Characterization of BRO enzymes and beta-lactamase transfer of Moraxella (Branhamella) catarrhalis isolated in Japan

Of the 68 strains of beta-lactamase-producing Moraxella (Branhamella) catarrhalis isolated in Japan that were studied, 62 (91%) produced the BRO-1-type beta-lactamase and 6 (9%) produced the BRO-2 type. There were no strains containing the BRO-3-type beta-lactamase. We compared the susceptibility of BRO-1- and BRO-2-producing strains to various oral beta-lactam antibiotics. We found that the BRO-1-producing strains were less susceptible than the BRO-2-producing strains. Although the BRO-1 and BRO-2 types showed a similar hydrolysis pattern, the specific activity of BRO-1 was 3-fold that of BRO-2. We examined the transfer of the BRO-1 and BRO-2 genes to non-beta-lactamase-producing M. catarrhalis No. 4020 and found that of the 13 donor strains producing BRO-1, 11 (85%) were able to transfer the gene for BRO-1 production by conjugation. Of the 6 donor strains producing BRO-2, 2 (33%) were able to transfer the gene for BRO-2 production by conjugation. For 3 of the 13 (23%) BRO-1-producing strains and 1 of the 6 (17%) BRO-2-producing strains, about 13 Mdalton of plasmids were detected. These plasmid-containing strains were used as donors, and in beta-lactamase-producing transconjugants the same size of plasmids was detected. However, when the total DNA is extracted from strains with the ability to transfer by conjugation, the transformation of the beta-lactamase-producing gene can occur regardless of the presence or absence of plasmids. Furthermore, even if purified plasmids are transformed, beta-lactamase-producing transformants are not obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Moraxella (Branhamella) catarrhalis: its isolation in the respiratory secretions of adult patients

OBJECTIVE: We have designed a retrospective study in order to know the clinical significance of the isolation of Moraxella (Branhamella) catarrhalis (MC) in respiratory specimens of adult hospitalized patients. METHODS: We performed a Gram stain and culture on blood-agar, MacConkey media and quantitative culture in chocolate-agar to all respiratory samples. In patients with a clinical diagnosis of pneumonia BCYE-alpha was added. During 2 years (1992-1993) MC was isolated in respiratory specimens from 52 patients. We revised the clinical history of all these patients. RESULTS: MC was isolated in 60 respiratory specimens (sputum and/or tracheobronchial aspirates) from 52 patients. The Gram stain showed gram-negative cocci in 77% and gram-positive cocci in 17% of the cases. MC grew in pure culture in 28 specimens (46.6%). In 23% of cases MC was isolated with Streptococcus pneumoniae and in 21% with Haemophilus influenzae. Fifty-two stocks (86.6%) produced beta-lactamase. Twelve patients had a clinical diagnosis of pneumonia, 8 of them had an underlying chronic respiratory disease. Other 24 patients with an underlying chronic respiratory disease had a bronchial infection as a cause of exacerbation of their respiratory disease. Seven patients without an underlying chronic respiratory disease had a clinical episode of acute bronchitis. Finally, in 9 patients the isolation of MC was considered a colonization. CONCLUSIONS: In 17% cases MC was identified as a gram-positive cocci in the Gram stain, which may cause false diagnosis. The etiological importance of MC in episodes of acute exacerbation of patients with an underlying chronic respiratory disease is high.     

Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases

Moraxella catarrhalis is an important pathogen of humans. It is a common cause of respiratory infections, particularly otitis media in children and lower respiratory tract infections in the elderly. Colonisation of the upper respiratory tract appears to be associated with infection in many cases, although this association is not well understood. Nosocomial transmission is being increasingly documented and the emergence of this organism as a cause of bacteremia is of concern. The widespread production of a beta-lactamase enzyme renders Moraxella catarrhalis resistant to the penicillins. Cephalosporins and beta-lactamase inhibitor combinations are effective for treatment of beta-lactamase producers, and the organism remains nearly universally susceptible to the macrolides, fluoroquinolones, tetracyclines and the combination of trimethoprim and sulfamethoxazole. Two major beta-lactamase forms, BRO-1 and BRO-2, have been described on the basis of their isoelectric focusing patterns. The BRO-1 enzyme is found in the majority of beta-lactamase-producing isolates and confers a higher level of resistance to strains than BRO-2. The BRO enzymes are membrane associated and their production appears to be mediated by chromosomal determinants which are transmissible by an unknown mechanism. The origin of these novel proteins is unknown.     

