Methods for Monitoring and Managing Fecal Microbial

Contamination of Beaches

By

Steve

A Major Research Paper
Submitted to the Faculty of Graduate Studies
through the Department of civil and Environmental engineering
in Partial Fulfillment of the Requirements for
the Degree of Master of Science
at the University of Windsor

Windsor, Ontario, Canada

2015

© 2015 Steve

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Methods for Monitoring and Managing Fecal Microbial
Contamination of Beaches

by

Steve Joe

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ABSTRACT
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Fecal or bacterial source tracking also referred to as Microbial Source Tracking
(MST) is a method used to identify sources of fecal indicator bacteria in the
environment. Human and livestock activity has been greatly responsible for fecal
contamination of beaches. Accumulation of pathogenic microorganisms in beaches has
given rise to multiple infections in human population. The present study aims at
highlighting environmental problems related to fecal contamination of beaches.
Furthermore the fecal monitoring methods are investigated in detail so as to identify the
problems regarding each method. Advanced detection and quality monitoring methods
are required to control the contamination of beaches. Several MST methods are used to
identify the source of fecal bacteria that are introduced into the water bodies. These
methods are mainly divided into two basic categories; one is microbial methods and
second is chemical methods. Microbial methods are further divided into library
dependent and library independent methods. Library dependent methods are based on
maintained databases from various bacterial isolates of known fecal sources. However,
library independent methods identify sources based on host-specific characteristics of
bacteria without the requirement of library. Currently no standardized methods are
available for molecular and biochemical methods. However, recent developments are
more focused on improving library independent methods by expanding the range of
fecal sources that can be identified. Further studies are still needed to advance the
existing monitoring methods of fecal contamination so as to reduce recreational water
contamination and multiple lethal infections caused by them.

4

DEDICATION
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ACKNOWLEDGEMENTS

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6

TABLE OF CONTENTS
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DECLARATION OF ORIGINALITY...............................................................................
ABSTRACT........................................................................................................................
DEDICATION.....................................................................................................................
ACKNOWLEDGEMENTS................................................................................................
LIST OF TABLES............................................................................................................
LIST OF FIGURES............................................................................................................
LIST OF APPENDICES......................................................................................................
LIST OF ABBREVIATIONS/SYMBOLS.........................................................................
NOMENCLATURE...........................................................................................................
CHAPTER 1 CHAPTER TITLE HERE..............................................................................
Section Title Here...............................................................................................
Sub-Section Title Here......................................................................................

REFERENCES/BIBLIOGRAPHY.....................................................................................
APPENDICES.....................................................................................................................
Appendix A.......................................................................................................

VITA AUCTORIS................................................................................................................

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LIST OF TABLES
[Where applicable]

8

LIST OF FIGURES
[Where applicable]

9

LIST OF APPENDICES
[Where applicable]

LIST OF ABBREVIATIONS/SYMBOLS
Fecal Indicator Bacteria

FIB

Escherichia coli

E. coli

International Organization for Standardization

ISO

10

Polymerase chain reaction

PCR

Fluorescence in situ hybridization

FISH

Microbial Source Tracking

MST

Repetitive Palindromic Polymerase Chain Reactions

repPCR

Pulsed filed gel electrophoresis

PFGE

Quantitative polymerase chain reaction

qPCR

Antibiotic Resistance Analysis

ARA

Carbon Utilization Profile

CUP

Nutrient Utilization Pattern

NUP

Optical brighteners

OBs

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Chapter 1: Introduction
1.1 Background

1.2 Problem definition
1

1.3 Objectives

2

The present study aims to address the health and environmental issue related to fecal
contamination of beaches. Different methods and techniques employed for microbial
source tracking are reviewed in this study. These methods will be categorized into
different groups based on their methodological techniques. The suitability of new
emerging methods to qualitatively analyze the microbial levels for health and safety
regulations will be studied. In addition, the limitations and scope of their applicability
will also be discussed in this paper. Further, the competency of each technique for
tracking specific microbial source will be explored.

