Waterborne Pathogens and Stateof-art Detection Methods
Professor Bharat Patel
Section I.
Indicators of Water
Pollution
SECTION 1CONTENT
1. Introduction
1.1 Microbes on our planet & their role
1.2 Water as an environment
1.3 Microbes & their role in water
1.4 Why monitor water supplies?
1.5 Ensuring the safety of drinking water.
2. Bacterial Indicators of Pollution
2.1 What are Bacterial indicators of pollution
2.2 Total coliforms
2.3 Changes in coliform definitions
3. Alternatives to Total Coliforms
Section II.
Risk Assessment Analysis
Framework and Pathogens
SECTION II CONTENT
1. Epidemiological data on some pathogens.
2.The current list of pathogens
3.How to monitor and assess the risk of pathogens?
SECTION III.
Molecular Biology
Databases and Tools
SECTION III CONTENT
Molecular Biology
Bioinformatics
Databases
Online tools
SECTION IV.
The Biology, Methods for
Detection, Identification
& Quantitation of Waterborne Pathogens
SECTION IV CONTENT
1. The Biomolecules & Molecular Biology of Cells
2. Biomolecule Based Technics
3. The Biology & Detection Methods of Some
Pathogens
4. Modern Techologies
a. Polymerase Chain Reaction (PCR)
b. Real Time PCR
c. Pulse Field Gel Electrophoresis
d. New High Throughput Methods
LECTURE BEGINS
Section I.
Indicators of Water
Pollution
1. INTRODUCTION
1.1 Microbes on our planet & their roles
60% of the organisms are microbial (more microbes than human cells)
Surive & thrive in virtually in all environments, often where no other
“higher forms” of life exist.
1% have been characterised (24 kingdoms) & 99% remain
uncharacterised (the tree of life has been generated using rRNA as
chronometers)
Efficient colonisers (rapid growth & doubling)
Provide a service to the planet:
Ecosystem servicing (biogeochemical cycle, flux)
Biotechnology (vitamins, amino acids)
Also produce harm:
Directly as pathogens
Indirectly producing byproducts (toxins)
Simple morphology provides very little clues to their identities
NEW
Water Microbiology as it Relates to Public Health
Human reservoir
Animal reservoirs
Wastewater
Groundwater
Aerosols
Domestic use
Crops
Surface water
Aerosol
Shellfish
Land surface
Recreation
Domestic use
Three main routes must be considered to prevent the spread of waterborne (& foodborne)
diseases. The particular pathogen, its reservoir and its mode of transmission. The figuree
shows the potential route(s) of transmission and the reservoirs. For examples, cows are
sources for crypotosporodiosis and poultry are sources for campylosis.
1.2 Water as a Changable Heterogeneous Environment
1.
2.
3.
4.
5.
6.
7.
8.
9.
Climate variability
Rainfall
Soil erosion
Catchment runoff
Reservoir
Environmental flows
Water allocation
Irrigation
Billabong (wetland)
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Drinking water Filtration plant
Constructed wetland
Urban run-offs
Wastewater treatment
Industrial use
Industrial Re-use
Bore
Water table
River sediment
Mangrooves
Estuary
Recreational use
8
1.3 Microbes & their role in water
In nature, microbes live as communities (compete, synergy, complement)
They can change the environment for their growth
Most natural ecosystems are pristine ie very little nutrients
What about reservoirs or dams (man made to maximise storage)
A case study of what goes on in a reservoir:
Activities affecting a reservoir
Danger
Donot enter
Farming
activities
Recreation
Forestry
activities
pump
stratification
C, N, S, O
fluxes &
transformation
s
Distribution system
ipe
Copper p
ad
e
L ipe
p
PVC p
ipe
Filtration
&
treatment
film
?
Bio ment
p
lo
eve
INTERACTIVITY &
INTERDEPENDENCY
Ecology, Environmental & Public
Health Microbiology Groups
Regulatory Group
Transparency Group
1.4 Why monitor water supplies?
Pathogens (produce disease):
Present in water due to human / animal fecal contamination
Bacteria, virus, protozoa, helminths
Diverse types present (eg 100 types of viruses)
• Chemical pollutants
Carcinogens, toxins, endocrine disruptors & treatment
byproducts
Present due to industry, microbial activities, geological
• Risk to Human Health
Dose, host resistance (age, immunity), length of exposure
1.5 Ensuring the safety of drinking water (management)
Primary assessment: Correct operation of water supply system
Verification: Proof that water is safe after supply. This includes
monitoring for compliance.
Risk assessment: Maximum Acceptable Concentration (MAC). Should
be zero but rarely technically & economically feasible. Compliance
parameters
Compliance & risk assessment may be different for countries,
states and applications.
Improved awareness: Flexible, transparent, achievable & realistic
outcomes
1.6 Ensuring the safety by monitoring & detection
Direct measurement of harmful agents
Microbes: Not usually undertaken. Difficult, expensive, time
consuming & lack of technology. Risk -> Acute & short-lived
Chemicals: Usually undertaken. Technology exists. Risk -> Chronic
exposure & delay between sampling, testing & acting on results is
okay
Monitoring water quality barriers (catchment activities, filtration,
disinfection)
Complete risk management system for health. Gaining popularity.
Currently used indicators of water quality
Inadequate, but will be used until “new” & “better” methods
tried, tested & ratified.
