Minimal criteria for identification of Moraxella (Branhamella) catarrhalis

A study was performed which aimed at testing the reliability of our routine diagnostic tests for identification of Moraxella (Branhamella) catarrhalis in clinical samples from the respiratory tract. A preliminary diagnosis of 122 isolates as Moraxella catarrhalis was obtained by using colony morphology and results of Gram stain and oxidase test as the sole diagnostic criteria. By using additional tests we could show that the preliminary diagnosis was incorrect for 21 isolates, which were classified as different Neisseria species. 20 of these were isolated from sputum samples. We propose that at least a test for DNA hydrolysis should be included in the routine procedure for identification of Moraxella catarrhalis in sputum.     

In vitro activity of cefuroxime against Moraxella (Branhamella) catarrhalis

Minimal inhibitory concentrations of cefuroxime were determined by an agar dilution procedure and compared with erythromycin and four other beta-lactam antibiotics (amoxycillin, amoxycillin + clavulanate, cefadroxil, cefaclor) on 76 strains of Moraxella (Branhamella) catarrhalis. Sixty four of them produced a beta-lactamase. Results show that the beta-lactamase of Moraxella (Branhamella) catarrhalis abrogates the activity of amoxycillin (MIC 90% = 4 mg/l) meanwhile the combination of amoxycillin-clavulanate is inhibitory at low concentration (MIC 90% = 0.25 mg/l). There are no difference in MICs between strains producing or not producing beta-lactamase with erythromycin (MIC 90% = 0.25 mg/l). All the strains evaluated in this investigation, producing or not producing beta-lactamase have MIC 90% less than or equal to 2 mg/l for cefuroxime lower than those obtained for the two other cephalosporins.     

Antigenic heterogeneity and molecular analysis of CopB of Moraxella (Branhamella) catarrhalis.

Outer membrane protein (OMP) CopB, an iron-repressible 81-kDa major OMP of Moraxella (Branhamella) catarrhalis has been a major focus of investigation. To assess CopB as a potential vaccine antigen, we elucidated the degree of antigenic and sequence heterogeneity in this protein among strains of M. catarrhalis. Two monoclonal antibodies, 1F5 and 2.9F, which bind to surface-exposed epitopes on CopB recognized 60 and 70% of the strains, respectively. The degree of sequence heterogeneity in CopB was assessed by cloning and sequencing the CopB gene from two different strains of M. catarrhalis and comparing with the published sequence. There was 92 to 96% homology between the sequences at the nucleotide level and 90 to 95% homology at the amino acid level. The variability in the protein sequence is confined mainly to three moderately variable regions. Restriction fragment length polymorphism (RFLP) analysis of the CopB genes obtained from 20 diverse strains by PCR was performed. Ninety percent of the potential restriction sites in the constant regions and 47% of the potential restriction sites in the variable regions were present in the 20 strains, indicating that the pattern of variable and constant areas in the CopB gene is a general pattern among strains of M. catarrhalis. We conclude that the CopB gene is largely conserved among strains of M. catarrhalis and contains discrete regions which show moderate heterogeneity among strains.     

Assessment of complement-mediated killing of Moraxella (Branhamella) catarrhalis isolates by a simple method.

Recently, we showed that complement resistance is an important virulence factor of Moraxella (Branhamella) catarrhalis. Our study used a serum bactericidal assay to determine complement resistance in M. catarrhalis. Although the serum bactericidal assay is considered the "gold standard" for determining complement resistance, it is laborious and time-consuming and therefore not well suited for large-scale studies. Using a large number (n = 324) of M. catarrhalis isolates obtained from the sputa of patients with lower respiratory tract infections (n = 200) and young carriers (n = 124), we assessed the value of a simple "culture-and-spot" test as an alternative to the serum bactericidal assay. For both groups of isolates, the degree of concordance between the two tests used was very significant (P < 0.0001). The agreement between the two assays was estimated to be "excellent beyond chance" (as determined by Cohen's kappa test). The culture-and-spot assay is a valuable alternative to the serum bactericidal assay, not only for screening purposes as shown here but also for studying the mechanism of complement resistance in M. catarrhalis at the molecular level.     

