Biofilm Formation by Nontypeable Haemophilus Influenzae and Resistance to Complement-Mediated Clearance.

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae.

Nasal Delivery of Haemophilus haemolyticus Is Safe, Reduces Influenza Severity, and Prevents Development of Otitis Media in Mice.

BACKGROUND: Despite vaccination, influenza and otitis media (OM) remain leading causes of illness. We previously found that the human respiratory commensal Haemophilus haemolyticus prevents bacterial infection in vitro and that the related murine commensal Muribacter muris delays OM development in mice. The observation that M muris pretreatment reduced lung influenza titer and inflammation suggests that these bacteria could be exploited for protection against influenza/OM. METHODS: Safety and efficacy of intranasal H haemolyticus at 5 × 107 colony-forming units (CFU) was tested in female BALB/cARC mice using an influenza model and influenza-driven nontypeable Haemophilus influenzae (NTHi) OM model. Weight, symptoms, viral/bacterial levels, and immune responses were measured. RESULTS: Intranasal delivery of H haemolyticus was safe and reduced severity of influenza, with quicker recovery, reduced inflammation, and lower lung influenza virus titers (up to 8-fold decrease vs placebo; P ≤ .01). Haemophilus haemolyticus reduced NTHi colonization density (day 5 median NTHi CFU/mL = 1.79 × 103 in treatment group vs 4.04 × 104 in placebo, P = .041; day 7 median NTHi CFU/mL = 28.18 vs 1.03 × 104; P = .028) and prevented OM (17% OM in treatment group, 83% in placebo group; P = .015). CONCLUSIONS: Haemophilus haemolyticus has potential as a live biotherapeutic for prevention or early treatment of influenza and influenza-driven NTHi OM. Additional studies will deem whether these findings translate to humans and other respiratory infections.

Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections.

Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.

cAMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence of Haemophilus influenzae.

Most naturally competent bacteria tightly regulate the window of the competent state to maximize their ecological fitness under specific conditions. Development of competence by Haemophilus influenzae strain Rd KW20 is stimulated by cAMP and inhibited by purine nucleotides, respectively. In contrast, cAMP inhibits cell growth, but nucleotides are important for KW20 growth. However, the mechanisms underlying the abovementioned reciprocal effects are unclear. Here, we first identified a periplasmic acid phosphatase AphAEc of Escherichia coli as a new cAMP-binding protein. We show cAMP competitively inhibits the phosphatase activities of AphAEc and its homolog protein AphAHi in the KW20 strain. Furthermore, we found cAMP inhibits two other periplasmic nonspecific phosphatases, NadNHi (which provides the essential growth factor V, NAD) and HelHi (eP4, which converts NADP to NAD) in KW20. We demonstrate cAMP inhibits cell growth rate, especially via NadNHi. On the other hand, the inhibitory effect of purine nucleotide AMP on competence was abolished in the triple deletion mutant ΔhelHiΔnadNHiΔaphAHi, but not in the single, double deletion or complemented strains. Adenosine, however, still inhibited the competence of the triple deletion mutant, demonstrating the crucial role of the three phosphatases in converting nucleotides to nucleosides and thus inhibiting KW20 competence. Finally, cAMP restored the competence inhibited by GMP in a dose-dependent manner, but not competence inhibited by guanosine. Altogether, we uncovered these three periplasmic phosphatases as the key players underlying the antagonistic effects of cAMP and purine nucleotides on both cell growth and competence development of H. influenzae.

A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers.

Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.

Substitutions in the nonactive site of the passenger domain on the activity of Haemophilus influenzae immunoglobulin A1 protease.

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the ß-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.

An infant mouse model of influenza-driven nontypeable Haemophilus influenzae colonization and acute otitis media suitable for preclinical testing of novel therapies.

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 105 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children.

Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

Tobacco use, self-reported professional dental cleaning habits, and lung adenocarcinoma diagnosis are associated with bronchial and lung microbiome alpha diversity.

