Induction of L Forms of Haemophilus influenzae in Culture and Their Demonstration in Human Bronchial Secretions

Transitional forms and round bodies of Haemophilus influenzae were identified in sputa from patients with chronic bronchitis who were receiving penicillin therapy for H. influenzae infections. In vitro growth of L forms of this organism was induced by penicillin and glycine and was studied for comparison with development in vivo. Variant forms demonstrated in sputum were similar to variant forms observed in penicillin-induced L colonies. Recurrence of infection after cessation of therapy was related to reversion of persisting L forms to bacillary forms. That these forms were derived from H. influenzae was established by direct staining with fluoresceinlabeled specific antibody. This demonstration that transitional forms and round bodies of H. influenzae occurred in vivo suggests that L forms of bacteria may be significant in chronic or recurrent infections.     

Culture and morphological characteristics of Haemophilus influenzae and pneumococcus in bronchial infection]

In the cultures obtained by inoculating sputum samples faken from patients with bronchial infection into solid agar medium prepared on Hottinger's hydrolysate with fresh rabbit blood added Haemophilus influenzae produced colonies varying in their from (dome-shaped, conical, trapeziform), as well as in the morphology of the organisms. Pneumococci produced mainly flat colonies surrounded by the zone of alpha hemolysis. Along-side with isolated H. influenzae and pneumococcal colonies, symbiotic colonies could be observed. In these colonies pneumococci were diffused among H. influenzae.     

Selective Culture Medium to Survey the Incidence of Haemophilus Species

A culture medium for the selective isolation of Haemophilus species is described. Bacitracin and nutritional supplements were incorporated in a rich basal agar medium to which rabbit blood was added to distinguish hemolytic species. Colony counts of seven typed strains of H. influenzae on this medium were within practical limits of counts on other media tested for clinical use. The bacitracin medium was as reliable as hemoglobin-agar for detecting H. influenzae and more sensitive for detecting other Haemophilus species in a clinical survey with the advantage of selectivity.     

Infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable Haemophilus influenzae

Infectious exacerbations of chronic obstructive pulmonary disease (COPD) have been reported to occur with both viral and bacterial pathogens. In this study, 35 exacerbations associated with the isolation of non-typeable Haemophilus influenzae from sputum were identified as part of a prospective longitudinal study. Samples from these patients were subjected to immunoassays to identify a new immune response to the homologous isolate of non-typeable H. influenzae to more accurately assess a bacterial etiology. These patients also were studied carefully for evidence of viral infection using viral culture, serology and polymerase chain reaction-based assays. Sixteen of 35 exacerbations (45.7%) were associated with evidence of acute viral infection and 11 of the 35 exacerbations (31.4%) were associated with the development of new serum IgG to homologous non-typeable H. influenzae. Overall, evidence of infection with a respiratory virus or non-typeable H. influenzae was seen in 24 of 35 exacerbations (68.6%). No association between viral infection and immune response to non-typeable H. influenzae was observed, although a trend toward an immune response to non-typeable H. influenzae and absence of viral infection was seen. The results show that exacerbations in adults with COPD were associated with infection caused by virus alone, non-typeable H. influenzae alone, or virus and non-typeable H. influenzae simultaneously.     

Etiology of suppurative bacterial meningitis

The results of the laboratory examination of 2034 patients with meningococcal infection and purulent meningitides, hospitalized during the period of June 1980 to October 1983, revealed that three main etiological agents were responsible for these diseases: meningococci, pneumococci and Haemophilus influenzae. The susceptibility of the patients to different etiological agents was found to depend on their age. Children aged up to 3 years constituted 75% of the patients with meningitis caused by H. influenzae; 50% of the patients with meningococcal infection were children aged up to 5 years; pneumococcal meningitis occurred more frequently in adults. Serogroup A meningococci were found to prevail in patients with meningococcal infection. Besides, in children serogroup C meningococci could be isolated in 24% of cases. Since 1983 the cases of the isolation of strains belonging to serogroup B increased in number. Among the pneumococci responsible for the disease serotypes 1, 19, 6 and in children serotype 12 occurred most frequently.     

Naturally occurring NAD-independent Haemophilus parainfluenzae

Four, NAD-independent, clinical isolates of Haemophilus parainfluenzae were recovered from a genital ulcer, a purulent skin lesion, a sputum specimen and a throat swab respectively. With the exception of NAD requirement, the strains exhibited the biochemical characteristics of H. parainfluenzae biotype II. The genetic relationship between these isolates and a standard strain of H. parainfluenzae was determined by testing transforming activities of two chromosomal markers, streptomycin resistance and nalidixic acid resistance. The clinical isolates were efficient donors and recipients in transformation. In addition, we demonstrated transfer of the genes conferring NAD independence to typical, NAD-requiring H. parainfluenzae and Haemophilus influenzae strains.     

Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients

The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.     

Dynamics of the shedding of bacterial and ultrafine forms of mycobacteria during the chemotherapy of patients with pulmonary tuberculosis

The specific features of bacterial excretion by patients with pulmonary tuberculosis in the process of chemotherapy, depending on the duration of treatment, have been studied, and the time-course of the excretion of ultramicro forms of mycobacteria by patients with and without caverns in the lungs in the process of chemotherapy has been followed. The results of the detection of M. tuberculosis ultramicro forms with the use of the biological and bacteriological methods indicate that both these methods are highly effective and informative. The method of the direct reversion of ultramicro forms into coccoid ones in Shkol'nikova's culture medium with 10% of plasma added has proved to be the simplest. The injection of sputum filtrates containing filter-passing (ultramicro) forms of mycobacteria into experimental animals induced the development of specific minor tuberculous inflammation of a productive character without the caseation of granulomas or progressing; such inflammation coursed as a latent lympho-hematogenous process.     

Early detection and rapid identification of Streptococcus pneumoniae and Haemophilus influenzae in acute pneumonias

The comparative study of the diagnostic value of the enzyme immunoassay (EIA), indirect immunofluorescence (IF) and countercurrent immunoelectrophoresis (CIE) was made. The serological identification of the isolated and reference pneumococci (19) and H. influenzae (38) strains revealed the possibility of using all three microanalytical methods for this purpose. The study of pneumococcal and H. influenzae antigens in native sputum obtained from 74 patients with acute pneumonia showed that EIA and indirect IF were highly sensitive, their sensitivity considerably exceeding that of the bacteriological analysis. Pneumococcal antigens were detected in 66.2% of patients by EIA and in 54.0% of patients by indirect IF, while H. influenzae antigens were detected in 58.1% of patients by EIA and in 67.6% of patients by indirect IF. The sensitivity of CIE proved to be considerably lower; in the detection of pneumococcal antigens it was level with the sensitivity of the bacteriological analysis (23.0%) and H. influenzae antigens could be detected only in 27.0% of patients.     

The characteristics of suppurative meningitis morbidity caused by Streptococcus pneumoniae and Haemophilus influenza type B

Of the 1,018 patients with purulent bacterial meningitis, hospitalized at the 2nd Clinical Infectious Hospital in Moscow during the period of 1980-1987, the diagnosis was confirmed in 54.7%; of these, meningitis of pneumococcal etiology was established in 44.8% and meningitis caused by H. influenzae, type b, in 23.8% of the patients. In meningitis of pneumococcal etiology high risk groups included mainly adults, especially those over 50 years, and children under 3 years of age. In meningitis of H. influenzae etiology high risk groups included mainly young children under 2 years of age. Meningitis of pneumococcal etiology was characterized by considerable death rate (on the average, 20%), while in meningitis of H. influenzae etiology death rate was 3 times lower. Pneumococci of serotypes 1, 3, 6, and 19 were found to be of the highest etiological importance for adults and pneumococci of serotypes 19, 6, 12, and 1, for children. In recent years greater etiological role of serotype 42 in adults was noted. The study of the spread of meningitides of different etiology is a high-priority task for this country.     

In Silico Metabolic Model and Protein Expression of Haemophilus influenzae Strain Rd KW20 in Rich Medium

The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.     

Etiology of pulmonary diseases in children in Khabarovsk Region

Microflora of upper respiratory tract in 658 children aged 1 month - 17 years hospitalized with acute pneumonia (AP), acute bronchitis (AB), recurrent obstructive bronchitis (ROB), malformation of lungs (ML) and broncho-alveolar dysplasia (BALD) were studied. Carriage rates of Streptococcus pneumoniae (up to 95%) and Haemophilus influenzae (up to 89%) in 240 children attending daycare centers and schools were determined. Etiology of infectious process was ascertained in 40% of cases. S. pneumoniae was isolated in 45% of acute cases (AP and AB) and in 25% of chronic cases (BALD). H. influenzae was isolated in 8 - 12% of acute cases and in 32% of chronic cases. In 23 - 29% of all cases of pulmonary pathology in children persistence of Enterococcus faecium was determined. There were 13 different serotypes among isolated pneumococci. In patients with pneumonia the rate of detection of S. pneumoniae and H. influenzae DNA fragments by PCR was significantly higher compared with rate of their isolation from sputum.     

Randomised controlled trial of antibiotics in patients with cough and purulent sputum.

