Article

Validation and field testing of library-independent microbialsource tracking methods in the Gulf of Mexico

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Abstract

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10�5–10�6 dilution). LODs for HPyVs and M. smithii assays were similar to each other (10�3–10�4), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no crossreactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited crossreactivity with cow, dog and seagull samples. An experts’ workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.

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... Volumes filtered for FIB, MST, and pathogen analysis were varied based on standard and previously published methods (2,64,107,133,(155)(156)(157)(158). Volumes filtered included 1 ml, 10 ml, and 100 ml for water samples. ...
... Water samples were processed using the previously published culture/PCR method for detection of the esp gene of Enterococcus faecium (88,89,133). Detection of HPyVs and HF183 were also performed using previously described, endpoint PCR methods (64,89 contamination. An extraction blank (empty PowerBead tube -no filter added) was subjected to all steps from DNA extraction through PCR as a negative control. ...
... At least two possible reasons for this discrepancy exist: (1) fewer samples and larger water volumes were analyzed for pathogens than for MST, so the samples were not directly comparable; and (2) with the exception of the enteric viruses, all of the pathogens tested can be shed by animals as well as humans (52,151,169). The MST markers used here were chosen in part because of their demonstrated ability to detect sewage pollution in ambient water volumes of several hundred mL or less (64,102,106,107,133); however, improved methods for sample filtration and purification, which could allow processing of larger sample volumes for MST assays without inhibiting the PCR, would increase the sensitivity of these assays and would probably result in a higher frequency of co-detection of pathogens and MST markers when the contamination is from a human source (98). Conversely, the inclusion of markers for fecal contamination from animals such as birds, dogs, and raccoons, all of which are plentiful in the Lake Carroll watershed, may have resulted in better agreement between marker and pathogen detection; however, these methods were not generally available when the study was performed. ...
Article
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The quality of recreational and shellfishing waters has historically been monitored using commensal, allochthonous bacteria shed in feces (fecal indicator bacteria, FIB). The fate of FIB in the environment should mimic that of bacterial, protozoan, and viral human pathogens, which may also be allochthonous (e.g. Salmonella, Cryptosporidium, or enteric viruses) or autochthonous (e.g. Vibrio spp.) to aquatic environments. FIB are contributed to water from human and animal sources; however, pollution source cannot be determined by conventional FIB measurements. Because fecal source determination is important for pollution remediation and assessment of human health risks, microbial source tracking (MST) methods are increasingly used in water quality studies. The host-specific genes (markers) used for MST include the 16S rRNA of Bacteroides HF183 and the T-antigen of human polyomaviruses (HPyVs). In my work, correlations among FIB, MST markers, and autochthonous pathogens were explored in the context of factors that may influence these relationships. Specifically, the effects of stormwater runoff, sediment resuspension, and survival/persistence of FIB on submerged aquatic vegetation were investigated in a recreational lake. Furthermore, the relationship between FIB and concentrations of the autochthonous pathogen, V. vulnificus, was investigated at water bodies surrounding Tampa Bay. I hypothesized that degraded water quality would influence the concentration and/or population structure of V. vulnificus, a potentially lethal human pathogen. Finally, I hypothesized that the gene encoding a sodium-phosphate transporter (nptA) would be differentially expressed in V. vulnificus strains under varying conditions of salinity and phosphate concentration. I hypothesized that stormwater infrastructure/runoff, SAV, and sediments would serve as reservoirs for FIB, human-associated microbes (HF183 and HPyVs), and allochthonous pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses). FIB concentrations in the water were positively associated with those in the sediment, SAV, and with 24hr antecedent rainfall. At least one MST marker or pathogen was found in 35% of samples following rain events. These data were incorporated into a Bayesian model, which predicted pathogen absence when fecal coliform concentrations were low. Stormwater was also shown to be an important reservoir/conveyance system for FIB, human-associated microbes, and pathogens. I hypothesized that polluted estuarine waters in Tampa Bay, and oysters harvested from them, would contain higher V. vulnificus concentrations, and that the population structure would be altered compared to unpolluted waters. Enumeration included direct plating, enrichment followed by plating, and quantitative PCR (qPCR). V. vulnificus colonies isolated directly on mCPC agar were rarely PCR-confirmed, although enrichment and qPCR methods yielded a higher confirmation frequency. Unconfirmed colonies resembling V. vulnificus were identified as V. sinaloensis via 16S rRNA sequence analysis and were more frequently detected in less polluted waters. Comparison of growth rates among V. vulnificus and V. sinaloensis strains in enrichment media and seawater showed that V. vulnificus had faster growth rates (µ) in enrichment media, but that µ of V. sinaloensis strains was greater in seawater. V. sinaloensis presence can therefore lead to overestimation of V. vulnificus concentrations when samples are directly plated. These results highlight a need for better understanding of the ecology and virulence potential of this newly-described species. Finally, I hypothesized that V. vulnificus strains with varying virulence potential would differentially express the nptA gene in response to changes in environmental conditions. Expression studies were performed on biotype 1, 2, and 3 strains, and strains more closely associated with environmental reservoirs (water or oysters) showed up to 100-fold greater nptA expression than strains isolated from clinical cases. Gene expression in environmentally-associated, but not clinically-isolated, strains was highest in media at pH 6.0 vs. those at pH ≥ 7.0 and at 10 / salinity. In contrast, expression was highest among clinical strains at 10 / salinity, pH 8.0 media. Sequence analysis of the nptA gene also divided strains into environmentally- and clinically-isolated groups. These results suggest that differences in gene expression may be related to host preference and may be associated with differential virulence of strains in humans. These studies demonstrate a relationship between water quality (determined by FIB concentrations) and the prevalence of allochthonous and autochthonous human pathogens, and reveal that many environmental habitats may serve as reservoirs for FIB and pathogens. Differences in water quality were further demonstrated to impact the community structure of Vibrio spp. and may affect the relative abundance of strains with greater virulence potential.
... Although several evaluation studies of molecular PCR markers have been conducted on the U.S. mainland (19)(20)(21) and elsewhere (22)(23)(24), no marker validation studies have been conducted in Hawaii or on other Pacific Islands. These validation studies have demonstrated that bacterial MST markers can be found in species of which they are not indicative, although at less frequently and at lower concentrations. ...
... The containers with the spiked freshwater (salinity, 0.3 ppt; pH 7.35) and seawater (salinity, 32.3 ppt; pH 7.96) were loosely closed, placed in the dark at 22°C, and continuously agitated using magnetic stirrers. All six microcosms were tested for concentrations of enterococci and C. perfringens (culture-based methods) and molecular markers (HF183TaqMan and HPyV) at days 0, 1, 3,5,8,11,15,19,24,30, and 40. The sampling frequency was determined based on the initial observed decay rates of FIB. ...
Article
Importance: This is the first in depth characterization of microbial source tracking (MST) markers in Hawaii. Distribution and concentrations of HF183TaqMan and HPyV markers in human and animal fecal samples and in wastewater, coupled with decay data obtained from sunlight-exposed and unexposed microcosms, supports concurrent application of HF183TaqMan and HPyV markers for sewage detection in Hawaii waters. Both markers are more conservative and more specific markers of sewage than fecal indicator bacteria (enterococci and Escherichia coli). Analysis of HF183TaqMan (or newer derivatives), is recommended for inclusion in future epidemiological studies concerned with beach water quality, while better concentrations techniques are need for HPyV. Such epidemiological studies can be used to develop new recreational water quality criteria, which will provide direct information on the absence or presence of sewage contamination in water samples as well as reliable measurements of risk of water borne disease transmission to swimmers.
... Microbial source tracking is a water quality management tool that identifies contaminating fecal input sources. Over the past several decades, MST tools have been developed for a range of targets and applied to a variety of waterbodies (Bernhard & Field, 2000;Hagedorn et al., 1999;Harwood et al., 2009a;Harwood et al., 2009b;Nguyen et al., 2018;Steele et al., 2018;Wiggins, 1996). MST tools have been validated to identify fecal input sources from human (Green et al., 2014a), cattle (Shanks et al., 2008), pig (Mieszkin et al., 2009), dog (Green et al., 2014b), chicken (Weidhaas et al., 2010), seagull , possum (Devane et al., 2013), and other animals by using quantitative PCR (qPCR) analysis of host-associated marker genes. ...
... For example, the CAT marker gene has been reported to be found in excreta from pigeons and other bird species (Ryu et al., 2012;Sinigalliano et al., 2013). Similarly, HF183 marker has been reported cross-react with dog, deer, and chicken fecal samples (Balleste et al., 2010;Gawler et al., 2007;Harwood et al., 2009a;Harwood et al., 2009b;Stea et al., 2015). Consequently, markers that exhibit low host-specificity in QMRA analysis will overestimate the risk from a specific source, and therefore, provide risk estimates with increased uncertainty. ...
Article
Full-text available
The use of microbial source tracking (MST) marker genes has grown in recent years due to the need to attribute point and non-point fecal contamination to specific sources. Quantitative microbial risk assessment (QMRA) is a modeling approach used to estimate health risks from exposure to feces-contaminated water and associated pathogens. A combination of these approaches [quantitative MST (qMST) and QMRA] can provide additional pathogen-related information for prioritizing and addressing health risks, compared to reliance on conventional fecal indicator bacteria (FIB). To inform expansion of this approach, a review of published qMST-QMRA studies was conducted to summarize the state of the science and to identify research needs. The reviewed studies primarily aimed to identify what levels of MST marker genes in hypothetical recreational waterbodies would exceed the United States Environmental Protection Agency (USEPA) risk benchmarks for primary contact recreators. The QMRA models calculated relationships between MST marker gene(s) and reference pathogens based on published data in the literature. The development of a robust, accurate relationship was identified as an urgent research gap for qMST-QMRA. This metric requires additional knowledge to quantify the relationship between MST marker genes and the degree of variability in decay of pathogens as a dynamic function of environmental conditions and combinations of fecal sources at multiple spatial and temporal scales. Improved characterization of host shedding rates of host-associated microorganisms (i.e., MST marker genes), as well as fate and transport of these microorganisms and their nucleic acids, would facilitate expansion of this approach to other exposure pathways. Incorporation of information regarding the recovery efficiency, and host-specificity of MST marker genes into QMRA model parameters, and the sensitivity analysis, would greatly improve risk management and site-specific water monitoring criteria.
