Prevalence of the hifBC, hmw1A, hmw2A, hmwC, and hia Genes in Haemophilus influenzae Isolates

Adherence of Haemophilus influenzae to respiratory epithelial cells is the first step in the pathogenesis of H. influenzae infection and is facilitated by the action of several adhesins located on the surface of the bacteria. In this study, prevalences of hifBC, which represent the pilus gene cluster; hmw1A, hmw2A, and hmwC, which represent high-molecular-weight (HMW) adhesin genes; and hia, which represents H. influenzae adhesin (Hia) genes were determined among clinical isolates of encapsulated type b (Hib) and nonencapsulated (NTHi) H. influenzae. hifBC genes were detected in 109 of 170 (64%) Hib strains and in 46 of 162 (28%) NTHi isolates (P = 0.0001) and were more prevalent among the invasive type b strains than invasive NTHi strains (P = 0.00003). Furthermore, hifBC genes were significantly more prevalent (P = 0.0398) among NTHi throat isolates than NTHi middle ear isolates. hmw1A, hmw2A, hmwC, and hia genes were not detected in Hib strains. Among NTHi isolates, the prevalence of hmw1A was 51%, the prevalence of hmw2A was 23%, the prevalence of hmwC was 48%, and the prevalence of hia was 33%. The hmw genes were significantly more prevalent among middle ear than throat isolates, while hia did not segregate with a respiratory tract site. These results show the variability of the presence of adhesin genes among clinical H. influenzae isolates and suggest that hemagglutinating pili may play a larger role in H. influenzae nasopharyngeal colonization than in acute otitis media whereas the HMW adhesins may be virulence factors for acute otitis media.     

Limited genetic diversity of recent invasive isolates of non-serotype b encapsulated Haemophilus influenzae

Invasive infections caused by non-type b encapsulated Haemophilus influenzae have increased in frequency in the last decade. This change prompted us to characterize the genetic relationships of 48 recently isolated invasive H. influenzae type a (Hia), e (Hie), and f (Hif) strains by comparison of restriction digest patterns (RDPs). Recent Hia isolates exhibited moderate genetic diversity, with the majority segregating into two major clonotypes. Recent Hie and, especially, Hif strains displayed considerably restricted genetic diversity. In particular, all but one Hif strain segregated into a single clonotype, and half of these isolates had identical RDPs. These results are consistent with the hypothesis that the increased incidence of disease due to non-type b encapsulated H. influenzae reflects the emergence of hypervirulent clones, especially in the case of Hif. Alternatively, it is possible that non-type b encapsulated H. influenzae strains have limited overall genetic diversity.     

Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains

The HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeable Haemophilus influenzae (NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified from Escherichia coli transformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains.     

Conservation and diversity of HMW1 and HMW2 adhesin binding domains among invasive nontypeable Haemophilus influenzae isolates

The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.     

Adhesin Expression in Matched Nasopharyngeal and Middle Ear Isolates of Nontypeable Haemophilus influenzae from Children with Acute Otitis Media

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable Haemophilus influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.     

Characterization of adherence of nontypeable Haemophilus influenzae to human epithelial cells

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.     

Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins

In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.     

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.     

Nontypeable Haemophilus influenzae strains with the capsule-associated insertion element IS1016 may mimic encapsulated strains

With the elimination of Haemophilus influenzae type b through vaccination, it has been suggested that other types of H. influenzae strains might acquire virulence traits and emerge as important pathogens. The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by nontypeable H. influenzae strains. To explore this further, 118 H. influenzae isolates, collected from both patients and healthy carriers, were investigated with PCR with reference to this gene sequence. Isolates positive for the insertion element were bio- and serotyped. The presence of hmw genes for adherence, the genetic profile, and the ability to form biofilm in vitro were investigated. A total of 15 isolates were IS1016-positive, whereof 12 were nontypeable. All 12 nontypeable isolates were obtained from healthy carriers, and 92% of the isolates were biotype I. They cross-reacted to some extent with type-specific antisera or exhibited a restricted genetic diversity like encapsulated strains. Furthermore, they lacked hmw-genes, and their ability to form biofilms was comparable with a capsule-deficient type b strain. Although this subset of strains mimicked traits usually exhibited by encapsulated strains, the isolation frequency did not seem to have been affected by vaccination.     

