Search Results (37)

Pulmonary hypertension complicating chronic obstructive pulmonary disease (COPD-PH) has a worse prognosis than airflow limitation alone but its pathogenesis is thought to be primarily due to alveolar hypoxia affecting pulmonary circulation. Two observations are not fully explained by this model. First, the majority of exacerbations of COPD are infectious and there is a stable frequent-exacerbator phenotype that is not fully accounted for by spirometric severity. Second, the airways of COPD are not sterile. They are home to a resident microbial community that changes as the disease progresses and the gut microbial community also undergoes disease-associated changes. This review examines whether microbial factors actively contribute to pulmonary vascular remodelling or merely reflect advanced disease. Three candidate exposures are considered: chronic airway colonisation, dysbiosis of the airway and gut communities and recurrent infectious exacerbation. We then examine the virulence mechanisms and host-recognition pathways of non-typeable Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, rhinovirus, influenza viruses, respiratory syncytial virus and SARS-CoV-2. We then trace the pathways linking microbial recognition to vascular injury: Toll-like receptor signalling and NF-κB activation; interleukin-6, tumour necrosis factor-α and interleukin-1β; endothelin-1 upregulation against nitric oxide depletion; reactive oxygen species and hypoxia-inducible factor signalling converging with hypoxic pulmonary vasoconstriction; and, along the gut–lung axis, lipopolysaccharide translocation, trimethylamine N-oxide and depletion of short-chain fatty acid-producing taxa. Therapeutically, there is evidence and a vascular rationale for interventions that decrease exacerbation frequency. Pulmonary vasodilators have a sound physiological rationale, although their clinical efficacy is frequently limited by worsening ventilation–perfusion mismatch. Current evidence supports biological plausibility rather than demonstrated causality: no direct longitudinal human study has established that microbial alterations or microbial exposure cause pulmonary vascular remodelling in COPD-PH. We identify study designs capable of directly testing this hypothesis. Full article

Background/Objectives: Re-emergence of Haemophilus influenzae serotype b (Hib) was reported in several European countries. We aimed to characterize the age distribution of H. influnezae carriage before and after Hib vaccination. Methods: We conducted a systematic review and meta-analysis to reassess H. influenzae carriage dynamics in the pre- and post-Hib vaccination eras, focusing on age-specific patterns in childhood. Searches were performed with no date restriction and included PubMed/MEDLINE, Scopus, Web of Science, WHO Global Index Medicus, and the Cochrane Library. Eligible studies reported nasopharyngeal and/or oropharyngeal carriage prevalence and serotype distribution. Pooled estimates with 95% confidence intervals (CIs) were calculated using random-effects models, with age-stratified analyses. Results: Twenty-two studies were included (12 pre- and 10 post-Hib vaccination). Pre-vaccination, pooled H. influenzae carriage prevalence was 24.3% (95% CI, 18.9–30.7%), including 6% (95% CI, 3.4–12.8%) for Hib and 17.5% (95% CI, 12.6–23.9%) for non–type b strains. Post-vaccination, overall carriage remained similar (21.8%; 95% CI, 14.6–31.2%), but Hib carriage declined markedly to 0.67% (95% CI, 0.26–1.71%), while non–type b strains predominated (16.7%; 95% CI, 10.4–25.6%). Meta-analysis showed that carriage peaked around 4–5 years of age and persisted into later childhood. Conclusions: Hib vaccination has reduced Hib carriage, but overall H. influenzae carriage persists due to non–type b strains. Age-related persistence of carriage may have implications for herd protection, particularly in the context of evolving vaccination schedules with early childhood boosters. Continued surveillance integrating carriage and immunological data is needed to inform optimization of vaccination strategies. Full article

