP, piperacillin/tazobactam; GEN, gentamicin; CIP, ciprofloxacin; TGC, tigecycline; CST, colistin; WT, wild-type; TC, transconjugant. aMLST was performed according to the protocols described at the MLST web sites for K. pneumoniae (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) and E. coli (http://mlst.ucc.ie/mlst/dbs/Ecoli). bThe antimicrobial susceptibility profiles of the isolates were determined using Etests according to the manufacturer's instructions (bioMérieux) and interpreted according to the clinical breakpoints set by EUCAST (http://www.eucast.org/clinical_breakpoints/). Relevant characteristics of OXA-48-like isolates and respective transconjugants MEM, meropenem; IPM, imipenem; ETP, ertapenem; CAZ, ceftazidime; CTX, cefotaxime; TZP, piperacillin/tazobactam; GEN, gentamicin; CIP, ciprofloxacin; TGC, tigecycline; CST, colistin; WT, wild-type; TC, transconjugant. aMLST was performed according to the protocols described at the MLST web sites for K. pneumoniae (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) and E. coli (http://mlst.ucc.ie/mlst/dbs/Ecoli). bThe antimicrobial susceptibility profiles of the isolates were determined using Etests according to the manufacturer's instructions (bioMérieux) and interpreted according to the clinical breakpoints set by EUCAST (http://www.eucast.org/clinical_breakpoints/). Analysis of the β-lactamase content of the isolates using PCRs followed by sequencing, as previously described,5–8 or a nucleic acid-based microarray (Check-Points, Wageningen, The Netherlands) revealed that two isolates harboured blaOXA-48 while one isolate contained both the blaOXA-181 variant and blaNDM-1 (Table 1). The coproduction of OXA-181 and NDM-1 has previously been described in K. pneumoniae from the Sultanate of Oman.9 Further, all isolates also possessed a CTX-M group 1 extended-spectrum β-lactamase (‘ESBL’), as is often found in isolates with blaOXA-48.2 All isolates were positive for the OXA-1 group, but negative for the OXA-2 group and OXA-9. The isolate that coproduced OXA-181 and NDM-1 showed high-level resistance to all carbapenems, while the two OXA-48-producing isolates were susceptible to both meropenem and imipenem, but resistant to ertapenem according to EUCAST clinical breakpoints (Table 1). High-level resistance to extended-spectrum cephalosporins was observed in all isolates due to the presence of blaNDM-1 and/or blaCTX-M. Other acquired antimicrobial resistance markers identified by PCR assays10 included the aac(6′)-Ib-cr variant found in all isolates, as well as qnrB in the OXA-48-positive K. pneumoniae isolate. The OXA-181- and NDM-1-producing isolate was also resistant to colistin, making this isolate an extremely drug-resistant variant. The two K. pneumoniae isolates were sequence typed as sequence type (ST) 405 and ST525, respectively, by multilocus sequence typing (MLST; Table 1). ST405 has recently been associated with OXA-48 in Spain.11 The E. coli isolate was assigned to ST405, a representative ST of the virulent phylogroup D lineage often associated with blaCTX-M-15.12 The blaOXA-48 gene has previously been found to be part of the composite transposon Tn1999 and variants thereof.2 Mapping of the genetic structure surrounding blaOXA-48-like was performed on our isolates using previously described primers.13 In both OXA-48-producing isolates, IS1999 was found on both ends of blaOXA-48, where the IS1999 element upstream of blaOXA-48 was interrupted by an IS1R, as described for the Tn1999.2 variant.2 In the blaOXA-181-positive isolate, ISEcp1 was identified upstream of blaOXA-181, as previously described.2 Conjugation experiments to E. coli J53-2 were successful for both blaOXA-48 isolates (Table 1). For the blaOXA-181-carrying isolate, only transconjugants positive for blaCTX-M and negative for blaOXA-181 and blaNDM-1 were obtained. The blaOXA-48 gene has mainly been associated with an ∼62 kb IncL/M plasmid harbouring conserved backbone genes (repA, traU and parA).13 Both blaOXA-48-positive isolates and the corresponding transconjugants were positive for repA, traU and parA by PCR. repA, traU and parA were not detected in the blaOXA-181 isolate. Plasmid analysis using S1 nuclease digestion of total genomic DNA followed by PFGE and Southern blot hybridization14 with the blaOXA-48 and parA probes revealed that blaOXA-48 was localized on an ∼50–60 kb plasmid that cohybridized with the parA probe (data not shown). The blaOXA-181 gene was localized on an ∼200 kb plasmid. In conclusion, this study further documents the ongoing global dissemination of blaOXA-48-like, as well as the coexistence of blaNDM-1 and blaOXA-181 in a single K. pneumoniae strain. The acquisition of blaOXA-48-like was related to colonization during foreign travel. These observations emphasize the necessity of rigorous screening of relevant patients upon entry after foreign travel, to prevent the further spread of these strains in low-prevalence countries. Part of this study was funded by a research grant from the Northern Norway Regional Health Authority. None to declare. We acknowledge the excellent technical assistance of Bjørg Haldorsen, Bettina Aasnæs, Runa Wolden and Cathrine Ramberg.