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Microbial interactions are ubiquitous in nature, and are equally as relevant to human wellbeing as the identities of the interacting microbes. However, microbial interactions are difficult to measure and characterize. Furthermore, there is growing evidence that they are not fixed, but dependent on environmental context. We present a novel workflow for inferring microbial interactions that integrates semi-automated image analysis with a colony stamping mechanism, with the overall effect of improving throughput and reproducibility of colony interaction assays. We apply our approach to infer interactions among bacterial species associated with the normal lung microbiome, and how those interactions are altered by the presence of benzo[a]pyrene, a carcinogenic compound found in cigarettes. We found that the presence of this single compound changed the interaction network, demonstrating that microbial interactions are indeed dynamic and responsive to local chemical context.

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We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.

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Haemophilus parainfluenzae isolates recovered from patients with respiratory diseases were studied for their ability to undergo genetic transformation by isogenic DNA. Two chromosomal markers, streptomycin resistance and nalidixic acid resistance, were tested for transformation efficiencies in H. parainfluenzae recipients from three biotypes. Most efficient in transformation was biotype II, followed by biotype I, while biotype III was nontransformable. Lack of transformation was not owing to poor donor activity of DNA, but to inability of the cells to develop competence. Strains that formed clumps in liquid media were nontransformable. Since the transformable biotype II is one of the prevalent biotypes world wide, one can speculate that DNA transformation probably plays a major role in the spread of drug resistance in H. parainfluenzae.

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The biochemical and serological properties of Haemophilus paragallinarum isolates recovered from 11 recent outbreaks of infectious coryza in layer hens and one case of swollen-head syndrome in broilers in Argentina are described. Twenty-four isolates had the typical biochemical properties of H. paragallinarum. All isolates were serotyped according to the Page scheme. Ten of the isolates were serovar A, 11 were serovar B, one was serovar C, and two isolates could not be serotyped. The isolates were also examined using a panel of monoclonal antibodies (MAbs) for Page serovars A (one MAb available) and C (three MAbs available). The serovar B isolates all failed to react with any MAb. The serovar C isolate reacted with all three serovar C MAbs but not with the serovar A MAb. Only six of the 10 serovar A isolates reacted with the serovar A MAb. These results indicate that H. paragallinarum isolates from Argentina are antigenically distinct from those examined in other countries, and it is suggested that coryza vaccines intended for use in Argentina may be more effective if based on local strains.

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Haemophilus parasuis strains isolated from the noses of apparently healthy animals and from animals with pathological conditions were examined for the presence of a capsule, for their ability to agglutinate in acriflavine or after boiling, and for their peptide profile after polyacrylamide gel electrophoresis (PAGE). The capsule was identified by precipitation against hexadecyl trimethylammonium bromide (Cetavlon), by demonstration of iridescence, and by means of a capsule-staining method. We found a group of capsulated strains showing a rather coccobacillary morphology compared with the morphology with polymorphism, varying from rod-like to filamentous, in strains without detectable capsules. The strains of the latter group were agglutinated by acriflavine or by boiling. Soluble antigens of capsulated strains reacting with Cetavlon were thermostable and resisted proteolytic enzymes, thus suggesting the presence of an acidic polysaccharide. A few of the capsulated strains did not precipitate with Cetavlon, which indicated that their chemical composition was different. Acriflavine-positive strains belonging to a definite PAGE pattern (type II) seemed to be associated with pathological conditions more frequently than were capsulated strains which were mostly isolated from nasal cavities of apparently healthy pigs. We put forward the hypothesis that the agglutinability in acriflavine, together with the PAGE profile type II, may be associated with particular structures responsible for virulence.

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Lesions induced in chickens by an encapsulated or nonencapsulated strain of Haemophilus paragallinarum were investigated. In terms of lesion severity, major differences in pathogenicity were observed between the encapsulated and nonencapsulated variants. The principal lesion manifested by the encapsulated variant was an acute catarrhal inflammation of the upper respiratory tract, mainly of the nasal cavity and paranasal sinus. Infiltration of a large number of mast cells into the lamina propria of the mucous membrane of the nasal cavity was also characteristic. Numerous organisms were found on the cilia or on the surface of the epithelial cells of the nasal mucosa. Chickens with these histologic lesions had severe clinical signs of coryza, and organisms were recovered in high numbers from the nasal cavity and paranasal sinus. In contrast, chickens that were given the nonencapsulated variant did not have clinical signs of coryza, and the organisms were recovered in low numbers from the inoculated sites; slight histopathologic lesions were observed in the nasal mucosa at postinoculation day 1. Mast cell infiltration in the chickens inoculated with the encapsulated variant indicated that mast cells may be responsible for producing clinical signs of coryza via the activation of pharmacologic mediators. Adherence to and colonization of the encapsulated variant on the nasal mucosa seems to be a first step of the infection.

