Genetic Diversity among Strains of Moraxella catarrhalis: Analysis Using Multiple DNA Probes and a Single-Locus PCR-Restriction Fragment Length Polymorphism Method

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce β-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.     

Antibiotic Susceptibilities Among Recent Clinical Isolates of Haemophilus influenzae and Moraxella catarrhalis from Fifteen Countries

 Between July 1998 and July 1999, 3,060 Haemophilus influenzae and 1,486 Moraxella catarrhalis strains were isolated in 31 centers in 15 countries in order to determine their antimicrobial susceptibilities and the presence of β-lactamase production in Haemophilus influenzae. Overall 17.1% of the Haemophilus influenzae isolates were β-lactamase positive, while more than 95% were susceptible to amoxicillin/clavulanate, cefaclor, loracarbef, cefuroxime, azithromycin and ciprofloxacin. Eleven (0.3%) isolates were β-lactamase positive and ampicillin resistant and 7 (0.2%) isolates were ciprofloxacin resistant. The minimum inhibitory concentrations for 90% of the isolates tested were lowest for ciprofloxacin (0.03) and highest for cefprozil (8) against Moraxella catarrhalis.     

Determinants of Moraxella catarrhalis colonization in healthy Dutch children during the first 14 months of life

Moraxella catarrhalis is an established bacterial pathogen, previously thought to be an innocent commensal of the respiratory tract of children and adults. The objective of this study was to identify significant risk factors associated with M. catarrhalis colonization in the first year of life in healthy Dutch children. This study investigated a target cohort group of 1079 children forming part of the Generation R Study, a population-based prospective cohort study following children from fetal life until young adulthood, conducted in Rotterdam, The Netherlands. Nasopharyngeal swabs for M. catarrhalis culture were obtained at 1.5, 6 and 14 months of age, with all three swabs being available for analyses from 443 children. Data on risk factors possibly associated with M. catarrhalis colonization were obtained by questionnaire at 2, 6 and 12 months. M. catarrhalis colonization increased from 11.8% at age 1.5 months to 29.9% and 29.7% at 6 and 14 months, respectively. Two significantly important colonization risk factors were found: the presence of siblings and day-care attendance, which both increased the risk of being positive for M. catarrhalis colonization on two or more occasions within the first year of life. Colonization with M. catarrhalis was not associated with gender, educational level of the mother, maternal smoking, breast-feeding, or antibiotic use. Apparently, crowding is an important risk factor for early and frequent colonization with M. catarrhalis in the first year of life.     

Comparison of Randomly Amplified Polymorphic DNA Analysis and Pulsed-Field Gel Electrophoresis for Typing of Moraxella catarrhalis Strains

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of M. catarrhalis.     

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.     

Analysis of the Immunological Responses to Transferrin and Lactoferrin Receptor Proteins from Moraxella catarrhalis

Moraxella catarrhalis expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with M. catarrhalis pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to M. catarrhalis proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA(-) mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of M. catarrhalis TbpB and LbpB in humans and support their potential as vaccine candidates.     

Isolation and Characterization of Two Proteins from Moraxella catarrhalis That Bear a Common Epitope

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.     

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.     

Cognitive,Neuroanatomical, and Genetic Predictors of Executive Function in Healthy Children and Adolescents

The Dimensional Change Card Sort (DCCS) is a measure of cognitive flexibility for children, which requires rule-use and shifting. Demographic, cognitive, regional cortical thickness, and genetic variables, including those related to language and executive function, were used to build predictive models of DCCS scores in 556 healthy pediatric participants. Gender, age, frontal, and temporal lobe regions of interest, and measures of sustained attention, inhibition, and word reading were selected as the best predictors of DCCS performance. Results indicated that DCCS performance is related to a broad range of cognitive functions and anatomic regions associated with various levels of cognitive function.     

