Serological studies of Bacteroides vulgatus from normal gut flora

A serological classification scheme for 48 strains of Bacteroides vulgatus was established by agglutination technique. Bacterial strains were isolated in our laboratory from gut flora of healthy persons. Absorbed antisera to 11 strains of B. vulgatus were prepared. All 48 strains tested reacted with one or more antisera. Among these strains, 22 serogroups were formed; 11 of them contained only one group component and 11 were made up of multiple group components.     

Characterization of black-pigmented Bacteroides strains isolated from animals

The aims of the study were: the isolation of strains of black-pigmented Bacteroides from the gingival sulcus of different animals, their biochemical and immunological characterization and comparison of their properties for classification within the genus. A total of 104 strains, isolated from cats, dogs, racoons and a jaguar, were characterized on the basis of fermentation of carbohydrates, metabolic end products, haemagglutination studies, enzymatic activities, catalase production and indirect immunofluorescence. No differences were observed between the strains regardless of their animal origin. The strains did not ferment carbohydrates, produce phenylacetic acid, show an array of enzyme activities or agglutinate sheep red blood cells. They were catalase-positive and so differed from the human oral strains of Bact. gingivalis. Immunofluorescence microscopy revealed that the animal strains shared at least one major antigen with Bact. gingivalis but none with Bact. asaccharolyticus. Apart from their catalase activity, the animal strains isolated were similar to those of human Bact. gingivalis strains.     

Characterization of black-pigmented Bacteroides strains isolated from animals

The aims of the study were: the isolation of strains of black-pigmented Bacteroides from the gingival sulcus of different animals, their biochemical and immunological characterization and comparison of their properties for classification within the genus. A total of 104 strains, isolated from cats, dogs, racoons and a jaguar, were characterized on the basis of fermentation of carbohydrates, metabolic end products, haemagglutination studies, enzymatic activities, catalase production and indirect immunofluorescence. No differences were observed between the strains regardless of their animal origin. The strains did not ferment carbohydrates, produce phenylacetic acid, show an array of enzyme activities or agglutinate sheep red blood cells. They were catalase-positive and so differed from the human oral strains of Bact. gingivalis. Immunofluorescence microscopy revealed that the animal strains shared at least one major antigen with Bact. gingivalis but none with Bact. asaccharolyticus. Apart from their catalase activity, the animal strains isolated were similar to those of human Bact. gingivalis strains.     

Draft genome sequence of Bacteroides vulgatus PC510, a strain isolated from human feces

Although Bacteroides vulgatus is one of the most prevalent microorganisms in the human gastrointestinal tract, little is known about the genetic potential of this species. Here, we describe the annotated draft genome sequence of B. vulgatus PC510 isolated from human feces.     

Enterotoxin-producing Bacteroides fragilis strains isolated from horses]

Seven Bacteroides fragilis strains were cultured from samples collected from horses. From all the tested strains, as well as from the reference B. fragilis strains: enterotoxigenic NCTC 11925 and nonenterotoxigenic IPL 323 strain, DNA was isolated using Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland). To detect the enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied, using the following starters: 404 (GAG CCG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in a thermocycler (Techne). The temperature profile was as follows: 1 cycle (4 min. 94 degrees C), 40 cycles (1 min. 94 degrees C, 1 min. 52 degrees C, 1 min. 74 degrees C). Amplification products were detected by electrophoresis in agarose gel (1%) with ethidium bromide added. The presence of the fragilysin gene was detected in two strains. Among the strains isolated from horses enterotoxin gene-possessing Bacteroides fragilis strains (ETBF) can be detected.     

Degradation of intestinal glycoproteins by Bacteroides vulgatus

Bacteroides vulgatus, isolated from a patient with Crohn's disease, produced in gnotobiotic rats 7 constitutive enzymes that might be concerned with the degradation of intestinal glycoproteins. Furthermore Bacteroides vulgatus caused an almost complete loss of blood group antigenicity of the intestinal glycoproteins. Enzymes with the potency to release toxic compounds from hepatic conjugates and plant glycosides, beta-glucuronidase and beta-glucosidase, respectively, were only detectable in small amounts. These findings indicate that Bacteroides vulgatus, which accounts for 40% of the total flora of patients with Crohn's disease, may play a role in the pathogenesis of Crohn's disease, by increasing the break-down of the mucus layer and therefore damaging its protective function.     

