RESUMEN

The Haemophilus cryptic genospecies is an important cause of maternal genital tract and neonatal systemic infections and initiates infection by colonizing the genital or respiratory epithelium. In recent work, we identified a unique Haemophilus cryptic genospecies protein called Cha, which mediates efficient adherence to genital and respiratory epithelia. The Cha adhesin belongs to the trimeric autotransporter family and contains an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a C-terminal membrane anchor domain. The passenger domain in Cha contains clusters of YadA-like head domains and neck motifs as well as a series of tandem 28-amino-acid peptide repeats. In the current study, we report that variation in peptide repeat number gradually modulates Cha adhesive activity, associated with a direct effect on the length of Cha fibers on the bacterial cell surface. The N-terminal 404 residues of the Cha passenger domain mediate binding to host cells and also facilitate bacterial aggregation through intermolecular Cha-Cha binding. As the tandem peptide repeats expand, the Cha fiber becomes longer and Cha adherence activity decreases. The expansion and contraction of peptide repeats represent a novel mechanism for modulating adhesive capacity, potentially balancing the need of the organism to colonize the genital and respiratory tracts with the ability to attach to alternative substrates, disperse within the host, or evade the host immune system.

Asunto(s)

Asunto(s)

RESUMEN

Nineteen isolates belonging to a cryptic genospecies of Haemophilus (referred to here as genital strains) isolated from genital tract infections (6 strains) and from neonatal infections (13 strains) were studied for fimbrial genes. Sixteen strains exhibit peritrichous fimbriae observed by electron microscopy. By PCR with primers corresponding to the extreme ends of the Haemophilus influenzae type b (Hib) hifA and hifD genes and Southern blotting, a hifA-like gene (named ghfA) and a hifD-like gene (named ghfD) were identified in 6 of the 19 strains. Five of these six strains were from the genital tracts of adults, and one was from a neonate. For each gene, the nucleotide sequence was identical for the six strains. A hifE-like gene (named ghfE) was amplified from only one of the 19 genital strains of Haemophilus, but the ghfE probe gave a signal in Southern hybridization with the five other strains positive for ghfA and ghfD. Therefore, these strains may carry a ghfE-like gene. The Hib fimbrial gene cluster is located between the purE and pepN genes as previously described. For the 13 genital Haemophilus strains that lack fimbrial genes, this region corresponds to a noncoding sequence. Another major fimbrial gene designated the fimbrin gene was previously identified in a nontypeable H. influenzae strain. A fimbrin-like gene was identified for all of our 19 genital strains. This gene is similar to the ompP5 gene of many Haemophilus strains. Therefore, other, unidentified genes may explain the piliation observed in electron microscopy on genital Haemophilus strains which do not possess LKP-like fimbrial genes. Fimbrial genes were significantly associated with strains isolated from the genital tract. They may confer on the strain the ability to survive in the genital tract.

Asunto(s)

RESUMEN

The high-molecular-weight (HMW) immunoglobulin binding proteins (IgBPs) of Haemophilus somnus and a 76-kDa surface protein (p76) are found in serum-resistant virulent strains but not in several serum-sensitive strains from asymptomatic carriers. For the first time, p76 was shown to be an IgBP also. This was done by competitive inhibition studies with affinity-purified antidinitrophenol (anti-DNP) and DNP to ensure that binding was not antigen specific. The HMW IgBPs, but not the p76 IgBP, were partially purified from concentrated culture supernatant in detergent by fluid-phase liquid chromatography with a gel filtration column. Membrane extraction studies showed that p76 predominated in the Sarkosyl-soluble fraction of the bacterial cell pellet. Since integral outer membrane (OM) proteins are Sarkosyl insoluble, this is consistent with our previous finding that implicated p76 as a peripheral OM protein. The HMW IgBPs were found predominantly in the Sarkosyl-soluble fraction of the culture supernatant. This suggests that they were not integral membrane proteins and that their presence in the supernatant was not due to OM blebbing. We then showed that two IgBP-positive serum-resistant virulent strains have a surface fibrillar network but that two IgBP-negative serum-sensitive H. somnus strains from asymptomatic preputial carriers do not. Fibrils on the surfaces of IgBP+ strains bound gold-labelled bovine immunoglobulin G2 (IgG2) anti-DNP, indicating that these fibrils have IgG2 binding activity. Therefore, this study shows that H. somnus has two IgBPs, including a peripheral membrane protein and a fibrillar surface network.

Asunto(s)

RESUMEN

In the electron microscope an additional layer (glycocalix) of the cell wall and fimbriae on Haemophilus parasuis were shown in thin sections of the infected CAM which have their origin on the CM of the Haemophilus parasuis-cells. No fimbriation was seen after conventional cultivation.