Functional characteristics of a protective monoclonal antibody against serotype A and C lipooligosaccharides from Moraxella catarrhalis

A monoclonal antibody (MAb), designated MAb 8E7 (immunoglobulin G3), specific for Moraxella catarrhalis lipooligosaccharide (LOS) was evaluated for its functional activity in vitro and in a mouse model of colonization. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the MAb 8E7 could be prepared to a high titer against LOS of the homologous strain 035E, and that it had bactericidal activity. MAb 8E7 reacted with M. catarrhalis serotype A and C LOSs but not serotype B LOS, as measured by ELISA and Western blotting. On the basis of published structures of LOSs, this suggests that the epitope recognized by MAb 8E7 is directed to a common sequence of either alpha-GlcNAc-(1-->2)-beta-Glc-(1--> at the branch substituting position 4 of the trisubstituted Glc residue or a terminal tetrasaccharide alpha-Gal-(1-->4)-beta-Gal-(1-->4)-alpha-Glc-(1-->2)-beta-Glc-(1--> at the branch substituting position 6 of the trisubstituted Glc residue. In a whole-cell ELISA, MAb 8E7 reacted with 70% of the 30 wild-type strains and clinical isolates tested. Immuno-electron microscopy demonstrated that MAb 8E7 reacted with a cell surface-exposed epitope of LOS on strain O35E. MAb 8E7 inhibited the adherence of strain O35E to Chang conjunctival epithelial cells by 90%. Passive immunization with MAb 8E7 could significantly enhance the clearance of strain O35E from mouse lungs in an aerosol challenge mouse model. This enhanced bacterial clearance was inhibited when MAb 8E7 was absorbed by M. catarrhalis serotype A LOS, indicating that the M. catarrhalis LOS-directed antibody may play a major role in the enhancement of M. catarrhalis clearance from lungs. These data suggest that MAb 8E7, which recognizes surface-exposed LOS of M. catarrhalis, is a protective antibody against M. catarrhalis.     

Roles of 3-deoxy-D-manno-2-octulosonic acid transferase from Moraxella catarrhalis in lipooligosaccharide biosynthesis and virulence

Lipooligosaccharide (LOS), a major outer membrane component of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of human infections caused by the organism. However, information about the roles of the oligosaccharide chain from LOS in bacterial infection remains limited. Here, a kdtA gene encoding 3-deoxy-D-manno-2-octulosonic acid (Kdo) transferase, which is responsible for adding Kdo residues to the lipid A portion of the LOS, was identified by transposon mutagenesis and construction of an isogenic kdtA mutant in strain O35E. The resulting O35EkdtA mutant produced only lipid A without any core oligosaccharide, and it was viable. Physicochemical and biological analysis revealed that the mutant was susceptible to hydrophobic reagents and a hydrophilic glycopeptide and was sensitive to bactericidal activity of normal human serum. Importantly, the mutant showed decreased toxicity by the Limulus amebocyte lysate assay, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. These data suggest that the oligosaccharide moiety of the LOS is important for the biological activity of the LOS and the virulence capability of the bacteria in vitro and in vivo. This study may bring new insights into novel vaccines or therapeutic interventions against M. catarrhalis infections.     

Survival of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis frozen in skim milk- tryptone-glucose-glycerol medium

In STGG (skim milk, tryptone, glucose, glycerol) medium at -80 degrees C, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates survived for at least 3 years, and the same species have survived in nasopharyngeal swabs for at least 1.5 years. At -20 degrees C, S. pneumoniae and M. catarrhalis survived for 1.5 years, but H. influenzae survived for only 2 months.     

Moraxella catarrhalis bacteremic pneumonia in adults: Two cases and review of the literature

Moraxella (formerlyBranhamella)catarrhalis is a gram-negative coccus now recognized as one of the common pathogens in respiratory infections. Documented cases of bacteremic pneumonia due to this organism, however, have been a rarity. Two cases ofMoraxella catarrhalis bacteremic pneumonia in immunosuppressed adult patients are reported. The clinical characteristics of these patients together with those of the seven adult and the six pediatric patients reported to data in the literature, are analyzed. All patients had an underlying condition and most were male. The mean age was 64.9 years. No adult patient had skin lesion, although purpuric rash was frequent in children. The overall mortality rate was only 13.3 %, in spite of the underlying diseases. In three patients the pneumonia was nosocomial. The seasonal recovery ofMoraxella catarrhalis in respiratory infections is significantly increased during the late fall through early spring period. Because most strains are beta-lactamase positive, empiric use of penicillin, ampicillin or amoxicillin for this organism can no longer be recommended.     