Enhancement of pulmonary clearance of Moraxella (Branhamella) catarrhalis following immunization with outer membrane protein CD in a mouse model

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.     

Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin

Moraxella (Branhamella) catarrhalis is a gram-negative human mucosal pathogen, which primarily causes otitis media in young children. However, this bacterium is also a common cause of lower respiratory tract infections in adults with underlying lung disease. Our previous data have shown that M. catarrhalis expresses iron-repressible outer membrane proteins in response to iron limitation. We have extended these observations to demonstrate that one of these proteins, termed outer membrane protein (OMP) B1, binds human transferrin. Using a newly developed monoclonal antibody to OMP B1, we determined that this protein is conserved in the iron-stressed outer membranes of all clinical isolates of M. catarrhalis tested to date. Furthermore, our data have confirmed that children infected with M. catarrhalis have immunoglobulin G antibodies to OMP B1 in their convalescent sera. These current data suggest that OMP B1 is immunogenic and expressed in vivo and may be involved in an iron uptake mechanism utilized by M. catarrhalis.     

The identification of response regulators of Branhamella catarrhalis using PCR

A variable-pressure scanning electron microscope (VP-SEM) equipped with a high-sensitive backscattered electron (BSE) detector of the YAG type was applied to studies of biological tissue samples. The rat kidney and trachea were fixed, dehydrated in ethanol, critical point-dried and examined in the VP-SEM under a specimen chamber pressure of 1 to 150 Pa. The high-resolution surface images of the non-coated specimens were obtained in a low-vacuum (1-20 Pa) environment at an accelerating voltage of 5 kV, while the images at 10-20 kV contained information beneath the surface of the specimens. The observation in the VP-SEM equipped with YAG detector in a low-vacuum (1-20 Pa) environment at low (3-5 kV) accelerating voltages is useful for the three-dimensional analysis of the surface morphology of biological non-coated samples.     

Cloning and expression of the Moraxella catarrhalis lactoferrin receptor genes

The lactoferrin receptor genes from two strains of Moraxella catarrhalis have been cloned and sequenced. The lfr genes are arranged as lbpB followed by lbpA, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame, orf3, is located one nucleotide downstream of lbpA. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The lbpB gene was PCR amplified and sequenced from a third strain of M. catarrhalis, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from Escherichia coli, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of M. catarrhalis strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.     

Outer membrane protein CD of Branhamella catarrhalis: sequence conservation in strains recovered from the human respiratory tract

Branhamella catarrhalis causes lower respiratory tract infections in patients with chronic obstructive pulmonary disease. The outer membrane protein CD (OMP-CD) of B. catarrhalis is a major, heat-modifiable OMP. The goals of this study are to characterize the degree of conservation of OMP-CD among strains and to investigate if OMP-CD maintains its homogeneity under the effect of host immune selective pressure. Isolates of B. catarrhalis were collected prospectively from patients with bronchiectasis and chronic bronchitis. We studied the OMP-CD gene by analysis of PCR restriction fragment length polymorphisms (PCR-RFLP) and further determined DNA sequence of the CD gene of eight selected isolates. Five patterns of PCR-RFLP of the OMP-CD gene were observed among all isolates when the gene was digested with Sau3AI. The sequence analysis revealed a high degree of homogeneity in OMP-CD among strains of B. catarrhalis. Three regions of OMP-CD with minimal sequence heterogeneity were identified. The sequences of the OMP-CD gene of isolates collected from patients colonized with the same strain for up to 6 months was identical. These observations establish that the OMP-CD of B. catarrhalis recovered from clinical isolates is highly conserved among strains.     