RATIONALE: The lung microbiome is an inflammatory stimulus whose role in the development of lung malignancies is incompletely understood. We hypothesized that the lung microbiome associates with multiple clinical factors, including the presence of a lung malignancy. OBJECTIVES: To assess associations between the upper and lower airway microbiome and multiple clinical factors including lung malignancy. METHODS: We conducted a prospective cohort study of upper and lower airway microbiome samples from 44 subjects undergoing lung lobectomy for suspected or confirmed lung cancer. Subjects provided oral (2), induced sputum, nasopharyngeal, bronchial, and lung tissue (3) samples. Pathologic diagnosis, age, tobacco use, dental care history, lung function, and inhaled corticosteroid use were associated with upper and lower airway microbiome findings. MEASUREMENTS AND MAIN RESULTS: Older age was associated with greater Simpson diversity in the oral and nasopharyngeal sites (p = 0.022 and p = 0.019, respectively). Current tobacco use was associated with greater lung and bronchus Simpson diversity (p < 0.0001). Self-reported last profession dental cleaning more than 6 months prior (vs. 6 or fewer months prior) was associated with lower lung and bronchus Simpson diversity (p < 0.0001). Diagnosis of a lung adenocarcinoma (vs. other pathologic findings) was associated with lower bronchus and lung Simpson diversity (p = 0.024). Last professional dental cleaning, dichotomized as ≤ 6 months vs. >6 months prior, was associated with clustering among lung samples (p = 0.027, R2 = 0.016). Current tobacco use was associated with greater abundance of pulmonary pathogens Mycoplasmoides and Haemophilus in lower airway samples. Self-reported professional dental cleaning ≤ 6 months prior (vs. >6 months prior) was associated with greater bronchial Actinomyces and lung Streptococcus abundance. Lung adenocarcinoma (vs. no lung adenocarcinoma) was associated with lower Lawsonella abundance in lung samples. Inhaled corticosteroid use was associated with greater abundance of Haemophilus among oral samples and greater Staphylococcus among lung samples. CONCLUSIONS: Current tobacco use, recent dental cleaning, and a diagnosis of adenocarcinoma are associated with lung and bronchial microbiome α-diversity, composition (ß-diversity), and the abundance of several respiratory pathogens. These findings suggest that modifiable habits (tobacco use and dental care) may influence the lower airway microbiome. Larger controlled studies to investigate these potential associations are warranted.

Semisynthetic glycoconjugates as potential vaccine candidates against Haemophilus influenzae type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-ß-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Trends in serotype distribution and antimicrobial susceptibility pattern of invasive Haemophilus influenzae isolates from Brazil, 2009-2021.

INTRODUCTION: Invasive Haemophilus influenzae (Hi) disease poses a significant global health challenge. With the relaxation of COVID-19 pandemic measures and declining H. influenzae serotype b (Hib) vaccination coverage, there is concern about a potential increase in Hi cases worldwide. METHODOLOGY: This study analyzed 1437 invasive Hi isolates in Brazil over 13 years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. RESULTS: The primary source of isolation for these invasive H. influenzae isolates was blood (54.4%), followed by cerebrospinal fluid (37.1%) and lung specimens (8.5%), respectively. Consequently, bacteremia (47%) was the most common clinical presentation, followed by meningitis (39.6%) and pneumonia (13.4%). Non-encapsulated Hi (NTHi) predominated among the isolates (51.4%), along with serotype a (22%) and serotype b (21.5%) among the encapsulated isolates. The majority of the encapsulated isolates were isolated from children under 14 years of age (76.7%), while NTHi isolates were identified in patients older than 15 years, particularly those ≥ 60 years old (40%). Ampicillin resistance was observed in 17.1% of cases, displaying ß-lactamase production as the principal resistance mechanism. MLST revealed a diverse NTHi population, whereas the encapsulated isolates presented a clonal structure. CONCLUSION: This study describes the prevalence of NTHi isolates circulating in Brazil after two decades of the Hib vaccine immunization program. Continuous universal surveillance is crucial for implementing prompt public health measures to prevent and control invasive Hi disease and monitor changes in antibiotic resistance profiles.

