Identification and characterization of conjugative transposons CTn86 and CTn9343 in Bacteroides fragilis strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Occurrence of free ceramides in Bacteroides fragilis NCTC 9343.

The occurrence of free ceramides at high concentrations was demonstrated in the chloroform-methanol extractable lipids of Bacteroides fragilis NCTC 9343. The long-chain bases were isolated from the free ceramides and identified as branched and normal saturated dihydroxy bases with carbon chains consisting of 17, 18, and 19 atoms. The major fatty acid was 3-hydroxy 15-methylhexadecanoic acid. The major molecular species of the ceramides were identified by gas chromatography-mass spectrometry and gas chromatography of the cleaved products as LCB-d-iso17: 0-3-OH iso17: 0 FA, LCB-d-anteiso17: 0-3-OH iso17: 0 FA, LCB-d-iso18: 0-3-OH iso17: 0 FA, and LCB-d-anteiso19: 0-3-OH iso17: 0 FA.     

Characterization of proteases formed by Bacteroides fragilis

Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease:arylamidase ratios of these enzymes indicated that they were probably endopeptidases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 degrees C. Proteolysis was temperature sensitive, however (90% reduction at 60 degrees C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaCl suggested that ionic interactions were involved in the association of these enzymes with the membranes.     

Characterization of an iron-regulated alpha-enolase of Bacteroides fragilis

This study describes the identification, cloning and molecular characterization of the alpha-enolase P46 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 virulence factors have been described that may be involved in the development of these infections. Many of these virulence factors are upregulated under conditions of iron- or heme-starvation. We found a major protein of 46 kDa (P46) that is upregulated under iron-depleted conditions. This protein was identified as an alpha-enolase. Alpha-enolases in several gram-positive bacteria and eukaryotic cells are located at the cell surface and function as plasminogen-binding proteins. Localization studies demonstrated that P46 is mainly located in the cytoplasm and partly associated with the inner membrane (IM). Under iron-restricted conditions, however, P46 is localized primarily in the IM fraction. Plasminogen-binding to B. fragilis cells did occur but was not P46 dependent. A 60-kDa protein was identified as a putative plasminogen-binding protein in B. fragilis.     

Structural studies of the polysaccharide part of the cell wall lipopolysaccharide from Bacteroides fragilis NCTC 9343

The structure of the polysaccharide part of the lipopolysaccharide from Bacteroides fragilis NCTC 9343 has been determined using sugar and methylation analysis as the principal tools. Phenol--water extraction followed by a phenol--chloroform--light petroleum extraction yielded a lipopolysaccharide suitable for structural analysis. Analysis of sugars using alditol acetates showed that the polysaccharide contained L-rhamnose, D-galactose and D-glucose in the approximate molar ratios of 1:5:1. After weak acid hydrolysis, two polysaccharide fractions were isolated by gel permeation chromatography: PSI and PSII with the sugar molar ratios 1:5:1 and 1:2:1 respectively. Chromium trioxide oxidation revealed that all galactosyl residues have the beta configuration, and that the rhamnosyl and glucosyl residues have the alpha configuration. From methylation analysis of lipopolysaccharide and the PS I and PS II fractions the following structures could be deduced.     

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.     

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.     

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.     

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。     

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.     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

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.     

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.     

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.     

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.     

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.     

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.