... The molecular description of bacterial communities of aquatic systems linked to affected sites from mining or industrial activities has brought very valuable information for the identification of biomonitoring organisms (Harwood et al. , 2009;Mondragón et al., 2011;Paul et al., 2013). At present, the genetic characterization and the metabolic routes of bacterial species with the ability to reduce or oxidize some metals and other pollutants, is one of the main goals in the biotechnological research in Mexico. ...
... Estos trabajos evidencian cómo ciertas características de las poblaciones, o de sus individuos, se utilizan para cuantificar los efectos de los factores ambientales sobre la fauna terrestre, y los cambios en conductas, patrones demográficos y tamaños poblacionales que originan. La descripción molecular de comunidades bacterianas de sistemas acuáticos asociados a sitios impactados por actividades mineras o industriales ha proporcionado información muy valiosa para la identificación de organismos biomonitores (Harwood et al., 2009;Mondragón et al., 2011;Paul et al., 2013). La caracterización genética y de las rutas metabólicas de especies bacterianas capaces de reducir u oxidar ciertos metales y otros contaminantes es uno de los objetivos primordiales de la investigación biotecnológica en México. ...
Article
Mexico has a unique biodiversity that places it within the list of megadiverse countries; it has three of the 34 ecoregions of the world and sites that are considered wilderness areas worldwide. The use of Genomics as a tool for research in Mexico began in the late 1930s with work aimed at the genetic improvement of commercial crops and to understand the ecological foundations of the genetic variation in Drosohpila pseudooscura, however, it wasn't until the decades of 1980-1990s that these tools were used for the study of natural populations of flora and fauna with purposes of conservation and management. Nonetheless, the potential that genomic tools have to improve the strategies and policies of management to ensure food production and conservation of wildlife in Mexico, these have not been widely applied. In this paper the areas of knowledge in wildlife where genomics have been applied in the study of natural populations of flora and fauna in Mexico were reviewed, and the practical applications of genomics for management and conservation of species of biological and commercial concern were discussed.
... In addition , certain FIB strains have adapted to the environment and therefore proliferate and persist in different habitats (Anderson et al. 2005). Moreover, FIB do not allow for differentiating between sources of fecal pollution (Harwood et al. 2009) and as a result the health risk associated with the use of a particular contaminated water source cannot be accurately assessed and remediation becomes more complicated (Harwood et al. 2014). Microbial source tracking (MST) and chemical source tracking (CST), which are described as a collection of methods and an investigation plan to identify possible sources of pollution in environmental waters (Harwood et al. 2014), have the potential to resolve some of the pitfalls associated with the use of FIB. ...
... Fecal Bacteroides strains show promise as MST markers as they have been found to be highly host specific (Scott et al. 2002; Seurinck et al. 2005) and may be utilized to differentiate between the fecal matter from dogs, ruminants, horses, pigs, and humans (Field and Samadpour 2007; Meays et al. 2004; Kildare et al. 2007; Ahmed et al. 2009; Layton et al. 2013). Previous studies have reported the successful utilization of Bacteroides as an indicator for sewage pollution in storm water runoff (Sidhu et al. 2013 ) and coastal waters used for recreational purposes (swimming and fishing) (Harwood et al. 2009). Bacteroides have also been found in high numbers in wastewater (Layton et al. 2013 ) and has shown high specificity in the identification of the source of pollution. ...
Article
Full-text available
Microbial source tracking (MST) and chemical source tracking (CST) markers were utilized to identify fecal contamination in harvested rainwater and gutter debris samples. Throughout the sampling period, Bacteroides HF183 was detected in 57.5 % of the tank water samples and 95 % of the gutter debris samples, while adenovirus was detected in 42.5 and 52.5 % of the tank water and gutter debris samples, respectively. Human adenovirus was then detected at levels ranging from below the detection limit to 316 and 1253 genome copies/μL in the tank water and debris samples, respectively. Results for the CST markers showed that salicylic acid (average 4.62 μg/L) was the most prevalent marker (100 %) in the gutter debris samples, caffeine (average 18.0 μg/L) was the most prevalent in the tank water samples (100 %) and acetaminophen was detected sporadically throughout the study period. Bacteroides HF183 and salicylic acid (95 %) and Bacteroides HF183 and caffeine (80 %) yielded high concurrence frequencies in the gutter debris samples. In addition, the highest concurrence frequency in the tank water samples was observed for Bacteroides HF183 and caffeine (60 %). The current study thus indicates that Bacteroides HF183, salicylic acid and caffeine may potentially be applied as source tracking markers in rainwater catchment systems in order to supplement fecal indicator analyses.
... A microbial assay with higher counts (CFU or GC) in fecal material has greater significance for water-quality monitoring; for example, such assay remains still detectable even after many folds of dilution in a surface water resource (Harwood et al., 2009(Harwood et al., , 2014Layton et al., 2013). Sensitivity refers to the proportion of known positive controls that are correctly identified as positive. ...
... In fact, multiple previous studies reported the cross-reaction of MST markers with the fecal materials from non-targeted species (Ryu et al., 2012;Boehm et al., 2013;Sinigalliano et al., 2013). Therefore, the performance characteristics, mainly specificity, related to the false-positive rate of the assays should be carefully evaluated in a new geographical location (Harwood et al., 2009;Stewart et al., 2013). ...
Article
Full-text available
For microbial source tracking (MST), the 16S ribosomal RNA genes (rDNA) of host-specific bacteria and mitochondrial DNA (mtDNA) of animal species, known to cause fecal contamination of water, have been commonly used as molecular targets. However, low levels of contamination might remain undetected by using these DNA-based qPCR assays. The high copy numbers of ribosomal RNA (rRNA) could offer a solution for such applications of MST. This study compared the performance of eight MST assays: GenBac3 (general Bacteroidales), HF183 (human), BacCan (dog), Rum-2-Bac (ruminant), Pig-2-Bac (swine), Gull4 (gull), GFD, and Av4143 (birds) between rRNA-based and rDNA-based approaches. Three mtDNA-based approaches were tested: DogND5, SheepCytB, and HorseCytB. A total of 151 animal fecal samples and eight municipal sewage samples from four regions of Finland were collected for the marker evaluation. The usability of these markers was tested by using a total of 95 surface water samples with an unknown pollution load. Overall, the performance (specificity, sensitivity, and accuracy) of mtDNA-based assays was excellent (95-100%), but these markers were very seldom detected from the tested surface water samples. The rRNA template increased the sensitivity of assays in comparison to the rDNA template. All rRNA-based assays (except Av4143) had more than 80% sensitivity. In contrast, only half (HF183, Rum-2-Bac, Pig-2-Bac, and Gull4) of rDNA-based assays reached this value. For markers targeted to bird feces, the use of the rRNA-based assay increased or at least did not change the performance. Regarding specificity, all the assays had >95% specificity with a DNA template, except the BacCan assay (71%). While using the RNA template for the assays, HF183 and BacCan exhibited only a low level of specificity (54 and 55%, respectively). Further, the HF183 assay amplified from multiple non-targeted animal fecal samples with the RNA template and the marker showed cross-amplification with the DNA template as well. This study recommends using the rRNA-based approach for MST assays targeting bird fecal contamination. In the case of Frontiers in Microbiology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 673306 Rytkönen et al. Performance of rRNA-Based MST Markers mammal-specific MST assays, the use of the rRNA template increases the sensitivity but may reduce the specificity and accuracy of the assay. The finding of increased sensitivity calls for a further need to develop better rRNA-based approaches to reach the required assay performance.
... The molecular description of bacterial communities of aquatic systems linked to affected sites from mining or industrial activities has brought very valuable information for the identification of biomonitoring organisms (Harwood et al. , 2009;Mondragón et al., 2011;Paul et al., 2013). At present, the genetic characterization and the metabolic routes of bacterial species with the ability to reduce or oxidize some metals and other pollutants, is one of the main goals in the biotechnological research in Mexico. ...
... Estos trabajos evidencian cómo ciertas características de las poblaciones, o de sus individuos, se utilizan para cuantificar los efectos de los factores ambientales sobre la fauna terrestre, y los cambios en conductas, patrones demográficos y tamaños poblacionales que originan. La descripción molecular de comunidades bacterianas de sistemas acuáticos asociados a sitios impactados por actividades mineras o industriales ha proporcionado información muy valiosa para la identificación de organismos biomonitores (Harwood et al., 2009;Mondragón et al., 2011;Paul et al., 2013). La caracterización genética y de las rutas metabólicas de especies bacterianas capaces de reducir u oxidar ciertos metales y otros contaminantes es uno de los objetivos primordiales de la investigación biotecnológica en México. ...
Article
Full-text available
La genómica en la investigación científica y en la gestión de la vida silvestre en México Resumen México alberga una diversidad biológica excepcional que lo coloca entre los principales países megadiversos, pues posee tres de las 34 ecorregiones del mundo y zonas consideradas áreas silvestres a nivel mundial, como los desiertos de Chihuahua, Sonora y California; su importancia radica en que reune alrededor de 70 % de su hábitat original en buenas condiciones y una densidad poblacional humana menor a 5 habitantes km-2. El uso de la genómica como herramienta en la investigación científica en este país tuvo sus inicios a finales de 1930 con trabajos encaminados al mejoramiento genético de cultivos comerciales y a entender los fundamentos ecológicos de la variación genética en Drosophila pseudooscura, pero hasta los años 80 y 90 comenzó el estudio de la flora y la fauna bajo esa perspectiva. Sin embargo, a pesar del potencial que las técnicas genómicas ofrecen para mejorar el desarrollo de estrategias y políticas de gestión que aseguren la producción de alimentos y la preservación de especies, no han sido extensamente utilizadas. Se presenta una revisión de las áreas del conocimiento en la vida silvestre en las que la genómica ha sido incorporada para abordar poblaciones naturales y se discuten los aspectos en los que puede incidir dentro del manejo y conservación de taxa de importancia biológica y comercial. Abstract Mexico has a unique biodiversity that places it within the list of megadiverse countries; it has three of the 34 ecoregions of the world and sites that are considered wilderness areas worldwide. The use of Genomics as a tool for research in Mexico began in the late 1930s with work aimed at the genetic improvement of commercial crops and to understand the ecological foundations of the genetic variation in Drosohpila pseudooscura, however, it wasn't until the decades of 1980-1990s that these tools were used for the study of natural populations of flora and fauna with purposes of conservation and management. Nonetheless, the potential that genomic tools have to improve the strategies and policies of management to ensure food production and conservation of wildlife in Mexico, these have not been widely applied. In this paper the areas of knowledge in wildlife where genomics have been applied in the study of natural populations of flora and fauna in Mexico were reviewed, and the practical applications of genomics for management and conservation of species of biological and commercial concern were discussed.
