Search Results (33)

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

Microbiota studies have dramatically increased over these last two decades, and the repertoire of microorganisms with potential health benefits has been considerably enlarged. The development of next generation probiotics from new bacterial candidates is a long-term strategy that may be more efficient and rapid with discriminative in vitro tests. Streptococcus strains have received attention regarding their antimicrobial potential against pathogens of the upper and, more recently, the lower respiratory tracts. Pathogenic bacterial strains, such as non-typable Haemophilus influenzae (NTHi), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), are commonly associated with acute and chronic respiratory diseases, and it could be interesting to fight against pathogens with probiotics. In this study, we show that a Streptococcus mitis (S. mitis) EM-371 strain, isolated from the buccal cavity of a human newborn and previously selected for promising anti-inflammatory effects, displayed in vitro antimicrobial activity against NTHi, P. aeruginosa or S. aureus. However, the anti-pathogenic in vitro activity was not sufficient to predict an efficient protective effect in a preclinical model. Two weeks of treatment with S. mitis EM-371 did not protect against, and even exacerbated, NTHi lung infection. Full article

Background: The decrease in the rate of meningitis due to Haemophilus influenzae type b after vaccine introduction and a possible change in epidemiology of H. influenzae disease highlights the need for continuous serotype surveillance. Methods: A single-tube multiplex PCR assay for serotyping of H. influenzae was developed and deployed. Results: During 2003–2020, 108 meningitis cases due to H. influenzae were notified; 86 (80%) were confirmed and serotyped by molecular methods. The overall specificity and sensitivity of the assay were estimated (100% PPV and NPV respectively). The overall mean annual reported incidence for H. influenzae was 0.02, while for Hib and non-b meningitis equaled 0.02 and 0.03 per 100 000, respectively. Analysis by age group revealed that H. influenzae peaks in toddlers and children 0–4 years and in adults >45 years old. Among the serotyped cases, 39.8% were identified as Hib, 46.3% as NTHi, and 0.9% and 2.8% as serotypes a (Hia) and f (Hif)) respectively. Conclusions: Low incidence due to Hib was observed while non-typeable H. influenzae (NTHi) and serotypes Hia and Hif seem to emerge. The application of the current assay discloses the ongoing change of invasive H. influenzae disease trends during the Hib post-vaccine era. Full article

BackgroundSince the appearance of COVID-19 in January 2020, invasive bacterial infections have decreased significantly worldwide. However, alterations in age and sex distributions, clinical forms, phenotypes, and genotypes of isolates have not been analyzed. Our goal is to present and discuss these data considering the current COVID-19 pandemic situation. Methods: The data of the national reference center for meningococci and Haemophilus influenzae in France were mined to examine the above aspects of invasive bacterial infection before (2018–2019) and after (2020–2021) the COVID-19 pandemic. Detailed epidemiological, clinical, and microbiological data were collected, and whole genome sequencing was carried out on meningococcal isolates (n = 1466). Results: In addition to the overall decline in the number of cases, various changes in age, sex, and phenotypes of isolates were also noted. As for N. meningitidis, more cases were observed in adults, as well as more invasive pneumopathies. Furthermore, fewer hyperinvasive meningococcal genotypes have circulated since COVID-19 emerged. The situation has been different for H. influenzae, as the number of invasive cases among adults decreased due to a reduction in non-typeable isolates. In contrast, cases due to serotypeable isolates, particularly serotypes a and b, increased in children <5 years-old. Conclusions: It is possible that measures implemented to stop COVID-19 may have reduced the circulation of N. meningitidis and H. influenzae isolates, but to a variable extent. This may be due to differences in circulation between these two species according to age groups. Vaccination schedules against these two species may have also influenced the evolution of these invasive bacterial infections since the emergence of the COVID-19 pandemic. Full article

Lung macrophage iron levels are increased in COPD patients. Lung macrophage iron levels are thought to be increased by cigarette smoke, but the role of red blood cells (RBCs) as a source of iron has not been investigated. We investigate RBCs as a potential source of alveolar iron in COPD, and determine the effect of RBC-derived iron on macrophage function. We used lung tissue sections to assess RBC coverage of the alveolar space, iron and ferritin levels in 11 non-smokers (NS), 15 smokers (S) and 32 COPD patients. Lung macrophages were isolated from lung resections (n = 68) and treated with hemin or ferric ammonium citrate (50, 100 or 200 μM). Lung macrophage phenotype marker gene expression was measured by qPCR. The phagocytosis of Non-typeable Haemophilus influenzae (NTHi) was measured by flow cytometry. Cytokine production in response to NTHi in iron-treated macrophages was measured by ELISA. Lung macrophage iron levels were significantly correlated with RBC coverage of the alveolar space (r = 0.31, p = 0.02). Furthermore, RBC coverage and lung macrophage iron were significantly increased in COPD patients and correlated with airflow obstruction. Hemin treatment downregulated CD36, CD163, HLA-DR, CD38, TLR4, CD14 and MARCO gene expression. Hemin-treated macrophages also impaired production of pro-inflammatory cytokines in response to NTHi exposure, and decreased phagocytosis of NTHi (200 μM: 35% decrease; p = 0.03). RBCs are a plausible source of pulmonary iron overload in COPD. RBC-derived iron dysregulates macrophage phenotype and function. Full article