Genetic and Biochemical Characterization of a Chromosome-Encoded Carbapenem-Hydrolyzing Ambler Class D β-Lactamase fromShewanella algae

Abstract

A chromosome-encoded ␤-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli. It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases. Although conferring a narrow-spectrum ␤-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S. algae KB-1. Very similar oxacillinases were found in other S. algae isolates.Bacteria of the genus Shewanella are gram-negative bacilli, facultatively anaerobic, belonging to the family Alteromonadaceae (25), that are widely distributed in marine and freshwater environments (23). Shewanella algae is a bacterial species recently identified (23, 24) that is closely related to Shewanella putrefaciens (formerly Pseudomonas putrefaciens) (25). Both S. putrefaciens and S. algae species are rare human pathogens (12,15,25).Recently, we have characterized ␤-lactamase OXA-54 from S. oneidensis and we have shown that this Ambler class D enzyme hydrolyzed imipenem significantly (19). Three S. algae isolates were obtained from clinical samples during the last 2 years from our hospital, and the aim of the present study was to determine their ␤-lactamase content. MATERIALS AND METHODSBacterial strains and plasmids. S. algae clinical isolate KB-1 was isolated from an arm wound of a 10-year-old girl hospitalized at the Bicêtre hospital in 2001 (Kremlin-Bicêtre, France). S. algae KB-2 and KB-3 were from a peritoneum aspiration and from a deep pus sample of the flank, respectively, from patients also hospitalized at Bicêtre hospital. These isolates were identified by the API 32GN system (bioMérieux, Marcy l'Etoile, France) and by sequencing of 16S rRNA genes using primers 16S 8-27 (5Ј-AGAGTTTGATCHTGGYTYAGA-3Ј; H is A, T, or C and Y is C or T) and 16S 1512-1491 (5Ј-ACGGYTACCTTGT TACGACTTC-3Ј; Y is C or T) (9,11,23).S. putrefaciens CIP 8040 (Institut Pasteur strain collection, Paris, France) was used as reference strain. Escherichia coli reference strain DH10B, and plasmid pBK-CMV (Stratagene, Amsterdam, The Netherlands) were used for cloning experiments, whereas rifampin-resistant E. coli JM109 was used for conjugation experiments (3).Antimicrobial agents and MIC determinations. The agents and their sources have been referenced elsewhere (17). MICs were determined by an agar dilution technique on Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Paris, France) with an inoculum of 10 4 CFU per spot and were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (14).Plasmid DNA content and conjugation. Plasmid DNA extraction of S. algae KB-1 was performed according to different methods as previously described (18). Direct transfer of ␤-lactam resistance markers into rifampin-resistant E. coli JM109 was attempted by liquid and solid mating-out assays and by electroporation of a putative plasmid DNA suspension of S. algae KB-1 into E. coli DH10B. Transconjugants and electroporants were...

Carbapenemases: the Versatile β-Lactamases

“…from France, Greece, Italy, Romania, Turkey, Argentina, and Kuwait (28,68,125,170,215). OXA-55 and OXA-SHE, both from Shewanella algae, form the fifth group (71). The OXA-48 enzyme forms the sixth subgroup, along with OXA-54 and additional oxacillinases found in the environmental bacteria Shewanella spp.…”

“…Several of the OXA carbapenemase genes such as OXA-50 and its variants in P. aeruginosa (56,96), OXA-51-like enzymes in A. baumannii (69,213), OXA-62 in Pandoraea pnomenusa (192), and OXA-54 and OXA-55 in Shewanella spp. (71,165) appear to be natural components of their respective bacterial chromosomes. In the case of the OXA genes in A. baumannii, insertion sequences of the ISAba1 type, carrying strong promoters, have been detected upstream of the chromosomal oxacillinase, resulting in increased expression and concomitant carbapenemase resistance (212).…”

Treatment Failure Due to Emergence of Resistance to Carbapenem during Therapy for Shewanella algae Bacteremia

“…We could detect a chromosome-encoded carbapenem-hydrolyzing Ambler class D ␤-lactamase from our S. algae isolates. This suggests that a carbapenem-hydrolyzing ␤-lactamase plays a central role in the emergence of resistance to imipenem in S. algae (7). When it is considered that the two isolates had the same PFGE pulsotype and that we had detected Ambler class D ␤-lactamase by performing PCR, it is likely that the emergence of resistance was due to a mutation that resulted in the derepression of Ambler class D ␤-lactamase synthesis.…”

“…We also demonstrated in vitro that S. algae organisms have a propensity toward resistance to imipenem. The mechanism of resistance to imipenem in the organism may be related to a carbapenemhydrolyzing Ambler class D ␤-lactamase, as described previously (7). PCR experiments were performed as described previously with the specific primers OXA-55/1 (5Ј-CATCTACCT TTAAAATTCCC-3Ј) and OXA-55/2 (5Ј-AGCTGTTCCTGC TTGAGCAC-3Ј) to amplify the bla OXA-55 gene from the imipenem-resistant S. algae isolate.…”

Human Infection with Shewanella putrefaciens and S. algae: Report of 16 Cases in Martinique and Review of the Literature

“…It is important to note that Shewanella can show resistance to imipenem by secreting an oxacillinase. 57 Infection with Shewanella spp. infections should be considered in a suggestive environmental context (tropical area, sea water exposure).…”