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.     

An assessment of Bacteroides fragilis group organisms as indicators of human faecal pollution

Membrane filtration techniques were used to enumerate Bacteroides fragilis group (BFG) organisms and Escherichia coli in a variety of natural waters, the influents and effluents from three types of sewage treatment plants and faeces of various animals. The results suggest that BFG organisms die off more rapidly than E. coli in water and that animal faeces are not a significant source of BFG. It is suggested that the ratio of BFG to E. coli in water may be used to indicate the proximity of a source of human faecal contamination.     

An assessment of Bacteroides fragilis group organisms as indicators of human faecal pollution

Membrane filtration techniques were used to enumerate Bacteroides fragilis group (BFG) organisms and Escherichia coli in a variety of natural waters, the influents and effluents from three types of sewage treatment plants and faeces of various animals. The results suggest that BFG organisms die off more rapidly than E. coli in water and that animal faeces are not a significant source of BFG. It is suggested that the ratio of BFG to E. coli in water may be used to indicate the proximity of a source of human faecal contamination.     

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.     

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.     

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.     

An evaluation of the use of 4-methylumbelliferyl-β-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods

The use of 4-methylumbelliferyl-β- D -glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 μg ml⁻¹), incubation temperatures (37 or 41·5°C) and incubation times (8, 12, 24 and 48 h). Different kinds of foods, both naturally and artificially contaminated, were analysed. The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation. In this case an incubation temperature of 37°C would be preferred and the MUG concentration could be reduced to 50 μg ml⁻¹. Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41·5°C) and MUG concentration (100 μg ml⁻¹).     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.     

Differential Effects of Oxygen and Oxidation Reduction Potential on the Multiplication of Three Species of Anaerobic Intestinal Bacteria

The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured. The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV). However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide. When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen. P. magnus was found to be the most sensitive. After 2 h of aerobic incubation, no viable organisms could be detected. B. fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation. C. perfringens was the least sensitive. Under conditions of aerobic incubation, viable organisms survived for 10 h. During the experiments with Clostridium, no spores were observed by spore staining.     

Use of reactions with Limulus amoebocyte lysate (LAL) to determine biological activity of lipopolysaccharides from reference and clinical strains of the Bacteroides fragilis group

The aim of this study was to determine and compare a biological activity of lipopolysaccharides (LPS) from reference and clinical strains of strictly anaerobic bacteria belonging to the Bacteroides fragilis group (BFG) by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides of five BFG species were extracted by Westphal and Jann method (1965) from eight reference and two clinical strains of B. fragilis group. Crude LPS preparations were purified according to the procedure described by Gmeiner (1975) with ultracentrifugation and nuclease treatment. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit, Charles River Endosafe Ltd., USA). Tests were performed according to the producer's recommendations. E. coli O55:B5 LPS was applied to compare its activity in reaction with LAL reagent with activities of LPS preparations from rods of the Bacteroides genus. Among examined bacterial compounds the most active in BET method was E. coli O55:B5 LPS. Activities of lipopolysaccharides from five species of BFG rods in reaction with Limulus amoebocyte lysate were differentiated. Greater ability to activate LAL proenzyme revealed lipopolysaccharides of these species of the Bacteroides genus, which are important from the clinical point of view--B. fragilis and B. thetaiotaomicron.     

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   

Presence and partial characterization of internal acid protease of Aspergillus oryzae.

A membrane filter procedure is described for the enumeration of Candida albicans in natural waters. Several hundred milliliters of sample can be examined by filtration through 1.2-micrometer membranes. Selectivity is achieved by the use of a defined (yeast-nitrogen base plus maltos-) agar medium inclusion of the antimicrobial agents chloramphenicol and cycloheximide, and incubation at 37 degrees C. C. albicans colonies are differentiated primarily through color by use of a bismuth salt indicator system. Average recovery of various strains of C. albicans stressed in seawater at 4 degrees C was 82%, compared with those of spread plate controls on a noninhibitory medium. With river water and raw sewage, 90% of typical C. albicans colonies were confirmed as such in a simplified germ tube test. Atypical colonies verified as C. albicans were infrequent (3%). C. tropicalis and Torulopsis candida were the most common false-positive colonies.     

