Detection of OXA-370, an OXA-48-Related Class D β-Lactamase, in Enterobacter hormaechei from Brazil

LETTER

The class D β-lactamase OXA-48 (1) and its variants have emerged as important determinants of carbapenem resistance in Enterobacteriaceae, representing a public health concern in some countries (2–4). The objective of this study was to report a new OXA-48 variant.

In May 2013, the 87F-2 strain, initially identified as Enterobacter cloacae complex by a Vitek 2 system (bioMeriéux), was recovered from a rectal swab collected from an adult patient in an intensive care unit of a tertiary-care hospital in Porto Alegre, Brazil, in a routine surveillance for carbapenem-resistant Enterobacteriaceae. As the strain presented reduced susceptibility to imipenem and resistance to ertapenem (Table 1), according to the CLSI (5), a multiplex real-time PCR for detecting bla_OXA-48, bla_GES, bla_KPC, bla_VIM, bla_NDM, and bla_IMP genes (6) was performed and revealed the presence of a bla_OXA-48-like gene.

TABLE 1.

MICs for Enterobacter hormaechei strain 87F-2, E. coli TOP10–p87F2 carrying bla_OXA-370, and E. coli TOP10^a

Antimicrobial	MIC (μg/ml)
	E. hormaechei (87F-2)	E. coli TOP10–p87F2 carrying blaOXA-370	E. coli TOP10
IMP	1.5	0.5	0.25
MEM	0.5	0.064	0.032
ERT	4	0.032	0.008
DOR	0.38	0.047	0.023
PTZ	>256	64	3
FEP	48	4	0.047
FEP/CLA	>4	0.064	0.064
CAZ	>256	2	0.38
CAZ/CLA	>4	0.50	0.19
CTX	>256	4	0.064
CTX/CLA	>1	0.094	0.064
CRO	>32	>32	0.094
ATM	96	4	0.0125
SAM	>256	48	3
CIP	0.5	0.003	0.006
LVX	0.5	0.006	NP
GEN	0.38	0.25	0.25
AMK	1.5	1.5	3
PMB	0.19	0.125	0.25
CST	0.38	0.125	0.19
TGC	0.38	0.125	0.25
FOS	24	1	0.5

^aMICs with were assessed by Etest. IMP, imipenem; MEM, meropenem; ERT, ertapenem; DOR, doripenem; PTZ, piperacillin-tazobactam; FEP, cefepime; FEP/CLA, cefepime plus clavulanic acid; CAZ, ceftazidime; CAZ/CLA, ceftazidime plus clavulanic acid; CTX, cefotaxime; CTX/CLA, cefotaxime plus clavulanic acid; CRO, ceftriaxone; ATM, aztreonam; SAM, ampicillin-sulbactam; CIP, ciprofloxacin; LVX, levofloxacin; GEN, gentamicin; AMK, amikacin; PMB, polymyxin B; CST, colistin; TGC, tigecycline; FOS, fosfomycin; NP, not performed.

Plasmidial DNA (7) was electroporated into Escherichia coli Top10 (Life Technologies). Transformants were selected on LB agar containing ceftazidime (0.5 μg/ml). The presence of bla_OXA-48-like was confirmed by amplification of a 755-bp fragment by PCR and sequencing using Sanger's chemistry and primers oxa-48-JS-fw (5′-GGCTGTGTTTTTGGTGGCAT) and oxa-48-JS-rv (5′-TGAGCACTTCTTTTGTGATGGC).

Species identification was also determined by partial sequencing of gyrB (8). When the obtained sequence (GenBank KJ488944.1) was compared to those from type strains made available at GenBank by Brady et al. (8), the highest similarity index (96.9%) was observed in comparison to Enterobacter hormaechei CCUG 27126 (GenBank accession JX424983.1).

Agarose gel electrophoresis of plasmid extraction (3) from transformants (E. coli TOP10–p87F2) evidenced a single plasmid of approximately 150 kbp. This plasmid was fully sequenced using Ion Torrent (Life Technologies) and preliminarily assembled using SeqMan NGen 4.0 (DNASTAR). Comparison of nucleotide sequences from contigs to those available at GenBank evidenced a tra operon with highest similarity to those found in IncF plasmids and also revealed the presence of bla_TEM-1, bla_CTX-M-8, and a new bla_OXA-48 variant gene which was designated bla_OXA-370 (http://www.lahey.org/studies/). The nucleotide sequences of bla_OXA-370 and its genetic context were deposited in GenBank. This gene differed from bla_OXA-48 by three nucleotides, but only substitutions 634T→G and 635C→A resulted in a unique amino acid substitution, S220E, according to class D β-lactamase numbering (9).

Contrasting to the bla_OXA-48 genetic environment, which has the IS1999 upstream of the open reading frame (ORF) at the promoter region, the bla_OXA-370 was flanked upstream by a Tn3 family tnpA gene truncated at 3′ by insertion of an IS5075-like insertion sequence (IS). The bla_OXA-370 was flanked downstream by a Tn4 family tnpA gene truncated at 5′ by insertion of an IS15-like IS (Fig. 1). Similarly to bla_OXA-48, predicted −10 and −35 promoter region signals were located 121 and 98 bp upstream of the bla_OXA-370 start codon, respectively.

FIG 1.

Genetic context of bla_OXA-370. Vertical bars represent the inverted repeats of IS5075. Note the absence of Tn1999.

MICs of the wild-type strain 87F-2, E. coli TOP10–p87F2, and E. coli TOP10 were assessed by Etest and are shown in Table 1.

A total of 10 OXA-48 variants, including OXA-370, have been described to date, some of them, e.g., OXA-163 and OXA-247, with no carbapenem-hydrolyzing activity (2–4, 10, 11). Although the hydrolytic profile of OXA-370 was not evaluated in this study, the transformant was susceptible to carbapenems and the MICs for ceftazidime, cefotaxime, and cefepime were reduced when clavulanate was added, suggesting that the increased MICs for these cephalosporins might be due to the expression of CTX-M-8. Indeed, only the piperacillin MIC was high in the transformant, even in the presence of tazobactam, which is compatible with the piperacillin-hydrolyzing activity of OXA-48 (1). Nonetheless, appropriate kinetic evaluation of OXA-370 must be further performed to define its hydrolytic profile.

In summary, we described a new OXA-48 variant, the OXA-370 variant, and this is the first report of an OXA-48-like variant in Brazil. The role of this new enzyme in the carbapenem-resistant phenotype should be further evaluated.

Nucleotide sequence accession numbers.

The nucleotide sequences of bla_OXA-370 and its genetic context were deposited in GenBank under accession numbers KF900153.1 and KJ488943.1.