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n = 81) and non-Aboriginal (n = 76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P < 0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P = 0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.     

High prevalence and molecular analysis of macrolide-nonsusceptible Moraxella catarrhalis isolated from nasopharynx of healthy children in China

Three hundred eighty-three isolates of Moraxella catarrhalis were collected from healthy children aged less than 2 years in China and assessed for antimicrobial resistance. We found that 92.2% (n=353) produced a β-lactamase. Nonsusceptibility rates to erythromycin and azithromycin, determined using Clinical Laboratory Standards Institute (CLSI) breakpoints, were 40.3% and 22.5%, respectively; nonsusceptibility rates determined using pharmacokinetics/pharmacodynamics breakpoints, however, were 59% and 60.1%. The minimal inhibitory concentration (MIC)(90) values were >256 μg/ml. Nonsusceptibility rates varied by region from 9.7% in Dongguan to 75.9% in Jinan. Further, concomitant resistance to β-lactam antibiotics was also observed. Pulsed-field gel electrophoresis analysis of 27/37 high-level macrolide-resistant M. catarrhalis isolates showed that closely related pulsotypes dominated, with a total of 11 different pulsotypes being observed. The closely related pulsotypes were observed in isolates originating from all six Chinese cities investigated, possibly as a consequence of the mobility of the Chinese population. Sixteen patterns of 23S rRNA mutations were found among 97 selected isolates using polymerase chain reaction and sequencing, but no known ermA, ermB, mefA, or mefE genes could be detected. Mutations A2982T and A2796T in 23S rRNA were related to high-level macrolide resistance (MICs ranging from 24 to >256 μg/ml), while an A2983T mutation was associated with low-level macrolide resistance (MICs ranging from 0.19 to 16 μg/ml).     

Workplace Stress and Working from Home Influence Depressive Symptoms Among Employed Women with Young Children

Background

Poor balance between work and family can be a major stressor for women with young children and have a negative impact on emotional well-being. Family-friendly workplace attributes may reduce stress and depressive symptoms among this population. However, few studies have analyzed the role of specific workplace attributes on mental health outcomes among women with young children because available data are limited.

Purpose

This study examines the impact of workplace attributes on changes in depressive symptoms among working women with young children between 6 and 24 months of age.

Method

This study uses data from the National Institute of Child Health and Human Development (NICHD) Study of Early Child Care and Youth Development (SECCYD) collected between 1991 and 1993 to examine the effects of work intensity, work schedule (night/day/variable), schedule flexibility, working from home, and work stress on changes in depressive symptoms among a national US sample of 570 women who returned to work within 6 months after childbirth. Depressive symptoms were assessed using the CES-D score. Treatment effects were estimated using fixed effects regression models.

Results

Working from home and work stress predicted within-individual changes in depressive symptoms between 6 and 24 months postchildbirth. Women who worked from home reported a statistically significant decrease in depression scores over time (β?=??1.36, SE?=?0.51, p?=?0.002). Women who reported a one-unit increase in job concerns experienced, on average, a 2-point increase in depression scores over time (β?=?1.73, SE?=?0.37, p?<?0.01). Work intensity, work schedule, and schedule flexibility were not associated with changes in depressive symptoms.

Conclusions

This study is one of the few to use longitudinal data and causal-inference techniques to examine whether specific workplace attributes influence depressive symptoms among women with young children. Reducing stress in the workplace and allowing women to work from home may improve mental health among women who transition back to work soon after childbirth.