Proteolytic activity of black-pigmented Bacteroides strains

Abstract The proteolytic activity of several black-pigmented Bacteroides species was measured. Bacteroides gingivalis was the only species having collagenolytic activity. General proteolytic activity on gelatin and Azocoll was shown in cultures of B. gingivalis, B. asaccharolyticus, B. endodontalis, B. intermedius, B. corporis and to a lesser extent B. melaninogenicus; B. loescheii did not show proteolytic activity. When culture filtrates were tested, B. gingivalis showed high cell free proteolytic activity, whereas the other species had only very weak cell free activity. Growth curves of B. gingivalis revealed two distinct proteolytic activities; general proteolytic activity was found during the logarithmic growth phase, whereas a second peak containing high collagenolytic activity was found after prolonged incubation of cells showing autolysis.     

The effects of deoxycholate and sodium dodecyl sulphate on the serological reactivity of antigens isolated from six Bacteroides reference strains

The detergents sodium dodecyl sulphate (SDS) and sodium deoxycholate (NaD) are frequently used as solvents for macromolecular polysaccharide complexes in immunochemical and serological techniques. The influence of the disaggregating surfactants on the serological reactivity of endotoxins isolated from six serotype specific reference strains of the Bacteroides fragilis group was investigated by comparing haemagglutinating and precipitating reactivities of antigen solutions in phosphate buffered saline (PBS), NaD and SDS. All antigens were phenol/water extracted endotoxins. Solutions of antigens isolated from serotypes A, B, C and D in PBS exhibited mainly serotype specificity and a few well known low-titer cross reactions; solutions in NaD showed additional cross reactivity, which was enhanced by solubilization of the antigens in SDS. In immunoelectrophoresis endotoxins isolated from serotypes A and C and dissolved in NaD or SDS showed additional precipitation lines compared to solutions of the same antigens in PBS. These changes in the serological reactivity are of relevance for investigations where the serological specificity of antigens is in question.     

Immunochemical and biological studies of antigens isolated from a strain of Bacteroides fragilis

Phenol/water-extracted lipopolysaccharide and a fraction HM, extracted with acetate buffer pH 2.0, from Bacteroides fragilis strain 62/73 are antigenically different as shown by immunodiffusion, passive haemagglutination, haemagglutination inhibition and preliminary chemical investigations. Biological activity, assessed with the local Shwartzmann reaction, was demonstrated for the lipopolysaccharide whereas antigen HM was almost inactive in this test. HM is immunogenic in rabbits. Antibodies against HM were detected in seven out of ten sera of healthy humans.     

Detection of plasmids in strains of Bacteroides ureolyticus isolated from the genital tract

Plasmids were detected in 17 of 24 clinical isolates of Bacteroides ureolyticus. All 17 strains harboured a single large plasmid of ≥24·5 MD but two also had smaller plasmids of 2·3 and 3·9 MD, respectively. The function of the plasmids is unknown.     

Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate

The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.     

Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn's disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF-COSY, TOCSY, ROESY, HSQC-TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: -->3)beta-D-Glcp(1-->6)[alpha-D-GalpNAc(1-->2)beta-D-Galp(1-->4)]beta-D-GlcpNAc(1-->3)alpha-D-Galp(1-->4)beta-D-Manp(1-->.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

Susceptibility to antimicrobial drugs of enterotoxin producing strains of Bacteroides fragilis isolated from different countries

Twenty two Bacteroides fragilis strains isolated from clinical samples in different countries (England, France, the Netherlands, Poland and USA) were used in the experiments. In all strains the presence of enterotoxin (fragilysin) gene was found by PCR with primers 404/407. Drug susceptibility of B. fragilis strains was determined with Etest (MICs for penicillin G, ceftriaxone, amoxicillin/clavulanic acid, imipenem, clindamycin and metronidazole). MICs were estimated in accordance to the NCCLS recommendations (1997). All tested strains were susceptible to imipenem and metronidazole. Twenty one strains were susceptible and one was intermediate susceptible to amoxicillin/clavulanic acid. Fourteen strains were resistant to ceftriaxone and five were found highly resistant to clindamycin. All examined strains were resistant to penicillin G. Four tested strains were simultaneously resistant to penicillin G, ceftriaxone and clindamycin (three French human strains isolated from postoperative wound, peritoneal fluid and bone inflammation, and one strain isolated from a pig).     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.