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Bactericidal monoclonal antibody against Moraxella catarrhalis lipooligosaccharide cross-reacts with Haemophilus Spp.

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.

Animals↗

Asialo-GM1 and asialo-GM2 are putative adhesion molecules for Moraxella catarrhalis.

Moraxella catarrhalis is an important pathogen of respiratory and middle ear infections. We previously reported that the attachment of M. catarrhalis to pharyngeal epithelial cells is mediated by ganglioside M2 (GM2). Several sets of adhesins or receptors are involved in such attachment process. In this study, we used the same strains and similar bacterial culture conditions as those in our previous study, and demonstrated by thin layer chromatography that M. catarrhalis can also bind to asialo-GM1 (Gg4Cer) and asialo-GM2 (Gg3Cer). GalNAcbeta1-->4Galbeta1 is a common sequence in both Gg4Cer and Gg3Cer, and in many respiratory bacteria, this sequence acts as a receptor for attachment to host cells. Treatment of human pharyngeal epithelial cells with anti-GM2 and anti-Gg4Cer antibodies significantly decreased attachment of M. catarrhalis to these cells; however, treatment with anti-Gg3Cer antibody did not decrease M. catarrhalis attachment. Immunofluorescence microscopy revealed that human pharyngeal epithelial cells are positive for GM2 and Gg4Cer, but not for Gg3Cer. Our results indicate that Gg4Cer on human pharyngeal epithelial cells, and Gg3Cer,possibly on other cells, could serve as molecules for attachment of M. catarrhalis.

Adhesins, Bacterial↗

Adhesion of Moraxella catarrhalis to human bronchial epithelium characterized by a novel fluorescence-based assay.

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. Adhesion of this pathogen to epithelial cells is critical for its pathogenicity. Although much work has been done on identifying surface molecules of M. catarrhalis as adhesins, several adhesion assays were used in these studies which has never been validated or compared to each other. In the present study, we have examined the capacity of M. catarrhalis to adhere to different human epithelial cells. By using the two most commonly used adhesion assays based on the enumeration of colony-forming units or on the counting of adherent bacteria per epithelial cell by light microscopy, we identified significant limitations of both methods. These arose either from differences in strain-specific adhesion pattern on the epithelial cell surface or the dependence on the state of confluence of the epithelial cell layer. We developed a new fluorescence-based adhesion assay and compared our results to the two conventional methods. We demonstrated that the fluorescence-based adhesion assay offers a reliable and convenient method for the quantification of M. catarrhalis adhesion to confluent epithelial cell monolayers.

Bacterial Adhesion↗

First definite case of aortic valve endocarditis due to Moraxella phenylpyruvica.

Described here is the first definite case of endocarditis due to Moraxella phenylpyruvica, which occurred in a 50-year-old male with a bicuspid aortic valve. The diagnosis was delayed because of the confounding positivity of the Widal and Wright tests. The patient was cured with surgical valve replacement and antibiotic treatment.

Aortic Valve↗

Antimicrobial activities of tosufloxacin against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella branhamella catarrhalis isolated from otolaryngological infectious diseases.

In 2003, the Japan Society for Infectious Diseases in Otolaryngology conducted its third nationwide survey of clinical isolates from otolaryngological infectious diseases. We selected three primary causative organisms of otolaryngological infectious diseases, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella Branhamella catarrhalis, and evaluated their sensitivities to tosufloxacin (TFLX), a new oral quinolone, because the survey revealed a rise in drug-resistant strains, suggesting potential problems with the antibiotics commonly used against these organisms. The minimum inhibitory concentration (MIC)(90) values of TFLX against S. pneumoniae, H. influenzae, and M. catarrhalis were 0.25 microg/ml, <or=0.06 microg/ml, and <or=0.06 microg/ml respectively, and TFLX was shown to be as effective as or superior to other new quinolones. In addition, TFLX showed sufficient antimicrobial effects against frequently detected drug-resistant bacteria such as penicillin-resistant S. pneumoniae (PRSP) and beta-lactamase-negative, ampicillin-resistant strains of H. influenzae (BLNAR). Furthermore, only a few strains of bacteria showed resistance to TFLX.

Adolescent↗

Bacteremic Moraxella catarrhalis pneumonia in a patient with immunoglobulin deficiency.

A-75-year old woman with agammaglobulinemia developed Moraxella catarrhalis bacteremic pneumonia. M. catarrhalis pneumonia is rarely associated with bacteremia, and neutrophils have been reported as a significant factor in the host defense system against this bacteria. This case suggests that immunoglobulin also plays a key role in the host defense system against M. catarrhalis.

Agammaglobulinemia↗

Moraxella nonliquefaciens endophthalmitis after trabeculectomy.

