Methods for Monitoring and Managing Fecal Microbial Contamination of Beaches

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

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Methods for Monitoring and Managing Fecal Microbial

Contamination of Beaches

By

Steve

A Major Research Paper

2014

201! "te#e

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Methods for Monitoring and Managing Fecal Microbial

Contamination of Beaches
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by

Steve Joe

APPR$%&' B()

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+ame of /nternal 'ept- Reader'epartment +ame

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Fecal or bacterial so,rce trac5ing also referred to as Microbial "o,rce 3rac5ing

9M"3: is a method ,sed to identify so,rces of fecal indicator bacteria in the

en#ironment- ,man and li#estoc5 acti#ity has been greatly responsible for fecal

contamination of beaches- Acc,m,lation of pathogenic microorganisms in beaches has

gi#en rise to m,ltiple infections in h,man pop,lation- 3he present st,dy aims at

highlighting en#ironmental problems related to fecal contamination of beaches-

F,rthermore the fecal monitoring methods are in#estigated in detail so as to identify the

problems regarding each method- Ad#anced detection and 7,ality monitoring methods

are re7,ired to control the contamination of beaches- "e#eral M"3 methods are ,sed to

identify the so,rce of fecal bacteria that are introd,ced into the 6ater bodies- 3hese

methods are mainly di#ided into t6o basic categories< one is microbial methods and

second is chemical methods- Microbial methods are f,rther di#ided into library

dependent and library independent methods- ibrary dependent methods are based on

maintained databases from #ario,s bacterial isolates of 5no6n fecal so,rces- o6e#er.

library independent methods identify so,rces based on host=specific characteristics of

bacteria 6itho,t the re7,irement of library- C,rrently no standardi>ed methods are

a#ailable for molec,lar and biochemical methods- o6e#er. recent de#elopments are

more foc,sed on impro#ing library independent methods by e8panding the range of

fecal so,rces that can be identified- F,rther st,dies are still needed to ad#ance the

e8isting monitoring methods of fecal contamination so as to red,ce recreational 6ater

contamination and m,ltiple lethal infections ca,sed by them-

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

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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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Appendi8 A.......................................................................................................

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Fecal /ndicator Bacteria F/B

+scherichia coli +. coli

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Polymerase chain reaction PCR

Fl,orescence in sit, hybridi>ation F/"

Microbial "o,rce 3rac5ing M"3

Repetiti#e Palindromic Polymerase Chain Reactions repPCR

P,lsed filed gel electrophoresis PF&

D,antitati#e polymerase chain reaction 7PCR

Antibiotic Resistance Analysis ARA

Carbon ;tili>ation Profile C;P

+,trient ;tili>ation Pattern +;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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3he present st,dy aims to address the health and en#ironmental iss,e related to fecal

contamination of beaches- 'ifferent methods and techni7,es employed for microbial

so,rce trac5ing are re#ie6ed in this st,dy- 3hese methods 6ill be categori>ed into

different gro,ps based on their methodological techni7,es- 3he s,itability of ne6

emerging methods to 7,alitati#ely analy>e the microbial le#els for health and safety

reg,lations 6ill be st,died- /n addition. the limitations and scope of their applicability

6ill also be disc,ssed in this paper- F,rther. the competency of each techni7,e for

trac5ing specific microbial so,rce 6ill be e8plored-

1.4 #tructure o$ t%e"i"

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Chapter 2: Literature Review

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ater is an important nat,ral reso,rce that f,lfills di#ersified needs of all the li#ing

organisms- /t is ,sed for drin5ing. agric,lt,re irrigation and recreation p,rposes- +at,ral

6aters 6hen contaminated thro,gh microbes can impact all the essential needs and 6ill

also ca,se serio,s health problems in h,man pop,lation- Beaches are considered as a

major commercial and recreational reso,rce that ann,ally attracts n,mero,s to,rists and

local #isitors mainly in s,mmer- Pathogenic microbes released into the 6ater thro,gh

fecal matter has p,t the li#es of s6immers at ris5 mainly h,mans 6ith a 6ea5er imm,ne

system 9Colford et al-. 200E:-

Controlling fecal microbial contamination in beach 6ater is therefore #ital to protect

the life of h,man pop,lation. birds and marine animals- &8pos,re to these pathogenic

microbes ca,ses serio,s diseases s,ch as nose. ear and eye infections along 6ith

hepatitis. diarrhea. #omiting. s5in rashes. encephalitis and respiratory illness-

Recreational 6ater ,sers can be s,bjected to 6ide range of diseases ca,sing

pathogens that either e8ist nat,rally in 6ater or res,lt from fecal contamination-

aterborne non=fecal pathogens are bacteria that nat,rally e8ist in recreational

6aters- aterborne illnesses that res,lt from these bacteria are not transmitted thro,gh

fecal oral path6ay- ,mans are affected either by ingestion or inhalation of the poll,ted

6ater- egionella. elicobacter pylori and Aeromonas are considered the most dangero,s

microorganisms that ca,se serio,s respiratory illness as pne,monia 9a, et al-. 200:-

aterborne fecal pathogens that occ,r in 6ater are the res,lt of fecal poll,tion from

h,man or animal so,rces- 3hro,gh the history. fecal bacteria incl,de &scherichia Coli.

