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Similar a Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes
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Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes
- 1. Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes Dr Asima Zehra School of Public Health and Zoonoses
- 2. Abstract Microbial source tracking (MST) Suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters Rely on the association of certain fecal microorganisms with a particular host MST is used to assess recreational water quality
- 3. MST rely on signature molecules (markers) DNA sequences of host-associated microorganisms Performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR)
- 4. History of MST Techniques In the 1960s early attempt to track bacterial sources included the use of fecal coliform-to-fecal streptococci ratios (EPA,2011)* First review of MST techniques Published in 1999 The Washington state department of Ecology`s MST field is still an emerging body of science**
- 5. Introduction RISK PREVENTION Protecting Public Health PREVENTION with RISK ASSESSMENT
- 7. Library dependent techniques Molecular (genotypic) 1. Ribotyping 2. PFGE 3. rep-PCR Biochemical (phenotypic) 1. Antibiotic resistance analysis (ARA) 2. Carbon utilization profile (CUP) and Nutrient utilization pattern (NUP)
- 8. Library Dependent Method Logistics
- 9. Evaluation of Library-Dependent Techniques performance • Evaluated the performance of library-dependent MST techniques Few studies • PFGE, rep-PCR and ribotyping • ARA and CUP In 2002 SCCWRP conducted an MST comparison study (SCCWRP, 2003) Southern California Coastal Water Research Project, 2003
- 10. Performance statistics when MST techniques were tested with reference samples to determine the ability or failure to detect the sole source of fecal contamination (Stoeckel and Harwood, 2007)
- 11. A statistical appraisal of disproportional versus proportional MST libraries Four of the most commonly used statistical matching algorithms in library-based MST maximum similarity (MS), average similarity (AS), discriminant analyses (DA), and k-means nearest neighbor (k-NN) Brian et al. 2007 No widespread consensus exists as to which is most appropriate
- 12. Library independent techniques Bacteriophages Bacterial PCR • Bacteroidales, bacteroidetes, bacteriodes • Bifidobacterium species • Methanobrevibacter species • Phodococcus coprophilus • E. coli • Enterococcus species Viral PCR
- 13. Library Independent Method Logistics
- 14. Evaluation of Library-Independent Techniques performance In 2002 SCCWRP conducted an MST comparison study (SCCWRP, 2003) • Human-specific Bacteroidetes sp. (several laboratories) and • The E. coli toxin gene. • F+ specific coliphages • Adenoviruses • Enteroviruses
- 15. Performance statistics for tests in which library-independent MST techniques were tested with reference samples to determine the ability or failure to detect the sole source of fecal contamination adapted from Stoeckel and Harwood, 2007)
- 16. Library Independent techniques: Variables Orin et al. 2016 Correlation Binary logistic regression ANOVA Multivariate regression analysis Non-parametric Friedman test….. Data Acceptance Criteria for Standardized Human Associated Fecal Source Identification Quantitative Real-Time PCR Methods
- 17. Establishing the need for MST Limitations of fecal indicator bacteria (FIB) Monitoring for all waterborne pathogens Monitoring for only one or a handful of pathogens Many pathogens are difficult and costly to culture and to identify Patchy distributions or low concentrations unrealistic false impression of safety
- 18. Limitations of fecal indicator bacteria (FIB) FIB concentrations correlated pathogens Due to the widely differing physiology and phylogeny of FIB and pathogens Comprised of bacteria, enteric viruses, and protozoans (Cryptosporidium spp. and Giardia spp.)