... These two PCR/qPCR assays have been most commonly used to determine the host-sensitivity and -specificity of HPyVs in human and nonhuman wastewater and faecal samples. Merging faecal and wastewater samples from studies (McQuaig et al., 2006;Harwood et al., 2009;McQuaig et al., 2009;Ahmed et al., 2010b;Kirs et al., 2011;Staley et al., 2012a;Hellein et al., 2011), the presence of HPyVs was tested by analysing 841 non-human faecal and wastewater samples. HPyVs were 100% specific to human wastewater and urine samples. ...
... Several factors such as types of water, dilution effect, turbidity, differences in analytical methods (culture-based vs. qPCR), sources of faecal inputs, differential decay and numbers of samples may account for the lack of correlations observed. Harwood et al., (2009) compared Enterococcus spp. concentrations in undiluted human wastewater and the dilution corresponding to the limit of detection of HPyVs by PCR, with the rationale that the observations should be correlated if the FIB and marker co-vary in the undiluted human wastewater . ...
... Human adenovirus (HAdV), human enterovirus (HEV), and human polyomavirus (HPyV) have been suggested as potential MST tools indicating human pollution sources (Harwood et al. 2009;Noble et al. 2003;Ahmed et al. 2010). Fong et al. (2005) characterized HAdVs and HEVs as sound library-independent indicators that can be used for the identification of water pollution sources. ...
... After analyzing pig slaughterhouse slurries, urban sewage, and river water samples, Hundesa et al. (2006Hundesa et al. ( , 2009 suggested that porcine adenoviruses' (PAdVs) detection provides a valuable MST approach. Also, HPyVs are highly human specific so that their detection provides a reliable indication of contamination from a human source (Harwood et al. 2009). Bovine adenovirus (BAdV) and bovine enterovirus (BEV) were proposed for use in identifying agricultural water pollution sources (Ahmed et al. 2010;Fong et al. 2005). ...
Article
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This paper reviews the state of knowledge regarding human viruses in water systems from an environmental engineer's perspective. The authors describe (1) viruses of concern and potential human diseases; (2) waterborne outbreaks related to viruses; (3) the sources, reservoirs, and fate of viruses in the environment; (4) the use of viruses as microbial source tracking tools; (5) virus survival and virus transport; (6) virus concentration and detection methods; (7) the fate of viruses in water treatment; (8) the removal of viruses in full-scale, bench, and pilot-scale conventional and membrane bioreactor (MBR) wastewater systems; and (9) other issues related to viruses in water systems such as the role of bacterial viruses (phages) and viral risk assessment. Occurrence of human pathogenic viruses in environmental waters (i.e., surface waters, groundwater, drinking water, recreational water, and wastewater) raises concerns regarding the possibility of human exposure and waterborne infections. Commonly observed waterborne viruses include adenoviruses, enteroviruses, noroviruses, and rotaviruses. Viruses are the smallest of all microorganisms, and their size facilitates transport in environmental media. In addition, viruses have very low die-off rates and low infectivity doses, increasing concern over outbreaks of disease related to waterborne or sludge-related virus exposures. Overall, virus presence in water and wastewater is a difficult problem for environmental engineers because of prevalence, infectivity, and resistance of viruses to disinfection. Environmental engineers should be aware that even state-of-the-art wastewater treatment plants may not be able to eliminate viruses from wastewater, and viruses potentially escaping from drinking water treatment plants because of technical and management deficiencies may lead to human exposure and disease. The knowledge and tools summarized in this paper provide basic information needed to make decisions for efficient water and wastewater management and reduction of risk of human exposure. (C) 2014 American Society of Civil Engineers.
... 32 As most natural wastewater systems have long retention times and provide habitat for a variety of wildlife (including birds, bats, turtles, and small mammals), fecal indicator bacteria from non-human origin (which do not necessarily indicate a threat to human health) may be added to the wastewater throughout the system. Microbial source tracking 33 is one technique that can help distinguish between animal and human fecal contamination in natural wastewater systems, and thus can provide a better understanding about actual microbial risks to humans. ...
Article
Natural wastewater treatment systems have been used for centuries to recover resources through agriculture and aquaculture water reuse. Because the management of wastewater using natural methods relies on the integration of environmental, engineered, economic, and social systems, pathogens cannot be effectively monitored and controlled in these systems using a single approach or a single indicator organism (e.g., monitoring for coliform indicator bacteria). Different types of pathogens are removed at different rates in natural wastewater treatment systems and certain diseases are more important in some regions than they are in others. For natural systems in tropical regions that incorporate water reuse for agriculture or aquaculture, parasites such as soil-transmitted helminths, Schistosoma, Taenia, or food-transmitted trematodes may be of greater public health concern than some bacterial pathogens. Professionals and practitioners must consider how social and environmental systems might be shaped by the use of natural wastewater management systems, and how, in turn, natural wastewater management may impact existing socio-environmental relationships. Because of this, effective pathogen monitoring and control in natural wastewater treatment systems requires coordinated participation from stakeholders in multiple sectors.For further resources related to this article, please visit the WIREs website.
... The persistence of the qPCR target of a surrogate should be more persistent than the reference pathogen, or else the pathogen risk will be underestimated. For example, there are mixed views as to the longevity of the Bacteroidales qPCR target in the environment (Harwood et al., 2009;Stapleton et al., 2009) versus the human enteric viruses that are thought to be the major health-related pathogen group in recreational waters (Sinclair et al., 2009). On the other hand, the use of a highly persistent qPCR marker (say C. perfringens spores) may overestimate the risk for rapidly inactivated pathogens. ...
Chapter
Microbial risk assessment (MRA) has traditionally utilized microbiological data that was obtained by culture-based techniques that are expensive and time consuming. With the advent of PCR methods there is a realistic opportunity to conduct MRA studies economically, in less time, and simultaneously targeting multiple pathogens and their sources. More importantly, recently developed qPCR assays provide the opportunity to estimate the densities of the reference pathogens and their sources, which is critical to quantitative MRA (QMRA) analyses. In this chapter we discuss the use of qPCR-based methods to identify risks associated with exposure to water, namely drinking and recreational waters. We discuss the advantages associated with the current qPCR approaches used in microbial water quality studies and critically evaluate some of the limitations as they relate to the use of QMRA in the assessment of microbial water quality and public health protection.
... Microbial source tracking (MST) methodology offers tools to diagnose and identify fecal contamination sources in the environment (1,2). Although it is not a standard environmental monitoring method used for routine regulatory purposes, it has been extensively studied and applied worldwide, especially for deciphering fecal pollution in surface and recreational waters (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). ...
Article
Modern man-made environments, including urban, agricultural and industrial, have complex ecological interactions among themselves and with the natural surroundings. Microbial Source Tracking offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study is intended to assess karst spring susceptibilities to different fecal sources using MST qPCR assays, targeting human, bovine and swine markers. It involved a dual time monitoring frame: monthly, throughout the calendar year, and daily, during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring, improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST markers quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event which was mirrored in others markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. The pig-pens were ascribed to the watersheds of Zuf, Shefa, and Giach springs, and omitted from Gaaton, and correlated with Pig-2-Bac marker partial detection in Gaaton spring compared to the other springs. Ruminant and swine markers were detected intermittently and their contamination potential was exposed during the wet samplings. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... The link between rainfall patterns, land runoff, and its affect on aquatic microbiomes warrants further investigation. Previous coastal water quality monitoring studies have focused on target organisms, usually coliforms and FIB (Finkl and Charlier 2003;Shibata et al. 2004;Brownwell et al. 2007;Hartz et al. 2008;Harwood et al. 2009;Wright et al. 2009;Abdelzaher et al. 2010;Staley et al. 2012), such as the work done by the FACE program, utilizing both culture-based and molecular quantitative PCR techniques (Carsey et al., unpubl. data). ...
Article
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Coastal waters adjacent to populated southeast Florida possess different habitats (reefs, oceanic inlets, sewage outfalls) that may affect the composition of their inherent microbiomes. To determine variation according to site, season, and depth, over the course of 1 year, we characterized the bacterioplankton communities within 38 nearshore seawater samples derived from the Florida Area Coastal Environment (FACE) water quality survey. Six distinct coastal locales were profiled - the Port Everglades and Hillsboro Inlets, Hollywood and Broward wastewater outfalls, and associated reef sites using culture-independent, high-throughput pyrosequencing of the 16S rRNA V4 region. More than 227,000 sequences helped describe longitudinal taxonomic profiles of marine bacteria and archaea. There were 4447 unique operational taxonomic units (OTUs) identified with a mean OTU count of 5986 OTUs across all sites. Bacterial taxa varied significantly by season and by site using weighted and unweighted Unifrac, but depth was only supported by weighted Unifrac, suggesting a change due to presence/absence of certain OTUs. Abundant microbial taxa across all samples included Synechococcus, Pelagibacteraceae, Bacteroidetes, and various Proteobacteria. Unifrac analysis confirmed significant differences at inlet sites relative to reef and outfalls. Inlet-based bacterioplankton significantly differed in greater abundances of Rhodobacteraceae and Cryomorphaceae, and depletion of SAR406 sequences. This study also found higher counts of Firmicutes, Chloroflexi, and wastewater associated SBR1093 bacteria at the outfall and reef sites compared to inlet sites. This study profiles local bacterioplankton populations in a much broader context, beyond culturing and quantitative PCR, and expands upon the work completed by the National Oceanic and Atmospheric Administration FACE program. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
... Host prevalence is also considered an important performance characteristic because it is likely that a highly host-prevalent marker will be more frequently detected in polluted water samples. Many studies have reported the host prevalence values of the HF183, HAdV, and HPyV markers by analyzing individual fecal and wastewater samples using binary PCR (positive/negative) (8,12,26,27,32). The host prevalence value of a particular marker in the host population may vary geographically due to uneven distribution. ...
