Microbial source tracking markers for detection of fecal contamination
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The document reviews microbial source tracking (MST) methods used to identify fecal contamination sources in water, emphasizing the limitations of traditional fecal indicator bacteria (FIB) and the development of library-independent methods. Key advancements in MST, including quantitative PCR techniques that target specific human-associated microorganisms, are highlighted alongside the health risks posed by fecal pollution from various animal sources. It concludes with a recognition that while MST shows promise, no single method can guarantee the identification of specific fecal pollution sources.
Microbial source tracking markers for detection of fecal contamination
- 1. Journal of FEMS (Federaton Of European Microbiological Societies)
- 2. Valerie J. Harwood - Department of Integrative Biology, University of South Florida, Tampa, FL, USA Christopher Staley- Department of Integrative Biology, University of South Florida, Tampa, FL, USA; University of Minnesota BioTechnology Institute, St. Paul, MN, USA
- 3. Brian D. Badgley- Department of Crop and Soil Environmental Science, Virgina Tech, Blacksburg, VA, USA Kim Borges- University of Maine Fort Kent, Fort Kent, ME, USA Asja Korajkic- National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH, USA
- 4. Microbial source tracking (MST) describes a 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 and associated human health risk
- 5. 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 performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR) is reviewed
- 7. Establishing the need for microbial source tracking (MST) Limitations of fecal indicator bacteria (FIB) Monitoring for all waterborne pathogens in environmental waters is currently unrealistic due to the great diversity of pathogens
- 8. monitoring for only one or a handful of pathogens may give a false impression of safety if pathogens other than those tested are present. many pathogens are difficult and costly to culture, difficult to identify have patchy distributions or low concentrations in environmental waters
- 9. Approach of Fecal indicator bacteria (FIB) has been used to overcome these problems low pathogenic potential, high levels in sewage and feces, relationship to pathogen presence The major FIB used worldwide include fecal coliforms, Escherichia coli, and enterococci
- 10. FIB concentrations have not been well correlated with pathogens in many studies Due to the widely differing physiology and phylogeny of FIB and pathogens comprised of bacteria, enteric viruses, and protozoans such as Cryptosporidium spp. and Giardia spp.)
- 11. 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
- 12. Much of the waterborne disease burden in developed countries is attributed to viral infections human sewage contamination is considered very risky Another issue is ‘near-ubiquitous’ distribution of FIB among host species that prohibits the identification of sources of contamination, which in turn interferes with remediation of polluted waters
- 13. MST emerged at the end of the 20th century This research area emerged in order to; determine the extent to which fecal source (e.g. human, dog, cattle) influences human health risk from contact with water attribute FIB loading in water bodies to the correct fecal sources
- 14. certain fecal microorganisms are strongly associated with particular hosts identified attributes of these host-associated microorganisms can be used as markers for fecal contamination from the host
- 15. The utility of two basic strategies have been tested: library-dependent analyses- collection and typing of many FIB isolates for some identifying attribute, including Antibiotic resistance carbon source utilization genetic type
- 16. library-independent analyses- that target a particular feature of a specific bacterial species or type For example, a variable region of the 16S rRNA gene of Bacteroidales
- 17. diverse, ranging from assessment of beach water quality) to source allocations for total maximum daily load plans food safety
- 18. 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
- 19. This review focuses on library-independent methods for human source contamination and includes a brief overview of methods for identifying fecal contamination from other animals A recent trend has been to adapt end-point PCR methods to the quantitative PCR (qPCR, also called real-time PCR) format
- 20. The validation of MST markers is accomplished by assessing certain performance criteria, Sensitivity- refers to the proportion of known positive samples Specifity- refers to the proportion of known negative samples The limit of detection (LOD) is a quantitative or semi quantitative expression of the lowest amount of target that can be detected
- 21. One of the first library-independent methods developed for the detection of human fecal contamination Based on end-point PCR detection of the 16S rRNA gene of human-associated Bacteroidales