A monoclonal antibody reactive with a common epitope of Moraxella (Branhamella) catarrhalis lipopolysaccharides.

A hybrid cell line producing a monoclonal antibody (MAb) against Moraxella (Branhamella) catarrhalis lipopolysaccharide (LPS) was established. The specificity of the MAb 1B12 to purified rough LPSs from six strains of M. catarrhalis was ascertained by enzyme-linked immunosorbent assay (ELISA), competitive-inhibition ELISA, and immunoblotting. MAb 1B12 bound to live bacterial cells and culture supernatants from a total of 34 strains of M. catarrhalis, including 12 strains with different LPS serotypes. No cross-reactions with smooth and rough LPSs from selected enterobacterial and nonenterobacterial strains, with other respiratory pathogens, or with Neisseria species were observed. These data suggest that MAb 1B12 recognizes a common epitope of M. catarrhalis LPS which differs from serotype determinants.     

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.     

Synthesis and Characterization of Lipooligosaccharide-Based Conjugates as Vaccine Candidates for Moraxella (Branhamella) catarrhalis

Moraxella (Branhamella) catarrhalis is an important cause of otitis media and sinusitis in children and of lower respiratory tract infections in adults. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and elicits bactericidal antibodies. Treatment of the LOS from strain ATCC 25238 with anhydrous hydrazine reduced its toxicity 20,000-fold, as assayed in the Limulus amebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled to tetanus toxoid (TT) or high-molecular-weight proteins (HMP) from nontypeable Haemophilus influenzae through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively. The antigenicity of the two conjugates was similar to that of the LOS, as determined by double immunodiffusion. Subcutaneous or intramuscular injection of both conjugates elicited a 50- to 100-fold rise in the geometric mean of immunoglobulin G (IgG) to the homologous LOS in mice after three injections and a 350- to 700-fold rise of anti-LOS IgG in rabbits after two injections. The immunogenicity of the conjugate was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced antisera had complement-mediated bactericidal activity against the homologous strain and heterologous strains of M. catarrhalis. These results indicate that a detoxified LOS-protein conjugate is a candidate for immunization against M. catarrhalis diseases.     

The macrophage response to bacteria. Modulation of macrophage functional activity by peptidoglycan from Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis organisms have been shown to be particularly efficient in inducing in a pure population of bone marrow-derived mononuclear phagocytes secretory and cellular activities. In the present study, the ability of peptidoglycan from this Gram-negative organism to trigger a macrophage response was compared with that elicited by peptidoglycan from Staphylococcus aureus and Bacillus subtilis. The results show that the three peptidoglycans were similarly active in triggering the secretion of tumour necrosis factor and tumouricidal activity but differed considerably in their ability to induce the generation of nitrite in macrophages; in this respect, peptidoglycan from M. catarrhalis was particularly potent. The impressive capacity of M. catarrhalis peptidoglycan to induce in low concentration the secretion of tumour necrosis factor and nitrite and tumouricidal activity may, in addition to its lipopolysaccharide, contribute to the extraordinary potential of this organism to trigger the functional activities of macrophages.     

Serum antibody response to proteins of Moraxella (Branhamella) catarrhalis in patients with lower respiratory tract infection.

We searched for antibodies against Moraxella (Branhamella) catarrhalis proteins in the sera of patients with lower respiratory tract infection. Sera from 48 patients with M. catarrhalis and 39 patients without M. catarrhalis in their lower respiratory tract specimens were studied by a gel electrophoresis-immunoperoxidase technique; sera from 23 healthy adult blood donors were also included. Immunoglobulin G (IgG) antibodies against a 28-kDa protein were found significantly more frequently in patients with M. catarrhalis in lower respiratory tract specimens (71%) than in patients without M. catarrhalis in lower respiratory tract specimens (28%) or healthy adult blood donors (22%). Seroconversion, from the acute to the convalescent stages, occurred in at least eight patients with M. catarrhalis and in one patient without detectable M. catarrhalis. IgG antibodies against other M. catarrhalis proteins were found in most sera, including those obtained from blood donors. By adsorption experiments the 28-kDa protein was demonstrated to be surface exposed. IgM antibodies against an 85-kDa protein were found in serum from one patient from whom M. catarrhalis and Streptococcus pneumoniae were isolated from the lower respiratory tract, while IgA antibodies against M. catarrhalis proteins could not be detected in any serum specimen.     

