Genetic and Biochemical Characterization of OXA-405, an OXA-48-Type Extended-Spectrum β-Lactamase without Significant Carbapenemase Activity

Abstract

The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a bla OXA-48 -type gene. Sequencing identified a novel OXA-48-type ␤-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The bla OXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M bla OXA-48 -carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like ␤-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.A mbler class D ␤-lactamases (oxacillinases) are widely disseminated among clinically relevant Gram-negative bacteria (1). They exhibit a high degree of diversity of hydrolysis activity ranging from narrow to broad-spectrum hydrolysis activity toward ␤-lactams (1). Among the class D ␤-lactamases, several enzymes hydrolyze carbapenems. Most carbapenemhydrolyzing class D ␤-lactamases (CHDLs) are from Acinetobacter spp. (e.g., OXA-23, OXA-40, OXA-58, OXA-143) (2, 3), whereas OXA-48-type enzymes are identified in Enterobacteriaceae only (4). The OXA-48-derived CHDLs have initially been identified in Turkey (5), first in Klebsiella pneumoniae and then in other enterobacterial species (4). The known OXA-48 variants are currently as follows: (i) OXA-162, identified from K. pneumoniae isolates in Turkey (6); (ii) OXA-163, identified from K. pneumoniae and Enterobacter cloacae isolates in Argentina (7, 8); (iii) OXA-181, identified from a K. pneumoniae isolate in India (9); (iv) OXA-204, identified from K. pneumoniae isolates in patients having a link with North Africa (10); (v) OXA-232, identified in France from a K. pneumoni...

Horizontal Transfer of Carbapenemase-Encoding Plasmids and Comparison with Hospital Epidemiology Data

“…Chen et al (41) describe the conjugative resistance plasmid pBK30683, and also pBK30661, which had lost many of the tra genes, providing evidence for the spread of resistance through both clonal spread and horizontal transfer. Dortet et al (29) also describe a conjugative resistance plasmid, pOXA-48, and a related plasmid with a deletion in the tra operon. The set of proteins in pOXA-48, a plasmid reported to show very efficient conjugation in both intraspecies and interspecies experiments (22), has a set of predicted transfer genes that differ from those of pKPC_UVA01 and from plasmids shown in the heatmap.…”

“…We analyzed plasmids from donor and recipient strains using Illumina MiSeq and single-molecule, real-time (SMRT) sequencing. We also included a few recently described plasmids from other studies that examined plasmids carrying carbapenemase genes, their conjugation ability, and HGT (29,41). Using a candidate gene approach, we selected a set of 77 genes that are potentially associated with plasmid transfer, replication, or maintenance based on current literature, realizing that this is not an exhaustive list.…”

“…One might speculate that the gastrointestinal (GI) environment within a patient, given its high bacterial densities, could potentially provide an ideal environment for gene transfer (26). However, despite this high potential for spread, there are relatively few reports of bla KPC interspecies transfer within patients (22,(27)(28)(29). Plasmid transfer between bacteria within the hospital or community environment adds an additional layer of complexity (21).…”

Rapid Identification of OXA-48 and OXA-163 Subfamilies in Carbapenem-Resistant Gram-Negative Bacilli with a Novel Immunochromatographic Lateral Flow Assay

“…The reported sensitivity and specificity were both 100%, with the result obtained in less than 10 min (6)(7)(8)(9)(10). Noteworthy is that new allelic variants of OXA-48 have emerged, namely, OXA-163 and the related variants OXA-247, OXA-405, and OXA-438 (11)(12)(13)(14), which are not recognized by the OXA-48 K-SeT test (6,8,10). OXA-163, which is the only variant of this subfamily that has spread to several hospitals in Argentina and Egypt (15,16), differs from OXA-48 by a single amino acid substitution and a four-amino-acid deletion.…”

Noncarbapenemase OXA-48 Variants (OXA-163 and OXA-405) Falsely Detected as Carbapenemases by the β Carba Test

“…However, OXA-163-producing Klebsiella pneumoniae and OXA-405-producing Serratia marcescens gave positive results at 30 min, 1 h, and 3 h with the ␤ Carba test, whereas they were not CPE. Indeed, the OXA-48 group is composed of enzymes with true carbapenemase activity (OXA-48, OXA-181, OXA-204, OXA-232, OXA-244, OXA-245, OXA-370, and OXA-484) and of enzymes devoid of any carbapenemase activity because of an amino acid deletion in their active site (OXA-163, OXA-247, and OXA-405) (14,15). Because of their close relationship with OXA-48, these three noncarbapenemase variants usually gave false-positive results with molecular methods (11).…”