Background: Non-typeable Haemophilus influenzae (NTHi) is a major cause of acute respiratory tract infections and chronic airway disease, despite its clinical importance, no licensed vaccine is available, largely due to the extensive genetic and antigenic diversity among circulating isolates. We previously identified conserved outer membrane proteins capable of inducing systemic protection against NTHi. Methods: In this study, we evaluated whether a multi-antigen protein vaccine composed of conserved NTHi antigens (P5 and P26) could protect against pulmonary infection. Transgenic mice expressing human transferrin and factor H were immunized via the intraperitoneal or intranasal route and challenged intranasally with a clinical NTHi isolate. Bacterial clearance, antigen-specific mucosal and systemic antibody responses, and recruitment of innate immune cells to the airways were assessed. Results: Both immunization routes significantly reduced bacterial loads compared with controls. Vaccination induced robust mucosal and systemic IgG and IgA responses and enhanced early recruitment of macrophages, monocytes, dendritic cells, and neutrophils to the airways. Intranasal immunization elicited strong mucosal antibody responses and was associated with improved local bacterial clearance. Conclusions: These findings demonstrate that multi-antigen vaccines targeting conserved NTHi proteins can elicit effective mucosal and systemic immunity and represent promising candidates for the prevention against NTHi respiratory infections. Full article

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease in young children, yet determinants of disease severity in otherwise healthy infants remain poorly understood. RSV disease severity has been linked to temporal differences in airway epithelial type I/III IFN and inflammatory responses, with defective viral genomes (DVGs) acting as potent inducers of type I/III IFNs. Additionally, an increased abundance of nontypeable Haemophilus influenzae (NTHi) is associated with more severe RSV disease, although the mechanisms underlying this association and the impacts of NTHi on DVG replication and DVG-driven host responses remain unclear. To address this knowledge gap, we modeled a clinically relevant but underexplored scenario of simultaneous transmission by performing concurrent RSV–NTHi co-infection experiments. Co-infection reduced titers of RSV but not of NTHi. Mechanistically, extracellular NTHi inhibited RSV particle binding to host cells. Using A549 cells and human precision-cut lung slices, we found that NTHi alone was a weak inducer of type I/III IFNs but a strong inducer of pro-inflammatory cytokines. Accordingly, RSV–NTHi co-infection suppressed DVG replication and DVG-driven IFN responses while enhancing inflammatory signaling, potentially driven by increased cell-associated NTHi. Together, these findings provide a mechanistic basis for how NTHi exacerbates RSV disease severity through dysregulated host immune responses rather than increased viral burden. Full article

Biofilms are involved in both acute and chronic respiratory infections. While cigarette smoke extract (CSE) has been shown to increase biofilm formation by certain respiratory pathogens, the impact of emerging heated tobacco products (HTPs) remains unclear. We compared the effects of CSE with two HTP aerosol extracts on biofilm biomass and metabolic activity of common respiratory pathogens. Reference strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, and non-typeable Haemophilus influenzae (NTHi), known respiratory pathogens, were grown as 24 h biofilms in 96-well plates (48 h for S. aureus and P. aeruginosa). These were exposed to CSE and HTP extracts from iQOS™ (Terea™ Turquoise, ILUMA™ device) and glo™ (neo™ Azure, HyperPro™ device), prepared in liquid culture media. Biofilm density was quantified by the crystal violet assay. Metabolic activity (planktonic and biofilm) was assessed by MTT reduction to formazan. At 24 h, CSE markedly reduced H. influenzae biomass versus iQOS™, glo™, and control, while K. pneumoniae, S. aureus, and P. aeruginosa showed no significant biomass differences. At 48 h, CSE significantly increased biomass in P. aeruginosa and S. aureus versus other exposures. Biofilm MTT assay measured metabolic activity increased in CSE exposure for K. pneumoniae versus iQOS™ and control, and for S. aureus versus control. Overall, HTP extracts showed limited, inconsistent effects compared with CSE, indicating combustion-derived constituents more strongly promote biofilm maturation in this model. Full article

