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Antimicrob Agents Chemother. Oct 2011; 55(10): 4896–4899.
PMCID: PMC3186949

Characterization of OXA-181, a Carbapenem-Hydrolyzing Class D β-Lactamase from Klebsiella pneumoniae[down-pointing small open triangle]

Abstract

Klebsiella pneumoniae KP3 was isolated from a patient transferred from India to the Sultanate of Oman. K. pneumoniae KP3 was resistant to all β-lactams, including carbapenems, and expressed the carbapenem-hydrolyzing β-lactamase OXA-181, which differs from OXA-48 by four amino acid substitutions. Compared to OXA-48, OXA-181 possessed a very similar hydrolytic profile. The blaOXA-181 gene was located on a 7.6-kb ColE-type plasmid and was linked to the insertion sequence ISEcp1. The ISEcp1-mediated one-ended transposition of blaOXA-181 was also demonstrated.

TEXT

Resistance to carbapenems in Enterobacteriaceae is related mainly to the production of carbapenem-hydrolyzing β-lactamases of Ambler class A, such as KPC or GES (30); class B, such as IMP, VIM, and NDM (5, 30); or class D, such as OXA-48 (24, 26). OXA-48 hydrolyzes penicillins and imipenem but spares expanded-spectrum cephalosporins (24). The genetic environment of blaOXA-48 is made up of two copies of the insertion sequence IS1999 located at its ends, giving rise to composite transposon Tn1999. The occurrence of OXA-48 producers has been extensively reported in the Mediterranean area (10). Recently, OXA-163 has been identified, which shares 98% amino acid identity with OXA-48 (21) and hydrolyzes expanded-spectrum cephalosporins but has impaired activity toward carbapenems.

Here we report on K. pneumoniae KP3, which was isolated from a wound of a 54-year-old Tanzanian patient hospitalized in the surgery unit of the Muscat hospital in the Sultanate of Oman in March 2010. Before her transfer to Muscat, she had been initially operated on for an open femur fracture in Tanzania (unknown city) and then transferred to Mumbai, India, where she was operated on again. Identification of K. pneumoniae KP3 was performed by using the API 20E system (bioMérieux, Marcy l'Etoile, France), and susceptibility testing was done according to the CLSI guidelines (7). K. pneumoniae KP3 showed resistance to β-lactamase inhibitor–penicillin combinations, broad-spectrum cephalosporins, and aztreonam and decreased susceptibility to cefoxitin and carbapenems (Table 1). In addition, K. pneumoniae KP3 was resistant to aminoglycosides, fluoroquinolones, sulfonamides, and tetracycline. The MICs of colistin, tigecycline, and fosfomycin were, respectively, 0.5, 1, and 8 μg/ml. Whole-cell DNA of K. pneumoniae KP3 was extracted as described previously (20) and used as a template under standard PCR conditions as previously reported (8, 23, 2628, 30, 31). K. pneumoniae KP3 possessed a blaOXA-48-like gene corresponding to blaOXA-181, the corresponding β-lactamase differing from OXA-48 by four amino acid substitutions, namely, Thr104Ala, Asn110Asp, Glu175Gln, and Ser179Ala, according to the class D β-lactamase nomenclature (14). K. pneumoniae KP3 isolate also possessed the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-15 together with the blaOXA-1, blaTEM-1, and blaSHV-11 genes. Isoelectric focusing analysis, performed as described previously (24), showed that K. pneumoniae KP3 produced β-lactamases with pI values of 5.4, 7.2, 7.4, 7.6, and 8.9, corresponding to TEM-1, OXA-181, OXA-1, SHV-11, and CTX-M-15, respectively.

Table 1.
MICs of β-lactams for various bacteriaa

This is another example of the spread of OXA-181 producers after that identified in France (27) and in The Netherlands (15) and the extensive spread of OXA-181 producers from India (5). Data available now suggest that although the blaOXA-48, blaOXA-163, and blaOXA-181 genes are structurally related, their ecological niches might be different, i.e., OXA-48 in the Mediterranean area, OXA-163 in South America, and OXA-181 at least in India.