A protective epitope of Moraxella catarrhalis is encoded by two different genes

The high-molecular-weight UspA protein of Moraxella catarrhalis has been described as being both present on the surface of all M. catarrhalis disease isolates examined to date and a target for a monoclonal antibody (MAb 17C7) which enhanced pulmonary clearance of this organism in a mouse model system (M. E. Helminen et al., J. Infect. Dis. 170:867-872, 1994). A recombinant bacteriophage that formed plaques which bound MAb 17C7 was shown to contain a M. catarrhalis gene, designated uspA1, that encoded a protein with a calculated molecular weight of 88,271. Characterization of an isogenic uspA1 mutant revealed that elimination of expression of UspA1 did not eliminate the reactivity of M. catarrhalis with MAb 17C7. In addition, N-terminal amino acid analysis of internal peptides derived from native UspA protein and Southern blot analysis of M. catarrhalis chromosomal DNA suggested the existence of a second UspA-like protein. A combination of epitope mapping and ligation-based PCR methods identified a second M. catarrhalis gene, designated uspA2, which also encoded the MAb 17C7-reactive epitope. The UspA2 protein had a calculated molecular weight of 62,483. Both the isogenic uspA1 mutant and an isogenic uspA2 mutant possessed the ability to express a very-high-molecular-weight antigen that bound MAb 17C7. Southern blot analysis indicated that disease isolates of M. catarrhalis likely possess both uspA1 and uspA2 genes. Both UspA1 and UspA2 most closely resembled adhesins produced by other bacterial pathogens.     

Some cultural characteristics of Branhamella ovis isolated from the conjunctival sac of sheep

The colonial morphology and other cultural characteristics of Branhamella ovis were studied. The current investigation showed that colonies could be designated R (rough) and S (smooth) dependent on their appearance on agar. The colonial variants were apparently stable and each produced distinct types of pitting when grown on agar. A CAMP-like reaction was also shown to be a characteristic of B. ovis.     

Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003-2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.     

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.     

Extended-spectrum beta-lactamases from Klebsiella pneumoniae strains isolated at an Italian hospital

Eighteen strains of Klebsiella pneumoniae recently isolated from hospitalized patients were resistant or moderately resistant to oxyimino-cephalosporins (ceftazidime and/or cefotaxime), aztreonam, cefoxitin and all but one were susceptible to imipenem. Analysis of enzymes produced by these clinical isolates revealed a wide pattern of extended-spectrum beta-lactamases. All isolates produced one or more beta-lactamases that were characterized preliminarily by their isoelectric point. Strains isolated early were from patients in the Intensive Care Unit and produced an ES beta-lactamase with an apparent pI of 7.6, whereas the later isolates were from surgical and medical wards of the same hospital and produced ES beta-lactamases with apparent pI of 8.2 and 8.4, respectively. This suggests the emergence of SHV-5 and MIR-1 beta-lactamases in our hospital. Agarose gel electrophoresis of plasmid DNA revealed the presence of a similar plasmid of approximate size 60 Kb in all isolates.     

Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E

The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). Isogenic uspA1, uspA2, and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, the uspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 by M. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and the uspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and the uspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis.     

Occurrence of extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical strains of gram-negative rods

Hairy cell leukemia (HCL) is clinically associated with severe T-cell dysfunction. Several new observations have given more insight into the abnormal T-cell responses seen in this disease. T-lymphocytes in the spleen of patients with HCL seem to be abnormally activated. On the other hand, they are non-responsive, possibly as a result of monocytopenia which may lead to inadequate antigen presentation. This, together with the lack of CD28 on T-cells, may cause T-cell dysfunction. Furthermore, there is a very restricted repertoire of the T-cell receptor-beta family, which may also result in non-responsiveness. Otherwise, T-cell clonal excess may be indicative for activated, possibly autoreactive T-cells.     

Recent trends in incidence of respiratory tract pathogens and antimicrobial susceptibilities of Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis isolated in 1994 and 1995

The incidence of pathogenic bacteria in respiratory tract infections in 1994 and 1995 was investigated using quantitative cultures of sputa from patients with the infections in our department. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were isolated at high rates (70.5% in 1994 and 73.8% in 1995) from the specimens of out-patients, and the incident rates were similar to the past data. The antimicrobial susceptibilities of these three pathogens were examined with the agar dilution method. The incidence of penicillin (Pc) resistant S. pneumoniae against which MIC of Pc-G was higher than 0.125 microgram/ml was markedly increased from 24% in 1994 to 34.9% in 1995. Most of the Pc resistant isolates were also resistant to other antibiotics including erythromycin, minocycline and tosufloxacin. Serotype of strains against which MIC of Pc-G was higher than 1.0 microgram/ml was 19. The ratios of beta-lactamase-producing strains among H. influenzae isolated in 1994 and 1995 were 20 and 15.8%, respectively, which were slightly higher than those in the past. One quinolone resistant strain was isolated in this study. Although the ratio of beta-lactamase-producing strains among M. catarrhalis was as high (96.7%) as in the past, no increased resistance against the drugs examined was observed.