A comparison of molecular typing methods for Moraxella catarrhalis

AIMS: Three molecular typing techniques were examined to determine which method was the most discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. METHODS AND RESULTS: Twenty-five Mor. catarrhalis isolates obtained from nasopharyngeal aspirates collected from Aboriginal and non-Aboriginal children were subjected to random amplified polymorphic DNA (RAPD) analysis, automated ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two Mor. catarrhalis types, automated ribotyping with PstI determined four Mor. catarrhalis ribogroups and PFGE analysis with NotI determined 21 pulse field groups within the 25 isolates examined. CONCLUSIONS: Analysis of discrimination index and typeability demonstrated that PFGE is the most discriminatory method for typing Mor. catarrhalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that PFGE is the most appropriate molecular tool for the epidemiological study of Mor. catarrhalis.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Construction of a Combined NotI/SmaI Physical and Genetic Map of Moraxella (Branhamella) catarrhalis Strain ATCC25238

Using pulsed field gel electrophoresis (PFGE) and Southern hybridization techniques, a physical map of Moraxella catarrhalis strain ATCC25238 was constructed to provide basic genetic knowledge of this bacterium that has attracted attention in recent years as a human pathogen. Restriction endonuclease NotI cut the genome into 10 fragments and SmaI into 9, and the molecular size of the genome was estimated to be 1,940 kilobases. Location of the 12 genes participating in the biosynthesis of purine, pyrimidine and nine kinds of amino acids were determined on the circular physical map of the strain.     

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.     

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.     

卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

为制备抗卡他莫拉菌(Moraxella catarrhalis,Mc)表面蛋白UspA1胞外结构域的多克隆抗体(PcAb),对UspA1蛋白进行生物信息学分析,获取胞外结构域中抗原表位最为丰富的肽段,找到其对应的基因序列并引入大肠杆菌偏好性密码子,对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体p ET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔,经4次免疫后,用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAbIgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定,抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。     

Attachment of Moraxella catarrhalis to pharyngeal epithelial cells is mediated by a glycosphingolipid receptor

Abstract Moraxella catarrhalis is one of the major pathogens of respiratory infections and has the ability to attach to the pharyngeal cells via fimbriae. We characterized the epithelial cell receptor to which fimbriate M. catarrhalis binds. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease of M. catarrhalis attachment, suggesting interaction with the sialic acid component. The attachment was not decreased in M. catarrhalis pretreated with 2 and 1 mg/ml of fucose, N -acetyl-neuraminic acid, N -acetyl-glucosamine, N -acetyl-galactosamine, acetyl-salicylic acid and colominic acid. However, M. catarrhalis treated with gangliosides M1, M2, D1a, D1b and T1a at a concentration of 2.5 μg/ml had significantly decreased the attachment compared to the control. In contrast treatment with gangliosides M3 and asialoganglioside M1 did not decrease the attachment of M. catarrhalis and thereby provided evidence for specificity of the inhibition. Concentration dependent effects of ganglioside M2 on the attachment were also observed. Other fimbriate isolates of M. catarrhalis showed decrease in attachment after treatment with ganglioside M2. However there was no effect on attachment when a nonfimbriate isolate was treated with ganglioside M2. This study indicates that the receptor of fimbriate M. catarrhalis on pharyngeal epithelial cells resides in the sequences of ganglioside M2.     

Moraxella (Branhamella) catarrhalis: Restriction enzyme analysis typing with HinfI, HaeIII and PstI

Abstract Restriction enzyme analysis typing with Hin fI, Hae III and Pst I was performed on Moraxella (Branhamella) catarrhalis strains consecutively collected from children suspected of respiratory tract infection and the type strain. Use of Hin fI gave the most distinct patterns. Great polymorphism was seen among strains.     

Identification of a Moraxella catarrhalis gene that confers adherence to various human epithelial cell lines in vitro

Moraxella catarrhalis is a pathogen of the human airways. We found that expression of the M. catarrhalis gene mcmA by Escherichia coli increases adherence to epithelial cells 100-fold. Furthermore, we discovered that disrupting mcmA decreases M. catarrhalis adherence to laryngeal and lung cells, which are relevant to pathogenesis by the bacterium.     