Article
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Recreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is utmost important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater is considered as an important factor because it is likely that a marker whose concentrations is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp., and HFMs Bacteroides HF183 (HF183), human adenoviruses (HAdVs) and polyomaviruses (HPyVs) in raw municipal wastewater influent from varying climatic zones in Australia. The mean concentrations of E. coli in pooled human wastewater datasets (from varying climatic zones) were the highest (3.2 × 106 gene copies per mL), followed by HF183 (8.0 × 105 gene copies per mL) and Enterococcus spp. (3.6 × 105 gene copies per mL). HAdV and HPyV concentrations were 2-3 orders of magnitude lower than FIB and the HF183. Strong positive and negative correlations were observed between FIB and HFMs concentrations within and across WWTPs. To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated that Bacteroides HF183 were more sensitive in detecting human fecal pollution compared to HAdVs and HPyVs. Since, the HF183 marker can be occasionally present in non-target animal fecal samples, it is recommended that for tracking human fecal pollution in Australian environmental waters, HF183 along with a viral marker (HAdVs or HPyVs) should be used.
... Other confounders related to the preanalytical process include poor sample quality either through lack of expertise or inappropriate sample handling, holding time/temperature, or processing. All analytical methods require strict adherence to quality assurance, quality control, and procedural requirements, therefore interlaboratory studies and future regulatory procedures require detailed standard operating procedures (e.g., 89,90). When considering molecular MST tools, one must consider both preanalytical (the source, location, timing, transport, and storage of the sample) and analytical processes (choice of method, accuracy, precision, and source-specific interfering substances, if any). ...
Chapter
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Field study design and implementation are critical to the success of fecal source tracking (FST) studies. Significant advances in the field of microbial and chemical source tracking (MST or CST) provide access to a variety of analytical tools, beyond traditional culture-based measurements of fecal indicator bacteria (FIB), in the identification and apportionment of pollution sources adversely impacting water quality and of public health concern. Execution of investigative studies employing these tools requires a phased approach: defining the questions and desired outcome, site assessment, field sampling/laboratory analysis, confirmatory testing, statistical analysis, interpretation of results, and translation of results into actionable items. A sound study design addresses spatial and temporal variability as well as geographic distribution of markers or target organisms used to develop statistically robust data sets from which sound conclusions are drawn. While FST tools may be cutting edge, and informative in their own right, multiple lines of evidence are necessary to adequately characterize pollutant source, loading, and human exposure risk. Assessments of environmental, meteorological and hydrological parameters may increase accuracy in the assignation of relative contributions (from multiple sources) in complex environments. In the absence of strong correlation between FIB, physical attributes and MST or CST markers, a weight of evidence approach may be used to target human exposure interventions, which may take the form of engineered, naturalized, or educational measures. A multi-barrier approach to protecting public health, cognizant of confounding factors impacting the analytical or remediation process, should be stressed and include stakeholder engagement throughout the process.
... Molecular human MST markers target unique sequences in bacterial, viral, coliphage, and mitochondrial DNA. Human MST, based on qPCR or PCR, identifies bacterial DNA sequences affiliated with (Ahmed and Katouli, 2008) Enterococcus faecium, ( Harwood et al., 2009) Methanobrevibacter smithii, (Balleste and Blanch, 2011) Bifidobacterium dentium. The 16S rRNA gene (Bernhard and Field, 2000;Layton et al., 2006;Haugland et al., 2010) is currently the most prominent amplification target among Bacteroidales assays. ...
Article
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The emerging Microbial Source Tracking (MST) methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes High Throughput Sequencing (HTS) to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU) analysis was then performed, from which sequences were retained for downstream qPCR marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae and other bacteria. Two assays (Av4143 and Av163) identified most of the avian fecal samples and demonstrated sensitivity values of 91% and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216) identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.
... However, bacteriophage is also questioned by some researchers because certain viruses such as EVs were detected when bacteriophage was below the detection limit (Hot et al. 2003). It is also suggested to use AdVs, EVs and polyomavirus (PyV) directly as potential indicators of waterborne pathogens (Hamza et al. 2011;Harwood et al. 2009). The way to identify or develop more suitable indicators of viral contamination may continuously be a hot topic for future studies. ...
Article
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Domestic wastewater contains various pathogens, which, if not sufficiently eliminated, may enter the receiving water bodies and cause water-transmitted diseases. Among the waterborne pathogens, viruses may occur, survive and/or decay much differently from bacteria in water. In many cases, the diseases caused by viruses are more severe. Therefore, research efforts are mainly directed at the behavior of viruses in water environments, as well as the elimination of viruses from wastewater. In this paper, an overview of the occurrence of viruses in wastewater is presented, together with their categories, methods of detection and potential to cause waterborne diseases. As wastewater treatment plants are critical nodes for the influx and termination of virus transmission, the behavior of viruses at each stage of treatment is reviewed. Particular attention is paid to the unit operations, which play crucial roles in virus removals, such as coagulation and membrane filtration, and that for virus inactivation, such as chemical disinfection and UV irradiation. Future needs for the development of new technologies for virus elimination, source control, and finding more suitable indicators of viral pathogens are also highlighted.
... All bacterial concentrations were log 10 transformed prior to analyses. Due to the frequent lack of detection of culturable V. vulnificus organisms, one-half the detection limit was used for "non-detect" observations (27). A general linear model with a full factorial design was performed with univariate tests for significance with sigma-restricted parameterization using Statistica version 12 for Windows (StatSoft Software, Tulsa, OK). ...
Article
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The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats, and is readily cultured from water and oysters in warm conditions, but infrequently at ambient conditions < 15° C. The presence of V. vulnificus in other habitats such as sediments and aquatic vegetation has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR), and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus were sampled from five sites around Tampa Bay, Florida. Levels determined by qPCR and culture were significantly correlated (P=0.0006; r=0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%), and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 - 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments, but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices, and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... The MST technology currently being developed will allow us not only to detect fecal contamination using indicators as well as the foodborne pathogen contamination, but also to simultaneously determine the source of contamination in both water and food samples. Potential applications of MST have been driving methods development, and we currently find ourselves with multiple library-dependent and library-independent approaches in various stages of development and validation (Hassan et al., 2007;Harwood et al., 2009;Lyautey et al., 2010); however, no one method has emerged as being superior to be adopted as a standard. Until individual MST methods are developed to the point of being accepted as both regulatory and management tools, one way to overcome the limitations of any one method is to perform multiple methods concurrently. ...
Chapter
Cross-contamination of foodborne pathogens or nonpathogenic spoilage bacteria in the food production environment can cause huge economic losses attributing to waste of raw food materials or a significant public health issue. Bacterial cross-contamination and recontamination episodes have been connected to the following factors: polluted raw materials, poor sanitation practices, poor equipment design, and deficient control of ingredients. This chapter provides an overview of types and diversity of bacteria, molecular methods for tracking bacterial contamination, and elimination of bacteria in the food production. Furthermore, future directions in the field are also discussed.
... Several studies have reported 100% host specificity of the HF183 marker for non-human fecal samples [51,[66][67][68][69][70]. In contrast, the occasional presence of the HF183 markers in non-human fecal samples has also been reported [52,57,61,71,72], particularly in dog, deer, and chicken feces. ...
Article
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Microbial source tracking (MST) endeavors to determine sources of fecal pollution in environmental waters by capitalizing on the association of certain microorganisms with the gastrointestinal tract and feces of specific animal groups. Several decades of research have shown that bacteria belonging to the gut-associated order Bacteroidales, and particularly the genus Bacteroides, tend to co-evolve with the host, and are, therefore, particularly suitable candidates for MST applications. This review summarizes the current research on MST methods that employ genes belonging to Bacteroidales/Bacteroides as tracers or “markers” of sewage pollution, including known advantages and deficiencies of the many polymerase chain reaction (PCR)-based methods that have been published since 2000. Host specificity is a paramount criterion for confidence that detection of a marker is a true indicator of the target host. Host sensitivity, or the prevalence of the marker in feces/waste from the target host, is necessary for confidence that absence of the marker is indicative of the absence of the pollution source. Each of these parameters can vary widely depending on the type of waste assessed and the geographic location. Differential decay characteristics of bacterial targets and their associated DNA contribute to challenges in interpreting MST results in the context of human health risks. The HF183 marker, derived from the 16S rRNA gene of Bacteroides dorei and closely related taxa, has been used for almost two decades in MST studies, and is well characterized regarding host sensitivity and specificity, and in prevalence and concentration in sewage in many countries. Other markers such as HumM2 and HumM3 show promise, but require further performance testing to demonstrate their widespread utility. An important limitation of the one-marker-one-assay approach commonly used for MST is that given the complexities of microbial persistence in environmental waters, and the methodological challenges of quantitative PCR (qPCR) in such samples, the absence of a given marker does not ensure the absence of fecal pollution in the source water. Approaches under development, such as microarray and community analysis, have the potential to improve MST practices, thereby increasing our ability to protect human and ecosystem health.
... Several studies have reported 100% host specificity of the HF183 marker for non-human fecal samples [51,[66][67][68][69][70]. In contrast, the occasional presence of the HF183 markers in non-human fecal samples has also been reported [52,57,61,71,72], particularly in dog, deer, and chicken feces. ...
Article
Full-text available
Microbial source tracking (MST) endeavors to determine sources of fecal pollution in environmental waters by capitalizing on the association of certain microorganisms with the gastrointestinal tract and feces of specific animal groups. Several decades of research have shown that bacteria belonging to the gut-associated order Bacteroidales, and particularly the genus Bacteroides, tend to co-evolve with the host, and are, therefore, particularly suitable candidates for MST applications. This review summarizes the current research on MST methods that employ genes belonging to Bacteroidales/Bacteroides as tracers or "markers" of sewage pollution, including known advantages and deficiencies of the many polymerase chain reaction (PCR)-based methods that have been published since 2000. Host specificity is a paramount criterion for confidence that detection of a marker is a true indicator of the target host. Host sensitivity, or the prevalence of the marker in feces/waste from the target host, is necessary for confidence that absence of the marker is indicative of the absence of the pollution source. Each of these parameters can vary widely depending on the type of waste assessed and the geographic location. Differential decay characteristics of bacterial targets and their associated DNA contribute to challenges in interpreting MST results in the context of human health risks. The HF183 marker, derived from the 16S rRNA gene of Bacteroides dorei and closely related taxa, has been used for almost two decades in MST studies, and is well characterized regarding host sensitivity and specificity, and in prevalence and concentration in sewage in many countries. Other markers such as HumM2 and HumM3 show promise, but require further performance testing to demonstrate their widespread utility. An important limitation of the one-marker-one-assay approach commonly used for MST is that given the complexities of microbial persistence in environmental waters, and the methodological challenges of quantitative PCR (qPCR) in such samples, the absence of a given marker does not ensure the absence of fecal pollution in the source water. Approaches under development, such as microarray and community analysis, have the potential to improve MST practices, thereby increasing our ability to protect human and ecosystem health.
