Efflux Pumps Contribute to Antibiotic Cross Resistance in Ciprofloxacin Selected Staphylococcus aureus

Staphylococcus aureus is a human pathogen that may become resistant to multiple antibiotics including fluoroquinolones such as ciprofloxacin (CIP). The overexpression of the NorA efflux pump, caused by a mutation in the norA promoter region, effluxes fluoroquinolones rendering S. aureus resistant to this class of antibiotics. We hypothesize that the spontaneous CIP mutants will have mutations in the norA promoter and structural gene sequence, and thus confer cross resistance to other fluoroquinolones. Spontaneous CIP resistant S. aureus ATCC25923 mutants were isolated from TSA medium supplemented with CIP 2 μg/ml. The CIP minimal inhibitory concentration (MIC) of 94 mutants was determined by plating the mutants on MHA containing two-fold dilutions of 0.25-32 μg/ml CIP. To screen mutants with a potential norA mutation, the mutants were plated on MHA containing CIP with or without 50 μg/ml of the known efflux pump inhibitor chlorpromazine (CPZ). The norA promoter and gene region of representative mutants that showed a decreased MIC in the presence of CPZ were amplified and sequenced using methods previously described. Using the Clinical and Laboratory Standards Institute disk diffusion assay, the CIP mutants were analyzed for antibiotic cross-resistance to tetracycline, chloramphenicol, gentamicin, erythromycin, rifampicin, clindamycin, vancomycin, norfloxacin, moxifloxacin, ceftazidime, ciprofloxacin, and trimethoprim. All of the CIP mutants had an increased in CIP MIC to at least 8 μg/ml compared to the wild type of 0.5 μg/ml. In the presence of CPZ; 91% of the mutants showed a decreased CIP MIC to 1 μg/ml. Of the 23 CIP mutant norA genes sequenced, CIP10 showed a mutation with an insertion in the amino acid sequence at position 108-110 and CIP1 showed multiple substitutions at NorA (F6L), (V87I), (H89Q), (S92L), (V93I). There were mutations in CIP1, 2, 6, and 27 in the norA promoter region. When tested for cross resistance, all the CIP mutants conferred resistance to second generation fluoroquinolone – norfloxacin. Mutants with CIP MIC of 32 μg/ml and above conferred resistance to fourth generation fluoroquinolone – moxifloxacin and mutants with CIP MIC of 8 μg/ml showed increased resistance to antibiotics from different classes such as erythromycin and ceftazidime. Our results show that the primary mechanism to become resistant when S. aureus is selected by CIP is by efflux pumps. Mutation in the norA promoter and gene is one of the mechanisms by which S. aureus becomes resistant to ciprofloxacin. The decrease of CIP MIC in the presence of CPZ supports the role of efflux pumps in mutant resistance. CPZ used in conjunction with ciprofloxacin renders the wild-type more susceptible. Cross resistance to moxifloxacin is particularly significant as this antibiotic is an antibiotic of last resort.