Complex role of hemoglobin and hemoglobin-haptoglobin binding proteins in Haemophilus influenzae virulence in the infant rat model of invasive infection

Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.     

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.     

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.     

A mutation in the sap operon attenuates survival of nontypeable Haemophilus influenzae in a chinchilla model of otitis media

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla beta-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.     

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.     

Protein sources of heme for Haemophilus influenzae.

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.     

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.     

Experimental otitis media in gerbils and chinchillas with Streptococcus pneumoniae, Haemophilus influenzae, and other aerobic and anaerobic bacteria.

To ascertain the usefulness of Mongolian gerbils as an inbred model for otitis media, 52 Mongolian gerbils (Meriones unguiculatus, strain MONT/Tum) were compared with 26 chinchillas (Chinchilla laniger) for susceptibility to Streptococcus pneumoniae type 3. Haemophilus influenzae type b, and a polymicrobic culture including anaerobes (Streptococcus intermedius, Propionibacterium acnes, Staphylococcus epidermidis, and Corynebacterium sp.). Organisms were inoculated percutaneously into the superior chamber of the middle ear bulla. The gerbils and chinchillas shared similar susceptibilities and responses to the inoculated organisms as determined by X-ray, otoscopic, histopathological, and microbiological determinations at 5 to 7 days. Koch's postulate studies proved the role of S. pneumoniae and H. influenzae in the pathology found in both animal models. The animals were also susceptible to the polymicrobic culture, although the relative virulence of the individual members of this mixture was low, suggesting that these species potentiated as a polymicrobic mixture. The Corynebacterium sp. appeared to elicit the greatest histopathological response in chronic (8-week) studies in gerbils. The gerbils were found to be useful as an alternative animal model for the study of otitis media of bacterial etiology.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

Haemophilus influenzae luxS mutants form a biofilm and have increased virulence

To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5alpha expressing a cloned luxSHi contained 350-fold more AI-2 activity per cell than E. coli DH5alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxSHi mutants. Two clinical H. influenzae and their luxSHi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxSHi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxSHi mutants tended to be more invasive. The luxSHi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.     

Determination of the epidemiology and transmission of nontypable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints.

It is assumed that the causative bacteria in children suffering from otitis media reach the middle ear via the eustachian tube. The purpose of this investigation was to use endonuclease restriction of bacterial chromosomal DNA to compare isolates of nontypable (NT) Haemophilus influenzae obtained from the nasopharynx and from middle ear (ME) effusions of patients with otitis media. Strains of NT H. influenzae were isolated from the nasopharynx (NP) and affected ME from a group of 13 unrelated children with otitis media with effusion (OME). For 12 of these children, identical strains were isolated from the NP and ME in a first episode of OME. Each of these 12 sets differed from the other 11. Six of these children suffered from a second episode of OME with NT H. influenzae. Five of these children with recurrence again had identical NP and ME strains. These results suggest that at the time of an episode of OME, there is one predominant strain of NT H. influenzae that colonizes both the NP and ME. The strains of NT H. influenzae isolated from all six of the second episodes were different from strains from the first episode, indicating turnover of the predominant strain in the NT H. influenzae population between episodes. When we investigated three siblings with concurrent episodes of OME, we found that they shared several similar strains of NT H. influenzae, thereby demonstrating that within a family, transmission of NT H. influenzae from child to child is possible. These results from DNA fingerprinting were essentially identical when compared with results from outer membrane protein subtyping performed on the same set of strains. The analysis of endonuclease restriction patterns of total genomic DNA provides a sensitive measure of genetic dissimilarity between strains and represents an easily applicable method for epidemiological and transmission studies of bacterial infections associated with NT H. influenzae.     

A mouse model for acute otitis media

To induce acute otitis media in the mouse and to describe the clinical and bacteriological course of the infection, middle ears of BALB/c, Swiss-Webster and C57BL/6 mice were inoculated with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Systemic and local changes were monitored by clinical observations, otomicroscopy, and analysis of bacterial samples from blood and middle ears. Agglutination of mouse erythrocytes by M. catarrhalis was also tested. Depending on bacterial strain, bacterial dose, and mouse strain three responses were identified: acute otitis media, otitis media with serous effusion, or no reaction. BALB/c mice were the most susceptible animals. On day 3, 76% of the BALB/c mice had developed middle ear infection, 50% had a positive middle ear culture, 56% were bacteremic, and 10% had succumbed to a disseminated infection. The local infections lasted approximately a week. Animals which survived recovered without permanent deterioration or otomicroscopically discernible changes. In no case did M. catarrhalis induce a culture-positive middle ear infection, possibly due to an inability to agglutinate the mouse erythrocytes. The mouse model can become a useful tool in studies of pneumococcal and H. influenzae-induced otitis media, but the bacterial dose has to be carefully titrated and adjusted to the chosen mouse strain.     