Does not take into account emerging risks (microbes,
chemicals). New risks, new ways.
2. Bacterial Indicators
of Water Pollution
2.1 What are bacterial indicators of pollution?
Direct pathogen identification / isolation is impractical and / or
impossible
Alternate indirect “indicator organism” based inference is
necessary:
•universally present in large nos. in warm blooded animal faeces
•readily detectable by simple methods
•do not grow in natural waters
•persistence in water treatment regimes is similar to that for
pathogens
2.2 Coliforms & E.coli as bacterial indicators (Pre 1948)
Coliforms (coli-like, 1880) fulfill these criteria as they indicate
fecal pollution and therefore “unsafe water”
Total coliforms (Enterobacteriaceae): Escherichia, Klebsiella,
Enterobacter & Citrobacter - Ferment lactose, 1% or 109/g human
faeces. Used as a standard for testing (assuming that total
coliforms = E. coli)
PROBLEMS WITH TOTAL COLIFORM RULE
•Proportion of E. coli ↓ & coliforms↑ as faeces leaves the body.
(Coliforms are are normal inhabitants of unpolluted soils &
water).
•Coliforms & waterborne disease outbreaks are not always
linked & does not necessarily indicate potential health risk.
The current guidelines for drinking water & freshwater
recreational waters are shown in the next table as comparisons
Table Bacteriological drinking water & recreational freshwater standards or
guidelines
Maximum no of indicated organisms permitted / 100 ml of water type
Total coliformsa
Source of
standard
WHO
Canada
European Economic
Community
a
Drinking
Recreational
Thermotolerant
coliforms
Drinking
1-10
0
<10
0
0
<10,000d
Recreational
Enterococci Turbidtyb
(recreational)
(NTU)
<1-5
200c
35
<1-5
0-4
< 1 out of the <40 monthly samples analysed or < 5% of the > 40 samples analysed monthly should be
Unitedpositive
Statesfor coliforms 0
200e
<2,000d
1
b
Nephlometric Turbidity Units
(monthly)
C
> 90% are E.coli
d
Compulsory limits, bathing is restricted if >20% samples over 14 day period are positive
e
If 5 samples taken over 30 days are positive
2.2 Coliforms & E. coli as bacterial indicators (Post 1948)
Rapid methods of identifying were E. coli developed
Specific & well known thermotolerant (faecal) coliform test developed.
The Total Coliform Rule has been revised, reviewed, reassessed but
not dropped (Criteria based on quality & compliance & health risk
assessment)
•Example 1. US Envrion. Protection agency (USEPA, 1990): The water
authority must not find coliforms in > 5% samples. If found, repeat samples
within 24 hrs. If repeat samples test positive then it must be analysed for
faecal coliforms and E. coli. A positive test signifies Maximum Coliform Limits
(MCC) violation & this neccessitates rapid state and public notification.
•Example 2 EU Directive, 1998: E. coli, Enterococci & Coliforms 0 / 100ml.
Aesthetic parameters (color, conductivity, chloride, taste & ordour). The
parameters should be taken in the context of health risk assessment.
2.3 Recent changes in coliform definition
Coliforms: Members of the family Enterobactericeae; produce acid & gas
from lactose (24-48 h @ 36±2oC)
Thermotolerant (fecal) coliforms: As above but were able to grow &
ferment lactose at 44.5±0.2oC and include E. coli < Klebsiella,
Enterobacter & Citrobacter (E. coli also produce indole from tryptophan).
SEE “TESTS FOR DIFFERENTIATING COLIFORMS” SLIDE
Report 71, 1994 Bacteriological Examination of Drinking water supplies:
biochemical definition changed to “acid-only production from lactose” &
therefore increased the numbers of species in the coliform category
Enzymes: Lactose fermentation by the presence of β-galactosidase is now
considered as another modification to the coliform definition.
Australiasia, UK, Europe & soon USEPA use commercial enyme
kits & these detect coliforms that are not traditionally picked up culture
media (Noncultural but viable) hence increasing the numbers of species in
the coliform group.
Table showing coliform members by evolving definition
Acid & Gas from
lactose
Escherichia
Acid from
lactose
Escherichia
β-galactosidase
Escherichia
Klebsiella, Enterobacter, Citrobacter
Yersinia, Serratia, Hafnia,
Pantoea, Kluyvera
Cedecea
Edwingella
Moellerella
Leclercia
Rahnella
Yokenella
Coliforms that can be present in the environment & in human
faeces (bold ) and coliforms that are only environmental (bold
& underlined)
Commercial kits based detection methods for
microbial indicators
Kit Manufacturers:
IDEXX: Enterolert, Colisure, Colilert
Hach: m-ColiBlue
BioControl: ColiComplete
Chromocult: Merck
Gelman: MicroSure
Indicator
group
Enzyme / (substrate)
Total
Coliforms
β-D-galactosidase (o-nitophenyl, 6-bromo-2-napthyl, 5bromo-4-chloro-3-indolyl linked to β-D-galactoside)
SEE NEXT SLIDE
E. coli
β-D-glucoronidase (5-bromo-4-chloro-3-indolyl, 4methylumbelliferyl linked to β-D-glucoronide) SEE NEXT
SLIDE
Enterococci
β-D-glucosidase (4-methylumbelliferyl, indoxyl- linked
to β-D-glucoroside)
Coliforms
E. coli
E. aerogenes
K.
