Relationships of nontypeable Haemophilus influenzae strains to hemolytic and nonhemolytic Haemophilus haemolyticus strains

Haemophilus influenzae is both a human respiratory pathogen and pharyngeal commensal, while H. haemolyticus, the closest phylogenetic relative of H. influenzae, is arguably a strict pharyngeal commensal. A hemolytic phenotype has historically differentiated H. haemolyticus from H. influenzae, but the recent recognition of significant nonhemolytic H. haemolyticus colonization has decreased this trait's resolvability. Given this and the potential of recombination between the species, we examined the distribution of microbiologic and molecular traits between collections of H. influenzae and H. haemolyticus strains separated within a dendrogram obtained by multilocus sequence analysis (MLSA). All strains hybridizing with a probe to iga, a gene encoding an immunoglobulin A protease of H. influenzae, clustered apart from strains that did not hybridize with the probe. Other traits also segregated significantly along this division, suggesting a separation of the species. Of note, the LOS genes licA, lic2A, and lgtC of H. influenzae were approximately 2, 6, and 54 times, respectively, more prevalent in H. influenzae than in H. haemolyticus. In contrast to species separation, interspecies recombination was evidenced by the inability of single gene sequences to phylogenetically separate the species and by the “fuzzy” distribution of some species-specific traits across the species dividing line. Together, these data support the historically accurate and pragmatic division of these species while recognizing their potential for recombination. Future comparative genomic studies identifying common and distinctive genes could be useful in evaluating their role in the commensal or virulent growth, respectively, of H. influenzae.     

Prevalence of the sodC Gene in Nontypeable Haemophilus influenzae and Haemophilus haemolyticus by Microarray-Based Hybridization

The sodC gene has been reported to be a useful marker for differentiating nontypeable (NT) Haemophilus influenzae from Haemophilus haemolyticus in respiratory-tract samples, but discrepancies exist as to the prevalence of sodC in NT H. influenzae. Therefore, we used a microarray-based, “library-on-a-slide” method to differentiate the species and found that 21 of 169 (12.4%) NT H. influenzae strains and all 110 (100%) H. haemolyticus strains possessed the sodC gene. Multilocus sequence analysis confirmed that the 21 NT H. influenzae strains were H. influenzae and not H. haemolyticus. An inactive sodC gene has been reported in encapsulated H. influenzae strains belonging to phylogenetic division II. Capsule-specific Southern hybridization and PCR and a lack of copper/zinc-cofactored superoxide dismutase (CuZnSOD) expression indicated that 6 of the 21 sodC-containing NT H. influenzae strains in our study were likely capsule-deficient mutants belonging to phylogenetic division II. DNA sequence comparisons of the 21 H. influenzae sodC genes with sodC from H. haemolyticus or encapsulated H. influenzae demonstrated that the sodC genes of the six H. influenzae capsule-deficient mutants were, on average, 99% identical to sodC from encapsulated H. influenzae but only 85% identical to sodC from H. haemolyticus. The sodC genes from 2/15 NT H. influenzae strains were similarly more closely related to sodC from encapsulated strains, while sodC genes from 13 NT H. influenzae strains were almost 95% identical to sodC genes from H. haemolyticus, suggesting the possibility of interspecies recombination in these strains. In summary, this study demonstrates that sodC is not completely absent (9.2%) in true NT H. influenzae strains.Haemophilus influenzae asymptomatically colonizes the pharyngeal cavity of humans but may cause either systemic or respiratory-tract infections. Those strains that possess a polysaccharide capsule, serotypes a through f, cause most H. influenzae invasive infections (11), whereas nonencapsulated or nonserotypeable (NT) H. influenzae strains are associated with localized respiratory-tract diseases, such as otitis media and exacerbation of chronic obstructive pulmonary disease (COPD) (19). In addition, a cryptic genospecies of NT H. influenzae is also associated with neonatal invasive infections and adult urogenital infections (36). Phylogenetically, however, the cryptic genospecies is more closely related to Haemophilus haemolyticus than to H. influenzae of the respiratory tract (26, 27).H. haemolyticus is a commensal of the pharyngeal cavity, and H. haemolyticus and NT H. influenzae isolated from the respiratory tract overlap taxonomically and phylogenetically, likely through the exchange of DNA by natural transformation (1, 20, 21, 32). The species have been traditionally differentiated by the ability of H. haemolyticus to hemolyze horse blood (11, 25), but recent studies have identified significant numbers of nonhemolytic H. haemolyticus in NT H. influenzae collections isolated from throat or sputum samples (21, 37). Since sputum samples are used to monitor COPD exacerbation, accurate differentiation of NT H. influenzae from H. haemolyticus is clinically important (5, 21).Fung et al. (9) recently suggested that the presence of the sodC gene or activity of its cognate protein, copper/zinc-cofactored superoxide dismutase (CuZnSOD), can differentiate H. haemolyticus from NT H. influenzae, as sodC was present in 20 H. haemolyticus isolates and absent in 20 NT H. influenzae isolates. In addition, a previous study failed to find the sodC gene in 45 NT H. influenzae disease isolates (18). Prior to these studies, however, 12 of 26 NT H. influenzae strains were found to hybridize weakly with a sodC gene probe, and these strains also displayed CuZnSOD activity (13). The discrepancy between these studies has never been explained. Aside from the questionable absence of sodC in NT H. influenzae of the respiratory tract, the gene and its associated CuZnSOD activity are present in the H. influenzae cryptic genospecies associated with neonatal invasive infections and adult urogenital infections (18). In addition, the gene is present among serotype e encapsulated strains and in strains that belong to the capsular phylogenetic division II, a multilocus enzyme electrophoresis (MLEE)-based division that contains some serotype a and b (Hib) strains and all serotype f (Hif) strains (13, 18, 22). When present, the sodC gene in encapsulated strains is adjacent to the bexA to -D genes of the capsule locus. The sodC gene of encapsulated sodC-containing strains shares significant sequence homology with the gene from Haemophilus parainfluenzae, but unlike H. parainfluenzae, encapsulated H. influenzae lacks CuZnSOD activity, presumably due to a substitution mutation that replaces an active-site histidine with tyrosine (13).In the present study, we further evaluated the potential of the sodC gene to differentiate a large collection of known NT H. influenzae and H. haemolyticus respiratory tract isolates using a whole-genome microarray hybridization technology called library on a slide (39).     

