Structural properties of Type I collagen isolated from chickens with scoliosis

This study examines biochemically the Type I collagen isolated from skin of chickens that develop idiopathic scoliosis. Previous studies indicate a defect in collagen exists in these chickens. Alpha 1 (I) and alpha 2 chains were separated by gel filtration and carboxymethyl cellulose column chromatography and were then subjected to the analytical techniques of sodium dodecyl sulfate gel electrophoresis, Staphylococcus aureus V8 protease digestion, cyanogen bromide peptide mapping and amino acid analyses. In all categories, the scoliotic alpha 1 (I) and alpha 2 chains were identical to alpha chains isolated from normal chickens. These data suggest that the altered properties of collagen solubility and connective tissue stress relaxation seen in these scoliotic chickens are not a manifestation of an altered primary structure of the alpha chains or post-translational modification affecting chromatographic elution profiles or electrophoretic migration patterns.     

Aeromonas species isolated from cases of acute gastroenteritis

A total of 67 Aeromonas strains were isolated as the sole bacterial pathogen from 1485 patients with acute gastroenteritis. A. hydrophila (64.2%) was the predominant isolate followed by A. sobria (28.4%) and A.caviae (7.4%). Majority of the isolates were sensitive to gentamicin, nalidixic acid but were resistant to ampicillin. Minimum inhibitory concentration (MIC) of resistant strains of Aeromonas to ampicillin ranged from 80-1280 microg/mL.     

Bovine lactoferrin binding to six species of coagulase-negative staphylococci isolated from bovine intramammary infections.

Bovine lactoferrin (BLf), an acute-phase iron-binding secretory protein present in secretions of the bovine udder, was demonstrated to bind to the following staphylococcal species associated with bovine intramammary infections: S. epidermidis, S. warneri, S. hominis, S. xylosus, S. hyicus, and S. chromogenes. The degree of 125I-labeled BLf uptake significantly varied among the blood agar-grown cells of all six species of coagulase-negative staphylococci tested. Isolates identified as S. xylosus demonstrated the highest (mean, 35.1 x 10(6) +/- 13.3 x 10(6) nmol) and S. hyicus the lowest (mean, 10.7 x 10(6) +/- 5.9 x 10(6) nmol) binding to 125I-BLf. BLf binding was optimum at an acidic pH, with time-dependent binding saturation ranging from 70 min for S. warneri to 240 min for S. hominis. The BLf-binding mechanism was specific, with affinity constants (Ka values) ranging between 0.96 x 10(6) and 11.90 x 10(6) liters/mol. The numbers of BLf-binding sites per cell, as determined by using Scatchard analysis, were as follows: S. epidermidis, 3,600; S. warneri, 1,900; S. hominis, 4,100; S. xylosus, 4,400; S. hyicus, 6,100; and S. chromogenes, 4,700. 125I-BLf binding to all species was inhibited by unlabled BLf and unlabeled human lactoferrin, whereas none of the various plasma, connective tissue, or mucosal secretory proteins or carbohydrates tested caused significant interference. BLf-binding receptors of the six coagulase-negative staphylococcal species demonstrated marked differences in patterns of susceptibility to proteolytic or glycolytic enzyme digestion and to heat or periodate treatment. These data suggest that the BLf-binding components in S. epidermidis and S. warneri are proteins containing glycosidyl residues. In the remaining four species, the proteinaceous nature of the BLf-binding component was evident, but the involvement of glycosidyl residues was not clear. Results of this study establish the presence of specific binding components for BLf on coagulase-negative staphylococci isolated from bovine intramammary infections.     

Binding of fibronectin, fibrinogen and type II collagen to streptococci isolated from bovine mastitis

Binding of 125I-labelled fibronectin, fibrinogen and type II collagen to group B (S. agalactiae), group C (S. dysgalactiae and S zooepidemicus), group E (S. uberis) and nontypable streptococci isolated from bovine mastitis was studied. S. agalactiae and S. uberis were found to bind low levels of all three proteins, while S. zooepidemicus bound high levels. Binding of the proteins to S. dysgalactiae varied, i.e. fibronectin was high, fibrinogen moderate and collagen low. Nontypable strains showed moderate or low binding of all proteins. Both hydrophobic and hydrophilic strains were found to bind fibronectin. For S. dysgalactiae the specific fibronectin binding ranged from 70% to 10% and for S. zooepidemicus it was more than 80% and this binding was sensitive to papain treatment. The binding of 29K-fibronectin fragment to one S. dysgalactiae strain showed an affinity of KD = 2.6 x 10(-8) M and the number of binding sites per colony forming unit (CFU) was calculated at 11,000.     

