Methods for Monitoring and Managing Fecal Microbial Contamination of Beaches.docx

7/24/2019 Methods for Monitoring and Managing Fecal Microbial Contamination of Beaches.docx

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Methods for Monitoring and Managing Fecal Microbial

Contamination of Beaches

By

Steve

A Major Research PaperSubmitted to the Faculty of Graduate Studies

through the Department of ciil and Environmental engineeringin Partial Fulfillment of the Re!uirements for

the Degree ofMaster of Science

at the "niersity of #indsor

#indsor$ %ntario$ Canada

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* &'() Stee

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Methods for Monitoring and Managing Fecal Microbial

Contamination of Beaches

by

Steve Joe

APPR%+,D B-.

//////////////////////////////////////////////

0ame of %utside Dept1 Reader$ e1g1 ]Department 0ame

//////////////////////////////////////////////

0ame of 2nternal Dept1 Reader

Department 0ame

//////////////////////////////////////////////

Adisor3s 0ame$ Adisor

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2nsert Defense Date 4ere
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D,C5ARA62%0 %F %R2G20A526-

his statement should be used when the thesis does notinclude material based on joint

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2 hereby certify that 2 am the sole author of this thesis and that no part of this

thesis has been published or submitted for publication1

2 certify that$ to the best of my 7no8ledge$ my thesis does not infringe upon

anyone3s copyright nor iolate any proprietary rights and that any ideas$ techni!ues$

!uotations$ or any other material from the 8or7 of other people included in my thesis$

published or other8ise$ are fully ac7no8ledged in accordance 8ith the standard

referencing practices1 Furthermore$ to the e9tent that 2 hae included copyrighted material

that surpasses the bounds of fair dealing 8ithin the meaning of the Canada Copyright Act$

2 certify that 2 hae obtained a 8ritten permission from the copyright o8ner:s; to include

such material:s; in my thesis and hae included copies of such copyright clearances to my

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2 declare that this is a true copy of my thesis$ including any final reisions$ as

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ABS6RAC6

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Fecal or bacterial source trac7ing also referred to as Microbial Source 6rac7ing

:MS6; is a method used to identify sources of fecal indicator bacteria in the

enironment1 4uman and liestoc7 actiity has been greatly responsible for fecal

contamination of beaches1 Accumulation of pathogenic microorganisms in beaches has

gien rise to multiple infections in human population1 6he present study aims at

highlighting enironmental problems related to fecal contamination of beaches1

Furthermore the fecal monitoring methods are inestigated in detail so as to identify the

problems regarding each method1 Adanced detection and !uality monitoring methods

are re!uired to control the contamination of beaches1 Seeral MS6 methods are used to

identify the source of fecal bacteria that are introduced into the 8ater bodies1 6hese

methods are mainly diided into t8o basic categories< one is microbial methods and

second is chemical methods1 Microbial methods are further diided into library

dependent and library independent methods1 5ibrary dependent methods are based on

maintained databases from arious bacterial isolates of 7no8n fecal sources1 4o8eer$

library independent methods identify sources based on host=specific characteristics of

bacteria 8ithout the re!uirement of library1 Currently no standardi>ed methods are

aailable for molecular and biochemical methods1 4o8eer$ recent deelopments are

more focused on improing library independent methods by e9panding the range of

fecal sources that can be identified1 Further studies are still needed to adance the

e9isting monitoring methods of fecal contamination so as to reduce recreational 8ater

contamination and multiple lethal infections caused by them1

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6AB5, %F C%06,06S

*If you omit any of the optional sections (e.g. Dedication, List of

Figures, etc.), simply delete the actual page and update the Table of

Contents by clicing on the Table of Contents and clic !"pdate

Table# in the upper left corner and select !"pdate $ntire Table.#

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Appendi9 A.......................................................................................................