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3otal Coliform and &nterococci 6ere #ery 5no6n to be the primary reason for

gastrointestinal illness 9Mot et al-. 2012< Anderson et al-. 200!:-

igh le#el of fecal bacteria is considered as a primary ca,se of recreational beach

ad#isories and closing in the ;"A. 6hich can res,lt in economic losses to the

s,rro,nding area 9;"&PA 200:- 3he ,n5no6n contamination so,rces incl,de ho,sehold

se6age. s,rface r,noff. combined se6er o#erflo6. domestic animals and 6ildlife-

/dentifying the right fecal poll,tion so,rce is #ery cr,cial for mitigating and pre#enting

f,t,re poll,tion-

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2.2.1 Human sources

3he o#erflo6 or lea5age of the se6erage and drainage system is the largest so,rce of

microorganism contaminant on beaches- 3hese o#erflo6s are d,e to clogged or lea5ing

piping system. 6hich spill ,ntreated se6erage 6aste into coastal areas- "imilarly. densely

pop,lated areas 6ith inade7,ate facilities and fast e8panding ,rban areas also add b,rden

on aged se6erage system that ca,sing o#erflo6s and lea5age near the 6ater bodies and

beaches- /n addition. poorly managed septic system at domestic le#el add e8tra b,rden on

the already declining drainage system that e#ent,ally empties its ,ntreated 6aste into

6ater bodies and beaches- F,rthermore. the h,man pop,lation and recreational acti#ities

on these recreational sites and beaches are the direct so,rce of contamination- ,man

bodies shed different 5ind of microorganism 6hich adds to these contaminants- ittering

and open 6astage disposal are the other so,rces of microbial contamination-

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2.2.2 Domestic Animal

"t,dies ha#e identified that agric,lt,ral animals are one of the largest contrib,tor of

the fecal contaminants- Con#ersion of con#entional small farms to ind,strial scale

prod,cti#e farmlands demands increase in the animals in#ol#ed in the agric,lt,re

process- arge n,mbers of animals are 5ept in small areas 6hich res,lts in increased

combine feeding- 3h,s. e8cess amo,nt of man,re is prod,ced that e8ceeds the crops

assimilati#e capacity for nat,ral fertili>er- As a res,lt. the e8cess 6aste is disposed into

near 6ater bodies contains high le#el animal 6aste 6hich reaches to recreational and

coastal areas-

2.2.3 Wildlie Waste

ery year a significant amo,nt of microbial contamination is added by 6ildlife

animals- 'ifferent 6ildlife animals add fecal contamination directly into 6ater bodies

incl,ding recreational as 6ell as fresh 6ater- Mostly fecal 6astage is ta5en to sea and

coastal areas from non=point so,rces thro,gh as rain 6ater or other 6ater bodies- /n

addition. the climatic changes and the seasonal transition ma5e 6ild animals to migrate

from one place to another and come in contact 6ith different 6ater bodies and coastal

areas- 3his res,lts in adding fecal contamination into different regions- 3h,s. controlling

fecal contamination ca,sed by 6ildlife is not easy and straightfor6ard 6hich poses

challenges in trac5ing microbial so,rces of 6ildlife fecal contamination- 3herefore. #ery

minimal st,dies and strategies ha#e been carried o,t to control the 6ildlife so,rces that

contaminate beach and other 6ater bodies-

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Fecal 6aste from h,man or animals poses serio,s threat to h,man health- Fecal

pathogens greatly affect the indi#id,als 6ho are imm,ne=compromised and imm,ne=

s,pressed s,ch as older ad,lts. infants and others 6ho are s,ffering from debilitating

illness li5e A/'" and pne,monia 9"inclair. Hones. I erba. 200:-

3he pathogenic organism present in h,man and animal feces comprise of di#erse gro,p

of #ir,ses. bacteria and proto>oa 5no6n for ca,sing different diseases- 3o date. there are

more than 1!0 different types of diseases and the n,mber 5eep increasing as ne6

pathogens are recogni>ed 9"andra et al-. 201G:- astrointestinal illness is one of the most

commonly identified 6aterborne related disease and therefore it become #ery important

criteria in determining the 6ater 7,ality 9orna et al-. 201!:- Respiratory infections. s5in.

ear and eye problems are also associated 6ith recreational 6ater ,se 9"andra et al-. 201Gymes are ,sed on the 6hole '+A genome and then

larger genomic fragments are generated that m,st not be bro5en d,ring sample

preparation- 3he genomic fragments are than parted by alternately p,lsed ,sing

perpendic,larly oriented electrical fields instead of ,sing standard gel electrophoresis-

Banding pattern emerges after electrophoresis and staining of gels- enerated patterns are

then compared 6ith the so,rces in the library database 9Rees et al-. 2010< ar6ood.

