Nontypeable H. Influenzae Strains Research Articles

Characterization of Serotypes and Molecular Drug Resistance Patterns of Haemophilus influenzae in Kunming Children.

The present study aimed to determine the capsular serotype distribution and antimicrobial drug resistance patterns of Haemophilus influenzae from children in the Kunming region of China. This information could guide policymakers in clinical treatment. In the present study, H. influenzae isolates were tested for their serotypes, antimicrobial susceptibility pattern, and presence of β-lactamases. One-hundred forty-eight H. influenzae strains isolated from children 0-2 years old were investigated for capsular types by glass slide agglutination and molecular methods, and biotyped by the biochemical reactions. The drug resistance-encoding genes TEM-1, ROB-1, and the ftsI gene mutations PBP3-3, and PBP3-BLN were detected with real-time quantitative polymerase chain reaction (qPCR). The prevalence of β-lactamase-producing strains (60.3%) was significantly higher (p < 0.05) than non-enzyme-producing strains. β-Lactamase-producing strains were multidrug resistant to various antibiotics such as ampicillin, tetracycline, sulfamethoxazole/trimethoprim, chloramphenicol, cefuroxime, and cefaclor. Among β-lactamase-producing strains, the detection rates of the TEM-1, PBP3-BLN, PBP3-s, and ROB-1 were 54.1%, 18.9%, 11.8%, and 6.9%, respectively. The biotyping results show that most H. influenzae strains were of type II and III. Non-typeable H. influenzae (NTHi) accounted for 89.3% of the strains. NTHi strains were the most prevalent in this region; most belonged to biological types II and III. β-Lactamase-positive ampi-cillin-resistant (BLPAR) strains were prevalent among H. influenzae isolates in this region.

Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines.

The human upper respiratory tract is colonized with a variety of bacterial microorganisms including Haemophilus influenzae. The species H. influenzae consists of typeable and nontypeable H. influenzae (NTHi) variants. Typeable H. influenzae are subdivided into types a through f, based on the polysaccharide capsule, whereas the NTHi strains do not express a polysaccharide capsule. In this review, we highlight the current advances in the field of H. influenzae, with the focus on bacterial virulence mechanisms that facilitate bacterial colonization and disease, particularly for NTHi. In the past decade, it has become apparent that NTHi has the ability to cause invasive infections. Recently, a number of adhesins have been shown to be crucial for bacterial colonization and invasion and these proteins were investigated as vaccine antigens. Although NTHi lacks a polysaccharide capsule, it expresses lipooligosaccharide that contribute to adhesion and evasion of complement-mediated killing, both contributing to bacterial virulence, which could potentially be targeted by novel antimicrobial drugs or vaccines. The unraveling of H. influenzae virulence mechanisms resulted in the identification of promising targets for novel antimicrobials and vaccine antigens aiming to prevent or treat both typeable and nontypeable H. influenzae infections.

Clinical and Molecular Epidemiology of Childhood Invasive Nontypeable Haemophilus influenzae Disease in England and Wales

In countries with established Haemophilus influenzae type b (Hib) immunization programs, nontypeable H. influenzae (NTHi) is now responsible for nearly all invasive H. influenzae cases across all age groups. Public Health England (PHE) conducts enhanced national surveillance of invasive H. influenzae disease in England and Wales. Invasive NTHi isolates submitted to Public Health England from children of ages 1 month to 10 years during 2003-2010 were characterized by multilocus sequence typing (MLST). Detailed clinical information was obtained for all laboratory-confirmed cases of invasive NTHi disease in children during 2009-2013. In England and Wales, there were 7797 cases of invasive H. influenzae disease diagnosed during 2000-2013 and 1585 (20%) occurred in children aged 1 month to 10 years, where NTHi was responsible for 31-51 cases (incidence, 0.53-0.92/100,000) annually. Detailed clinical follow-up of 214 confirmed NTHi cases diagnosed in this age-group during 2009-2013 revealed that 52% (n = 111) occurred in <2-year-old and 52% (n=110) had comorbidity. Bacteremic pneumonia was the most common clinical presentation (n = 99, 46%), 16% (n = 34) required intensive care and 11% (n = 23) died. Characterization by biotyping and MLST of 316 NTHi strains from children with invasive disease during 2003-2010 revealed a genetically heterogeneous population (155 MLSTs) with diverse biotypes and no association with comorbidity status, clinical disease or outcome. The high level of genetic diversity in invasive NTHi strains highlights the difficulties in developing an effective vaccine against this pathogen.