pneumoniae
assay for
all
ferment
Lactose
Enzyme
at
If growth
at
named
35 C
o
Elevated temperature
uses
of
Enzyme
44.5 oC
designate
as
named
β-galactosidase
β-glucoronidase
detected
with
MUG
designate
as
E. coli
Fecal coliforms
detected
with
ONPG
designate
as
Total coliforms
Tests for differentiating coliforms
3. Alternatives to
Coliforms as indicators of
water pollution
Faecal coliform absence indicates enetric pathogens most likely
absent but does not guarantee absence of viruses & protozoal cysts
(survive longer in water & more resistant to disinfection)
Enterococci, sulfite-reducing clostridia, Bacteroides fragilis,
Bifidobacteria, bacteriophages & non-microbiological indicators (faecal
sterols) have been proposed as alternatives to fecal coliforms
Entercocci is the most preferred (also as alternative to E. coli)
•Common commensals in warm blooded guts
•19 species (faecium, faecalis, durans, hirae dominate)
•Survive longer & do not grow in the environment
•An order of magnitude less than coliforms
•Commercial test available
Section II.
Risk Assessment
Analysis Framework
and Emerging
Pathogens
1.Epidemiology of some waterborne pathogens.
2.The current list of pathogens
3.How to monitor and assess the risk of pathogens?
1.
Epidemiology of some
pathogens.
Information modified
1. 90% water related illness are microbial
2. Canada (1974 – 1987): 32 waterborne outbreaks - Giardia:10,
Norwalk & HAV: 5, 17 unknown origin. 2000: E. coli O157, 2001
Cryptosporidium.
3. USA (1993 – 1998): Cryptosporidium (Milwaukee, Las Vegas, Nevada)
2001: Microcystin & cylindrospermopsin found in Florida drinking water
plant (5 times WHO guideline)
4. Europe (1980 –1990): Cryptosporidium (UK)
6. Vibrio cholera surveillance in India: 34 k (33 deaths) Flood related
since July 2001
5. E. coli 0157 ->feces contaminated soil, to irrigation water, to food
(Both E. coli 0157:H7 and VT6 gene strains isolated)
Swaziland: 1992 (20k), Missouri: 1989, UK: several outbreaks
reported, Wyoming: 1998, NY: 1999 (1k involved, 2 deaths,
Campylobacter also implicated), Canada: 2001 (2K involved, 7 deathsheavy rainfall & inadequate treatment)
6. Northern Ireland: 2001 Cryptosporidium
7. Portugal: 2001 Cyanobacteria toxins reported
8. Multiagent waterborne disease outbreaks:
- Switzerland: 2001, coinfection of small round structured
virus (SSRV) + Shigella + Campylobacter
- Canada: 2001, E. coli 0157:H7 & Campylobacter -> 2300 ill, 27
developed haemolytic uraemic syndrome complications (HUS), 7 deaths.
2.
Common Waterborne
Pathogens
Waterborne Pathogens:
are classified as members of domains Bacteria, Eucarya or virus.
they differ in:
morphologies
growth
physiology & metabolism
fine genetic details
• Both classification & Identification is now increasingly based on their
molecular events & molecular details (see next figure).
• The pathogens listed in the following tables have been detected in
water and / or in outbreaks. An attempt has been made to provide
their classification on the newly introduced molecular trend.
• The biology of a number of the pathogens will be described and the
possible targets sites for their identification highlighted.
EUKARYA (7)
ARCHAEA (3)
BACTERIA (21)
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Brown algae
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Thermococcus
Animals
Dinoflagellates
Ciliates
Green algae
Plants
Red algae
Fung
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2. A list of bacterial waterborne pathogens
Bacterial pathogen
Phylum
Feces
Urine
H
H
A
Disease
A
Sphingomonas
α
Potential
Burkeholdaria
β
Potential
E. coli 0157:H7 (hemorrhagic)
E. coli (enteroinvasive)
E. coli (enterpathogenic)
E. coli (enteroitoxigenic)
Salmonellae species
Salmonella enterica (serovar
typhi)
Shigella (S.flexneri, S. sonnei,
S. dysenteriae, S. boydii)
Plesiomonas shigelloides
Vibrio cholera 01
Vibrio cholera non-01
Legionella
Pseudomonas
Aermomonas hydrophila
Desulfovibrio species
P
r
o
t
e
o
b
a
c
t
e
r
i
a
χ Enterobacterales
+
+
+
+
+
+
+
+
-
+
+
+
-
χ Enterobacterales
+
-
-
-
Watery, bloody diarrhea
Typhoid, enteric fever, abdominal
pain
Shigellosis (bacillary dysentery)
χ Enterobacterales
?
?
?
?
Fish & crustaceans
χ Vibrionales
+
+
-
-
-
-
Cholera (Asiatic flu, Indian, El Tor)
+
+
-
-
Water diarrhea
χ Enterobacterales
χ Legionellales
χ Pseudomanadales
χ Aeromoandales
δ Desulovibrionales
Campylobacteria
ε
Arcobacter
ε
Duration
of disease is between 1 to 42
Helicobacteria
ε days
Strain dependent cramps, vomit,
diarrhea, fever
Legionellosis
Potential
Stomach colitis (?)
