A mosaic transposon encoding OXA-48 and CTX-M-15: towards pan-resistance

Sir, Reports of OXA-48-producing Enterobacteriaceae have recently dramatically increased worldwide, in particular in the Middle East and Europe.1 The dissemination of the blaOXA-48 gene among various enterobacterial species is mainly related to the dissemination of a single 62 kb IncL/M conjugative plasmid, which carries the blaOXA-48 gene as the only resistance determinant.2 The co-production of an extended-spectrum β-lactamase (ESBL) in 80% of the OXA-48 producers (L. Poirel and P. Nordmann, unpublished results) is therefore due to the acquisition of additional plasmids. The blaOXA-48 gene was first identified as part of Tn1999, a composite transposon made of two copies of the insertion sequence IS1999 (Figure 1).3 Two other Tn1999-derivative structures have been reported.3,4 Transposon Tn1999.2, identified in various enterobacterial isolates from Turkey, differs from Tn1999 by the insertion of insertion sequence IS1R into the left-hand IS1999 copy (Figure 1).4 Transposon Tn1999.3 has been identified from a single Escherichia coli isolate recovered in Italy, being similar to Tn1999.2 but harbouring a second copy of IS1R (Figure 1).5 We report here the identification of two enterobacterial isolates harbouring the blaOXA-48 gene encoded by a novel Tn1999 transposon derivative that additionally contains the ESBL gene blaCTX-M-15. Linear map of the blaOXA-48-bearing Tn1999-like transposon structures. Open reading frames are shown as arrows or horizontal boxes with an arrow indicating the orientation of the coding sequence. Target site duplications are represented by black bars. E. coli ESS-1 and Enterobacter cloacae ESS-2 were isolated in France in September 2011 from a rectal swab of a 70-year-old patient previously hospitalized in Algeria. Identification of those two isolates was performed using the API 20E system (bioMérieux, La Balme-les-Grottes, France). Susceptibility testing done by the disc diffusion method and MICs determined by Etest (bioMérieux) and interpreted according to the CLSI guidelines6 revealed that the two isolates were resistant to penicillins, penicillin/β-lactamase inhibitor combinations and broad-spectrum cephalosporins, and had decreased susceptibility to carbapenems. The MICs of imipenem, ertapenem and meropenem were 0.5, 0.5 and 0.12 mg/L, respectively, for E. coli ESS-1 and 0.75, 3 and 0.38 mg/L, respectively, for E. cloacae ESS-2. The patient did not develop any clinical infection and these organisms were considered to be colonizers. PCR analysis and sequencing, performed as described previously,7 identified the blaOXA-48 carbapenemase gene and the blaCTX-M-15 ESBL gene in both isolates. E. coli ESS-1 additionally harboured the blaTEM-1 gene. Conjugation experiments using E. coli ESS-1 and E. cloacae ESS-2 as donors and E. coli J53 as recipient were performed as described,4 using ertapenem (0.5 mg/L) and azide (100 mg/L) for selection. A single type of transconjugant was obtained for both donor strains. The E. coli transconjugants displayed the exact same resistance profile and were resistant to penicillins, penicillin/β-lactamase inhibitor combinations and broad-spectrum cephalosporins, and were of decreased susceptibility to carbapenems. For these two transconjugants, the MICs of imipenem, ertapenem and meropenem were 0.5, 0.38 and 0.12 mg/L, respectively. PCR experiments showed that both transconjugants co-harboured the blaOXA-48 and blaCTX-M-15 genes. Plasmid analysis showed that both E. coli transconjugants harboured a single ∼66 kb plasmid. By using specific primers designed from the blaOXA-48-positive reference plasmid pOXA-48a from Klebsiellapneumoniae 11978,2 we showed that the single plasmid of both E. coli transconjugants belonged to the IncL/M group, as did most of the OXA-48-positive isolates.2 Mapping of the blaOXA-48-flanking regions by PCR revealed a peculiar genetic structure. The blaOXA-48 gene was part of a novel transposon named Tn1999.4, consisting of Tn1999.2 truncated by another transposon. This transposon, named Tn2015, was 4603 bp long and consisted of ISEcp1, blaCTX-M-15 and a truncated Tn2-type transposase gene (Figure 1). Transposon Tn2015 was bracketed by two imperfect 14 bp inverted-repeat sequences, the inverted repeat left (IRL) of ISEcp1 and an inverted repeat right (IRR)-like sequence sharing 6 out of 14 bp with the IRL of ISEcp1 (Figure 1). Tn2015 was inserted into the LysR-encoding gene of Tn1999 and a 5 bp duplication of the target site (TTTAA) was identified as a signature of the ISEcp1-mediated transposition event (Figure 1). Mapping of the Tn1999.4-flanking regions revealed that this transposon was inserted into the tir gene, as observed for Tn1999 in plasmid pOXA-48a.2 Since the insertion of Tn1999.4 was identified at the same position as Tn1999, this suggested that Tn1999.4 likely evolved from Tn1999.2 by the insertion of another transposon element into Tn1999.2, once it had been incorporated onto the plasmid backbone. We describe here a novel Tn1999 derivative, which actually corresponds to a mosaic structure in which the transposon carrying the carbapenemase gene blaOXA-48 is associated with that carrying the ESBL gene blaCTX-M-15. This association gave rise to a complex genetic structure capable of compromising the efficiency of all available β-lactams. To our knowledge, this is the first identification of a transposon carrying both an ESBL and a carbapenemase gene, being a significant evolutionary step towards pan-resistance. Considering that at least one copy of IS1999 and subsequently its transposase gene is still intact, we may speculate that the whole structure might still be functional and might mobilize the two β-lactamase genes as a whole. The nucleotide sequence of the Tn1999.4 transposon has been submitted to the EMBL/GenBank nucleotide sequence database under accession number JX423832. This work was supported by a grant from the Ministère de la Recherche, Université Paris XI, Paris and by the INSERM, France. The research leading to these results has also received funding from the European Community's Seventh Framework Programme FP7/2007-2013 (TEMPOtest-QC and R-GNOSIS). None to declare.