Genes encoding high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae are part of gene clusters.

We previously reported the cloning and sequencing of genes designated hmw1 and hmw2 from a prototype nontypeable Haemophilus influenzae strain. The genes encode proteins which are related to filamentous hemagglutinin of Bordetella pertussis and promote attachment of the nontypeable H. influenzae strain to human epithelial cells (J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). Subcloning studies suggested that correct processing of these high-molecular-weight proteins required the products of additional downstream genes. In the present study we analyzed the 3'-flanking regions of the hmw1A and hmw2A structural genes and found that both genes are flanked by two additional downstream open reading frames (ORFs), designated B and C, respectively. The B ORFs are 1,635 bp long. Their derived amino acid sequences are 99% identical and demonstrate similarity to the derived amino acid sequences of two genes that encode proteins required for secretion and activation of hemolysins of Proteus mirabilis and Serratia marcescens. The C ORFs are 1,950 bp long, and their derived amino acid sequences are 96% identical. In Escherichia coli transformants, interruption of the hmw1C or both the hmw1B and hmw1C genes resulted in defective processing of the hmw1A structural gene product and loss of the ability of the transformants to adhere to human epithelial cells. The precise interactions of the proteins encoded by these gene clusters are yet to be defined, but their elucidation may further our understanding of the biology of nontypeable H. influenzae bacteria and the interaction of these organisms with the human host.     

Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization.

Four hundred thirty-eight strains of Haemophilus influenzae were examined for production of and sensitivity to haemocin, a bacteriocin produced by some members of this species. Whereas 199 of 212 (94%) type b isolates produced haemocin, 131 of 134 (98%) nontypeable and 91 of 92 (99%) encapsulated non-type b isolates were sensitive to haemocin. Among strains previously genetically characterized by multilocus enzyme electrophoresis, haemocin production was detected in type b isolates belonging to 25 of 29 (86%) clonally distinct electrophoretic types. None of 60 clonally distinct nontypeable strains produced this substance, and all were sensitive to it in vitro. The genes encoding haemocin production were transformed independently of the genes necessary for capsule expression from a prototypic type b strain to a nontypeable strain. After intranasal inoculation of infant rats with an equal mixture of a non-haemocin-producing strain and its haemocin-producing transformant, organisms capable of haemocin production predominated in both nasopharyngeal and blood cultures. These data demonstrate that haemocin production is strongly associated with type b encapsulated members of this species and suggest a mechanism by which haemocin might play a role in host nasopharyngeal colonization by this pathogen.     

Serotype and ampicillin susceptibility of Haemophilus influenzae causing systemic infections in children: 3 years of experience.

Over a 3-year period, 96% of systemic infections in children caused by Haemophilus influenzae were of serotype b. Of 346 invasive infections, 15 (4%) were caused by non-type b H. influenzae. The monthly prevalence of ampicillin resistance in all isolates was highly variable (0 to 63%). Ampicillin resistance in H. influenzae causing invasive disease occurred in 13% of non-type b and 21.8% of type b isolates. There was no significant difference (x2 - 0.21; p greater than 0.10) in the rate of ampicillin resistance between type b and non-type b H. influenzae causing systemic illness in children over a 3-year period.     

Human antibodies specific for the high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae mediate opsonophagocytic activity