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52S6 %F APP,0D2C,S

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52S6 %F ABBR,+2A62%0SS-MB%5S

Fecal 2ndicator Bacteria F2B

+scherichia coli +. coli

2nternational %rgani>ation for Standardi>ation 2S%

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Polymerase chain reaction PCR

Fluorescence in situ hybridi>ation F2S4

Microbial Source 6rac7ing MS6

Repetitie Palindromic Polymerase Chain Reactions repPCR

Pulsed filed gel electrophoresis PFG,

uantitatie polymerase chain reaction !PCR

Antibiotic Resistance Analysis ARA

Carbon "tili>ation Profile C"P

0utrient "tili>ation Pattern 0"P

%ptical brighteners %Bs

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Chapter 1: Introduction

1.1 Background

1.2 Problem defnition

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1.3 Obecti!e"

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6he present study aims to address the health and enironmental issue related to fecal

contamination of beaches1 Different methods and techni!ues employed for microbial

source trac7ing are reie8ed in this study1 6hese methods 8ill be categori>ed into

different groups based on their methodological techni!ues1 6he suitability of ne8

emerging methods to !ualitatiely analy>e the microbial leels for health and safety

regulations 8ill be studied1 2n addition$ the limitations and scope of their applicability

8ill also be discussed in this paper1 Further$ the competency of each techni!ue for

trac7ing specific microbial source 8ill be e9plored1

1.4 #tructure o$ t%e"i"

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#ater is an important natural resource that fulfills diersified needs of all the liing

organisms1 2t is used for drin7ing$ agriculture irrigation and recreation purposes1 0atural

8aters 8hen contaminated through microbes can impact all the essential needs and 8ill

also cause serious health problems in human population1 Beaches are considered as a

major commercial and recreational resource that annually attracts numerous tourists and

local isitors mainly in summer1 Pathogenic microbes released into the 8ater through

fecal matter has put the lies of s8immers at ris7 mainly humans 8ith a 8ea7er immune

system :Colford et al1$ &'';1

Controlling fecal microbial contamination in beach 8ater is therefore ital to protect

the life of human population$ birds and marine animals1 ,9posure to these pathogenic

microbes causes serious diseases such as nose$ ear and eye infections along 8ith

hepatitis$ diarrhea$ omiting$ s7in rashes$ encephalitis and respiratory illness1

Recreational 8ater users can be subjected to 8ide range of diseases causing

pathogens that either e9ist naturally in 8ater or result from fecal contamination1

#aterborne non=fecal pathogens are bacteria that naturally e9ist in recreational

8aters1 #aterborne illnesses that result from these bacteria are not transmitted through

fecal oral path8ay1 4umans are affected either by ingestion or inhalation of the polluted

8ater1 5egionella$ 4elicobacter pylori and Aeromonas are considered the most dangerous

microorganisms that cause serious respiratory illness as pneumonia :5au et al1$ &''E;1

#aterborne fecal pathogens that occur in 8ater are the result of fecal pollution from

human or animal sources1 6hrough the history$ fecal bacteria include ,scherichia Coli$

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6otal Coliform and ,nterococci 8ere ery 7no8n to be the primary reason for

gastrointestinal illness :Mot et al1$ &'(&< Anderson et al1$ &'');1

4igh leel of fecal bacteria is considered as a primary cause of recreational beach

adisories and closing in the "SA$ 8hich can result in economic losses to the

surrounding area :"S,PA &''E;1 6he un7no8n contamination sources include household

se8age$ surface runoff$ combined se8er oerflo8$ domestic animals and 8ildlife1

2dentifying the right fecal pollution source is ery crucial for mitigating and preenting

future pollution1

&1& S%"RC,S %F P%55"62%0

2.2.1 Human sources

6he oerflo8 or lea7age of the se8erage and drainage system is the largest source of

microorganism contaminant on beaches1 6hese oerflo8s are due to clogged or lea7ing

piping system$ 8hich spill untreated se8erage 8aste into coastal areas1 Similarly$ densely

populated areas 8ith inade!uate facilities and fast e9panding urban areas also add burden

on aged se8erage system that causing oerflo8s and lea7age near the 8ater bodies and

beaches1 2n addition$ poorly managed septic system at domestic leel add e9tra burden on

the already declining drainage system that eentually empties its untreated 8aste into

