OXA-46, a New Class D β-Lactamase of Narrow Substrate Specificity Encoded by abla_VIM-1-Containing Integron from aPseudomonas aeruginosaClinical Isolate

Abstract

A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a bla VIM-1 metallo-␤-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D ␤-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of bla OXA-46 in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D ␤-lactamases.

Establishing Clonal Relationships between VIM-1-Like Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains from Four European Countries by Multilocus Sequence Typing

“…The structures of the variable regions of integrons carrying the bla VIM gene cassette were determined for strains PA66, AK5493, Ps100, PA555, 67MG, 85MG, 105MG, and 134MG (for the other strains, it had previously been determined) (12,20,31).…”

“…The P. aeruginosa strains investigated in this study are listed in Table 1. They included (i) two representatives of a VIM-1-producing dominant Italian clone spreading in different hospitals since 1997 (the index strain VR-143/97 and strain PPV97, which were epidemiologically unrelated to each other and slightly different by PFGE profiling and which carried bla VIM integrons with different structures) (12,31); (ii) four additional VIM-1-producing P. aeruginosa isolates from Italy (representative of those spreading more recently in a long-term care and rehabilitation facility in northern Italy [L. Pagani et al, unpublished results]); and (iii) four VIM-4-producing isolates from Greece (n ϭ 1), Sweden (n ϭ 1) (11), and Hungary (n ϭ 2) (20). In addition, a VIM-2-producing isolate from Sweden (AK5493), obtained from a Greek national patient who had recently been hospitalized in Greece, was also included in the comparison.…”

Diversity, Epidemiology, and Genetics of Class D β-Lactamases

“…It hydrolyzes mostly penicillins, oxacillin, and narrow-spectrum cephalosporins. Interestingly, it is inactivated by tazobactam and carbapenems but exhibits low susceptibility to NaCl (57).…”

“…We have observed that this specific property is shared by other class D ␤-lactamases, such as those belonging to the OXA-10 subgroup (unpublished data). Also, the production of some OXAs whose activity is inhibited by clavulanate or tazobactam, such as OXA-12 from A. jandaei (139) and OXA-18, OXA-45, and OXA-46 from P. aeruginosa (57,118,153), may be identified by synergy tests using clavulanic acid-containing disks. However, the result of these synergy tests may be misinterpretated and the corresponding isolates may be considered to be class A ESBL producers.…”

Modes and Modulations of Antibiotic Resistance Gene Expression

“…More recently identified cassettes are associated with genes that mediate resistance to rifampin and quinolones and increasingly also with genes encoding extended-spectrum ␤-lactamases and carbapenemases (82,88,136,200,203,208,261,264). Transcriptional control of resistance gene expression in class 1 integrons.…”