Prevalence and serotype distribution of encapsulated Haemophilus influenzae isolates from patients with lower respiratory tract infections in Poland

Among 1,272 H. influenzae isolates obtained from patients with lower respiratory tract infections, 5.6% were found to be encapsulated. The most common were serotypes b (40.3%) and e (38.9%) followed by serotypes f (16.7%) and d (4.1%). All encapsulated isolates within a particular serotype showed high genetic homogeneity and did not produce beta-lactamases.     

Binding of Haemophilus influenzae to purified mucins from the human respiratory tract.

Mucins are high-molecular-weight glycoproteins and major constituents of the mucus layer which covers the airway surface. We have studied the interactions between bacteria, mucins, and epithelial cells from the human respiratory tract. Nontypeable strains of Haemophilus influenzae were found to bind to purified airway mucins in suspension and on solid phase. Mucins in suspension inhibited the attachment of these strains to nasopharyngeal epithelial cells, while mucin coating of the cells enhanced their binding. In contrast, strains of Streptococcus pneumoniae and encapsulated and other nontypeable H. influenzae strains failed to interact with mucins. These H. influenzae strains used other strategies for adherence to epithelial cells. The type b strain 770235 attached via fimbriae but also expressed a subcapsular adhesin that was detected in a capsule- and fimbria-defective mutant. Mucin pretreatment of these bacteria did not inhibit adherence, but mucin pretreatment of epithelial cells inhibited adherence, probably by shielding of the receptors for these adhesins. Non-mucin-binding nontypeable and encapsulated H. influenzae strains would, therefore, adhere only after disruption of the mucus layer and exposure of cellular receptors. Differences in tissue toxicity and invasiveness among H. influenzae strains may also be influenced by the mucin interactions of the strains.     

Comparative molecular analysis of Haemophilus influenzae isolates from young children with acute lower respiratory tract infections and meningitis in Hanoi, Vietnam

Thirty-seven Haemophilus influenzae strains from nasopharyngeal swabs (NP) and 44 H. influenzae strains from cerebrospinal fluid (CSF) were investigated. Of the 37 H. influenzae isolates from NP, the serotypes of 30 isolates were nontypeable, 4 were type b, 2 were type c, and 1 was type a, whereas all of the 44 isolates from CSF were type b. The MICs of 16 antibiotics for the H. influenzae isolates from NP and CSF were similar, and no beta-lactamase-negative ampicillin-resistant strain was found. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that the 37 H. influenzae strains from NP had 22 PFGE patterns, with none predominating, and the 44 H. influenzae strains from CSF had 9 PFGE patterns, with patterns alpha (22 isolates) and beta (12 isolates) predominating. Our results indicate that two predominant types of H. influenzae type b strains have the potential to spread among children with meningitis in Hanoi, Vietnam.     

Comparison of methods for serotyping isolates of Haemophilus influenzae.

This study compared four methods for serotyping isolates of Haemophilus influenzae. Slide agglutination with commercial antisera (Difco Laboratories and Wellcome Diagnostics), coagglutination (Phadebact Haemophilus Test [Pharmacia Fine Chemicals]), latex agglutination with affinity-purified anticapsular antibody, and counterimmunoelectrophoresis with multiple antisera were used to serotype 80 isolates of H. influenzae. Coagglutination and counterimmunoelectrophoresis correctly identified all 80 isolates as either type b or not type b. Slide agglutination and latex agglutination each successfully identified 76 of the 80 isolates; however, each of these two methods failed to type four isolates because of agglutination of controls. We recommend slide agglutination or coagglutination as the serotyping methods of choice in most laboratories because they are simple, accurate, and rapid. Slide agglutination with Difco antiserum can be performed at the lowest cost.     

Selective media for recovery of Haemophilus influenzae from specimens contaminated with upper respiratory tract microbial flora

Isolation of Haemophilus influenzae from specimens contaminated with upper respiratory tract microbial flora was attempted with three different media: enriched chocolate agar, chocolate agar plus vancomycin, and chocolate agar plus vancomycin, bacitracin, and clindamycin. Recovery rates of H. influenzae from 852 pediatric pharyngeal swab specimens were 6.0, 28.5, and 59.9%, respectively.     

Biochemical characteristics of 130 recent isolates from Haemophilus influenzae meningitis.

A total of 130 Haemophilus strains, comprising virtually all isolates from Danish and Norwegian cases of Haemophilus meningitis occurring in the period from October 1975 through September 1976, were examined by biochemical and serological means. All isolates were identified as H. influenzae and, except for one noncapsulated strain, possessed a capsule of serotype b. The vast majority of strains (93%) belonged to biotype I, which, in contrast to biotypes II and III, is rarely encountered as a commensal of the upper respiratory tract. This finding is a strong incentive for studies of possible additional virulence factors associated with biotype I organisms. The results are discussed in the light of North American reports, which have suggested changes in the etiology of Haemophilus meningitis.     

Multi locus sequence type comparison of invasive and commensal Haemophilus influenzae isolates from Delhi

Haemophilus influenzae is a major public health concern in the developing world. The most virulent strain is H. influenzae Type b (Hib). Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. Through dendogram based on composite gene sequences of seven multi locus sequence type genes, it was observed that invasive and commensal isolates made two completely separate clusters which are indicative of independent evolution of these two groups of H. influenzae in the Indian subcontinent.     

Haemophilus influenzae in respiratory pathology in adults

Fifty patients were diagnosed bronchopulmonary Haemophilus infections, because of the production of a purulent sputum, containing at least 10(8) Haemophilus influenzae per ml. Among them were 36 males (average 52 years old) and 14 females (average 58 years old). There was a high percentage (64%) of smokers (over 30 packs/year) within this population, which also included heavy drinkers. The top incidence occurred in winter and spring. Most cases were related to an acute infection in a chronic bronchitis (26 cases). The other cases included 6 cancers, 6 sequelae of tuberculosis, 4 bronchiectasis, 4 asthma, and only 3 pulmonary consolidations. There usually was a low grade fever (only 8 cases reached or went beyond 38 degrees, while in 29 cases the body temperature kept below 38 degrees). The return to a normal temperature was obtained after 4 to 10 days of ampicillin therapy, with no fatal case within this series. The 50 strains were studied by the microbiology laboratory. The minimum inhibitory concentrations showed a peculiar response to ampicillin and erythromycin, and a less dramatic response to chloramphenicol and tetracyclin. Some strains were proved resistant (MIC over 4 micrograms per ml) to cefoxitine and cefamandole.     

Subtype distribution of Haemophilus influenzae isolates from north India

A total of 120 Haemophilus influenzae isolates from blood, cerebrospinal fluid, sputum and throat swabs of patients and carriers in North India was characterised by biotyping, ribotyping and random amplification of polymorphic DNA (RAPD)-PCR. Of these, 77 isolates (64%) were serotype b; the other 43 (36%) were non-typable. Biotype I was the most predominant among the typable strains and biotype II among the non-typable strains. Ribotyping with restriction endonucleases HaeIII and EcoRI differentiated the isolates into three and six ribotypes, respectively. However, RAPD fingerprints generated by the application of arbitrary primers AP1 and AP2 provided a higher level of discrimination. RAPD typing revealed distinct polymorphism among the serologically typable isolates. This study is the first report that stratifies the subtypes of H. influenzae strains from India by molecular techniques.     

Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates.

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein.     

Haemophilus influenzae: antibiotic susceptibility.

Ampicillin resistance was first reported among clinical isolates of Haemophilus influenzae in 1972. Reports of chloramphenicol resistance followed shortly thereafter. The principal mechanism of resistance to these two antibiotics is enzymatic. Although other mechanisms have been described, they are found in comparatively few strains. The genetic information for the inactivating enzymes is plasmid mediated and therefore readily transmissible to susceptible strains. Consequently, effective therapy for invasive disease caused by this pathogen has been seriously compromised. As antibiotic susceptibility became less predictable, in vitro testing became increasingly important. Unfortunately, the standardization of methods for laboratory testing has been slow and complicated by the fastidious nature of the organisms. This review traces the development of antibiotic resistance in H. influenzae, discusses the mechanisms which appear to be important in mediating resistance, explores newer antimicrobial agents which might be useful in the treatment of infection, and analyzes the various approaches to in vitro testing.     

Simple screening method for beta-lactamase-positive and -negative ampicillin-resistant Haemophilus influenzae isolates.

A simple, inexpensive method for screening for beta-lactamase-positive and beta-lactamase-negative ampicillin-resistant Haemophilus influenzae isolates was developed. Disks containing 10 micrograms of cloxacillin yielded no zone of inhibition when placed on chocolate agar plates inoculated with beta-lactamase-positive (16 strains) or ampicillin-resistant (greater than or equal to 1 microgram/ml) beta-lactamase-negative (10 strains) H. influenzae, whereas ampicillin-susceptible (less than or equal to 0.5 microgram/ml; 36 strains) H. influenzae almost always (92%) showed a zone of inhibition.     

Abnormalities in cell-mediated immune functions to Haemophilus influenzae chronic purulent infections of the upper respiratory tract

Delayed hypersensitivity (dh) skin test reactivity to a somatic antigen of Haemophilus influenzae was studied in 21 patients with unexplained, chronically relapsing, purulent upper respiratory tract infections. Only 2 showed a dh reactivity comparable to that of healthy controls. A majority--15 patients--had a defective dh response, whereas 4 showed exaggerated reactivity leading to necrosis of the test site and general feelings of malaise. Not only was the dh reactivity to somatic H. influenzae antigen affected, but also that to streptokinase/streptodornase and candidal antigen in most cases, though to a lesser extent. Skin test reactivity to the mitogen PHA was normal as were the dh skin test reactivities in 4 out of 5 control patients with mucous atopic rhinitis/sinusitis and 2 cases of nasal suppuration due to disturbed mucociliary transport. Delayed hypersensitivity skin test disorders were associated with elevated ratios of OKT4 + /OKT8 + peripheral lymphoid cells. In addition a high incidence of atopy and thyroid autoimmunity was evident in patients as well as in their first-degree relatives. A negative lymphocyte proliferative response to somatic H. influenzae antigen was found in 3 of our patients. These results suggest that unexplained, chronically relapsing upper respiratory tract infections might be based on restricted T-cell defects to H. influenzae, streptococcal, and candidal antigens. Such defects are reminiscent of the T-cell immune disorders to fungi playing a role in some cases of chronic mucocutaneous candidiasis.     

Pilus-mediated adherence of Haemophilus influenzae to human respiratory mucins

Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeable H. influenzae bind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeable H. influenzae to human respiratory mucins. We used isogenic H. influenzae strains with a mutation in the structural gene for pilin (hifA), a laboratory H. influenzae strain transformed with a type b pilus gene cluster (from strain C54), antibodies raised against H. influenzae HifA, and Escherichia coli strains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliated H. influenzae to mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence. E. coli strains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding of H. influenzae to human respiratory mucins.     

Occult bacteremia with nontypeable Haemophilus influenzae.

A 2-year-old boy had occult bacteremia with nontypeable Haemophilus influenzae 6 weeks after receiving H. influenzae type b polysaccharide vaccine. Evaluation of his host defense was normal. As determined by outer membrane protein electrophoresis and Southern hybridization analysis, this strain was not related to type b strains. Its virulence in rats was similar to that of another nontypeable strain and less than that of a type b strain.