Objectives:

A total of 101 clinical isolates of Klebsiella pneumoniae (67), Escherichia coli (14), Enterobacter cloacae (10) and Citrobacter freundii (10) resistant to third-generation cephalosporins (TGC) were isolated in Ruzinov University Hospital in Bratislava (Slovakia) during a discontinuous survey performed during a few months of 1998, 1999, 2001 and 2002. The aim of this study was to determine the mechanism(s) of resistance to TGC and to investigate the epidemiology of ESBL producers.

Methods:

Susceptibility testing was performed by disk diffusion on Mueller–Hinton agar, and the double-disk synergy test (on medium with and without oxacillin) was used for detection of ESBL production. The determination of the pI of the b-lactamases was performed by isoelectric focusing. The relatedness of the isolates was investigated by pulsed-field gel electrophoresis (PFGE). One isolate of each pulsotype was used for analysis of the bla gene by PCR using primers specific for TEM, SHV and CTX-M enzymes.

Results:

The double-disk synergy test was positive for all isolates of K. pneumoniae, 12 isolates of E. coli and only for two isolates of E. cloacae and two isolates of C. freundii. The isolates with negative double-disk synergy test were assumed to be mutants that overproduced their chromosomally encoded cephalosporinase. PFGE revealed respectively 10, 8, 7 and 5 pulsotypes among the isolates of K. pneumoniae, E. coli, E. cloacae and C. freundii. SHV-type ESBLs with pI 7.6 were found in all species, SHV-type ESBLs with pI 8.2 were present in isolates of K. pneumoniae and E. cloacae. TEM-type ESBLs with pI 5.2 and 6.0, as well as a CTX-M-type ESBL with pI 8.4 were produced by isolates of K. pneumoniae. Two strains of K. pneumoniae produced two different ESBLs simultaneously (one with a TEM-type of pI 6.0 plus SHV-type of pI 7.6, and one with a SHV-type of pI 8.2 plus a CTX-M-type of pI 8.4).

Conclusion:

The production of ESBL was the major mechanism of resistance to TGC in Ruzinov University Hospital in Bratislava. At least five different ESBLs were encountered and a few clonal strains disseminated in different wards.