Background: Haemophilus influenzae (Hi), a Gram-negative bacterium, is divided into two broad categories: encapsulated and non-capsulated isolates, also called non-typeable Hi isolates (NTHi). NTHi has become prevalent since the introduction of the vaccine against Hi of serotype b. Hi can cause local infections on respiratory mucosal surfaces and urogenital infections, which can lead to septic abortion in pregnant women. It can also cause invasive infections such as meningitis and septicemia. Moreover, NTHi isolates are becoming increasingly resistant to antibiotics. Vaccines targeting NTHi are not yet available. As these NTHi isolates are not encapsulated, vaccines should target proteins at the bacterial surface. However, vaccine development is hindered by the high variability of these proteins. We aimed to identify conserved outer membrane proteins (OMPs) for vaccines against NTHi. Methods: We analyzed core-genome multilocus sequence typing (cgMLST) of 1144 genomes of Hi collected between 2017 and 2022 and, of these, identified 514 conserved genes that encoded OMPs. We focused on two specific OMPs: Haem1295, encoding the protein P5 (P5), and Haem1040, encoding the protein 26 (P26). P5 is known to bind human complement regulatory protein factor H (FH), while both P5 and P26 are involved in enhancing immune responses. The genes encoding these proteins were cloned, overexpressed, purified, and tested in both active and passive protection models using systemic infection in mice. Results: P5 and P26 were found to be immunogenic during human infections. Vaccination with these proteins conferred protection against both homologous and heterologous NTHi isolates in mice, suggesting broad cross-protection. Conclusions: P5 and P26 are promising vaccine candidates showing cross-protection against NTHi and offering the additional benefit of targeting bacterial virulence factors, enhancing vaccine efficacy against NTHi isolates. Full article

Purpose: The adenoid microbiota plays a key role in adenoid hypertrophy (AH). This study explored the molecular epidemiology and antimicrobial resistance of Haemophilus. Influenzae (H. influenzae) strains in pediatric AH patients. Methods: Retrospective analysis of pediatric AH patients undergoing endoscopic adenoidectomy. Adenoid tissue samples were cultured to screen for pathogens. H. influenzae strains were identified by 16S rRNA sequencing and serotyped via q-PCR. Multilocus sequence typing (MLST) and ftsI gene analysis were conducted using PubMLST. β-lactamase genes (bla_TEM-1, bla_ROB-1) were detected by PCR, and antibiotic susceptibility testing (AST) was performed using the Etest method. For imipenem-resistant strains, the acrRAB efflux pump gene cluster and ompP2 porin gene were sequenced and compared with those of the wild-type strain Rd KW20. Results: Over 8 months, 56 non-duplicate H. influenzae strains were isolated from 386 patients. The detection rate was highest in children under 5 years (30.5%) compared to those aged 5–10 years (13.4%) and 10–15 years (8.7%). Of 49 sub-cultured strains, all were non-typeable H. influenzae (NTHi). MLST identified 22 sequence types (STs) and 13 clonal complexes (CCs), with CC11 (26.5%), CC3 (14.3%), and CC107 (14.3%) being predominant. Common STs included ST103 (22.4%), ST57 (10.2%), and ST107 (10.2%). Most strains belonged to the ftsI group III-like+ (57.1%). β-lactamase positivity was 98.0% (48/49), with bla_TEM-1 (95.9%) and bla_ROB-1 (18.4%) detected. AST showed low susceptibility to ampicillin (10.2%), amoxicillin–clavulanate (34.7%), azithromycin (12.2%), and trimethoprim–sulfamethoxazole (14.3%). Among the β-lactamase-positive strains, 44/48 were β-lactamase-positive ampicillin-resistant (BLPAR); none were β-lactamase-negative ampicillin-resistant (BLNAR). Imipenem susceptibility was 91.8% (45/49). No carbapenemases were found in the imipenem-resistant strains, but mutations in acrRAB (88.12–94.94% identity) and ompP2 (77.10–82.94% identity) were observed. Conclusions: BLPAR NTHi strains of CC11 are major epidemic strains in pediatric AH. Imipenem resistance in H. influenzae likely results from porin mutations rather than carbapenemase activity. Enhanced surveillance of H. influenzae’s role in AH and its resistance patterns is warranted. Full article

Bacterial meningitis remains a critical public health issue globally due to its high morbidity and mortality. Understanding regional epidemiological trends is essential to inform vaccination strategies and public health interventions. This observational, retrospective study analyzed cerebrospinal fluid (CSF) isolates collected from 731 confirmed cases of bacterial meningitis between 2014 and 2024 in Lombardy, Italy. Pathogen identification and serotyping of Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), and Haemophilus influenzae (HI) were conducted using culture-based and molecular techniques. Trends were assessed across age groups and time using Kruskal–Wallis and chi-square tests. Results: SP was the predominant pathogen (78.4%), followed by NM (13.0%) and HI (8.6%). Significant temporal variation was observed for SP and NM, while HI trends remained stable. The impact of COVID-19-related restrictions was evident in a reduction in cases during 2020–2021. SP serotypes 3 and 8, HI non-typeable strains, and NM serogroup B were most frequent. No major shifts in serotype distribution were observed. Long-term surveillance data from Lombardy underscore the dominance of vaccine-targeted serotypes, ongoing circulation of resilient clones, and post-pandemic epidemiological shifts. These findings support continuous surveillance and inform vaccine strategy adjustments at the regional and national levels. Full article

