Spread of Klebsiella pneumoniae isolates producing OXA-48  -lactamase in a Tunisian university hospital

Sir, The emergence and dissemination of Klebsiella pneumoniae isolates harbouring carbapenemases is a serious problem. Since the initial report of the OXA-48 enzyme in a K. pneumoniae isolate from Turkey in 2001,1 OXA-48 producers have been reported in many countries of the world.2–4 Current reports indicate that OXA-48 producers are widespread, mostly from Mediterranean countries as well as other countries in Europe.2–4 In North Africa, OXA-48 producers have been identified in Morocco and Tunisia.3,4 The outbreaks of OXA-48-producing K. pneumoniae isolates have been described in several cities in Turkey, once in the UK and recently in France.4 In the present report, we describe the spread of OXA-48 associated with CMY-4- and CTX-M-14-producing K. pneumoniae clinical isolates in Sfax University Hospital. During a 6 month period (October 2009–March 2010), 153 clinical isolates of K. pneumoniae with reduced susceptibility to extended-spectrum cephalosporins and/or imipenem were recovered in Sfax University Hospital. Among these isolates, 21 (13.7%) produced the blaOXA-48 gene. These isolates were recovered from patients in eight different wards. The antibiogram determined by the disc diffusion method and MICs determined by agar dilution and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) revealed that all isolates were resistant to ticarcillin (MICs > 2048 mg/L) (Table 1). All but one isolate were resistant to extended-spectrum cephalosporins, including cefotaxime (MICs 4–1024 mg/L), ceftazidime (MICs 0.5–256 mg/L) and cefepime (MICs 2–64 mg/L). All isolates but one (Kp11) were susceptible to imipenem (MIC90 = 2 mg/L; MIC range = 0.5–8 mg/L). This isolate, Kp11, was intermediate to imipenem but susceptible to extended-spectrum cephalosporins. However, all isolates were resistant to ertapenem (MIC90 = 8 mg/L; MIC range = 2–32 mg/L). Characteristics of the K. pneumoniae strains and their respective transconjugants TIC, ticarcillin; TIM, ticarcillin/clavulanic acid (2 mg/L); CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; ETP, ertapenem. MICs were determined by susceptibility testing. Characteristics of the K. pneumoniae strains and their respective transconjugants TIC, ticarcillin; TIM, ticarcillin/clavulanic acid (2 mg/L); CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; ETP, ertapenem. MICs were determined by susceptibility testing. The β-lactamase genes detected by PCR as described previously and sequencing in the 21 OXA-48-positive K. pneumoniae isolates were as follows: blaOXA-48 (1 isolate); blaOXA-48 + blaCMY-4 + blaCTX-M-14 (14 isolates); blaOXA-48 + blaCMY-4 + blaCTX-M-14 + blaOXA-1 (3 isolates); blaOXA-48 + blaCMY-4 + blaCTX-M-14 + blaTEM-1 (1 isolate); blaOXA-48 + blaCTX-M-14 + blaOXA-1 (1 isolate); and blaOXA-48 + blaCMY-4 + blaCTX-M-15 (1 isolate). Transferability of the blaOXA-48 gene to Escherichia coli J53 was observed in 20 OXA-48-producing isolates. However, conjugation and electroporation experiments failed for Kp4, suggesting that in this isolate the blaOXA-48 gene might be chromosomally located. However, the blaOXA-48 gene was shown to be mostly plasmid-borne and associated with insertion sequence IS1999 but not integrons.2 Using a series of PCR primers, two IS1999 insertion sequences were found surrounding the blaOXA-48 gene in all our isolates, as found in the prototype OXA-48-positive K. pneumoniae 11978 isolate from Turkey.1,2 Molecular analysis of E. coli transconjugants showed that the blaOXA-48 and blaCMY-4 genes were detected on the same plasmid, explaining the resistance to β-lactams of the K. pneumoniae isolates and their transconjugants. Plasmid analysis showed that 19 isolates harboured a plasmid of ∼70–80 kb belonging to the same incompatibility group, IncA/C. This is the first description of the localization of blaOXA-48 in an A/C plasmid. In fact, the majority of studies reported that the blaOXA-48 gene was carried by a similar 70 kb plasmid for which the incompatibility group was indeterminate by the PCR-based replicon typing method. However, it was demonstrated recently that this plasmid would be of incompatibility group P.2 CMY-4 was reported to be located only in the A/C replicon.5 In our hospital CMY-4 was described, associated with VIM-4 metalloenzyme, in an epidemic K. pneumoniae clone.6 In our study, it may be hypothesized that the CMY-4-encoding plasmid had acquired OXA-48 in a single replicon IncA/C, as it was described in IncA/C plasmid pCC416 encoding VIM-4 and CMY-4 β-lactamases.5 PFGE revealed four clones (Table 1). The majority of isolates (18 K. pneumoniae isolates) were genetically related and belonged to clone A. Among these isolates, 13 produced OXA-48 + CMY-4 + CTX-M-14, 3 produced OXA-48 + CMY-4 + CTX-M-14 + OXA-1, 1 produced OXA-48 + CMY-4 + CTX-M-14 + TEM-1 and 1 produced OXA-48 + CTX-M-14 + OXA-1. The three remaining isolates were sporadic and they produced OXA-48, OXA-48+CMY-4 + CTX-M-14 and OXA-48 + CMY-4 + CTX-M-15. The present work indicates that the spread of the blaOXA-48 gene is not driven by the dissemination of a single K. pneumoniae clone that harboured the same A/C replicon (isolates belonging to clone A), but by the diffusion of this same replicon in different clones [two genetically unrelated isolates producing OXA-48 + CMY-4 (Kp10) and OXA-48 + CMY-4 + CTX-M-15 (Kp12)] or by the localization of blaOXA-48 in a different replicon [one isolate harbouring a plasmid with an indeterminate incompatibility group (Kp11)] and probably in the chromosome for another isolate, Kp4. OXA-48-producing isolates co-expressed several β-lactamases, including the class A extended-spectrum β-lactamase SHV-2a and especially CTX-M-15 and the narrow-spectrum β-lactamases TEM-1, OXA-1 and OXA-47.2–4 The Tunisian OXA-48-producing K. pneumoniae isolated in France co-produced TEM-1 and CTX-M-15.3 All our OXA-48-producing isolates, except one, co-produced at least two β-lactamases, such as CMY-4, CTX-M-14, OXA-1, TEM-1 and CTX-M-15. This study demonstrates the alarming diffusion of the blaOXA-48 gene among expanded-spectrum cephalosporin-resistant K. pneumoniae (13.7%), mainly associated with the blaCMY-4 and blaCTX-M-14 genes. The emergence of carbapenem resistance among Enterobacteriaceae is worrying since carbapenems are often the last resort for treating infections due to third-generation cephalosporin-resistant isolates. Thus, there is a clear need for a structured nationwide prevalence study of OXA-48-producing Enterobacteriaceae in Tunisia. This study was supported by the Ministry of Scientific Research Technology and Competence Development of Tunisia. None to declare. We would like to thank Professor G. Arlet (Tenon Hospital, Paris, France) for control strains.