- 19. Outcome Vs FIB Host specificity correlation between human health outcomes and FIB levels can not be find out particularly when the pollution is not from a known point source This is due to the fact that E. coli, enterococci, and other FIB are shed in the feces of many different animals ‘near-ubiquitous’ distribution of FIB among host species*
- 20. What makes a good MST marker
- 21. The limit of detection (LOD) Joel et al. 2016 Sensitivity- is the ability to detect the source when it is present The number of samples needed to determine sensitivity has not been determined but recent studies have included 20 or more samples*. Specificity- is the ability to detect the source when it is not present Presently there is no universal criterion for a specificity measure but method with less than 80% specificity are not useful in most cases
- 22. Correlation with pathogens and risks of waterborne illness An important step in MST is to predict human health risks due to waterborne pathogens* Four epidemiological studies published to date that include MST methods- USA Enterococcus copy numbers were significantly associated with GI illness** This study attempted to determine correlations between indicators of water quality and rates of swimming-related illnesses
- 23. Only skin rash and diarrhea were significantly more frequent in swimmers compared with non swimmers No correlations between rates of illness and FIB levels or rates of detection of human Bacteroides or somatic coliphage, was found
- 24. Correlations observed in studies between MST markers and risks for various types of waterborne illnesses Harwood et al 2014
- 25. Correlation with pathogens Studies: MST marker effectiveness by correlating markers and human pathogens Human health risk from animal fecal contamination is generally assumed to be less severe than from human sources Waterborne zoonotic infections caused by pathogens shed in domestic/agricultural animal feces, such as Salmonella, E. coli O157:H7, Campylobacter jejuni … Still pose a definite health risk
- 26. Swine markers Two TaqMan methods for pig feces (Pig-1-Bac and Pig- 2-Bac) target the 16S rRNA gene of porcine- associated Bacteroidales A TaqMan method targeting porcine adenovirus (PAdV) was evaluated using pig slurries, urban and slaughterhouse sewage, and river water SOP VITAL 015, 2010
- 27. Bovine markers A TaqMan qPCR method specific for cow feces was developed This assay was 100% sensitive to cow fecal material and showed 95% specificity Wolf et al. 2010
- 28. MST and applications This research area emerged in order to determine the extent to which fecal source influences human health risk from contact with water attribute FIB loading in water bodies to the correct fecal sources *Identified attributes of the host-associated microorganisms can be used as markers for fecal contamination from the host
- 29. MST useful To supplement sanitary surveys: Identify sources of beach contaminants. Identify sources of TMDL violations For risk analyses: Human versus non-human Human versus domestic animal
- 30. Conclusions Overall, there is no single method that is capable of identifying specific sources of fecal pollution in the environment with absolute certainty Some markers are not completely host specific Some strongly host specific markers are less concentrated in sewage The promise of MST should not be under estimated, even in light of the challenges
- 31. The Future of Source Tracking New methods will arise Some methods will become obsolete
- 32. References Ritter, K. J., Carruthers, E., Carson, C. A., Ellender, R. D., Harwood, V. J., Kingley, K., Nakatsu, C., Sadowsky, M., Shear, B., West, B., Whitlock, J. E., Wiggins, B. A. & Wilbur, J. D. 2003. Assessment of statistical methods used in library-based approaches to microbial source tracking. J. Wat. Health. 1, 209–223. Anicet R. Blanch,1 Lluı´s Belanche-Mun˜oz, Xavier Bonjoch, James Ebdon,Integrated . 2006. Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, p. 5915–5926 Orin C. Shanksa, Catherine A. Keltya, Robin Oshirob, Richard A. Hauglanda, Tania Madic, Lauren Brooksd, Katharine G. Fieldd and Mano Sivaganesana. Appl. Environ. Microbiol. May 2016 vol. 82 no. 9 2773-2782 EPA, 2007d. Report of the Experts Scientific Workshop on Critical Research Needs for the Development of New or Revised Recreational Water Quality Criteria. EPA/823-R-07-006. U.S. Environmental Protection Agency, Cincinnati, OH. http://water.epa.gov/scitech/swguidance/standards/criteria/health/recreation/index.cfm EPA, 2011. Using Microbial Source Tracking to Support TMDL Development and Implementation. Prepared for U.S. Environmental Protection Agency, Region 10, Watersheds Unit by Tetra Tech Inc. and Herrera Environmental Consultants, Seattle, WA. www.epa.gov/region10/pdf/tmdl/mst_for_tmdls_guide_04_22_11.pdf Southern California Coastal Water Research Project, 2003. Journal of Water and Health, IWA Publishing, Vol 01, Issue 4, December 2003. Stoeckel, Donald M. and V J. Harwood, 2007. Performance, Design, and Analysis in Microbial Source Tracking Studies. Applied and Environmental Microbiology, Vol. 73: No. 8: 2405-2415.
- 33. Thank you
Notas del editor
- Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters.