+
+
+
+
+
+
-
-
Diarrhea
Diarrhea
Stomach ulcers
Problems associated with bacterial identification
Phylum Cyanobacteria (blue green algae):
Some 50 to 60 genera; some produce oligopeptide toxins& are of increasing
concern (dermal, cytotoxin, mutantion causing and carcinogens). Lifelong exposure vs
short term acute exposure
Toxins are produced by (a) nonribosomal peptide synthetase (NRPS) which have
iterative catalytic domains. Overproduction of one or several sets up a catalytic
reaction leading to production of the toxins. (b) Peptide kinase synthetase (PKS).
MALDI-TOF MS shows a large spectrum of oligopeptides & other poorly undertood
metabolities from cyanobacteria.
Microcystis exist as toxigenic organism in reservoirs & form blooms (summer to late
autumn) but reports of non-toxicogenic strains have been reported.
Some 60 toxins (collectively called Microcystin) are produced; these are thought
to react with chlorine to produce other toxin bye-products
They have been traditionally classified on the basis of morphology & physiology
which has created confusion. Based on 16S rRNA and DNA homology studies, the 23
species have now been identified as belonging to M. aeruginosa
Toxin production in strains vary based on growth conditions (in vivo and in situ)
causing more confusion.
"Calothrix desertica" PCC 7102.
Cylindrospermopsis raciborskii str. AWT205.
"Anabaena variabilis" IAM M-3.
Nostoc muscorum PCC 7120.
Planktothrix rubescens str. BC-Pla 9303.
"Oscillatoria agardhii" str. CYA 18.
"Oscillatoria corallinae" str. CJ1 SAG8.92.
Nostoc punctiforme PCC 73102.
"Anabaena cylindrica" str. NIES19 PCC 7122.
Pseudoanabaena biceps PCC 7367.
Lyngbya confervoides PCC 7419.
10%
Trichodesmium species
Spirulina subsalsa str. M-223.
Prochloron didemni.
Cyanobacterium stanieri PCC 7202.
"Oscillatoria rosea" str. M-220.
Merismopedia glauca str. B1448-1.
Gloeothece membranacea.
Microcystis wesenbergii.
Microcystis novacekii str. TAC20.
Microcystis viridis.
Microcystis ichthyoblabe str. TAC48.
Microcystis aeruginosa.
Chamaesiphon subglobosus PCC 7430.
Octopus Spring microbial mat DNA Yellow
Leptolyngbya boryanum PCC 73110.
"Plectonema boryanum" UTEX 485.
Leptolyngbya foveolarum str. Komarek 1964/112.
Gloeochaete wittrockiana str. SAG B 46.84
Glaucocystis nostochinearum str. SAG 45
Cyanophora paradoxa (colorless flagellate alga) -- cyanelle.
"Oscillatoria limnetica" str. MR1
Phormidium mucicola str. M-221.
Phormidium ambiguum str. M-71.
Microcystis holsatica.
Microcystis elabens.
Prochlorococcus marinus PCC 9511.
Synechococcus elongatus.
Prochlorothrix hollandica.
"Oscillatoria neglecta" str. M-82
Phormidium "ectocarpi" str. N182.
Phormidium minutum str. D5.
The identification of cyanobacteria,
the causative agents for a number of
toxin-producing illnesses, is in a state
of flux. The previous identification by
morphology & / or toxin production
does not reflect the rRNA based
molecular phylogeny.
2. A list of protozoal waterborne pathogens
Protozoa
Source
Disease
Animal
feces
Nonfecal
Entamoeba
histolytica
Rare
No
Amebiasis (dysentry, enetritis, colitis)
Giardia lamblia
Yes
No
Giardiasis (hikers disease)
Cryptosporidium
parvum
Yes
No
Cryptosporodiosis (cramp, vomit, fever,
diarrhea)
Microsporidia:
Enterocytozoon
Septata
Yes
?
Cyclospora
cayatenensis
?
?
Toxoplasma gondii
Yes
No
Acanthamoeba
No
Yes
Blantidium coli
Yes
Yes
Cramp, vomit, fever, diarrhea
Abdominal pain, bloody diarrhea
2. A List of viral waterborne pathogens
Virus
Cytopathogenic human
orphan (ECHO),
polio,
coxsackies
Hepatitis A Virus (HAV)
Hepatitis E Virus (HEV)
Rotavirus A
Rotavirus B
Nowalk virus
Snow mountain
Astrovirus
100’s of others (Developing
new method to work with
them?) Small small round
structured virus (SSRV)
Group
Entero
Hepatitis
Faecal Source
Human Animal
Yes
No
Calicivirus
Yes
Yes
Yes
Yes
Yes
No
Pigs ?
Yes
Yes
?
Astrovirus
Yes
No
Picorna,
Corona,
parvo,
picobirna,
picotrirna
?
?
Rotavirus
Disease
Aseptic meningitis, infantile
diarrhea, polio
Infectious Hepatitis
Acute gastroenteritis
Acute gastroenteritis
Acute gastroenteritis
Uncertain
Viruses:
Role of some human enteric & respiratory viruses (&
some animal viruses) as waterborne pathogens has
been well established
Most are nonenveloped (except corona & picobirnaviruses) – more ressistant to physical & chemical
agents then the lipid containing enveloped viruses
Potential transmission route directly or indirectly
from animal → human & this is of concern
3. How to prioritise the list of pathogens
for further studies?
