Application of direct plaque assay for detection and enumeration of bacteriophages of Bacteroides fragilis from contaminated-water samples.

The direct double-agar-layer plaque assay for the detection and enumeration of specific bacteriophages of Bacteroides fragilis from contaminated-water samples was performed. Several factors that affect the methods, such as conditions of the bacterial culture, composition of the assay medium, addition of divalent cations, and decontamination techniques applied to the sample, were evaluated. The results obtained show that the direct assay technique proved to be more efficient than the most-probable-number technique. A higher recovery of bacteriophages was obtained from 17 of 24 samples with the direct assay. The two methods only showed similar results from samples with a low degree of pollution.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin, penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 micron pores were significantly more efficient than those with 0.45 micron pores in the isolation of BFG. A preliminary incubation period of 4 h at 30 degrees C prior to 44 h at 37 degrees C yielded significantly higher numbers of BFG than direct incubation at 37 degrees C for 48 h.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A llsop K. & S tickler D. J. 1984. The enumeration of Bacteroides fragilis group organisms from sewage and natural waters. Journal of Applied Bacteriology 56 , 15–24.
A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin. penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 μm pores were significantly more efficient than those with 0.45 μm pores in the isolation of BFG. A preliminary incubation period of 4 h at 30C prior to 44 h at 37C yielded significantly higher numbers of BFG than direct incubation at 37C for 48 h.     

Comparison of three types of reagents for detection of Bacteroides fragilis by direct immunofluorescence

Three different methods were used to prepare conjugates for the detection of rods of the Bacteroides fragilis group by direct immunofluorescence. Lyophilized conjugates were prepared. Three sets (five in each) of monovalent conjugates against serotype strains of B. fragilis (including conjugate E/E1 + E2) and polyvalent conjugate (A + B + C + D + E1 + E2) were obtained. Each conjugate was prepared in two variants: 1. unabsorbed, 2. absorbed with tissue powder prior to lyophilization. Conjugates obtained by precipitation of sera with 50% ethanol and direct coupling of gammaglobulins with stain were found to meet the requirement for good fluorescence reagents and are well suited for the detection of B. fragilis by direct immunofluorescence. Absorption of the conjugates with tissue powder before lyophilization did not affect their quality.     

Human origin of Bacteroides fragilis bacteriophages present in the environment.

Bacteroides fragilis HSP40 phages have been detected in waters with various levels of fecal contamination of human origin. The average numbers of B. fragilis phages present in sewage water reached 5.3 x 10(3) per 100 ml of water. We found a number 1,000 times lower in a river contaminated with domestic sewage only, in which the levels of fecal coliforms and fecal streptococci were 10,000 times lower than those found in raw sewage. In addition, B. fragilis phages were not found in significant numbers in slaughterhouse wastewaters. They were not present in fecal-polluted waters containing fecal contamination from wildlife only. Although the number of B. fragilis phages present in contaminated waters was lower than the number of coliphages, their presence indicated human fecal contamination. It is also shown that Bacteroides phages are only able to multiply under anaerobic conditions in the presence of nutrients, and they cannot multiply in natural waters and sediments.     

A reverse hemolytic plaque assay for the detection and the enumeration of immunoglobulin allotype-secreting cells

We describe a reverse hemolytic plaque assay to enumerate rabbit immunoglobulin allotype-secreting cells. This technique makes use of sheep red blood cells (SRBC) sensitized with goat anti-rabbit IgG and rabbit anti-allotypic sera as revealing antisera. We have used the assay to compare at the IgG molecule level and at the immunoglobulin allotype-secreting cell level, the preferential expression (pecking order), in heterozygous rabbits, of one of the two alleles either at the a or the b locus, respectively, governing the a series allotypic specificities carried by the variable region of the heavy chains and the b series allotypic specificities essentially carried by the constant region of the K light chains.     

Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage.

A new medium has been adapted for the growth of Bacteroides fragilis so that its phages can be recovered from environmental samples, and its efficiency has been assessed. Polyvinylidene difluoride membranes allow significantly higher recoveries among different membrane filters used to decontaminate the samples. In all cases, a number of phages remain in the filters and a percentage of them can be recovered by treatment with an eluant.     

