Method for Isolation of Bacteroides Bacteriophage Host Strains Suitable for Tracking Sources of Fecal Pollution in Water

Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe.     

Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.     

Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides- specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.     

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.     

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.     

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.     

Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization.

A dot blot hybridization procedure with 32P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization

A dot blot hybridization procedure with 32P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.     

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.     

Specific Strains of Bacteroides Species in Human Fecal Flora as Measured by Deoxyribonucleic Acid Homology

Membrane competition homology experiments were used to compare Bacteroides uniformis and Bacteroides vulgatus isolates obtained from fecal samples from different individuals and isolates obtained from fecal samples of single individuals. Isolates of B. uniformis, when isolated from different individuals, had interstrain deoxyribonucleic acid homology values that ranged from 63 to 95%, with most of the values being in the 70 to 85% range. When isolates obtained from a single individual were compared, each species was represented by one or two groups of very closely related organisms, with each group having essentially 100% interstrain homology. When strains from two groups were compared with each other, the homology values were in the same range as when organisms were isolated from different individuals. Isolates which have nearly 100% homology with each other persisted in fecal samples collected over a 5- to 6-month period. It appears that the colon of each person may be populated by bacterial strains that are specific for that individual. Somatic antigen serotyping has been used as an indicator for specific Escherichia coli strains in fecal samples. Two isolates having the same O, K, and H antigens had 99% homology, but when only O and H antigens were in common, the homology values were in the 70 to 85% range. It seems that isolates of a given serotype, when isolated from a single individual, may represent a unique strain, but isolates of a given serotype, when isolated from different individuals, probably do not.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Re-assessment of phenotypic identifications of Bacteroides putredinis to Alistipes species using molecular methods

Alistipes (previously Bacteroides) are strictly anaerobic gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive; however, they are only weakly saccharolytic and most species produce light brown pigment only on laked rabbit blood agar. In this retrospective study, we re-identified 18 organisms previously identified phenotypically as "Bacteroides putredinis-like", but that did not produce pigment on routine media. The strains were identified with 16S rDNA sequencing and pigment production was evaluated on several different culture media. Only 12/18 strains had molecular identifications of Alistipes species, while the remaining strains phylogenetically resembled Butyricimonas and Odoribacter spp. Pigment production was not a reliable test for those Alistipes strains that are described as pigment producers.     

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.     

Bacterial metabolism of natural and synthetic sex hormones undergoing enterohepatic circulation

Steroids undergoing enterohepatic circulation are exposed to bacterial metabolism particularly by obligate anaerobes which account for 99.99% of the fecal flora. The most common transformation is hydrolysis of conjugated steroids. The glucuronidases are synthesized by Escherichia coli and Bacteroides species. The bacterial catabolism of unconjugated steroids may be considered under several headings: 1. Reduction of ring-A due to clostridia species synthesizing specific enzymes; C. paraputrificum, 3 alpha,5 beta-reductase; C. innocuum, 3 beta,5 beta-reductase; and a new species C.J-1, 3 beta,5 alpha-reductase. 2. Reduction of the delta 5 bond by human fecal flora. The specific strain(s) synthesizing the enzyme have not yet been identified. 3. Reduction of 17-keto estrogens by the above mentioned ring-A reducing clostridia and by Eubacterium lentum. 4. Reduction of 17-keto androstenes by Bacteroides fragilis. 5. Desmolase mediated side chain cleavage at C17-C20 position of 17 alpha-hydroxysteroids by two new species Clostridium scindens and Eubacterium desmolans isolated from human and cat fecal flora respectively and by Clostridium cadavaris isolated from New York City sewage. 6. And 16 alpha- and 21-dehydroxylase by E. lentum a normal inhabitant of the human gut; it is the only organism known to synthesize 16 alpha- or 21-dehydroxylases. Due to the high specificity of the enzymes and the simplicity of extracting the metabolites, biosynthesis of reference compounds and radioimmunoassay reagents is practical and inexpensive. The enzymes can also be used for titration of specific bacterial strains in fecal flora and as markers for bacterial identification in particular for the strains difficult to be defined by regular biochemical reactions.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.