By using risk assessment
analysis frame work
1. Case of illness detected
Yes
No
Severity?
Numbers of cases?
In general or specific population?
Secondary spread of disease?
Medical treatment available
1.
Lab technique poor (sensitivity,
specificity, lack of use of good
technology
2.
Diagnosed but not reported (improve
surveillance activities)
2. Water transmission plausible? suspected? (see next slide)
Fecal /oral, person-person, foodborne, waterborne)
Yes
No
3. Water borne transmission demonstrated?
No
Recognition
Investigation
Reporting
Yes
Outbreak
Yes
How many cases/outbreak
Under what circumstances
Communicable / noncommunicable
Type source/treatment
Epidemiological studies
No
Recognition
Investigation
Reporting
Yes
Attributable risk
to water high or
low due to type
of water
No
Methodology problem
Table 1 Public health significance framework for waterborne pathogens
Table 2 Ecology / occurrence framework for waterborne pathogens
Occurrence determinants:
Incidence,
Lifecycle(s),
Epidemiological data – worldwide,
reservoirs of agents (animal / human),
geological distribution
Detection:
General,
viable?,
temperature (water pollution)
Water-based vs water borne:
Secondary hosts
Biofilm
Treatment barriers:
Source water quality
Watershed management
Treatment process configuration (driven by source water quality)
Distribution concerns
Microbial adaptation:
Treatment chain
Distribution system
Pathways:
Ingestion
Dermal
Inhalation
Table3 Treatment framework for waterborne pathogens
Organism properties & origins:
Physical: Size, surface properties, (charge, hydrophobicity, affinity for adsobtion),
surface structure, settling rate, aggregration, spore-formation
Oxidant: Mechanism of action
Origin: human, animal, naturally occurring
Disinfection kinetics:
Disinfection sensitivity (chlorine/chloroamine, chlorine dioxide, ozone, advanced oxidation
processes (AOP), UV, pottasium permanganate
Synergistic / sequential
Contact time
Organism survivability:
Survival/transport
Inactivation/injury/culturability
Survival in sludges
Organism growth / regrowth:
Regrowth
Growth in filters
Microbial protection / antagonism:
Engineering Plant operation:
Source basin (size, settling rate, residence time, turnover)
intake characteristics (level, position, hydrology),
filter operations,
line breaks / replacements
Maintenance practices (flushing)
Water Quality Characteristics:
Particulates
Chemical & physical (pH, temp, NOM, hardness, alkalinity)
Watershed management:
Human activity (sewage inputs)
Animal & environmental sources
Table3 Methods framework for waterborne pathogens
Objectives:
Key criteria for relevant microorganism
Objective for assay (qualitative vs quantitative)
Potential for transferability
Evaluation:
Sensitivity
Specificity
Positive predictive valu
Negative predicitive value
Rapidity
Throughput
Cost
Validation by collaborative study:
Reproducibility
Affordability
Purpose of method
Standard method:
Research training:
Training researchers in methodology approaches
Training technologist/analyst level
Criteria for defining potential risk posed by organisms:
Source & level of shedding
Susceptible population & infectious dose
Persistence & survivability in environment
Severity of disease
Mode of transmission
Potential of secondary spread
Treatability of disease
Ecology context
Conclusions from discussion on pathogens
Many pathogens cause water-borne diseases
Complex habitats for their growth
Pathogenic bacteria, virus & protozoa may co-exist
Symptoms similar but causative agents may be different.Therefore
assisted diagnosis is not always possible
Identification essential as patient treatment regimes depend on the
type of causative agent (bacteria vs virus vs protozoa)
Alternative methods to assess the risk of the pathogens present in
water are necessary which can be achieved by using various
frameworks
The Need for Molecular methods for the identification &
detection of pathogens
• Current US$380 million market & a 20% annual increase is expected
• Emerging sophisticated gene technologies (indicators & pathogens)
• Skilled (bioinformatics, genomics, phenomics) staff required.
• Multicomprehension (ecology, environmental etc) required
• Method rapid flexible, reproducible & can be ariticulated to particular
needs of different countries
• Initial research & development outlay is expensive (research costs)
Next?
Finding molecular biology
“information libraries”
Understanding the principles of
molecular biology
Finding & using tools for molecular
methods
SECTION III.
Molecular Biology
Databases and Tools
CONTENT
Molecular Biology
Bioinformatics
Databases
Online tools
Microbial Genomes
Molecular Biology DataBases
A. Biologists have been very successful in finding DNA & protein sequences:
- high-speed automated DNA sequencing equipme
- the Microbial Genomes (and eucaryotic genomes
- bulk sequences of cDNAs (ESTs) especially for eucaryotic genomes.
Why?
- Bioinformatics scientists collect, organize and make sequence data that is
generated, available to all biologists
- Today data is shared and integrated between the three major data
depositories, namely, GenBank, which forms part of the NCBI,
European Molecular Biology Laboratory (EMBL) and the
DNA Database of Japan (DDBJ).