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Indirect evidence suggests that immunoglobulin A1 (IgA1) proteases may be factors in the pathogenesis of certain infectious diseases, including meningitis, gonorrhoea, and destructive periodontitis. Bacterial IgA1 proteases are therefore potential candidates as vaccines. In this study, IgA1 proteases from 166 clinical isolates and reference strains of Haemophilus influenzae and Haemophilus aegyptius were compared with regard to specific activity and pattern of enzyme inhibition by antisera raised against IgA1 protease from nine selected strains of H. influenzae. A total of 93% of H. influenzae strains and all H. aegyptius strains had detectable IgA1 protease activity. The majority of strains cleaved a prolyl-seryl or a prolyl-threonyl peptide bond in the alpha 1 hinge region, whereas occasional H. influenzae strains possessed two separate IgA1 proteases with these two specific activities. Of the 155 IgA1 protease-producing strains, all except 12 could be assigned to one of 14 IgA1 protease "inhibition types," each defined by a characteristic pattern of inhibition by the nine antisera. There was no correlation between IgA1 protease type and biotype of the strains. However, among 92 encapsulated H. influenzae strains, a close correlation between capsular serotype and IgA1 protease type was observed. With the exception of serotype f, strains of all capsular serotypes produced an exclusive antigenic type of IgA1 protease. All 38 strains of serotype b produced IgA1 protease of inhibition type 1, which was never demonstrated in non-encapsulated H. influenzae strains. These results facilitate the detection of an antibody response against specific IgA1 proteases and are of practical value for a possible future vaccine against H. influenzae serotype b infections.

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This study aimed at clarifying the relationship of Haemophilus aegyptius and Haemophilus influenzae isolated from acute conjunctivitis in Egypt. Twenty-nine freshly isolated strains selected from a large clinical material were examined for morphological and growth characteristics, biochemical properties and susceptibility to selected antibiotics. H. aegyptius strains were clearly differentiated from strains of H. influenzae by their inability to grow on tryptic soy agar containing X + V factors, by their susceptibility to trooleandomycin, by a distinct bacillary morphology, and, in part, by not fermenting xylose. The results confirm that H. aegyptius is distinct from H. influenzae and provides reproducible means of differentiating the two species.

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The effect of a commercially available, chemically defined enrichment (Iso-VitaleX; BBL Microbiology Systems, Cockeysville, Md.) on the growth of 10 strains of Haemophilus somnus was studied. A 6- to 10-fold increase in growth, as measured turbidimetrically, was observed when Iso VitaleX was added to a basal medium of brain heart infusion broth to a final concentration of 1% (vol/vol). Thiamine pyrophosphate (cocarboxylase), a constituent component of Iso VitaleX, was found to be the only growth-promoting factor, and it could be used as a substitute for Iso VitaleX. An equimolar concentration (2.2 microM) of thiamine monophosphate promoted growth equal to that of thiamine pyrophosphate. Thiamine was nonstimulatory for all 10 strains tested. When alkaline thermal-treated brain heart infusion broth was used as the basal medium, 7 of the 10 strains had an absolute requirement for thiamine monophosphate or thiamine pyrophosphate. The three remaining strains showed minimal growth when thiamine was added to this basal medium; however, excellent growth was observed when thiamine monophosphate or thiamine pyrophosphate was utilized. Factor X (hemin) was found to further enhance the growth when concentrations of 5 to 10 micrograms/ml were coupled with thiamine pyrophosphate. No increase in growth was observed when factor V (nicotinamide adenine dinucleotide) was coupled with thiamine pyrophosphate. This is the first report of a growth factor requirement for H. somnus.

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Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.

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An infection model in laboratory mice for studying the bacterium (proposed name Haemophilus equigenitalis) causing contagious equine metritis is described. Small porous chambers were implanted subcutaneously into mice and after 1 to 3 weeks were inoculated with H equigenitalis. Infections that persisted for > 30 days were established by direct transfer of infective chamber fluid or by injection of laboratory-grown cultures. Immunization of mice with formaldehyde-treated cells induced significant, strain-related immunity to infection and did not appear to require complement as a protection mediator. Substantial differences in staining characteristics and cell morphology were observed between bacteria grown in mouse chambers and those grown on a laboratory medium. These differences were more apparent in smears of the organism stained by a modified Gimenez method than in smears stained by the conventional Gram stain. This murine model may be a convenient and inexpensive method for studying the immunobiology of this newly discovered animal pathogen.

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Isolation and microbiological features of a Haemophilus paraphrophilus strain which caused a subacute bacterial endocarditis are described. The isolate conformed to the first description by ZINNEMANN et al., except that it was sensitive to ampicillin.

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The saccharolytic capacity in respect to 14 carbohydrates, the lipolytic activity, the presence of urease, catalase, galactosidase, the formation of hydrogen sulphide and indol and also serological properties with the species agglutinating sera were studied in the representatives of the genus of hemoglobinophilic microbes: H. influenzae, H. parainfluenzae, H. aegiptius, H. haemolyticus, H. aphrophilus. There were revealed differences in the individual representatives of the genus by the enzymatic activity and serological properties. Thus, representatives of H. influenzae possessed urease activity, but all of them lacked galactosidase. H. aegiptius possessed urease and galactosidase, and H. parainfluenzae, H. haemolyticus and H. aphrophilus--galactosidase of high activity, but no urease. Representatives of each of the species were agglutinated by homologous sera only.

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Haemophilus vaginalis 594 has a trilaminar cell wall, ribosomes, a fibrillar nucleoid, and both convoluted and simple mesosome-like bodies. Polar bulbous enlargements of the cell and multiple cross wall formations may explain its pleomorphism.

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The identity of haemophili isolated in our laboratories from eye swabs was investigated. Ten out of 114 strains belonged to capsulated types of H. influenzae. Seventy-six strains were submitted to tests for the identification of H. aegyptius (the Koch-Weeks bacillus), but none was proved to belong to this species. An unexpectedly high proportion of the haemophilus strains, including most of those with capsules, came from patients with lachrymal duct obstruction.

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