Isolation of a ferritin from Bacteroides fragilis

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Characterization of restriction endonuclease BfrBI from Bacteroides fragilis strains BE3 and AIP 10006

A new type II restriction endonuclease, named BfrBI, was detected in two strains of Bacteroides fragilis, BE3 and AIP 10006 (NCTC 9343T). The enzyme BfrBI, an isoschizomer of NsiI and AvaIII, recognized the hexanucleotide sequence [5'-ATG decreases CAT-3'], with a cleavage site generating blunt ends.     

脆弱拟杆菌的研究进展

脆弱拟杆菌是定殖于哺乳动物肠道中的共生菌,同时也是临床感染病例中常见的条件致病菌。本文从致病及益生特性两大方面综述脆弱拟杆菌的研究现状,着重讨论了脆弱拟杆菌作为潜在益生菌在预防和治疗糖尿病及免疫性疾病中所起的重要作用,从而为筛选及应用益生脆弱拟杆菌菌株提供一定的参考。     

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.     

脆弱类杆菌属菌株的培养及免疫血清的制备

本文报告了从国外引进的五种类杆菌培养,鉴定及其抗血清的制备。结果表明这些类杆菌在牛心脑培养基中容易生长。均为厌氧性革兰氏阴性无芽孢多形态杆菌。经生化反应鉴定结果发现脆弱类杆菌对红霉素(60μg)、利福平(15μg)敏感,而对多粘菌素B(10μg)、青霉素(2IU)、卡那霉素(1000μg),万古霉素(5μg)耐受。用不同接种途径免疫家兔所获得的脆弱类杆菌抗血清的结果表明皮下加不完全佐剂组血清中的抗体滴度明显高于一般皮下组及静脉注射组。     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.     

Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.     

Cloning and expression of the Bacteroides fragilis YCH46 neuraminidase gene in Eschirichia coli and Bacteroides uniformis

Abstract A neuraminidase-encoding gene nanH of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The nanH gene was subcloned from the cosmid and was located within a 2.2-kb Xho I- Kpn I fragment. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial chromosome. Neuraminidase activity expressed in the initial Escherichia coli clone was approximately 3600-fold lower than that expressed in B. fragilis YCH46. However, when nanH was transferred from E. coli to B. uniformis by mobilization of a shuttle plasmid, the transconjugant expressed 1100-fold higher activity than the E. coli donor did. These results suggest that modes of nanH expression in E. coli and Bacteroides are heterologous.     

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.     

Cytokine release and expression induced by OmpA proteins from the Gram-negative anaerobes, Porphyromonas asaccharolytica and Bacteroides fragilis

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.     

A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation

High-level carbapenem-resistant (CpmR) mutants, with MICs for imipenem and carbapenem of greater than 128 micrograms/ml, were selected in vitro from four carbapenem-susceptible (CpmS) clinical isolates of Bacteroides fragilis. The CpmS strains produced very low levels of beta-lactamase activity, which was increased approx. 50- to 100-fold in the CpmR mutants. Isoelectric focussing and enzyme kinetic analysis (Km and Vrel) of the 'carbapenemases' from the CpmR mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584-2593) hybridized with DNA from the CpmR mutants, their CpmS parental strains as well as clinical CpmR isolates, but not from randomly chosen carbapenem-susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.     

Effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of Bacteroides fragilis

The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.     

Electron microscopic visualization of the outer membrane permeability of Bacteroides fragilis

Abstract Electron microscopy of negatively stained Bacteroides fragilis cells treated with hypertonic solutions of di- or trisaccharides showed the shrinkage of whole cells, whereas the pentose- or hexose-treated cells shrank less significantly. Electron microscopy of thin sections of the monosaccharide-treated cells showed plasmolysis. Determination of the diffusion rate by the liposome-swelling method suggested that the apparent exclusion limit of the outer membrane pore is close to the size of uncharged saccharides of approx. M _r 250. These results suggested that the outer membrane of B. fragilis contains small diffusion pores.     

The use of bacteriophages of Bacteroides fragilis as indicators of the efficiency of virucidal products

The potential use of bacteriophage B40-8 of Bacteroides fragilis for the evaluation of the virucidal activity of antiseptics or disinfectants was investigated. The antiviral activity of two antiseptics and two disinfectants was evaluated according to a standard guideline. The effect of the virucidal agents was assessed on (i) viruses usually spread by direct contact with surfaces with contaminated secretions, i.e. herpes virus 1 and 2, and vaccinia virus, and (ii) viruses transmitted by the fecal-oral route, i.e. hepatitis A virus, poliovirus, adenovirus and rotavirus. The survival of B40-8 always equalled or exceeded that of the animal viruses tested. Our data suggest the use of bacteriophage B40-8 to complement the information furnished by some standardized methods in ascertaining the antiviral activity of virucidal preparations.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

A new subtype of the metalloprotease toxin gene and the incidence of the three bft subtypes among Bacteroides fragilis isolates in Japan

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.     

The inactivation of bacteriophages infecting Bacteroides fragilis by chlorine treatment and UV-irradiation

Abstract The sensitivity to chlorination and to UV-irradiation of bacteriophage B40-8, which infects Bacteroides fragilis , was evaluated in comparison to that of Escherichia coli, Streptococcus faecalis , poliovirus 1, simian rotavirus 11 and coliphage f2. The results indicated that viruses persisted longer than bacteria in the presence of both disinfectants. Phage B40-8 was the most resistant microorganism to chlorination while coliphage f2 was the most resistant to UV-irradiation. In the latter, phage B40-8 was nevertheless as resistant as poliovirus and rotavirus. As expected, all microorganisms were more resistant to inactivation in sewage water than in tapwater.