Serological Activity of 'Bacteroides fragilis' Endotoxins—Strains Isolated in Poland

Antigens were extracted by the phenol-water method from each of the 11 strains of ' Bacteroides fragilis ' species, isolated from clinical material. Nine of these strains had been identified by the direct immunofluorescence test (IFT) as ' B. fragilis spp. fragilis ', and two as ' B. fragilis ssp. thetaiotaomicrori '.
These antigens were then used in the immunodiffusion test (ID), performed with antisera of six serotypes. Results of the ID test agreed in nine cases with those of IFT. An antigenic heterogeneity among strains of ' B. fragilis ssp. fragilis ' strains was noted. Antigens prepared in the same way from two strains did not react in the ID test with any of the antisera used although cells were positively stained in the IFT by specific anti-' B. fragilis ' serotype conjugates.     

Rapid Identification and Serotyping of 'Bacteroides fragilis' in Clinical Material by Direct Immunofluorescence

Material from 32 patients, mostly after abdominal surgery, was examined by the direct immunofluorescence test (IFT) using the conjugates against six ' Bacteroides fragilis ' serotypes. In most cases ' B. fragilis ' strains could be detected in direct smears of the clinical material. There was good agreement between the results obtained by IFT and cultural methods.     

Serological Activity of 'Bacteroides fragilis' Culture Supernatants

Culture supernatants of 17 strains of the ' Bacteroides fragilis ' group were treated with four volumes of acetone. The precipitates, after dialysis and lyophilization, were used as antigens in the double diffusion test with antisera against serotype strains of ' B. fragilis '. In the culture supernatant of one strain we did not demonstrate the presence of serologically active substances. Sixteen preparations reacted in immunodiffusion with antiserum against ' B. ovatus ' serotype B. Ten preparations reacted with antiserum B only and six preparations gave, additionally, precipitation lines with other serotype antisera (A, E₂).     

Characterization of the Batl (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems

YT135.2.8, a Tn4400' insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild-type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild-type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur-containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non-sulphur reducing agent Tris-(2-carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400' insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the Batl operon; Tn4400' inserted within batD. All of the hypothetical proteins possess one or more membrane-spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2-4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The Batl complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.     

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.     

Rapid differentiation of the species of the genus Bacteroides sensu stricto by capillary gas chromatography of cellular carbohydrates

Whole-cell hydrolysates of Bacteroides fragilis , the type species of the genus Bacteroides Castellani and Chalmers 1919, and the genetical closely related species B. vulgatus, B. ovatus, B. eggerthii, B. distasonis, B. uniformis, B. thetaiotaomicron, B. stercoris, B. merdae , and B. caccae were used to determine characteristic carbohydrate patterns by capillary gas chromatography. On the basis of the chemical derivatization of the carbohydrates seven characteristic peaks for peracetylated aldononitriles and nine characteristic peaks for peracetylated o -methyloximes were selected from the carbohydrate fingerprints of the reference strains to prepare a dichotomous identification key. The classification of an unknown strain supposed to belong to the formerly called ' Bacteroides fragilis group'is possible with this key. Some of the advantages of the technique were that the identification of Bacteroides fragilis -like strains requires only 4–5 h after primary isolation and that the bacteria can be exposed to oxygen because viability of the organisms is not necessary. Sophisticated anaerobic techniques can therefore be avoided for identification.     

Isolation and characterization of cLV25, a Bacteroides fragilis chromosomal transfer factor resembling multiple Bacteroides sp. mobilizable transposons

Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis. To further our understanding of DNA transfer in B. fragilis, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from B. fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400DeltaBglII. Similar to other Bacteroides sp. transfer factors, cLV25 was mobilized in E. coli by the conjugative plasmid R751. Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B. fragilis mobilizable transposon Tn4399. A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3' end of orf22. This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399. Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399. In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520.     

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。     

Susceptibility trending of blood isolates of the Bacteroides fragilis group over a 12-year period to clindamycin, ampicillin-sulbactam, cefoxitin, imipenem, and metronidazole

Numerous reports have described a steady overall increase in resistance among clinical isolates of the Bacteroides fragilis group to several antimicrobial agents, particularly clindamycin. Determination of resistance rates is significantly influenced by the number of isolates of each species within the B. fragilis group tested. Historically, the B. fragilis species has remained the most susceptible to most antimicrobials when compared to non-B. fragilis species. This study compares the effect of a gradually changing ratio of blood isolates of B. fragilis to non-B. fragilis species tested by broth micro-dilution over a 12-year period on selected antimicrobial agents. In 1987, the ratio of blood isolates of B. fragilis to non-B. fragilis was 68% to 32%; in 1991 it was 59% to 41%; and in 1999 it was 51% to 49%. Both metronidazole and imipenem showed the least changes because of their inherent high activity against all species. For clindamycin, decreases in susceptibility ranged from 84% to 64% for B. fragilis compared to 58% to 67% for non-B. fragilis species. Ampicillin-sulbactam showed a decrease in susceptibility in B. fragilis and non-B. fragilis species, but was highest in 1999 when the ratio of non-B. fragilis species was the highest. Overall resistance rates to cefoxitin varied from 8% to 25% during the testing years and was consistently higher among the non-B. fragilis species. These comparisons indicate that the ratio of B. fragilis group species isolated from the blood has changed over the last 12 years and has appreciably affected the resistance rates to some commonly used anti-anaerobic agents. Whether the noted changes in species isolation rates are a result of selective antibiotic pressure or other factors is yet to be determined.     

