Introduction
Microbial source tracking is a process of identifying a particular source (such as human, cattle, or bird) of fecal contamination in water, which is generally measured through fecal indicator bacteria, like Es- cherichia coli (E.coli) or Enterococci. The basic assumption of microbial source tracking is that there are characteristics unique to the fecal bacteria from a particular host and these characteristics allow re-searchers to identify the source of the contamination. Most of these target key genes that can be “fingerprinted” or tied to a type of mammal, human or bird.
Maintenance of the microbiological quality and safety of water systems used for drinking, for recreating, and in the harvesting of seafood is imperative, as contamination of these systems can exact high risks to human health as well as result in significant economic losses due to closures of beaches and shellfish harvesting areas. Animals are major reservoirs for a variety of enteric pathogens (e.g. Salmonella, E.coli, and Cryptosporidium spp.). Traditional and alternative indicator microorganisms have been used for many years to predict the presence of fecal pollution in water. Due to the ubiquitous nature of these organisms, the effectiveness of using traditional indicators to predict the presence of human or animal waste impact and subsequent health risks is limited. The usefulness of the microbial indicators as tools for risk assessment can be significantly enhanced by the development of testing methods and analysis techniques that can define specific sources of these organisms.
The concept that the origin of fecal pollution can be traced using microbiological, genotypic, phenotypic, and chemical methods has been termed microbial source tracking. This essay will provide an overview of microbial source tracking methods that are currently being used to predict and identify sources of fecal pollution in the environment as well as provide insight into future directions in the field (Troy M. Scott 2002)
How is Microbial Source Tracking Done?
Indicator microorganisms are used to predict the presence of and/or minimise the potential risk associated with pathogenic microbes. Ideally, indicators are nonpathogenic, rapidly detected, easily enumerated, have survival characteristics that are similar to those of the pathogens of concern, and can be strongly associated with the presence of pathogenic microorganisms. Total and fecal coliforms have been used extensively for many years as indicators for determining the sanitary quality of surface, recreational, and shellfish growing waters. In recent years, scientists have learned more about the ways in which the coliforms’ ecology, prevalence, and resistance to stress differ from those of many of the pathogenic microorganisms they are proxy for (Desmarais, T. R. 2002).These differences are so great that they limit the utility of the coliforms as indicators of fecal pollution. Therefore, additional microbes have been suggested for use as alternative indicators, including E. coli, enterococci, and Clostridium perfringens.(Griffin, D. W. et.al 2016)
There are several different methods for microbial source tracking:
1.Library-dependent, culture based: Samples are collected from all over a watershed and researchers grow bacteria in the lab to create a library from a variety of source organisms. Then, water samples are collected from rivers, lakes, or beaches and the bacteria in the samples are also grown in the lab. The results of the water sample are compared to the library to determine sources of contamination.
2.Library-independent, culture based: Water samples are collected and the bacteria and viruses in the samples are grown or cultured in the lab. The bacteria and viruses grown are known to be from specific hosts or sources of fecal contamination so there is no need to compare results to a library.
3. Library-independent, culture independent: Water samples are collected and molecular techniques are used to isolate and identify DNA directly from the sample without first having to grow or culture the bacteria and viruses in the sample.
Microbial indicators of fecal pollution
E. coli
E. coli has long been used as an indicator of fecal pollution. It has good characteristics of a fecal indicator, such as not normally being pathogenic to humans, and is present at concentrations much higher than the pathogens it predicts.(Geldreich, E. E. 1966)
Enterococcus spp
The enterococcus group is a subgroup of the fecal streptococci including: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus gallinarum, and Enterococcus avium. They are differentiated from other streptococci by their ability to grow in at high pH and temperature. E. faecalis and E. faecium are the species most frequently found in humans. Enterococci have been used successfully as indicators of fecal pollution and are especially reliable as indicators of health risk in marine environments and recreational waters.(Cabelli, V. J. 1983) .It is known, however, that environmental reservoirs of enterococci exist and that regrowth of these organisms may be possible once they are introduced into the environment.(Desmarais, T. R. et.al 2002)
C. perfringens.
C. perfringens is an enteric, gram-positive, anaerobic, spore-forming, pathogenic bacterium found in human and animal feces. Although there is considerable controversy surrounding the use of C. perfringens as a water quality indicator (J. Vierheilig, 2013) because of its persistence in the environment, a number of scientists continue to recommend its use, particularly in situations where the prediction of the presence of viruses or remote fecal pollution is desirable.