Association of IS1016 with the hia adhesin gene and biotypes V and I in invasive nontypeable Haemophilus influenzae

A subset of invasive nontypeable Haemophilus influenzae (NTHI) strains has evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. We examined IS1016-positive invasive NTHI isolates collected as part of Active Bacterial Core Surveillance within the Georgia Emerging Infections Program for the presence or absence of hmw1 and hmw2 (two related adhesin genes that are common in NTHI but absent in encapsulated H. influenzae) and hia (homologue of hsf, an encapsulated H. influenzae adhesin gene). Isolates were serotyped using slide agglutination, confirmed as NTHI strains using PCR capsule typing, and biotyped. Two hundred twenty-nine invasive NTHI isolates collected between August 1998 and December 2006 were screened for IS1016; 22/229 (9.6%) were positive. Nineteen of 201 previously identified IS1016-positive invasive NTHI isolates collected between January 1989 and July 1998 were also examined. Forty-one IS1016-positive and 56 randomly selected IS1016-negative invasive NTHI strains were examined. The hia adhesin was present in 39 of 41 (95%) IS1016-positive NTHI strains and 1 of 56 (1.8%) IS1016-negative NTHI strains tested; hmw (hmw1, hmw2, or both) was present in 50 of 56 (89%) IS1016-negative NTHI isolates but in only 5 of 41 (12%; all hmw2) IS1016-positive NTHI isolates. IS1016-positive NTHI strains were more often biotype V (P < 0.001) or biotype I (P = 0.04) than IS1016-negative NTHI strains, which were most often biotype II. Pulsed-field gel electrophoresis revealed the expected genetic diversity of NTHI with some clustering based on IS1016, hmw or hia, and biotypes. A significant association of IS1016 with biotypes V and I and the presence of hia adhesins was found among invasive NTHI. IS1016-positive NTHI strains may represent a unique subset of NTHI strains, with characteristics more closely resembling those of encapsulated H. influenzae.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment.     

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.     

Turnover of nonencapsulated Haemophilus influenzae in the nasopharynges of otitis-prone children.

Restriction enzyme analysis of total genomic DNA was applied to study the epidemiology of nontypeable Haemophilus influenzae (NTHI) isolated from the nasopharynges of children with recurrent acute otitis media (AOM). The turnover of strains, as judged from genetic fingerprinting of a total of 213 H. influenzae isolates collected prospectively during a 2-year study period from 38 children under 3 years of age, was examined in relation to episodes of AOM as well as to courses of antibiotic treatment. The children were selected if they had had at least one episode of AOM before 1 year of age and if more than two nasopharyngeal isolates of H. influenzae were recovered. The 213 H. influenzae isolates (90% NTHI) recovered corresponded to 128 different DNA fingerprints. Fifty-eight percent of the fingerprints were observed only once, whereas 42% appeared on two or more occasions in isolates from the same individual or in close relatives, i.e., brothers and sisters. Sixty-seven percent of these strains had a minimum colonization period of 2 months or less. Intermittent nasopharyngeal colonization periods longer than 5 months could be demonstrated for 13% of the strains. The present data suggest that intermittent colonization is due to endogenous reinfections. Genetically identical NTHI strains from unrelated individuals were never identified. As expected from the observation of a relatively high proportion of persistent colonizations, no correlation was found between episodes of AOM and the acquisition of new strains of H. influenzae, nor was any direct relation between antimicrobial therapy and the elimination of nasopharyngeal colonization with a particular strain of H. influenzae observed.     

Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244?H. influenzae and 33?H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.     