Multilocus sequence typing, performed as described previously (12), identified K. pneumoniae KP3 as an ST11 strain that corresponds to a single-locus mutant of the widespread ST258 clone, which is known to be the main vehicle for the spread of blaKPC genes (9). K. pneumoniae clone ST11 has been reported to be the main K. pneumoniae clone producing CTX-M-15 in Asia and Hungary (11, 18), SHV-5 or DHA-1 in the Czech Republic (6), KPC in China and Brazil (1, 29), and VIM-4 in Hungary (16).

For comparison of hydrolytic activities, the blaOXA-181 and blaOXA-48 genes were amplified using preOXA-48A (5′-TATATTGCATTAAGCAAGGG-3′) and preOXA-48B (5′-CACACAAATACGCGCTAACC-3′) and K. pneumoniae KP3 and 11978 as templates. The amplicons were cloned into the vector pTOPO (Qiagen, Courtaboeuf, France) and expressed in E. coli TOP10 by following the manufacturer's recommendations. The resulting E. coli strains, harboring recombinant plasmids pTOPO-OXA-48 and pTOPO-OXA-181 and expressing OXA-48 and OXA-181, respectively, displayed the exact same β-lactam resistance phenotype (Table 1). The specific activities of the β-lactamases OXA-181 and OXA-48 for carbapenems, measured as described previously (24), were very similar, at 230 and 240 mU mg−1 protein for imipenem, 9 and 10 mU mg−1 protein for ertapenem, and 10 and 10 mU mg−1 protein for meropenem, respectively.

Plasmid DNA analysis showed that K. pneumoniae KP3 possessed two plasmids, pKP3-A and pKP3-B, ca. 7 and 70 kb in size, respectively (data not shown). Electroporation into E. coli TOP10 using imipenem (0.5 μg/ml) for selection gave E. coli TOP10(pKP3-A) harboring a ca. 7-kb plasmid bearing the blaOXA-181 gene (Table 1). Plasmid pKP3-A was not typeable by PCR-based replicon typing (PBRT) as described previously (4). Mating-out assays gave only one type of transconjugant that corresponded to E. coli J53(pKP3-B) expressing the ESBL CTX-M-15. Plasmid pKP3-B also remained untypeable by PBRT (4). Attempts to transfer the pKP3-A plasmid by mating-out assays were unsuccessful, suggesting that this plasmid is not self-conjugative.

The insertion sequence ISEcp1 was identified upstream of the blaOXA-181 gene, as reported for other β-lactamase resistance genes, such as blaCTX-M, blaCMY, and blaACC (13, 19, 22, 25). The blaOXA-181 gene was part of a novel 3,139-bp potential transposon, named Tn2013, flanked by a 5-bp duplication of the target site (ATATA), the signature of a transposition event. This novel transposon Tn2013 was bracketed by two imperfect 14-bp inverted-repeat sequences, namely, the inverted long repeat (IRL) of ISEcp1 and an IRR2 sequence with 7 out of 14 bp identical to those of the original IRR1 of ISEcp1 (Fig. 1). This observation is in accordance with previous studies showing that ISEcp1 is able to use as IRRs sequences sharing weak identity with its original IRR1 sequence, corresponding to a one-ended transposition mechanism (17). This is the first report of the ISEcp1-related acquisition of a class D β-lactamase-encoding gene. As described previously (22), −35 (TTGAAA) and −10 (TACAAT) promoter sequences were identified inside ISEcp1 near its IRR; therefore, it is very likely to be involved in the expression of blaOXA-181, as demonstrated for blaCTX-M genes (22).

Fig. 1.
Schematic map of the structure of transposon Tn2013. Open reading frames are shown as arrows or horizontal boxes with an arrow indicating the orientation of the coding sequence. The IRL, IRR1, and IRR2 motifs are indicated (black base pairs are identical, ...