1.4 Structure of thesis

3

Chapter 2: Literature Review
2.1 MICROBIAL CONTAMINATION OF RECREATIONAL WATER

4

Water is an important natural resource that fulfills diversified needs of all the living
organisms. It is used for drinking, agriculture irrigation and recreation purposes. Natural
waters when contaminated through microbes can impact all the essential needs and will
also cause serious health problems in human population. Beaches are considered as a
major commercial and recreational resource that annually attracts numerous tourists and
local visitors mainly in summer. Pathogenic microbes released into the water through
fecal matter has put the lives of swimmers at risk mainly humans with a weaker immune
system (Colford et al., 2007).
Controlling fecal microbial contamination in beach water is therefore vital to protect
the life of human population, birds and marine animals. Exposure to these pathogenic
microbes causes serious diseases such as nose, ear and eye infections along with
hepatitis, diarrhea, vomiting, skin rashes, encephalitis and respiratory illness.
Recreational water users can be subjected to wide range of diseases causing
pathogens that either exist naturally in water or result from fecal contamination.
Waterborne non-fecal pathogens are bacteria that naturally exist in recreational
waters. Waterborne illnesses that result from these bacteria are not transmitted through
fecal oral pathway. Humans are affected either by ingestion or inhalation of the polluted
water. Legionella, Helicobacter pylori and Aeromonas are considered the most dangerous
microorganisms that cause serious respiratory illness as pneumonia (Lau et al., 2009).
Waterborne fecal pathogens that occur in water are the result of fecal pollution from
human or animal sources. Through the history, fecal bacteria include Escherichia Coli,

5

Total Coliform and Enterococci were very known to be the primary reason for
gastrointestinal illness (Mot et al., 2012; Anderson et al., 2005).
High level of fecal bacteria is considered as a primary cause of recreational beach
advisories and closing in the USA, which can result in economic losses to the
surrounding area (USEPA 2009). The unknown contamination sources include household
sewage, surface runoff, combined sewer overflow, domestic animals and wildlife.
Identifying the right fecal pollution source is very crucial for mitigating and preventing
future pollution.
2.2 SOURCES OF POLLUTION
2.2.1 Human sources

The overflow or leakage of the sewerage and drainage system is the largest source of
microorganism contaminant on beaches. These overflows are due to clogged or leaking
piping system, which spill untreated sewerage waste into coastal areas. Similarly, densely
populated areas with inadequate facilities and fast expanding urban areas also add burden
on aged sewerage system that causing overflows and leakage near the water bodies and
beaches. In addition, poorly managed septic system at domestic level add extra burden on
the already declining drainage system that eventually empties its untreated waste into
water bodies and beaches. Furthermore, the human population and recreational activities
on these recreational sites and beaches are the direct source of contamination. Human
bodies shed different kind of microorganism which adds to these contaminants. Littering
and open wastage disposal are the other sources of microbial contamination.

6

2.2.2 Domestic Animal

Studies have identified that agricultural animals are one of the largest contributor of
the fecal contaminants. Conversion of conventional small farms to industrial scale
productive farmlands demands increase in the animals involved in the agriculture
process. Large numbers of animals are kept in small areas which results in increased
combine feeding. Thus, excess amount of manure is produced that exceeds the crops
assimilative capacity for natural fertilizer. As a result, the excess waste is disposed into
near water bodies contains high level animal waste which reaches to recreational and
coastal areas.
2.2.3 Wildlife Waste

Every year a significant amount of microbial contamination is added by wildlife
animals. Different wildlife animals add fecal contamination directly into water bodies
including recreational as well as fresh water. Mostly fecal wastage is taken to sea and
coastal areas from non-point sources through as rain water or other water bodies. In
addition, the climatic changes and the seasonal transition make wild animals to migrate
from one place to another and come in contact with different water bodies and coastal
areas. This results in adding fecal contamination into different regions. Thus, controlling
fecal contamination caused by wildlife is not easy and straightforward which poses
challenges in tracking microbial sources of wildlife fecal contamination. Therefore, very
minimal studies and strategies have been carried out to control the wildlife sources that
contaminate beach and other water bodies.