Functional Characterization of Type IV Pili Expressed on Diarrhea-Associated Isolates of Aeromonas species

Our past work has shown that long, flexible type IV pili (single or in bundles) are the predominant pili expressed on fecal isolates of diarrhea-associated species of Aeromonas (Aeromonas veronii biovar sobria and A. caviae). They represent a family of type IV pili which we have designated Bfp (for bundle-forming pili). Reports from Japan suggest that Bfp are intestinal colonization factors. This study presents compelling evidence to support this conclusion. Aeromonas bacteria and/or Bfp purified from a strain of A. veronii biovar sobria were shown to adhere to epithelial and intestinal cell lines, freshly isolated human enterocytes, and fresh and fixed human and rabbit intestinal tissues, as determined by light and electron microscopy and immunohistochemical detection. Removal of Bfp by mechanical means decreased adhesion to cell lines by up to 80%. Purified Bfp blocked adhesion of the test strain to intestinal cells in a dose-dependent manner. Adhesion was also blocked by the Fab fraction of anti-Bfp immunoglobulin G. Moreover, ultrastructural studies (ruthenium red staining and transmission and scanning electron microscopy) demonstrated for the first time that Aeromonas adhesion to human enterocytes is pilus mediated and suggested that Bfp may also promote colonization by forming bacterium-to-bacterium linkages. Bfp-positive isolates examined for type IV pilus-mediated twitching motility in agar and slide culture assays developed for Pseudomonas aeruginosa did not, however, exhibit this function.     

Phenotypic characteristics of Aeromonas species isolated from adult humans.

The phenotypic characteristics of 89 Aeromonas strains, most of which had been isolated from feces, were examined. Eighty-two percent of the isolates could be placed into one of four groups on the basis of five tests. The relationship of these groups to the three motile species of Aeromonas (Aeromonas caviae, A. hydrophila, and A. sobria) that have been isolated from humans is unclear. Because the means for identification of Aeromonas strains to the species level appear to be imprecise and because the role of each of these species in human diarrhea is unclear at this time, we recommend that identification of enteric Aeromonas isolates to the species level not be done routinely.     

Skin and soft-tissue infections caused by Aeromonas species

This study investigated the clinical characteristics of patients with skin and soft-tissue infections (SSTIs) due to Aeromonas species. Patients with SSTIs caused by Aeromonas species during the period from January 2009 to December 2011 were identified from a computerized database of a regional hospital in southern Taiwan. The medical records of these patients were retrospectively reviewed. A total of 129 patients with SSTIs due to Aeromonas species were identified. A. hydrophila (n?=?77, 59.7 %) was the most common pathogen, followed by A. veronii biovar sobria (n?=?22, 17.1 %), A. veronii biovar veronii (n?=?20, 15.5 %), A. caviae (n?=?9, 7.0 %), and A. schubertii (n?=?1, 0.8 %). The most common isolates obtained from patients with polymicrobial infections were Klebsiella species (n?=?33), followed by Enterococcus spp. (n?=?24), Enterobacter spp. (n?=?21), Escherichia coli (n?=?17), Staphylococcus spp. (n?=?17), Streptococcus spp. (n?=?17), and Acinetobacter spp. (n?=?15). Liver cirrhosis and concomitant bacteremia were more common among patients with monomicrobial Aeromonas SSTIs than among patients with polymicrobial SSTIs. Nine (7 %) patients required limb amputations. The in-hospital mortality rate was 1.6 %. In conclusion, Aeromonas species should be considered as important causative pathogens of SSTIs, and most infections are polymicrobial. In addition, the clinical presentation differs markedly between patients with monomicrobial and those with polymicrobial Aeromonas SSTIs.     

Clinical significance of Aeromonas species isolated from patients with diarrhea.

A total of 248 strains of Aeromonas spp. were isolated from 3,334 human fecal specimens submitted to a state public health laboratory over a 2-year period to be cultured for enteric pathogens. Cary-Blair transport medium, blood ampicillin agar, and alkaline peptone water enrichment provided optimal recovery of Aeromonas spp. A questionnaire requesting clinical and epidemiological information was sent to physicians, who submitted stool samples for testing, with each laboratory report for 107 consecutive stool isolates of Aeromonas spp. The 56 questionnaires which were completed and returned were analyzed to determine the seasonal distribution of illness and the age and sex distribution of patients; characteristic symptoms; and predisposing factors for gastrointestinal disease caused by Aeromonas spp. It was concluded that some A. hydrophila, A. sobria, and A. caviae strains are capable of causing diarrhea and that antibiotic therapy and the drinking of untreated water are significant risk factors for susceptible hosts.     

