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Ultrastructure of Bacteroides species: Bacteroides asaccharolyticus, Bacteroides fragilis, Bacteroides melaninogenicus subspecies melaninogenicus, and B. melaninogenicus subspecies intermedius.

Representative strains of two subspecies of Bacteroides melaninogenicus (subspecies melaninogenicus and subspecies intermedius) and Bacteroides asaccharolyticus as well as B. asaccharolyticus strain 536B isolated from a human perirectal abscess and Bacteroides fragilis ATCC 25285 were examined by glutaraldehyde-osmium fixation, ruthenium red fixation and staining, and thorium hydroxyde staining as well as by the physical preparative techniques of critical point drying--transmission electron microscopy (CPD--TEM) and scanning electron microscopy (SEM). All strains, with the exception of B. fragilis 25285, possessed an electron-dense material external to their outer membranes. Ruthenium red staining further revealed a layer, external to the surface of the outer membrane, that was distinct for each species examined. Thorium hydroxide, as well as CPD--TEM and SEM, showed the cells to be interconnected by thin fibers that not only connected adjacent cells but also traversed several microns to connect cell aggregates.

Bacteroides↗

Bacteroides barnesiae sp. nov., Bacteroides salanitronis sp. nov. and Bacteroides gallinarum sp. nov., isolated from chicken caecum.

Eight bacterial strains isolated from the caecum of chicken, BL2(T), BL66, EG3, EG6, M27, BL78(T), C35(T) and C43, were characterized by determining their phenotypic characteristics, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Bacteroides. One group of five strains (BL2(T), BL66, EG3, EG6 and M27) was related most closely to Bacteroides coprocola JCM 12979(T), with approximately 93 % 16S rRNA gene sequence similarity, and to Bacteroides plebeius JCM 12973(T), with about 92 % similarity, and shared >or=99.6 % similarity with each other. Strain BL78(T) exhibited 90.5 % similarity to B. plebeius JCM 12973(T) and 89.8 % similarity to B. coprocola JCM 12979(T) and differed from the above group of five strains at >or=10 % sequence divergence. Strains C35(T) and C43 were related most closely to Bacteroides eggerthii JCM 12986(T), with 95.1 % sequence similarity, to Bacteroides stercoris JCM 9496(T), with 94.6 % similarity, and to Bacteroides uniformis JCM 5828(T), with 94.4 % similarity, and shared 100 % similarity with each other. From results of phenotypic examination, cellular fatty acid composition analysis, menaquinone composition analysis and DNA G+C contents, the group of five strains as well as strain BL78(T) were shown to differ from the type strains of B. coprocola and B. plebeius. Strain BL78(T) differed from the others based on its menaquinone composition, which included MK-11 and MK-12. Strains C35(T) and C43 could also be differentiated from the type strains of B. eggerthii, B. stercoris and B. uniformis. The group of five strains, strain BL78(T), B. coprocola JCM 12979(T) and B. plebeius JCM 12973(T) showed low levels of DNA-DNA relatedness (<35 %) with each other. High levels of DNA-DNA relatedness were obtained within the group of five strains (>75 %). Strains C35(T) and C43 exhibited a high level of DNA-DNA relatedness (>88 %) with each other, but low levels with B. eggerthii JCM 12986(T) (<40 %), B. stercoris JCM 9496(T) (<37 %) and B. uniformis JCM 5828(T) (<16 %). On the basis of these data, three novel Bacteroides species are proposed: Bacteroides barnesiae sp. nov. (type strain BL2(T)=JCM 13652(T)=DSM 18169(T)), Bacteroides salanitronis sp. nov. (type strain BL78(T)=JCM 13657(T)=DSM 18170(T)) and Bacteroides gallinarum sp. nov. (type strain C35(T)=JCM 13658(T)=DSM 18171(T)).

Animals↗

Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov.