- During Oct. 1996, GenBank contained 1,021,211 sequence records =
652,000,000 bases of DNA sequence = 3.1 gigabytes of computer
storage space. In June 1997 this escalated to 1,491,000 records and
967,000,000 bases. Check the sequence record out for for 2000
- The contents of GenBank are now doubling in less than a year, and the
doubling rate is accelerating ie the data generated and collected is
growing exponentially.
- Whole genome data has been generated with
32 microbial genomes sequenced. A list of completely sequenced genomes
and ongoing genome projects are maintained at
Genomes Online Database (GOLD).
- Even simple computation or searching these enormous database requires a
huge amount of computer power. What will be needed in 5 to 10 years time
is hard to image.
B. The Resources at NCBI
Established in 1988 as a national resource for molecular biology information.
It creates public databases, conducts research in computational biology,
develops software tools for analyzing genome data, and disseminates
biomedical information - all for the better understanding of molecular
processes affecting human health and disease.
The NCBI can be summarised as having 3 arms:
•GenBank Data Base: The GenBank Database is a sequence database and
has a collecti on of publically available sequence data. It is part of
National Institute of He alth (NIH), USA. GenBank, DataBank of Japan
(DDBJ) and European Molecular Biolog y Laboratory (EMBL) have formed
the International Nucleotide Sequence Database C ollaboration project
under which the 3 organisations exchange data on a daily basis.
•In this database, new protein and nucleic acids sequences are
deposited by researchers. These sequences are annotated and placed in
the sequence database for access and public viewing. The database can
also be searched.
• Literature Data Base: This is refered to as PubMed. The database
holds the abstracte of published articles.
The various Sequence Data Bases and PubMed literature Data Base are
linked via ENTREZ. ENTREZ is at the core of the search and retrieval
system that integrates and links th e various databases. In order to
maximise the benfits of the various databases it is imperative that you
read and learn from the ENTREZHELP FILE
•Bioinformatics Tools: The most commonly used tool is known as
BLAST and enables the user to input a sequence and search for the
most similar sequences in the Data Base.
C The Ribosomal DataBase Project (RDP):
Contains downloadable GenBank formatted aligned and unaligned small
subunit ribosomal rRNA sequences. Mainly extracted from the
GenBank Data Base - is a GenBank subset specialist Data Base. It
also conatins a set of integrated online analysis bioinformatics tools
useful for aligning user input sequences based on rRNA secondary
structural constraints and for constructing phylogeny.
D. KEGG Data Base:
Some Useful Online Molecular Biology Tools
1. Search launchers at http://searchlauncher.bcm.tmc.edu/
2.
Computational Biology at EMBL:
http://www.embl-heidelberg.de/Services/index.html
3. National Centre for Genome Research:
http://www.ncgr.org/
4. UC Sac Diego Motif Search & alignment tools:
http://meme.sdsc.edu/meme/website/
5. The tools at InfoBiogen, France:
http://www.infobiogen.fr/services/deambulum/english/index.html
6. The tools at the University of Pennsylvania:
http://www.cbil.upenn.edu/
7. Compilation of tools & references at the University of
California, Santa Cruz:
http://www.cse.ucsc.edu/~karplus/compbio_pages.html
Microbial Genomes
Why study microbial genomes?
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until whole genome analysis became viable, life sciences
have been based on a reductionist principle – dissecting cell
and systems into fundamental components for further study
studies on whole genomes and whole genome sequences in
particular give us a complete genomic blueprint for an
organism
we can now begin to examine how all of these parts operate
cooperatively to influence the activities and behavior of an
entire organism – a complete understanding of the biology of
an organism
microbes provide an excellent starting point for studies of
this type as they have a relatively simple genomic structure
compared to higher, multicellular organisms
studies on microbial genomes may provide crucial starting
points for the understanding of the genomics of higher
organisms
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analysis of whole microbial genomes also provides insight
into microbial evolution and diversity beyond single protein
or gene phylogenies
in practical terms analysis of whole microbial genomes is
also a powerful tool in identifying new applications in for
biotechnology and new approaches to the treatment and
control of pathogenic organisms
History of microbial genome sequencing
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1977 - first complete genome to be sequenced was
bacteriophage φX174 - 5386 bp
first genome to be sequenced using random DNA
fragments - Bacteriophage λ - 48502 bp
1986 - mitochondrial (187 kb) and chloroplast (121 kb)
genomes of Marchantia polymorpha sequenced
early 90’s - cytomegalovirus (229 kb) and Vaccinia (192
kb) genomes sequenced
1995 - first complete genome sequence from a free living
organism - Haemophilus influenzae (1.83 Mb)
late 1990’s - many additional microbial genomes
sequenced including Archaea (Methanococcus jannaschii 1996) and Eukaryotes (Saccharomyces cerevisiae - 1996)
Genomes sequenced to date
Go to the Gold database for an up to date information at
the URL- http://www.genomesonline.org/
Laboratory tools for studying whole genomes
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conventional techniques for analysing DNA are designed for the
analysis of small regions of whole genomes such as individual
genes or operons
many of the techniques used to study whole genomes are
conventional molecular biology techniques adapted to operate
effectively with DNA in a much larger size range. An example
is that of pulsed field gel electrophoresis (PFGE), the principle
of which will be discussed in detail under Molecular Methods
section.
