engleski

Prevalence and molecular epidemiology of CTX-M beta-lactamases in Croatia

Out of the non-TEM-non SHV-ESBL types, CTX-M b-lactamases have become recently a very high epidemiologic significance. These include PER-1, PER-2, VEB-1, GES-1, BES-1 and the CTX-M group. It is a small, but constantely growing family of plasmid-encoded ESBLs, that preferentially hydrolyze cefotaxime. Recently first E. coli strains with high resistance to cefotaxime and ceftriaxone have been isolated in three University hospitals in Croatia. The aim of this study was to characterize β -lactamases produced by these isolates. Five E. coli isolates were identified at University Hospital Split, five were obtained from Clinical Hospital Center Zagreb and a single isolate was recovered in Sisters of Mercy Hospital in Zagreb. Extended-spectrum β -lactamases (ESBL) were detected by double-disk synergy test and CLSI combined disk test. Susceptibility to a wide range of antibiotics was determined by the broth microdilution method according to CLSI. The transfer of the cefotaxime resistance determinants was determined by the broth mating method. ESBLs were characterized by isoelectric focusing, substrate profile determination, PCR and sequencing of blaCTX-M genes. The two latter approaches were also used to study the genetic environment of the genes. Genetic relatedness between the strains was tested by PFGE. Results Despite some similarities, susceptibility testing revealed significant differences between the groups of isolates identified in the three hospitals. The isolates from the University Hospital Center in Zagreb were almost uniform in their resistance patterns and levels, and they were in general more resistant with significantly increased MICs of ceftazidime (MICs 8-32 mg/L). At the same time they all were highly resistant to aminoglycosides and ciprofloxacin. The remaining isolates were susceptible to ceftazidime and more phenotypically diversified. All of the isolates produced β -lactamases with high pI values, either 8.9 in the case of the isolates from the University Hospital Center Zagreb or 8.4 in the case of the all remaining isolates. PCR for blaCTX-M genes was positive and sequencing these identified the pI 8.9 enzymes as CTX-M-15, whereas those with a pI of 8.4 as CTX-M-3 β -lactamase. An insertion sequence ISFcp1 was identified 48 bp upstream of all blaCTX-M-15 producers and 128bp upstream of one blaCTX-M-3 gene, whereas the remaining blaCTX-M-3 genes were accompanied by the IS26 element. The five CTX-M-15-producing isolates were indistinguishable in the PFGE analysis, and different from the all remaining isolates. The group of CTX-M-3 producers from Split was much more diverse with only two isolates that were clonally related to each other. The isolate from the Sisters of Mercy Hospital in Zagreb was unrelated to any other study isolate. This study reported the appearance of CTX-M-3 and CTX-M-15 in clinical isolates of E. coli from Croatia. Despite the high similarity of these enzymes, differences in the genetic context of their genes indicated that they emerged independently on each other. Moreover, it is possible that there were also two different origins of CTX-M-3, which altogether is striking considering the relative small geographic region of the isolates identification. CTX-M-15 producers disseminated in the hospital in Zagreb by clonal spread, whereas in Split the blaCTX-M-3 gene was probably horizontally transmitted between non-related strains. CTX-M β -lactamases are still rare in Croatia in contrast to some other countries.

CTX-M beta-lactamases; cefotaxime; ceftriaxone; E. coli

Clinical Microbiology and Infection 13 (2007)suppl. P862