A review of the role of Haemophilus influenzae in community-acquired pneumonia.

In an era when Haemophilus influenzae type b (Hib) conjugate vaccine is widely used, the incidence of Hib as a cause of community-acquired pneumonia (CAP) has dramatcally declined. Non-typeable H. influenzae (NTHi) strains and, occasionally, other encapsulated serotypes of H. influenzae are now the cause of the majority of invasive H. influenzae infectons, including bacteraemic CAP. NTHi have long been recognised as an important cause of lower respiratory tract infecton, including pneumonia, in adults, especially those with underlying diseases. The role of NTHi as a cause of non-bacteraemic CAP in children is less clear. In this review the evidence for the role of NTHi and capsulated strains of H. influenzae will be examined.

Nontypeable Haemophilus influenzae Genetic Islands Associated with Chronic Pulmonary Infection

Background Haemophilus influenzae (Hi) colonizes the human respiratory tract and is an important pathogen associated with chronic obstructive pulmonary disease (COPD). Bacterial factors that interact with the human host may be important in the pathogenesis of COPD. These factors, however, have not been well defined. The overall goal of this study was to identify bacterial genetic elements with increased prevalence among H. influenzae strains isolated from patients with COPD compared to those isolated from the pharynges of healthy individuals.Methodology/Principal FindingsFour nontypeable H. influenzae (NTHi) strains, two isolated from the airways of patients with COPD and two from a healthy individual, were subjected to whole genome sequencing using 454 FLX Titanium technology. COPD strain-specific genetic islands greater than 500 bp in size were identified by in silico subtraction. Open reading frames residing within these islands include known Hi virulence genes such as lic2b, hgbA, iga, hmw1 and hmw2, as well as genes encoding urease and other enzymes involving metabolic pathways. The distributions of seven selected genetic islands were assessed among a panel of 421 NTHi strains of both disease and commensal origins using a Library-on-a-Slide high throughput dot blot DNA hybridization procedure. Four of the seven islands screened, containing genes that encode a methyltransferase, a dehydrogenase, a urease synthesis enzyme, and a set of unknown short ORFs, respectively, were more prevalent in COPD strains than in colonizing strains with prevalence ratios ranging from 1.21 to 2.85 (p≤0.0002). Surprisingly, none of these sequences show increased prevalence among NTHi isolated from the airways of patients with cystic fibrosis.Conclusions/SignificanceOur data suggest that specific bacterial genes, many involved in metabolic functions, are associated with the ability of NTHi strains to survive in the lower airways of patients with COPD.

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

The expression and characterization of a putative adhesin B from H. influenzae

In the H. influenzae type b (Hib) genome, two putative adhesin B genes, HI0119 and HI0362, have been identified on the basis of homology to the adhesin B (FimA) of Streptococcus parasanguis. We expressed and characterized one of them, HI0119, from a non-typeable H. influenzae strain (NTHI). This 37 kDa protein was selectively isolated from an H. influenzae surface protein (water) extract by elution from a celite matrix with EDTA. The adhesin B protein is 97.7% identical to that of H. influenzae, strain Rd, has 23.7% identity and 47.8% similarity to FimA of Streptococcus parasanguis but is distinguished from the FimA family by the absence of the N-terminal lipid anchor consensus sequence LXXC, the presence of a C-terminal disulfide-bonded domain, and a central histidine-rich domain. Recombinant fusion protein bound specifically to celite. Antisera raised against fusion protein recognized a 37 kDa protein from whole cell extracts of H. influenzae on Western blots. A truncated mutant lacking the C-terminal disulfide-bonded domain and a Cys 308 to Ser mutant were constructed and expressed as fusion proteins. Both mutants retained celite binding. However, purified fusion proteins could not, unlike H. influenzae, bind Hep2 cells, suggesting that HI0119 may not be an adhesin in this organism.

