Development of a Methanogen Marker for Detection of Porcine Fecal Pollution in Surface Waters

The goal of this study was to evaluate methanogen diversity in animal hosts to develop the first swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed five distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine-specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%), but did not amplify a product in 47 bacterial and archaeal stock cultures, 477 environmental bacterial isolates, sewage, and water samples from a bovine waste lagoon and polluted creek. Amplification was only observed in 1 sheep out of 260 human and non-swine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10-6 g of wet pig feces in 500 mL of PBS and 10-4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker

Contribution of Sediment to High Enterococcus Counts Along the Northern Gulf of Mexico

Enumeration of enterococci (EN) bacteria in water is an USEPA approved indicator of fecal pollution and the possible presence of enteric pathogens. Along the northern Gulf of Mexico, the water is shallow with a high organic and particulate load because of the Mississippi River discharge. Disturbance of coastal sediments during wind/wave action caused either by the weather or human activities may increase bacterial counts as a result of increased EN persistence in the water column and/or resuspension of EN in the sediment. The goals of this project are to determine the relationship between organic content and EN counts in the water and whether bacterial resuspension from the sediment contributes to elevated EN counts. We found that EN counts in the water were correlated with wave conditions at seven sites along the Mississippi Gulf Coast. During calm wave conditions, low bacterial levels (1.0 – 227 CFU/100mL) were observed in the water with higher counts in the sediment; the reverse was observed (10 – 351 CFU/100mL) during rough wave conditions. EN counts were positively correlated with organic content of the sediment. Wave activity to keep EN in suspension was apparently critical for high counts. EN counts decreased by 50% in 4 hr from 38 to 17 CFU/100mL in the absence of resuspension and decreased to 1 CFU/100mL after 48 hr. EN in the sediment are not stationary as genetic fingerprinting using REP-PCR showed low persistence of specific isolates over time. Jackknife analysis revealed low similarity among EN isolates from the water and sediment collected on the same day and site during calm wave conditions. This shows that EN are not persisting for long periods in the same area but instead are resuspended and redistributed along the coast. Results from this study provide evidence that high organic content and resuspension of isolates from the sediment during periods of strong wave action contribute to high EN counts. Current research on the survival of EN in estuarine habitats will provide insight on the balance between environmental persistence and fecal pollution in causing high EN counts along beaches in the northern Gulf of Mexico

Methanogens as Ruminant-Specific Indicators of Fecal Pollution

Tracking the source of fecal pollution in surface waters has traditionally focused on the origin of enteric indicators including coliforms, enterococci, or E. coli. Recently, questions of genetic variability and environmental persistence have encouraged researchers to investigate additional animal specific indicators of fecal pollution. To date only eubacteria have been utilized as markers of human and animal-specific pollution. Here we report domestic ruminant-specific markers of fecal pollution utilizing methanogens found in the rumen. PCR primers for the mcrA gene of Methanomicrobium mobile (MMmcrA) and the nifH gene of Methanobrevibacter ruminantium (Mrnif-1) were designed, tested, and used to detect ruminant-specific pollution in fecal and environmental samples. The MMmcrA amplicons were detected in expected fecal and environmental samples (71% cow, 92% sheep, 50% goat, 100% cow lagoon samples, and 100% creek contaminated with cow lagoon waste), and were observed in only 5% of human and deer fecal samples. Mrnif-1 amplification was seen in 80% cow, 100% sheep, and 54% goat fecal samples; only 2% individual human samples were positive. No PCR amplification was observed when the MMmcrA and Mrnif-1 primers were tested against 47 bacterial stock cultures and fecal samples from 134 non-ruminant animals. More importantly, no amplification was observed in sewer samples using either primer pair. Sensitivity assays using MMmcrA primers demonstrated a detection limit of 0.01ng total DNA in bovine feces, 10ng in fecally contaminated surface water, and 5ng in cow lagoon samples

Evaluation of Methanobrevibacter smithii as a Human-Specific Marker of Fecal Pollution

Microbial source tracking has historically focused on the origin of traditional enteric indicators including coliforms, enterococci, or Escherichia coli. Recently, questions of genetic variability and environmental persistence have encouraged researchers to search for additional animal specific indicators of fecal pollution. To date only eubacteria have been utilized as markers of human and animal-specific pollution. In this study we developed a molecular primer pair specific for Methanobrevibacter smithii, a methanogen found only in the human intestine. PCR primers for the nifH gene of M. smithii were designed, tested, and used to detect the presence or absence of the organism in fecal and environmental samples. Product amplification was observed in 28.6% of all human fecal samples and 93% of sewer samples, and water contaminated with human sewage. No amplification was observed when primers were tested against 43 bacterial stock cultures and fecal samples from 204 animals. Sequencing of PCR products from sewers and M. smithii cells demonstrated that the 222bp product amplified was the nifH gene of M. smithii. Sensitivity assays demonstrated a detection limit of 10ng in human feces, 10ng in fecally contaminated water, and 5 ng in sewer samples. In addition, samples seeded with M. smithii established a lower detection limit of 13 cells/ml. The Mnif method for M. smithii detection appears to be a rapid, inexpensive, and reliable test for determining the presence or absence of human fecal pollution in recreational waters

\u3ci\u3eMethanobrevibacter ruminantium\u3c/i\u3e as an Indicator of Domesticated-Ruminant Fecal Pollution in Surface Waters

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples

Influence of Coastal Processes On High Fecal Coliform Counts in the Mississippi Sound

Microbial source tracking efforts have historically focused on the input of fecal bacteria from sources such as storm drains, sewers, and runoff. Fecal coliform levels in the Mississippi Sound have been analyzed and compared with physical factors in an attempt to characterize possible nonpoint sources of pollution. Results from this study show that a primary factor in elevated levels of fecal coliform is a change in wind direction. The passage of warm and cold fronts through the northern Gulf of Mexico causes numerous 90 degrees-180 degrees shifts in wind directions over a period of 6-8 days. Commonly, a rise in fecal coliform counts is observed at coastal monitoring stations after an abrupt shift in wind direction and wind speed. When these trends of increased fecal coliform levels occur before rainfall, it is inferred that the sediment could be a source of the fecal coliform observed in the water column. The changes in wind direction and velocity might induce more energetic conditions at the shoreline (e.g., increased wave heights and increased longshore current velocities). Fecal coliform counts collected from five monitoring stations along the Harrison County, Mississippi, coast during 2002-2003 have been compared with wind, wave, and current records from within the Mississippi Sound. The occurrence of high fecal coliform counts at multiple stations can be correlated with higher energy events in the Sound. Statistically, wind direction and high bacterial counts are correlated, and higher counts are most likely to occur when the winds are out of the west or southwest at most stations

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes▿

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6 g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen \u3ci\u3emcrA\u3c/i\u3e Genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6 g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker