engleski

Rare occurence of Pseudomonas aeruginosa isolates carrying VIM-2 metallo-β-lactamase in Croatia

Objectives: Reports of aquired metallo-β-lactamases (MBL) in P. aeruginosa explaining the resistance to carbapenems are increasing worldwide. After the first description of two strains harboring the VIM-2 MBL in P. aeruginosa in Croatia we determined the occurence of MBL producing strains among Pseudomonas aeruginosa isolates with reduced susceptibility to carbapenems isolated in a 7-year period (2001-2007). Methods: Minimal inhibitory concentrations to common antipseudomonal antibiotics were determined in 169 isolates, including the MICs to imipenem after addition of EDTA, and MICs to ceftazidim and imipenem after addition of boronic acid, respectively. O-serotypes were determined by slide-agglutination. PFGE was peformed after restriction of genomic DNA with XbaI enzyme and profiles analysed by GelCompare software. Potential clonal relatedness was determined at an ≥80% level of similarity. PCR using primers amplifying VIM and IMP genes was done for all strains. For MBL positive strains the structure of the variable regions of the blaVIM-containing integrons was determined by a PCR mapping and sequencing approach. Results: The majority of isolates (49.7%) were multiresistant, ie. resistant to representatives of all five classes of antipseudomonal antibiotics, eg. ureidopenicillins, cephalosporines, carbapenems, aminoglycosides and fluoroquinolones. The most potent antibiotic was amikacin with 82.8% susceptible strains (MIC50 4 mg/L), followed by colistin (50% of susceptible strains ; MIC50 4 mg/L), and ceftazidime (30.8% of strains found susceptible ; MIC50 16 mg/L). Median value of MICs to imipenem was 16 mg/L, while in addition of EDTA it was 8 mg/L. Only in 6 isolates EDTA significantly (≥4 times) lowered MICs to imipenem. The addition of boronic acid significantly lowered MICs to ceftazidime and imipenem in 59.7% of isolates. The most common serotype was O12 (99 isolates ; 58.6%), followed by O11 (29 ; 17.1%) and O1 (7 ; 4.1%). Majority of O12 isolates belonged to the multiresistant group (77.7%), as well as O11 (44.8%). PFGE analysis determined 9 main clones with O12 isolates belonging mostly to predominant clone A, and O11 isolates belonging mostly to second most prevalent clone B. blaVIM-2 gene was found in only 6 isolates (3.5%) and was located in the first gene cassette of a novel class 1 integron sized 3277 bp and followed by OXA-10, qac-F, aacA4, and qacEdelta1 gene cassettes. All except one (being O12) MBL positive strains were O11 serotype. Four MBL positive strains belonged to the same clone. Conclusion: Metallo-β-lactamases are still a rare cause of carbapenem resistance in Croatia. The VIM-2 genes were found in a novel class one integron. The majority of strains were significantly inhibited by boronic acid suggesting derepression of ampC as a main mechanisms of resistance to beta-lactams and possibly, to carbapenems.

Pseudomonas aeruginosa; metalo-beta-lactamases; VIM beta-lactamases

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