The acylated form of protein D of Haemophilus influenzae is more immunogenic than the nonacylated form and elicits an adjuvant effect when it is used as a carrier conjugated to polyribosyl ribitol phosphate.

The nonacylated form of protein D (PDm) of Haemophilus influenzae has been shown to induce the production of antibodies that are bactericidal to homologous and heterologous nontypeable H. influenzae (NTHi) strains. In this study, immunization of rats with lipoprotein D (LPD) induced higher levels of anti-protein D immunoglobulin G and A serum antibodies than immunization with PDm, and the bactericidal activities of sera from LPD-immunized rats were greater than those of sera from PDm-immunized rats. Immunization with LPD or PDm did not prevent the development of acute otitis media (AOM) when rats were challenged with 10(4) CFU of an NTHi strain. However, on the eighth day of bacterial challenge, 50% (5 of 10) of LPD-immunized rats had recovered from otitis media and 30% (3 of 10) had negative middle ear cultures, whereas only 30% (3 of 10) of PDm-immunized rats had recovered, though none was culture positive. Immunization with an inactivated homologous bacterial strain elicited 70% protection (i.e., 7 of 10 rats) in the rat otitis media model. LPD and PDm were also conjugated to the H. influenzae type b (Hib) capsular polysaccharide, polyribosyl ribitol phosphate (PRP), to test protein D-conjugated PRP vaccine's potential for protection against Hib infection. When two LPD-conjugated and two PDm-conjugated PRP vaccines, each containing a different protein concentration, and a tetanus toxoid-conjugated vaccine (ACT-HIB) were tested in the experimental model of rat otitis induced with a Hib strain (Minn A), both of the LPD-conjugated and one of the PDm-conjugated vaccines induced significant protection from AOM, the level of protection being highest in animals given the vaccine with the highest LPD content. Sera from these rats also manifested the highest anti-PRP and anti-LPD antibody levels and the highest bactericidal activities against a Hib strain and an NTHi strain.

A resolved pneumococcal infection protects against nontypeable Haemophilus influenzae: an evaluation of different routes of whole cell immunization in protection against experimental acute otitis media

A conferred cross-protection between Haemophilus influenzae type b (Hib) and non-typeable H. influenzae (NTHi) was demonstrated in a previous study of experimental recurrent otitis media. To explore cross-protection further, and to compare oral administration of whole cells with two more conventional routes for vaccination against acute otitis media (AOM), a total number of 79 rats were immunized perorally, subcutaneously and intrabullarly with H. influenzae or pneumococci and thereafter challenged in the middle ear with NTHi or Hib 4 or 9 weeks later. Otomicroscopic changes, bacterial cultures, and serum IgG antibody levels were monitored. The study demonstrated that while peroral administration did not elicit any protection, a resolved pneumococcal AOM could reduce the susceptibility to reinfection with NTHi. In the latter case no cross-reacting antibodies were detected, but the protective rate was 50% or more, and it was comparable with that found after subcutaneous or intrabullar immunization with homologous NTHi or Hib strains. The results suggest that the protection of the rat middle ear mucosa may involve unspecific responses.

Identification of a locus involved in the utilization of iron by Haemophilus influenzae.

Haemophilus influenzae has an absolute requirement for heme for aerobic growth. This organism can satisfy this requirement by synthesizing heme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme. In contrast, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme. A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DL42 the ability to grow on iron and PPIX. Nucleotide sequence analysis revealed that this NTHI DNA insert contained three genes, designated hitA, hitB, and hitC, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmic binding protein-dependent iron transport system in this enteric organism. The NTHI HitA protein also was 69% identical to the ferric-binding protein of Neisseria gonorrhoeae. Inactivation of the cloned NTHI hitC gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth deficiency of Hib DL42. Construction of an isogenic NTHI TN106 mutant lacking a functional hitC gene revealed that this mutation prevented this strain from growing on a medium containing iron and PPIX in place of heme. This NTHI hitC mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the products encoded by the hitABC genes are essential for the utilization of iron by NTHI.