7

2.3 MICRO-ORGANISMS AND DISEASES
Fecal waste from human or animals poses serious threat to human health. Fecal
pathogens greatly affect the individuals who are immune-compromised and immunesupressed such as older adults, infants and others who are suffering from debilitating
illness like AIDS and pneumonia (Sinclair, Jones, & Gerba, 2009).
The pathogenic organism present in human and animal feces comprise of diverse group
of viruses, bacteria and protozoa known for causing different diseases. To date, there are
more than 150 different types of diseases and the number keep increasing as new
pathogens are recognized (Sandra et al., 2013). Gastrointestinal illness is one of the most
commonly identified waterborne related disease and therefore it become very important
criteria in determining the water quality (Lorna et al., 2015). Respiratory infections, skin,
ear and eye problems are also associated with recreational water use (Sandra et al., 2013;
Lorna et al., 2015).
2.3.1 Bacteria
The most common microbiological contaminants of natural water are fecal Bacteria.
These Bacteria including pathogenic E. coli, Salmonella, Shigella, Yersinia and
Campylobacter are well known for casing gastrointestinal illness (Margaret et al., 2013).
2.3.1.1 Pathogenic E. coli
E.coli usually reside in the digestive tracks of warm-blooded animals and human, and are
excreted in feces. Harmless E. coli are a part of normal digestive system; however the
pathogenic E. coli O157:H7 is responsible for causing diarrhea that ranges in severity
8

from mild and self-limiting to severe and life-threatening (Percival et al., 2004; AWWA
2006) and hemolytic uremic syndrome (Margaret et al., 2013).
2.3.1.2 Salmonella and Shigella
These types of Bacteria are mainly identified as causing gastrointestinal illness that
belongs to the same family as E. coli, Enterobacteriaceae. Both of these Bacteria also
cause symptoms of diarrhea vomiting and abdominal pain (AWWA, 2006).
2.3.1.3 Yersinia and Campylobacter
Yersinia and Campylobacter are pathogenic bacteria mainly found in the feces of
domestic and wild animals. Gastroenteritis caused by Campylobacter bacteria associated
with flu-like symptoms, abdominal pain, watery diarrhea, vomiting and fever (AWWA,
2006).
2.3.2 Virus
One of the important viruses found among fecal pathogens is Polioviruses causing
poliomyelitis which remain a major health threat in underdeveloped and developing
countries. Other viruses considered as fecal contaminant are enteroviruses (encephalitis),
adenoviruses (respiratory tract infections), Rotaviruses (gastroenteritis) and hepatitis A
and E causing virus.
2.3.3 Protozoa
Different pathogenic protozoa found in fecal contaminated water are Entamoeba
histolytica (amoebic dysentery), Giardia lamblia (gastroentritis) and Acanthamocba
castellani (meningoencephalitis) (Santo & Ashbolt, 2008).
9

2.5 need for Monitoring FECAL CONTAMINATION ( HEALTH AND SAFETY
ASPECT) SOURCE TRACKING FOR MAANAGEMENT
2.6 Traditional methods for detecting fecal contaminations (logic)
2.6.1 Culture based methods (Sandra et al,2013)
2.6.1.1 Most probable number
2.6.1.2 Membrane filtration
2.6.2 Chemical based methods
2.7 Need for advanced methods ( difficient for human health/
persistence correlation management perspective)

Chapter 3: Advanced Methods of microbial source
tracking
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MST methods are typically divided into two major classes: Library-Dependent Method
(LDM) and Library Independent Method (LIM). Each method has its own strengths and
weaknesses and can be applied depending on the testing scenarios and other constraints.
LDM identify fecal sources from water samples by comparing the fingerprints of
Genotypic or phenotypic for fecal sources with already stored database. Since one need to
build a database of the fingerprints of all potential fecal source, this method is call library
dependent method. On the other hand, the library independent methods identify the fecal
sources based on the characteristics of the bacteria or virus. In this methods, one does not
required to stored fingerprints of the fecal sources and thus it is called library independent
method.
Library-dependent methods
Culture-dependent Methods
Phenotypic

