Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.     

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

Background

Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring.

Methodology/Principal Findings

Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and α-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC∶A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies.

Conclusions/Significance

This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.     

Survival of Mycobacterium avium subsp. paratuberculosis in Dam Water and Sediment

In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.     

Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.     

Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.     

Discrimination Efficacy of Fecal Pollution Detection in Different Aquatic Habitats of a High-Altitude Tropical Country, Using Presumptive Coliforms, Escherichia coli, and Clostridium perfringens Spores

The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa.     

Distribution and Significance of Fecal Indicator Organisms in the Upper Chesapeake Bay

Total viable aerobic, heterotrophic bacteria, total coliforms, fecal coliforms, and fecal streptococci were enumerated in samples collected at five stations located in the Upper Chesapeake Bay, December 1973 through December 1974. Significant levels of pollution indicator organisms were detected at all of the stations sampled. Highest counts were observed in samples collected at the confluence of the Susquehanna River and the Chesapeake Bay. The indicator organisms examined were observed to be quantitatively distributed independently of temperature and salinity. Counts were not found to be correlated with concentration of suspended sediment. However, significant proportions of both the total viable bacteria (53%) and fecal indicator organisms (>80%) were directly associated with suspended sediments. Correlation coefficients (r) for the indicator organisms examined in this study ranged from r = 0.80 to r = 0.99 for bottom water and suspended sediment, respectively. Prolonged survival of fecal streptococci in most of the sediment samples was observed, with concomitant reduction of the correlation coefficient from r = 0.99, fecal streptococci to total coliforms in water, to r = 0.01, fecal streptococci to fecal coliforms in sediments. The results of this study compared favorably with fecal coliforms: fecal streptococci ratios for the various sample types. Characterization of organisms beyond the confirmed most-probable-number procedure provided good correlation between bacterial indicator groups.     

Identification of Fecal Escherichia coli from Humans and Animals by Ribotyping

Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.     

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”     

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Degradation of Pectins with Different Degrees of Esterification by Bacteroides thetaiotaomicron Isolated from Human Gut Flora

A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids.     

Biotic and Abiotic Factors Influencing In Vitro Growth of Escherichia coli O157:H7 in Ruminant Digestive Contents

The gastrointestinal tract (GIT) of ruminants is the main reservoir of enterohemorrhagic Escherichia coli, which is responsible for food-borne infections in humans that can lead to severe kidney disease. Characterization of biotic and abiotic factors that influence the carriage of these pathogens by the ruminant would help in the development of ecological strategies to reduce their survival in the GIT and to decrease the risk of contamination of animal products. We found that growth of E. coli O157:H7 in rumen fluid was inhibited by the autochthonous microflora. Growth was also reduced when rumen fluid came from sheep fed a mixed diet composed of 50% wheat and 50% hay, as opposed to a 100% hay diet. In fecal suspensions, E. coli O157:H7 growth was not suppressed by the autochthonous flora. However, a probiotic strain of Lactobacillus acidophilus inhibited E. coli O157:H7 growth in fecal suspensions. The inhibitory effect was dose dependent. These lactic acid bacteria could be a relevant tool for controlling O157:H7 development in the terminal part of the ruminant GIT, which has been shown to be the main site of colonization by these pathogenic bacteria.     

Microbial Species Involved in Production of 1,2-sn-Diacylglycerol and Effects of Phosphatidylcholine on Human Fecal Microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.     

Lack of absorption and rapid elimination of Rifampicin as factors precluding tests against nuclear polyhedrosis in Spodoptera littoralis larvae

Rifampicin administered per os in artificial medium to first-instar larvae of Spodoptera littoralis together with nuclear polyhedrosis virus did not affect virus infection and resulted in total larval mortality. A pharmacokinetic study of this antibiotic was carried out by means of a microbiological assay. Using Bacillus thuringiensis var. thuringiensis as the test organism for assaying the antibiotic, as little as 0.5 μg of Rifampicin could be detected in the presence of hemolymph, and no such detectable amounts could be found in the hemolymph of sixth instar larvae which had been fed the antibiotic. Most if not all of the antibiotic was in the feces, excreted in active form. When given parenterally, Rifampicin was detected in the hemolymph 30 min after injection but not 24 hr thereafter, when active antibiotic was found concentrated in the first fecal pellets evacuated after injection. This disappearance is ascribed to excretion via the Malpighian tubules. The larval responses detailed above preclude the testing of Rifampicin as an antiviral drug against nuclear polyhedrosis.     