The HMW1- and HMW2-like adhesion proteins of nontypeable Haemophilus influenzae are expressed by 75% of these strains, and antibodies directed against these proteins are protective in animal models of infection. The purpose of the present study was to define the functional activity of human antibodies specific for these proteins in an in vitro complement-dependent opsonophagocytic assay. Human promyelocytic cell line HL-60 served as the source of phagocytic cells, and a commercial preparation of intravenous immunoglobulin (IVIG) served as the source of human antibodies. High-molecular-weight (HMW) proteins were purified from four prototype nontypeable H. influenzae strains and used to prepare solid-phase affinity columns. IVIG was adsorbed on each column to remove strain-specific anti-HMW antibodies and to allow recovery of affinity-purified anti-HMW antibody fractions. Unadsorbed IVIG killed each of the prototype strains at titers of 1:80 to 1:320. HMW-adsorbed sera demonstrated fourfold decreases in opsonophagocytic titer against the homologous strains compared to unadsorbed IVIG. Affinity-purified anti-HMW antibody preparations demonstrated opsonophagocytic titers of 1:20 to 1:80 against the respective homologous strains and opsonophagocytic titers as high as 1:80 against heterologous strains. None of the affinity-purified anti-HMW antibody preparations was opsonophagocytic for a representative nontypeable H. influenzae strain that did not express HMW1- or HMW2-like proteins. These data demonstrate that human antibodies specific for the HMW1/HMW2-like adhesion proteins of nontypeable H. influenzae are opsonophagocytic and that such antibodies recognize epitopes shared by the HMW proteins of unrelated nontypeable H. influenzae strains. These results argue for continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for prevention of disease due to nontypeable H. influenzae.     

Presence in bovine enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli of genes encoding for putative adhesins of human EHEC strains

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells. The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium. A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E. coli (NTEC). In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups. Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC. Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.     

Mapping of binding domains of nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins

Nontypeable Haemophilus influenzae is an important cause of localized respiratory tract disease, which begins with colonization of the upper respiratory mucosa. In previous work we reported that the nontypeable H. influenzae HMW1 and HMW2 proteins are high-molecular-weight nonpilus adhesins responsible for attachment to human epithelial cells, an essential step in the process of colonization. Interestingly, although HMW1 and HMW2 share significant sequence similarity, they display distinct cellular binding specificities. In order to map the HMW1 and HMW2 binding domains, we generated a series of complementary HMW1-HMW2 chimeric proteins and examined the ability of these proteins to promote in vitro adherence by Escherichia coli DH5alpha. Using this approach, we localized the HMW1 and HMW2 binding domains to an approximately 360-amino-acid region near the N terminus of the mature HMW1 and HMW2 proteins. Experiments with maltose-binding protein fusion proteins containing segments of either HMW1 or HMW2 confirmed these results and suggested that the fully functional binding domains may be conformational structures that require relatively long stretches of sequence. Of note, the HMW1 and HMW2 binding domains correspond to areas of maximal sequence dissimilarity, suggesting that selective advantage associated with broader adhesive potential has been a major driving force during H. influenzae evolution. These findings should facilitate efforts to develop a subcomponent vaccine effective against nontypeable H. influenzae disease.     

Participation of complement in host defense against encapsulated Haemophilus influenzae types a, c, and d.

Each of the serotypes of Haemophilus influenzae (types a to f) may colonize the respiratory tract of humans, but only type b strains commonly cause invasive systemic infections. To investigate the role of complement in immunity to encapsulated non-type b strains, rats were depleted of C3 with cobra venom factor and challenged with representative serotypes of H. influenzae (type a, b, c, or d) by different routes. After intravenous challenge, rats depleted of C3 had a greater incidence and magnitude of bacteremia with each of the serotypes when compared with normal controls. Intraperitoneal inoculation of type b organisms resulted in meningitis in normal and C3-depleted rats, but only C3-depleted, and not normal, rats developed meningitis after inoculation of serotype a, c, or d. In contrast to systemic inoculation, intranasal challenge with the different serotypes resulted in bloodstream invasion and meningitis only after challenge with type b organisms. These data suggest that complement plays a significant role in immunity to encapsulated, non-type b H. influenzae through its effect on bloodstream clearance.     

Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 10⁸-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 10⁷- to 10⁹-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.Otitis media remains a significant health problem for children in this country and elsewhere in the world (15, 16). Most children in the United States have had at least one episode of otitis by the third birthday, and one-third have had three or more episodes (51). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (50). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for prevention of otitis media in young children (26).Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of cases of acute otitis media (20, 53). Streptococcus pneumoniae has been the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (20, 53). Nontypeable Haemophilus influenzae (NTHi) is the second most common bacterium recovered and accounts for 20% to 30% of cases of acute otitis media and a larger percentage of cases of chronic and recurrent disease (37). Interestingly, since introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccination schedule, nontypeable Haemophilus influenzae has become an even more common cause of acute and recurrent middle ear disease, often surpassing Streptococcus pneumoniae in frequency of recovery from middle ear fluid specimens (12, 18).Many different antigens have been suggested as possible nontypeable Haemophilus influenzae vaccine candidates (1, 5, 23, 43, 44, 63). In our early work, we demonstrated that development of bactericidal antibody in the sera of children who had recovered from acute NTHi otitis media was associated with the appearance of serum antibodies directed against highly immunogenic high-molecular-weight (HMW) proteins (7). This work led subsequently to the identification and characterization of the HMW1 and HMW2 (HMW1/HMW2) family of proteins (8). The HMW1/HMW2 proteins have subsequently been shown to be major adhesins of nontypeable Haemophilus influenzae (57) as well as targets of opsonophagocytic (65, 66) and protective (6) antibodies. The HMW1/HMW2-like proteins are expressed by approximately 75% of NTHi strains (8, 58). The 25% of NTHi strains that do not express HMW1/HMW2-like proteins also express immunogenic high-molecular-weight proteins that are recognized by human convalescent-phase serum antibodies (11). Almost all the HMW1/HMW2-negative strains have subsequently been shown to express a second distinct class of adhesins known as Hia proteins (11). The Hia proteins are members of a large family of bacterial proteins known as autotransporters that are found in many Gram-negative bacteria (28, 69). The Hia proteins have also recently been shown to serve as targets for opsonophagocytic antibodies (64). Nearly all NTHi strains that lack HMW1/HMW2 proteins contain a hia gene and express a Hia protein, and conversely, strains that express HMW1/HMW2 proteins lack a hia gene (11, 58).Several groups have begun exploring mucosal and, in particular, nasopharyngeal immunization strategies to stimulate a protective immune response in the upper respiratory tract and middle ear (19, 24), and results to date have been encouraging (4, 29, 32, 47). Intranasal immunization has a number of potential advantages over traditional parenteral immunization approaches for prevention of otitis media. The presence of abundant microvilli in the nasal cavity greatly increases the available surface area of this anatomical site, thereby generating a large absorptive surface (34). Immunization via the nasal cavity also allows direct delivery of immunogens of interest to inductive/effector sites (i.e., nasal mucosa-associated lymphoid tissue [NALT] and Waldeyer''s ring), which can result in both a systemic and a mucosal response (14, 33, 62). Furthermore, the nasal cavity is readily accessible and allows for noninvasive delivery of antigens or vaccines, thus eliminating the need for trained staff or the use of sterile needles and syringes. The option of treating or immunizing against human disease with such a noninvasive technique would almost certainly result in increased patient compliance if intranasal vaccines advance to the clinic.A number of mucosal vaccination strategies continue to be actively explored (41, 49). Adenoviruses have been identified as promising live recombinant vaccine vectors based upon their ability to induce high levels of heterologous gene expression (13, 25, 55, 60) and to stimulate mucosal immunity in the upper respiratory tract, their natural site of replication. E1-deletion-containing replication-defective adenoviral recombinants based on human serotype 5 (Adhu5) have been tested as vaccine candidates for prevention of a number of infectious diseases (21, 60). Studies of adenoviral vaccines based upon the H5N1 hemagglutinin protein (30), surface proteins of Streptococcus pneumoniae (3), the rabies virus glycoprotein (68), the circumsporozoite protein of Plasmodium falciparum (54), and the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) (27) have demonstrated that E1-deletion-containing vaccines induce excellent B-cell and CD8⁺-T-cell responses in experimental animals, even if given at moderate doses.Recombinant adenovirus (rAd) vectors have yet to be investigated as potential vaccine candidates for prevention of otitis media. However, they are particularly attractive candidates for the reasons noted above. The objective of the present study was to construct E1-deletion-containing replication-defective recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia adhesion protein of nontypeable Haemophilus influenzae and to assess their immunogenicity in the chinchilla experimental model.     

