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Characterization of extended-spectrum β-lactamases in the isolates of Enterobacter cloacae from Split, Croatia

The development of highly stable expanded-spectrum cephalosporins at the beginning of 1980s was quickly followed by the emergence of extended-spectrum β-lactamases (ESBL) in Klebsiella pneumoniae and other Enterobacteriaceae including Enterobacter spp. Recently an increase in the prevalence of ESBL positive Enterobacer cloacae was observed at the University hospital in Split, Croatia. During 2009-2010. thirty ceftazidime resistant isolates of E. cloacae were collected at the University Hospital in Split, Croatia from various specimens and hospital units. Production of extended-spectrum β-lactamases was tested by double-disk synergy test and confirmed by CLSI combined disk test. Susceptibility to a wide range of antibiotics was tested by disk-diffusion and broth microdilution method. Transfer of cefotaxime resistance was determined by conjugation (broth mating method) using E. coli A15R- strain resistant to sodium azide. The frequency of conjugation was expressed relatively to the number of donor cells. Molecular characterization of ESBLs was performed by PCR with primers specific for TEM, SHV and CTX-M β-lactamases. Multiplex PCR was used to determine the group of CTX-M β-lactamases. Plasmids encoding ESBLs were extracted with Macherey Nagel mini kit. There was 60% of the strains resistant to ceftriaxone, 56% to ceftazidime, 50% to cefotaxime and 40% to cefepime. All strains were susceptible combination of cefotaxime with clavulanate and piperacillin with tazobactam. No resistance to carbapenems was observed. Among non β-lactam antibiotics high resistance rate was found for gentamicin (70%). Only 33% of the isolates were resistant to ciprofloxacin. The most potent antibiotics were meropenem and imipenem with MIC90 of 0.06 and 1 mg/L respectively. The least potent antibiotics were ceftazidime, cefotaxime and ceftriaxone with MIC90 of 256 mg/L. All strains transferred cefotaxime resistance to E. coli recipient strain with the frequency ranging from 1 to 5 x10-6. Tranconjugants showed similar resistance phenotypes to β-lactam antibiotics as their respective donors. All strains were shown to possess CTX-M β-lactamases by PCR. Multiplex PCR revealed group 9. Sequencing of the amplicons will be done to determine the exact type of CTX-M β-lactamase. CTX-M β-lactamases were encoded on the large plasmids of approximately 150 kb which were transferable to E. coli recipient. Plasmid extractions yielded PCR products with primers specific for CTX-M β-lactamases. This study demonstrated clonal outbreak of CTX-M group 9 positive E. cloacae at the University hospital in Split. Genotyping of the isolates by pulsed-field gel electrophoresis will be performed to determine the possible clonal relatedness. Sequencing of blaCTX-M genes will identify the exact type of CTX-M β-lactamase. Carbapenems are the antibiotics of choice for the treatment of infection caused by our ESBL positive E. cloacae strains. According to CLSI all cephalosporins should be reported as resistant for ESBL producing organisms regardless of in vitro susceptibility testing. Finally, the spread of CTX-M producing Enterobacteriaceae in Croatia in a manner similar to that observed in other countries may indicate difficulty in controlling this emerging resistance determinances. This demonstrates the need to monitor hospitalized patients for the further emergence of transferable resistance to expanded-spectrum cephalosporins.

Enterobacter cloacae; CTX-M beta-lactamases; cefotaxime

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