Enumeration of Enterococcus sp. using a modified mE method

A modified mE medium (mEI) containing the chromogenic substrate indoxyl-β- D -glucoside to detect β- D -glucosidase activity was evaluated with respect to specificity and recovery of enterococci from environmental waters. Extending incubation from 24 to 48 h improved enterococci recovery but 77% of the colonies classified as non-target were confirmed as enterococci. Randomly chosen enterococcal isolates from sewage, exposed in microcosms containing 0·22 μm membrane filtered fresh or estuarine water, exhibited differences in persistence as a function of exposure treatment. Decreasing the concentration of or eliminating indoxyl-β- D -glucoside from mE did not significantly affect recovery of purified isolates.     

Col V plasmids and the resistance of Escherichia coli to divalent copper ions

Free organisms of both plasmid-free and plasmid-carrying strains of Escherichia coli were killed by incubation in water containing low levels of cupric ions. Sensitivity was temperature-dependent with killing being more marked at 20° or 25°C than at 10° or 15°C. In contrast to the effects of other inhibitors from natural waters (which affect free Col V⁺organisms more than Col^-ones), free Col^-and Col V⁺organisms were equally sensitive to kill by Cu²⁺. Attachment to glass beads essentially abolished sensitivity to cupric ions with full survival after exposure to 15 μ g/ml. This applied to both p⁺and p^-strains but attachment would have more effect on the survival of p⁺organisms in natural waters because some plasmids markedly enhance attachment.     

Bacteriophages active against Bacteroides fragilis in sewage-polluted waters.

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Bacteriophage resistance of attached organisms as a factor in the survival of plasmid-bearing strains of Escherichia coli

Unattached organisms of plasmid-free and plasmid-bearing strains of Escherichia coli showed marked sensitivity to phages T4, Tula and K3 on incubation in broth at 37°C but organisms attached to glass beads or sand were resistant. Phage T4 sensitivity of free organisms and resistance of glass bead-attached ones were also observed when incubation was in broth at 15° or 20°C or anaerobically at 37°C. In contrast, free and attached organisms were resistant at 37°C or lower temperature when incubation was in poor media. It seems likely that the presence of phage will be a major factor reducing survival in the intestine and in sewage and that attachment (which is more significant for strains bearing certain plasmids) will protect. In contrast, survival of either free or attached organisms in polluted water will probably not be significantly influenced by the presence of phage.     

Bacteriophages active against Bacteroides fragilis in sewage-polluted waters

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Growth of Bacteraides fragilis group in agar and broth media

B rook , I. 1990. Growth of Bacteroides fragilis group in agar and broth media. Journal of Applied Bacteriology 69 , 697–700.
The rate of bacterial growth of four . Bacteroides fragilis group organisms was determined in agar and broth media. Exponential bacterial growth occurred in agar media within 4 to 8 h, while such growth was delayed in broth media and occurred within 12–24 h after inoculation. This phenomenon may explain why antimicrobials which manifest an 'inoculum effect' may show increased resistance to antimicrobials when tested in agar media.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Selective enumeration of Bacteroides vulgatus and B. distasonis organisms in the predominant human fecal flora by using monoclonal antibodies.

The genus Bacteroides represents about one-third of the isolates from human fecal samples. The proportions of the different species are difficult to estimate because there is no method for rapid identification of mixtures of anaerobes. Monoclonal antibodies against Bacteroides vulgatus and B. distasonis were prepared. They did not react with the other Bacteroides species of the B. fragilis group. These reagents allowed direct enumeration of B. vulgatus and B. diastasonis organisms in human fecal samples. Anaerobic bacteria resistant to 1-h contact with air were enumerated in fecal human samples, a filter was layered on the colonies, and then B. vulgatus colonies were identified by an immunoassay performed with the prepared monoclonal antibodies. Healthy human adult volunteers were tested. Most of them harbored B. vulgatus at high levels, while the B. distasonis levels were always lower. Kinetic studies suggested that time variations for each volunteer were small. The simplified quantification of Bacteroides strains at the species level described here will prove useful in complementing our knowledge of the factors which may influence the predominant human fecal flora.