Airway inflammation and etiology of acute exacerbations of chronic bronchitis.

STUDY OBJECTIVES: The etiologic role of bacterial pathogens isolated from sputum culture in 40 to 50% of acute exacerbations of chronic bronchitis (AECB) is controversial. If bacterial pathogens cause these AECB, they should be associated with greater neutrophilic airway inflammation than pathogen-negative exacerbations. DESIGN: This hypothesis was tested by comparing levels of interleukin (IL)-8, tumor necrosis factor (TNF)-alpha, and neutrophil elastase (NE) in 81 sputum samples obtained from 45 patients with AECB. Four groups were compared. In the first three groups, nontypable Haemophilus influenzae (n = 20), Haemophilus parainfluenzae (n = 27), and Moraxella catarrhalis (n = 14) were isolated as sole pathogens, respectively. In the fourth group, only normal flora was isolated (n = 20). Paired samples, obtained from individual patients at different times, that differed in their culture results were also compared. SETTING: An outpatient research clinic at a Veterans Affairs Medical Center. PATIENTS: These patients were participating in a prospective, longitudinal study of the dynamics of bacterial infection in chronic bronchitis, for which they were seen in the study clinic on a monthly basis as well as when they were experiencing symptoms suggestive of AECB. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: H influenzae exacerbations were associated with significantly higher sputum IL-8, TNF-alpha, and NE. M catarrhalis exacerbations demonstrated significantly higher sputum TNF-alpha and NE when compared to pathogen-negative exacerbations. H parainfluenzae-associated exacerbations had an inflammatory profile similar to pathogen-negative exacerbations. Sputum elastase level distinguished bacterial from nonbacterial AECB and correlated with clinical severity of the AECB. CONCLUSIONS: Increased airway inflammation associated with isolation of H influenzae and M catarrhalis supports an etiologic role of these pathogens in AECB.

Simultaneous respiratory tract colonization by multiple strains of nontypeable haemophilus influenzae in chronic obstructive pulmonary disease: implications for antibiotic therapy.

Nontypeable Haemophilus influenzae often causes exacerbations of chronic obstructive pulmonary disease (COPD), and these exacerbations are frequently treated with oral antibiotics. The goals of this study were to determine the frequency of the simultaneous presence of multiple strains of H. influenzae in sputum and to measure the MICs of antibiotics for the isolates. In a prospective study, adults with COPD were seen monthly. Sputum cultures were obtained, and individual colonies were subjected to genomic DNA typing and MIC determinations. Multiple strains of H. influenzae were present simultaneously in the sputum of 26.3% of adults with COPD. In 64.5% of these, MICs of >/=1 antibiotic varied by >/=4-fold among the strains. Therefore, multiple strains of H. influenzae are frequently present simultaneously in the sputum of adults with COPD, and the antimicrobial susceptibility of different strains in the same sputum sometimes differs.

Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method.

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein.

We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

Dynamics of respiratory tract colonization by Branhamella catarrhalis in bronchiectasis.

Branhamella catarrhalis is increasingly recognized as a lower respiratory tract pathogen, particularly in chronic lung diseases. This project defines a population of patients in whom the dynamics of colonization and infection caused by this organism could be studied. A method employing pulsed field gel electrophoresis (PFGE) of genomic DNA was developed. Twenty-eight patients with bronchiectasis followed prospectively for 26.8 mo (mean) were seen monthly or bimonthly and at the time of a purulent exacerbation. Quantitative bacterial cultures were performed on sputum obtained at each visit. Six of 28 had B. catarrhalis isolated repeatedly. Viable numbers of B. catarrhalis were similar to other bacterial pathogens. Restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA using PFGE was performed on 37 of the 47 isolates recovered. Each patient was colonized by two to four strains with different RFLP patterns. Duration of colonization by the same strain was 2.3 mo (mean). Strain acquisition did not correlate with exacerbation, antibiotic therapy, or season. We conclude that (1) a subset of bronchiectatic patients is colonized with B. catarrhalis, (2) RFLP is a sensitive tool to study strain acquisition, and (3) acquisition and clearance of B. catarrhalis from the respiratory tract is a dynamic process.

Purification and characterization of a high-molecular-weight outer membrane protein of Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis is an important bacterial cause of otitis media in children and lower respiratory tract infections in adults. In this study, we describe the presence of a novel high-molecular-weight outer membrane protein (HMW-OMP). This protein varies from 350 to 720 kDa in apparent molecular mass among strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was detected on SDS-PAGE in 13 of 14 strains tested. We developed a monoclonal antibody and polyclonal antisera to this protein. In immunoblot assays, the protein was present in all 14 strains tested. The immunoblot assays suggest that the protein has at least one epitope that is conserved among strains. A purification method using anion-exchange chromatography is described. Treatment of outer membrane preparations and purified protein by heat and reducing agents did not change the apparent molecular mass of the HMW-OMP. Formic acid treatment of outer membrane preparations and purified HMW-OMP produced a single band with an apparent molecular mass of 120 to 140 kDa. We postulate that this may be the monomer of an oligomeric protein. The HMW-OMP, which varies in molecular mass among strains and is antigenically conserved, will be studied further to determine its role in the human immune response and may be useful as a marker in studying strain acquisition in patients.

Nearest neighbor analysis of outer membrane proteins of nontypeable Haemophilus influenzae.

The arrangement of outer membrane proteins on the surface of nontypeable Haemophilus influenzae was investigated with cleavable and noncleavable bis-imidate cross-linking agents. Whole organisms were subjected to cross-linking agents, and oligomers of proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and immunoblot assay, using monoclonal antibodies to outer membrane proteins. The major outer membrane protein (P2) formed dimers and trimers detected by all three methods. Oligomers of other outer membrane proteins were not detected. These data indicate that P2 exists as a trimer on the outer membrane and suggest that other outer membrane proteins exist as monomers on the outer membrane.