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A new Moraxella strain was isolated from angular conjunctivitis. It is characterized by the ability to produce acid from several carbohydrates, by its ability to produce indole, and by a lack of catalase activity.
A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis. The transformation assay clearly distinguishes M. osloensis from Acinetobacter. Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis.
A Moraxella strain, A-43, produced a compound inhibitory to the outgrowth of Clostridium botulinum type E spores. The inhibitor could be produced in various laboratory media, and the outgrowth of germinated spores was inhibited by a 1/10th dilution of the A-43 spent medium. Germination was not affected. Molecular weight of the inhibitor was estimated at 800 to 1,000. The inhibitor was dialyzable and could be concentrated by lyophilization. It was stable at 37, 25, and 5 C, but was 70% inactivated when heated at 65 C for 10 min. The inhibitor was not volatile and could not be vacuum-distilled at 40 C. Solutions of acids with pH values below 2.0 destroyed the activity. The A-43 inhibitor appears to be similar, in molecular weight and inhibition characteristics, to tylosin.
An enzyme-linked immunosorbent assay (ELISA) for determination of serum immunoglobulin G (IgG) antibodies to Moraxella catarrhalis was developed, with an ultrasonic extract of M. catarrhalis immobilized on polystyrene microtiter plates serving as the antigen. The specificity was determined by adsorption tests. All of the 541 women tested showed a high level of maternal IgG antibodies to M. catarrhalis in umbilical cord blood specimens. One hundred eighty-nine children aged 0 to 15 years were examined. A low level of IgG antibodies to M. catarrhalis in serum was found in children aged up to 1 year; in older children, the levels increased with age. Levels in the same range as maternal IgG antibody levels were reached at the age of 10 years. The level of antibodies in children did not correlate with the state of colonization with M. catarrhalis or with the state of acute lower respiratory tract infection. Pairs of acute-phase and convalescent-phase serum samples did not discriminate between the children with M. catarrhalis in pure culture and those with mixed cultures of M. catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae. In adult women, high IgG antibody levels and low colonization rates with M. catarrhalis were found, whereas in small children, low IgG antibody levels and high colonization rates were found.
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.
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.
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 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.
A Western blot (immunoblot) method for detecting antibodies against outer membrane protein (OMP) epitopes of Moraxella (Branhamella) catarrhalis was evaluated. Paired serum samples from patients suspected of M. catarrhalis (n = 38) and non-M. catarrhalis (n = 25) bronchopulmonary infection were examined for the presence of antibodies of the immunoglobulin M (IgM), IgG, and IgA classes to OMPs from M. catarrhalis by a gel electrophoresis-immunoperoxidase technique (Western blotting); sera from 40 healthy adult blood donors were also included. A significantly (P = 0.004) more frequent occurrence of IgM-class antibodies and/or an increase in the number of IgG-class antibodies against different M. catarrhalis OMPs from acute- to convalescent-phase serum samples was found for patients with M. catarrhalis (79%) than for patients without M. catarrhalis (40%). IgM-class antibodies against OMPs of M. catarrhalis were found in acute- and/or convalescent-phase serum samples form 58% of patients with M. catarrhalis and 32% of patients without M. catarrhalis. Fifty percent of patients with M. catarrhalis and 16% of patients without M. catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgG-class antibodies directed against different OMPs. A total of 34% of patients with M. catarrhalis and 4% of patients without M. catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgA-class antibodies directed against different OMPs. The present study indicates that M. catarrhalis is one of the bacteria involved in acute exacerbations of chronic bronchitis.
Moraxella catarrhalis (formerly known as Branhamella catarrhalis) has emerged as a significant bacterial pathogen of humans over the past two decades. During this period, microbiological and molecular diagnostic techniques have been developed and improved for M. catarrhalis, allowing the adequate determination and taxonomic positioning of this pathogen. Over the same period, studies have revealed its involvement in respiratory (e.g., sinusitis, otitis media, bronchitis, and pneumonia) and ocular infections in children and in laryngitis, bronchitis, and pneumonia in adults. The development of (molecular) epidemiological tools has enabled the national and international distribution of M. catarrhalis strains to be established, and has allowed the monitoring of nosocomial infections and the dynamics of carriage. Indeed, such monitoring has revealed an increasing number of B-lactamase-positive M. catarrhalis isolates (now well above 90%), underscoring the pathogenic potential of this organism. Although a number of putative M. catarrhalis virulence factors have been identified and described in detail, their relationship to actual bacterial adhesion, invasion, complement resistance, etc. (and ultimately their role in infection and immunity), has been established in a only few cases. In the past 10 years, various animal models for the study of M. catarrhalis pathogenicity have been described, although not all of these models are equally suitable for the study of human infection. Techniques involving the molecular manipulation of M. catarrhalis genes and antigens are also advancing our knowledge of the host response to and pathogenesis of this bacterial species in humans, as well as providing insights into possible vaccine candidates. This review aims to outline our current knowledge of M. catarrhalis, an organism that has evolved from an emerging to a well-established human pathogen.
Moraxella catarrhalis strains can express either a UspA2 protein or a UspA2H protein. The latter protein is encoded by a gene that possesses a homopolymeric nucleotide tract containing eight adenine (A) residues [i.e., a poly(A) tract] which is located near the 5' end. A spontaneous UspA2H-negative variant of M. catarrhalis strain O46E, designated O46E.U2V, was found to have a uspA2H poly(A) tract that contained seven A residues. Northern blot analysis of total RNA from the O46E parent strain revealed a readily detectable uspA2H mRNA transcript, whereas little or no uspA2H transcript was detectable in total RNA from the UspA2H-negative variant O46E.U2V. The 5' end of the uspA2H genes from both the O46E parent strain and the O46E.U2V variant were ligated to a promoterless lacZ gene to prepare translational fusions for use as reporter constructs. The level of beta-galactosidase activity expressed by the fusion construct containing eight A residues in its poly(A) tract was 200-fold greater than that obtained with the construct that had seven A residues. Site-directed mutagenesis of the 5' end of the uspA2H gene confirmed that translation was initiated at a GTG codon located 21 nucleotides (nt) upstream of the poly(A) tract. Primer extension analysis determined that the transcriptional start site of the uspA2H gene was located 291 nt upstream from the GTG translational start codon. This poly(A) tract was also found to be present in the uspA2H genes of other M. catarrhalis strains.