Viable Intestinal Passage of a Canine Jejunal Commensal Strain Lactobacillus acidophilus LAB20 in Dogs

The strain Lactobacillus acidophilus LAB20 with immunomodulatory properties was previously found dominant in the jejunal chyme of four dogs, and the novel surface layer protein of LAB20 suggested its competitive colonization in canine gut. To evaluate the persistence and survival of LAB20 in healthy dogs, LAB20 was fed to five healthy pet dogs for 3 days, at a dosage of 10⁸ CFU daily as fermented milk supplement. The fecal samples, from 1 day prior to feeding, three continuous feeding days, and on day 5, 7, 14, and 21, were collected for strain-specific detection of LAB20 using real-time PCR. We found that LAB20 count was significantly increased in dog fecal samples at the second feeding day, but rapidly decreased after feeding ceased. The fecal samples from prior to feeding, during feeding, and post-cessation days were plated onto mLBS7 agar, from where LAB20 was recovered and distinguishable from other fecal lactobacilli based on its colony morphotype. Using strain-specific PCR detection, the colonies were further verified as LAB20 indicating that LAB20 can survive through the passage of the canine intestine. This study suggested that canine-derived strain LAB20 maintained at high numbers during feeding, viably transited through the dog gut, and could be identified based on its colony morphotype.     

Longitudinal Study of Escherichia coli O157 in a Cattle Finishing Unit

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (≤0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.     

The abundance of fecal Faecalibacterium prausnitzii in relation to obesity and gender in Chinese adults

The influence of gender and obesity on the abundance of human colonic Feacalibacterium prausnitzii is currently unclear. We collected fecal samples from 54 obese and 54 sex- and age-matched normal-weight Chinese adults and quantified the fecal F. prausnitzii as percentage of 16S rRNA gene copies of F. prausnitzii accounting to that of total gut bacteria with quantitative PCR. The fecal F. prausnitzii amount was not significantly different between obese and lean subjects. Men possessed significantly lower level of fecal F. prausnitzii than women, and the significant and positive correlation of fecal F. prausnitzii quantity with fasting glucose level was observed in men, not in women. Our results suggest that the gender effect, in addition to other factors including the geographic location, ethnicity, diet and gut transit times of study subjects, has to be considered when studying the relationship between gut F. prausnitzii and diseases.     

Microplate Subtractive Hybridization To Enrich for Bacteroidales Genetic Markers for Fecal Source Identification

The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).     

Evaluating the Membrane Fecal Coliform Test by Using Escherichia coli as the Indicator Organism

The fecal coliform membrane filter method (MFC) currently used in water pollution analysis was evaluated by using two strains of Escherichia coli, a known fecal coliform, as the indicator organism. A large relative error in the results obtained with this method was found to be dependent upon the brand of membrane filter employed, the medium, and the temperature of incubation. MFC densities varied between 10 and 60% of the densities determined by means of total bacteria counts and total coliform counts performed at 35 C. Due to the large relative error encountered, the MFC method cannot be recommended as an analytical tool for the laboratory enumeration of E. coli. The results do show that the MFC method can be used at 35 C for enumeration of E. coli and for differential counts of E. coli and Enterobacter aerogenes.     

Analysis of human and animal fecal microbiota for microbial source tracking

Microbial compositions of human and animal feces from South Korea were analyzed and characterized. In total, 38 fecal samples (14 healthy adult humans, 6 chickens, 6 cows, 6 pigs and 6 geese) were analyzed by 454 pyrosequencing of the V2 region of the 16S rRNA gene. Four major phyla, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were identified in the samples. Principal coordinate analysis suggested that microbiota from the same host species generally clustered, with the exception of those from humans, which exhibited sample-specific compositions. A network-based analysis revealed that several operational taxonomic units (OTUs), such as Lactobacillus sp., Clostridium sp. and Prevotella sp., were commonly identified in all fecal sources. Other OTUs were present only in fecal samples from a single organism. For example, Yania sp. and Bifidobacterium sp. were identified specifically in chicken and human fecal samples, respectively. These specific OTUs or their respective biological markers could be useful for identifying the sources of fecal contamination in water by microbial source tracking.