Antibiotic

resistance analysis
Carbon utilization

Library-independent methods
Culture-independent

Genotypic

Phenotypic or


PFGE
Ribotypin

Genotypic
 Bacteriophage
 Bacterial culture

g
Rep-PCR

Methods
Genotypic

Host-specific

bacterial PCR
Host-specific

viral PCR
Host-specific qPCR

Table 01: Common types of MST Methods
3.1 Library Dependent Methods (LDM)
Library-dependent methods are based upon choosing a fecal indicator microorganism
(e.g., Escherichia coli) and establishing a reference library of characteristics of individual
11

isolates of the selected microorganism obtained from diverse bacterial isolates belonging
to potential fecal pollution sources. Library-dependent methods are typically culture
based techniques. The target organisms are cultured from the target water samples, and a
source identifier is used to determine the sources of potential fecal indicator. Different
types of microbial isolates are used for library-dependent methods and the commonly
used microbial isolates are: Escherichia coli, fecal streptococci and fecal coliforms (Santo
Domingo, & Sadowsky, 2007). Library-dependent method uses either molecular or
biochemical discrimination of isolates and the corresponding methods are called
Genotypic and Phenotypic methods, respectively.
3.1.1 Genotypic methods
Molecular methods uses molecular analysis of the DNA or RNA of viral and bacterial
organism. The concept behind the application of the method is that unique strains of a
bacteria species are adopted to their specific environment. Therefore, different bacterial
strains can be found in different host species. Different genotypic techniques are given
below and discussed further in the following subsection.
1. Pulsed Field Gel Electrophoresis (PFGF)
2. Ribotyping
3. repetative extragenic palindromic polymerase chain reaction (rep_PCR)

3.1.1.1 Pulsed Field Gel Electrophoresis (PFGF)
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It is a technique almost similar to ribotyping. The only difference is that in ribotyping
rRNA portion of genome is used whereas in PFGE whole DNA genome is used instead.
However, the initial steps for getting the bacterial isolates are same for both the
techniques. Cutting restriction enzymes are used on the whole DNA genome and then
larger genomic fragments are generated that must not be broken during sample
preparation. The genomic fragments are than parted by alternately pulsed using
perpendicularly oriented electrical fields instead of using standard gel electrophoresis.
Banding pattern emerges after electrophoresis and staining of gels. Generated patterns are
then compared with the sources in the library database (Rees et al., 2010; Harwood,
Staley, Badgley, Borges, & Korajkic, 2014).
It is a very advantageous method that classifies isolates from multiple sources. It is a
highly reproducible method if performed skillfully and is among the most discriminatory
genotyping methods. However, some of the limitations of the method are that it is time
consuming and highly costly. It demands high degree of technical skill. Furthermore, a
large geographically-specific source library is required.

3.1.1.2 Ribotyping
It is one of the widely used method in library-dependent Microbial Source Tracking
applications. It is based on the recognition of genetic diversity in the genomic sequences
within or flanking the 16S and 23S rRNA genes. These ribosomal ribonucleic genes are
highly conserved among bacterial species. The selected bacterial group for ribotyping is
cultured from the fecal samples implementing standard methods. Enterococci or E. coli
13

are commonly isolated and a certain percentage of bacterial count is selected for
genotypic characterization. Genomic DNA is separately isolated for each selected strain.
Restriction enzymes are used to digest the fragments of bacterial DNA. In the next step
gel electrophoresis is used to separate the DNA fragments with respect to their size.
Separated fragments are then moved to a gel blot where a labeled probe is used to attach
to certain portions of the rRNA genes. Probe binding to the DNA fragments creates a
banding pattern as the genome contains numerous copies of rRNA genes distributed
throughout the chromosome. The banding pattern obtained is further visualized by
chemical development or autoradiography. These banding patterns are captured through
digital
location and size of the banding pattern is are then matched with known sources in
the library database. Furthermore, commercially available software are used for image
analysis to compare binding patterns (Rees et al., 2010).
Two variables are used in ribotyping. First variable is the type of fecal indicator
bacteria that is selected to generate library. Second variable is the type and amount of
restriction enzymes used to fragment the DNA. Studies suggest that two restriction
enzymes are useful in increasing the method’s discriminatory ability.
Ribotyping is advantageous in classifying the samples from multiple sources and is
highly reproducible if performed skillfully. However, there are some limitations to this
method. It is a demanding method that has multiple steps and requires specialized
equipment. Furthermore, individuals need proper training, high supply costs and time is
needed to complete the procedure. Expertise in the field of statistics is also needed to

