Genotypic detection of resistance to fluoroquinolones by PCR-RFLP in Acinetobacter baumannii clinical isolates
Abstract number: p1746
García-peñuela E., Alarcón T., Pérez de Ayala A., Arenal N., Navarro J.L., López-Brea M.
To detect the resistance to fluoroquinolones in 30 Acinetobacter baumannii strains by a PCR-RFLP assay.
Thirty A. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.). The MICs (Minimal Inhibitory Concentrations) for ofloxacin were determined by agar dilution following standard methodology.
A PCR-RFLP method using one primer pair for amplification of a 344 bp fragment related to gyrA gene (which codifies subunity A of DNA-gyrase) and using one restriction enzyme Hinf I was developed to study the resistance to ofloxacin in the different A. baumannii strains. When an A. baumannii strain is resistant to fluoroquinolones, a mutation in the position Ser 83 of the DNA-gyrase has been detected, decreasing the affinity for the antimicrobial. Agarosa gel was used to determine the DNA pattern: 2 fragments of 289 bp and 55 bp when there is not mutation and 1 fragment of 344 bp when the Ser 83 to Leu 83 mutation is present.
The relationship between the PCR-RFLP pattern and the MIC to ofloxacin is shown in the table 1. The results of PCR-RFLP analysis of most strains were in agreement with the results of MIC. One isolate was susceptible to ofloxacin by agar dilution (MIC = 0.25 mg/l) whereas by PCR-RFLP this isolate seems to be resistant because it presents the mutation in gyrA gene. Two isolates with intermediate MIC (4 mg/l) showed mutation in gyrA.
The genotypic study by PCR-RFLP proved that ofloxacin resistant A. baumannii strains showed a punctual mutation in gyrA gene, in the same position inside the sequence of gene.
|Session name:||XXIst ISTH Congress|
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