Role of p38 mitogen-activated protein kinase in middle ear mucosa hyperplasia during bacterial otitis media

Hyperplasia of the middle ear mucosa contributes to the sequelae of acute otitis media. Understanding the signal transduction pathways that mediate hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. Endotoxin derived from bacteria involved in middle ear infection can contribute to the hyperplastic response. The p38 mitogen-activated protein kinase (MAPK) is known to be activated by endotoxin as well as cytokines and other inflammatory mediators that have been documented in otitis media. We assessed the activation of p38 in the middle ear mucosa of an in vivo rat bacterial otitis media model. Strong activity of p38 was observed 1 to 6 h after bacterial inoculation. Activity continued at a lower level for at least 7 days. The effects of p38 activation were assessed using an in vitro model of rat middle ear mucosal hyperplasia in which mucosal growth is stimulated by nontypeable Haemophilus influenzae during acute otitis media. Hyperplastic mucosal explants treated with the p38 alpha and p38 beta inhibitor SB203580 demonstrated significant inhibition of otitis media-stimulated mucosal growth. The results of this study suggest that intracellular signaling via p38 MAPK influences the hyperplastic response of the middle ear mucosa during bacterial otitis media.     

Genomic subtraction followed by dot blot screening of Streptococcus pneumoniae clinical and carriage isolates identifies genetic differences associated with strains that cause otitis media

Streptococcus pneumoniae strains are the leading cause of bacterial otitis media, yet little is known about specific bacterial factors important for this disease. We utilized a molecular epidemiological approach involving genomic subtraction of the S. pneumoniae serogroup 19 middle ear strain 5093 against the laboratory strain R6. Resulting subtraction PCR (sPCR) products were used to screen a panel of 93 middle ear, 90 blood, 35 carriage, and 58 cerebrospinal fluid isolates from young children to identify genes found more frequently among middle ear isolates. Probe P41, similar to a hypothetical protein of Brucella melitensis, occurred among 41% of middle ear isolates and was found 2.8 (95% confidence interval [CI], 1.32 to 6.5), 3.3 (95% CI, 1.9 to 5.7), and 1.8 (95% CI, 1.1 to 3.0) times more frequently among middle ear strains than carriage, blood, or meningitis strains, respectively. sPCR fragment H10, similar to an unknown Streptococcus agalactiae protein, was present in 31% of middle ear isolates and occurred 3.6 (95% CI, 1.2 to 11.2), 2.8 (95% CI, 1.5 to 5.4), and 2.6 (95% CI, 1.2 to 5.5) times more often among middle ear isolates than carriage, blood, or meningitis strains, respectively. These studies have identified two genes of potential importance in otitis media virulence. Further studies are warranted to outline the precise role of these genes in otitis media pathogenesis.     

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin–haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.     

Protection by serum antibodies in experimental nontypable Haemophilus influenzae otitis media.

The chinchilla experimental model of otitis media was used to examine the importance of serum antibodies in protection against disease caused by nontypable Haemophilus influenzae. An immune serum pool was prepared by immunizing chinchillas with killed bacterial cells of nontypable H. influenzae 3245. Pooled preimmune or immune serum from these immunized animals was administered intravenously to a group of nonimmune chinchillas 1 day before intrabullar challenge with strain 3245. Of 5 animals receiving preimmune serum, 5 developed otitis media compared with 0 of 10 animals receiving immune serum (P = 0.008). The immune serum pool contained antibodies directed against both surface-exposed outer membrane proteins and lipopolysaccharide (LPS). The 39-kilodalton major outer membrane protein was the immunodominant surface protein. Anti-LPS antibodies were removed from the immune serum pool by affinity chromatography, and affinity-purified anti-LPS antibodies were recovered. Immune serum, immune serum absorbed of LPS antibodies, or affinity-purified LPS antibodies were then administered to another group of experimental animals 1 day before bacterial challenge. Of four animals that received the affinity-purified LPS antibodies, four developed otitis compared with zero of four animals that received the immune serum or zero of four animals that received the LPS-absorbed immune serum (P = 0.028). These studies indicate that passive immunization with immune serum is protective in experimental nontypable H. influenzae otitis media and that bacterial outer membrane proteins may be the principal targets of protective antibody.     

Role of sialic acid and complex carbohydrate biosynthesis in biofilm formation by nontypeable Haemophilus influenzae in the chinchilla middle ear

Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.     

A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b.

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.     

Detection rates of bacteria in chronic otitis media with effusion in children

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.