14

identify the sources present in the data. The geographic distribution of isolated bacteria,
the library size and the occurrence of replicate isolates in bacterial source library affect
the quality of ribotyping to discriminate between different bacteria at the host-species
level. Additionally, the phenotypic and genotypic methods both would nearly break down
in intricate watersheds with several sources.
3.1.1.3 rep-PCR
Polymerase Chain Reaction method allows for quick amplification of target DNA
sequences. It is used for cultivation dependent and independent approaches. In rep-PCR
method, the intervening sequences present between repetitive portions of microbial DNA
are amplified using rep-PCR method and one primer that marks each end of the
repetitive, palindromic sequence. Discriminatory patterns are generated by the repetitive
elements of bacterial genome which are parted by the distances specific to each bacterial
specie. Agarose gel is then used to amplify DNA fragments which generates a fingerprint
which also discriminates among diverse bacterial strains.
It is a quicker method to classify isolates from multiple sources. It is easier to use, less
costly and faster compared to the other methods. However, the results produced by this
method are less reproducible compared to ribotyping and PFGE (Rees et al., 2010;
Harwood, Staley, Badgley, Borges, & Korajkic, 2014).

3.1.2 Phenotypic Methods

15

Phenotypic techniques mainly exploit the biochemical and physical properties of an
organism.
Two commonly used phenotypic methods are:
1. Antibiotics resistance Analysis (ARA)
2. Carbon utilization profile and nutrient utilization pattern
And further discussed in the following subsection.
3.1.2.1 Antibiotic Resistance analysis (ARA)
In Antibiotic Resistance Analysis (ARA) the sources of fecal contamination are
identified using patterns of antibiotic resistance. The main concept behind this method is
that animals and humans are exposed to certain antibiotics and thus this selective pressure
will affect and change the antibiotic resistance profile of their fecal bacteria. These
differences are useful in distinguishing among different fecal bacterial sources. A source
library is maintained using fecal samples from potential contributors in water shed. In the
next step the known sources are analyzed for resistance patterns and antibiotic resistance.
Predictive equations using the antibiotic resistance patterns are then generated to classify
the unknown samples by source (Rees et al., 2010; Harwood, Staley, Badgley, Borges, &
Korajkic, 2014).
This method is rapid, less costly and requires less technical expertise than the genotypic
methods. It assists in discriminating among multiple sources including animals and
humans. However, complex statistical analysis used to determine the type of sources
demands a good working knowledge of statistics.
16

3.1.2.2 Carbon Utilization Profile (CUP) and Nutrient Utilization Pattern (NUP)
Different bacteria use different amount of carbon and nitrogen sources for growth and
energy. This difference is used by CUP and NUP where rapid calculations are performed
and results are tabulated for each utilization tests per isolate. Similar to ARA, the patterns
obtained can be used to determine the sources by comparing them to the known source
database. The method has some limitations and is impractical for field determination as
environmental factors greatly influence the bacterial nutrient requirements.
It is a fast method that demands less technical skills. Equipment and supplies are costly
and the method sill requires more testing (Simpson, Santo Domingo, & Reasoner, 2002).
3.1.3 Limitations of Library Dependent Techniques
There are certain limitations associated with library-dependent methods. For instance,
biochemical and molecular library-dependent techniques assume that bacterial strains are
associated with specific animal species. However, studies have proved that at times
bacterium changes with respect to time, rainfall, geography and habitat. Therefore, in
order to cover this limitation the database must comprise of large number of isolates.
Furthermore, another major concern for all library-based techniques is the stability of
fecal indicator populations in altering environmental conditions.
Several studies have provided evidence that smaller source libraries generated from
the data collection of specific area are accurate when dealing with local area compared to
when they are applied in other regions. Subsequently, temporal variability may also
contribute to error in the outcome.