8ater bodies and beaches1 Furthermore$ the human population and recreational actiities

on these recreational sites and beaches are the direct source of contamination1 4uman

bodies shed different 7ind of microorganism 8hich adds to these contaminants1 5ittering

and open 8astage disposal are the other sources of microbial contamination1

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2.2.2 Domestic Animal

Studies hae identified that agricultural animals are one of the largest contributor of

the fecal contaminants1 Conersion of conentional small farms to industrial scale

productie farmlands demands increase in the animals inoled in the agriculture

process1 5arge numbers of animals are 7ept in small areas 8hich results in increased

combine feeding1 6hus$ e9cess amount of manure is produced that e9ceeds the crops

assimilatie capacity for natural fertili>er1 As a result$ the e9cess 8aste is disposed into

near 8ater bodies contains high leel animal 8aste 8hich reaches to recreational and

coastal areas1

2.2.3 Wildlie Waste

,ery year a significant amount of microbial contamination is added by 8ildlife

animals1 Different 8ildlife animals add fecal contamination directly into 8ater bodies

including recreational as 8ell as fresh 8ater1 Mostly fecal 8astage is ta7en to sea and

coastal areas from non=point sources through as rain 8ater or other 8ater bodies1 2n

addition$ the climatic changes and the seasonal transition ma7e 8ild animals to migrate

from one place to another and come in contact 8ith different 8ater bodies and coastal

areas1 6his results in adding fecal contamination into different regions1 6hus$ controlling

fecal contamination caused by 8ildlife is not easy and straightfor8ard 8hich poses

challenges in trac7ing microbial sources of 8ildlife fecal contamination1 6herefore$ ery

minimal studies and strategies hae been carried out to control the 8ildlife sources that

contaminate beach and other 8ater bodies1

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&1 M2CR%=%RGA02SMS A0D D2S,AS,S

Fecal 8aste from human or animals poses serious threat to human health1 Fecal

pathogens greatly affect the indiiduals 8ho are immune=compromised and immune=

supressed such as older adults$ infants and others 8ho are suffering from debilitating

illness li7e A2DS and pneumonia :Sinclair$ ones$ H Gerba$ &''E;1

6he pathogenic organism present in human and animal feces comprise of dierse group

of iruses$ bacteria and proto>oa 7no8n for causing different diseases1 6o date$ there are

more than ()' different types of diseases and the number 7eep increasing as ne8

pathogens are recogni>ed :Sandra et al1$ &'(;1 Gastrointestinal illness is one of the most

commonly identified 8aterborne related disease and therefore it become ery important

criteria in determining the 8ater !uality :5orna et al1$ &'();1 Respiratory infections$ s7in$

ear and eye problems are also associated 8ith recreational 8ater use :Sandra et al1$ &'(ation1 Genomic D0A is separately isolated for each selected strain1

Restriction en>ymes are used to digest the fragments of bacterial D0A1 2n the ne9t step

gel electrophoresis is used to separate the D0A fragments 8ith respect to their si>e1

Separated fragments are then moed to a gel blot 8here a labeled probe is used to attach

to certain portions of the rR0A genes1 Probe binding to the D0A fragments creates a

banding pattern as the genome contains numerous copies of rR0A genes distributed

throughout the chromosome1 6he banding pattern obtained is further isuali>ed by

chemical deelopment or autoradiography1 6hese banding patterns are captured through

digital

location and si>e of the banding pattern is are then matched 8ith 7no8n sources in

the library database1 Furthermore$ commercially aailable soft8are are used for image

analysis to compare binding patterns :Rees et al1$ &'(';1

68o ariables are used in ribotyping1 First ariable is the type of fecal indicator

bacteria that is selected to generate library1 Second ariable is the type and amount of

restriction en>ymes used to fragment the D0A1 Studies suggest that t8o restriction

en>ymes are useful in increasing the method3s discriminatory ability1

Ribotyping is adantageous in classifying the samples from multiple sources and is

highly reproducible if performed s7illfully1 4o8eer$ there are some limitations to this

method1 2t is a demanding method that has multiple steps and re!uires speciali>ed

e!uipment1 Furthermore$ indiiduals need proper training$ high supply costs and time is

needed to complete the procedure1 ,9pertise in the field of statistics is also needed to