Endophthalmitis caused by Moraxella nonliquefaciens developed five years after trabeculectomy in a 67-year-old man and 15 months after trabeculectomy in a 62-year-old women. Symptoms in both cases included pain, inflammation, and decreased visual acuity. Aggressive treatment with antibiotics and corticosteroids led to resolution within a few days.

Aged↗

A polysaccharide produced by a mucoid strain of Moraxella nonliquefaciens with a 2-acetamido-2-deoxy-5-O-(3-deoxy-beta-D-manno-octulopyranosyl)-beta-D- galactopyranosyl repeating unit.

A capsular polysaccharide, isolated from the mucoid Moraxella nonliquefaciens strain 3828/60, has been investigated by component analyses, periodate oxidation, methylation analyses, mass spectrometry, 1H and 13C NMR spectroscopy, and hydrolysis to give a disaccharide that was isolated and characterised. The results showed that the polysaccharide has the repeating unit-->3)-beta-D- GalpNAc-(1-->5)-beta-Kdo p-(2-->, with approximately 40% of O-8 of Kdo being acetylated.

Carbohydrate Conformation↗

Structural studies of the O-antigen oligosaccharides from two strains of Moraxella catarrhalis serotype C.

The oligosaccharide parts from Moraxella (Branhamella) catarrhalis serotype C lipooligosaccharides were isolated by mild acid hydrolysis followed by gel permeation chromatography. Four different oligosaccharides could be identified from strain RS26 and two from strain RS10. The structures of the O-oligosaccharides were established by methylation analyses, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide O-antigens from RS26 are a mixture of octa-, deca-, and undeca-saccharides, and most likely a heptasaccharide. Strain RS10 contains the deca- and the undeca-saccharide only. The structures for the oligosaccharides are shown below. [formula: see text] OS(7) [formula: see text] OS(8) [formula: see text] OS(10) [formula: see text] OS(11) Methylation analysis of the intact lipooligosaccharides showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. It also showed that they consisted of a lipid A portion with 6-substituted glucosamine residues.

Carbohydrate Sequence↗

Structural studies of the O-polysaccharide from the lipopolysaccharide of Moraxella (Branhamella) catarrhalis serotype A (strain ATCC 25238).

The polysaccharide of the Moraxella (Branhamella) catarrhalis serotype A lipopolysaccharide was prepared by mild acid hydrolysis followed by gel permeation chromatography. The structure was established by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the O-antigenic polysaccharide has the following structure. [formula see text] Methylation analysis of the intact lipopolysaccharide showed that the lipid A portion consisted of 6-substituted glucosamine residues. Methylation followed by methanolysis showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. A terminal Kdo thus substitutes the branch-point Kdo in the 4-position.

Carbohydrate Conformation↗

Synthesis of 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6-tri-O-beta-D- glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2-ulopyrano sid onic acid, a highly branched pentasaccharide corresponding to structures found in lipopolysaccharides from Moraxella catarrhalis.

Syntheses of the pentasaccharide 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6- tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2- ulopyranosidonic acid and of the tetrasaccharide 3,4,6-tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside, both as its methyl and 2-(4-trifluoro-acetamidophenyl)ethyl glycoside, are described. These oligosaccharides correspond to structures found in the lipopolysaccharide of Moraxella catarrhalis and were needed for biological experiments aimed at producing antibodies against the bacteria. The best way to introduce the glucopyranosyl groups into the 3-, 4-, and 6-positions of the branched target compounds was found to be a one-step reaction using a 3,4,6-triol as acceptor and 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl bromide as donor in a silver trifluoromethanesulfonate-promoted coupling. The spacer arm, necessary for the formation of immunoactive glycoconjugates, was introduced into the glucose moiety via a dimethyl(methylthio)sulfonium trifluoromethanesulfonate-promoted reaction using the ethyl thioglucoside as donor, whereas for Kdo, the acetylated glycal derivative, methyl 4,5,7,8-tetra-O-acetyl-2,6-anhydro-3-deoxy-D-manno-oct-2-enonate, was used as donor and phenylselenyl trifluoromethanesulfonate as a stereocontrolling promoter.

Carbohydrate Conformation↗

Scanning electron microscope studies on preparations of bovine cornea exposed to Moraxella bovis.

Preparations of bovine cornea were studied by scanning electron microscopy before and after in vitro exposure to Moraxella bovis. The bacteria were observed in association with the surface of the corneal epithelium; they showed a predilection for dark cells relatively devoid of surface structure. Pit-like depressions related to the presence of individual bacteria were produced in these cells. An evident association with corneal epithelium was shown by 2 strains known to be pathogenic experimentally in cattle but not by 2 non-pathogenic strains.

