Neuraminidase in Bacteroides fragilis.

A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin.     

Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Antibiotic susceptibility of Bacteroides fragilis group strains in Hungary

Resistance rates to different antibiotics of 495 Bacteroides fragilis group strains were followed between 1987 and 1994 in Hungary. In 1992 the strains were collected in three different laboratories, whereas during the other periods strains were isolated in one centre. Metronidazole, chloramphenicol, imipenem and amoxicillin/clavulanic acid were the most active drugs. A high level of resistance was observed in 1987 for ampicillin (88% at > 4 mg/L), erythromycin (51% at > 4 mg/L), tetracyclin (53% at > 8 mg/L) and clindamycin (27% at > 4 mg/L). The same level of resistance was seen during the further years for clindamycin and ampicillin. Resistance to cefoxitin increased from 6% to 11% between 1987 and 1993/1994. No differences in resistance rates were observed between the strains collected in the three centers. For 100 strains, the results of the E test were compared with those of the micro-broth dilution test, both being used routinely for testing the antibiotic susceptibility of Bacteroides fragilis group strains in this period.     

Nutritional Features of Bacteroides fragilis subsp. fragilis

Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B₁₂, minerals, bicarbonate-carbon dioxide buffer, NH₄Cl, and sulfide. The vitamin B₁₂ requirement of 0.1 ng/ml was replaced with 7.5 μg of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, β-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B₁₂, or related compounds, must be available during much of the time.     

Transferable 5-nitroimidazole resistance in the Bacteroides fragilis group

We report the characterization of a strain of Bacteroides vulgatus, BV17, that exhibits a moderate resistance to 5-nitroimidazoles and carries plasmids of 4.5, 5, 7.7, and 56 kb. A genetic determinant involved in this resistance is carried by the 7.7 +/- 0.2-kb plasmid (pIP417). This plasmid can be introduced and replicated in a sensitive strain of B. fragilis 638R by transformation or by conjugation. In the latter case, the transfer may involve mobilization by the 56-kb conjugative plasmid (pIP418) regularly found in transconjugants but not in transformants.     

Growth of Bacteroides fragilis group in agar and broth media

The rate of bacterial growth of four Bacteroides fragilis group organisms was determined in agar and broth media. Exponential bacterial growth occurred in agar media within 4 to 8 h, while such growth was delayed in broth media and occurred within 12-24 h after inoculation. This phenomenon may explain why antimicrobials which manifest an 'inoculum effect' may show increased resistance to antimicrobials when tested in agar media.     

Recovery of Bacteroides fragilis group from clinical specimens following antimicrobial therapy.

Over a period of 14 years (1973-1987), 3165 specimens submitted to the microbiology laboratory demonstrated the recovery of anaerobic bacteria. A total of 988 Bacteroides fragilis group isolates were recovered (0.3 isolates per specimen). Bacteroides fragilis accounted for 62% of the total of all B. fragilis group isolates, Bacteroides thetaiotaomicron for 15%, Bacteroides vulgatus for 8%, Bacteroides ovatus for 7%, Bacteroides distasonis for 6%, and Bacteroides uniformis for 2%. Of the 988 B. fragilis group isolates, 310 (31%) were recovered after the administration of antimicrobial therapy, and 129 (13%) were the single isolate recovered from the infected site at that time. The recovery rate of all members of B. fragilis group after the administration of antimicrobial therapy, when isolated alone or when mixed with other bacteria, was similar. The data illustrate the equal ability of all members of the B. fragilis group to persist in and to contribute to the inflammatory process; and provide further support for their pathogenic role.     

Evaluation of the Pathogenicity of the Bacteroides fragilis Toxin Gene Subtypes in Gnotobiotic Mice

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.     

Intracellular polysaccharide of Bacteroides fragilis.

Formation of iodophilic polysaccharide (IPS) from glucose was demonstrated in 27 strains of Bacteroides fragilis. Synthesis was dependent on the glucose concentration of the medium, the pH and the growth phase. When glucose was in short supply the cellular polysaccharide was degraded rapidly at pH 4.5 to 6.5 and fatty acids accumulated in the medium. Storage of IPS was not responsible for the low carbon recoveries observed in fermentation balance studies. In electron micrographs of thin sections, the IPS was observed as cytoplasmic granules dispersed throughout the whole cell. After extraction and purification the IPS was characterized as a glycogen.     

Bacteriophage-associated spherical bodies in Bacteroides fragilis.

Unique spherical bodies with multilayered walls were observed by electron microscopy in cells of a single strain of Bacteroides fragilis subsp. fragilis. Phage-like particles were present in the same cells, both free in the cytoplasm and within the spheres. The proportion of cells containing the phage-associated spherical structures ranged from less than 0.01% to about 7% depending on the culture conditions. Phage particles of morphological type B and spherical bodies were also found free in the medium surrounding the cells. Spherical bodies with discontinuities in their walls, through which phage-like particles sometimes appeared to be escaping, were also found both intra- and extracellularly. The biological significance of these distinctive spherical structures is a matter of conjecture.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Prevalence of the Bacteroides fragilis Group and Enterotoxigenic Bacteroides fragilis in Immunodeficient Children

Members of the Bacteroides fragilis group are indigenous to the human and animal intestinal microbiota and they are responsible for several endogenous infections. Enterotoxigenic B. fragilis (ETBF) has been associated with acute diarrhea in children and farm animals. Immunodeficient patients are more predisposed to different opportunistic infections, including anaerobic infections. In this study, 130 stool samples were analysed from 56 immunodeficient and 74 healthy children. Enterotoxin production was detected by cytotoxicity assay on HT-29 cells and by PCR. B. fragilis sensu strictu was prevalent in both groups and ETBF species was detected from a single stool sample belonged to an immunodeficient child with AIDS.     

Haemagglutination of Bacteroides fragilis

Abstract The relationship between the ability to cause haemagglutination (HA) and the presence of capsules and/or pili was examined for 50 clinical isolates of Bacteroides fragilis . HA was tested using a slide technique, and bovine, porcine, guinea pig, rat, rabbit, horse, human, chicken and pigeon erythrocytes. Chicken and pigeon erythrocytes were the best indicators for HA with 43 (86%) of the strains tested causing HA and 39 (78%) with strong reactions. Capsule staining showed that the same 43 strains causing HA also produced a demonstrable capsule. No pili were found on either encapsulated or non-encapsulated strains using transmission electron microscopy. These results suggest that adherence of B. fragilis is related to the presence of capsular material, not pili.     

Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

Enumeration of enterotoxigenic Bacteroides fragilis in municipal sewage.

Of 237 isolates of Bacteroides fragilis from sewage influent at the Bozeman, Mont., wastewater treatment plant, 22 (9.3%) were enterotoxigenic, as indicated by the ability to elicit fluid accumulation in the lamb ileal loop test. It appears that enterotoxigenic B. fragilis is endemic in the human population at a moderately high level.     

Carbohydrate repression of catalase synthesis in Bacteroides fragilis.

Catalase formation by Bacteroides fragilis was immediately stopped upon addition of glucose to a culture growing in peptone medium. Each of eight other carbohydrates fermented by the organism also repressed catalase formation. Without added carbohydrate, the strains produced relatively large amounts of catalase (25 to 50 U/mg of protein).     

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin, penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 micron pores were significantly more efficient than those with 0.45 micron pores in the isolation of BFG. A preliminary incubation period of 4 h at 30 degrees C prior to 44 h at 37 degrees C yielded significantly higher numbers of BFG than direct incubation at 37 degrees C for 48 h.     

Bacteriophages active against Bacteroides fragilis in sewage-polluted waters.

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.