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identify the sources present in the data1 6he geographic distribution of isolated bacteria$

the library si>e and the occurrence of replicate isolates in bacterial source library affect

the !uality of ribotyping to discriminate bet8een different bacteria at the host=species

leel1 Additionally$ the phenotypic and genotypic methods both 8ould nearly brea7 do8n

in intricate 8atersheds 8ith seeral sources1

1(1(1 rep=PCR

Polymerase Chain Reaction method allo8s for !uic7 amplification of target D0A

se!uences1 2t is used for cultiation dependent and independent approaches1 2n rep=PCR

method$ the interening se!uences present bet8een repetitie portions of microbial D0A

are amplified using rep=PCR method and one primer that mar7s each end of the

repetitie$ palindromic se!uence1 Discriminatory patterns are generated by the repetitie

elements of bacterial genome 8hich are parted by the distances specific to each bacterial

specie1 Agarose gel is then used to amplify D0A fragments 8hich generates a fingerprint

8hich also discriminates among dierse bacterial strains1

2t is a !uic7er method to classify isolates from multiple sources1 2t is easier to use$ less

costly and faster compared to the other methods1 4o8eer$ the results produced by this

method are less reproducible compared to ribotyping and PFG, :Rees et al1$ &'('e of library must be

enough to truly represent the geographical area for 8hich library is cultured1 6he direct

trac7ing methods of different contaminant microbes 8ithout the use of culture libraries

are referred as library independent methods1 2nstead of libraries$ the microbes are directly

cultured from 8ater samples as 8ell as the analy>ing the genetic components of the

sample to tract the sources of different microbial contaminants1 6he library independence

ma7es this method simpler and economical as resources needed to generate large libraries

can be aoided1 68o common types of techni!ues used in independent library methods

are. culture methods 8hich basically comprise of bacterial and bacteriophage culture$ and

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molecular methods comprising of using PCR 8ith mar7er genes in order to carry out the

host= specific PCR1

3.2.1 Bacteriophage C!lt!re

Bacteriophage is a 7ind of iruses that utili>es bacteria as a host body1 6he

bacteriophages are 7no8n for their specificity as they inade specific bacterial cells and

in particular cases specific strain also1 Bacteriophage causes disruption and disintegration

of bacterial cell1 Coliphage is a special 7ind of bacteriophage 8hich uses specifically ,1

coli as a host thus can be used to identify the human and non=human source of

contamination in drin7ing and recreational 8ater1 Coliphage is further diided into

categories on the basis of genome type that is either D0A or R0A based bacteriophage1

Another important 7ind of differentiation used is on the basis of attachment targetsP)

3.1.2.2 P'(

3.1.2.3 ?bot?ing

3.2 +ibrar? indeendent met%od"

3.2.1 (!olution o$ met%od" /"ecifcit? "en"iti!it? limit o$

detection

3.2.2 #ource identifcation

3.2.3 *lication $or + met%od" $or "ource tracking

3.2.4 *bilit? o$ - to redict t%e ri"k o$ illne""

3.2.5 )orrelation o$ marker" and %uman at%ogen"

3.2.1 P) /(, PO,-

o"t "ecifc Bacteria /(-( &*, O *,&*+

o"t "ecifc !iru"

&itoc%ondrial ,*

3.2.2 @>P) /(*+ -&(

o"t "ecifc Bacteria

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o"t "ecifc !iru"

&itoc%ondrial ,*

3.2.2 ->P) /,*

3.3 enetic marker"

3.3.1 uman marker

3.3.2 *nimal &arker"

Pig marker

og marker

)oA marker

Bird marker

3.3.3 )ombined &arker

1 i"k @uantifcation

2 #ource tracking

Chapter %: Recommendations and Conclusion

e$erence"

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(ppendi (

?2f applicable$ include copyright permission for preiously published material1@

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