Target protein mutation in the analysis of bactericidal antibiotic action and resistance

Abstract Future antimicrobial drug design and resistance minimisation, requires an understanding of current antimicrobial action and resistance. This study investigated bactericidal action and resistance within two antimicrobial groups; the fluoroquinolones and the aminoglycosides. The importance of target protein mutation to fluoroquinolome resistance was characterised in an outbreak of fluoroquinolone resistant Escherichia coli (n=17), and allowed a direct comparison with previous data deduced solely from in vitro laboratory selected mutants. Resistance to ciprofloxacin ranged from (MICs) 0.25—>256mg/L, with 7/17 isolates highly resistant (MICs=>32mg/L). A genotypic analysis by Random Amplified Polymorphic DNA (RAPD) typing, demonstrated that clonal spread of two strains was responsible for the highly resistant isolates. Enterobacterial intergenic concensus (ERIC) PCR typing supported RAPD results whereas PFGE and AFLP analysis were unsuccessful in typing isolates. A sequential increase in resistance was observed in both strains providing a unique opportunity to study resistance acquisition in the clinic. Five isolates (TYPE-I) showed the following increase in resistance: MICs of ciprofloxacin 8-» 32—> 128—>256 mg/L. Gene amplification and sequencing showed that all isolates contained mutations Ser83->Leu and Asp87—>Asn/Tyr in DNA Gyrase GyrA, and mutation Ser80->Ile in Topoisomerase IV ParC. Isolates with ciprofloxacin MICs of 128 and 256 mg/L, had the additional mutation Glu84—>Lys in ParC. Four isolates (RAPD type II) showed an increase in resistance from a clinically sensitive (MIC=lmg/L) to a highly resistant (MIC=:32mg/L) phenotype. The sensitive isolates (n=3) contained mutations Ser83—>Leu in GyrA and Ser80—>Ile in ParC. With progression to clinical resistance this strain additionally acquired mutations Asp87—>Asn in GyrA, and Glu84—>Val in ParC. This indicates that selection of high-level resistance is dependent upon second step mutations in both DNA gyrase and topoisomerase IV enzymes, as loss of Ser83 in GyrA and Ser80 in ParC do not, in themselves, confer the highly resistantFuture antimicrobial drug design and resistance minimisation, requires an understanding of current antimicrobial action and resistance. This study investigated bactericidal action and resistance within two antimicrobial groups; the fluoroquinolones and the aminoglycosides. The importance of target protein mutation to fluoroquinolome resistance was characterised in an outbreak of fluoroquinolone resistant Escherichia coli (n=17), and allowed a direct comparison with previous data deduced solely from in vitro laboratory selected mutants. Resistance to ciprofloxacin ranged from (MICs) 0.25—>256mg/L, with 7/17 isolates highly resistant (MICs=>32mg/L). A genotypic analysis by Random Amplified Polymorphic DNA (RAPD) typing, demonstrated that clonal spread of two strains was responsible for the highly resistant isolates. Enterobacterial intergenic concensus (ERIC) PCR typing supported RAPD results whereas PFGE and AFLP analysis were unsuccessful in typing isolates. A sequential increase in resistance was observed in both strains providing a unique opportunity to study resistance acquisition in the clinic. Five isolates (TYPE-I) showed the following increase in resistance: MICs of ciprofloxacin 8-» 32—> 128—>256 mg/L. Gene amplification and sequencing showed that all isolates contained mutations Ser83->Leu and Asp87—>Asn/Tyr in DNA Gyrase GyrA, and mutation Ser80->Ile in Topoisomerase IV ParC. Isolates with ciprofloxacin MICs of 128 and 256 mg/L, had the additional mutation Glu84—>Lys in ParC. Four isolates (RAPD type II) showed an increase in resistance from a clinically sensitive (MIC=lmg/L) to a highly resistant (MIC=:32mg/L) phenotype. The sensitive isolates (n=3) contained mutations Ser83—>Leu in GyrA and Ser80—>Ile in ParC. With progression to clinical resistance this strain additionally acquired mutations Asp87—>Asn in GyrA, and Glu84—>Val in ParC. This indicates that selection of high-level resistance is dependent upon second step mutations in both DNA gyrase and topoisomerase IV enzymes, as loss of Ser83 in GyrA and Ser80 in ParC do not, in themselves, confer the highly resistant