- 22. The obligate anaerobic Bacteroides-Prevotella taxon was targeted due to high concentrations in feces and tendency to coevolve with the host A forward primer targeting apparently human-specific members of the Bacteroidales was paired with a general reverse primer
- 23. Two ‘human-specific’ forward primers were designed HF134F and HF183F , which was most useful due to its sensitivity and specificity sensitivity, was over 84% (n = 13) in human fecal samples and 100% (n = 3) in sewage The LOD was 1012 g DNA or 105 gene copies
- 24. This marker, which is frequently termed HF183 in the literature and whose sequence has been found in the 16S rRNA gene of the cultured Bacteroides dorei has since been used in many field tests. Many qPCR methods targeting the 16S rRNA gene of human-associated Bacteroidales have been developed
- 25. Members of the genus Bifidobacterium are anaerobic, gram-positive rods that are abundant in intestinal microbial communities of humans and some animals Subsequent research has demonstrated that B. adolescentis is not confined to the human gastrointestinal tract
- 26. a set of B. adolescentis primers amplified fecal DNA from cows, as well as from pigs, sheep, and chicken A TaqMan qPCR assay targeting Bifidobacterium adolescentis(ADO) showed high sensitivity in human fecal samples
- 27. An end-point PCR method targeting the enterococcal surface protein (esp) gene of E. faecium was developed to identify human fecal contamination method utilized a culture step to increase target cell numbers
- 28. Although the method was 97% sensitive and 100% specific in this study, other studies have detected some level of Ent. faecium esp in the feces of animals A SYBR green qPCR method targeting the esp gene not requiring a culture step was developed and tested in Australia
- 29. The method employed the previously utilized forward primer specific to esp of Ent. faecium and a reverse primer containing a sequence shared by Ent. faecium and Ent. Faecalis Sensitivity was high, particularly in domestic sewage Specificity was 100% when tested against twelve types of fecal samples from domestic animals and wildlife in Australia
- 30. Genotyping of F+ (F-specific) RNA coliphages has been suggested as a possible tool for discriminating between human and animal sources of fecal contamination The differential host distribution of these coliphages was first demonstrated by serotyping
- 31. Among the four subtypes, groups II and III are more prevalent in human sewage, while groups I and IV are generally associated with animal feces A multiplex qPCR for simultaneously typing and quantifying F+ RNA coliphages was developed
- 32. HPyVs are highly specific to humans with a high prevalence in human populations The viruses are shed primarily in urine, but also in feces, and high titers have been reported in municipal sewage An end-point PCR method was developed for the detection of HPyVs in environmental waters
- 33. A qPCR assay that targets only polyomavirus has been developed; however, sensitivity and specificity were not tested for this method
- 34. The first ‘fecal source tracking’ method based on a eukaryotic genetic marker end-point PCR assay targeting the human mitochondrial NADH dehydrogenase subunit mtDNA was proposed as a marker based upon the premise that it should be abundant in feces
- 35. A multiplex qPCR method targeting the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene from humans, cattle, and pigs was developed
- 36. An important step in MST is to predict human health risks due to waterborne pathogens 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
- 37. Four epidemiological studies published to date that include MST methods The first study was conducted at two freshwater beaches in the U.S Great Lakes that were impacted by WWTP effluent
- 38. 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 Enterococcus copy numbers were significantly associated with GI illness
- 39. A second epidemiological study took place at multiple beaches within Mission Bay in coastal California This study attempted to determine correlations between indicators of water quality and rates of swimming-related illnesses
- 40. 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
- 41. 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
- 43. Some studies have offered additional insight into MST marker effectiveness by correlating markers and human pathogens in separate studies conducted in California, two groups analyzed the presence of human-associated Bacteroides HF183 by end-point PCR the presence of enteroviruses via quantitative reverse transcriptasePCR in surface water samples
- 44. 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
- 45. 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
- 46. a TaqMan qPCR method specific for cow feces was developed This assay was 100% sensitive to cow fecal material and showed 95% specificity
- 47. 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