Intercontinental travels of patients and dissemination of plasmid-mediated carbapenemase KPC-3 associated with OXA-9 and TEM-1

Sir, Among β-lactam antibiotics, carbapenems are those that have the highest stability towards the hydrolytic activity of most of the innate natural and acquired extended-spectrum β-lactamases prevalent within bacterial species involved in clinical practice. However, emergence of acquired carbapenem-hydrolysing β-lactamases, i.e. carbapenemases, which were first recognized in the early 1990s in some isolates of opportunistic Gram-negative bacilli, has been increasingly reported from various parts of the world during the last decade.1 Carbapenem-hydrolysing β-lactamases can be metallo-β-lactamases, expanded-spectrum oxacillinases or Ambler class A enzymes.1,2 KPC β-lactamases are class A carbapenemases, which were first detected in 2001 in a Klebsiella pneumoniae isolate from North Carolina.3 Soon afterwards, several reports documented the emergence of Enterobacteriaceae strains from various species producing KPC-2 and KPC-3 β-lactamase variants in the eastern USA and recently disseminated in other countries worldwide, such as France, Columbia, Israel and China.2 A 31-year-old man was admitted to the intensive care unit of the Institut Gustave-Roussy in November 2005 with sepsis related to intra-abdominal suppuration. The patient had previously undergone a total gastrectomy for intractable gastric bleeding episodes during a trip to New York and had stayed for 3 weeks in the intensive care unit of a local hospital. Blood cultures taken at admission in our hospital yielded a K. pneumoniae isolate fully susceptible to antimicrobial agents usually active against this species. Specimens taken during subsequent surgical debridement of abdominal abscesses yielded an Enterobacter cloacae isolate resistant to all β-lactam antibiotics including imipenem (Table 1), aminoglycosides, sulphonamides and fluoroquinolones and an Enterococcus faecium isolate resistant to ampicillin and vancomycin, and which carried the vanA gene (detected by PCR). MICs of β-lactams for a clinical strain of E. cloacae, a transconjugant strain (Tc E. coli C600), E. coli C600, recombinant E. coli DH10B producing KPC-3 enzyme (KPC-3 clone), recombinant E. coli DH10B producing TEM-1 enzyme (TEM-1 clone), recombinant E. coli DH10B producing OXA-9 enzyme (OXA-9 clone) and E. coli DH10B aCLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB, tazobactam at a fixed concentration of 4 mg/L. MICs of β-lactams for a clinical strain of E. cloacae, a transconjugant strain (Tc E. coli C600), E. coli C600, recombinant E. coli DH10B producing KPC-3 enzyme (KPC-3 clone), recombinant E. coli DH10B producing TEM-1 enzyme (TEM-1 clone), recombinant E. coli DH10B producing OXA-9 enzyme (OXA-9 clone) and E. coli DH10B aCLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB, tazobactam at a fixed concentration of 4 mg/L. A double disc diffusion assay with imipenem and amoxicillin/clavulanate discs showed a small increase in imipenem activity against the E. cloacae isolate, whereas no synergistic effect was observed with EDTA (data not shown), thus excluding the presence of a metallo-β-lactamase. Using ceftazidime (2 mg/L)/sodium azide (400 mg/L) as selective agent and azide-resistant Escherichia coli C600 as recipient strain, a conjugative experiment was performed. It allowed the transfer en bloc of resistance to β-lactam antibiotics including imipenem (Table 1), aminoglycosides and sulphonamides. Plasmid analysis showed that both the clinical and the transconjugant strains carried a unique plasmid, more than 126 kb in size. The gene encoding for KPC β-lactamase was detected by PCR in both the donor and recipient strains. Sequencing of the amplified fragment showed it to display 100% identity with blaKPC-3 (GenBank accession number AF395881). PCR amplification with specific primers and sequencing analysis showed that both the clinical isolate and the transconjugant carried blaTEM-1. The genetic environment of these β-lactamase-encoding genes was further investigated using a cloning approach. Total DNA of the transconjugant strain was partially digested with the endonuclease Sau3A, and fragments were ligated into a pACYC184 vector (Fermentas, St Rémy Les Chevreuse, France) before transformation of the recipient strain, E. coli DH10B. By selection on medium supplemented with ceftazidime (2 mg/L), we obtained a clone resistant to imipenem (Table 1) that produced the KPC-3 enzyme. Analysis of the blaKPC-3 cloning fragment (GenBank accession number AM774409) showed that blaKPC-3 displayed the same genetic environment as blaKPC-2 in Salmonella Cubana 4707 (GenBank accession number AF481906). By selection on ticarcillin (50 mg/L), we obtained two different phenotypes of resistance to β-lactams. Sequencing of the inserts showed the presence of blaTEM-1 or blaOXA-9 (Table 1). Accurate intragenic region amplifications of the OXA-9 fragment suggested blaTEM-1 and blaOXA-9 to be part of Tn1331, as has been previously described for the multiresistance plasmid pJHCMW1 of K. pneumoniae (GenBank accession number AF479774).4 This is the first KPC-3 β-lactamase-producing E. cloacae strain recovered from a patient in France. Interestingly, a KPC-2-producing K. pneumoniae strain has been recently recovered from another patient admitted to a hospital in Paris.5 A common feature of these two patients was that they stayed in intensive care units of New York City hospitals before their admission to a French hospital. More recently, K. pneumoniae and E. coli isolates harbouring blaKPC-2 were reported in Columbia, Israel and China, with no discernible linkage to the USA.2 Furthermore, as recently described, our KPC-producing strain was found to accumulate other β-lactam resistance enzymes (OXA-9 and TEM-1).2 Emergence of KPC β-lactamase-producing strains of Enterobacteriaceae and, recently, of Pseudomonaceae is worrisome, because they are resistant to all β-lactam antibiotics and often also to most other classes of the available antibacterial agents.2 Moreover, some reports have focused on the ability of some KPC β-lactamase-producing K. pneumoniae strains to cause outbreaks in medical centres in New York City.6 Evidence of intercontinental transfer of KPC β-lactamase-producing strains through patient travel stresses the risk for the rapid worldwide dissemination of these recently recognized carbapenemases. This work was partially funded by grants from the Faculté de Médecine Pierre et Marie Curie, Université Paris 6 and from the European Community (6th PCRD Contract: LSHM-CT 2003-503335). None to declare.