While alternative microbial indicators can be useful for predicting the possible presence of fecal contamination in water, their shortcomings as tools for risk assessment are apparent. The advent of microbial source tracking technologies has enhanced the ability of these and traditional indicator organisms to be used as tools for predicting potential sources of fecal pollution as well as health risks associated with impaired water systems.
METHODS USED FOR MICROBIAL SOURCE TRACKING
Fecal coliform/fecal streptococcus ratio.
To meet the challenge of identifying sources of fecal pollution there are various microbiological methods used. The first method is to use the ratio of fecal coliforms to fecal streptococci eg. where a ratio of >4.0 would indicate human pollution and a ratio of ≤0.7 would indicate nonhuman pollution (Pourcher, A. M et.al 1981). The rationale behind the use of this method was the observation that human feces contain higher fecal coliform counts, while animal feces contain higher levels of fecal streptococci.
The advantage of using this method is its ability to provide rapid results and requires little skill or expertise to carry out. However, this approach has proven to be unreliable due to variable survival rates of fecal streptococci species, variations in detection methods, and variable sensitivity to water treatments and has been abandoned as a viable approach to fecal source tracking.
Bacteroides fragilis bacteriophage.
B. fragilis is an obligately anaerobic, gram-negative, pleomorphic rod-shaped bacterium. The Bacteroides group of bacteria is present in high numbers in both human and animal intestines. Tartera and Jofre examined 40 human fecal samples for the presence of different Bacteroides spp. and determined that one B. fragilis strain, was found in 10% of the human samples but was not detected in samples from any other animal species. Puig et al. attempted to identify additional host strains of Bacteroides in order to detect additional phage originating from the human gut or the guts of different animal species that may have more far-reaching capacity than the HSP40 bacteriophage. They identified an additional strain of B. fragilis, which was very similar to strain HSP40 and which showed similar sensitivity to infection by bacteriophage. Overall, the detection of B. fragilis bacteriophage has the advantage of being a highly specific method for tracking the source of human fecal pollution. In addition, these phage do not replicate in the environment, and their presence in the environment has been found to significantly correlate with the presence of human enteric viruses . The absence of B. fragilis phage in highly polluted waters and sewage in some areas (such as the United States) and the inherent difficulty in performing the assay limit the usefulness of this method. (Jagals, P. et al 1995)
Human enteric viruses.
Over 100 different enteric viruses which are the commonest causes of gastroenteritis worldwide, and are often transmitted via the faecal-oral route, with transmission by direct human contact and via fomites being common. The infective dose can be very low and many of these viruses are not easily cultivated in environmental samples; however, methods have been developed to concentrate and cultivate these organisms and are useful for directly detecting the presence of human fecal contamination and public health risk. Studies have shown that outbreaks of gastroenteritis have been associated with water supplies with acceptable coliform counts (Craun, G. F. 1991). By monitoring directly for human enteric viruses, any uncertainty associated with the use of fecal indicators can be avoided. Jiang et al. Monitoring directly for human pathogens provides valuable information as to the quality of the water system being evaluated. However, many viruses can be present in a water system, while only a few can be detected by cultivation methods that distinguish viable from nonviable organisms. Molecular methods (reverse transcription-PCR) can be used to detect non cultivable viruses, however, nonviable viruses are also detected by this procedure (Abbaszadegan, M et al. 1999). Therefore, this method should not be used solely when trying to predict fecal contamination.