Background/Objectives: Acute otitis media is a common pediatric infection caused primarily by nontypeable Haemophilus influenzae. With rising antibiotic resistance, vaccines are essential for combating this public health issue. Although the PD-1/PD-L1 pathway has been extensively studied for its role in tumor immunity, its impact on mucosal immunity, particularly in vaccine responses, is unclear. Methods: BALB/c mice were intranasally immunized with nontypeable H. influenzae outer membrane protein and treated with anti-PD-L1 antibodies. Immune responses were evaluated in middle ear mucosa (MEM), the cervical lymph node, and the spleen using an enzyme-linked immunosorbent assay, an enzyme-linked immunospot assay, and flow cytometry. The effects on CD4⁺ T cells, T follicular helper (Tfh) cells, and B-cell differentiation were analyzed. Results: Anti-PD-L1 antibody treatment increased CD3⁺CD4⁺CD185⁺ (CXCR5⁺) Tfh cells in MEM, which play a crucial role in supporting B-cell activation and antibody production. This correlated with a significant increase in IgA- and IgG-producing cells in MEM, which enhanced local bacterial clearance. Although B-cell activation and differentiation into plasmablasts were observed in MEM, no significant changes were noted in the cervical lymph node and spleen, suggesting a localized enhancement of mucosal immunity. Conclusions: Anti-PD-L1 antibodies promoted Tfh cell expansion and B-cell differentiation in MEM, leading to enhanced antibody production and improved bacterial clearance. These findings suggest that PD-L1 blockade can potentiate mucosal vaccine-induced immunity by strengthening local humoral responses. This supports its potential application in developing intranasal vaccines for acute otitis media. Full article

Non-typeable Haemophilus influenzae (NTHi) is often asymptomatically carried in the upper airways but can cause a wide spectrum of disease conditions, from local respiratory tract infections to invasive disease such as sepsis or meningitis. The factors driving NTHi’s transition from benign carriage to severe systemic disease remain poorly understood. It is unknown whether NTHi strains associated with invasive or non-invasive disease differ in their capacity to trigger inflammatory responses in innate immune cells. To address this question, we have used an in vitro infection model of human THP-1 cells differentiated to macrophages. To evaluate inflammatory responses, we studied the expression of 3 prototypic pro-inflammatory molecules, ICAM-1, TNF-α, and IL-1β. The role of lipooligosaccharide in triggering inflammatory responses was assessed using inhibition of Toll-like receptor 4 signaling. Our experiments demonstrated that NTHi strains isolated from cases of invasive and non-invasive infections were similarly able to induce strong activations of macrophage pro-inflammatory responses. Our findings support the hypothesis that the development of invasive versus non-invasive NTHi disease may be more significantly influenced by the adaptive immune response than the innate immune response. Full article

Otitis media is the most frequently diagnosed disease and a leading cause of hearing loss in young children. However, genetic contributors to susceptibility and pathogen–host–environment interactions in otitis media remain to be identified. Such knowledge would help identify at-risk individuals and effectively monitor, diagnose, and treat patients with otitis media. Through exome and Sanger sequencing, we identified a rare, deleterious splice variant SLPI c.394+1G>T co-segregating with otitis media in a large pedigree, with a genome-wide significant maximum LOD score of 4.59. Alternative splicing of SLPI was observed in saliva RNA of variant carriers. In bulk mRNA-seq data from an independent cohort of children with otitis media, SLPI was co-expressed with genes involved in infection, immune response, inflammation, and epithelial cell organization. After inoculation of non-typeable Haemophilus influenzae, Slpi was upregulated in polymorphonuclear leukocytes and epithelial cells of mouse middle ears. Furthermore, in the human middle ear, Haemophilus was significantly enriched in non-carriers, whereas Family-XI-Incertae-Sedis and Dialister were significantly enriched in variant carriers. Given the role of SLPI in immune modulation and host defense in mucosal epithelia, our findings support the SLPI variant as modulating susceptibility to otitis media. Full article