... Enumeration of fecal coliforms and enterococci was by standard membrane filtration methods for water and oyster homogenates (American Public Health Association, 1995; United States Environmental Protection Agency, 2002). The presence of human polyomavirus (HPyVs) and human-associated Bacteroidales was determined by PCR as described previously to determine whether human fecal contamination was present (Harwood et al., 2009). ...
... The molecular techniques available today are being continuously refined in order to make them applicable to a diversity of pathogens and conditions, to increase their sensitivity and to reduce the time and steps required in the analytical process [13] . This refinement, standardization and validation of protocols is considered a very important requirement for the implementation of molecular techniques in the pathological, clinical or the environmental fields [14][15][16][17][18]. Present paper describes standardization of polymerase chain reaction (PCR) technique for detection of myxozoan parasites and identification of two exotic species of Myxobolus Butschli, 1882 from an Indian freshwater fish Clarias batrachus Linnaeus, 1758. ...
Article
The international trade of live aquatic animals has gained a momentum in recent years. To facilitate this trade disease/pathogen related risks associated with fish movement have to be minimized. Tests done through molecular tools for confirming presence/absence of a pathogen in consignment have been approved most appropriate by International Organization of Epizootics (OIE), WTO, FAO and NACA, Bangkok. With the help of Polymerase Chain Reaction (PCR) amplification of RNA/DNA, it has become possible to rapidly detect the pathogen even if the quantity of DNA/RNA is very little. In India, there is a paucity or we can say total lack of work on molecular diagnostics of fish parasites particularly 'Protozoans'. Hence we developed and standardized the DNA/RNA polymerase chain reaction (PCR) amplification technique for detection of Myxozoan (Protozoan) parasites. Further, two exotic species of Myxobolus Butschli, 1882 from an Indian catfish Clarias batrachus have also been detected and identified with the help of present PCR technique which are reported here.
... The molecular techniques available today are being continuously refined in order to make them applicable to a diversity of pathogens and conditions, to increase their sensitivity and to reduce the time and steps required in the analytical process [13] . This refinement, standardization and validation of protocols is considered a very important requirement for the implementation of molecular techniques in the pathological, clinical or the environmental fields [14][15][16][17][18] . Present paper describes standardization of polymerase chain reaction (PCR) technique for detection of myxozoan parasites and identification of two exotic species of Myxobolus Butschli, 1882 from an Indian freshwater fish Clarias batrachus Linnaeus, 1758. ...
Article
The international trade of live aquatic animals has gained a momentum in recent years. To facilitate this trade disease/pathogen related risks associated with fish movement have to be minimized. Tests done through molecular tools for confirming presence/absence of a pathogen in consignment have been approved most appropriate by International Organization of Epizootics (OIE), WTO, FAO and NACA, Bangkok. With the help of Polymerase Chain Reaction (PCR) amplification of RNA/DNA, it has become possible to rapidly detect the pathogen even if the quantity of DNA/RNA is very little. In India, there is a paucity or we can say total lack of work on molecular diagnostics of fish parasites particularly 'Protozoans'. Hence we developed and standardized the DNA/RNA polymerase chain reaction (PCR) amplification technique for detection of Myxozoan (Protozoan) parasites. Further, two exotic species of Myxobolus Butschli, 1882 from an Indian catfish Clarias batrachus have also been detected and identified with the help of present PCR technique which are reported here.
... The choice of targeting mtDNA for source tracking in this particular study resulted from several premises: (i) mtDNA markers target specifically eukaryote cell DNA from the target animals rather than targeting specific bacteria or viruses associated to a specific animal; (ii) mtDNA evolves faster than nuclear DNA therefore providing an adequate number of sequence variations for the design of species-associated PCR assays; (iii) a large number of exfoliated cells are released in the feces of animals each containing a high number of mitochondrion and respective DNA sequences, which increases largely the sensitivity of the assays; (iv) specific strains of Bacteroides spp. may not be present in all of their specific hosts, as is the case for instance of the HF183 marker which is not present in the intestinal microbiome of all humans (Harwood et al., 2009); and (v) Bacteroides spp. are obligatory anaerobes and in our particular setting this could conduct rather immediately to their lysis and subsequent DNA degradation. ...
Article
Full-text available
Rainfall and associated urban runoff have been linked to an increased deterioration of environmental waters, carrying several pollutants including pathogenic microorganisms. Such happens because fecal matter is washed into storm drainage pipes that are afterward released into environmental waters. Stormwater has not been extensively characterized as it is, because most studies are performed either on drainage pipes that are often impacted by sewage leakage or directly in environmental waters following a rain event. In this study, stormwater collected directly from the streets, was monitored for the presence of fecal indicator bacteria (FIB) and three potential important sources of fecal contamination in urban environments (human, cats, and dogs) in three distinct basins in Lisbon, Portugal. Stormwater was collected in sterilized plastic boxes inserted in the storm drains, therefore collecting only runoff. High concentration of fecal contamination was detected with a high percentage of the samples displayed at least one source of contamination. A strong relationship was found between the number of detected sources and the precipitation levels. Although no statistical correlation was found between the locations and the presence of FIB or source markers, the results show a trend in geographical information on the type of urban use in each basin. To the best of our knowledge, this is the first study analyzing the runoff collected directly from the streets. This study suggests that, in urban areas, stormwater runoff is highly impacted by fecal matter, not only from domestic animals but also from human origin, before any cross-contamination in the drainage system and may, by itself, pose a high risk to human health and the environment, particularly if water reuse of this water without further disinfection treatment is the final goal.
... M. smithii may be a useful indicator of sewage or human fecal pollution because of its host's specificity and high abundance in the human intestine [95]. Investigating sewage-associated nifH gene marker of M. smithii in recreational waters [96][97][98][99], showed a 29% sensitivity among individual human fecal samples, but a 93% sensitivity among sewage samples and a 100% specificity in tested samples of Mississippi and Missouri and concluded that this result appears more representative of real-world problems [100]. ...
Article
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Prokaryotes forming the domain of Archaea, named after their first discovery in extreme environments, are acknowledged but still neglected members of the human digestive tract microbiota. In this microbiota, cultured archaea comprise anaerobic methanogens: Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter massiliense, Methanosphaera stadtmanae, Methanobrevibacter arboriphilus,Methanobrevibacter millerae and Methanomassiliicoccus luminyensis; along with the non-methanogen halophilic Archaea Halopherax massiliense. Metagenomic analyses detected DNA sequences indicative of the presence of additional methanogenic and non-methanogenic halophilic Archaea in the human intestinal tract and oral cavity. Methanogens specifically metabolize hydrogen produced by anaerobic fermentation of carbohydrates into methane; further transforming heavy metals and metalloids into methylated derivatives, such as trimethylbismuth which is toxic for both human and bacterial cells. However, the role of Archaea as pathogens remains to be established. Future researches will aim to increase the repertoire of the human digestive tract Archaea and to understand their possible association with intestinal and extra-intestinal infections and diseases including weight regulation abnormalities.
... Face à l'abondance des techniques proposées pour tenter d'identifier l'origine d'une contamination fécale dans l'environnement, aucun consensus n'est aujourd'hui défini quant à la meilleure stratégie à adopter. Des études multi-laboratoires sont régulièrement menées en aveugle afin d'évaluer ces différentes approches, de les standardiser et d'identifier les plus efficaces (Blanch et al., 2006;Ebentier et al., 2013;Griffith et al., 2003;Harwood et al., 2013Harwood et al., , 2009Shanks et al., 2016). Les données les plus récentes tendent à considérer l'ensemble de ces méthodes comme une boite à outils et la recherche concomitante de différents marqueurs est souvent préconisée puisque certains marqueurs s'avèrent plus ou moins pertinents selon le contexte. ...
Thesis
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Les virus entériques sont à l’origine de pathologies liées au péril fécal et dans l’état actuel des connaissances, la recherche des indicateurs de pollution fécale conventionnels (i.e. Escherichia coli, entérocoques) peut s’avérer inefficace pour évaluer le danger viral. La définition d’autres indicateurs pour gérer le danger lié à la présence des virus entériques dans les matrices hydriques et alimentaires est aujourd’hui nécessaire. Parmi eux, les bactériophages ARN F-spécifiques (FRNAPH) présentent plusieurs intérêts. Ces virus d’origine entérique sont présents en quantité importante dans les eaux usées. Très proches des virus entériques en termes de structure, ces microorganismes présentent l’avantage d’être facilement cultivables. Ils sont enfin souvent étudiés pour déterminer l’origine d’une pollution fécale (i.e. humaine ou animale). Certaines limites leur sont cependant fréquemment associées, que ce soit en termes de corrélation avec les pathogènes entériques ou concernant leur potentiel pour discriminer l’origine d’une pollution. Dans ce contexte, l’objectif du travail présenté ici était de préciser l’intérêt des FRNAPH en tant qu’indicateurs de pollution fécale mais aussi en tant qu’indicateurs de pollution virale dans l’environnement et les coquillages. Ces travaux ont permis dans un premier temps d’améliorer la capacité des FRNAPH à identifier les contaminations d’origine humaine. Nos résultats soulignent par ailleurs la plus-value apportée par la recherche des FRNAPH en cas de pollution fécale massive, en particulier si on s’intéresse à la contamination des coquillages. En effet, contrairement aux indicateurs bactériens, l’accumulation des FRNAPH ainsi que leur persistance dans ces aliments est très comparable à celles des virus entériques (i.e. norovirus). Enfin, en utilisant des méthodes de détection comparables, une forte corrélation entre la présence des FRNAPH d’origine humaine et celle des norovirus a été observée dans les coquillages. Compte tenu de ces résultats, une méthode de détection assurant la détection sensible des FRNAPH infectieux d’origine humaine dans différents types de matrices hydriques ou alimentaires (e.g. eaux de surface, fruits de mer, fruits rouges, salades) est proposée pour améliorer la gestion du danger viral
... Human pathogens, like Giardia, Cryptosporidium , and norovirus genogroup I (NoVGI), are increasingly included in microbial water quality studies because conventional FIB do not always correlate with their presence ( Ashbolt et al., 2010 ;Boehm et al., 2009 ). Microbial source tracking (MST) methods have been developed to determine fecal pollution sources ( Chase et al., 2012 ;Harwood et al., 2009 ). Microorganisms associated with human feces and wastewater are frequent targets of MST assays, including the 16S rRNA gene of a Bacteroides species (HF183) ( Seurinck et al., 2005 ) and pepper mild mottle virus (PM-MoV) ( Symonds et al., 2018( Symonds et al., , 2016. ...
