engleski

False positive phenotypic detection of metallo- beta- lactamases in acinetobacter baumaniia

ackground and aim: Acquired carbapenem resistance in A. baumannii can be mediated by acquired carbapenemases of class A (KPC), class B metallo- β-lactamases (MBLs) of IMP, VIM, SIM and NDM family and class D carbapenem- hydrolyzing oxacillinases (CHDL) (OXA-23-like, OXA-40-like, OXA-58-like, OXA-143-like and OXA- 238-like. Inhibitor based test with EDTA are often used to detect MBLs in A. baumannii. The aim of the study was to investigate the sensitivity and specificity of inhibitor based tests with EDTA in detection of MBLs in A. baumannii. Material and methods: In total 100 carbapenem- resistant Acinetobacter baumanii strains collected in General hospital Pula (27), University Hospital Zagreb (39), General Hospital Varaždin (4), General Hospital Sisak (3), University Hospital Merkur (1), Clinic for Infectious diseases (6), General Hospital Bjelovar (1) and Godan nursing home in Zagreb (19) during 2009-2014 from various clinical specimens, were analyzed. The susceptibility to a wide range of antibiotics was determined by disk-diffusion and broth microdilution method. Phenotypic detection of metallo-β- lactamases was performed by combined disk test with addition of EDTA and by broth microdilution test with addition of fenantroline and EDTA (0.5 mM). Augmentation of inhibition zone aroung imipenem and meropenem disk for at least 5 mm in the presence of EDTA was considered as a positive results in disk diffusion while 8 fold (three dilutions) reduction of imipenem or meropenem MIC by EDTA was indicative for production of MBL in dilution test. Genes encoding metallo-β- lactamases (IMP, VIM, NDM, SIM) and carbapenem hydrolyzing oxacillinases were detected by PCR. Results: All strains were found to be resistant to carbapenems with MICs of imipenem and meropenem ranging from 8 ->128 and from 16- >128, respectively. Twenty seven strains were positive for blaOXA-23-like, 52 for blaOXA- 40- like and 21 for blaOXA-58-like genes. Eleven of blaOXA-23-like positive isolates coharboured blaVIM-1 gene. Sequencing of representative bla genes revealed the presence of blaOXA-23 , blaOXA- 40, blaOXA-72, blaOXA-58 and blaVIM-1 genes. Combined disk test with EDTA and dilution test with EDTA were positive in all 11 isolates possessing VIM-1 MBL. However, phenotypic testing was positive in 14 out of 16 isolates positive only for OXA-23 (87%), in 11 of 21 strain positive for OXA-58 (52%) and in all OXA-40 harbouring isolates (100%). The augmentation of inhibition zone in the presence of EDTA ranged from 10 to 22 mm. Conclusions: False positive results in inhibitor based tests can occur because in the presence of EDTA oxacillinases can be converted to a less active state leading to the augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem MICs. This phenomenon was previously reported in A. baumannii and P. aeruginosa. The rate of false positive results was the highest in OXA- 40 positive isolates and the lowest in OXA-58 positive isolaes which can be explained by the better hydrolysis of carbapenem substrates by OXA-40-like β-lactamases. The study showed high sensitivity but low specificity of phenotypic methods in detection of MBLs in A. baumanii and points out the necessity of molecular detection of MBL genes in A. baumannii by PCR.

Acinetobacter baumannii, metallo-beta-lactamase, detection, inhibitor based test

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