Inhbitor-resistant beta-lactamases in clinical isolates of Enterobacteriaceae from Croatia

Inhibitor-resistant β-lactamases are derivatives of TEM, SHV and OXA-β-lactamases and confer resistance to beta-lactam combinations with inhibitors such as amoxacillin/clavulanate and ampicillin/sulbactam. Plasmid-mediated AmpC β-lactamases also confer resistance to β-lactam/inhibitor combinations. They pose therapeutic problem in the treatment of urinary tract infections. Recently emergence of resistance to amoxycillin/clavulante was noticed among Escherichia. coli and Klebsiella pneumoniae isolates in Croatia. The aim of the study was to determine the phenotype of resistance and the mechanisms of resistance to β-lactam/inhibitor combinations in clinical isolates of E. coli and K. pneumoniae isolates in Croatia. In total, 56 isolates (52 E. coli and 4 K. pneumoniae) were collected in thee large medical centers (University Hospital Center Zagreb, Sisters of Mercy University Hospital and Public Health Institute of Osijek-Baranja County) from urine. The susceptibility to a wide range of antibiotics was determined by disk-diffusion and broth microdilution method. The transferability of resistance to amoxycillin/clavulante was determined by conjugation (broth mating method). PCR was used to detect genes encoding resistance to β-lactam/inhibitor combinations (blaTEM, blaSHV, blaOXA, blaampC). All strains showed resistance to amoxycillin, piperacillin and amoxacillin/clavulanate. 39% of the strains were resistant to cefotaxime and ceftriaxone, 31% to cefepime 27% to cefoxitin and 20% to ceftazidime. 10% of the strains were resistant to gentamicin and 4% to ciprofloxacin. No resistance to carbapenems was observed. Fourteen strains (25%) transferred resistance to co-amoxyclav to E. coli recipient strain. TEM type of β-lactamase was identified in 28 strains (50%) and OXA-1 in 19 strains (34%). Twelve strains coproduced TEM and OXA-1 β-lactamase. Plasmid-mediated AmpC β-lactamases were found in 30% of the isolates. CMY type of β-lactamase in 13 strains (23%) and DHA-2 in four strains (7%). Sequencing of the representative amplicons revealed TEM, OXA-1, CMY-2 and DHA-1 β-lactamase. This is the first investigation of inhibitor-resistant β-lactamases in Croatia. The results showed that TEM and OXA variants are the most frequent inhibitor-resistant β-lactamases in Croatia. Combination of amoxycillin with clavulanate was more affected by production of inhibitor-resistant beta-lactamases than the combination of piperacillin with tazobactam. Resistance to expanded-spectrum cephalosporins and cefoxitin was associated with production of plasmid-mediated AmpC beta-lactamases. Inhibitor-resistant variants pose serious therapeutic problem in the treatment of community-acquired-urinary tract infections. Accurate and quick laboratory identification of such isolates is imporant to improve antimicrobial therapy.