Prevalence and distribution of adhesins in invasive non-type b encapsulated Haemophilus influenzae

Adhesion to the respiratory epithelium plays an important role in Haemophilus influenzae infection. The distribution of H. influenzae adhesins in type b and nontypeable strains has been characterized, but little is known about the prevalence of these factors in non-type b encapsulated strains. We analyzed 53 invasive type a, type e, and type f strains for the presence of hap, hia, hmw, and hif genes; Hap, Hia, and HMW1/2 adhesins; and hemagglutinating pili. The hap gene was ubiquitous, and homologs of hmw and hia were present in 7 of 53 (13.2%) and 45 of 53 (84.9%) strains, respectively. Hap was detected in 28 of 45 (62.2%) hap(+) strains, HMW1/2 was detected in 5 of 7 (71.4%) hmw(+) strains, and Hia was detected in 31 of 45 (68.8%) hia(+) strains. The hif gene cluster was present in 26 of 53 strains (49.1%), and 21 of 26 hif(+) strains (80.8%) agglutinated (HA) red blood cells. Nine isolates exhibited HA but lacked the hif gene cluster. The distribution of adhesin genes correlated with the genetic relatedness of the strains. Strains belonging to one type a clonotype and the major type e clonotype possessed hia but lacked the hif cluster. Strains belonging to the second type a clonotype possessed both hia and hif genes. All type f strains belonging to the major type f clonotype possessed hia and lacked hifB. Although the specific complement of adhesin genes in non-type b encapsulated H. influenzae varies, most invasive strains express Hap and Hia, suggesting these adhesins may be especially important to the virulence of these organisms.     

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.     

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.     

Low rate of nasopharyngeal carriage and high rate of ampicillin resistance for Haemophilus influenzae among healthy children younger than 5 years old in northern Taiwan.

BACKGROUND AND PURPOSE: Surveillance data of colonization by Haemophilus influenzae in Taiwan are lacking. This study aimed to define the nasopharyngeal carriage rate of H. influenzae among children younger than 5 years in northern Taiwan, and to determine the antibiotic susceptibility, serotype and the clonal relationship of these isolates. METHODS: Nasopharyngeal specimens were obtained from 511 healthy children younger than 5 years. All H. influenzae isolates were serotyped. The minimal inhibitory concentrations for various antibiotics were determined. Pulsed-field gel electrophoresis (PFGE) was used for clonal analysis. RESULTS: Among 511 children, 269 (52.6%) had been vaccinated with at least one dose of H. influenzae type b (Hib) conjugate vaccine, 236 (46.2%) were unvaccinated and 6 (1.2%) had no vaccination records available. Twenty six H. influenzae strains were isolated. There were three Hib isolates and the others were nontypeable H. influenzae (NTHi). The carriage rate for Hib was 0.6% (3/511) and of NTHi was 5% (23/511). Three (1.27%) of the 236 unvaccinated children were carriers of Hib, whereas none of the 269 vaccinated children carried Hib. Two out of the three Hib isolates and 14 (60.9%) of 23 NTHi isolates were ampicillin-resistant. Multidrug resistance was found in 7 (26.9%) of the isolates. Among the isolates, 61.5% were beta-lactamase producers; there were no beta-lactamase-negative ampicillin-resistant isolates. The PFGE restriction patterns showed a wide diversity of genotypes. CONCLUSIONS: There is very low nasopharyngeal carriage of Hib among children younger than 5 years in northern Taiwan. This may explain why the incidence of invasive Hib disease is also low in Taiwan. In addition, we found a high prevalence of beta-lactamase-positive ampicillin-resistant nasopharyngeal H. influenzae isolates.     

Capsule gene analysis of invasive Haemophilus influenzae: accuracy of serotyping and prevalence of IS1016 among nontypeable isolates

We evaluated the accuracy of serologic capsule typing by analyzing capsule genes and related markers among invasive Haemophilus influenzae isolates before and after the introduction of H. influenzae serotype b (Hib) conjugate vaccines. Three hundred and sixty invasive H. influenzae isolates were collected as part of Active Bacterial Core surveillance within the Georgia Emerging Infections Program between 1 January 1989 and 31 July 1998. All isolates were biotyped, serotyped by slide agglutination serotyping (SAST), and evaluated using PCR capsule typing. Nontypeable H. influenzae (NTHi) isolates were probed with Hib cap-gene-containing plasmid pUO38 and with IS1016; a subset was examined with phosphoglucose isomerase (pgi) genotyping and pulsed-field gel electrophoresis (PFGE). Discrepancies between SAST and PCR capsule typing were found for 64/360 (17.5%) of the isolates; 48 encapsulated by SAST were NTHi by PCR, 8 NTHi by SAST were encapsulated by PCR, 6 encapsulated by SAST were a different capsule type by PCR, and 2 encapsulated by SAST were capsule-deficient Hib variants (Hib-minus). None of the PCR-confirmed NTHi isolates demonstrated homology with residual capsule gene sequences; 19/201 (9.5%) had evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. The majority of IS1016-positive NTHi were biotypes I and V and showed some genetic relatedness by PFGE. In conclusion, PCR capsule typing was more accurate than SAST and Hib-minus variants were rare. IS1016 was present in 9.5% of NTHi isolates, suggesting that this subset may be more closely related to encapsulated organisms. A better understanding of NTHi may contribute to vaccine development.     

