Detection Of GyrA And ParC Mutations In Quinolone-Resistant Clinical Isolates Of Enterobacter Cloacae

Fluoroquinolones are broad-spread antimicrobial agents which have effective antimicrobacterial activity against gram-positive and gram- negative bacteria. However, widespread of this class of agents has resulted in an increasing incidence of fluoroquinolones resisance in many bacterial species including Enterobacter cloacae. E.cloacae is an important conditional pathogen which may cause infections of lower respiratory tract and urinary tract. Recently, the emergency of fluoroquinolone resistance and the increasing development of resistant strains in E.cloacae isolates have been reported. The resistance of this organism has become important in clinical, because it was associated with therapeutic failure.Studies on the mechanisms of quinolone resistance in gram-negative bacteria have demonstrated that alterations in the GyrA subunit of DNA gyrase are primarily responsible for high level resistance to nalidixic acid and the decreased susceptibility to fluoroquinolones, and that mutations in parC, which encodes the ParC subunit of toposomerase IV, play a secondary role in enhancing resistance to fluoroquinolones. However ,there was not enough investigation in E.cloacae.In this study, 59 E.cloacae clinical isolates were randomly collected from clinical specimens of sputum and urine from patients with lower respiratory tract and urinary tract infections at four hospital in Tianjin from June 2000 to June 2001. gyrA aqd parC point mutations in 30 E.cloacae quinolone resistant isolates were examined with polymerase chain reaction(PCR), combination ofrestriction fragment length polymorphism(RFLP) analysis and single-strand conformation polymorphism(SSCP) analysis. Meanwhile, we analysed the correlations of gyrA and parC with resistance phenotypes to quinolones.In this study, alterations in GyrA located at amino acid positions corresponding to Ser-83 in the E.coli GyrA protein were found by PCR-RFLP analysis in 30 quinolone-resistant strains for which MICs of nadilixic acid were 32 u g/ml. There were 29 strains for which MICs of nalidixic acid (MIC 10241 g/ml) and 25 strains being cross-resistant to ciprofloxacin(MIC4u g/ml) and ofloxacin(MIC 811 g/ml) in all quinolone-resistant isolates; there were other point mutation besides Ser-83 in the gyrA gene, we found two kinds of mutation in the parC gene by PCR-SSCP analysis from 12 quinolone-resistant strains for which MICs of ciprofloxacin were 8 P g/ml-16 V- g/ml, while in the other quinolone-resistant strains no strain we found had mutation in parC gene. In two quinolone-susceptible clinical isolates, for which MICs of nadilixic acidwas 41 g/ml, MICs of ciprofloxacin was 0.25 u g/ml, we found no mutation in gyrA and parC gene. This study demonstrated that the alterations in GyrA and ParC were responsible for the resistance phenotypes in E.cloacae. That is, the alterations in the GyrA are primary responsible for resistant to quinolones, and the alterations in the ParC may play a complemental role in enhancing resistant to fluoroquinolones.