Fluoroquinolone resistance in Haemophilus influenzae is associated with hypermutability

Abstract

Forty-three percent (12/28) of ciprofloxacin (CIP)-nonsusceptible respiratory isolates of Haemophilus influenzae were hypermutable, compared with 8.5% (3/35) in the CIP-susceptible control group (P=0.002). CIP-nonsusceptible mutants were obtained with hypermutable strains only; these mutants developed three resistance mechanisms in a step-by-step process: target modifications, loss of a porin protein, and increased efflux.

FIG. 1.

Distribution of H. influenzae strains according to their mutation frequency in response to rifampin and according to the presence of amino acid changes in the quinolone resistance-determining region of GyrA. The control group comprises strains with no amino acid changes in the quinolone resistance-determining region of GyrA and MICs for CIP of <0.12 μg/ml. The study group comprises strains with amino acid changes in the quinolone resistance-determining region of GyrA and MICs for CIP of 0.12 μg/ml to 32 μg/ml. Horizontal bars represent the average mutation frequency of the study group (4 × 10⁻⁶ CFU/ml) and the control group (7 × 10⁻⁸ CFU/ml) (P = 0.017; Mann-Whitney U test).

FIG. 2.

Profile of outer membrane proteins in the H. influenzae wild strains A (lane 1) and D (lane 6) and their in vitro mutants A1, A2, A3, and A4 (lanes 2 to 5) and D1, D2, and D4 (lanes 7 to 9). Outer membrane protein porin P2 is indicated. KD and M, molecular mass markers in kDa.

FIG. 3.

Accumulation of norfloxacin in three pairs of isogenic strains in the absence (basal) and presence (0.1 mM) of MC-207,110. Vertical lines separate wild strains (A, C, and D) from their in vitro mutants (A4, C3, and D4). The data shown are the averages ± standard deviations of triplicate experiments. *, increase over basal accumulation of 2,126% (21 times); **, increase over basal accumulation of 140% (1.4 times).