Respiratory tract carrier rates of Moraxella (Branhamella) catarrhalis in adults and children and interpretation of the isolation of M. catarrhalis from sputum.

Nonselective media and previously described selective media were used to study the occurrence of Moraxella (Branhamella) catarrhalis in sputum samples of good and poor quality and in samples taken from different sites of the upper respiratory tracts of healthy subjects. It was found that in healthy adults the carrier rate was 5.4%, as opposed to 50.8% in children and 26.5% in people older than 60 years. M. catarrhalis was recovered significantly more often from sputum samples of good quality (5%) than from poor quality samples (0.5%), and when present, it was found mostly in the presence of high inocula. From these data gathered from healthy and diseased subjects, it is concluded that the presence of M. catarrhalis in the sputum of adults is rarely due to oronasopharyngeal contamination of the sputum. Similar findings reported by others are discussed, and the origins of the currently held concept that M. catarrhalis is a common commensal organism of the human upper respiratory tract are traced.     

Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay.

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.     

Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and lactoferrin receptor orf3 isogenic mutants

Pathogenic members of the family Neisseriaceae produce specific receptors to acquire iron from their host's lactoferrin and transferrin. Recently, putative Moraxella catarrhalis lactoferrin receptor genes and a third open reading frame (lbpB, lbpA, and orf3) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.     

Pseudogonococcal ophthalmia neonatorum. Branhamella (Neisseria) catarrhalis conjunctivitis.

A culture from conjunctivitis occurring in a neonate in association with a recurrent fever yielded a nearly pure growth of Branhamella (Neisseria) catarrhalis. The conjunctivitis was not appreciated and effectively treated until a second hospitalization in the fourth week of life. Partial suppression of symptoms had followed short-term parenteral antibiotic therapy during the first admission. Resolution quickly occurred in response to instillation of sodium sulfacetamide ophthalmic solution. Although B. catarrhalis is considered a non-pathogen, the literature reviewed included a number of diverse infections, but no previous instance of conjunctivitis. The organism's close similarities to Neisseria gonorrhoeae necessitate isolation and correct biochemical differentiation. Misdiagnosis of gonococcal conjunctivitis carries obvious social, psychological and medical impact.     

Characterisation of hospital isolates of Moraxella (Branhamella) catarrhalis by SDS-PAGE of whole-cell proteins, immunoblotting and restriction-endonuclease analysis.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP), immunoblot analysis and DNA restriction-endonuclease analysis (REA) were applied as potential typing methods to 31 clinically significant strains of Moraxella (Branhamella) catarrhalis, five of which came from a suspected outbreak of nosocomial infection in a respiratory-diseases ward. Twelve of 31 isolates were placed in four groups, each of which contained strains indistinguishable by the three typing techniques used. Each of a further two groups contained two strains, and they were similar by at least one technique; the remaining 15 strains were unique by all three methods. Four of five strains from the suspected outbreak were indistinguishable by SDS-PAGE of WCP, immunoblotting and REA. Results show that SDS-PAGE of WCP, immunoblotting and REA are suitable techniques for characterising M. catarrhalis and that there is a considerable degree of strain heterogeneity. Nosocomial infection with M. catarrhalis may be relatively common and further epidemiological studies with a combination of typing techniques are indicated.     

Branhamella (Neisseria) catarrhalis: criteria for laboratory identification.

Eleven clinically significant isolates of Branhamella catarrhalis grew well on modified Thayer-Martin medium and produced beta-lactamase, but did not grow on nutrient agar at 22 degrees C. Minimum inhibitory concentrations of vancomycin, colistin, and trimethoprim were found to be higher than the concentrations of these antibiotics in modified Thayer-Martin medium. The criteria necessary for laboratory identification of B. catarrhalis are discussed.     

Otitis media caused by beta-lactamase-producing Branhamella (Neisseria) catarrhalis.

A beta-lactamase-producing Branhamella catarrhalis was isolated in pure culture from the right middle ear aspiration of an otitis media in a 3-month-old girl. The patient responded well to cefamandole treatment.     