PHENOTYPIC METHODS USED IN MICROBIAL SOURCE TRACKING
Numerous phenotypic methods have been suggested for use in discriminating among various groups of bacteria. These include biochemical tests, phage susceptibility, outer membrane protein profiles and antibody reactivity. However, these systems have their disadvantages, including unstable phenotypes, low sensitivity at the intraspecies level, and limited specificity there have been a few phenotypic methods have been used successfully as bacterial source tracking (BST) methodologies.
MAR analysis.
MAR analysis is a method that has been used to differentiate bacteria (usually E. coli or fecal streptococci) from different sources using antibiotics commonly associated with human and animal therapy, as well as animal feed. The use of this method is based on the underlying principle that the bacterial flora present in the gut of various types of animals are subjected to different types, concentrations, and frequencies of antibiotics. Over time, selective pressure within a specific group of animal selects for flora that possess specific “fingerprints” of antibiotic resistance.(Cooke, M. D. 1976.)
This procedure involves the isolation and culturing of a target organism, then replicating the isolates on media containing various antibiotics at various concentrations. The plates are then incubated and the organisms are scored according to their susceptibilities to various antibiotics to generate an antibiotic resistance profile. These fingerprints are then characterised, analysed by discriminate analysis, and compared to a reference database to identify an isolate as being either human or animal derived.
GENOTYPIC METHODS USED IN MICROBIAL SOURCE TRACKING
Repetitive element PCR and Ribotyping.
Repetitive element poly chain reaction (PCR) uses primers corresponding to interspersed repetitive DNA elements present in various locations within the prokaryotic genome to generate highly specific genomic fingerprints. PCR contains several bands, which can subsequently be analysed, categorised by host source, and used to construct a database to which fingerprints from unknown isolates can be compared. Successful identification of an unknown bacterial isolate also requires that a reference database be established, and additional known isolates must be fingerprinted from a large geographic region in order to assess the potential universal application of this procedure. Questions have also arisen as to the reproducibility of this method. (J. Vierheilig, 2013)
Ribotyping is a method of DNA fingerprinting whereby highly conserved rRNA genes are identified using oligonucleotide probes after treatment of genomic DNA with restriction endonucleases. The method is a labor-intensive procedure that involves bacteriological culture and identification, DNA extraction, gel electrophoresis, Southern blotting, and discriminant analysis of the resulting DNA fingerprints. Ribotyping has proven to be a very useful epidemiological technique for use with various bacteria, including E. coli, S. enterica, and Vibrio cholerae.
Host-specific molecular markers.
Detection of host-specific molecular markers in raw water samples holds promise as an effective method for characterising a microbial population without first culturing the organisms in question. Rapid tests that discriminate human fecal pollution from human and bovine fecal pollution are currently in the literature and use length heterogeneity PCR and terminal restriction fragment. This approach offers the advantage of eliminating the need for a culturing step, which allows a more rapid identification of target organisms. In addition, the use of Bacteroides spp. is desirable, as anaerobic bacteria are less likely to reproduce once introduced in the environment. However, little is known about the survival and persistence of Bacteroides spp. in the environment, which raises questions as to its utility as an indicator organism(Bernhard, A. E., and K. G. Field. 2000). Assaying for toxin or adhesion genes has not been thoroughly investigated and is complicated by the fact that many organisms do not contain these genes regardless of their host specificity.
CHEMICAL METHODS USED IN MICROBIAL SOURCE TRACKING
Caffeine.
Caffeine is present in several beverages, including coffee, tea, soft drinks, in many pharmaceutical products and consumed globally. It is excreted in the urine of individuals who have ingested the substance, and subsequently, it has been suggested that the presence of caffeine in the environment would indicate the presence of human sewage. Levels of caffeine in domestic wastewater have been measured to be between 20 and 300 μg/liter and levels in receiving waters are much lower due to significant dilution, and little is known about the fate of caffeine in the environment once it has been deposited (Burkhardt, M. R. 1999).