PFGE is utilised routinely for epidemiological studies and for
fingerprinting of E. coli and Neisseria meningitidis genomes. A
potential useful tool for studying species, strain and
serovariants
Characteristics of sequenced genomes
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the 32 complete genome sequences available in 1998
covered a diverse range in terms of phylogeny and
environments (eg. human pathogens, plant pathogens,
extremophiles etc.)
what conclusions can be made by comparing the genomes
of these organisms regarding specific adaptations to
proliferation in remarkably different environments?
What conclusions can be made about evolutionary
relationships between these organisms?
Horizontal gene transfer
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before microbial genome sequences became available most of
the focus of microbial evolution was on ‘vertical’ transmission of
genetic information – mutation recombination and rearrangement
within the clonal lineage of a single microbial population
genome sequences have demonstrated that horizontal transfer
of genes (between different types of organisms) are widespread
and may occur between phylogentically diverse organisms
generally speaking, essential genes (such as 16S rRNA) are
unlikely to be transferred because the potential host most likely
already contains genes of this type that have co-evolved with
the rest of its cellular machinery and and cannot be displaced
genes encoding non-essential cellular processes of potential
benefit to other organisms are far more likely to be
transferred (eg. those involved in catabolic processes)
clearly, lateral transfer of genomic information has enormous
potential in improving an microorganisms ability to compete
effectively - this may explain why horizontally transferred
genes appear so frequently and ubiquitously in microbial genomes
an example of this is horizontally transferred genes has been
found in pathogenic microbes
Whole genome phylogenetic analysis
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most of the evolutionary relationships between microorganisms
are inferred by comparison of single genes – usually 16s rRNA
genes
although extremely effective, single gene phylogenetic trees
only provide limited information which can make determining
broad relationships between major groups difficult
phylogenetic relationships can be determined by whole genome
comparisons of the observed absence or presence of protein
encoding gene families
in effect this is similar to using the distribution of
morphological characteristics to determine phylogeny – without
the problem of convergent evolution
trees produced using this method are similar to 16s rRNA
trees, however, as more genome sequences become available
more detailed conclusions can be drawn using this method
Species and strain specific genetic diversity
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although genome sequencing and analysis is very useful when
comparing phylogenetically distant taxa, it is also of
interest to examine the genomes of very closely related
microorganisms
this allows a more quantitative approach for examining the
relationships between genotype and phenotype
complete genome sequences have been determined for two
species of the genus Chlamydia (pneumoniae and
trachomatis)
although the overall genome structure was quite similar,
C.pneumoniae contained an additional 214 genes most of
which have an unknown function
two strains of the bacterium Helicobacter pylori have been
completely sequenced (26695 and J99)
overall the two strains were very similar genetically with
only 6% of genes being specific to each strain
Case study - Neisseria meningitits
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N. meningititis causes bacterial meningitis and is therefore
an important pathogen
genome is 2.2 megabases in size
2121 ORF’s were identified with many having extremely
variable G+C% (recently acquired genes)
many of these recently acquired genes are identified as cell
surface proteins
there is a remarkable abundance and diversity of repetitive
DNA sequences
nearly 700 neisserial intergenic mosaic elements (NIME’s) 50 to 150 bp repeat elements
these repeat elements may be involved in enhancing
recombinase specific horizontal gene transfer
Case study - Borellia
burgdorferi
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B. burgdorferi is a spirochaete which causes Lyme disease
it has a 0.91 megabase linear genome and at least 17 linear
and circular plasmids which total 0.53 megabases
853 predicted ORF’s identified - these encode a basic set
of proteins for DNA replication, transcription, translation
and energy metabolism
no genes encoding proteins involved in cellular biosynthetic
reactions were identified - appears to have evolved via gene
loss from a more metabolically competent precursor
there is significant amount of genetic redundancy in the
plasmid sequences although a biological role has not been
determined
it is possible the these plasmids undergo frequent homologous
recombination in order to generate antigenic variation in
surface proteins
E. coli genome studies:
Comparative Genomics: Multiple Pathogenecity Associated Islands (PAI) of 4
uropathogenic E.coli strains against the backdrop of E. coli strain K-12. The PAIs
of 25 to 190 k, are inserted within or adjacent to tRNA genes & contain a
different % GC content to the genomic DNA. Transfer mechanism(s)?
leuX
190 kb 97 min
pheR 94 min
~25 kb
selC 82 min
70 kb
pheV
>170
kb
64 min
thrw
5.6 min ~25 kb
Strain #
535
536
J96
E. coli K-12
Chromosome
metV
27 min 60 kb
44 min asnT
45 kb
CFT073
Summary
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Microbial genome sequencing and analysis is a rapidly
expanding and increasingly important strand of microbiology
important information about the specific adaptations and
evolution of an organism can be determined from genome
sequencing
however, genome sequencing merely a strong starting point
on road to completely understanding the biology of
microorganisms
further characterisation of ORF’s of unknown function, in
combination with gene expression analysis and proteomics is
required
SECTION IV.