Moraxella catarrhalis is only a weak activator of the mannose-binding lectin (MBL) pathway of complement activation

A hemolytic bystander assay was used to assess the functional serum mannose-binding lectin (MBL) activating capacity of five isolates of Moraxella catarrhalis obtained from children who suffered recurrent acute otitis media episodes. Results showed that this organism is only a poor activator of the lectin pathway of complement activation, with subsequent consequences for the etiology of otitis media by this organism.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.     

Polymorphism of the major surface epitope of the CopB outer membrane protein of Moraxella catarrhalis

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.     

Characterization of antimicrobial resistant Escherichia coli isolated from processed bison carcasses

AIM: To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. METHODS AND MATERIALS: The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. RESULTS: Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16.7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13.0%), sulfamethoxazole (7.9%) and streptomycin (5.8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. CONCLUSION: This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.     

Transmission electron microscopy studies of Moraxella (Branhamella) catarrhalis

A trypsin-sensitive 200-kDa protein has been reported to be exclusively associated with haemagglutinating isolates of Moraxella (Branhamella) catarrhalis. Transmission electron microscopy studies revealed that haemagglutination by M. catarrhalis to both human and rabbit erythrocytes was mediated by a trypsin-sensitive outer fibrillar coat. This fibrillar layer was absent on non-haemagglutinating isolates examined. Immuno-electron microscopy, using a polyclonal antiserum containing antibodies to the 200-kDa protein as a probe, showed that the 200-kDa protein is present on the outer fibrillar layer of the bacterium. These findings suggest that the haemagglutinin of M. catarrhalis is a 200-kDa protein present on the outer fibrillar coat.     

Human serum and mucosal antibody responses to outer membrane protein G1b of Moraxella catarrhalis in chronic obstructive pulmonary disease

Moraxella catarrhalis is an important human pathogen that causes otitis media, sinusitis, and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. Outer membrane protein G1b is a approximately 29-kDa protein that has a high degree of homology among strains, contains surface-exposed epitopes, and is a potential vaccine candidate. The ompG1b gene was cloned, expressed in Escherichia coli, and purified. To assess the expression of outer membrane protein G1b during human infection, paired serum and sputum supernatants from patients with chronic obstructive pulmonary disease followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant outer membrane protein G1b to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 39% of patients developed either a serum IgG (28.6%) or a sputum supernatant IgA (19.2%) response to outer membrane protein G1b following 100 episodes of acquisition and clearance of M. catarrhalis. A sputum supernatant IgA response was more likely following exacerbations compared with asymptomatic colonizations, whereas a serum IgG response occurred at similar rates. Serum IgG antibodies following natural infection were directed toward surface-exposed epitopes of outer membrane protein G1b. Overall, these studies show that outer membrane protein G1b is expressed during infection of the human respiratory tract and that human antibodies bind to outer membrane protein G1b epitopes on the bacterial surface. These observations indicate that outer membrane protein G1b should be evaluated further as a vaccine antigen.     

Tissue culture adherence and haemagglutination characteristics of Moraxella (Branhamella) catarrhalis

The haemagglutination and tissue culture adherence properties of 20 isolates of Moraxella catarrhalis obtained from the sputum of elderly patients with lower respiratory tract infections were compared with those of 20 isolates of M. catarrhalis obtained from the nasopharynx of elderly persons colonised by the organism. Eighty percent of isolates from the infected group as opposed to 5% of isolates from the colonised group haemagglutinated human erythrocytes (P < 0.001), indicating that the haemagglutinin might be a marker of pathogenicity for M. catarrhalis. There was a significant difference in the adherence to HEp-2 cells of isolates from the infected group in comparison to isolates from the colonised group (P = 0.03). Haemagglutination and tissue culture adherence properties were unrelated, indicating that separate adhesin systems are involved. The adherence of M. catarrhalis to HEp-2 cells was unaffected following pronase and trypsin treatment, however, sodium periodate pre-treatment of the bacteria significantly reduced the tissue culture adherence index, indicating that the adhesin by which the bacteria bind to HEp-2 cells may have a carbohydrate moiety. Transmission electron microscopy studies revealed that adherence of M. catarrhalis to HEp-2 cells was mediated by trypsin-resistant 'tack-/spicule-like' structures protruding from the surface of the bacteria.