The fecal microbiome of cattle plays a critical role not only in animal health and productivity but also in food safety, pathogen shedding, and the performance of fecal pollution detection methods. Unfortunately, most published molecular surveys fail to provide adequate detail about variability in the community structures of fecal bacteria within and across cattle populations. Using massively parallel pyrosequencing of a hypervariable region of the rRNA coding region, we profiled the fecal microbial communities of cattle from six different feeding operations where cattle were subjected to consistent management practices for a minimum of 90 days. We obtained a total of 633,877 high-quality sequences from the fecal samples of 30 adult beef cattle (5 individuals per operation). Sequence-based clustering and taxonomic analyses indicate less variability within a population than between populations. Overall, bacterial community composition correlated significantly with fecal starch concentrations, largely reflected in changes in the Bacteroidetes, Proteobacteria, and Firmicutes populations. In addition, network analysis demonstrated that annotated sequences clustered by management practice and fecal starch concentration, suggesting that the structures of bovine fecal bacterial communities can be dramatically different in different animal feeding operations, even at the phylum and family taxonomic levels, and that the feeding operation is a more important determinant of the cattle microbiome than is the geographic location of the feedlot.The enteric microbiota of cattle affects animal health and food safety and is used as an indicator of fecal pollution, which can affect the types and concentrations of indicator organisms in recreational surface waters. The presence of pathogenic bacteria such as Escherichia coli O157:H7 in the bovine gastrointestinal tract has been linked to disease outbreaks due to the consumption of contaminated beef, milk, and drinking water (3). The average feedlot steer produces 1.62 kg of feces (dry matter) per day (2), resulting in more than 18 million metric tons of feces (dry matter) per year in the United States alone. When bovine fecal waste is moved from feedlot operations for land application as fertilizer or is accidentally discharged into the environment due to severe storms, hazardous events, or failure of onsite waste management practices, pathogenic members of this microbial community, such as E. coli O157:H7, Campylobacter jejuni, Salmonella spp., Leptospira interrogans, and Cryptosporidium parvum (5,14,22,41,44), can pose a serious public health risk.Because of the enormous influence the fecal bacterial community of cattle has on the beef and dairy industry, the economy, and public health, a great deal of research has been conducted to characterize the effects of animal age, disease state, feeding practices, and antibiotic treatments on cattle fecal microorganisms. Many of the most comprehensive studies use DNA-based methodologies, such as sequencing of the full-length 16S rRNA gen...

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based on initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools.

h Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. N umerous host-associated indicators are used to identify human fecal pollution in ambient surface waters, with many relying on molecular methods such as quantitative real-time PCR (qPCR). The most widely used methods target the HF183 16S rRNA gene cluster of members of the genus Bacteroides initially identified in human fecal samples collected from the United States Pacific Northwest (1) and targeted by PCR to distinguish humanassociated contamination (2). Over the past decade, researchers have developed (2-9) and implemented (10-15) many PCR-based methods targeting the HF183 cluster worldwide.In most HF183 PCR applications, the forward primer targets the Bacteroides HF183 cluster, reported to include Bacteroides dorei (3). The reverse primer typically hybridizes to a wider diversity of Bacteroidales so as not to further restrict the range of targeted bacteria. Researchers have employed this strategy for endpoint PCR applications (2), as well as a variety of quantitative real-time PCR chemistries, such as the SYBR green (8, 16) and TaqMan (4, 5, 9, 17) chemistries.In recent performance evaluation studies, PCR-based methods targeting the HF183 cluster, in particular, the TaqMan HF183/ BFDrev assay (3), consistently outperformed other tested approaches in terms of specificity and sensitivity (18-21). TaqMan chemistry utilizes an internal oligonucleotide probe, greatly reducing the chance of false-positive results due to the accumulation of unintended amplification by-products (e.g., primer dimerization [PD] molecules). The TaqMan HF183/BFDrev qPCR assay was recently tested in a five-laboratory repeatability study and shown to be highly reproducible across laboratories when key components of the protocol were standardized (22). However, the ...

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Aims: To characterize the composition of microbial populations in a distribution system simulator (DSS) by direct sequence analysis of 16S rDNA clone libraries. Methods and Results: Bacterial populations were examined in chlorinated distribution water and chloraminated DSS feed and discharge water. Bacterial strains isolated from DSS discharge water on R2A medium were identified using 16S rDNA sequence analysis. The majority of the bacteria identified were a-proteobacteria, ranging from approx. 34% in the DSS discharge water to 94% of the DSS isolates. Species richness estimators Chao1 and ACE (abundance-based coverage estimators) indicated that the chlorinated distribution water sample was representative of the total population diversity, while the chloraminated DSS feed water sample was dominated by Hyphomicrobium sp. sequences. The DSS discharge water contained the greatest diversity of a-, b-, cproteobacteria, with 36% of the sequences being operational taxonomic units (OTUs, sequences with >97AE0% homology). Conclusions: This work demonstrated the dominance of a-proteobacteria in distribution system water under two different disinfectant residuals. The shift from chlorine to monochloramine residual may have played a role in bacterial population dynamics. Significance and Impact of the Study: Accurate identification of bacteria present in treated drinking water is needed in order to better determine the risk of regrowth of potentially pathogenic organisms within distribution systems.