- MST rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms Human sewage pollution is among the greatest concerns for human health
- *While this approach is no longer considered informative due to widely varying survival rates of the bacterial groups in the environment, scientists were encouraged to develop and evaluate new tools to discriminate among different sources of bacterial pollution. **Although this field is mainly experimental at this phase of its development, there is strong pressure on natural resource managers to use these techniques to identify bacterial sources of pollution .
- Potential pollution sources: host species implicated as potential pollution sources in various water bodies range from human to agricultural animals (to pets and wild animals such as gulls. In 2008 there were over 400 waterbodies in Washington that did not meet designated use criteria due to fecal colifrom levels that exceed (do not meet) water quality standards. In recent years, nonpoint sources of bacterial pollution have surpassed point sources as the major source of fecal contamination to surface water (EPA,2005).
- Molecular and biochemical techniques rely on the close association of certain microorganisms (generally bacteria or viruses), with a specific host and genetic or phenotypic differences that allow host specific microbes to be discriminated from others. Chemicals methods generaly rely on the detection of chemicals associated with anthropogenic activities. Library dependent tech identify fecal sources from water samples based on a library or database of bacteria isolated from known fecal sources. The library is developed by collecting microbial isolates from known potential sources. The molecular or biochemical pattern of the individual microbial isolates is sometimes referred to as a fingerprint. These identifying patterns may be discerned by a variety of methods e.g, ribotyping, PFGE, ARA etc Library independent tech do not require the development of source library database. These tech rely on a species specific genotype or characteristics.
- Evaluation of library dependent techn: page no. 17 Ref: review n critique of current MST tech. 2011
- Advantages page no. 17 Many of the library-dependent techniques use typical fecal indicator bacteria. This can be an advantage when comparing MST results to fecal indicator concentrations. Library-dependent techniques can be used to classify isolates from multiple fecal sources. Disadvantages Both molecular and biochemical library-dependent techniques are based on the assumption that subspecies or strains of specific bacteria are associated with specific animal species. Recent E. coli studies suggest that subspecies of this bacterium change considerably with respect to geography, time, rainfall, and habitat. For this reason, the library would need to contain a very large number of isolates (Kuntz et al., 2003). The stability of fecal indicator populations over time, geography, and differing environmental conditions is a major concern for all library-based techniques. Various studies show that small known-source libraries (i.e., fewer than 2000 isolates) collected in an area have higher accuracy in the local area than when they are applied in other areas. In addition, temporal variability can contribute to errors in studies (Stoeckel and Harwood, 2007). Furthermore, most E. coli and Enterococcus strains are not host-specific; rather they are “cosmopolitan” and occur in the gastrointestinal tract and feces of many host species (EPA, 2005; Stoeckel and Harwood, 2007). Other disadvantages of the MST techniques that use a library are: the need for large library sizes, the complexity of statistical analyses for some techniques, and, for techniques that require bacterial culture, species identification is necessary (Rees et al., 2010). There is no consensus about the minimum number of fecal isolates needed for reliable source identification in a library. The number is likely quite large. In 2002 SCCWRP conducted an MST comparison study (SCCWRP, 2003). Twenty-two researchers employing different techniques were given sets of identically prepared water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull, or sewage), and the fecal source was blinded to the researchers. In addition, source material was provided for the libraries (Griffith et al., 2003). For this study, several of the genotypic library-based techniques performed better than the phenotypic techniques.
- 1 Sensitivity is the ability to detect a source when it is present (calculated by dividing the number of true-positive results by the number of samples that should contain the target). 2 Specificity is the ability to detect a source when it is not present (calculated by dividing the number of true negative results by the number of samples that should not contain the target). 3 Multiple Antibiotic Resistance (similar to ARA).
- Four of the most commonly used statistical matching algorithms in library-based MST when applied to disproportionately-represented known source libraries A statistical appraisal of disproportional versus proportional microbial source tracking libraries. Brian J. Robinson, Kerry J. Ritter and R. D. Ellender Journal of Water and Health | 05.4 | 2007 give prediction bias between above mentioned algorithms. The success of the library-based approach depends on the distribution of fingerprint patterns among source candidates, how well each source candidate is represented in the library, and which statistical algorithm or matching criterion is used to match unknowns (Ritter et al. 2003). The concern is that libraries that are heavily “loaded” toward a particular source may bias predictions toward the dominant library source.