Asunto(s)

RESUMEN

The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4-9) each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears.

Asunto(s)

RESUMEN

Two separate experiments were conducted using modifications of protein A-gold immunoelectron microscopy (PAGIEM) to evaluate the ability of sera from calves vaccinated against Haemophilus somnus to bind virulent organisms (experiment I) and to detect differences in the antibody accessible antigenic sites on the outer membrane of selected strains of H. somnus having different virulence attributes using a high IgG2 titre specific bovine hyperimmune serum (experiment II). The results of experiment I demonstrated that the direct opsonisation of H. somnus by specific antisera was related to its IgG2 titre. In experiment II, strain-dependent differences in the labelling of antigenic sites by specific IgG2 antibodies were observed. The virulent strains of both septicaemic and genital isolates of H. somnus showed higher protein A-gold labelling than their non-virulent counterparts. The results from a comparison of pathogenic and non-pathogenic respiratory isolates did not reveal the same difference in labelling intensity. The studies demonstrated the PAGIEM technique to be a sensitive, versatile and a reliable laboratory method to analyse antigen-antibody interactions of H. somnus.

Asunto(s)

RESUMEN

Gardnerella vaginalis has a very thin cell wall with a characteristic gram-negative staining pattern and an apparent lamellar structure when viewed at an oblique angle by electronmicroscopy. Examination at right angles to the cell-wall plane and by freeze-etching showed absence of an outer membrane or any other lamellar structure. Cell-wall extracts made by methods specific for lipopolysaccharide (LPS) gave negative reactions by silver staining and for endotoxin in the limulus amoebocyte lysate assay. 2-Keto-3-deoxy-D-manno-2-octonoic acid (KDO), heptose and hydroxy fatty acids specific for LPS were not detected in the extracts. G. vaginalis cell walls are unequivocally gram-positive in their ultrastructural characteristics and chemical composition.

Asunto(s)

RESUMEN

Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, IgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goat IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but all other isolates appeared to have identically migrating proteins.

Asunto(s)

Asunto(s)

RESUMEN

Fourteen recently isolated strains and two laboratory strains of Gardnerella vaginalis were examined by electronmicroscopy for the presence of pili. All strains isolated recently from both men and women were heavily pilated. In contrast only a few pili were seen on organisms of the two laboratory strains, with many of the organisms having no pili. The importance of multiple subculture in this loss was supported by the observation that the degree of pilation of one freshly isolated strain decreased on repeated subculture. Other findings suggested that this was probably due to gradual loss of pili and not to selection of organisms that were non-pilate originally.

Asunto(s)

RESUMEN

Capsular structure and biochemical composition varied between two isolates (virulent and avirulent) of Haemophilus pleuropneumoniae serotype 5. The presence of capsule was determined by transmission electron microscopy with glutaraldehyde-osmium, ruthenium red, alcian blue, and phosphotungstic acid staining procedures. The virulent isolate of H. pleuropneumoniae had a distinct, adherent capsule. The avirulent isolate had a fragile, easily removed capsule. Capsular material (CM) and a lipopolysaccharide (LPS) were isolated from each bacterial isolate and were compared biochemically and biologically. CM from both isolates contained carbohydrates, no detectable protein, and no detectable to trace amounts of lipid A. Each LPS contained heptose, hexose, galactose, glucosamine, 2-keto-3-deoxyoctonate, and lipid A. Biological responses to CM and LPS from both isolates were demonstrated in the proclotting enzyme of Limulus polyphemus amebocyte lysate activation and in serological cross-reactions by immunofluorescence and immunodiffusion precipitation. The virulent isolate contained approximately 10 mg of LPS per g more on an original dry weight basis than the avirulent isolate. LPS from the virulent isolate contained approximately 13 times more galactose than LPS from the avirulent isolate. The differences of capsular structure and biochemical composition may contribute to the role of CM in porcine H. pleuropneumoniae infections.