Use of automated riboprinter and pulsed-field gel electrophoresis for epidemiological studies of invasive Haemophilus influenzae in Taiwan

A total of 87 invasive isolates of Haemophilus influenzae isolated throughout Taiwan from 1994 to 1998 was collected; 57 were from children <14 years old. In all, 60.9% of isolates were resistant to ampicillin and produced beta-lactamase. Ribotyping revealed six different profiles in 55 isolates of type b, nine profiles in 10 isolates of non-type b and 12 profiles in 22 isolates of non-typable H. influenzae. Among isolates from 35 cases of meningitis, 30 (86%) were in ribogroups 1, 2 and 3 with >90% genetic similarity. Compared with all the other ribogroups, ribogroups 1, 2 and 3, which encompassed all H. influenzae type b, were significantly more prevalent as a cause of meningitis in children <14 years old. Further subtyping of the predominant ribogroup by pulsed-field gel electrophoresis (PFGE) identified differences of 0-6 bands among these isolates of ribogroup 1, which indicated distant relatedness. Automated ribotyping was found to be a useful method and was less time-consuming for molecular epidemiology studies of H. influenzae. PFGE is suggested as an addition to ribotyping to improve discrimination if H. influenzae type b is involved. Differentiating ribogroups between type b and non-type b H. influenzae by genotyping may help to understand the molecular characteristics of outbreaks, endemicity and value of vaccination. According to the results of ribotyping and PFGE, it seems possible that spread of invasive H. influenzae type b had occurred and ribotyping confirmed that there was no clonal spread of non-type b H. influenzae in Taiwan.     

The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate

Nontypeable Haemophilus influenzae is a major causative agent of bacterial otitis media in children. H. influenzae Hap autotransporter protein is an adhesin composed of an outer membrane Hapbeta region and a moiety of an extracellular internal 110-kDa passenger domain called Hap(S). The Hap(S) moiety promotes adherence to human epithelial cells and extracellular matrix proteins, and it also mediates bacterial aggregation and microcolony formation. A recent work (D. L. Fink, A. Z. Buscher, B. A. Green, P. Fernsten, and J. W. St. Geme, Cell. Microbiol. 5:175-186, 2003) demonstrated that Hap(S) adhesive activity resides within the C-terminal 311 amino acids (the cell binding domain) of the protein. In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of Hap(S) from H. influenzae strains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native Hap(S) purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of Hap(S) is capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeable H. influenzae diseases.     

Characterization of fusidic acid-resistant Staphylococcus aureus isolates in the community of Casablanca (Morocco)

Resistance to fusidic acid in Staphylococcus aureus is caused by mutation of the elongation factor G (EF-G) encoded by fusA or by expression of a protein, encoded by fusB or fusC, that protects the drug target. Other mechanisms involved in this resistance are mutations in the riboprotein L6 operon within rplF. The aim of this study was to determine the prevalence and mechanisms of resistance to fusidic acid in clinical isolates of S. aureus in Casablanca (Morocco) and to define the phenotypic and genotypic traits of these isolates and their clonal relationship. All fusidic acid-resistant S. aureus (FAR-SA) isolates were tested for fusB and fusC genes and were evaluated for the detection of mutations in fusA and fusE (rplF). fusB-positive strains were tested for a cadDX operon, encoding cadmium resistance. The agr group and the presence of toxin genes were monitored to characterize all FAR-SA isolates which were typed by pulsed-field gel electrophoresis (PFGE) and spa typing. Among 140 clinical S. aureus isolates collected in 2007 and 2008, 18 (～13%) exhibited resistance to fusidic acid. The most common resistance determinant was fusC, found in 16 isolates. Molecular typing showed that 14 of them harboured an agr group III and belonged to the same clonal complex (CC) spa type 127 and identical clonotype (cluster labelled A). These isolates also possessed the staphylococcal enterotoxin H gene. The second resistance determinant was fusB found in two isolates. These two isolates lacked cadDX gene and were found to belong to two unrelated clusters and spa types. While no isolate carrying mutations in rplF was found, 15 expressed a silent mutation in fusA (nucleotide 342). Only acquired fusidic acid resistance genes (mainly fusC) were prevalent among FAR-SA isolates with almost all of the clinical specimens belonging to CC-spa type 127. This study provides valuable data on the prevalence of fusidic acid-resistant S. aureus with the associated molecular mechanisms of resistance and the genetic background of the strains in Casablanca.