The characteristics of three Bacteroides species, Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae, were examined. 16S rRNA gene sequence analysis showed that B. distasonis, B. goldsteinii and B. merdae should not be classified as species within the genus Bacteroides. Although B. distasonis, B. goldsteinii and B. merdae were phylogenetically related to Tannerella forsythensis, the ratios of anteiso-C(15 : 0) to iso-C(15 : 0) in whole-cell methanolysates of the three species were different from that of T. forsythensis. In addition, whereas the major menaquinones of T. forsythensis were MK-10 and MK-11, the major menaquinones of B. distasonis, B. goldsteinii and B. merdae were MK-9 and MK-10. The three species were phenotypically similar to Bacteroides sensu stricto, but phylogenetically distinct. Furthermore, B. distasonis, B. goldsteinii and B. merdae could be differentiated from Bacteroides sensu stricto (predominant menaquinones: MK-10 and MK-11) by the menaquinone composition. This is an important chemotaxonomic characteristic of the three species. On the basis of these data, a novel genus, Parabacteroides gen. nov., is proposed for B. distasonis, B. goldsteinii and B. merdae, with three species, Parabacteroides distasonis gen. nov., comb. nov. (the type species), Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. The type strains of P. distasonis, P. goldsteinii and P. merdae are JCM 5825(T) (=CCUG 4941(T)=DSM 20701(T)=ATCC 8503(T)), JCM 13446(T) (=CCUG 48944(T)) and JCM 9497(T) (=CCUG 38734(T)=ATCC 43184(T)), respectively.

Bacterial Typing Techniques↗

Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncI alpha R64 drd-ll transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDP1, pCG30, and pEG920 could be mobilized from B. uniformis donors to both E. coli and Bacteroides recipients by a conjugative Bacteroides Tcr (Tcr ERL) element which was originally found in a clinical Bacteroides fragilis strain (B. fragilis ERL). However, the shuttle vector pE5-2, which contains pB8-51 cloned in a restriction site that prevents its mobilization by IncP or IncI alpha plasmids, also was not mobilized at detectable frequencies from Bacteroides donors by the Tcr ERL element. The mobilization frequencies of pCG30, pDP1, and pEG920 by the Tcr ERL element in B. uniformis donors to E. coli recipients was about the same as those to isogenic B. uniformis recipients. Transfer of the shuttle vectors from B. uniformis donors to E. coli occurred at the same frequencies when the matings were done aerobically or anaerobically. Growth of the B. uniformis donors in tetracycline (1 microgram/ml) prior to conjugation increased the mobilization frequencies of the vectors to both E. coli and Bacteroides recipients 50 to 100 times.

Bacteroides↗

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis.

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.

Bacteroides↗

[Frequency of isolation and antibiotic sensitivity of Bacteroides related to Bacteroides bivius and Bacteroides oralis].

274 strains of the Bacteroides oralis-bivius group are studied: 112 are identified to Bacteroides bivius or B. disiens, 73 to Bacteroides oralis and 49 to Bacteroides oris or Bacteroides buccae. These strains are isolated from clinical sample: gynecologic suppurations or respiratory tract infections. The susceptibility of 63 strains to 7 antibiotics were determined. Tested antibiotics were: cefalotin, cefoxitine, cefotaxime, mezlocillin, clindamycin, metronidazole and colistine. Cefalotine has a poor activity against these strains.

Anti-Bacterial Agents↗

Chemical composition, serological reactivity and endotoxicity of lipopolysaccharides extracted in different ways from Bacteroides fragilis, Bacteroides melaninogenicus and Bacteroides oralis.

Lipopolysaccharides (LPS) extracted from strains of Bacteroides fragilis, Bacteroides melaninogenicus and Bacteroides oralis with phenol-water, trichloroacetic acid, EDTA or liquid phenol-chloroform-petroleum ether (PCP) and isolated by ultracentrifugation, varied considerably in their quantitative chemical composition. Negligible yields of LPS were obtained by PCP-extraction. All preparations were more or less serologically active. All methods (except PCP) extracted the same O-antigenic determinants from B. fragilis. Endotoxic activity, as measured by primary skin inflammations in rabbits, was low but was present in all preparations. Proteins (and/or lipoproteins) co-precipitated with LPS in the ultracentrifuge.

Bacteroides↗

Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in clinical specimens by immunofluorescence with a monoclonal antibody to B. fragilis lipopolysaccharide.

A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroides fragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group.

Antibodies, Bacterial↗

Characterization of cephalosporinases from Bacteroides fragilis, Bacteroides thetaiotaomicron and Bacteroides vulgatus.