The Biology, Methods for
Detection, Identification
& Quantitation of Waterborne Pathogens
CONTENT
1. The Biomolecules & Molecular Biology of Cells
2. Biomolecule Based Technics
3. The Biology & Detection Methods of Some
Pathogens
4. Modern Techologies
a. Polymerase Chain Reaction (PCR)
b. Real Time PCR
c. Pulse Field Gel Electrophoresis
d. New High Throughput Methods
1. The biomolecules & molecular
biology of cells
DNA
RNA
TOTAL DNA:
•Mol%G+C
•Restriction Patterns
(RFLP, PFGE)
•Genome size
•DNA homology
DNA SEGMENTS:
• PCR based fingerprinting
(ribotyping, ARDDRA, RAPD,
AFLP, AP-PCR, rep-PCR)
•DNA probes
•DNA sequencing
• rRNA
sequencing
23
•LMW RNA
profiles
S
16
S
5
S
tRN
A
Plasmid DNA
DN
A
•Electrophoretic patterns of
total cellular or cell envelope
proteins (1D or 2D)
PROTEIN
S
•Multienzyme patterns
(multilocus enzyme
electrophoresis)
CHEMOTAXONOMIC
MARKERS
•Cellular fatty acids (FAME)
•Mycolic acids
•Polar lipids
•Quinones
•Polyamines
•Cell wall compounds
•Exopolysaccharides
mRNA
EXPRESSED
FEATURES
•Morphology
•Physiology (Biolog, API, …)
•Enzymololgy (APIzyme)
•Serology (monoclonal,
polyclonal)
DIFFERENT TARGETS FOR MICROBIAL
IDENTIFICATION
New
Selection of Different Targets
1. Cell surface:
a. proteins (receptors, porins, siderophores): 200,000 / cell
b. Polysaccharides (LPS): 2 million in Gram –ve cells
2. Cytoplasmic:
a. Ribosomes (rproteins & rRNA): 20,000 in dividing cells.
b. Non-ribosomal RNA: 100 – 1,000 / cell (depending on rate
of transcription or rate of degradation)
c. Non-iobosomal proteins (RNA polymerase): 3,000 / cell
The target concentrations in a 1 ml sample will be 0.03
attomolar(3,000 molecules / cell) to 20 attomolar (2 million /
cell)
2. Biomolecule based Technology
F
s
ie
us
c
n
e
Ge Sp
n
ily
St
ra
i
Technique
am
Restriction Fragment Length Polymorphism (RFLP)
Low frequency restriction fragment analysis (PFGE)
Phage and bacteriocin typing
Serological techniques
Ribotyping
DNA amplification (AFLP, AP-PCR, RAPD)
Zymograms (multilocus enzymes)
Total cellular protein electrophoretic patterns
DNA homology
Mol% G+C
DNA amplification (ARDRA)
tDNA-PCR
Chemotaxonomic markers
Cellular fatty acid fingerprinting (FAME)
rDNA / rRNA sequencing
DNA probes
DNA sequencing
Highthrougput assays (Microarrays, Cantilever arrays)
The limits of resolution of various techniques in microbial identification
3. The biology & detection
methods of some pathogens
Virulence Factors (VF) of Water-borne Pathogens
Virulence Factors:
•VF encoded by genes
•their presence makes the microbe pathogenic
•Most E. coli in human/animals not pathogenic as VF genes are absent
•Aquatic environment may be reservoir where “virulence breed” by
Plasmids/phage transmissision of VF (E. coli, Y.eneterocolitica & A. hydrophila)
Viruses:
• Virus multiplication
•Most non-enveloped. Antigenic shift & drift in capsid proteins
Bacteria:
•Salmonella – O (in LPS, endotoxin) & Vi (capsule) antigens
•E. coli may contain > 1 VFs:
-EIEC enteroinvasive: Shiga-like toxin (SLT),
-ETEC enterotoxigenic: Vibrio like heat labile/stable toxin (ST, LT), ID
> 1
million cells. Interfere with Na & Cl across CM, travelers diarrhea.
-EPEC, enteropathogenic: Adhesive VF for GI epithelia., infantile diarrhea in
developing countries
-EHEC, enterohemorrhagic: Shiga-like toxin (SLT), ID < 1000 cells, Since
1982, strain O157:H7 has affected 20,000 in US (>100 deaths), Found in ground
beef & now in cider & fruit juices.
• Vibrio cholera:
Cholera txin resides on plasmids which are transferred by phage
Protozoal Parasites:
Detection in water supplies is a challenge
Biology remains unstudied, biomarkers unavailable
Methods have limitation & cannot differentiate:
•human species form animal species
•infectious forms from noninfectious forms
Techniques such as Microscopy, PCR & RFLP of limited use for diagnostics
Characteristics:
• Entamoeba histolytica:
a long history as a waterborne pathogen (no US major
outbreaks reported for decades, no major nonhuman reservoir)
Cryptosporidium parvum: Major problem.
•
• Microsporidia:
unknown.
Ubiquitous parasite of insects, human & animals. Significance
Diagnostic Methods
1. Recovery and Concentration:
To increase pathogen concentration by physical, chemical or enrichments.
2. Purification & Separation:
Methods use knowledge of pathogen size, shape, density etc surface
properties (hydrophilicity, reactivity, receptors), growth stages (spores,
capsules, ooocytes) for this.
3. Assay & Characterisation:
Differentiate pathogens from all others: Qualitative / quantitative,
viable / nonviable. Cultural, immunological and NA based [ NA amplification
(PCR), NA identification & characterisation methods (hybridisation by gene
probes, RFLP & nucleotide sequencing)]. NA based methods are specific &
sensitive but incapable of differentiating live but inactivated cells from
dead / noninfectious ones.