P868 Occurrence of OXA-58 and an OXA-58 variant in Acinetobacter baumannii isolates from blood cultures in a university hospital in Athens, Greece

Oligonucleotide primers used in multiplex PCR Woodford et al, 2006. Int J Antimicrob Agents; 27: 351–353 Gene or gene region Oligonucleotide sequence (5'  3') Product size (bp) blaOXA-23-like 5’-GAT CGG ATT GGA GAA CCA GA-3’ 501 bp 5’-ATT TCT GAC CGC ATT TCC AT -3’ blaOXA-24-like 5’-GGT TAG TTG GCC CCC TTA AA -3’ 246 bp 5’-AGT TGA GCG AAA AGG GGA TT -3’ blaOXA-51-like 5’-TAA TGC TTT GAT CGG CCT TG -3’ 353 bp 5’-TGG ATT GCA CTT CAT CTT GG -3’ blaOXA-58-like 5’-AAG TAT TGG GGC TTG TGC TG -3’ 599 bp 5’-CCC CTC TGC GCT CTA CAT AC -3’ Phenotypic characterization of the 27 isolates Antibiotic % S % I % R MIC50 MIC90 Imipenem 100 >32 >32 Meropenem 100 >32 >32 Ertapenem 100 >32 >32 Ampicillin/ Sulbactam 33 41 26 16 128 Colistin 100 0,5 0,5 Objectives: Imipenem (IMP)-resistant A. baumannii isolates, collected from blood cultures of hospitalized patients in the University General Hospital “Attikon”, between September 2004 May 2005, were studied for their clonality and carbapenemase content. Methods: Susceptibility to antimicrobials was determined using a broth microdilution method and E-test. Bacterial clones were identified by PFGE with ApaI. Metallo-betalactamases were detected by the IMP-EDTA disc synergy test and by PCR with primers specific for blaVIM. Multiplex PCR with primers amplifying blaOXA-23-like, blaOXA-24-like, blaOXA51-like and blaOXA-58-like, was performed according to Woodford et al (Int J Antimicrob Agents 2006; 27:351–353). Sequencing of cloned PCR products was performed by MWGTHE Genomic Company. Sequence similarity searches were carried out with the BLAST program found at the website of the NCBI. Results: Twenty-seven non-repetitive IMPresistant isolates, 70.2% of all A. baumannii strains isolated from blood cultures, were tested. All isolates presented a multi-resistance pattern, with all being susceptible to colistin (MICs: 0,125-0,75) and 33% being susceptible also to ampicillin/sulbactam. A. baumannii isolates were distributed into seven genotypes by PFGE profiles, with 12, 4, 4, 3, 2, 1, and 1 strains in each group. The three genotypegroups of 12 and 4 isolates were further divided into 2 subgroups. None of the isolates was positive for metallo-beta-lactamase production by IMP-EDTA synergy test or PCR. Multiplex PCR for OXA-type carbapenemases revealed the presence of blaOXA-51-like, which is believed to be intrinsic to A. baumannii and blaOXA-58-like in all isolates. Sequencing of blaOXA-58 PCR amplicon revealed the presence of the blaOXA-58 in most of the isolates but also the existence of a novel variant of OXA-58 in which an A to G substitution revealed a Thr to Ala amino acid change. Conclusions: Several clones of IMP-resistant A. baumannii producing both blaOXA-58 and naturally occurring blaOXA-51-like have emerged as important bloodstream pathogens in our hospital. This observation emphasizes the importance of both the restriction of carbapenem usage as well as of the strict implementation of hand hygiene techniques for the containment of this emerging threat. Contact Information: Irene Galani, PhD ATTIKON,Unv Hospital, 1 Rimini Street, 124 62 Athens, GREECE TEL.: +30 210 5831984 FAX: +30 210 5326446 e-mail: egalani@med.uoa.gr Acinetobacter baumannii: an opportunistic pathogen  outbreaks of pneumonia, urinary tract and blood stream infections (BSI),  patients with more severe illness  increasingly rates of multidrug, especially carbapenem resistant isolates, probably as a consequence of extensive use of antimicrobial agents. Carbapenem resistance in A. baumannii class D carbapenem-hydrolysing oxacillinases (CHDLs): the most common  class B metallo-β-lactamases (MBLs): powerful carbapenemases, identified in a wide variety of Gram-negative species, but only rarely in A. baumannii Carbapenem-hydrolysing oxacillinases (CHDLs)  Intrinsic, chromosomally encoded type of oxacillinase OXA-51 ⁄ 69 variants (to date 11 variants): first reported in 2004, ubiquitous in all A. baumannii isolates, only a weak ability to hydrolyze carbapenems, may sometimes be overexpressed, resulting in a decreased level of susceptibility to carbapenems  Acquired oxacillinases: OXA-23 variants: first reported in 1995, shares 56% amino-acid identity with OXA-51 /69 OXA-24 variants: shares 63% amino-acid identity with OXA-51 /69 OXA-58: first reported in 2005, shares 59% amino-acid identity with OXA-51 /69  Significant ability to hydrolyze imipenem (but not always meropenem)  Several strains harbour more than one OXA-encoding gene β-lactamases Other rare mechanisms  porin loss or modification  penicillin-binding proteins (PBPs)  efflux pump over expression Twenty-seven non-repetitive IMPresistant A. baumannii isolates were collected from blood cultures of hospitalized patients of the University General Hospital “Attikon”, between September 2004 May 2005. Susceptibility to antimicrobials was determined using a broth microdilution method according to CLSI guidelines (M100-S14, 2005) and E-test (AB Biodisk, Solna Sweden) according to manufacturer’s instructions. Clonal relationship was assessed with Pulsed field gel electrophoresis. Total DNA in agarose plugs was digested with 30U of ApaI (BioLabs, New England) restriction endonuclease and electrophoresis was performed in 1.5% agarose gels using a Gene-Navigator system (Pharmacia, Uppsala, Sweden) over 20 h at 10oC with 5-50s linear ramping at six V/cm. Multiplex PCR with primers amplifying blaOXA-23-like, blaOXA-24-like, blaOXA-51-like and blaOXA-58-like, was performed according to Woodford et al. The amplification conditions were, initial denaturation at 94oC for 5 min, 30 cycles of 94oC for 25 s, 52oC for 40 s and 72oC for 50 s, and a final elongation at 72oC for 6 min. Metallo-beta-lactamase production was screened by the IMP-EDTA disc synergy test and by PCR with primers specific for blaVIM and blaIMP genes. Ge e or e e region li cle ti e se e ce (5' 3') Product size (bp) blaOXA-23-like 5’-GAT CGG ATT GGA GAA CCA GA-3’ 501 bp 5’-ATT TCT GAC CGC ATT TCC AT -3’ blaOXA-24-like 5’-GGT TAG TTG GCC CCC TTA AA -3’ 246 bp 5’-AGT TGA GCG AAA AGG GGA TT -3’ blaOXA-51-like 5’-TAA TGC TTT GAT CGG CCT TG -3’ 353 bp 5’-TGG ATT GCA CTT CAT CTT GG -3’ blaOXA-58-like 5’-AAG TAT TGG GGC TTG TGC TG -3’ 599 bp 5’-CCC CTC TGC GCT CTA CAT AC -3’ Distribution of A. baumannii Palsotypes in Different Departments of our Hospital Hospital Department Pulsotypes