Binding of Heme-Hemopexin Complexes by Soluble HxuA Protein Allows Utilization of This Complexed Heme by Haemophilus influenzae

Utilization of heme-hemopexin as a source of heme by Haemophilus influenzae type b is dependent on expression by this bacterium of the 100-kDa HxuA protein, which is both present on the bacterial cell surface and released into the culture supernatant (L. D. Cope, R. Yogev, U. Muller-Eberhard, and E. J. Hansen, J. Bacteriol. 177:2644–2653, 1995). Radioimmunoprecipitation analysis showed that the soluble HxuA protein present in H. influenzae type b culture supernatant bound heme-hemopexin complexes in solution. An isogenic H. influenzae type b hxuA mutant was unable to utilize soluble heme-hemopexin complexes for growth in vitro unless soluble HxuA protein was provided exogenously. Soluble HxuA protein secreted by a nontypeable H. influenzae strain also allowed growth of this H. influenzae type b hxuA mutant. These results indicated that the heme present in heme-hemopexin complexes is rendered accessible to H. influenzae when these complexes are bound by the soluble HxuA protein.     

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n = 81) and non-Aboriginal (n = 76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P < 0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P = 0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.     

Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.     

Potential of a Novel Protein, OMP26, from Nontypeable Haemophilus influenzae To Enhance Pulmonary Clearance in a Rat Model

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of Haemophilus influenzae. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable H. influenzae (NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer’s patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of H. influenzae type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the H. influenzae Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.     

Duplicate copies of lic1 direct the addition of multiple phosphocholine residues in the lipopolysaccharide of Haemophilus influenzae

The genes of the lic1 operon (lic1A to lic1D) are responsible for incorporation of phosphocholine (PCho) into the lipopolysaccharide (LPS) of Haemophilus influenzae. PCho plays a multifaceted role in the commensal and pathogenic lifestyles of a range of mucosal pathogens, including H. influenzae. Structural studies of the LPS of nontypeable H. influenzae (NTHI) have revealed that PCho can be linked to a hexose on any one of the oligosaccharide chain extensions from the conserved inner core triheptosyl backbone. In a collection of NTHI strains we found several strains in which there were two distinct but variant lic1D DNA sequences, genes predicted to encode the transferase responsible for directing the addition of PCho to LPS. The same isolates were also found to express concomitantly two PCho residues at distinct positions in their LPS. In one such NTHI isolate, isolate 1158, structural analysis of LPS from lic1 mutants confirmed that each of the two copies of lic1D directs the addition of PCho to a distinct location on the LPS. One position for PCho addition is a novel heptose, which is part of the oligosaccharide extension from the proximal heptose of the LPS inner core. Modification of the LPS by addition of two PCho residues resulted in increased binding of C-reactive protein and had consequential effects on the resistance of the organism to the killing effects of normal human serum compared to the effects of glycoforms containing one or no PCho. When bound, C-reactive protein leads to complement-mediated killing, indicating the potential biological significance of multiple PCho residues.     

Comparative Analysis of Haemophilus influenzae hifA (Pilin) Genes

Adherence of Haemophilus influenzae to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with AluI and RsaI. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained only nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly H. influenzae type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected hifA genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against H. influenzae type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable H. influenzae identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity. These regions of HifA amino acid sequence heterogeneity may explain the immunologic diversity seen in intact pili.     

Molecular Epidemiology of Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae

Resistance to ampicillin without β-lactamase production is not a frequent occurrence among Haemophilus influenzae strains. This kind of resistance is encountered in unencapsulated strains isolated from bronchial secretions and ear, nose, and throat specimens and is exceptional in H. influenzae type b. We studied 29 of these strains from various areas in France and 2 reference strains. Strains were compared by using ribotyping, arbitarily primed PCR with two primers, and pulsed-field gel electrophoresis. Each technique enabled the identification of 20 to 23 different patterns among the 31 strains. The combination of the different patterns for the strains obtained by the different techniques provided 27 distinct profiles. According to these results, it seems that the clonal propagation of these resistant strains does not occur.     

Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Identification and analysis of potential virulence-associated genes in Haemophilus parasuis based on genomic subtraction

Haemophilus parasuis is a commensal bacterium in the swine upper respiratory tract, but virulent strains can invade host and cause Glässer’s disease under certain conditions. In order to explore the virulence difference in H. parasuis strains, a virulent isolate, SH0165 (serotype 5), and an avirulent isolate, 7140 (serotype 4), were selected to investigate genetic differences by genomic subtraction. A total of 560 clones were screened by dot-blot and PCR, and 33 fragments present in SH0165 but absent from 7140 were identified. They encode eight gene categories, the sclB family, restriction modification system, phage products, transport system, outer membrane proteins, metabolism, and DNA synthesis and insertion sequences. We investigated the distribution of 21 fragments in 15 reference strains. Six fragments were more prevalent in virulent strains than avirulent strains based on statistical analysis; three fragments were SH0165-specific, and the remaining 12 were diversely distributed. Having analyzed these significantly differential fragments, including the sclB family, ABC-type transporter, fhuA and nhaC, we presume that these genes are associated with virulence in H. parasuis.     

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.