Two hundred and twelve adults with cough and purulent sputum of up to one week''s duration were allocated randomly to treatment with doxycycline or placebo capsules for up to 10 days. Cough, purulent sputum, feeling "off colour," and time off work lasted as long in treatment and control groups, but running nose persisted for a shorter time in the doxycycline group. The number of new episodes of lower respiratory tract infections, vaginal infections, gastrointestinal upsets, and otitis media over the next six months were the same in both groups, but fewer new upper respiratory infections were experienced by the doxycycline-treated patients. There is no consensus among doctors about using antibiotics in patients with cough and purulent sputum, and these results indicate that otherwise healthy people with these symptoms will usually get better without antibiotic treatment.     

Duration of preservation of Streptococcus pneumoniae and Haemophilus influenzae in the sputum and bronchial washings of patients with acute and chronic pneumonias

The study of 108 samples of pathological material stored in a common refrigerator revealed that S. pneumoniae survived, on the average, for 7.6 days in sputum samples and for 4.2 days in bronchial washings, while Haemophilus influenzae could be detected in these materials, on the average, only within 48 hours. After 24-hour storage at 4-8 degrees C pneumococci were detected in all sputum samples and in 88.2% of bronchial washings, H. influenzae was detected ins 85% of these samples; the concentration of the above-mentioned microorganisms in the pathological material stored at 4-8 degrees C for 24 hours decreased, on the average, by 2.3-2.6 lg per ml.     

Proteases from purulent sputum. Purification and properties of the elastase and chymotrypsin-like enzymes.

A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.     

A Rapid Procedure for the Large Scale Purification of Elastase and Cathepsin G from Human Sputum

A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatography on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum a re identical to the same enzymes isolated directly from the leukocytes of human blood.     

A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum.

A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatogrphy on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum are identical to the same enzymes isolated directly from the leukocytes of human blood.     

Specific latex preparations in the etiological diagnosis of suppurative bacterial meningitis

The results of the evaluation of the diagnostic latex preparations Bactigen, manufactured by Wampole Laboratories (USA) and intended for the detection of meningococcal antigens, serogropus A, B, C, Y, pneumococcal polyantigens and type b Haemophilus influenzae antigens in the spinal fluid and blood of patients with meningococcal infection and purulent bacterial meningitides, are presented. The pathological material was studied by traditional methods and by the latex agglutination (LAG) test. 522 LAG tests were made, including 414 tests for meningococcal infection, 60 tests for pneumococcal infection and 48 tests for type b H. influenzae. The results of this study revealed that the latex preparations were highly specific with respect to type b H. influenzae antigens and meningococcal antigens (false positive reactions constituted 0.96%). The simplicity of the test and the rapid techniques making it possible to obtain results within 30-40 minutes indicate good prospects of using the LAG test in laboratory practice.     

Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients

Serial sputum isolates of Haemophilus influenzae (n = 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.     

Haemophilus influenzae immunoglobulin A1 protease genes: cloning by plasmid integration-excision, comparative analyses, and localization of secretion determinants.

Many bacteria which establish infections after invasion at human mucosal surfaces produce enzymes which cleave immunoglobulin A (IgA), the primary immunoglobulin involved with protection at these sites. Bacterial species such as Haemophilus influenzae which produce IgA1 proteases secrete this enzyme into their environment. However, when the gene encoding this protein was isolated from H. influenzae serotype d and introduced into Escherichia coli, the activity was not secreted into the medium but was localized in the periplasmic space. In this study, the IgA1 protease gene (iga) from an H. influenzae serotype c strain was isolated and the gene from the serotype d strain was reisolated. The IgA1 proteases produced in E. coli from these genes were secreted into the growth medium. A sequence linked to the carboxyl terminus of the iga gene but not present in the original clone was shown to be necessary to achieve normal secretion. Tn5 mutagenesis of the additional carboxyl-terminal region was used to define a 75- to 100-kilodalton coding region required for complete secretion of IgA1 protease but nonessential for protease activity. The iga genes were isolated by a plasmid integration-excision procedure. In this method a derivative of plasmid pBR322 containing a portion of the protease gene and the kanamycin resistance determinant of Tn5 was introduced into H. influenzae by transformation. The kanamycin resistance gene was expressed in H. influenzae, but since pBR322 derivatives are unable to replicate in this organism, kanamycin-resistant transformants arose by integration of the plasmid into the Haemophilus chromosome by homologous recombination. The plasmid, together with the adjoining DNA encoding IgA1 protease, was then excised from the chromosome with DNA restriction enzymes, religated, and reintroduced into E. coli. Comparisons between the H. influenzae protease genes were initiated which are useful in locating functional domains of these enzymes.