Characterization of Aeromonas schubertii strains recently isolated from traumatic wound infections.

Recent studies have resulted in the proposal of a new species, Aeromonas schubertii (mannitol, sucrose, and indole negative), formerly termed Enteric Group 501, on the basis of the study of seven strains isolated from the southeastern and southwestern United States and Puerto Rico. We have isolated two phenotypically similar A. schubertii strains from infected human wounds sustained in the Chesapeake Bay area. Their identification was confirmed by DNA-DNA hybridization to the Centers for Disease Control definition strain 2446-81 (ATCC 43700) for group 12. The strains were further examined for the presence of virulence-associated markers: hemolysin, hemagglutinins, cytotoxin production, agglutination in acriflavine, resistance to normal human serum, and autoagglutination phenotype. Both strains were positive for hemolysin by the plate assay, cytotoxin production at 1:10, and DNase and protease. They were resistant to human serum and negative for acriflavine agglutination, and only one of the strains was autoagglutination positive. Both strains were negative for cell-free hemolysin, hemagglutinins, pectinase, and chitinase. These isolations of A. schubertii further extend its previously described geographic distribution and reinforce its role as a primary causative agent of cellulitis with possible increased antimicrobial resistance.     

Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis.

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (Fn) [corrected], whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (Cn) [corrected] type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 125I-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 125I-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.     

Ultrastructural differences in isolated mast cells from various species

Conclusions The lymphocyte activation observed by moderate doses of H₂ antagonists was assumed to be a direct action, which made it difficult to verify whether an H₂ receptor was specifically involved in the histamine inhibition of lymphocyte activation. This, however, does not preclude the possibility that locally released histamine could well be a chemical modulator of lymphocyte function in vivo.     

Ultrastructural differences in isolated mast cells from various species

Conclusions The different types of granules appeared either jointly within one single cell (e.g. guinea-pig) or in separate cells from the same source (e.g. man), possibly indicating various stages of maturity.     

Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections.

The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binding EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subepithelial matrix proteins and carbohydrates tested (in 10(4)-fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (Ka) of 1.4 x 10(-7) M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli.     

Mechanisms of quinolone resistance in Aeromonas species isolated from humans,water and eels

Mechanisms of resistance were determined in 33 quinolone-resistant isolates of the species Aeromonas hydrophila, Aeromonas caviae, Aeromonas media, Aeromonas salmonicida, Aeromonas popoffii and Aeromonas veronii, recovered from humans, freshwater and eels. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were sequenced in these resistant strains, as well as in 8 quinolone-sensitive Aeromonas used as controls. All quinolone-resistant Aeromonas carried point mutations in the gyrA QRDR at codon 83, respectively giving rise to substitutions Ser⁸³ → Ile (32 strains) or Ser⁸³ → Val (1 strain). Almost half of these isolates (48%) carried additional point mutations in the gyrA QRDR at codon 92 and/or in the parC QRDR at codon 80 corresponding to substitutions Leu⁹² → Met and Ser⁸⁰ → Ile. In all cases, MICs of quinolones were determined in the presence and absence of the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN). Addition of PAβN had no effect on the levels of resistance observed in these isolates. In conclusion, the mechanism of quinolone resistance in the Aeromonas isolates studied was related to mutations in QRDR regions of gyrA and parC genes, with little obvious involvement of pumps inhibited by PAβN.     

Aeromonas trota sp. nov., an ampicillin-susceptible species isolated from clinical specimens.

