Escherichia coli from Italy Producing OXA-48 Carbapenemase Encoded by a Novel Tn1999 Transposon Derivative

LETTER

OXA-48 is an emerging class D carbapenemase originally identified in isolates from Turkey (14) and subsequently detected in several European and north African countries (10). Klebsiella pneumoniae is the most common host for OXA-48, but the enzyme has also been detected in Escherichia coli and Enterobacter cloacae (10). The bla_OXA-48 gene is carried by the composite transposon Tn1999 or a variant thereof, named Tn1999.2 (1), and is plasmid mediated. A 60-kb IncL/M-related conjugative plasmid, named pOXA-48a, which carries the carbapenemase gene as the only resistance determinant, was found to mediate the horizontal dissemination of bla_OXA-48 among strains of different species and from different sources (13).

In this report we describe the first case of a strain from Italy producing OXA-48, encoded by a pOXA-48a-like plasmid which carries a new variant of Tn1999.

E. coli ECBZ-1 was isolated in April 2011 from a urinary tract infection (UTI) of a 54-year-old female patient living in northern Italy. Susceptibility testing, performed by Etest (bioMérieux, Marcy l'Etoile, France), revealed that the isolate was resistant to trimethoprim-sulfamethoxazole, penicillins (including β-lactamase inhibitor combinations), narrow-spectrum cephalosporins, and ertapenem (even by disk diffusion), while it remained susceptible to other carbapenems, expanded-spectrum cephalosporins, aminoglycosides, fluoroquinolones, and fosfomycin (Table 1). Initially treated empirically with trimethoprim-sulfamethoxazole without improvement, the infection was successfully treated with oral fosfomycin (3 g per day for 2 days). A modified Hodge test (9) suggested production of carbapenemase, but neither EDTA nor boronic acid affected meropenem susceptibility in combination disk tests (16). PCR analysis (3, 4, 6, 11, 12, 14), and sequencing revealed the presence of a bla_OXA-48 carbapenemase gene and of a bla_TEM-1b gene. This β-lactamase profile was consistent overall with the resistance phenotype of ECBZ-1, since OXA-48 hydrolyzes carbapenems (although weakly), penicillins, and narrow-spectrum cephalosporins but not expanded-spectrum cephalosporins and is not inhibited by clavulanate and sulfones (5, 14). The peculiar resistance phenotype of ECBZ-1 underlines the importance of surveillance of isolates showing resistance only to ertapenem even in the presence of susceptibility to expanded-spectrum cephalosporins.

Table 1.

MICs for E. coli ECBZ-1, J53(pECBZ-1), and J53

Antibiotic	MIC (μg/ml) (category)a
	ECBZ-1	J53 (pECBZ-1)	J53
Ampicillin	>32 (R)	>32	0.5
Amoxicillin-clavulanate	>32 (R)	>32	0.5
Cefotaxime	0.75 (S)	0.75	0.25
Ceftazidime	1 (S)	0.38	0.25
Cefepime	0.75 (S)	0.125	0.047
Imipenem	1b (S)	0.5	0.064
Ertapenem	2b (R)	1	0.016
Meropenem	1b (S)	0.5	0.008
Piperacillin-tazobactam	256 (R)	8	0.25
Levofloxacin	0.064 (S)	ND	ND
Ciprofloxacin	0.023 (S)	ND	ND
Amikacin	2 (S)	ND	ND
Gentamicin	0.064 (S)	ND	ND
Tobramycin	1 (S)	ND	ND
Trimethoprim-sulfamethoxazole	256 (R)	0.023	0.023
Tigecycline	0.75 (S)	ND	ND
Colistin	0.19 (S)	ND	ND

^aMICs were determined for E. coli ECBZ-1, E. coli transconjugant J53(pECBZ-1), and recipient strain E. coli J53. Category classification was based on updated EUCAST breakpoints (http://www.eucast.org/clinical_breakpoints/ [version 2.0, January 1 2012]). R, resistant; S, susceptible; ND, not determined.

^bDiameters of inhibition: imipenem, 23 mm (S); meropenem, 22 mm (S); ertapenem, 16 mm (R).

Analysis of clinical records revealed a history of recurrent UTIs and a previous vacation (in November 2010) in Egypt, a country where OXA-48-producing isolates have been reported (10), suggesting a cross-border source for the OXA-48-producing strain.

ECBZ-1 belongs in phylogenetic group D (2) and in sequence type ST2076 (http://mlst.ucc.ie/mlst/dbs/Ecoli) related to clonal complex 394, which also includes ST471, which was found to be associated with diffusion of KPC-2 carbapenemase in Israel (7).

A conjugation experiment (8) using E. coli J53 (pro met Rif^r Nal^r) (17) as the recipient and selection with ertapenem (0.5 μg/ml), nalidixic acid (32 μg/ml), and rifampin (250 μg/ml) yielded J53 transconjugants (frequency, 7 × 10⁻⁵ ± 1.5 × 10⁻⁵ transconjugants per recipient) containing the bla_OXA-48 but not the bla_TEM-1b gene, as assessed by PCR, and showing a resistance phenotype resembling that of the donor except for resistance to trimethoprim-sulfamethoxazole (Table 1). Analysis of the plasmid (15) from a randomly selected transconjugant revealed a ca. 60-kb plasmid, named pECBZ-1 (data not shown), whose backbone was confirmed to be identical or closely related to that of the pOXA-48a IncL/M type plasmid by PCR analysis of the repA, traU, and parA genes (13).

Mapping of the bla_OXA-48-flanking regions and sequencing of the amplicons revealed an original genetic context that was similar overall to Tn1999.2 (1) but differed from the latter by the presence of a second copy of IS1R inserted 199 bp downstream bla_OXA-48 (Fig. 1). In this new Tn1999 transposon variant, named Tn1999.3, bla_OXA-48 could have the advantage of overexpression driven by the upstream IS1R copy (as in Tn1999.2 [1]), while the two copies of IS1R flanking bla_OXA-48 define a new putative composite transposon that might further mobilize the carbapenemase gene (Fig. 1). Mapping of the Tn1999.3-flanking regions revealed that the transposon was inserted into a tir gene, at the same position reported for Tn1999 in plasmid pOXA-48a (13) (Fig. 1), suggesting that that Tn1999.3 evolved from Tn1999 by stepwise insertion of the IS1R elements into the latter transposon, once it had been incorporated into the plasmid backbone. This hypothesis for the evolution of Tn1999.3 is also consistent with the fact that no direct repeats are present flanking the putative composite transposon made up of the two IS1R elements.

Fig 1.

Linear map of the transposon Tn1999.3 carried by ECBZ-1, compared with the other Tn1999 variants. Small arrows show primer positions. Primers used to characterize Tn1999.3 were OXA-48A and OXA-48B (14), IS1999 (5′-TGATGTTGTGCTTGGTTCGG-3′), LysR_Rev (5′-GCTAGTGCCAATCTTACAGG-3′), Tir_1 (5′-GCCCAACAGTAAACCCAGC-3′), and Tir_2 (5′-AGTTTATGCTGGTTCTGCTGG-3′). The putative composite transposon formed by the two IS1R insertion sequences, carrying bla_OXA-48, is underlined.

Nucleotide sequence accession number.

The nucleotide sequence of the bla_OXA-48 gene and flanking regions has been submitted to the GenBank/EMBL database and assigned accession no. HE617182.