The transposition ability of Tn2013 was investigated as described previously (17), by using mating-out assays with E. coli RZ211 harboring plasmid-located Tn2013 (Ticr) and the gentamicin-resistant plasmid pOX-38 (Genr) as the donor and E. coli J53 (Azr) as the recipient strain. The recombinant pTOPO-OXA-181 plasmid harboring transposon Tn2013 used for these experiments was obtained as described previously (24), by using primers preTn2013A (5′-CAAAAGCATCAGACACCTCC-3′) and preTn2013B (5′-ATATAAGTCTCTCTAGTCGG-3′). PCR experiments confirmed that those transconjugants possessed the entire Tn2013 sequence. Plasmid DNAs of several Ticr, Genr, and Azr transconjugants were extracted, but sequencing could not confirm the location of transposon Tn2013 on plasmid pOX38, suggesting that Tn2013 had been integrated into the E. coli J53 chromosome. These E. coli transconjugants were susceptible to kanamycin, ruling out the possibility that this could correspond to an azide-resistant donor strain and ruling out the integration of plasmid pTOPO-OXA-181 into the chromosome of E. coli J53.

A primer-walking approach was used to fully sequence plasmid pKP3-A. Its size was 7,605 bp, and it contained nine open reading frames, MobC, MobA, MobB, MobD, TnpA of ISEcp1, OXA-181, RepA, ΔEre-like, and ΔLysR-like (Fig. 2). The GC content of pKP3-A was 48.6%. Overall, the backbone of pKP3-A showed features of those of ColE-type plasmids, with a divergent scaffold sequence. The RepA protein showed 61% amino acid identity with that of the broad-host-range plasmid pETEC58 that had been identified in E. coli (Table 2) (8). Several Mob proteins (mobA, mobB, mobC, and mobD) forming a plasmid mobilization system were identified that show the highest similarities to those encoded by plasmid pAsa2 from Aeromonas salmonicida (Table 2) (3). Downstream of the blaOXA-181 gene, an 81-bp fragment of a gene encoding a putative LysR-type transcriptional regulator showing 96% nucleotide identity with that identified downstream of the blaOXA-48 gene was identified (Table 2) (2, 24). Comparison of the two blaOXA-lysR loci suggested that the blaOXA-181 and blaOXA-48 genes were acquired in two separate and independent genetic events and that both were likely from two Shewanella species. Indeed, the blaOXA-48-like–lysR locus had been identified in the chromosome of Shewanella oneidensis, which is considered to be a progenitor of blaOXA-48-like genes (23).

Fig. 2.
Map of natural plasmid pKP3-A harboring the blaOXA-181 gene. Coding regions are indicated by arrows showing the direction of transcription.
Table 2.
Open reading frames identified in pKP3-A

The broadness of plasmid pKP3-A's host range was assessed by obtaining electrotransformants using Pseudomonas aeruginosa PU21 and Acinetobacter baumannii CIP7010 as recipient strains as described previously (Table 1) (21). PCR experiments confirmed the presence of the blaOXA-181 gene in those transformants, and plasmid DNA analysis confirmed the presence of a ca. 7-kb plasmid in the P. aeruginosa transformants (data not shown) but not in the A. baumannii transformants, suggesting the integration of blaOXA-181 into the chromosome in the latter case.

This study characterized the β-lactamase OXA-181, which confers a pattern of resistance similar to that conferred by OXA-48. Interestingly, the acquisition of the blaOXA-181 gene was linked to ISEcp1, which is a very efficient genetic vehicle for spreading clinically significant expanded-spectrum β-lactamases such as plasmid-encoded cephalosporinases and ESBLs. Disturbingly, the blaOXA-181 gene has already been reported in different enterobacterial species, all from India (5, 15, 27), suggesting that this part of the world might be an important reservoir of this carbapenemase gene, in addition to blaNDM-1 (with which it can be associated in the same strain).

Nucleotide sequence accession number.

The nucleotide sequence data reported in this work have been deposited in the GenBank database under accession no. JN205800.

Acknowledgments

This work was partially funded by a grant from the INSERM (U914) and the Université Paris XI, Paris, France, and by grants from the European Community (TROCAR HEALTH-F3-2008-223031 and TEMPOtest-QC HEALTH-2009-241742).

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 July 2011.

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