- Evaluation of library independent tech page no. 23
- Advantages The most significant advantage of library-independent techniques is that they do not require development of a library database; this saves time and resources. Furthermore, the techniques that do not require culture of microorganisms can be quite rapid (i.e., completed and reported in the same day in which sampling occurred). Disadvantages One of the major limitations of library-independent techniques is the lack of techniques for host species beyond humans and a few domestic animal species. While some of the markers have been thoroughly vetted, and their error rate is fairly well-established (e.g., human Bacteroides HF183), other markers require much more substantial validation before they can receive general use (Harwood, 2011). The distribution of many markers has not been thoroughly characterized; the expectation that host-associated markers are present in a specific host population and not present in non-host populations should be confirmed. In addition, the relative abundance of many of these MST molecular markers has not been assessed. Validation studies need to include assessment of the abundance of these markers in the environment. All markers (with the exception of HPyVs) tested in validation studies do show some cross reactivity with non-target host species (Harwood, 2011). Validation studies would enable quantification of cross-reactivity, thus helping to determine usability of MST data. In 2002 SCCWRP conducted an MST comparison study (SCCWRP, 2003). Twenty-two researchers employing different techniques were given sets of identically prepared water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull, or sewage), and the fecal source was blinded to the researchers.
- 1 Sensitivity is the ability to detect a source when it is present (calculated by dividing the number of true-positive results by the number of samples that should contain the target). 2 Specificity is the ability to detect a source when it is not present (calculated by dividing the number of truenegative results by the number of samples that should not contain the target).
- Monitoring for all waterborne pathogens in environmental waters is currently unrealistic due to the great diversity of pathogens Monitoring for only one or a handful of pathogens may give a false impression of safety if pathogens other than those tested are present Patchy distributions or low concentrations in environmental waters
- FIB concentrations have not been well correlated with pathogens in many studies
- *that prohibits the identification of sources of contamination, which in turn interferes with remediation of polluted waters Many diseases in developed countries is attributed to viruses
- The validation of MST markers is accomplished by assessing certain performance criteria: Sensitivity- refers to the proportion of known positive samples. More sensitive marker will be more frequently detected in a polluted water sample than a less sensitive marker. *key to sensitivity determination is obtaining fecal samples from the study area to determine sensitivity of the marker you propose to use. This shld be done in advance of water testing to ensure the marker is detectable in the study area. Specificity- refers to the proportion of known negative samples. That is to rule out the target when it is present. MST marker shld be host specific, it shld not be found in non-host species. EPA document recommends that atleast 10 animals per host type are sample for specificity. Composite sampling would be an efficient means of testing. Page no. 26 The limit of detection (LOD) is a quantitative or semi quantitative expression of the lowest amount of target that can be detected Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR. 2016. Joel P. Stokdyka, c, Aaron D. Firnstahla, c, Susan K. Spencerb, c, Tucker R. Burchb, c, Mark A. Borchardtb, c. water research Volume 96, 1 June 2016, Pages 105–113
- *because many of the MST methods previously described have been developed recently, few studies to date have investigated the ability of these methods to predict the risk of illness **A cohort of beachgoers were surveyed on the sampling day and in follow-up interviews (n = 5667) to determine how rates of gastrointestinal (GI) illness correlated with water-quality analyses, including qPCR for Enterococcus A cohort of beachgoers was surveyed for tracking GI, respiratory, dermatological, and other symptoms Human-associated Bacteroides (HF183) was quantified via qPCR somatic and F+ coliphages were quantified, MPN estimates of adenovirus and norovirus concentrations were determined via reverse transcriptase PCR
- A plus (+) sign indicates that a significant correlation was found; a (−) sign indicates that it was tested, but no significant correlation was found; (n.d.) = no data, which indicates that data were collected, but the results were not published due to methodological difficulties or sampling limitations. References are given for each case.
- Some studies have offered additional insight into MST marker effectiveness by correlating markers and human pathogens Human health risk from animal fecal contamination is generally assumed to be less severe than from human sources
- determine the extent to which fecal source (e.g. human, dog, cattle) influences human health risk from contact with water *certain fecal microorganisms are strongly associated with particular hosts
- Most of the reviews focuses on library-independent methods for human and animal source contamination and a recent trend has been to adapt end-point PCR methods to the quantitative PCR (qPCR)