The changing epidemiological profile of invasive Haemophilus influenzae infections (IIHi) is noted in the post-vaccination era. The aim of this study was to characterize phenotypically and genotypically invasive Haemophilus influenzae (Hi) isolates detected in Tunisian pediatric patients. A retrospective study was conducted in the microbiology laboratory of the Children’s Hospital of Tunis over ten years (2013–2023). All IIHi cases were included. Molecular identification and serotyping were conducted through qPCR. Molecular typing and analysis of resistance genes were extracted from whole genome sequencing data. Fifty-three IIHi cases were collected. Children under five years old were the most affected (81%). Non-typable isolates (NTHi) were predominant (79%) followed by serotype b (17%) and serotype a (4%). Genetic diversity was observed, essentially among NTHi isolates. Resistance of Hi isolates to ampicillin, amoxicillin–clavulanic acid and cefotaxime (CTX) were 42%, 20% and 4%, respectively. Thirteen isolates (29%) produced a beta-lactamase and 14 carried the blaTEM-1 gene (kappa = 0.95). For non-enzymatic resistance, group 3 (n = 12) showed resistance to ampicillin. Groupe 4 (n = 9, NTHi) showed discordances with resistance to CTX. The emergence of resistance to CTX is concerning. Continuous surveillance through molecular tools in conjunction with phenotypic and clinical data is necessary to ensure better management of these infections. Full article

Biofilms, aggregates of bacteria enclosed in a self-produced matrix, have been implicated in various pediatric respiratory infections, including acute otitis media (AOM), otitis media with effusion (OME), adenoiditis, protracted bacterial bronchitis, and pulmonary exacerbations in cystic fibrosis. These infections are prevalent in children and often associated with biofilm-producing pathogens, leading to recurrent and chronic conditions. Biofilms reduce antibiotic efficacy, contributing to treatment failure and disease persistence. This narrative review discusses biofilm production by respiratory pathogens such as Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Pseudomonas aeruginosa, and Staphylococcus aureus. It examines their mechanisms of biofilm formation, antibiotic resistance, and the challenges they present in clinical treatment. Various antibiofilm strategies have shown promise in vitro and in animal studies, including the use of N-acetylcysteine, enzymes like dispersin B, and agents disrupting quorum sensing and biofilm matrix components. However, their clinical application, particularly in children, remains limited. Traditional treatments for biofilm-associated diseases have not significantly evolved, even with biofilm detection. The transition from experimental findings to clinical practice is complex and requires robust clinical trials and standardized biofilm detection protocols. Addressing biofilms in pediatric respiratory infections is crucial for improving treatment outcomes and managing recurrent and chronic diseases effectively. Full article

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition. Full article

This study aimed to investigate the molecular epidemiology and antibiotic resistance of Haemophilus influenzae in Guangzhou, China. A total of 80 H. influenzae isolates were collected from the First Affiliated Hospital of Guangzhou Medical University from January 2020 to April 2021. Species identification, antimicrobial susceptibility, molecular capsular typing, multilocus sequence typing and the clinical characteristics analysis of patients were performed. For all recruited isolates, the majority of H. influenzae strains from patients with respiratory symptoms were found to be non-typeable H. influenzae (NTHi). The isolates were relative susceptible to third- and fourth-generation cephalosporins, quinolones and chloramphenicol, despite having a high ampicillin resistance rate (>70%). The genotyping results reveal a total of 36 sequence types (STs), with ST12 being the most prevalent ST. Remarkably, the 36 STs identified from 80 NTHi isolates within a short period of 15 months and in a single medical setting have revealed a high genetic diversity in NTHi isolates. In comparison, it is noteworthy that the most prevalent STs found in the present study have rarely been found to overlap with those from previous studies. This is the first study on the molecular epidemiology of NTHi isolates in Guangzhou, a city that is representative of southern China. Full article