Antimicrobial efficacy and amino acid substitutions associated with susceptibility to the tellurium compound AS101 against Haemophilus influenzae and Haemophilus parainfluenzae.

The tellurite toxicity in Haemophilus influenzae and H. parainfluenzae remains unclear. To understand the potential of tellurite as a therapeutic option for these bacteria, we investigated the antimicrobial efficacy of AS101, a tellurium compound, against H. influenzae and H. parainfluenzae and the molecular basis of their differences in AS101 susceptibility. Through broth microdilution, we examined the minimum inhibitory concentration (MIC) of AS101 in 51 H. influenzae and 28 H. parainfluenzae isolates. Whole-genome sequencing was performed on the H. influenzae isolates to identify genetic variations associated with AS101 susceptibility. The MICs of AS101 were ≦ 4, 16-32, and ≧ 64 µg/mL in 9 (17.6%), 12 (23.5%), and 30 (58.8%) H. influenzae isolates, respectively, whereas ≦ 0.5 µg/mL in all H. parainfluenzae isolates, including multidrug-resistant isolates. Time-killing kinetic assay and scanning electron microscopy revealed the in vitro bactericidal activity of AS101 against H. parainfluenzae. Forty variations in nine tellurite resistance-related genes were associated with AS101 susceptibility. Logistic regression, receiver operator characteristic curve analysis, Venn diagram, and protein sequence alignment indicated that Val195Ile substitution in TerC, Ser93Gly in Gor (glutathione reductase), Pro44Ala/Ala50Pro in NapB (nitrate reductase), Val307Leu in TehA (tellurite resistance protein), Cys105Arg in CysK (cysteine synthase), and Thr364Ser in Csd (Cysteine desulfurase) were strongly associated with reduced AS101 susceptibility, whereas Ser155Pro in TehA with increased AS101 susceptibility. In conclusions, the antimicrobial efficacy of AS101 is high against H. parainfluenzae but low against H. influenzae. Genetic variations and corresponding protein changes relevant to AS101 non-susceptibility in H. influenzae were identified.

Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in Portugal.

We report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.

Haemophilus influenzae endocarditis: a case report and literature review.

INTRODUCTION: Haemophilus influenzae (HI) is an exceedingly rare cause of infective endocarditis (IE). CASE PRESENTATION/METHODS: We present a case of a 90-year-old female diagnosed with HI-IE involving the native tricuspid valve in the absence of traditional risk factors for right-sided endocarditis. She was treated with a 5-week course of IV Ampicillin from negative cultures and suffered no complications. We also conducted a thorough literature review through PubMed and Google Scholar, which yielded a mere 15 reported cases of HI-IE. RESULTS: Fourteen of the reported HI-IE cases included epidemiological data, showing no gender predominance. The mean age of the subjects was 39.5, with the mitral valve being the most implicated (64%) and tricuspid valve involvement being rare (21%). CONCLUSION: Native tricuspid valve IE is an uncommon entity, especially in the absence of IV drug use. Haemophilus influenzae is an extremely rare cause of IE, with a literature review showing merely 15 reported cases. This article cites the 16th case of HI-IE published in the literature.

First global report about the prevalence of multi-drug resistant Haemophilus influenzae: a systematic review and meta-analysis.

