16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: A Bayesian approach

Abstract

We report the design and validation of new TaqMan^® assays for microbial source tracking based on the amplification of fecal 16S rRNA marker sequences from uncultured cells of the order Bacteroidales. The assays were developed for the detection and enumeration of non-point source input of fecal pollution to watersheds. The quantitative “universal” Bacteroidales assay BacUni-UCD detected all tested stool samples from human volunteers (18 out of 18), cat (7 out of 7), dog (8 out of 8), seagull (10/10), cow (8/8), horse (8/8), and wastewater effluent (14/14). The human assay BacHum-UCD discriminated fully between human and cow stool samples but did not detect all stool samples from human volunteers (12/18). In addition, there was 12.5% detection of dog stool (1/8), but no cross-reactivity with cat, horse, or seagull fecal samples. In contrast, all wastewater samples were positive for the BacHum-UCD marker, supporting its designation as 100% sensitive for mixed-human source identification. The cow-specific assay BacCow-UCD fully discriminated between cow and human stool samples. There was 38% detection of horse stool (3/8), but no cross-specificity with any of the other animal stool samples tested. The dog assay BacCan-UCD discriminated fully between dog and cow stool or seagull guano samples and detected 62.5% stool samples from dogs (5/8). There was some cross-reactivity with 22.2% detection of human stool (4/18), 14.3% detection of cat stool (1/7), and 28.6% detection of wastewater samples (4/14). After validation using stool samples, single-blind tests were used to further demonstrate the efficacy of the developed markers; all assays were sensitive, reproducible, and accurate in the quantification of mixed fecal sources present in aqueous samples. Finally, the new assays were compared with previously published sequences, which showed the new methodologies to be more specific and sensitive. Using Bayes’ Theorem, we calculated the conditional probability that the four assays would correctly identify general and host-specific fecal pollution in a specific watershed in California for which 73 water samples had been analyzed. Such an approach allows for a direct comparison of the efficacy of different MST methods, including those based on library-dependent methodologies. For the universal marker BacUni-UCD, the probability that fecal pollution is present when the marker is detected was 1.00; the probability that host-specific pollution is present was 0.98, 0.84, and 0.89 for the human assay HF160F, the cow assay BacCow-UCD, and the dog assay BacCan-UCD, respectively. The application of these markers should provide meaningful information to assist with efforts to identify and control sources of fecal pollution to impaired watersheds.

Introduction

The water quality of many waterways and coastal waters is deteriorating due to point and non-point sources of fecal contamination caused by human and animal sources (Handler et al., 2006). Microbial source tracking (MST) is an increasingly used approach to determine host-specific contributions of fecal contamination to waterbodies, thus helping resolve these unknown sources. This information can, in turn, guide decisions regarding the appropriate corrective measures for affected waters. One emerging MST method is the detection of host-specific 16S rRNA markers that target the order Bacteroidales, which are found exclusively in feces, animal rumen, and other cavities of humans and animals (Paster et al., 1994), often in greater abundance than traditionally used coliform bacteria.

Kreader developed PCR-based assays to amplify genes from three cultivated strains of Bacteroides to monitor human fecal pollution in water (Kreader, 1995), and evaluated their persistence in the environment (Kreader, 1998). Others further advanced this approach by identifying host-specific Bacteroidales 16S rDNA markers for humans and cows based on DNA sequences representing uncultivated fecal diversity. Initially, researchers relied on length heterogeneity-PCR (LH-PCR) and terminal restriction length polymorphism (T-RFLP) analysis to screen fecal bacterial DNA extracts from environmental waters (Bernhard and Field, 2000a). With information obtained from this study, they identified cluster-specific primer sets that are useful in discriminating human and ruminant feces (Bernhard and Field, 2000a). The system of qualitative assays for presence/absence detection was further tested and confirmed to reliably and specifically detect host-specific markers from feces and polluted water samples (Field et al., 2003). These PCR targets were classified as belonging to the phylum Bacteroidetes, of which the cultivated fecal members are in the order Bacteroidales and the genera Bacteroides and Prevotella (Dick et al., 2005a). Early advances by Field and colleagues were followed by (i) a quantitative PCR (qPCR) assay for the detection of general fecal pollution based on a 16S rRNA marker specific for many fecal Bacteroidales sequences (Dick and Field, 2004), (ii) further conventional PCR assays for the detection of pig- and horse-specific fecal pollution (Dick et al., 2005a) as well as dog- and elk-specific fecal pollution (Dick et al., 2005b), and (iii) a real-time PCR assay using SYBR Green 1 for the detection of the previously identified human-specific marker sequence (Seurinck et al., 2005). Recently, additional qPCR assays have been developed for “total” (Layton et al., 2006), bovine-associated (Layton et al., 2006; Okabe et al., 2007; Reischer et al., 2006; Stricker et al., in press), pig-associated (Okabe et al., 2007), and human-associated Bacteroidales 16S rRNA genes (Layton et al., 2006; Okabe et al., 2007; Reischer et al., 2007; Stricker et al., in press).

To date, the development of molecular target detection assays for Bacteroidales has provided a fast, reliable, and relatively inexpensive means by which to diagnose the source(s) of fecal contamination to natural waters. A comparison study showed Bacteroidales to be the most accurate MST method for discriminating human vs. non-human impacts using tests of mixed fecal sources in aqueous samples (Griffith et al., 2003). There has been some discussion concerning the benefits in terms of sensitivity and specificity of TaqMan over SYBR Green assays, and the relative sensitivities of the two technologies have been debated (Morrison et al., 1998; Wittwer et al., 1997). With regard to specificity, TaqMan assays are considered the better choice for differentiation of host-specific DNA sequences when environmental samples are considered. This is because non-specific products and mRNAs with high sequence identity may be detected with the SYBR Green chemistry. Melting curve analysis can assist in proper identification of these false-positive sequences, but accurate quantification may be compromised, and a comparison of numerical data generated by SYBR Green with quantitative results obtained by TaqMan analysis has not been performed for Bacteroidales. By combining multiple species-specific TaqMan PCR assays, specificity can be expanded within a genus if new sequence information becomes available.

The ultimate goal of MST is to determine the relative amounts of host-specific fecal contributions to a water sample. In the case of Bacteroidales methodologies, the goal may be to directly quantify the relative contributions of specific hosts (e.g. humans) based on a relationship between host-specific markers and the total concentration of Bacteroidales DNA sequences detected. These data would provide watershed managers enhanced information with which to formulate more accurate plans to reduce the loading of fecal pollution to receiving waters. Therefore, there is a need for Bacteroidales methodologies to not only become more quantitative with respect to the amplified target sequences, but also for such data to be analyzed statistically to calculate conditional probabilities of correctly identifying sources of fecal pollution in a watershed, given that a particular host-specific assay tests positive in environmental samples. The specific objectives of the present study were the design of a new, more inclusive, quantitative universal Bacteroidales assay, as well as three new quantitative host-specific Bacteroidales assays, allowing the relative determination of the amount of human-, dog-, and cow-specific fecal contaminations over space and time. Further, we developed a conditional probability approach to estimate the likelihood of correctly identifying fecal sources of contamination in a water sample given the detection of the new markers.