Moraxella catarrhalis immunoglobulin D-binding protein (MID) is a complex antigen with unique immunoglobulin D (IgD)-binding, adhesion, and hemagglutination properties. Previous studies have shown that antibodies raised against MID764-913 in rabbits inhibited M. catarrhalis adhesion to human alveolar epithelial cells, and immunization with MID764-913 resulted in an increased pulmonary clearance in a murine model. Strong immune responses against MID have also consistently been shown in humans. Here, the MID-specified IgG responses were compared to those of ubiquitous surface proteins A1 and A2 (UspA1/A2) using a series of recombinant fragments that spanned all three proteins. Sera were obtained from young children, aged 6 months to 1 year (n=8) and 2 to 3 years (n=15), and healthy adults (n=16). Acute- and convalescent-phase sera from chronic obstructive pulmonary disease (COPD) patients with M. catarrhalis infective exacerbations (n=23) were also analyzed. Young children, who are at risk of M. catarrhalis infection, had low levels of anti-MID and anti-UspA1/A2 antibodies. Healthy adults and the majority of COPD patients (16/23) had high levels of antibodies directed against, among others, the adhesive domain of MID and the fibronectin- and C3-binding domains of UspA1/A2. Among eight COPD patients in whom a rise in antibody levels could be detected, these functional domains were also the main regions targeted by the antibodies. In addition, human IgG directed against MID was bactericidal and anti-MID antibodies were additive to antibodies targeting UspA1/A2. Hence, the functional domains in these three antigens may have significant potential in a future vaccine against M. catarrhalis.
Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.
Pregnant mice, guinea pigs, rats, and rabbits responded to injections of Moraxella bovis strain EPP-63(300) with abortion, death, embryo resorption, and production of small litters. The nature of response appeared to depend primarily on the number of viable cells injected and to some extent on the species of animal and stage of pregnancy. Intraperitoneal injection of mice with 3 X 10(5) viable cells induced 100% abortion and no deaths. Embryo resorption and smaller litters were induced with injection of lower doses of M. bovis. None of the rats used in this study aborted; however, resorption rates were higher in rats than mice. Bacteria-free filtrates prepared from aqueous extracts of blood agar on which M. bovis had been grown induced abortion in mice and guinea pigs, at a rate similar to that caused by viable cells. The lyophilized filtrate could be diluted to produce 100% abortion with no death of injected mice. Abortion-inducing, nonlethal doses of M. bovis and lyophilized filtrates appeared to have no effect on nonpregnant female or male mice.
The pilin gene of Moraxella bovis Dalton 2d was isolated by cloning in Pseudomonas aeruginosa. The nucleotide sequence of this gene encodes a prepilin of 156 amino acid residues. When high levels of pilin were expressed from the gene in P. aeruginosa, by using the pL promoter of bacteriophage lambda inserted upstream of the coding sequence, pili which were indistinguishable from pili of M. bovis were produced.
Immunoblotting with polyclonal rabbit antibodies raised against pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoeae, Dichelobacter nodosus, and Vibrio cholerae was used to demonstrate that these polypeptides display conserved antigenic and, in most cases, immunogenic determinants. These determinants appear to be localized to the highly homologous amino-terminal domains (residues 1 to 25).
A surface polysaccharide antigen of Moraxella nonliquefaciens, reported to be cross-reactive with the capsular polysaccharides of group B Neisseria meningitidis and Escherichia coli K1 (K. Błvre, K. Bryn, O. Closs, N. Hagen, and L. O. Froholm, NIPH Ann. 6:65-73, 1983), was isolated, purified, and characterized chemically, immunologically, and by nuclear magnetic resonance. This polysaccharide was shown to be a linear homopolymer of alpha (2----8)-linked N-acetylneuraminic acid, identical to the capsular polysaccharide of group B N. meningitidis and O-acetyl-negative variants of E. coli K1.
Bovine erythrocytes (RBCs) exposed to Moraxella bovis culture supernatants exhibited rapid leakage of intracellular K+ (95% in 10 min), slower cell swelling (1.20-fold increase in mean corpuscular volume in 20 min), and subsequent lysis (76% leakage of hemoglobin in 25 min). Incubation media made hypertonic by the addition of 75 mM carbohydrates with molecular diameters of 0.72 to 1.32 nm prevented hemolysin-induced RBC swelling, but incubation media made hypertonic by the addition of carbohydrates with molecular diameters of less than 0.72 nm did not protect against hemolysin-induced RBC swelling. Raffinose (75 mM; molecular diameter, 1.14 nm) did not block hemolysin-induced K+ leakage but did block hemolysis. These findings support the hypothesis that hemolysin-induced lysis occurs by colloid-osmotic swelling and are compatible with M. bovis hemolysin acting as a pore-forming cytolysin. Assuming that M. bovis hemolysin acts as a transmembrane molecular sieve, then the functional size of the hemolysin transmembrane pores in bovine RBCs is approximately 0.9 nm, the molecular size of sucrose. Hemolytic activity was inhibited by the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), but hemolysin-induced K+ leakage was not affected by EGTA.