The susceptibility of 80 Bacteroides fragilis group strains isolated from clinical specimens to beta-lactam antibiotics was investigated by agar dilution method. Twenty strains showed high resistance to the antibiotics. The resistance level of the isolates to cephaloridine was related to the amount of beta-lactamase activity (cephalosporinase; CSase) produced. B. fragilis GN-11477, B. thetaiotaomicron GN11478 and B. vulgatus GN11479 were selected from among the CSase producing strains, and the enzymes were purified about 300-fold by affinity chromatography employed ampicillin as ligand bound to activated CH Sepharose 4B. The enzyme preparations gave a single protein band on polyacrylamide gel electrophoresis. The molecular weights of the three enzymes were estimated to be approximately 32,000 and their isoelectric points were 5.2, 4.9 and 4.5, respectively. The optimal pH and the optimal temperature of the enzymes were 7.2 and 37 degrees C, respectively. The enzyme activities were inhibited by iodine, some divalent ions, p-chloromercuribenzoate, clavulanic acid, cephamycin derivatives and cloxacillin. The enzymes showed hydrolytic activity against cephaloridine, cephalothin, cefazolin, cefuroxime and also newly introduced cephalosporins such as cefotaxime, cefoperazone and cefmenoxime. Each mouse antisera obtained against the purified enzymes showed cross-reactions with its each enzyme and others in neutralization test.

Ampicillin↗

Mobilization of Bacteroides plasmids by Bacteroides conjugal elements.

A 4.2-kilobase cryptic Bacteroides plasmid, pB8-51, is found in several colonic Bacteroides species. To determine whether pB8-51 is mobilized by any of the known Bacteroides conjugal elements, we constructed an Escherichia coli-Bacteroides shuttle vector, pVAL-1, which contains pB8-51. We constructed Bacteroides uniformis 0061 derivatives which carry pVAL-1 and various Bacteroides conjugal elements. The Bacteroides conjugal elements tested were six conjugal tetracycline resistance (Tcr) elements (which appear to be chromosomal), i.e., Tcr ERL, Tcr V479, Tcr Emr ERL, Tcr Emr 12256, Tcr Emr DOT, and Tcr Emr CEST, and the conjugal erythromycin resistance (Emr) plasmid pBF4. These Tcr conjugal elements have not been extensively characterized, except for Tcr ERL. All six Tcr elements tested mobilized pVAL-1 at high frequency (10(-3) to 10(-5)) from one Bacteroides strain to another or from a Bacteroides strain to E. coli. Pregrowth of the donors (containing one of the Tcr elements and pVAL-1) in 1 microgram of tetracycline per ml enhanced the transfer of pVAL-1 by 20- to 10,000-fold, depending on which Tcr element was present in the donor. An Ems derivative of pBF4 (pBF4 delta E2) mobilized pVAL-1 from one Bacteroides strain to another at a frequency of 10(-4) but did not mobilize pVAL-1 from a Bacteroides strain to E. coli as efficiently. Thus the Tcr conjugal elements and pBF4 recognize a mobilization region on pB8-51.

Bacteroides↗

Effect of encapsulated Bacteroides asaccharolyticus and Bacteroides fragilis on the growth of aerobic and facultative bacteria in mixed infections.

The effects of encapsulation of Bacteroides asaccharolyticus and Bacteroides fragilis on aerobic or facultative bacteria were evaluated using a subcutaneous abscess model in mice. The change in number of aerobic and facultative bacteria (AFB) was studied by comparing their number when injected with nonencapsulated, encapsulated Bacteroides species, and capsular material of Bacteroides species. In 14 combinations of AFB mixed with nonencapsulated Bacteroides species, an increase in the number of nonencapsulated Bacteroides species and an increase in the number of AFB occurred in two instances. An increase in the number of AFB occurred in 11 of the 14 combinations with encapsulated Bacteroides species. No change in the bacterial numbers was observed when the AFB were inoculated with capsular material of Bacteroides species. These data demonstrate the ability of viable encapsulated Bacteroides species to enhance the growth of aerobic or facultative bacteria.

Abscess↗

"Bacteroides nordii" sp. nov. and "Bacteroides salyersae" sp. nov. isolated from clinical specimens of human intestinal origin.