BACKGROUND: In recent decades, the prevalence of antibiotic resistance is increasing in Haemophilus influenzae (Haemophilus influenzae), which poses important challenges to global health. This research offers a comprehensive meta-analysis of the global epidemiology of multi-drug resistant (MDR) H. influenzae. METHODS: In this study, we conducted a meta-analysis based on PRISMA checklist. Electronic databases including PubMed, ISI Web of Science, Scopus, EMBASE, and Google Scholar were reviewed using keywords related to H. influenzae and antibiotic resistance. Eligible studies were selected based on stringent inclusion and exclusion criteria. Then, data from these studies were analyzed using the Comprehensive Meta-Analysis (CMA) software. RESULTS: Of 375 retrieved articles, 16 met the inclusion criteria. These studies were conducted from 2003 to 2023 and analyzed data from 19,787 clinical isolates of H. influenzae. The results showed different levels of resistance of H. influenzae to different antibiotics: ampicillin (36%), azithromycin (15.3%), ceftriaxone (1.4%), etc. The global prevalence for beta-lactamases producing H. influenzae and MDR H. influenzae was measured 34.9% and 23.1%, respectively. The prevalence rate of MDR H. influenzae was higher in Asian countries (24.6%) compared to Western regions (15.7%). MDR H. influenzae had the highest prevalence in meningitis cases (46.9%) and the lowest prevalence in acute otitis media (0.5%). CONCLUSIONS: The prevalence of MDR H. influenzae has been increasing worldwide, especially in Asian regions. This highlights the urgent need for monitoring and implementation of effective antibiotic stewardship programs globally.

Comparison of the adherence of nontypeable haemophilus influenzae to lung epithelial cells.

OBJECTIVE: Nontypeable Haemophilus influenzae (NTHi) plays an important role in respiratory tract infections, and adherence to lung epithelial cells is the first step in lung infections. To explore the role of NTHi in childhood lung infections, a comparative study was conducted on the adherence of strains isolated from sputum culture and bronchoalveolar lavage fluid to A549 lung epithelial cells. METHODS: Haemophilus influenzae strains were obtained from the sample bank of Shenzhen Children's Hospital, and identified as NTHi via PCR detection of the capsule gene bexA. NTHi obtained from healthy children's nasopharyngeal swabs culture were selected as the control group, and a comparative study was conducted on the adherence of strains isolated from sputum culture or bronchoalveolar lavage fluid of patients to A549 cells. RESULTS: The adherence bacterial counts of NTHi isolated from the nasopharyngeal cultures of healthy children to A549 cells was 58.2 CFU. In patients with lung diseases, NTHi isolated from bronchoalveolar lavage fluid was 104.3 CFU, and from sputum cultures was 115.1 CFU, both of which were significantly higher in their adherence to A549 cells compared to the strains isolated from the healthy control group. There was no significant difference in adherence between the strains isolated from sputum cultures and bronchoalveolar lavage fluid (t = 0.5217, p = 0.6033). CONCLUSION: NTHi played an important role in childhood pulmonary infections by enhancing its adherence to lung epithelial cells.

Bacterial antigens and asthma: a comparative study of common respiratory pathogenic bacteria.

Objective: In a previous study we have shown that, in the presence of interleukin (IL)-33, repeated, per-nasal challenge of murine airways with Streptococcus pneumoniae (S. pneumoniae) organisms induces human asthma-like airways inflammation. It is not clear, however, whether this effect is unique or manifest in response to other common respiratory pathogens.Methods: To explore this, airways of BALB/c mice were repeatedly challenged per-nasally with formaldehyde-inactivated bacterial bodies in the presence or absence of murine recombinant IL-33. Serum concentrations of S.pneumoniae, Moraxella catarrhalis (M.catarrhalis) and Haemophilus influenzae (H.influenzae) lysates-specific IgE were measured in patients with asthma and control subjects.Results: We showed that in the presence of IL-33, repeated, per-nasal airways exposure to the bodies of these bacteria induced airways hyperresponsiveness (AHR) in the experimental mice. This was accompanied by cellular infiltration into bronchoalveolar lavage fluid (BALF), eosinophilic infiltration and mucous hypertrophy of the lung tissue, with elevated local expression of some type 2 cytokines and elevated, specific IgG and IgE in the serum. The precise characteristics of the inflammation evoked by exposure to each bacterial species were distinguishable.Conclusions: These results suggest that in the certain circumstances, inhaled or commensal bacterial body antigens of both Gram-positive (S. pneumoniae) and Gram-negative (M. catarrhalis and H. influenzae) respiratory tract bacteria may initiate type 2 inflammation typical of asthma in the airways. In addition, we demonstrated that human asthmatic patients manifest elevated serum concentrations of M.catarrhalis- and H.influenzae-specific IgE.