Identification of the Metallo-β-Lactamase Gene from Clinical Isolates of Bacteroides fragilis

Bacteroides fragilis is one of the organisms known to produce carbapenem-hydrolysing metallo-β-lactamase, which can confer resistance to a wide variety of β-lactams. The purpose of this study was to identify carbapenem-hydrolysing metallo-β-lactamase-producing B. fragilis strains by means of PCR assay, nucleotide sequencing and enzyme inhibition studies. Ten β-lactam-resistant B. fragilis isolates were investigated. Four imipenem-resistant strains among the 10 isolates gave positive reactions in the PCR assay. The nucleotide sequences of the PCR products from two imipenem-resistant strains shared >98% similarity with the metallo-β-lactamase gene from B. fragilis TAL 3636, which was used as a control. The amino acid sequence homology between the two imipenem-resistant strains and B. fragilis TAL 3636 was 99.2%. These strains produced high amounts of Zn²⁺-dependent β-lactamases which were inactivated by EDTA.     

Detection ofBacteroides fragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis

The ability of counter immunoelectrophoresis (CIE) to detectBacteroides fragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection ofB. fragilis endotoxin in amniotic fluid in a concentration of 40 ng/ml or more. The sensitivity threshold was reduced to 2 ng/ml by using a highly reactive IgG-fraction isolated from rabbit anti-B. fragilis IPL E 323 antiserum.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

Enterotoxin-producing Bacteroides fragilis (ETBF) Strains in Stool Samples Submitted for Testing ofClostridium difficile and its Toxins

Fifty faecal samples of patients suspected of having diarrhoea associated with Clostridium difficile were studied. Toxins of C. difficile were tested in vivo directly from the faecal sample using Toxin Detection Kits (Oxoid) to detect toxin A and primers for detection genes of Toxin A and B in a PCR test. The same samples were tested for B. fragilis enterotoxin gene directly from the faecal sample using special primers and a PCR test. Samples were inoculated onto selective media for C. difficile (CCCA) and B. fragilis (BBE) for isolation of bacteria.In vitro Toxin A of C. difficile in culture was tested using a C. difficile toxin A immunoassay (Oxoid, U.K. test and Toxin B of C. difficile was tested by using the McCoy cell line. C. difficile toxin A and B genes were determined in DNA of isolated strains using special primers and a PCR reaction. The enterotoxin production in B. fragilis strains was tested on the human carcinoma cell line HT29/C1. The presence of fragilysin gene was detected using a special pair of primers and a PCR reaction. Toxinogenic strains of C. difficile and enterotoxigenic Bacteroides fragilis (ETBF) strains were isolated from the same samples.     

Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination

A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination.     

Mechanisms of tetracycline and rifampicin resistance inBacteroides fragilis mutants obtained by ionizing radiation

Summary Based on a dose-survival curve, a radiation dose of 3.99 C/kg was used to induce antibiotic-resistant mutants inBacteroides fragilis. Escherichia coli B/r membrane fragments were employed as a reducing agent. Antibiotic-resistant mutants ofB. fragilis were utilized to study the mechanism by which these organisms become resistant to selected chemotherapeutic agents. Decreased accumulation of tetracycline by resistant mutants ofB. fragilis suggests that the resistance to this antibiotic is associated with the outer membrane permeability. There is a marked difference in the inhibitory action of rifampicin on RNA polymerase activity in rifampicin-sensitive and-resistant strains ofB. fragilis. This enzyme is, therefore, the likely target for inhibition of bacterial growth in this anaerobe by rifampicin.     

Homogeneity of the Morphological Groups of Bacteriophages Infecting <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> Strain HSP40 and Strain RYC2056

Bacteriophages infecting Bacteroides fragilis strains RYC2056 and HSP40 have been proposed as indicators of water quality. To accomplish this function, homogeneity of the group of phages detected by these strains is necessary to ensure that the final results are not due to the different kinetics of inactivation of the phages. To evaluate homogeneity, we observed by electron microscopy bacteriophages isolated from sewage with two Bacteroides fragilis strains (HSP40 and RYC2056). A predominant group of phages was observed, Siphoviridae with slightly curved tails. Detection of other minority groups, which could be present in the sample, was done with neutralization experiments by using antiserum against the majority group and with host mutants resistant to infection with the predominant phage. Although two other minority groups were observed, results showed that bacteriophages infecting B. fragilis strain HSP40 and strain RYC2056 form a homogeneous group, Siphoviridae with slightly curved tails being the most predominant in sewage. Received: 7 March 2002 / Accepted: 5 August 2002     

Comparison of Cytokine Induction by Lipopolysaccharide of Bacteroides fragilis with Salmonella typhimurium in Mice

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor-alpha, interferon-gamma and interleukin-6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 μg/mouse) was much weaker compared with S. typhimurium LPS (50 μg/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 μg/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD₅₀ for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.     