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.     

Identification of new genetic regions more prevalent in nontypeable Haemophilus influenzae otitis media strains than in throat strains

Nontypeable (NT) Haemophilus influenzae strains cause significant respiratory illness and are isolated from up to half of middle ear aspirates from children with acute otitis media. Previous studies have identified two genes, lic2B and hmwA, that are associated with NT H. influenzae strains isolated from the middle ears of children with otitis media but that are not associated with NT H. influenzae strains isolated from the throats of healthy children, suggesting that they may play a role in virulence in otitis media. In this study, genomic subtraction was used to identify additional genetic regions unique to middle ear strains. The genome of NT H. influenzae middle ear strain G622 was subtracted from that of NT H. influenzae throat strain 23221, and the resultant gene regions unique to the middle ear strain were identified. Subsequently, the relative prevalence of the middle ear-specific gene regions among a large panel of otitis media and throat strains was determined by dot blot hybridization. By this approach, nine genetic regions were found to be significantly more prevalent in otitis media strains. Classification tree analysis of lic2B, hmwA, and the nine new potential otitis media virulence genes revealed two H. influenzae pathotypes associated with otitis media.     

Identification of the lipooligosaccharide biosynthesis gene lic2B as a putative virulence factor in strains of nontypeable Haemophilus influenzae that cause otitis media

Nontypeable (NT) strains of Haemophilus influenzae are an important cause of acute otitis media (OM). The pathogenic process by which NT H. influenzae strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT H. influenzae middle ear isolate G622 against H. influenzae strain Rd was conducted and the resulting subtraction products were used to screen a panel of H. influenzae isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene lic2B (originally identified in an H. influenzae type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The lic2B gene cloned from NT H. influenzae strain G622 was 99% identical at the amino acid level to that of the H. influenzae type b strain RM7004. The lic2B gene was used to screen a larger panel of H. influenzae isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT H. influenzae throat isolates from children attending day care, and 32 NT H. influenzae nasopharyngeal clinical isolates. The lic2B gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT H. influenzae gene is associated with OM.     

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.     

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.     

Naturally Acquired HMW1- and HMW2-Specific Serum Antibodies in Adults and Children Mediate Opsonophagocytic Killing of Nontypeable Haemophilus influenzae

The HMW1 and HMW2 proteins are highly immunogenic adhesins expressed by approximately 75% of nontypeable Haemophilus influenzae (NTHi) strains, and HMW1- and HMW2-specific antibodies can mediate opsonophagocytic killing of NTHi. In this study, we assessed the ability of HMW1- and HMW2-specific antibodies in sera from healthy adults and convalescent-phase sera from children with NTHi otitis media to mediate killing of homologous and heterologous NTHi. The serum samples were examined pre- and postadsorption on HMW1 and HMW2 affinity columns, and affinity-purified antibodies were assessed for ability to mediate killing of homologous and heterologous strains. Adult serum samples mediated the killing of six prototype NTHi strains at titers of <1:10 to 1:1,280. HMW1- and HMW2-adsorbed sera demonstrated unchanged to 8-fold decreased opsonophagocytic titers against the homologous strains. Each affinity-purified antibody preparation mediated the killing of the respective homologous strain at titers of <1:10 to 1:320 and of the five heterologous strains at titers of <1:10 to 1:320, with most preparations killing most heterologous strains to some degree. None of the acute-phase serum samples from children mediated killing, but each convalescent-phase serum sample mediated killing of the infecting strain at titers of 1:40 to 1:640. HMW1- and HMW2-adsorbed convalescent-phase serum samples demonstrated ≥4-fold decreases in titer. Three of four affinity-purified antibody preparations mediated killing of the infecting strain at titers of 1:20 to 1:320, but no killing of representative heterologous strains was observed. HMW1- and HMW2-specific antibodies capable of mediating opsonophagocytic killing are present in the serum from normal adults and develop in convalescent-phase sera of children with NTHi otitis media. Continued investigation of the HMW1 and HMW2 proteins as potential vaccine candidates for the prevention of NTHi disease is warranted.     

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.     

Epidemiology and evolution of antibiotic resistance of Haemophilus influenzae in children 5?years of age or less in France, 2001–2008: a retrospective database analysis

Trends in the evolution of antimicrobial resistance and mechanisms of resistance of Haemophilus influenzae to β-lactam antibiotics in France were assessed through a retrospective database review. The antimicrobial resistance of 2,206?H. influenzae strains from children aged ≤5 years was studied between 2001 and 2008. Strains were isolated from blood or cerebrospinal fluid (n?=?170), bronchial secretions (n?=?188), middle ear fluid, and nasopharynx or conjunctiva (n?=?1,848). A proportion of 95.1?% (n?=?2,097) were non-typeable H. influenzae (NTHi). β-lactamase production was identified in 27.5?% of NTHi isolates (all TEM-1), while β-lactamase-negative ampicillin resistance and β-lactamase-negative amoxicillin-clavulanate resistance among NTHi was 16.9 and 6.4?%, respectively. Over time, a statistically significant decrease in β-lactamase-producing strain prevalence (p?相似文献   

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.     

