Evaluating the occurrence of host-specific , general fecal indicators, and bacterial pathogens in a mixed-use watershed.

Fecal contamination of water is very common, and, in the United States, prevention is complicated by the colossal span of waterways (>3.5 million miles), heterogeneous sources of pollution, and competing interests in water monitoring. The focus of this study was the Upper Sugar Creek Watershed, a mixed-use watershed with many headwater streams and one of the most contaminated waterways in Ohio. Quantitative polymerase chain reaction (qPCR) and host-specific PCR for were evaluated for the potential to discern sources of fecal contamination. Pathogen-specific qPCR and culturable by most probable number (MPN) were compared at 21 established water quality monitoring sites in the watershed headwaters. Lower numbers of ruminant-specific markers were detected in the base flow water samples compared with the human-specific marker, suggesting the presence of hotspots of human fecal contamination. qPCR and MPN showed significant correlation ( = 0.57; < 0.001). Correlation between general fecal indicator and pathogen concentrations was weak or nonexistent. Coexistence of and human-specific was common ( = 0.015). qPCR may have a greater potential for predicting fecal contamination due to its sensitivity, rapid analysis, and availability of host-specific assays. However, the lack of a strong correlation between pathogens and general fecal indicators suggests that assessment of health risk associated with fecal contamination will require a complement of approaches.

Genetic markers for rapid PCR-based identification of gull, Canada goose, duck, and chicken fecal contamination in water.

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.

Relative decay of Bacteroidales microbial source tracking markers and cultivated Escherichia coli in freshwater microcosms.

Fecal indicator bacteria (FIB), commonly used to regulate sanitary water quality, cannot discriminate among sources of contamination. The use of alternative quantitative PCR (qPCR) methods for monitoring fecal contamination or microbial source tracking requires an understanding of relationships with cultivated FIB, as contamination ages under various conditions in the environment. In this study, the decay rates of three Bacteroidales 16S rRNA gene markers (AllBac for general contamination and qHF183 and BacHum for human-associated contamination) were compared with the decay rate of cultivated Escherichia coli in river water microcosms spiked with human wastewater. The following five sets of microcosms were monitored over 11 days: control, artificial sunlight, sediment exposure, reduced temperature, and no autochthonous predation. Decay was characterized by estimation of the time needed to produce a 2-log reduction (t(99)). No treatment-associated differences in the decay of the 4 targets were evident except with reduced predation, where E. coli, qHF183, and BacHum markers had lower levels of decay by day 3. However, there were substantial target-associated differences. Decay curves for the AllBac marker indicated a larger persistent population than those of the other targets. Exposure to sunlight, sediment, and reduced predation resulted in more rapid decay of the human-associated markers relative to cultivable E. coli, but there were no differences in t(99) values among the 4 targets under control conditions or at reduced temperatures. Further evaluation of epidemiological relationships will be needed in order to relate the markers directly to health risk. These findings suggest that the tested human-associated markers can complement E. coli as indicators of the human impact on sanitary water quality under the constrained conditions described in this paper.

Evaluation of two spike-and-recovery controls for assessment of extraction efficiency in microbial source tracking studies.

Quantitative PCR (qPCR), applied to complex environmental samples such as water, wastewater, and feces, is susceptible to methodological and sample related biases. In this study, we evaluated two exogenous DNA spike-and-recovery controls as proxies for recovery efficiency of Bacteroidales 16S rDNA gene sequences (AllBac and qHF183) that are used for microbial source tracking (MST) in river water. Two controls--(1) the plant pathogen Pantoea stewartii, carrying the chromosomal target gene cpsD, and (2) Escherichia coli, carrying the plasmid-borne target gene DsRed2--were added to raw water samples immediately prior to concentration and DNA extraction for qPCR. When applied to samples processed in replicate, recovery of each control was positively correlated with the observed concentration of each MST marker. Adjustment of MST marker concentrations according to recovery efficiency reduced variability in replicate analyses when consistent processing and extraction methodologies were applied. Although the effects of this procedure on accuracy could not be tested due to uncertainties in control DNA concentrations, the observed reduction in variability should improve the strength of statistical comparisons. These findings suggest that either of the tested spike-and-recovery controls can be useful to measure efficiency of extraction and recovery in routine laboratory processing.

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).

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.

Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes.

Assessment of health risk associated with fecal pollution requires a reliable fecal indicator and a rapid quantification method. We report the development of a Taq nuclease assay for enumeration of 16S rRNA genes of Bacteroidetes. Sensitivity and correlation with standard fecal indicators provide experimental evidence for application of the assay in monitoring fecal pollution.

A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking.

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.