Two groups of unknown bacteria, which phenotypically resemble members of the Bacteroides fragilis group but phylogenetically display >5% 16S rRNA gene sequence divergence from their nearest validly described species, Bacteroides thetaiotaomicron, were characterized by phenotypic and molecular taxonomic methods. Phylogenetically and phenotypically, the unidentified bacteria displayed a relatively close association with each other. However, a 16S rRNA gene sequence divergence of approximately 4% between the two unknown bacteria, as well as distinguishable biochemical characteristics, demonstrates that these organisms are genotypically and phenotypically distinct, and each group may represent a previously unknown subline within the Bacteroides phylogenetic cluster. Subsequent DNA-DNA hybridization studies confirmed that the two novel organisms were indeed distinct from each other. The previously described species closest to both of them is B. thetaiotaomicron (approximately 94% sequence similarity), but they can be differentiated easily from B. thetaiotaomicron by virtue of not utilizing trehalose. DNA-DNA pairing studies also documented the separateness of the unknown species and B. thetaiotaomicron. Based on the phenotypic and phylogenetic findings, two new species, "Bacteroides nordii" sp. nov. and "Bacteroides salyersae" sp. nov, are proposed. The G+C content of the DNA is 41.4 mol% for Bacteroides nordii and 42.0 mol% for Bacteroides salyersae. The type strains of Bacteroides nordii and Bacteroides salyersae are WAL 11050 (ATCC BAA-998 or CCUG 48943) and WAL 10018 (ATCC BAA-997 or CCUG 48945), respectively.

Bacterial Typing Techniques↗

In vitro susceptibility of clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron in Japan.

A nationwide survey of the susceptibility of 433 isolates of Bacteroides fragilis and 149 isolates of Bacteroides thetaiotaomicron was conducted from December 1986 through November 1989 in Japan. These strains were collected from 16 university hospitals and one metropolitan hospital. Metronidazole was the most active drug against both species, with no resistant isolates found. The activity of imipenem and sulbactam-cefoperazone was good, with very low resistance rates determined in Bacteroides fragilis (1.4% and 1.6%, respectively) and in Bacteroides thetaiotaomicron (3.4% for both drugs), and was comparable to that of metronidazole. Cefoxitin, cefmetazole, cefotetan, cefbuperazone, latamoxef and ceftizoxime were found to be more active against Bacteroides fragilis, for which resistance rates were 3.2 to 9.5%, than against Bacteroides thetaiotaomicron, for which resistance rates were 18.1 to 21.8%. Rates of piperacillin resistance in the two species were 12.9% and 26.8%, respectively. Clindamycin was very active at a low concentration (MIC50 of 0.39 to 1.56 mg/l), but 24% and 27.5% of Bacteroides fragilis and Bacteroides thetaiotaomicron isolates, respectively, were resistant to this agent.

Anti-Bacterial Agents↗

German multicentre study on the in vitro susceptibility of Bacteroides species. The German Bacteroides Study Group.

In 1990 the first German multicentre study on the in vitro susceptibility of Bacteroides species was completed. Employing a commercially prepared microbroth dilution assay, nine participating institutions evaluated approximately 100 consecutive isolates of Bacteroides species from relevant clinical specimens. A total of 911 strains (449 Bacteroides fragilis, 201 Bacteroides thetaiotaomicron, 79 Bacteroides ovatus, 78 Bacteroides vulgatus, 77 Bacteroides distasonis, 25 Bacteroides uniformis, 2 others) were tested. Most of the isolates came from surgical patients (72%); other sources included gynaecological patients (9%) and medical patients (5%). Seventy-eight percent of the anaerobes were found in mixed culture together with at least one aerobic organism (Escherichia coli 36%, streptococci 15%, or enterococci 13%), while in 22% of the cases, the anaerobes were the only bacteria grown from the specimens. The results showed that many of the strains were potent beta-lactamase producers (as judged by resistance to amoxicillin). However, all but one of the isolates demonstrated susceptibility in vitro when clavulanic acid was added to amoxicillin or ticarcillin. At the same time, 13% of the organisms were resistant to mezlocillin, 5% to cefoxitin and 4% to clindamycin. Three strains were reported resistant to imipenem and one strain to metronidazole.

Anti-Bacterial Agents↗

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.

Aerobiosis↗