Molecular epidemiology of Haemophilus influenzae type b in the Gambia.

One hundred two invasive and 64 noninvasive isolates of Haemophilus influenzae were collected in the course of a 2-year prospective field study on the epidemiology of H. influenzae meningitis in The Gambia. The isolates were serotyped, biotyped, and subtyped by outer membrane protein (OMP) profile analysis (OMP subtyping). H. influenzae meningitis was found to be caused by serotype b (95%). In invasive disease, serotype a, although present in the throat of healthy children, caused only occasionally (5.9%) disease. The distribution of biotypes of H. influenzae appeared to be very similar to that found outside The Gambia. A distinct pattern of OMP subtypes, different from other parts of the world, is prevalent in H. influenzae type b (Hib) in The Gambia. OMP subtypes 2, 4, 5, 8, and 9 were observed to be predominant. These subtypes, except subtype 2, have not been described. L subtypes (subtypes 2, 4, and 8) were associated with invasive disease, whereas non-L subtypes (subtypes 5 and 9) were found more often in healthy carriers (P less than 0.001). A significant difference in geographical distribution was found in subtypes of noninvasive Hib strains (P less than 0.05). We conclude that in The Gambia H. influenzae invasive disease is caused mainly by type b strains with a limited number of OMP subtypes, which are different from the subtypes found elsewhere in the world. These data are important for the surveillance of Hib disease in developing countries and are baseline data for a Hib polyribosyl-ribitolphosphate-conjugated vaccine trial in The Gambia. Alternative Hib OMP vaccines should include a set of representative OMPs.     

Clonal spread of an invasive strain of Haemophilus influenzae type b among nursery contacts accompanied by a high carriage rate of non-disease-associated strains

Haemophilus influenza carriage was examined in unvaccinated nursery contacts of a patient with H. influenzae type b (Hib) meningitis and isolates were typed by pulsed-field gel electrophoresis (PFGE). Nasopharyngeal isolates were classified into eight PFGE patterns. Seven Hib carriers were found among 15 nursery contacts. The isolates from the carriers showed a PFGE pattern identical to that of the meningitis strain. The carrier rate of non-disease-associated strains was also high (47%, 7 of 15). This study suggests that the clonal spread of invasive (serotype b) H. influenzae strains is accompanied by a high carriage rate of non-disease-associated strains.     

Characteristics of Haemophilus influenzae type b responsible for meningitis in Poland from 1997 to 2004

Two hundred forty-five H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of beta-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.     

Localization of high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae by immunoelectron microscopy.

A family of high-molecular-weight (HMW) surface-exposed proteins important in the attachment of nontypeable Haemophilus influenzae (NTHi) to human epithelial cells was previously identified (J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). In the present investigation, indirect immunogold labeling and electron microscopy were used to localize these proteins on three clinical isolates of NTHi, mutants deficient in expression of one or both HMW proteins, and embedded sections of human oropharyngeal cells after incubation with NTHi strain 12. The filamentous material comprising the proteins was labeled with monoclonal antibodies directed against two prototype HMW proteins (HMW1 and HMW2) of prototype NTHi strain 12. Gold labeling was observed as a cap or discrete aggregate off one pole or centrally along one long axis of the bacterial cell. Heavily labeled, non-bacterial-cell-associated, disk-like aggregates of the HMW proteins were frequently noted in both bacterial preparations as well as in association with the oropharyngeal cell surface and intracellularly. Mutants demonstrated diminished labeling or an absence thereof, respectively, which correlated well with their previously demonstrated reduced ability or inability to adhere to Chang conjunctival epithelial cells in vitro. The Haemophilus HMW proteins share antigenic determinants with and demonstrate amino acid sequence similarity to the filamentous hemagglutinin protein of Bordetella pertussis, a critical adhesin of that organism. The studies presented here demonstrate that the Haemophilus proteins and B. pertussis filamentous hemagglutinin show impressive morphologic and perhaps additional functional similarity.     

Emergence of nonencapsulated and encapsulated non-b-type invasive Haemophilus influenzae isolates in Portugal (1989-2001)

Phenotypes and genetic relatedness of invasive Haemophilus influenzae strains were evaluated from 1989 through 2001. Among 119 isolates, multidrug resistance decreased (from 50 to 0%), the level of H. influenzae serotype b (Hib) strains declined (from 81 to 16%), the level of noncapsulated strains rose (from 19 to 80%), and the first invasive H. influenzae serotype f strain was described. This study documents changes in invasive H. influenzae infections in Portugal, i.e., the emergence of non-type-b strains that are genetically diverse and unrelated to Hib.