A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.     

Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.     

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis

The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP). bftP is 1191 nucleotides coding for a 397 amino acid protein of 44.4 kDa. The toxin has a signal peptide of 18 amino acids that is typical of many lipoproteins followed by a 379 amino acid protoxin. The portion of the protoxin found in culture filtrates and stools begins at amino acid 212. An additional open reading frame located immediately upstream shows some sequence identity with cobra cytotoxins. If expressed, the ORF protein product could also play a role in the virulence of B. fragilis.     

Toxins of Bacteroides fragilis and Bacteroides thetaiotaomicron rods as stimulators of adhesion molecule expression on the surface of vascular endothelial cells

Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF), and from three B. thetaiotaomicron strains were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the procedure of van Tassell et al. (1992). The influence of the examined toxins on the expression of adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human dermal microvascular endothelial cells) was assayed in ELISA test with monoclonal antibodies. Four concentrations of toxins were applied: 0.01, 0.1, 1.0 and 10.0 (micrograms/ml). Endothelial cells were activated for 24 hours (ICAM-1 and VCAM-1 expression) and for 4 hours (E-selectin expression). The coloured product of immunoenzymatic reaction was measured by reading the absorbance at wavelength 492 nm. Two controls were performed in each experiment: with resting HMEC-1 and E. coli O55:B5 LPS (Sigma, USA). Bacteroides fragilis and B. thetaiotaomicron lipopolysaccharides stimulated three adhesion molecules under investigation. Their activity was comparable, but weaker than the activity of E. coli O55:B5 LPS. ICAM-1 was the most stimulated molecule. B. fragilis enterotoxin induced two adhesion molecules: VCAM-1 and E-selectin demonstrating weaker stimulatory activity than E. coli LPS. Stimulation of adhesion molecules on vascular endothelial cells should be considered to be a biological activity of B. fragilis and B. thetaiotaomicron endotoxins and B. fragilis enterotoxin.     

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

AIMS: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. METHODS AND RESULTS: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. CONCLUSIONS: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.     

Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting

PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.     

Isolation or group B streptococci defective for the type antigen Ia by transposon-mediated mutagenesis

Transposon-mediated mutability has been used to isolate the isogenic strains of the group B serotype Ia streptococci with the mutations in the genes coding for the production of capsular antigen. The transconjugants have lost the ability to bind type Ia antiserum as demonstrated by immunoblotting technique. The loss of type-specific antigen by the strains has resulted in a dramatic decrease in virulence for mice while the absorbtion indexes of transconjugants increased 2-3 fold. The mutant clones deficient in capsule expression had the increased buoyant density in the percoll gradient as compared with the parent strains of group B. The stable mutants impaired in ability to produce the polysaccharide capsule on the cell surface were obtained as a result of site-specific insertion of transposon Tn916 into the genome of group B serotype I streptococcus.     

Anaerobic gram-negative faecal flora in patients with Crohn's Disease and healthy subjects

The anaerobic gram-negative faecal flora of five patients with Crohn's Disease (CD) was identified and compared with that of healthy subjects. For isolation and cultivation of the anaerobic gram-negative rods a non-selective medium was used. There were no significant differences in numbers of Bacteroides and Fusobacterium spp. between patients with CD and healthy subjects. However, the numbers of the "Bacteroides fragilis" group were significantly higher in patients than in controls. The high numbers of the "B. fragilis" group in the faeces of patients were particularly due to B. vulgatus which was 6 times more frequent in patients than in healthy subjects. This indicated that B. vulgatus was responsible for the higher numbers of anaerobic gram-negative rods in the faecal flora of patients with CD.     

Pbp, a cell-surface exposed plasminogen binding protein of Bacteroides fragilis

The Gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine, but is also frequently found in severe intra-abdominal infections. Several B. fragilis virulence factors have been implicated in the development of these infections. A B. fragilis protein of circa 60-kDa was identified as a putative plasminogen binding protein (Pbp). The corresponding gene was located, cloned, sequenced and the subcellular localization of the protein was investigated. Pbp was both determined in the outer membrane of B. fragilis and of E. coli that expressed the cloned protein. Protease accessibility studies showed that the protein is expressed at the cell surface. Importantly, we demonstrated that Pbp is sufficient and required for plasminogen binding to whole cells in both E. coli and B. fragilis. Pbp-like proteins were also detected in some other Bacteroides subspecies. The role of this potential B. fragilis virulence factor in pathogenicity is discussed.     

Bacteroides fragilis adherence to Caco-2 cells

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.     

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.     

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60 masculine C, for 30 min, and among them, 12 (18.75%) were resistant at 60 masculine C, for 60 min. Also, none strain was resistant at 100 masculine C. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.     

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.     

Identification of Bacteroides fragilisby a modified immuno polymerase chain reaction (mIPCR), using a specific monoclonal antibody

Bacteroides fragilis is the anaerobic species most commonly isolated from human clinical specimens, and is resistant to many antimicrobial agents. A monoclonal antibody, mAb4H8 (IgG3), reacting with a specific epitope in the lipopolysaccharide (LPS) isolated from most of the B. fragilis strains, was produced and employed with modified Immuno Polymerase Chain Reaction (mIPCR) for identification of B. fragilis with a detection limit of 10(4) cfu/mL bacterial suspension. A number of bacterial strains were examined, including B. fragilis, Bacteroides spp. other than B. fragilis and other genera. All the B. fragilis strains with the immunodominant (beta1,6-linked D-galactosyl chain) epitope were positive. None of the other strains showed the positive reaction. The results indicate that mIPCR assay with mAb4H8 has a high specificity and high sensitivity.     

Bacteroides and appendicitis (author's transl)]

Bacteroides fragilis is frequently recovered from cases of appendicitis with perforation and from infections developing secondary to appendicitis. In order to assess the part played by B. fragilis in the aetiology of appendicitis, quantitative aerobic and anaerobic culture studies of the contents of 49 inflammated appendices were performed. Anaerobic gram-negative non-sporing rods were cultivated from 43 appendices in the range 10(3)-10(9)/g. A total of 1,473 isolates was differentiated by biochemical methods, and 1,374 cultures were found to belong to the saccharolytic species of the genus Bacteroides (B. fragilis, B. thetaiotaomicron, B. vulgatus, B. distasonis etc.). B fragilis was detected in 31 appendices; the species predominated in 18 samples. B theraiotamicron, recovered from 27 samples, was prevalent in 4 appendices. In one sample, B. fragilis and B. thetaiotaomicron outnumbered the other appendicular bacterial. B. vulgatus was cultivated from 12 appendices, but did once constitute the prevalent group. It has been previously shown that B. vulgatus (43% of intestinal isolates) and B. thetaiomicron predominate in the normal narge bowel flora. On the other hand, approximately 80% of pyrogenic Bacteroides strains belong to B. fragilis, B. thetaiotaomicron accounting for 19% and B. vulgatus being virtually absent. From these striking differences in species distribution the conclusion was drawn that B. fragilis possesses the highest virulence for man. Species distribution within the 1,374 appendicular isolates of saccharolytic Bacteroides (percentages of 62, 19 and 4.3 for B. fragilis, B. thetaiotaomicron, and B. vulgatus, respectively) was very similar to that encountered in clinical specimens. From the results obtained it becomes evident that pyrogenic Bacteroides, in particular B. fragilis, plays an important role in nearly 50% of cases of appendicitis.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.     

Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.