Dynamics of long-term colonization of respiratory tract by Haemophilus influenzae in cystic fibrosis patients shows a marked increase in hypermutable strains

Abstract

The persistence and variability of 188 Haemophilus influenzae isolates in respiratory tract of 30 cystic fibrosis (CF) patients over the course of 7 years was studied. Antibiotic susceptibility testing, DNA fingerprinting, and analysis of outer membrane protein profiles were performed on all isolates. A total of 115 distinct pulsed-field gel electrophoresis profiles were identified. Ninety percent of patients were cocolonized with two or more clones over the studied period. A third of the patients were cross-colonized with one or two H. influenzae strains; 11% of the clones persisted for 3 or more months. Biotype, outer membrane protein profiles, and resistance profiles showed variation along the studied period, even in persisting clones. Four isolates (2.1%) recovered from 3 patients were type f capsulate, with three of them belonging to the same clone. beta-Lactamase production was detected in 23.9% of isolates while 7% of the beta-lactamase-negative isolates presented diminished susceptibility to ampicillin (beta-lactamase-negative ampicillin resistance phenotype). Remarkably, 21.3% of the H. influenzae isolates presented decreased susceptibility to ciprofloxacin, which was mainly observed in persisting clones. Of the H. influenzae isolates from CF patients, 18 (14.5%) were found to be hypermutable in comparison with 1 (1.4%) from non-CF patients (P < 0.0001). Ten patients (33.3%) were colonized by hypermutable strains over the study period. A multiresistance phenotype and long-term clonal persistence were significantly associated in some cases for up to 7 years. These results suggest that H. influenzae bronchial colonization in CF patients is a dynamic process, but better-adapted clones can persist for long periods of time.

FIG. 1.

PFGE fingerprints of 15 strains from patient 1 in chronological order. The patient harbors three patterns: pattern 1A (lanes 1, 3, 5, 6, 10, 11, 12, 13, 14, and 15), pattern 1B (lanes 2, 4, 7, and 8), and pattern 2 (lane 9). Pattern 1B strains showed the same biotype (VI) as six strains (lanes 1, 3, 5, 6, 11, and 15) of pattern 1A and one strain of pattern 2 (lane 9). (A) SmaI PFGE fingerprints; (B) Bsp120 I (ApaI) PFGE fingerprints. M is the molecular size marker (PFGE marker).

FIG. 2.

PFGE fingerprints of 18 strains from patient 3 in chronological order. The patient harbors four clones: clone 4 (lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 16), clone 6 (lanes 14 and 18), clone 7 (lane 15), and clone 8 (lane 17). Clones 6 and 8 showed the same biotype (III). (A) SmaI PFGE fingerprints; (B) Bsp120 I (ApaI) PFGE fingerprints. M is the molecular size marker (PFGE marker). PFGE patterns of the same strains with Bsp120 I (ApaI) are shown in the second photo.

FIG. 3.

PFGE fingerprints of 27 strains from patient 28 in chronological order. The patient harbors five clones: clone 101 (lanes 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 24, 25, 26, and 27), clone 102 (lane 5), clone 103 (lane 18), clone 104 (lane 22), and clone 105 (lane 23). Three strains of clones 102 (lane 5), 103 (lane 18), and 104 (lane 22) and 20 strains (lanes 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14, 16, 17, 19, 20, 21, 24, 25, and 26) of clone 101 showed the same biotype (I). Clone 105 and two isolates of clone 101 (lanes 10 and 27) showed the same biotype (II). (A) SmaI PFGE fingerprints; (B) Bsp120 I (ApaI) PFGE fingerprints. M is the molecular size marker (PFGE marker).

FIG. 4.

Variants of OMP profiles of seven H. influenzae strains belonging to two PFGE patterns (1A and 1B) from patient 1 isolated at different periods of time. Letters a and b indicate variations in the OMPs.

FIG. 5.

Mutation frequency of the CF and non-CF H. influenzae strains for rifampin resistance. Each dot represents the mean mutation frequency calculated from 3 experiments for 1 strain. Most strains in both groups yielded mutation frequencies to rifampin resistance of <10⁻⁷. Strains with mutation rates of >10⁻⁷ were considered to display a hypermutable phenotype.