Sex effects in the association between airway microbiome and asthma.

BACKGROUND: Sex differences exist in asthma susceptibility and severity. Accumulating evidence has linked airway microbiome dysbiosis to asthma, and airway microbial communities have been found to differ by sex. However, whether sex modifies the link between airway microbiome and asthma has not been investigated. OBJECTIVE: To evaluate sex effects in the association between airway microbiome and asthma. METHODS: We analyzed induced sputum samples from 47 subjects (n = 23 patients with asthma and n = 24 normal controls) using 16S ribosomal RNA gene sequencing methods. The bacterial composition was analyzed for sex differences. Bacterial associations with asthma were assessed for each sex at the core taxa and genus levels. RESULTS: The microbiome in induced sputum differed in women vs men at the community level. A total of 5 core bacterial taxa were found in all samples. No sex-specific core taxa were detected. The most abundant core taxon, Streptococcus salivarius, was significantly enriched in women than in men (P = .02). Within each sex, individuals with relatively lower abundance of S salivarius were more likely to have asthma (P = .006). For both sexes, increased Lactobacillus species were found in sputum samples of patients with patients compared with normal controls (adjusted P = .01). Haemophilus species were associated with asthma in men and not in women. CONCLUSION: The airway microbiome differed by sex, and sex effects exist in the association of airway microbial markers and asthma. Future airway microbiome studies may yield better resolution if the context of specific sex is considered. The airway microbiome is a potential mechanism driving sex differences in asthma.

Anti-Haemophilus Activity of Selected Essential Oils Detected by TLC-Direct Bioautography and Biofilm Inhibition.

Essential oils (EOs) are becoming increasingly popular in medical applications because of their antimicrobial effect. Direct bioautography (DB) combined with thin layer chromatography (TLC) is a screening method for the detection of antimicrobial compounds in plant extracts, for example, in EOs. Due to their lipophilic character, the common microbiological assays (etc. disk diffusion) could not provide reliable results. The aim of this study was the evaluation of antibacterial and anti-biofilm properties of the EO of cinnamon bark, clove, peppermint, thyme, and their main components against Haemophilus influenzae and H. parainfluenzae. Oil in water (O/W) type Pickering nano-emulsions stabilized with silica nanoparticles from each oil were prepared to increase their water-solubility. Samples with Tween80 surfactant and absolute ethanol were also used. Results showed that H. influenzae was more sensitive to the EOs than H. parainfluenzae (except for cinnamon bark oil). In thin layer chromatography-direct bioautography (TLC-DB) the ethanolic solutions of thyme oil presented the best activity against H. influenzae, while cinnamon oil was the most active against H. parainfluenzae. Pickering nano-emulsion of cinnamon oil inhibited the biofilm formation of H. parainfluenzae (76.35%) more efficiently than samples with Tween80 surfactant or absolute ethanol. In conclusion, Pickering nano-emulsion of EOs could inhibit the biofilm production effectively.

Haemophilus Protein F Orthologs of Pathogens Infecting the Airways: Exploiting Host Laminin at Heparin-Binding Sites for Maximal Adherence to Epithelial Cells.

Haemophilus influenzae protein F (PF) is an important virulence factor interacting with laminin, an extracellular matrix protein ubiquitously expressed in the respiratory tract. Here we defined PF orthologs in Pseudomonas aeruginosa, Moraxella catarrhalis, and Staphylococcus aureus, bacteria that occasionally colonize and infect the human airways. Despite low sequence homology (48.2%-77.3% similarity), all orthologs (Paf, AfeA, and MntC) interacted with laminin. Interestingly, all proteins bound at the heparin-binding sites of laminin, including the globular domains, and also attached to laminin expressed on respiratory epithelial cells. Laminin is thus a highly important target for PF orthologs of the bacterial species examined.

Zero-inflated negative binomial mixed model: an application to two microbial organisms important in oesophagitis.

Altered microbial communities are thought to play an important role in eosinophilic oesophagitis, an allergic inflammatory condition of the oesophagus. Identification of the majority of organisms present in human-associated microbial communities is feasible with the advent of high throughput sequencing technology. However, these data consist of non-negative, highly skewed sequence counts with a large proportion of zeros. In addition, hierarchical study designs are often performed with repeated measurements or multiple samples collected from the same subject, thus requiring approaches to account for within-subject variation, yet only a small number of microbiota studies have applied hierarchical regression models. In this paper, we describe and illustrate the use of a hierarchical regression-based approach to evaluate multiple factors for a small number of organisms individually. More specifically, the zero-inflated negative binomial mixed model with random effects in both the count and zero-inflated parts is applied to evaluate associations with disease state while adjusting for potential confounders for two organisms of interest from a study of human microbiota sequence data in oesophagitis.

