Detection of Pseudomonas aeruginosa isolates of the international clonal complex 11 carrying the blaPER-1 extended-spectrum β-lactamase gene in Greece

Abstract

Objectives: The extended-spectrum β-lactamase (ESBL) PER-1 initially disseminated among Pseudomonas aeruginosa strains in Turkey. Despite reports from other European countries, such strains have not been detected in Greece until now. We describe the first bla(PER-1)-positive P. aeruginosa isolates from Greece and their genetic environment.

Methods: From January 2008 to December 2009, 287 consecutive non-duplicate P. aeruginosa isolates with reduced susceptibility or resistance to ceftazidime (MIC >8 mg/L) were screened for ESBL production with a modified boronic acid-based double-disc synergy test. Phenotypically ESBL-positive isolates were subjected to agar dilution, PFGE and multilocus sequence typing (MLST). Broad-spectrum bla genes were identified by PCR and sequencing. Plasmid analysis and conjugation experiments were performed. The location of the bla(PER-1) gene was detected by Southern blotting and its genetic environment was characterized using inverse PCR.

Results: Five isolates were phenotypically positive for ESBL production, exhibited resistance to cefepime, ceftazidime, aztreonam and meropenem, and carried the bla(PER-1) gene. MLST showed that they belonged to sequence type (ST) 235, which belongs to the international clonal complex 11. Four isolates had the same PFGE pattern. Southern blotting revealed the chromosomal location of the bla(PER-1) gene. Analysis of the bla(PER-1) flanking regions showed identity to transposon Tn1213 downstream and 1406 bp upstream of bla(PER-1). Further upstream, an orfA gene and ISPa12 were identified; both were truncated by the insertion of IS6100.

Conclusions: This study confirmed the presence of PER-1-producing P. aeruginosa strains in Greece. The chromosomal location of bla(PER-1), as part of a truncated transposon, suggests clonal expansion rather than horizontal gene transfer.