Pattern III Non-toxigenic Bacteroides fragilis (NTBF) Strains in Brazil

Bacteroides fragilis strains isolated from faeces of diarrhoeic and healthy children were studied by polymerase chain reaction (PCR), in order to characterise them as enterotoxigenic B. fragilis -ETBF—if they have one of the three bft gene alleles (pattern I) or as non-toxigenic B. fragilis—NTBF—if there was an absence of bft gene alleles and specific sites (flanking region of B. fragilis Pathogenicity Island—BfPAI) (pattern II NTBF) or absence of alleles, but the presence of this specific sites (pattern III NTBF). All strains were previously screened for cytotoxic activity. ETBF was detected in 1.5% (1/66) of the samples, in which we could verify, concomitantly, the presence of Escherichia coli enteroaggregative (EAEC). Due to these data, ETBF could not be associated with diarrhoea. A large number of pattern III NTBF strains were observed, which could suggest future changes in the phenotype of enterovirulence of B. fragilis species in our country. These populations were also analysed by using AP-PCR and a great heterogeneity could be observed. We were not able to make a correlation between enterovirulence patterns and genetic types.     

Close encounters of the type‐six kind: injected bacterial toxins modulate gut microbial composition

Bacteria of the phylum Bacteroidetes constitute a substantial portion of the human gut microbiota, including symbionts and opportunistic pathogens. How these bacteria coexist and provide colonization resistance to pathogenic strains is not well understood. In this issue of EMBO Reports, Hecht and colleagues describe a mechanism by which strains of Bacteroides fragilis compete with each other for an intestinal niche 1 . Prompted by the observation that B. fragilis populations appear to be dominated by either commensal, non‐toxigenic strains, or by enterotoxigenic, potentially pathogenic strains, the authors investigated mechanisms of competition between these two subsets. In agreement with two recent studies 2 3 , Hecht et al 1 found that competition between B. fragilis strains is dependent on a type‐6 secretion system (T6SS) apparatus, secreted effectors, and immunity genes. They identify a T6SS effector–immunity gene pair that enables a non‐toxigenic strain to competitively exclude enterotoxigenic B. fragilis, thus providing a proof of principle for the use of T6SS‐mediated killing as a therapeutic strategy to eradicate pathogenic strains.     

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll‐like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF‐κB‐α) and mitogen‐activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin‐6, chemokine (C‐X‐C motif) ligand 1 (CXCL1) and chemokine (C‐C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2‐deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF‐κB‐α and c‐Jun N‐terminal kinase mitogen‐activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2‐deficient cells,. Inhibitor assay revealed that NF‐κB and MAPKs are essential for B. fragilis‐induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.     

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.     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

Plasmid-Related Resistance to Cefoxitin in Species of the Bacteroides fragilis Group Isolated from Intestinal Tracts of Calves

Species of the Bacteroides fragilis group are considered the most common anaerobe in human and animal infections and also harbor plasmids conferring resistance to several antibiotics. In this study, resistance to cefoxitin, plasmid profile and β-lactamase production in species of the B. fragilis group isolated from intestinal tracts of calves were evaluated. One hundred sixty-one B. fragilis group bacteria isolated from calves with and without diarrhea were analyzed. Cefoxitin susceptibility was performed using an agar dilution method, β-lactamase production by using a nitrocefin method, and plasmid extraction by using a commercial kit. Minimal inhibitory concentration values for cefoxitin ranged from 32 to > 512 μg/ml, and 47 bacteria (29.2%) were resistant to cefoxitin (breakpoint 16 μl). Only seven isolates harbored plasmids varying from 6.0 to 5.0 kb, and a 5.5-kb plasmid in B. vulgatus Bd26e and B. fragilis Bc5j might be related to cefoxitin resistance. β-lactamase was detected in 33 (70.2%) isolates. The cepA gene was observed in total DNA and in the 5.5-kb plasmid. The plasmid presence in organisms isolated from cattle may be important in ecologic terms, and it needs further study.     

Strain competition restricts colonization of an enteric pathogen and prevents colitis

The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non‐toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis. We observed strain‐specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector–immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non‐toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non‐toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.     

Purification and Characterization of a Fibrinogen-Degrading Protease in Bacteroides fragilis Strain YCH46

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425-fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, K_m, and V_max for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg²⁺, Cu²⁺, Zn²⁺, diisopropyl fluorophosphate, N-ethylmaleimide or p-chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine-thiol-like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl-alanine p-nitroanilide, L -valyl-alanine p-nitroanilide, alanyl-alanyl-valyl-alanine p-nitroanilide, and glycyl-proline p-nitroanilide, but was inert toward L -alanine p-nitroanilide, alanyl-alanyl-alanine p-nitroanilide, and N-α-benzoyl-DL -arginine p-nitroanilide. The protease completely hydrolyzed the α-chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease-treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.     

Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).     

A Simple Bile-Disk Method for the Identification of Bacteroides fragilis

A simple method for the separation and identification of Bacteroides fragilis from other Bacteroides species through the use of oxgall-impregnated filter paper disks is described. As in the antibiotic sensitivity test, filter paper disks containing defined amounts of bile salt were placed on a lawn of bacterial cells on Gifu anaerobic medium agar plates. Bile-resistant bacteria were identified as strains of Bacteroides fragilis after incubation in anaerobic jars containing hydrogen and carbon dioxide generators at 35 C for 24 hr. The optimum concentration of bile salt, yielding results very similar to those of the conventional tube method, was determined to be 25 mg per disk, empirically. Since this method is easy to perform, time saving, economical and gives clear results, it may be readily used as a daily routine test in the clinical microbiology laboratory.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.