The Association of Chronic Hepatitis C with Respiratory Microbiota Disturbance on the Basis of Decreased Haemophilus Spp. Colonization.

BACKGROUND Haemophilus species are the most common microbiota in humans. The aim of this paper was to investigate Haemophilus spp., mainly H. parainfluenzae prevalence, in the upper respiratory tract of chronic hepatitis C (CHC-positive) patients with or without therapy using pegylated interferon alfa and ribavirin. MATERIAL AND METHODS We collected 462 samples from 54 healthy people and 100 CHC-positive patients at various stages: before (group A), during (group B), and after (group C) antiviral therapy. Identification of bacterial isolates including biotypes and antimicrobials susceptibility was accomplished by means of standard microbiological methods. RESULTS In 70.4% of healthy people (control group) and in 27.0% of CHC-positive patients, the presence of haemophili, mainly H. parainfluenzae was observed, and those differences were statistically significant (p<0.0001). Statistically significant differences in Haemophilus spp. colonization were also observed among healthy people and CHC-positive patients from group A (p=0.0012) and from B or C groups (p<0.0001). Resistance to ampicillin in beta-lactamase-positive isolates and multidrug resistance (MDR) of H. parainfluenzae was detected mainly in group A. CONCLUSIONS The obtained data suggest that chronic hepatitis C, together with antiviral therapy, may influence the respiratory tract microbiota composition as found using haemophili, mainly H. parainfluenzae.

Classification, identification, and clinical significance of Haemophilus and Aggregatibacter species with host specificity for humans.

The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology practice. However, identification of some strains will still be problematic, necessitating DNA sequencing of multiple housekeeping gene fragments or full-length 16S rRNA genes.

Haemophilus responses to nutritional immunity: epigenetic and morphological contribution to biofilm architecture, invasion, persistence and disease severity.

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.

The Haemophilus cryptic genospecies Cha adhesin has at least two variants that differ in host cell binding, bacterial aggregation, and biofilm formation properties.

The Haemophilus cryptic genospecies (HCG) causes genital tract infections in pregnant and postpartum women and respiratory infections in neonates. The major surface adhesin in HCG is called Cha, which mediates bacterial adherence to cultured human epithelial cells. In this study, we report that there are two antigenically distinct variants of Cha, dubbed Cha1 and Cha2. These variants are encoded by the same genetic locus in diverse strains and have nearly identical N-terminal export and C-terminal surface anchoring domains but significantly different internal adhesive domains. Based on the comparison of derivatives of a laboratory strain of Haemophilus influenzae expressing either surface-associated Cha1 or surface-associated Cha2, Cha1 mediates a higher level of adherence to cultured human epithelial cells and Cha2 mediates a higher level of adherence to abiotic surfaces. We hypothesize that variation in the Cha1 and Cha2 internal region results in changes in binding specificity or binding affinity and may be associated with adaptation to different host environments during colonization and disease.

Seven-valent pneumococcal conjugate vaccine and nasopharyngeal microbiota in healthy children.

Seven-valent pneumococcal conjugate vaccine (PCV-7) is effective against vaccine serotype disease and carriage. Nevertheless, shifts in colonization and disease toward nonvaccine serotypes and other potential pathogens have been described. To understand the extent of these shifts, we analyzed nasopharyngeal microbial profiles of 97 PCV-7-vaccinated infants and 103 control infants participating in a randomized controlled trial in the Netherlands. PCV-7 immunization resulted in a temporary shift in microbial community composition and increased bacterial diversity. Immunization also resulted in decreased presence of the pneumococcal vaccine serotype and an increase in the relative abundance and presence of nonpneumococcal streptococci and anaerobic bacteria. Furthermore, the abundance of Haemophilus and Staphylococcus bacteria in vaccinees was increased over that in controls. This study illustrates the much broader effect of vaccination with PCV-7 on the microbial community than currently assumed, and highlights the need for careful monitoring when implementing vaccines directed against common colonizers.

Adhesive activity of the haemophilus cryptic genospecies cha autotransporter is modulated by variation in tandem Peptide repeats.

The Haemophilus cryptic genospecies is an important cause of maternal genital tract and neonatal systemic infections and initiates infection by colonizing the genital or respiratory epithelium. In recent work, we identified a unique Haemophilus cryptic genospecies protein called Cha, which mediates efficient adherence to genital and respiratory epithelia. The Cha adhesin belongs to the trimeric autotransporter family and contains an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a C-terminal membrane anchor domain. The passenger domain in Cha contains clusters of YadA-like head domains and neck motifs as well as a series of tandem 28-amino-acid peptide repeats. In the current study, we report that variation in peptide repeat number gradually modulates Cha adhesive activity, associated with a direct effect on the length of Cha fibers on the bacterial cell surface. The N-terminal 404 residues of the Cha passenger domain mediate binding to host cells and also facilitate bacterial aggregation through intermolecular Cha-Cha binding. As the tandem peptide repeats expand, the Cha fiber becomes longer and Cha adherence activity decreases. The expansion and contraction of peptide repeats represent a novel mechanism for modulating adhesive capacity, potentially balancing the need of the organism to colonize the genital and respiratory tracts with the ability to attach to alternative substrates, disperse within the host, or evade the host immune system.

