Effect of subinhibitory concentrations of ciprofloxacin on Mycobacterium fortuitum mutation rates.

OBJECTIVES: Fluoroquinolones have found a place in the management of mycobacterial diseases including tuberculosis. It has been previously shown that subinhibitory concentrations of quinolones increase the mutation rate in Escherichia coli and staphylococci. The purpose of this study is to extend this observation to mycobacteria and to quantify mutation rates. METHODS: The mutation rate in Mycobacterium fortuitum to ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, erythromycin and gentamicin resistance was determined when grown with and without various sub-MIC concentrations of ciprofloxacin. RESULTS: M. fortuitum exposed to 1/2 MIC ciprofloxacin had an increase in the mutation rate of between 72- and 120-fold when selected on quinolones or other antimycobacterial antibiotics. Smaller, but significant increases in mutation rate were seen when the organism was exposed to lower concentrations (1/4 MIC and 1/8 MIC). CONCLUSIONS: These data show that sub-MIC concentrations of fluoroquinolone significantly increase mutation rates and these data suggest that care must be taken to ensure that bacteria are not exposed to subinhibitory concentrations when adding quinolones to a regimen used to treat mycobacterial infection.

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110.

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.

Fluoroquinolone resistance in Streptococcus pneumoniae: evidence that gyrA mutations arise at a lower rate and that mutation in gyrA or parC predisposes to further mutation.

Fluoroquinolones are being increasingly used for acute lower respiratory tract infection where Streptococcus pneumoniae is the most important bacterial pathogen. S. pneumoniae becomes resistant to quinolone antibiotics by mutations in a small section of the parC and gyrA genes. In this study, we investigated the mutation rates and spectrum of resistance when ciprofloxacin and gemifloxacin were the selective agents. When ciprofloxacin was the selective agent, parC mutants arose at a rate of 1.1 x 10(-9) mutations per cell division. There were two double mutants: parC + gyrA and parC + gyrB, and these mutations arose in as few as five generations. When gemifloxacin was the selective agent, all but one of the colonies growing on the x2 MIC plate had no mutations in gyrA or parC. The only mutation identified was in gyrA, and it appeared at a rate of 1.6 x 10(-11). When the gemifloxacin MIC of strains with mutations in parC was determined, there was no change from the susceptible parent. These data indicate that S. pneumoniae becomes resistant to gemifloxacin through mutation in gyrA rather than parC. Because gyrA mutations arise at a lower rate than parC mutations, it is likely that resistance to gemifloxacin will emerge more slowly than is seen with those quinolones that become resistant through an initial mutation in parC. The rate at which second-step mutants emerged was 1.3 x 10(-8) for parC Serine 79 Tyrosine and 7.2 x 10(-9) for gyrA Serine 81 Phenylalanine, 12 and 450 times higher, respectively, than for first-step rates, suggesting that mutation in either gene readies the genome for further mutation.

Evolutionary barriers to quinolone resistance in Streptococcus pneumoniae.

It is assumed that bacteria always pay a significant physiological price for the acquisition of resistance to antibiotics. To test whether this was the case for a strain of Streptococcus pneumoniae that develops resistance to fluoroquinolone antibiotics, we selected resistance to these agents in a wild-type strain and measured their fitness in comparative growth experiments. The relative growth rate of a mutant strain selected on ciprofloxacin (parC Serine 79 to Tyrosine) was compared with its susceptible isogenic parent and no significant deficit was found (relative fitness 1.15 95% C.I. +/- 0.2.). A double mutant, however, had a relative fitness of 0.81 (parC Serine 79 to Tyrosine gyrA Serine 81 to Tyrosine). Mutant strains selected on gemifloxacin had only a modest increase in minimum inhibitory concentration; thus, second-round mutants were competed with a first-round gyrA Serine 81 to Tyrosine or the susceptible isogenic parent. The growth rate of three double-mutant strains parC Serine 79 to Tyrosine gyrA Serine 81 to Phenylanine, parC Serine 79 to Tyrosine, and Asparagine 83 to Phenylalanine were similar to the isogenic susceptible parent 1.16 (95% C.I. +/- 0.17), 0.99 (95% C.I. +/- 0.05), and 0.95 (95% C.I. +/- 0.05), respectively. These data suggest that mutation in the parC and gyrA genes may, on some occasions, not be associated with a physiological deficit.