Plasmid-encoded OXA-48 carbapenemase in Escherichia coli from Israel

Abstract

Sir, Carbapenem resistance among Enterobacteriaceae in Israel emerged in 2004 and was observed mainly in Klebsiella pneumoniae, but also in Enterobacter species and Escherichia coli. Since its emergence, carbapenem resistance in these species, both in clinical and colonizing isolates, has been rendered by the production of plasmid-mediated K. pneumoniae carbapenemase (KPC). In late 2007, a woman in her early thirties previously diagnosed with acute lymphoblastic leukaemia, was admitted to the haematology ward in Tel Aviv Sourasky Medical Center. The patient had arrived in Israel from Jordan in order to undergo chemotherapy and, later, bone marrow transplantation. During the first month after transplantation the patient was intermittently febrile and was treated with various antimicrobials, including piperacillin/tazobactam, amikacin, ciprofloxacin, vancomycin, imipenem and voriconazole. Two months after admission, while being treated with imipenem and voriconazole, the patient suffered from fever, dyspnoea and renal failure, and was transferred to the intensive care unit. During that period a carbapenem-resistant E. coli strain (E. coli 1736) was isolated from a Hickman catheter, leading to removal of the catheter and further treatment with ceftazidime and colistin. Treatment with these antibacterial agents cleared the OXA-48-producing E. coli, yet unfortunately the patient died 3 months later from a systemic Pseudomonas infection. E. coli 1736 was multidrug resistant, showing resistance to penicillins, piperacillin/tazobactam, aminoglycosides, quinolones and carbapenems, but susceptibility to all cephalosporins, aztreonam, tigecycline and colistin (Table 1). Analytical isoelectric focusing (IEF) performed on crude enzyme preparations revealed the presence of two b-lactamases with pIs of 5.4 and 7.2 (data not shown). PCR screening for the presence of b-lactamases in E. coli 1736 indicated the presence of blaTEM-1 and blaOXA-48 genes corresponding to the pIs of the b-lactamases visualized by IEF. Plasmid analysis of E. coli 1736 revealed four plasmids, three of around ≤50 kb in size and a larger plasmid of around 100 kb. Plasmid DNA was purified and transformed into E. coli DH10B. Transformant colonies that were screened positive for blaOXA-48 by PCR harboured the 50 kb plasmid. Southern analysis of plasmid DNA derived from these transformants using a blaOXA-48-labelled probe demonstrated the presence of blaOXA-48 on the acquired 50 kb plasmid. Acquisition of this plasmid increased the MICs of imipenem, meropenem and ertapenem without conferring full resistance (Table 1). PCR mapping of the genetic environment surrounding blaOXA-48 was performed in collaboration with the laboratory of Professor P. Nordmann (Hospital de Bicetre, Paris, France). blaOXA-48 was found to be located inside Tn1999.2, similar to the structure described for other enteric strains, such as the E. coli strain from Turkey and the K. pneumoniae strain from Lebanon. E. coli 1736 was not resistant to all b-lactam antibiotics as reported for other OXA-48-producing strains, yet it presented a high level of resistance to the commonly used carbapenems (MICs ≥16 mg/L), higher than usually seen in KPC-producing E. coli strains isolated in our hospital. These high carbapenem MICs suggested the presence of additional resistance mechanisms together with OXA-48 carbapenemase. Outer membrane protein (OMP) produced by E. coli 1736 was determined by PCR and sequencing of ompA, ompC and ompF genes. Further OMP analysis was performed by protein extraction and separation on Tris–Tricine gels using SDS-PAGE followed by mass spectrometry (performed in the Biological Mass Spectrometry Facility at the Weizmann Institute of Science). Both methods indicated the absence of at least one major porin, OmpC. Until the isolation of E. coli 1736, carbapenem resistance in E. coli in our country was exclusively attributed to the Ambler class A carbapenemase KPC. This is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. To seek the possible origin of this strain we performed multilocus sequence typing (MLST; http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi. html), which genotyped the strain as sequence type (ST) 2, an E. coli ST that has never been recorded previously in our Research letters