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Moreover, some bacterial strains such as E. coli and Enterococcus are cosmopolitan
rather than host-specific strains. These bacteria reside in the gastrointestinal tract and
feces of various host species. Bigger databases that carry large information of
cosmopolitan strains reduce the specificity of source identification. On the contrary
smaller databases that may appear accurate cannot classify the isolates that are not part of
library (Stoeckel and Harwood, 2007).
Some other limitations of Microbial Source Tracking techniques specifically those which
use libraries are that they need larger databases, advanced statistical analysis and
compulsory specie identification. For instance, if multiple fecal sources are present then a
larger library consisting of thousands of isolates I required to get a good representation.
3.2 Library Independent Methods (LIM)
Due to the limitations of library generation, the trend is shifting toward methods in which
library is not required hence library independent methods. The size of library must be
enough to truly represent the geographical area for which library is cultured. The direct
tracking methods of different contaminant microbes without the use of culture libraries
are referred as library independent methods. Instead of libraries, the microbes are directly
cultured from water samples as well as the analyzing the genetic components of the
sample to tract the sources of different microbial contaminants. The library independence
makes this method simpler and economical as resources needed to generate large libraries
can be avoided. Two common types of techniques used in independent library methods
are: culture methods which basically comprise of bacterial and bacteriophage culture, and

18

molecular methods comprising of using PCR with marker genes in order to carry out the
host- specific PCR.
3.2.1 Bacteriophage Culture
Bacteriophage is a kind of viruses that utilizes bacteria as a host body. The
bacteriophages are known for their specificity as they invade specific bacterial cells and
in particular cases specific strain also. Bacteriophage causes disruption and disintegration
of bacterial cell. Coliphage is a special kind of bacteriophage which uses specifically E.
coli as a host thus can be used to identify the human and non-human source of
contamination in drinking and recreational water. Coliphage is further divided into
categories on the basis of genome type that is either DNA or RNA based bacteriophage.
Another important kind of differentiation used is on the basis of attachment targets;
somatic coliphage attach to the cell wall of the bacteria whereas F+ coliphage attach to
sex pilus of bacteria. Furthermore, the differentiation between F+RNA and F+DNA is
carried out by isolating in the presence of DNAse. Using methods of serotyping and
genotyping the coliphage is assigned to particular group. Uptill now four groups of
F+RNA have been identified through antigenic and genetic evaluation. In these groups, 2
and 3 is specific to human fecal sources whereas 1 and 4 is specific to animal fecal
sources. Bacteriophage has the capability to distinguish between sources as animal or
human feces but unable to differentiate between different animal sources (Unit, 2011).
3.2.2 Bacterial Polymerase Chain Reaction
The use of PCR for the extraction and analysis of bacterial genome has been
recognized technique. Through the use of PCR, the absence or presence of genetic
material specific to fecal source can be detected. The common types of bacterial
19

contaminants identified using bacterial PCR are Enterococcus and bacteroides. The
ability to identify the bacteroides in sample by PCR has solved the problem of cultivation
due to anaerobic nature. Bacteriods are found abundantly in the gut of mammals thus are
great source to indicate the human fecal contamination. But due to anaerobic nature,
number of problems is encountered in their culture including the complexity and time
required. Multiple human and non-human specific probes and markers have made the
analysis of bacteriode possible through the use of PCR.
The bacterial genetic segment usually DNA is isolated from the test water sample and
amplified in PCR using known primers of bacterial origin. The amplified segments are
then analyzed to identify the fecal contaminant sources. Special care has to be carried out
to exclude the dead and unviable cells from the sample to avoid any false positive results.
Due to its successive use, multiple markers have been developed for identification of
human and animals like goats, sheep and cattle. But improvement is required in the
identification of wild animals as very less markers are present for these animals.
Multiple enterococcus strains and species have been identified using PCR method.
Genetic markers have been developed for Enterococcus faecalis, Enterococcus faecium
and Enterococcus hirae (Sheridan, Masters, Shallcross, & Mackey, 1998).