Previous DNA hybridization studies established 12 Aeromonas genospecies, from which nine phenotypic species have been proposed: Aeromonas hydrophila, A. sobria, A. caviae, A. media, A. veronii, A. schubertii, A. salmonicida, A. eucrenophila, and A. jandaei. We have delineated a new Aeromonas genospecies, A. trota, on the basis of 13 strains isolated primarily from fecal specimens from southern and southeastern Asia. All strains were highly related to the proposed type strain, AH2 (ATCC 49657T): 51 to 100% (60 degrees C) and 49 to 99% (75 degrees C), with 0.2 to 2.2 divergence. AH2 was only 16 to 41% (60 degrees C) related to all other Aeromonas type strains and DNA group definition strains. The unique profile of A. trota includes negative reactions for esculin hydrolysis, arabinose fermentation, and the Voges-Proskauer test, positive reactions for cellobiose fermentation, lysine decarboxylation, and citrate utilization, and susceptibility to ampicillin, as determined by the broth microdilution MIC method and the Bauer-Kirby disk diffusion method (10 micrograms). Nine of the A. trota strains were from a single study of 165 geographically diverse aeromonads. This finding questions the efficacy of screening fecal specimens for Aeromonas spp. with ampicillin-containing media and suggests a previously unrecognized prevalence of this new species.     

Production and characterization of a monoclonal antibody to human Type IV collagen.

We have produced a monoclonal antibody to human basement membrane Type IV collagen. The antibody reacts with the pepsin-resistant, collagenase-sensitive domain of Type IV collagen isolated from placental membranes, but not with human collagens of Types I, II, III, V, 1alpha, 2alpha, and 3alpha. The antibody precipitates biosynthetically labeled human Type IV procollagen, and the precipitate contains both the alpha1 (IV) and alpha2 (IV) chains, suggesting the occurrence of both of these chains within the same triple-helical molecule. When used in indirect immunofluorescence, the antibody gives brilliant staining of basement membranes from a variety of human tissues but does not stain tissues of bovine, canine, rabbit, rat, or mouse origin. It is suggested that this antibody will be of value in research on the structure of human basement membrane collagen, on the distribution of this collagen in various basement membranes, and particularly for the study of basement membranes in normal human development and pathologic processes.     

Type I and type IV collagen promote adherence and spreading of human type II pneumocytes in vitro

Human lung tissue was enzymatically digested with dispase and type II pneumocytes were subsequently separated on a discontinuous metrizamide gradient. In primary adherence assays with freshly isolated cells, mean adherence values of 21.7, 19.6, 13.4, 6.8 and 7.0% were obtained after 48 hours on type I collagen (CI), type IV collagen (CIV), fibronectin (FN), laminin (LM) and tissue culture plastic, respectively. Secondly, readherence and spreading assays were performed with type II pneumocytes, previously cultured on CI, FN, matrix of murine EHS cells, and culture plastic. After a culture period of 48 to 72 hours, the cells were replated and tested on CI, CIV, FN, LM, CI + FN, CI + LM, CIV + FN and CIV + LM substrata in order to find out whether preculture substrata can modulate the adherence response of these cells. Readherence values, 1 hour after replating, were 3.3- to 8.6-fold higher than primary adherence values after 48 hours. The highest readherence values were always obtained on collagen-containing substrata after preculture on fibronectin. Spreading values after replating ranged between 35.4 and 6.6% on collagen-containing substrata, the highest values were again obtained after preculture on FN. Less than 6.9% and 0.5% of the cells spread on pure FN and pure LM, respectively. The data of the present investigation indicate that human type II pneumocytes adhere and spread preferentially on collagenous substrata rather than on other components of the extracellular matrix. This might be important in helping us to understand the functional in vivo activity of these cells, especially as regards re-epithelialization of the injured pulmonary alveolus.     

Helicobacter pylori and neutrophils: sialic acid-dependent binding to various isolated glycoconjugates

Helicobacter pylori has been shown to agglutinate erythrocytes in a sialic acid-dependent manner. However, very few studies have examined relevant target cells in the human stomach. Neutrophils are required for the onset of gastritis, and the inflammatory reaction may be induced on contact between bacteria and neutrophils. In the present work, glycolipids and glycoproteins were isolated from neutrophils and were studied for binding by overlay with radiolabeled bacteria on thin-layer chromatograms and on membrane blots. There was a complex pattern of binding bands. The only practical binding activity found was sialic acid dependent, since treatment of glycoconjugates with neuraminidase or mild periodate eliminated binding. As shown before for binding to erythrocytes and other glycoconjugates, bacterial cells grown on agar bound to many glycoconjugates, while growth in broth resulted in bacteria that would bind only to polyglycosylceramides, which are highly heterogeneous and branched poly-N-acetyllactosamine-containing glycolipids. Approximately seven positive bands were found for glycoproteins, and the traditional ganglioside fraction showed a complex, slow-moving interval with very strong sialic-acid-dependent binding, probably explained by Fuc substitutions on GlcNAc.