Asunto(s)

RESUMEN

Phagocytosis of Haemophilus somnus by bovine blood neutrophils required opsonization of the bacteria with antibodies against H. somnus. Few bacteria were phagocytosed in the absence of serum; in addition, absorption of immune serum with Formalin-killed H. somnus significantly reduced ingestion of H. somnus. Heat inactivation of antiserum (56 degrees C for 30 min) to eliminate complement activity had little effect on its ability to opsonize H. somnus for uptake by bovine neutrophils. Antiserum from an H. somnus-immunized calf and autologous sera from adult cattle supported equivalent phagocytosis of H. somnus by bovine neutrophils, suggesting that normal, healthy cattle may contain sufficient antibodies in their sera to facilitate phagocytosis of H. somnus. Although bovine neutrophils readily ingested H. somnus, they were unable to kill the bacterium in vitro. These same neutrophils readily killed opsonized Escherichia coli and Staphylococcus epidermidis, suggesting that H. somnus is able to survive and perhaps multiply within bovine neutrophils. Because bovine neutrophils stimulated with opsonized H. somnus demonstrated a reduced oxidative burst (as measured by chemiluminescence) compared with neutrophils stimulated by opsonized E. coli, we suggest that reduced production of reactive oxygen intermediates during the phagocytosis of H. somnus may account, in part, for the enhanced survival of H. somnus in bovine neutrophils.

Asunto(s)

RESUMEN

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.

Asunto(s)

RESUMEN

Transformation pathways in two closely related bacterial species, Haemophilus parainfluenzae and Haemophilus influenzae, were studied. Both organisms rapidly take up transforming DNA within minutes into specialized membranous structures on the cell surface (transformasomes). DNA within transformasomes is in a protected state, inaccessible to external DNase or internal restriction and modification enzymes. However, the subsequent processing of donor DNA differs in these two organisms. In H. influenzae, linear DNA immediately undergoes degradation from one end at a constant rate, leaving a lower-molecular-weight intermediate in the transformasome. The end undergoing degradation is searching for homologous regions of the chromosome. Once pairing is initiated, the remaining lower-molecular-weight DNA exits from the transformasome, and a single strand undergoes efficient integration. In contrast, in H. parainfluenzae little degradation of donor DNA is observed, with the majority remaining intact within the transformasomes after 1 h. Thus, whereas only 10% of donor DNA molecules leave the protected state after 1 h, portions of each molecule appear to become quantitatively integrated.

Asunto(s)

RESUMEN

Eight strains of Gardnerella vaginalis were examined by electron microscopy for the presence of pili. Narrow pili ranging from 3.0 to 7.5 nm in diameter were seen on bacteria from five of the strains studied.

Asunto(s)

RESUMEN

Twenty-two clinical isolates of Haemophilus species were studied within two passages of their original isolation for the presence of fimbriae by negative-staining electron microscopy. Six isolates were identified as fimbriated, including three strains of nontypable Haemophilus influenzae, one strain of Haemophilus parainfluenzae, and two strains of Haemophilus haemolyticus. In fresh isolates of fimbriated strains of nontypable H. influenzae, approximately 40%-50% of cells had fimbriae; after five passages in vitro, less than 1% of the cells had fimbriae. Thus, a variety of Haemophilus species that colonize the human respiratory tract can be fimbriated, and this fimbriation is rapidly lost on passage in vitro.

Asunto(s)

RESUMEN

Three colony variants (translucent, small opaque, and large opaque) of Haemophilus somnus recovered during infection studies of chicken embryos were examined using electron microscopy. Ruthenium red-strained H somnus preparations revealed no capsule in any variants. Pili were not seen on phosphotungstate-negative stained preparations. The translucent variant was thin-walled and had an irregular, pleomorphic rod shape. The small opaque variant had a thicker cell wall and an even rod shape. The large opaque variant had the thickest, most rigid wall of the 3, with uniform rod morphologic features. When each of the 3 variants was allowed to contact bovine epithelial turbinate cells, the translucent and small opaque were significantly (P less than 0.01) more adherent than was the large opaque variant.

Asunto(s)

RESUMEN

Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.

Asunto(s)

RESUMEN

Equine neutrophils were combined with Haemophilus equigenitalis (contagious equine metritis organism; CEMO) or Escherichia coli in low- and high-antibody-titer serum to evaluate the neutrophils ability to phagocytize and kill these bacteria. More E. coli than CEMO were phagocytized at each time period. After 120 min in low-antibody-titer serum, 56.3% of the E. coli and 34.3% of the CEMO were phagocytized. A total of 45% of CEMO and 74.9% of E. coli were phagocytized by 120 min when neutrophils were in high-antibody-titer serum. More than 75% of the ingested E. coli and 90% of the ingested CEMO were killed within 210 min of incubation. Fewer E. coli than CEMO were killed at any given time period. Ultrastructural examination showed CEMO to be degraded in the neutrophil. Degradation was the most extensive in neutrophils in high-titer serum. It is suggested that CEMO is a pathogenic extracellular bacterium incapable of prolonged intracellular survival and that it is slower to be phagocytized than a nonpathogenic E. coli.

Asunto(s)