3.2.3 Viral Polymerase Chain Reaction
As viruses is very specific with respect to the host, thus can be used accurately identify
fecal contamination sources. For this purpose, isolation, extraction and amplification of
viral genetic segments is carried out through PCR. Some human specific viruses like
20

enteroviruses and adenoviruses have been successfully used as human fecal indicator.
Similarly bovine specific enteroviruses and adenoviruses are used to identify the
livestock contamination sources. Similarly many non-pathogenic viruses are also
employed like human specific polyomaviruses are easier to identify due to their higher
prevalence than pathogenic viruses in human population.
The quantitative polymerase chain reaction uses the same method as that of bacterial and
viral PCR method but additionally incorporates the use of fluorescent probes to allow the
quantitative analysis of amplification step of the process. The signal observed from the
fluorescent probes can easily be quantitatively measured and analyzed. This quantitative
analysis is a great way to identify the contaminants population number in the sample.
This method is independent of libraries as well as culture thus it is not only economically
effective but also reduce complexity. It is also referred as real time polymerase chain
reaction due to collection of result in minimal time (Kildare et al., 2006).
3.2.4 Quantitative Polymerase Chain Reaction
The quantitative polymerase chain reaction uses the same method as that of bacterial
and viral PCR method but additionally incorporates the use of fluorescent probes to allow
the quantitative analysis of amplification step of the process. The signal observed from
the fluorescent probes can easily be quantitatively measured and analyzed. This
quantitative analysis is a great way to identify the contaminants population number in the
sample. This method is independent of libraries as well as culture thus it is not only
economically effective but also reduce complexity. It is also referred as real time
polymerase chain reaction due to collection of result in minimal time (Kildare et al.,
2006).
21

3.2.5

Limitation of Library Independent Method

Many studies provide different limitations and disadvantages of the library
independent methods. These limitations need to be improved in order to obtain better and
efficient result of microbial fecal contamination.
As most of the methods use the polymerase chain reaction, thus its implementation
and utility is limited to human and fewer animals for which the markers have been
developed. All those animals which mostly include the wild animals no markers have
been developed up till now thus these fecal sources cannot be identified from library
independent methods.
Similarly use of markers has to be assessed carefully as the chances of false positive
are observed to be high in markers which are not usually validated thoroughly. There are
many markers whose specificity are still to be determined thus limiting the usage of these
markers. Many studies have been carried out in the human specific population markers
but very fewer studies have targeted the marker for non-human population.
Correspondingly the use of viral polymerase chain reaction has to be assessed very
carefully to avoid any chances of cross reactivity. Most of the viral markers except the
non-pathogenic viruses show the issue of cross reactivity to some extent. Thus the further
assessment and validation process has to be carried out to quantify and minimize the
cross reactivity of viral markers.

22

3.1 Biochemical/PHENOTYPIC
3.1.1Antibiotics resistance + LIMITATIONS
3.1.2 Carbon Utilization
3.2 Molecular/GENOTYPIC
3.1.2.1Rep-PCR
3.1.2.2 PFGE
3.1.2.3 Rybotyping
3.2 Library independent methods

3.2.1 Evolution of methods (specificity, sensitivity, limit of
detection)
3.2.2 Source identification
3.2.3 Application for LI methods for source tracking
3.2.4 Ability of MST to predict the risk of illness
3.2.5 Correlation of markers and human pathogens

3.2.1 PCR (END POINT)
Host specific Bacteria (EITHER HUMAN OR ANIMAL)
Host specific virus
Mitochondrial DNA
3.2.2 q-PCR (REAL TIME)
Host specific Bacteria
23

Host specific virus
Mitochondrial DNA

3.2.2 RT-PCR (RNA)
3.3 Genetic markers
3.3.1 Human marker
3.3.2 Animal Markers
Pig marker
Dog marker
Cow marker
Bird marker
3.3.3 Combined Marker
1 Risk quantification
2 Source tracking

Chapter 4: Recommendations and Conclusion
References

24

REFERENCES/BIBLIOGRAPHY

[Ensure all citations are formatted in the same style. The style you choose is based on
your departmental/discipline standard]

25

APPENDICES
Appendix A
[If applicable, include copyright permission for previously published material.]

26

VITA AUCTORIS

NAME:
PLACE OF BIRTH:

Mary Scott
Windsor, ON

YEAR OF BIRTH:

1976

EDUCATION:

Assumption High School, Windsor, ON, 1995
University of Windsor, B.Sc., Windsor, ON, 1999
University of Windsor, M.Sc., Windsor, ON, 2002

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