Purification and characterization of OXA-23 from Acinetobacter baumannii.

Although the existence of bla(OXA-23) is reported in various parts of the world, the product of bla(OXA-23) gene, OXA-23, has not been purified and its kinetic properties are not known. In this study, OXA-23 of Acinetobacter baumannii isolated from Kocaeli University intensive care unit was characterized after purification using recombinant methods. Preliminary results showed that conventional protein purification methods were not effective for purification of OXA-23. Therefore, OXA-23 was fused to maltose-binding protein of Escherichia coli, the fused protein was expressed and purified to homogeneity. Kinetic properties of the pure protein were then studied with substrates e.g., imipenem, meropenem, cefepime, ceftazidime, ampicilline, piperacillin, penicillin G, and nitrocefin. Also clavulanic acid, tazobactam, and sulbactam concentrations that inhibit 50% of OXA-23 enzyme activity were calculated. Modelling of OXA-23 revealed its ionic surface structure, conformation in the fused form and its topology allowing us to make predictions for OXA-23 substrate specificity.

OXA-162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem, meropenem and doripenem.

CONTEXT: Isolation and characterization of OXA-162, a novel variant of OXA-48. OBJECTIVES: Klebsiella pneumoniae isolate with decreased susceptibility to carbapenems was recovered from a Turkish patient. This study aimed at characterizing the carbapenem resistance determinants of this isolate. MATERIALS AND METHODS: Antibiotic susceptibility tests, analytic isoelectric focusing (IEF), cloning and sequencing were performed. Cloned ß-lactamase was purified by means of preparative gel electrophoresis and the kinetic constants were determined under initial rate conditions. RESULTS: The identified bla(OXA-162) gene was located on a ca. 45-kb plasmid carrying a transposon consisted of two IS1999-2 elements. OXA-162 differed from OXA-48 by a single amino acid substitution (Thr213Ala) which increased the catalytic efficiency (k(cat)/K(M)) of OXA-162 towards imipenem and meropenem. Also this substitution caused a gain of hydrolysis ability towards doripenem. Analysis of OXA-162 model implied that the amino acid change might generate an extension in the opening of the substrate entry site and might cause extended hydrolytic activity towards imipenem, meropenem and doripenem. DISCUSSION AND CONCLUSION: OXA-162, a derivative of OXA-48 has enhanced catalytic properties.

CTX-M type extended spectrum beta-lactamases in Escherichia coli isolates from community acquired upper urinary tract infections at a university in the European part of Turkey.

Extended spectrum beta-lactamase (ESBL) producing Escherichia coli has been an emerging etiologic agent in the community acquired infections. We investigated the occurrence of ESBL producing E. coli isolated from patients admitted with community acquired urinary tract infection (UTI) to the hospital of the Trakya University, Turkey during 2006. Eleven single patient isolates of E. coli harboring ESBL were identified among 30 E. coli isolated from patients admitted with symptoms corresponding to upper UTI. CTX-M type ESBLs were detected in all 11 ESBL-producers by isoelectric focusing and polymerase chain reaction screening. Sequence analysis revealed CTX-M-1 in one isolate, CTX-M-3 in three isolates and CTX-M-15 in seven isolates. ESBL-producing E. coli isolated from community acquired UTIs are widespread in the European part of Turkey.

Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria.

BACKGROUND: We studied the beta-lactamases of an E. aerogenes isolate recovered from the blood of a two-year-old patient. The isolate demonstrated a disk-diffusion phenotype typical for an AmpC-ESBL co-producer. METHODS: Microbiology studies were performed according to standard protocols. The resistance gene was identified by transconjugation and cloning experiments. RESULTS: By transconjugation only a narrow spectrum beta-lactamase (TEM-1) encoded on a small plasmid was transmitted. The ESBL was cloned and expressed in an E. coli host. Sequence analysis of the recombinant plasmid revealed blaSHV-12 associated to the insertion sequence, IS26. CONCLUSION: This is the first study demonstrated the occurrence of SHV-12 in Nigeria.

High prevalence of OXA-51-type class D beta-lactamases among ceftazidime-resistant clinical isolates of Acinetobacter spp.: co-existence with OXA-58 in multiple centres.

OBJECTIVES: This study was designed to demonstrate the prevalence of the newly discovered carbapenem-hydrolysing class D enzymes, OXA-51-type and OXA-58, among clinical isolates of Acinetobacter spp. METHODS: A total of 72 isolates from six centres were studied. Isolates were screened by PCR with specific primers for bla(OXA-51-type) and bla(OXA-58). PCR products were sequence-analysed. Plasmids were digested with EcoRV and genomic DNAs were digested with PvuII. Hybridization experiments were done with digoxigenin-labelled specific probes. Macro-restriction analysis was done on SmaI-digested genomic DNAs. RESULTS: A total of 56 (77.8%) isolates were positive for bla(OXA-51-type) genes. Sequence analysis of the products from 23 selected isolates revealed the occurrence of multiple alleles in all contributing centres. The bla(OXA-58) gene was detected among 10 isolates from five centres. All were also positive for bla(OXA-51-type) genes. Among the bla(OXA-58)-positive isolates, two from the same centre were positive for a novel OXA-51 allele (OXA-86). Southern hybridization of plasmids and of genomic DNAs suggested that bla(OXA-51-type) genes are located on chromosomes whereas bla(OXA-58) genes are plasmid borne in these 10 isolates. Plasmid profiles and pulsed-field gel electrophoresis patterns indicated the spread of the bla(OXA-58) gene among multiple clones. The bla(OXA-51-type) and bla(OXA-58) co-carrier strains were mostly associated with a pandrug-resistant phenotype. CONCLUSIONS: This study indicated that bla(OXA-58)-bearing plasmids are readily spreading among multiple clones of the bla(OXA-51-type)-bearing clinical isolates of Acinetobacter spp. Since these isolates are highly resistant to antibiotics this finding indicates the existence of a significant problem in Turkish hospitals.