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An unusual case of beta-lactamase positive Haemophilus influenzae type b infection is reported. Clinical manifestations included meningitis, a left ankle subcutaneous abscess, and bilateral hand cellulitis. Discussion and review of literature are presented for the previously unreported association of this common childhood pathogen.
Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x 10(-4) to 1 x 10(-6). The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.
Haemophilus influenzae type b is a rare cause of adult lobar pneumonia and we are unaware of any reported British cases. However in the United States this condition is well described and the incidence is increasing (Levin et al. 1977).
Haemophilus influenzae type b was more resistant to killing by lipopolysaccharide (LPS) antibody and complement after growth in defined medium than in conventional broths. Resistance correlated with decreased binding of LPS antibody, as determined by whole-cell enzyme-linked immunosorbent assay. An inhibition radioimmunoassay was used to determine that bacteria grown in defined medium contained about 2.5 times more capsule than bacteria grown in conventional broth. No major differences were noted in the electrophoretic patterns of outer membrane proteins or LPS. The defined medium did not increase the resistance of a capsule-deficient mutant. Resistance and increased encapsulation could be reproduced after growth in conventional broth supplemented with magnesium, glutamic acid, and aspartic acid. Thus, the growth medium may influence the content of capsule on H. influenzae type b, and may in turn, influence the binding and bactericidal activity of LPS antibody to the cells.
Haemophilus influenzae type b acquires transferrin-bound iron via a siderophore-independent mechanism involving direct contact between the human iron-binding glycoprotein and the bacterial cell surface. Evidence has accumulated to show that the transferrin receptor consists of at least two iron-regulated outer membrane transferrin-binding proteins (TBPs), of which one has a molecular mass of around 100 kDa (TBP1) and the other has a molecular mass of 60 to 90 kDa (TBP2). In H. influenzae type b strain Eagan, proteins of 76, 90, and 107 kDa appear to be involved in transferrin binding. To determine whether these TBPs are expressed during growth in vivo, strain Eagan was recovered without subculture from the intraperitoneal cavities of infected infant rats. By using a dot blot assay, outer membranes prepared from these in vivo-grown bacteria, unlike those grown in iron-sufficient broth, bound human transferrin and produced the 76-, 90-, and 107-kDa TBPs. Immunoblotting experiments using convalescent sera from infected rats also revealed the presence of antibodies to the 76- and 90-kDa strain Eagan TBPs. In addition, convalescent sera from three of four patients recovering from H. influenzae type b meningitis contained antibodies to the 90- and 105-kDa TBPs of the corresponding infecting strain. Furthermore, fresh clinical isolates of H. influenzae type b recovered from blood and cerebrospinal fluid showed constitutive expression of the TBPs, which became iron regulated only after prolonged in vitro subculture on iron-sufficient medium. This contrasted with the laboratory-adapted Eagan strain, in which the TBPs remained iron regulated even after animal passage. These findings indicate that the H. influenzae type b transferrin receptor is expressed during experimental animal and human infections.
Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.
Haemophilus influenzae type b is the leading cause of meningitis in children younger than 5 years of age in the United States. The incidence of infection with H influenzae type b in certain populations, such as Apache and Navajo Indians and Alaskan Eskimos, is 10 to 20 times higher than in the general US population. Another important feature of H influenzae type b infections in these populations is that more than 80% of the cases occur during the first year of life, with 35% to 45% occurring during the first 6 months. One of the currently licensed vaccines that contains the capsular polysaccharide of the H influenzae type b organism is not reliably immunogenic in infants younger than 18 months of age. A number of new H influenzae type b vaccines prepared by covalently coupling the H influenzae type b capsular polysaccharide with a protein carrier antigen are undergoing clinical evaluation. One of these conjugate vaccines was shown to be efficacious in preventing disease caused by H influenzae type b in Finnish infants when they were immunized at 3, 4, and 6 months of age. Unfortunately, in a recently concluded trial, the same vaccine was not found to be efficacious in preventing such disease in infants younger than 1 year of age among the Alaskan Eskimo population. We have evaluated an alternative approach for protecting high-risk infants.(ABSTRACT TRUNCATED AT 250 WORDS)
Haemophilus influenzae type b is an encapsulated bacterium that initiates infection by colonizing the upper respiratory epithelium. In vitro studies indicate that H. influenzae type b is capable of expressing two morphologically distinct filamentous adhesive structures, referred to as pili and fibrils, respectively. In this study, we examined adherence to a variety of human epithelial-cell types and demonstrated that pili and fibrils have separate cellular binding specificities. In addition, we found that capsular material inhibits fibril recognition of the host-cell surface. This inhibitory effect was reduced when bacteria were grown to stationary phase, reflecting diminished encapsulation. However, when growth medium was supplemented with Mg2+, stationary-phase organisms were relatively heavily encapsulated and non-adherent. These observations suggest that encapsulation can be modulated in response to growth phase or environmental signals. It is possible that encapsulation is down-modulated early in the infectious process in order to avoid interfering with colonization. In contrast, encapsulation may be up-modulated between hosts and during bacteremia, where it appears to confer a selective advantage. We speculate that this model may also apply to other encapsulated pathogens.
Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) induces meningitis in an established rabbit model of experimental meningitis. To evaluate the inflammatory ability of Hib LOS in its native state as part of outer membrane of Hib, the CSF inflammatory response to Hib outer membrane vesicles (OMV) was compared with that produced by Hib LOS. A significant dose-dependent meningeal inflammatory response, as evidence by increased concentrations of white blood cells and protein and lactate concentration in CSF, was found after intracisternal injection of Hib OMV containing 0.02-200 ng of LOS. On a LOS weight basis, purified LOS and LOS in OMV did not differ significantly with regard to the CSF inflammatory response. Preincubation of Hib OMV with monoclonal antibodies directed against cell surface-exposed epitopes on Hib LOS did not affect the inflammatory ability of OMV, whereas preincubation of OMV with polymyxin B significantly reduced both pleocytosis and lactate concentration. Thus, Hib OMV represents a relevant nonreplicating vehicle in which Hib LOS might interact with CNS tissues to produce meningitis.
Haemophilus influenzae type b strains deposited in the National Reference Collections of the United States, United Kingdom and Sweden are derived from strains isolated in the United States during the 1940s. With respect to both the genetic locus for capsulation and to virulence, assessed in the infant rat bacteremia model, these strains are no longer representative of clinical isolates which currently cause meningitis and other serious infections in children all over the world. Alternative well-characterized strains resembling current common clinical isolates may often be more suitable for the study of Haemophilus influenzae pathogenicity.
Haemophilus influenzae type b, a causative agent of bacterial sepsis and meningitis in young children, contains a single superoxide dismutase (SOD), a cytoplasmic MnSOD. To study the role of this enzyme, a chromosomal sodA::lacZ mutant (M-2) was constructed. M-2 had an increased sensitivity towards oxygen and the redox-active agent paraquat. A 3.4-fold increase in sodA-lacZ expression was found in M-2 grown with oxygen supply rates between 3 and 36 mmol of O2/liter/h. In similar experiments with the wild type, assaying SodA activity, a 3.1-fold increase was found. Both the wild type and M-2 grew best at the lowest oxygen supply rate tested, consistent with the notion that H. influenzae prefers a more anaerobic environment. In the infant rat model of infection, the ability of M-2 to colonize the nasopharynx was found to be impaired, but its ability to cause invasive disease was unaffected. This suggests that after invasion, the growth disadvantage imposed by a SodA- phenotype is not limiting.
Haemophilus influenzae type B, strain W-2, is highly resistant to ampicillin (MIC, 12.5 mug/ml). The ampicillin resistance of strain W-2 was transferred to an antibiotic-sensitive strain TF-2 (RifR, SmR) during mixed incubation on membrane filters at 36 C(transfeer frequency, 4.6 times 10(-5) per donor). Resistance was also transferred from the primary recipient to a secondary one (TF-3, EryR, NovR). The transfer frequency between these derivative strains was 10(-4) after incubation for 30 min. Resistance in strain W-2 remained even after growth in the presence of ethidium bromide or at an elevated temperature, although ampicillin resistance was lost from 13%-25% of transcipient cells after growth in broth. Strain W-2 and transcipients of ampicillin resistance had equivalent levels of beta-lactamase activity, while sensitive segregants and recipient strains demonstrated little or no enzyme activity. Transfer of ampicillin resistance between strains of H. influenzae is probably mediated by conjugation since transfer (1) requires cell-to-cell contact, (2) remains unchanged in the presence of DNase I, and (3) occurs in the absence of demonstrable bacteriophage.