Article
Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms’ concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users’ health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches.
... relaying, depuration or high-pressure processing) before consumption. FIB presence poorly correlates with enteric viruses owing to the occurrence of FIB in diverse faecal sources and their different persistence rates in BMS [34,35]. Indeed, studies have reported enteric virus disease outbreaks associated with the consumption of microbiologically approved BMS [3,26,36]. ...
Article
Consumption of bivalve molluscan shellfish (BMS) grown in faecally contaminated waters is a public health risk. While faecal contamination in BMS and water can originate from multiple sources (i.e. human, livestock, wildlife and crop production), accurate identification of the source (s) is important to establish an appropriate, cost-effective plan to minimise the potential public health risks. Microbial source tracking (MST) targets (genes) are more source-specific than faecal indicator bacteria. Application of MST methods could eliminate the requirement of multiple testing regime for bacterial and viral pathogen in BMS. This approach therefore, can assist to develop an accurate, reliable faecal pollution management plan that reduces the cost for BMS farmers as well as public health risks associate with BMS consumption. However, validation of MST targets, their recovery from BMS and correlation with other bioaccumulated pathogens in BMS is required prior to its field application.
... None of the bacterial markers exhibit absolute host specificity; they occasionally are detected in nontarget animal species, probably due to physiology, colonization, similar diets, and cohabitation. For example, HF183, BacHum-UCD, and Methanobrevibacter smithii nifH marker genes have shown limited cross-reactivity with chicken, dog, and other animals (19)(20)(21). In contrast, certain viruses, such as human adenovirus (HAdV) and human polyomavirus (HPyV), have shown absolute host specificity, although other viruses currently used for MST field studies, such as pepper mild mottle virus (PMMoV) and crAssphage, have been detected sporadically in nonhuman fecal samples (22,23). ...
Article
Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.
... Therefore, all data were log-transformed to improve normality before statistical analysis ( Ahmed et al., 2015). The quantitative values for the non- detect (i.e., samples that were below ALOD for FIB, potentially pathogenic bacteria, and protozoa) samples were predicted by using the one-half of the detection limit ( Chase et al., 2015;Harwood et al., 2009). The nonparametric Spearman rank correlation with a two- tailed p value was used to establish the relationship between concentrations of bacteria/FIB with potential pathogens. ...
Article
We investigated the abundance of marker genes of two fecal indicator bacteria (FIB) and eight potential pathogens in fecal samples (n = 14) of humans and 10 domestic and native wild animals (n = 134). For each target animal, between 10 and 14 individual fecal samples were collected (n = 148 individual fecal samples in total). The abundance of FIB and potential pathogens within each sample was tested using quantitative PCR (qPCR) assays. All animals tested were positive for Escherichia coli (EC) and the concentrations ranged from 6.13 (flying fox) to 8.87 (chicken) log10 GC/g of feces. These values for Enterococcus spp. (ENT) were 5.25 log10 GC/g for flying fox and 8.12 log10 GC/g of feces for chicken. Moderate correlations were observed between EC with P. aeruginosa, EC O157 and Cryptosporidium parvum, whereas weak correlations were observed between EC and Salmonella and Giardia lamblia, Mycobacterium avium complex (MAC) and Campylobacter spp. The prevalence of MAC and P. aeruginosa were low in dog (14.3% each) and moderate (57.2%, MAC; 42.9% P. aeruginosa) in Eastern grey kangaroo fecal samples. C. parvum was detected in one cattle and one human fecal sample, while G. lamblia was detected in one dog, one flying fox, and one pig fecal samples. Among the eight potential pathogens tested, five pathogens were detected in chicken and dog fecal samples. The remaining animal species contained up to three potential pathogens in their feces. The data generated in this study will aid in the calculation of pathogen loads in the environment, and hence to assess the risks from fecal contamination of source waters.
... Such markers have successfully differentiated human fecal pollution sources from various animal sources including ruminants, dogs and birds (e.g. geese) (Bernhard and Field, 2000b;Kildare et al., 2007;Green et al., 2012) in a wide range of geographic regions that include Canada (Wilkes et al., 2013), the US Gulf of Mexico (Harwood et al., 2009), Japan (Okabe et al., 2007), Kenya (Jenkins et al., 2009), Tanzania (Pickering et al., 2011(Pickering et al., , 2012Mattioli et al., 2013), and New Zealand (Green et al., 2012). ...
Article
The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively.
... Among these performance characteristics, hostsensitivity and host-specificity are the two most important factors. To date, none of the MST markers in the research literature has exhibited absolute host-specificity except human adenoviruses (HAdV) and human polyomaviruses (HPyV) (Ahmed et al., 2010;Harwood et al., 2009). However, a drawback of these two markers is the concentrations tend to be low in untreated sewage because their occurrence (especially adenovirus) depends on illness rates in the community. ...
Article
Considerable efforts have been made in recent years in developing novel marker genes for fecal pollution tracking in environmental waters. CrAssphage are recently discovered DNA bacteriophage that are highly abundant in human feces and untreated sewage. In this study, we evaluated the host-sensitivity and -specificity of the newly designed crAssphage qPCR assays (Stachler et al., 2017) CPQ_056 and CPQ_064 (i.e., marker genes) in fecal samples collected from various human and several animal host groups in Australia. We also investigated the utility of these marker genes to detect sewage pollution in an urban recreational lake (i.e., Lake Parramatta) in Sydney, NSW. The mean concentrations of CPQ_056 and CPQ_064 marker genes in untreated sewage were 9.43 ± 0.14 log10 GC/L and 8.91 ± 0.17 log10 GC/L, respectively, 2 to 3 orders of magnitude higher than other sewage-associated viruses used in microbial source tracking studies. Among 177 animal fecal samples tested from 11 species, the host-specificity values for CPQ_056 and CPQ_064 marker genes were 0.95 and 0.93, respectively. Limited cross-reactivity was observed with cat fecal and cattle wastewater samples. Abundance of crAssphage markers were monitored in an urban lake that receives stormwater runoff. The concentrations of both markers were higher (CPQ_056 ranging from 3.40 to 6.04 log10 GC/L and CPQ_064 ranging from 2.90 to 5.47 log10 GC/L) in 20 of 20 (for CPQ_056) and 18 of 20 (for CPQ_064) samples collected after storm events with gauged sewer overflows compared to dry weather event (10 of 10 samples were qPCR negative for the CPQ_056 and 8 of 10 were negative for the CPQ_064 marker genes) suggesting sewage pollution was transported by urban stormwater runoff to Lake Parramatta. The results of the study may provide context for management of sewage pollution from gauged overflow points of the sewerage system in the catchment.
... The majority of research to identify host-specific genetic markers (i.e., a specific sequence within the DNA of fecal bacteria) have focused on Bacteroidales (Bernhard and Field, 2000;Dick et al., 2005;Reischer et al., 2006;Kildare et al., 2007;Fremaux et al., 2009). Geographic variability and host-specificity can factor into the efficacy of markers in discrimination of host sources, (Bernhard and Field, 2000;Harwood et al., 2009;. Only a few studies have explored the applicability of these markers in underdeveloped regions (Jenkins et al., 2009;Ahmed et al., 2010;Reischer et al., 2013). ...
Article
Untreated sewage discharges and limited agricultural manure management practices contribute to fecal pollution in rural Brazilian waterways. Few microbial source tracking studies have tested host-specific indicators in underdeveloped regions and have mostly focused on Bacteroidales. Sequencing of sewage, human feces, and animal feces with Illumina HiSeq revealed Prevotellaceae as the most abundant family in humans, with Lachnospiraceae and Ruminococcaceae also comprising a large proportion of the microbiome. These same families were also dominant in animals. Bacteroides, the most commonly utilized human-specific marker in the US, was present in very low abundance. We used oligotyping to identify Prevotella and Blautia sequences that can distinguish human fecal contamination. Thirty-five of 61 Blautia oligotypes and 13 of 108 Prevotella oligotypes in humans were specific to or highly abundant (i.e. host-preferred) compared to pig, dog, horse, and cow sources. Certain human Prevotella and Blautia oligotypes increased more than an order of magnitude along a polluted river transect in rural Brazil, but traditional fecal indicator levels followed a steady or even decreasing trend. While both Prevotella and Blautia oligotypes distinguished human and animal fecal pollution in Brazil surface waters, Blautia appears to contain more discriminatory and globally applicable markers for tracking sources of fecal pollution.
... This excretion route for polyomaviruses has already been documented in the literature (Egli et al., 2009;Shinohara et al., 1993). For this reason, the group has been widely used as a specific indicator of human excreta in water (Harwood et al., 2009). In recent years, new polyomaviruses have been described, including up to 13 human polyomaviruses (Mishra et al., 2014). ...
Article
The application of next-generation sequencing (NGS) techniques for the identification of viruses present in urban sewage has not been fully explored. This is partially due to a lack of reliable and sensitive protocols for studying viral diversity and to the highly complex analysis required for NGS data processing. One important step towards this goal is finding methods that can efficiently concentrate viruses from sewage samples. Here the application of a virus concentration method based on skimmed milk organic flocculation (SMF) using 10 L of sewage collected in different seasons enabled the detection of many viruses. However, some viruses, such as human adenoviruses, could not always be detected using metagenomics, even when quantitative PCR (qPCR) assessments were positive. A targeted metagenomic assay for adenoviruses was conducted and 59.41% of the obtained reads were assigned to murine adenoviruses. However, up to 20 different human adenoviruses (HAdV) were detected by this targeted assay being the most abundant HAdV-41 (29.24%) and HAdV-51 (1.63%). To improve metagenomics' sensitivity, two different protocols for virus concentration were comparatively analysed: an ultra-centrifugation protocol and a lower-volume SMF protocol. The sewage virome contained 41 viral families, including pathogenic viral species from families Caliciviridae, Adenoviridae, Astroviridae, Picornaviridae, Polyomaviridae, Papillomaviridae and Hepeviridae. The contribution of urine to sewage metavirome seems to be restricted to a few specific DNA viral families, including the polyomavirus and papillomavirus species. In experimental infections with sewage in a rhesus macaque model, infective human hepatitis E and JC polyomavirus were identified. Urban raw sewage consists of the excreta of thousands of inhabitants; therefore, it is a representative sample
... This marker has consistently been identified as the "gold standard" in detecting urban sewage intrusion in surface waters (Harwood et al., 2014); however, detection has proven more challenging in lower population watersheds, even when sewage contamination is a known issue (Cantor et al., 2017). This is likely at least partially because not all humans carry the HF183 in their intestinal microbiome (Harwood et al., 2009). Detection in groundwater is further complicated by a limited understanding of the movement and persistence of genetic ST markers in biologically complex soil environments, as has been noted previously (Tambalo et al., 2012). ...