Animals↗

Novel transformations of i-cholesterol and 6 beta-methoxy-i-cholesterol by Moraxella sp.

A soil microorganism was isolated by the enrichment culture technique using cholesterol as the sole source of carbon. The organism has been identified as belonging to the genus Moraxella. With this organism two novel biotransformations of sterols were observed viz. (1) isomerization of 3 alpha,5 alpha-cyclocholestan-6 beta-ol (i-cholesterol) to cholesterol, (2) demethylation of 6 beta-methoxy-3 alpha,5 alpha-cyclocholestane (6 beta-methoxy-i-cholesterol) to i-cholesterol with subsequent isomerization to cholesterol. The enzymes responsible for these transformations were shown to be inducible. The pH optimum of the partially purified i-cholesterol isomerase was found to be 8.4. The apparent Km value for i-cholesterol was 1.43 microM. A plausible mechanism for the i-cholesterol isomerization has been discussed.

Biotransformation↗

Infectious bovine keratoconjunctivitis: bacteriologic, immunologic, and clinical responses of cattle to experimental exposure with Moraxella bovis.

The bacteriologic, immunologic, and clinical responses of 3- to 4-month old Holstein-Friesian calves to experimental exposure with Moraxella bovis type 10900 has been investigated. After u.v. radiation and intraconjunctival exposure with 1.9 X 10(7) microorganisms, each eye of 16 calves exhibited signs of blepharospasm, photophobia, and increased lacrimation. Bacteria were recovered from exposed eyes for 2-7 consecutive weeks before maximal clinical response occurred. The severity of the cases varied from eyes that exhibited mild signs to severe clinical cases with profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. By 70 days after exposure, M. bovis could not be recovered from any conjunctival swabs, and clinical signs were not observed. Four non-exposed control animals did not develop clinical signs nor was M. bovis recovered from conjunctival swabs. Lacrimal secretions collected at the time of and 1 week after maximal clinical response had significantly elevated levels of total protein as compared to those collected 3, 2, and 1 week before, and 2 and 3 weeks after maximal clinical response. A passive hemagglutination test, using tanned formalized sheep erythrocytes sensitized with M. bovis sonicate antigen, detected antibody in lacrimal secretions from 22 of 32 eyes. The appearance of specific antibody in lacrimal secretions correlated with the amelioration of clinical signs and the decline in numbers of M. bovis microorganisms recovered from conjunctival swabs.

Animals↗

Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144.

A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced. The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772. This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility. The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.

Amino Acid Sequence↗

A prospective study of isolation of Moraxella catarrhalis in a hospital during the winter months.

Sputum samples submitted to the microbiology laboratory from general medical and respiratory wards were monitored for Moraxella catarrhalis on a prospective basis. All isolates were typed by restriction endonuclease typing. Nosocomial spread was found both by the clustering of cases and typing of isolates. Sampling of the environment of some cases was performed. Seven out of 37 samples revealed environmental contamination. Sampling for persistence of the organism in the environment was positive on one occasion out of 13. Evaluation of acquisition of M. catarrhalis in relation to length of stay showed that the average length of stay of a case with M. catarrhalis was considerably longer than average patient stay without M. catarrhalis. Four patients had two isolates available for typing. The type of M. catarrhalis was different on the second occasion to that on the first. Nosocomial spread of M. catarrhalis in the setting of general medical and respiratory wards was found to occur in the winter months.

Bacterial Typing Techniques↗

Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144.

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.

Cloning, Molecular↗

Growth requirements for Moraxella bovis.

Moraxella bovis isolates do not necessarily require complex growth media. Minimal growth requirements were approached by selecting mutants that grew on progressively simpler solid media. Isolate 137T was selected until it could be cultured on a medium containing sodium L-glutamate, with thiamine, nicotinamide, pyridoxine and inorganic salts. All 5 field strains isolated in 1983 showed the same requirements and no prior selection was needed. One of these 5 field strains (H388) was selected to require nicotinamide as the only added vitamin, and culture 137H was selected to grow without added vitamins. In contrast, type strains NCTC 8561 and NCTC 9426 could not be cultured on glutamate medium supplemented with a mixture of 8 growth factors. However, 35 out of 38 M. bovis cultures collected in this laboratory grew on that medium. A more complex medium (TG) contained 8 g glutamate and 2 g tryptone per litre of basal salts. Medium TG supported growth by both type strains and all 38 laboratory cultures. Growth on TG medium was promoted by adding 0.1 mg l-1 of pyridoxine, nicotinamide and thiamine. M. bovis strains from type-culture collections or those that have been subcultured on complex media only, may not have retained their native nutritional requirements.

Culture Media↗