BACKGROUND: As a result of the decline in Haemophilus influenzae type b (Hib) disease caused by the widespread use of conjugate vaccines, non-type b H. influenzae will become a more important cause of H. influenzae (Hi) disease. Characterization of the clinical and epidemiologic features of non-b Hi disease is needed in the Hib vaccine era. METHODS: A prospective active surveillance study of invasive Hi disease involving pediatricians in the United Kingdom and Republic of Ireland. For the first phase of the study (October 1, 1992, to October 31, 1995) pediatricians were asked to report any child who had invasive Hi disease and who had received Hib conjugate vaccine. For the second phase of the study (November 1, 1995. To December 31, 1998) pediatricians were asked to report any child with invasive Hi disease regardless of vaccination status. RESULTS: During the study period 102 cases of invasive non-type b Hi disease and 106 cases of invasive Hib disease were reported in children who had been fully vaccinated against Hib. Children with non-type b disease were younger (16 vs. 22 months of age, P = 0.08), less likely to have meningitis and epiglottitis (P < or = 0.001) and more likely to have pneumonia and bacteremia (P < or = 0.001) than children with type b disease. For the last 2 years of the study invasive Hi disease occurring in a fully vaccinated child was more likely to be caused by a non-b strain than by a type b strain (58 vs. 38). In 1998 the incidence of non type-b Hi disease in all children <5 years of age in the UK was 1.3/100,000 as compared with an incidence of Hib disease of 0.6/100,000. The majority (88%) of non-b strains isolated in children were nontypable strains. CONCLUSIONS: Non-b Hi is a rare cause of disease in children, but in the Hib vaccine era it has become more common than type b as a cause of Hi disease in fully vaccinated children.
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.
Haemophilus influenzae infections are preceded by airway colonization, a process facilitated by fimbriae. Here, we identified the complete fimbrial gene cluster of H. influenzae type b. HifA forms the major subunit. HifB, a periplasmic chaperone, and HifC, an outer membrane usher, are typical assembly genes; their inactivation abolished fimbriae formation. HifD and HifE are putative minor subunits, both participating in fimbriae biogenesis. Inactivation of either one drastically reduced fimbriae expression. HifD represents a novel type of fimbrial subunit with lipoprotein characteristics, pointing to a membrane-associated function of HifD. Transcription of all fimbrial genes is coregulated through two clustered promoters. The flanking of the fimbrial gene cluster by repetitive extragenic palindromic sequences together with a partial duplication of an adjacent unrelated operon indicated that the cluster was once inserted in the H. influenzae genome as a mobile virulence unit.
Haemophilus influenzae is a heme-dependent bacterium. However, little is known of the heme-iron uptake mechanism in this organism. By using a batch ligand affinity chromatography method, a hemin-binding protein of 39,500 molecular weight was isolated from total membranes derived from H. influenzae type b grown under iron-depleted but not under iron-sufficient conditions. Detection of the hemin-binding protein in a whole-cell binding assay demonstrated a surface-exposed location. Competition binding experiments indicated that this hemin-protein interaction was specific, since only hemin or heme-containing proteins, such as human hemoglobin and bovine catalase, but not protoporphyrin IX, iron-loaded human lactoferrin, or transferrin, could abrogate binding. In a limited survey of other H. influenzae strains, an identical hemin-binding protein was isolated, implying that this polypeptide may be structurally and functionally conserved among strains.
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.
Haemophilus influenzae has been a major cause of infectious diseases in children and has been attributed as a significant cause of septic arthritis and osteomyelitis in children. With the advent of widespread vaccination, the incidence of Haemophilus influenzae meningitis and other infections has been well documented. This is thought to be the first report that documents the effect of vaccination on bone and joint infections. One hundred sixty-five cases of acute hematogenous osteomyelitis or septic arthritis treated at the Department of Orthopaedics at Vanderbilt University in the years before and after the advent of the Haemophilus influenzae vaccine to assess whether vaccination affected the incidence of these diseases. The data indicate that the Haemophilus influenzae vaccine has reduced to near 0 the incidence of bone and joint infections because of Haemophilus influenzae. These findings suggest that coverage of Haemophilus influenzae as part of the empiric antibiotic coverage may be no longer needed in the management of acute hematogenous osteomyelitis and septic arthritis in children.