Article
The past three decades' data on outbreaks in the United States indicate that homes dependent on untreated groundwater (e.g. wells) for household drinking water that are also reliant on onsite treatment of household wastewater (e.g. septic systems) may be at greater risk for waterborne disease. While groundwater quality monitoring to protect public health has traditionally focused on the detection of fecal indicator bacteria, the application of emerging source tracking strategies may offer a more efficient means to identify pollution sources and effective means of remediation. This study compares the movement of common fecal indicator bacteria (E. coli and enterococci) with a chemical (optical brighteners, OB) and a molecular (Bacteroides HF183) source tracking (ST) target in small scale septic drainfield models in order to evaluate their potential utility in groundwater monitoring. Nine PVC column drainfield models received synchronized doses of primary-treated wastewater twice daily, with influent and effluent monitored bi-weekly over a 7-month period for all targets. Results indicate that E. coli and enterococci concentrations were strongly associated (Spearman's rank, p<0.05), and correlations between enterococci and optical brighteners were moderately strong. Bacteroides HF183 was significantly, but not strongly, associated with optical brighteners and both indicator bacteria (Point-biserial correlation, p<0.05), most likely due to its sporadic detection. Application of human ST marker monitoring in groundwaters at risk of contamination by human sewage is recommended, although consistent interpretation of results will rely on more detailed evaluation of HF183 incidence in source contamination waters.
... Host prevalence is also considered an important performance characteristic because it is likely that a highly host-prevalent marker will be more frequently detected in polluted water samples. Many studies have reported the host prevalence values of the HF183, HAdV, and HPyV markers by analyzing individual fecal and wastewater samples using binary PCR (positive/negative) (8,12,26,27,32). The host prevalence value of a particular marker in the host population may vary geographically due to uneven distribution. ...
... Within the last decade, molecular source techniques using a combination of more advanced DNA gene markers to identify fecal bacteria have been developed with a fair measure of success from estuarine to tropical aquatic systems (Bernhard and Field 2000;Meays et al. 2004;Lee et al. 2014;Neave et al. 2014). These methods have potential to drastically improve monitoring since they include library independent techniques that target various strains of harmful bacterial sequences with significantly more accuracy than traditional tests (Gerber et al. 2001;Harwood et al. 2009;Shanks et al. 2009;AWWA et al. 2012;Toledo-Hernandez et al. 2013). For example, a host-associated marker that targets animal gut microbiota (i.e., Bacteroidetes) has shown some success in case studies (Scott et al. 2002;Fremaux et al. 2009). ...
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Water quality impairment by fecal waste in coastal watersheds is a public health issue. The present study provided evidence for the use of a mitochondrial (mtDNA) marker to detect animal fecal sources in surface water. The accurate identification of fecal pollution is based on the notion that fecal microorganisms preferentially inhabit a host animal’s gut environment. In contrast, mtDNA host-specific markers are inherent to eukaryotic host cells, which offers the advantage by detecting DNA from the host rather than its fecal bacteria. The present study focused on sampling water presumably from non-point sources (NPS), which can increase bacterial and nitrogen concentrations to receiving water bodies. Stream sampling sites located within the Piscataqua River Watershed (PRW), New Hampshire, USA, were sampled from a range of sites that experienced nitrogen inputs such as sewer and septic systems and suburban runoff. Three mitochondrial (mtDNA) gene marker assays (human, bovine, and canine) were tested from surface water. Nineteen sites were sampled during an 18-month period. Analyses of the combined single and multiplex assay results showed that the proportion of occurrence was highest for bovine (15.6%; n = 77) compared to canine (5.6%; n = 70) and human (5.7%; n = 107) mtDNA gene markers. For the human mtDNA marker, there was a statistically significant relationship between presence vs. absence and land use (Fisher’s test p = 0.0031). This result was evident particularly for rural suburban septic, which showed the highest proportion of presence (19.2%) compared to the urban sewered (3.3%), suburban sewered (0%), and agricultural (0%) as well as forested septic (0%) sites. Although further testing across varied land use is needed, our study provides evidence for using the mtDNA marker in large watersheds.
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We investigated the usefulness of the β-d-glucuronidase gene variance in Escherichia coli as a microbial source tracking tool using a novel algorithm for comparison of sequences from a prescreened set of host-specific isolates using a high-resolution PhP typing method. A total of 65 common biochemical phenotypes belonging to 318 E. coli strains isolated from humans and domestic and wild animals were analysed for nucleotide variations at 10 loci along a 518 bp fragment of the 1812 bp β-d-glucuronidase gene. Neighbour-joining analysis of loci variations revealed 86 (76.8%) human isolates and 91.2% of animal isolates were correctly identified. Pairwise hierarchical clustering improved assignment; where 92 (82.1%) human and 204 (99%) animal strains were assigned to their respective cluster. Our data show that initial typing of isolates and selection of common types from different hosts prior to analysis of the β-d-glucuronidase gene sequence improves source identification. We also concluded that numerical profiling of the nucleotide variations can be used as a valuable approach to differentiate human from animal E. coli. This study signifies the usefulness of the β-d-glucuronidase gene as a marker for differentiating human faecal pollution from animal sources.
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Poor sanitation in rural infrastructure is often associated with high levels of fecal contamination in adjacent surface waters, which presents a community health risk. Although microbial source tracking techniques have been widely applied to identify primary remediation needs in urban and/or recreational waters, use of human-specific markers has been more limited in rural watersheds. This study quantified the human source tracking marker Bacteroides-HF183, along with more general fecal indicators (i.e. culturable Escherichia coli and a molecular Enterococcus marker), in two Appalachian streams above and below known discharges of untreated household waste. Although E. coli and Enterococcus were consistently recovered in samples collected from both streams, Bacteroides-HF183 was only detected sporadically in one stream. Multiple linear regression analysis demonstrated a positive correlation between the concentration of E. coli and the proximity and number of known waste discharge points upstream; this correlation was not significant with respect to Bacteroides-HF183, likely due to the low number of quantifiable samples. These findings suggest that, while the application of more advanced source-targeting strategies can be useful in confirming the influence of substandard sanitation on surface waters to justify infrastructure improvements, they may be of limited use without concurrent traditional monitoring targets and on-the-ground sanitation surveys.
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Microbial water quality is currently assessed by fecal indicator bacteria that are poor representatives of viral pathogens in the environment. Viruses are predicted to account for the majority of infectious risk from exposure to sewage contaminated water. Previously developed viral indicators suffer from a lack of human-specificity, low concentrations in sewage, or both. In this commentary review, we discuss recent advances in developing Cross-Assembly Phage (crAssphage) and Pepper Mild Mottle Virus (PMMoV) as viral water quality indicators. CrAssphage and PMMoV are abundant in and highly associated with human sewage, correlate with viral pathogens in sewage contaminated environments, and globally present. Future work is necessary to describe crAssphage and PMMoV fate in the environment, local variation in abundance and genetic makeup, and relationship between molecular detections and pathogen viability, among other areas. These developments will allow the integration of crAssphage and PMMoV into quantitative microbial risk assessment and water quality regulation.
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Fecal pollution at coastal beaches in the Northeast, USA requires management efforts to address public health and economic concerns. Concentrations of fecal-borne bacteria are influenced by different fecal sources, environmental conditions, and ecosystem reservoirs, making their public health significance convoluted. In this study, we sought to delineate the influences of these factors on enterococci concentrations in southern Maine coastal recreational waters. Weekly water samples and water quality measurements were conducted at freshwater, estuarine, and marine beach sites from June through September 2016. Samples were analyzed for total and particle-associated enterococci concentrations, total suspended solids, and microbial source tracking markers for multiple sources. Water, soil, sediment, and marine sediment samples were also subjected to 16S rRNA sequencing and SourceTracker analysis to determine the influence from these environmental reservoirs on water sample microbial communities. Enterococci and particle-associated enterococci concentrations were elevated in freshwater, but suspended solids concentrations were relatively similar. Mammal fecal contamination was significantly elevated in the estuary, with human and bird fecal contaminant levels similar between sites. A partial least squares regression model indicated particle-associated enterococci and mammal marker concentrations had the most significant positive relationships with enterococci concentrations across marine, estuary, and freshwater environments. Freshwater microbial communities were significantly influenced by underlying sediment while estuarine/marine beach communities were influenced by freshwater, high tide height, and estuarine sediment. We found elevated enterococci levels are reflective of a combination of increased fecal source input, environmental sources, and environmental conditions, highlighting the need for encompassing MST approaches for managing water quality issues. IMPORTANCE Enterococci have long been the federal standard in determining water quality at estuarine and marine environments. Although enterococci are highly abundant in the fecal tracts of many animals they are not exclusive to that environment and can persist and grow outside of fecal tracts. This presents a management problem for areas that are largely impaired by non-point source contamination, as fecal sources might not be the root cause of contamination. This study employed different microbial source tracking methods to delineate influences from fecal source input, environmental sources, and environmental conditions to determine which combination of variables are influencing enterococci concentrations in recreational waters at a historically impaired coastal town. Results showed that fecal source input, environmental sources and conditions all play a role in influencing enterococci concentrations. This highlights the need to include an encompassing microbial source tracking approach to assess the effects of all important variables on enterococci concentrations.