Gender Variations in the Oral Microbiomes of Elderly Patients with Initial Periodontitis.

Periodontitis is a globally prevalent disease that imposes a functional and aesthetic burden on patients. The oral microbiome influences human health. The aim of this study was at assessing gender variation in the subgingival bacterial microbiome of elderly patients with initial periodontitis and to determine the causes of this variation. Twelve males and twenty females (range 50-68 years old) with initial periodontitis provided subgingival plaque samples. 16S rRNA gene sequencing, QIIME-based data processing, and statistical analyses were carried out using several different analytical approaches to detect differences in the oral microbiome between the two groups. Males had higher Chao1 index, observed species, and phylogenetic diversity whole tree values than females. Analysis of ß-diversity indicated that the samples were reasonably divided by the gender. The linear discriminant analysis effect size showed that the most representative biomarkers were the genus Haemophilus in males, whereas the dominant bacteria in females were Campylobacter. Kyoto Encyclopedia of Genes and Genomes analysis showed that predicting changes in the female oral microbiota may be related to the immune system and immune system diseases are the main factor in males. These data suggest that gender may be a differentiating factor in the microbial composition of subgingival plaques in elderly patients with initial periodontitis. These results could deepen our understanding of the role of gender in the oral microbiota present during initial periodontitis.

The amino acid selected for generating mutant TbpB antigens defective in binding transferrin can compromise the in vivo protective capacity.

Haemophilus parasuis is the causative agent of the Glässer's disease (GD), one of the most important bacterial diseases that affect young pigs worldwide. GD prevention based on vaccination is a major concern due to the limited cross-protection conferred by the inactivated whole cell vaccines used currently. In this study, vaccines based on two mutant recombinant proteins derived from transferrin binding protein B of H. parasuis (Y167A-TbpB and W176A-TbpB) were formulated and evaluated in terms of protection against lethal challenge using a serovar 7 (SV7) H. parasuis in a high susceptibility pig model. Our results showed that H. parasuis strain 174 (SV7) is highly virulent in conventional and colostrum-deprived pigs. The Y167A-TbpB and W176A-TbpB antigens were immunogenic in pigs, however, differences in terms of antigenicity and functional immune response were observed. In regard to protection, animals immunized with Y167A-TbpB antigen displayed 80% survival whereas the W176A-TbpB protein was not protective. In conjunction with previous studies, our results demonstrate, (a) the importance of testing engineered antigens in an in vivo pig challenge model, and, (b) that the Y167A-TbpB antigen is a promising antigen for developing a broad-spectrum vaccine against H. parasuis infection.

An isolate of Haemophilus haemolyticus produces a bacteriocin-like substance that inhibits the growth of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) frequently colonises the upper respiratory tract and is an important cause of respiratory infections. Resistance to antibiotics is an emerging trend in NTHi and alternative prevention or treatment strategies are required. Haemophilus haemolyticus is a common commensal occupying the same niche as NTHi and, if able to produce substances that inhibit NTHi growth, may have a role as a probiotic. In this study, ammonium sulphate extracts from broth culture of 100 H. haemolyticus isolates were tested for the presence of substances inhibitory to NTHi using a well diffusion assay. One isolate produced a substance that consistently inhibited the growth of NTHi. The substance was inactivated by protease enzymes and had a molecular size of ca. 30 kDa as determined by size exclusion chromatography. When the substance was tested against bacteria from eight Gram-negative and three Gram-positive genera, only Haemophilus spp. were inhibited. Quantitative PCR testing showed the substance to be different to 'haemocin', the previously described bacteriocin of H. influenzae type b. These molecular characteristics, together with narrow-spectrum activity, suggest the substance may be a novel bacteriocin, and there is potential for this H. haemolyticus isolate to function as a probiotic for reduction of colonisation and subsequent infection with NTHi.

Growth-altering microbial interactions are responsive to chemical context.

Microbial interactions are ubiquitous in nature, and are equally as relevant to human wellbeing as the identities of the interacting microbes. However, microbial interactions are difficult to measure and characterize. Furthermore, there is growing evidence that they are not fixed, but dependent on environmental context. We present a novel workflow for inferring microbial interactions that integrates semi-automated image analysis with a colony stamping mechanism, with the overall effect of improving throughput and reproducibility of colony interaction assays. We apply our approach to infer interactions among bacterial species associated with the normal lung microbiome, and how those interactions are altered by the presence of benzo[a]pyrene, a carcinogenic compound found in cigarettes. We found that the presence of this single compound changed the interaction network, demonstrating that microbial interactions are indeed dynamic and responsive to local chemical context.

Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1ß, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease.

Utilization of a continuous streptococcal surface to measure interbacterial adherence in vitro and in vivo.

Cell-to-cell interactions are essential for the formation of dental plaque. A continuous layer of Streptococcus sanguis SA-1 cells fixed to a solid surface has been used to evaluate interactions among this bacterium, Haemophilus parainfluenzae, and Streptococcus sobrinus. S. sanguis cells were attached to a Falcon 3001 tissue culture plates or bovine enamel chips, coated with a biological adhesive. Scanning electron microscopy of the chips showed the streptococci as a contiguous surface. Radiolabeled bacteria were used to measure a second-species interbacterial adherence to the streptococcal-coated culture plates. Strains of H. parainfluenzae known to coaggregate (strain HP-28) and not to coaggregate (strains HP-42 and HP-80), in suspension with S. sanguis strain SA-1, were studied for adherence. Ten-fold-higher numbers of coaggregating strain HP-28 adhered in vitro to the streptococcal layer than did the non-coaggregating strains. S. sobrinus strain 6715 did not show appreciable adherence to the S. sanguis surface. Saliva did not affect the adherence of coaggregating or non-coaggregating H. parainfluenzae strains to S. sanguis strain SA-1. Bovine enamel chips, coated with streptococci, mounted on modified orthodontic appliances and placed in the mouths of three volunteers, facilitated the measurement of interbacterial adherence in vivo of streptomycin-resistant strains of H. parainfluenzae (HP-28R or HP-42R). Suspensions of bacteria were placed into the mouth, distributed throughout, and expectorated. After 15 or 120 minutes, the appliance with the chips was removed, the chips sonified, and colony-forming units (CFU) of streptomycin-resistant haemophili determined per chip.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of activated lymphocyte killing by bacteria associated with periodontal disease.

Complex interactions occur among host defense cells during bacterial infection. Bacteria and bacterial products may enhance or inhibit the effector and regulatory activity of human lymphocytes. Accordingly, we tested the ability of human periodontal pathogens to activate peripheral blood lymphocytes using standard chromium-release assays to measure lymphocyte-mediated cytolysis. Human adherent-cell depleted peripheral blood lymphocytes (PBL) with the addition of glutaraldehyde-fixed bacteria at a 5:1 bacteria:lymphocyte ratio were incubated at 37 degrees C for 24 hr in RPMI 1640 medium. Six of eight bacteria tested significantly augmented lymphocyte killing of the natural killer (NK) cell-sensitive human erythroleukemia cell line K562. E. corrodens, representing activating bacteria, was also able to induce the killing of NK-resistant targets (M14, Raji), comparable with induction by interleukin-2. Lipopolysaccharides extracted from A. actinomycetemcomitans strains, when incubated with PBL, were able to enhance cytotoxicity without the presence of whole bacteria. A majority of cytotoxicity was mediated by NK cells bearing Leu-11 and NKH-1 markers.

Binding of human high-molecular-weight salivary mucins (MG1) to Hemophilus parainfluenzae.

In human saliva, two different mucin populations can be distinguished, viz., high-molecular-weight mucins (MG1, mol. wt > 1 x 10(6)) and low-molecular-weight mucins (MG2, mol. wt approximately 125 kD). The carbohydrate moiety of MG1 displays a wide spectrum of oligosaccharide structures, varying in composition, length, branching, and acidity. The biological significance of the heterogeneity in carbohydrate structures of mucins is unclear. The present investigation focused on the question whether MG1, because of its diverse carbohydrate side-chain population, can bind to a large variety of oral micro-organisms. A replica plate technique, in combination with immunochemical detection with monoclonal antibodies against MG1, was used to screen in vivo human oral microflora for the presence of micro-organisms which could bind the high-molecular-weight salivary mucin MG1. Binding to purified MG1 was established for Hemophilus (para)influenzae species, whereas other species, including Streptococcus and Staphylococcus, were negative. MG1 binding to Hemophilus parainfluenzae could be abolished by protease treatment of MG1. In contrast, periodate acid treatment, partial deglycosylation, or addition of monosaccharides did not affect MG1 binding to H. parainfluenzae, indicating that MG1 carbohydrate side-chains were not directly involved in the binding. The binding was pH-dependent, showing an increase when the pH was lowered from 8.0 to 4.0. These data indicate that MG1 can be bound in a selective manner by Hemophilus spp. and suggest that the 'naked' unglycosylated polypeptide moiety of MG1 is involved in its binding to Hemophilus parainfluenzae.