Adenovirus serotype 1 does not act synergistically with Moraxella (Branhamella) catarrhalis to induce otitis media in the chinchilla.

A chinchilla model of otitis media in which adenovirus compromise of the tubotympanum facilitates the subsequent induction of middle ear disease was used to investigate Moraxella (Branhamella) catarrhalis pathogenesis. Intranasally inoculated M. catarrhalis did readily colonize the nasopharynx of this host; however, despite evidence of viral infection and tubotympanal compromise, M. catarrhalis did not induce culture-positive otitis media in this model.     

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.

The display of single-chain Fv (scFv) or Fab antibody fragments on the surface of filamentous phage particles and the selection of recombinant phages by binding to a target antigen provide a novel means of isolating antibodies with predetermined specificity (for reviews, see references 5 and 39). Libraries may be assembled from the variable (V) regions expressed by B lymphocytes from either an individual with a particular immune response or a nonimmunized individual in an attempt to recruit the diversity generated by the natural immune system. An alternative approach to create diverse libraries exploits the use of large collections of cloned V genes to which randomized CDR3 regions are fused in vitro by PCR. We recently constructed a large semisynthetic phage library of human scFv fragments with partially randomized heavy-chain CDR3 regions, designed to encode a high frequency of functional antibody molecules (8).Selection of phage antibodies (PhAbs) of desired specificities is conventionally performed by panning of libraries on solid-phase-coated antigens and eluting bound phages with high- or low-pH buffers. Alternative strategies have also been successfully used. For example, PhAbs have been obtained by direct selection on structures expressed on the membranes of eukaryotic and bacterial cells expressing the target antigen as a recombinant fusion protein (3, 9, 25, 30). Furthermore, phage libraries may be preabsorbed to remove unwanted specificities, or alternatively, selections may be performed in the presence of a homologous competitor antigen to enrich for phages directed to nonhomologous regions of the target antigen (6, 8, 25). Finally, phages bound to the target structure may be eluted by competition with ligand or a conventional monoclonal antibody (MAb) (5, 26, 39).In this study, we used our library in a competitive panning procedure to demonstrate the feasibility of obtaining antibodies specific for an unknown target structure predicted to be differentially expressed on two strains of the same bacterial species. Selections were performed on Moraxella (Branhamella) catarrhalis, a gram-negative bacterium that may cause upper respiratory tract disease in children and lower respiratory tract disease in elderly people and patients with chronic obstructive pulmonary disease (2, 7, 12, 28). Resistance against complement-mediated lysis is considered an important virulence factor of this bacterium (15, 17). Complement-resistant bacteria were coated onto a solid support, and phage selections were performed in the presence of a complement-sensitive strain as a particulate competitor antigen. After three rounds of selection, a collection of 10 different monoclonal PhAbs (MPhAbs) with binding specificity for complement-resistant but not complement-sensitive M. catarrhalis isolates was obtained. The molecule recognized was identified as the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis (22).     

Phenotypic characteristics of Branhamella catarrhalis strains.

Isolates of Branhamella catarrhalis from 13 patients with pneumonia, 6 patients with tracheobronchitis, and 8 patients who were colonized with the organism were studied with respect to susceptibility to the bactericidal action of normal human serum (NHS), glass slide hemagglutination (HA) of group O human erythrocytes, beta-lactamase production, and susceptibility to selected antimicrobial agents and laboratory drugs. A total of 18 of 27 isolates were serum resistant, 22 of 27 produced HA, and 21 of 27 were beta-lactamase positive. Statistically significant correlations were found between susceptibility to NHS and susceptibility to trypsin (r = +0.47; P = 0.01) and between susceptibility to NHS and HA (r = -0.48; P = 0.009). Significant correlations were also observed among several pairs of antimicrobial drugs. Analysis of variance showed that mean ampicillin MICs correlated with isolate group (r = -0.49; P = 0.03) in that the pneumonia isolates had higher MICs. Some phenotypic characteristics appeared to be independent of each other. These data suggest that important differences exist among clinically significant B. catarrhalis strains and that these differences may be due to differences in the cell wall envelope of the organism.