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Water is critical for life, but many people do not have access to clean and safe drinking water and die because of waterborne diseases. The analysis of drinking water for the presence of indicator microorganisms is key to determining microbiological quality and public health safety. However, drinking water-related illness outbreaks are still occurring worldwide. Moreover, different indicator microorganisms are being used in different countries as a tool for the microbiological examination of drinking water. Therefore, it becomes very important to understand the potentials and limitations of indicator microorganisms before implementing the guidelines and regulations designed by various regulatory agencies. This review provides updated information on traditional and alternative indicator microorganisms with merits and demerits in view of their role in managing the waterborne health risks as well as conventional and molecular methods proposed for monitoring of indicator and pathogenic microorganisms in the water environment. Further, the World Health Organization (WHO) water safety plan is emphasized in order to develop the better approaches designed to meet the requirements of safe drinking water supply for all mankind, which is one of the major challenges of the 21st century
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Enterococci have long been the federal standard in determining water quality at estuarine and marine environments. Although enterococci are highly abundant in the intestines of many animals, they are not exclusive to that environment and can persist and grow outside fecal tracts. This presents a management problem for areas that are largely impaired by nonpoint source contamination, as fecal sources might not be the root cause of contamination. This study employed different microbial source tracking methods for delineating the influences from fecal source input, environmental sources, and environmental conditions to determine which combination of variables are influencing enterococcal concentrations in recreational waters at a historically impaired coastal town. The results showed that fecal source input, environmental sources, and conditions all play roles in influencing enterococcal concentrations. This highlights the need to include an encompassing microbial source tracking approach to assess the effects of all important variables on enterococcal concentrations.
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Environmental waters are monitored for fecal pollution to protect public health. Many previously developed human-specific fecal pollution indicators lack adequate sensitivity to be reliably detected in environmental waters or do not correlate well with viral pathogens. Recently, two novel human sewage-associated source tracking qPCR markers were developed based on the bacteriophage crAssphage, CPQ_056 and CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-based, suggesting great promise for environmental application as human fecal pollution indicators. A 30-day sampling study was conducted in an urban stream impacted by combined sewer overflows to evaluate the crAssphage markers' performance in an environmental system. The crAssphage markers were present at concentrations of 4.02-6.04 log10 copies/100 mL throughout the study period, indicating their high abundance and ease of detection in polluted environmental waters. In addition, the crAssphage assays were correlated with rain events, molecular markers for human polyomavirus and HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064 assay correlated strongly to a greater number of biological indicators than the CPQ_056 assay. This study is the first to evaluate both crAssphage qPCR assays in an extended environmental application of crAssphage markers for monitoring of environmental waters. It is also the first study to compare crAssphage marker concentration with other viral-based indicators.
Chapter
Detailed knowledge of contamination sources is needed for efficient and cost-effective strategies to minimize fecal pollution in water, foods and the environment, and for evaluation and risk assessment. This chapter analyzes the concept of viruses as indices and indicators of fecal pollution and the applicability of specific viral groups as source markers for fecal contamination, usually referred to as microbial source-tracking (MST) tools. Specific human and animal viruses, bacteriophages and plant viruses are described and critically evaluated. Future prospects for technical and scientific developments are considered.
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Multiple host-specific molecular markers were used to detect the sources of faecal pollution in a mixed land use non-sewered catchment in Southeast Queensland, Australia. These markers included human-specific Bacteroides (HF183 and HF134), cattle-specific Bacteroides (CF128), dog-specific Bacteroides (BacCan) and human-specific enterococci surface protein (esp) markers. The sensitivity and specificity of these markers were determined by testing 197 faecal samples from 13 host groups. The overall sensitivity and specificity of these markers was high (sensitivity>/=85% and specificity>/=93%) indicating their suitability for detecting the sources of faecal pollution. Of the 16 samples collected from the study area, 14 (87%) were positive for at least one of the molecular marker tested. Amongst all the markers, cattle-specific CF128 was more prevalent than others, followed by human-specific HF183 which was consistently detected in samples collected from sites within close proximity to urban development. Significant correlations were found between E. coli and enterococci concentrations with the positive/negative results of human-specific Bacteroides HF183 (p<0.001, p<0.0001) and HF134 (p<0.001, p<0.004) markers. No correlations were found between faecal indicators (E. coli or enterococci) with the CF128 or BacCan markers. A significant correlation was also found between enterococci concentrations and the presence/absence of the esp marker (p<0.02). Based on the results, it appears that the host-specific markers such as HF183 and esp are a sensitive measure of sources of human faecal pollution in surface waters in Southeast Queensland, Australia.
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This study identified possible dose-response relationships among bathers exposed to marine waters contaminated with domestic sewage and subsequent risk of nonenteric illness. Four intervention follow-up studies were conducted within the United Kingdom. Healthy volunteers (n = 1273) were randomized into bather and nonbather groups. Intensive water-quality monitoring was used to assign five bacteriological indices of water quality to individual bathers. Illnesses studied were acute febrile respiratory illness, and eye, ear, and skin ailments. Fecal streptococci exposure was predictive of acute febrile respiratory illness, while fecal coliform exposure was predictive of ear ailments. Estimated thresholds of effect occurred at bather exposures above 60 fecal streptococci and 100 fecal coliform per 100 ml of water, respectively. Although no relationship was found between eye ailments and indicator organism exposure, compared with nonbathers, bathers were at higher risk for eye ailments. Nonenteric illness can be transmitted via recreational contact with marine waters contaminated with sewage. These results argue against the use of a single indicator to establish water quality standards.
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We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and theBacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium andBacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotellamarkers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides orPrevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10−5 to 2.8 × 10−7 g (dry weight) of feces/liter and 6.8 × 10−7 g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.
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Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.
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The human endogenous intestinal microflora is an essential “organ” in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.
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Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.
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Standard methods to measure recreational water quality require at least 24 hr to obtain results, making it impossible to assess the quality of water within a single day. Methods to measure recreational water quality in <or=2 hr have been developed. Application of rapid methods could give considerably more accurate and timely assessments of recreational water quality. We conducted a prospective study of beachgoers at two Great Lakes beaches to examine the association between recreational water quality, obtained using rapid methods, and gastrointestinal (GI) illness after swimming. Beachgoers were asked about swimming and other beach activities and 10-12 days later were asked about the occurrence of GI symptoms. We tested water samples for Enterococcus and Bacteroides species using the quantitative polymerase chain reaction (PCR) method. We observed significant trends between increased GI illness and Enterococcus at the Lake Michigan beach and a positive trend for Enterococcus at the Lake Erie beach. The association remained significant for Enterococcus when the two beaches were combined. We observed a positive trend for Bacteroides at the Lake Erie beach, but no trend was observed at the Lake Michigan beach. Enterococcus samples collected at 0800 hr were predictive of GI illness that day. The association between Enterococcus and illness strengthened as time spent swimming in the water increased. This is the first study to show that water quality measured by rapid methods can predict swimming-associated health effects.
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Despite numerous studies, uncertainty remains about how water quality indicators can best be used in the regulation of recreational water. We conducted a systematic review of this topic with the goal of quantifying the association between microbial indicators of recreational water quality and gastrointestinal (GI) illness. A secondary goal was to evaluate the potential for GI illness below current guidelines. We screened 976 potentially relevant studies and from these identified 27 studies. From the latter, we determined summary relative risks for GI illness in relation to water quality indicator density. Our results support the use of enterococci in marine water at U.S. Environmental Protection Agency guideline levels. In fresh water, (Italic)Escherichia(/Italic) coli was a more consistent predictor of GI illness than are enterococci and other bacterial indicators. A log (base 10) unit increase in enterococci was associated with a 1.34 [95% confidence intervals (CI), 1.00-1.75] increase in relative risk in marine waters, and a log (base 10) unit increase in E. coli was associated with a 2.12 (95% CI, 0.925-4.85) increase in relative risk in fresh water. Indicators of viral contamination were strong predictors of GI illness in both fresh and marine environments. Significant heterogeneity was noted among the studies. In our analysis of heterogeneity, studies that used a nonswimming control group, studies that focused on children, and studies of athletic or other recreational events found elevated relative risks. Future studies should focus on the ability of new, more rapid and specific microbial methods to predict health effects, and estimating the risks of recreational water exposure among susceptible persons.
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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.”
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We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.
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Microbial source tracking (MST) includes a group of methodologies that are aimed at identifying, and in some cases quantifying, the dominant source(s) of fecal contamination in resource waters, including drinking, ground, recreational, and wildlife habitat waters. MST methods can be grouped into two major types. Library-dependent methods are culture based and rely on isolate-by-isolate typing of bacteria cultured from various fecal sources and from water samples These isolates are matched to their corresponding source categories by direct subtype matching (41, 70) or by statistical means (23, 37, 40, 41, 80, 83, 102). In contrast, library-independent methods frequently are based on sample-level detection of a specific, host-associated genetic marker in a DNA extract by PCR (6, 11, 26, 88). Analyses of certain chemicals associated with sewage, including fecal sterols (29, 30, 47), optical brighteners (29, 30, 68), and host mitochondrial DNA (67), have also been utilized for what can be more broadly termed fecal source tracking; however, in this review we compare the performance of only fecal source tracking studies in which the target(s) is microbial.
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Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.
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Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools-Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers-were evaluated in a selected reach of Plum Creek in south-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to flowing surface water.
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In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193′), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.
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The human gut is home to trillions of microbes, thousands of bacterial phylotypes, as well as hydrogen-consuming methanogenic archaea. Studies in gnotobiotic mice indicate that Methanobrevibacter smithii, the dominant archaeon in the human gut ecosystem, affects the specificity and efficiency of bacterial digestion of dietary polysaccharides, thereby influencing host calorie harvest and adiposity. Metagenomic studies of the gut microbial communities of genetically obese mice and their lean littermates have shown that the former contain an enhanced representation of genes involved in polysaccharide degradation, possess more archaea, and exhibit a greater capacity to promote adiposity when transplanted into germ-free recipients. These findings have led to the hypothesis that M. smithii may be a therapeutic target for reducing energy harvest in obese humans. To explore this possibility, we have sequenced its 1,853,160-bp genome and compared it to other human gut-associated M. smithii strains and other Archaea. We have also examined M. smithii's transcriptome and metabolome in gnotobiotic mice that do or do not harbor Bacteroides thetaiotaomicron, a prominent saccharolytic bacterial member of our gut microbiota. Our results indicate that M. smithii is well equipped to persist in the distal intestine through (i) production of surface glycans resembling those found in the gut mucosa, (ii) regulated expression of adhesin-like proteins, (iii) consumption of a variety of fermentation products produced by saccharolytic bacteria, and (iv) effective competition for nitrogenous nutrient pools. These findings provide a framework for designing strategies to change the representation and/or properties of M. smithii in the human gut microbiota.