An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages

This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis . Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage–host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.     

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.     

Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B.?fragilis group and similar species. B.?fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B.?fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value?相似文献   

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.     

Detection of Bacteroides fragilis by PCR assay targeting the neuraminidase-encoding gene

T. KUWAHARA, S. AKIMOTO, H. UGAI, T. KAMOGASHIRA, T. KINOUCHI AND Y. OHNISHI. 1996. Oligonucleotide primers were designed on the basis of the sequence of the neuraminidase-encoding gene ( nanH ) of Bacteroides fragilis and used for the specific detection of this anaerobe by the nested PCR assay. Fifty-nine of 60 representative strains of Bact. fragilis were detected, while none of 45 strains of other species generated visible PCR products. The detection limits of Bact. fragilis cells and DNA by the nested PCR were 10 colony-forming units and 10 fg of chromosomal DNA, respectively. The PCR assay targeting the nanH gene has the potential for the detection of Bact. fragilis .     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.     

脆弱拟杆菌β—内酰胺酶的纯化及免疫学特性

The beta-lactamase crude extract of Bacteroides fragilis 55 was chromatographed with DEAE-sepharose CL-6B and sephadex G-100. The partial purified enzyme proteins was further purified by cutting the band on PAGE in which the beta-lactamase was distinguishable from other proteins by our method of fluorescent staining. Using purified preparations to be mixed with liposome-CPS-K, prepared specific antisera against the purified beta-lactamase. Serological reactions were carried out by IgG-ELISA together with western blotting. The results revealed that Bacteroides fragilis beta-lactamase possessed its species-specificity.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Evaluation of a fluorescent antibody technique for the rapid enumeration of Bacteroides fragilis group of organisms in water

The Bacteroides fragilis group has been evaluated as a prospective rapid indicator of faecal contamination of water. Fluorescent antibody (FA) stained B. fragilis group bacteria were enumerated microscopically and compared with faecal coliform or Escherichia coli counts as indicators of faecal contamination. Environmental samples included surface waters (raw drinking water and known contaminated water). Laboratory disinfection experiments with ozone, chlorine and u. v. radiation were also performed. Bacteroides FA counts specifically detected recent human faecal contamination in field samples in 2–3 h. Samples with a high content of particulates or debris limited the sensitivity to about 10 FA counts/ml. Viable counts showed that the sensitivity to all three disinfection agents was essentially the same for Bacteroides and E. coli. Fluorescent antibody counts of Bacteroides , conversely, were not altered by any of the agents. Therefore, the Bacteroides FA method is not recommended for routine monitoring but may be useful for cases where extensive human faecal contamination is suspected (e.g. pipeline rupture or pollution of recreational water) and where rapid remedial action must be taken to protect the public health.     

Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis

In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.     

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4⁺ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4⁺ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4⁺ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4⁺ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.     

Evaluation of a fluorescent antibody technique for the rapid enumeration of Bacteroides fragilis group of organisms in water

The Bacteroides fragilis group has been evaluated as a prospective rapid indicator of faecal contamination of water. Fluorescent antibody (FA) stained B. fragilis group bacteria were enumerated microscopically and compared with faecal coliform or Escherichia coli counts as indicators of faecal contamination. Environmental samples included surface waters (raw drinking water and known contaminated water). Laboratory disinfection experiments with ozone, chlorine and u.v. radiation were also performed. Bacteroides FA counts specifically detected recent human faecal contamination in field samples in 2-3 h. Samples with a high content of particulates or debris limited the sensitivity to about 10 FA counts/ml. Viable counts showed that the sensitivity to all three disinfection agents was essentially the same for Bacteroides and E. coli. Fluorescent antibody counts of Bacteroides, conversely, were not altered by any of the agents. Therefore, the Bacteroides FA method is not recommended for routine monitoring but may be useful for cases where extensive human faecal contamination is suspected (e.g. pipeline rupture or pollution of recreational water) and where rapid remedial action must be taken to protect the public health.