To investigate the epidemiology of nontypeable Haemophilus influenzae in the respiratory tract of cystic fibrosis (CF) patients, H. influenzae isolates from sputum specimens of 40 CF patients were analyzed longitudinally for 2 years. The isolates were characterized by analysis of the major outer membrane protein (MOMP) patterns. MOMP variant H. influenzae strains were discriminated from distinct strains by randomly amplified polymorphic DNA analysis of genomic DNA. Multiple H. influenzae strains and MOMP variant strains were isolated from single sputum specimens of 29 patients. In 22 patients, a distinct H. influenzae strain persisted over time (median persistence, 8 months; range 2-24). In general, the appearance of MOMP variant strains did not coincide with the occurrence of exacerbations.

No abstract

Summary. Non-capsulate strains of Haemophilus injluenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing : (i) the fingerprints of 16 colonies of a single H. injluenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. injluenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. injluenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. injluenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H . injluenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.

We investigated the relationship between susceptibility to β-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulatedHaemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinctH. influenzae strains from two patients extensively treated with β-lactam antibiotics. The variants differed in their susceptibilities to at least two β-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to β-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences ofompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.