The bactericidal mechanisms of polymorphonuclear leukocytes against Bacteroides fragilis: significance of the oxygen-dependent system

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen-radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2- and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen-dependent and oxygen-independent mechanisms.     

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.     

The effect of human bile on Bacteroides fragilis in health and disease

Bacteroides fragilis is generally believed to be stimulated by bile. Although B. fragilis is rarely found in the human duodenum, it is relatively frequent in gall bladder infections. To investigate this paradox, the growth of B. fragilis in the human bile in both health and disease was studied, and compared with the effect of bovine and porcine bile. B. fragilis was stimulated by the bovine bile and inhibited by the porcine bile. The normal human bile was either bacteriostatic or inhibitory whereas bile from cholelithiatic patients, in 50% of cases, stimulated the growth of B. fragilis. This explains the relative prevalence of B. fragilis in cholelithiatic cholecystitis patients although it is not a resident flora of the duodenum.     

Detection of Dientamoeba fragilis in fresh stool specimens using PCR

Dientamoeba fragilis is a trichomonad parasite that causes human gastrointestinal disease. Currently microscopy is considered to be the gold standard for diagnosis of D. fragilis infection. However, this method is time-consuming and relatively insensitive. A PCR assay based on the small-subunit ribosomal RNA gene of D. fragilis for the specific detection of D. fragilis DNA in fresh unpreserved stool samples was developed. The D. fragilis PCR was positive in 29/31 samples with positive microscopy and did not cross-react with other protozoan parasites. The PCR protocol showed a specificity of 100% and a sensitivity of 93.5% and the entire procedure can be performed in one day.     

The human commensal Bacteroides fragilis binds intestinal mucin

The mammalian gastrointestinal tract harbors a vast microbial ecosystem, known as the microbiota, which benefits host biology. Bacteroides fragilis is an important anaerobic gut commensal of humans that prevents and cures intestinal inflammation. We wished to elucidate aspects of gut colonization employed by B. fragilis. Fluorescence in situ hybridization was performed on colonic tissue sections from B. fragilis and Escherichia coli dual-colonized gnotobiotic mice. Epifluorescence imaging reveals that both E. coli and B. fragilis are found in the lumen of the colon, but only B. fragilis is found in the mucosal layer. This observation suggests that physical association with intestinal mucus could be a possible mechanism of gut colonization by B. fragilis. We investigated this potential interaction using an in vitro mucus binding assay and show here that B. fragilis binds to murine colonic mucus. We further demonstrate that B. fragilis specifically and quantitatively binds to highly purified mucins (the major constituent in intestinal mucus) using flow cytometry analysis of fluorescently labeled purified murine and porcine mucins. These results suggest that interactions between B. fragilis and intestinal mucin may play a critical role during host-bacterial symbiosis.     

Prevalence of enterotoxigenic Bacteroides fragilis strains (ETBF) in the gut of children with clinical diagnosis of antibiotic associated diarrhoea (AAD)

Prevalence of enterotoxin producing Bacteroides fragilis (ETBF) strains in faecal samples of children with clinical diagnosis antibiotic associated diarrhoea (AAD) was investigated. Out of faecal samples collected from sixty children, thirty C. difficile strains were isolated. Enterotoxigenic B. fragilis (ETBF) strains were cultured from four children what made 6.7% of investigated faecal samples. Out of two samples toxinogenic C. difficile strains [tox A(+) tox B(+)] were cultured together with enterotoxinogenic B. fragilis. From sample of one child C. difficile A negative/B positive strains [tox A(-) tox B(+)] was found together with B. fragilis (ETBF). From faecal sample of one child enterotoxinogenic B. fragilis only was isolated. It was shown that in the gut of children with clinical diagnosis of (AAD) enterotoxinogenic B. fragilis (ETBF) can be present. B. fragilis (ETBF) can be observed in concomitance with toxinogenic C. difficile.     

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.     

Classification of Kobresia fragilis C. B. Clarke (Cyperaceae) and related taxa

The classification of Kobresia fragilis C. B. Clarke and related taxa were studied based on gross morphology and nutlet epidermal micromorphology. About 13 names have been involved in the species group. They could not be clearly defined using the morphological characters employed by previous authors. Thus, more than 200 sheets of 89 specimens of related taxa were studied thoroughly, and the taxonomic significance of morpbological characters was re-evaluated. In order to further understand the classification of K. fragilis and its allies, micromorphology of nutlet epidermis of these taxa was observed under scanning electron microscope. Based on the results, K. curvata C. B. Clarke and K. fragilis were recognized. Other names were treated as synonymies of K. fragilis. K. clarkeana (Kükenthal) Kükenthal, K. clarkeana var. megalantha Kükenthal and K. curticeps (C. B. Clarke) Kükenthal var. gyirongensis Y. C. Yang were reduced to new synonymies of K. fragilis. K. curvata was distinct from K. fragilis on both morphological and micromorphological basis. One specimen of K. fragilis collected from Luqu, Gansu, China, represents anew distribution record of this species in Gansu.