"taley. Badgley. Borges. I oraj5ic. 2014:-

/t is a #ery ad#antageo,s method that classifies isolates from m,ltiple so,rces- /t is a

highly reprod,cible method if performed s5illf,lly and is among the most discriminatory

genotyping methods- o6e#er. some of the limitations of the method are that it is time

cons,ming and highly costly- /t demands high degree of technical s5ill- F,rthermore. a

large geographically=specific so,rce library is re7,ired-

G-1-1-2 Ribotyping

/t is one of the 6idely ,sed method in library=dependent Microbial "o,rce 3rac5ing

applications- /t is based on the recognition of genetic di#ersity in the genomic se7,ences

6ithin or flan5ing the 1J" and 2G" rR+A genes- 3hese ribosomal ribon,cleic genes are

highly conser#ed among bacterial species- 3he selected bacterial gro,p for ribotyping is

c,lt,red from the fecal samples implementing standard methods- &nterococci or &- coli

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are commonly isolated and a certain percentage of bacterial co,nt is selected for

genotypic characteri>ation- enomic '+A is separately isolated for each selected strain-

Restriction en>ymes are ,sed to digest the fragments of bacterial '+A- /n the ne8t step

gel electrophoresis is ,sed to separate the '+A fragments 6ith respect to their si>e-

"eparated fragments are then mo#ed to a gel blot 6here a labeled probe is ,sed to attach

to certain portions of the rR+A genes- Probe binding to the '+A fragments creates a

banding pattern as the genome contains n,mero,s copies of rR+A genes distrib,ted

thro,gho,t the chromosome- 3he banding pattern obtained is f,rther #is,ali>ed by

chemical de#elopment or a,toradiography- 3hese banding patterns are capt,red thro,gh

digital

location and si>e of the banding pattern is are then matched 6ith 5no6n so,rces in

the library database- F,rthermore. commercially a#ailable soft6are are ,sed for image

analysis to compare binding patterns 9Rees et al-. 2010:-

36o #ariables are ,sed in ribotyping- First #ariable is the type of fecal indicator

bacteria that is selected to generate library- "econd #ariable is the type and amo,nt of

restriction en>ymes ,sed to fragment the '+A- "t,dies s,ggest that t6o restriction

en>ymes are ,sef,l in increasing the methods discriminatory ability-

Ribotyping is ad#antageo,s in classifying the samples from m,ltiple so,rces and is

highly reprod,cible if performed s5illf,lly- o6e#er. there are some limitations to this

method- /t is a demanding method that has m,ltiple steps and re7,ires speciali>ed

e7,ipment- F,rthermore. indi#id,als need proper training. high s,pply costs and time is

needed to complete the proced,re- &8pertise in the field of statistics is also needed to

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identify the so,rces present in the data- 3he geographic distrib,tion of isolated bacteria.

the library si>e and the occ,rrence of replicate isolates in bacterial so,rce library affect

the 7,ality of ribotyping to discriminate bet6een different bacteria at the host=species

le#el- Additionally. the phenotypic and genotypic methods both 6o,ld nearly brea5 do6n

in intricate 6atersheds 6ith se#eral so,rces-

G-1-1-G rep=PCR

Polymerase Chain Reaction method allo6s for 7,ic5 amplification of target '+A

se7,ences- /t is ,sed for c,lti#ation dependent and independent approaches- /n rep=PCR

method. the inter#ening se7,ences present bet6een repetiti#e portions of microbial '+A

are amplified ,sing rep=PCR method and one primer that mar5s each end of the

repetiti#e. palindromic se7,ence- 'iscriminatory patterns are generated by the repetiti#e

elements of bacterial genome 6hich are parted by the distances specific to each bacterial

specie- Agarose gel is then ,sed to amplify '+A fragments 6hich generates a fingerprint

6hich also discriminates among di#erse bacterial strains-

/t is a 7,ic5er method to classify isolates from m,ltiple so,rces- /t is easier to ,se. less

costly and faster compared to the other methods- o6e#er. the res,lts prod,ced by this

method are less reprod,cible compared to ribotyping and PF& 9Rees et al-. 2010P) /,*

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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