Waterborne Pathogens and State-of-art Detection Methods

Professor Bharat Patel

Section I.

Indicators of Water Pollution

1. Introduction1.1 Microbes on our planet & their role1.2 Water as an environment1.3 Microbes & their role in water1.4 Why monitor water supplies?1.5 Ensuring the safety of drinking water. 1.5 Ensuring the safety of drinking water.

2. Bacterial Indicators of Pollution2.1 What are Bacterial indicators of pollution2.2 Total coliforms2.3 Changes in coliform definitions

3. Alternatives to Total Coliforms

SECTION 1CONTENT

Section II.

Risk Assessment Analysis Framework and Pathogens

1. Epidemiological data on some pathogens.

2.The current list of pathogens

3.How to monitor and assess the risk of pathogens?

SECTION II CONTENT

SECTION III. Molecular Biology

Databases and Tools

Molecular Biology

Bioinformatics

Databases

Online tools

SECTION III CONTENT

SECTION IV.

The Biology, Methods for Detection, Identification & Quantitation of Water-

borne 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

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 pathogensIndirectly producing byproducts (toxins)

Simple morphology provides very little clues to their identities

1.1 Microbes on our planet & their roles

Water Microbiology as it Relates to Public Health

Animal reservoirsHuman reservoir

Domestic use

Land surfaceGroundwater

ShellfishAerosolDomestic use Recreation

Wastewater

Surface water

Aerosols Crops

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.

NEW

1.2 Water as a Changable Heterogeneous Environment

1. Climate variability 2. Rainfall3. Soil erosion4. Catchment runoff5. Reservoir6. Environmental flows7. Water allocation8. Irrigation9. Billabong (wetland)

10. Drinking water Filtration plant11. Constructed wetland12. Urban run-offs13. Wastewater treatment14. Industrial use15. Industrial Re-use16. Bore17. Water table18. River sediment19. Mangrooves20. Estuary21. Recreational use

8

1.3 Microbes & their role in waterIn 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

Farming activities

C, N, S, O fluxes &

transformations

stratificationForestry activities

pump

Filtration &

treatment

Copper pipe

Lead

pipe

PV

C p

ipe

Distribution system

Recreation

Danger Donot enter

INTERACTIVITY & INTERDEPENDENCY

Ecology, Environmental & Public Health Microbiology Groups

Regulatory Group

Transparency Group

Biofilm

development ?

Pathogens (produce disease): Present in water due to human / animal fecal contaminationBacteria, virus, protozoa, helminths Diverse types present (eg 100 types of viruses)

• Chemical pollutantsCarcinogens, toxins, endocrine disruptors & treatment byproductsPresent due to industry, microbial activities, geological

• Risk to Human HealthDose, host resistance (age, immunity), length of exposure

1.4 Why monitor water supplies?

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

1.5 Ensuring the safety of drinking water (management)1.5 Ensuring the safety of drinking water (management)

1.6 Ensuring the safety by monitoring & detection1.6 Ensuring the safety by monitoring & detection

Direct measurement of harmful agents Direct measurement of harmful agents

Microbes: Not usually undertaken. Difficult, expensive, time Microbes: Not usually undertaken. Difficult, expensive, time consuming & lack of technology. Risk -> Acute & short-livedconsuming & lack of technology. Risk -> Acute & short-lived

Chemicals: Usually undertaken. Technology exists. Risk -> Chronic Chemicals: Usually undertaken. Technology exists. Risk -> Chronic exposure & delay between sampling, testing & acting on results is exposure & delay between sampling, testing & acting on results is okayokay

Monitoring water quality barriers (catchment activities, filtration, Monitoring water quality barriers (catchment activities, filtration, disinfection)disinfection)

Complete risk management system for health. Gaining popularity. Complete risk management system for health. Gaining popularity.

Currently used indicators of water qualityCurrently used indicators of water quality

Inadequate, but will be used until “new” & “better” methods Inadequate, but will be used until “new” & “better” methods tried, tested & ratified. tried, tested & ratified.

Does not take into account emerging risks (microbes, Does not take into account emerging risks (microbes, chemicals). New risks, new ways. chemicals). New risks, new ways.

2. Bacterial Indicators of Water 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

2.1 What are bacterial indicators of pollution?

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

2.2 Coliforms & E.coli as bacterial indicators (Pre 1948)

Source of standard

Maximum no of indicated organisms permitted / 100 ml of water type

Total coliformsa Thermotolerant coliforms Turbidtyb

(NTU)Drinking Recreational Drinking Recreational

WHO 1-10 0 <1-5

Canada <10 0 200c 35 <1-5

European Economic Community

0 <10,000d 0-4

United States 0 200e <2,000d 1 (monthly)

Enterococci(recreational)

Table Bacteriological drinking water & recreational freshwater standards or guidelines

a < 1 out of the <40 monthly samples analysed or < 5% of the > 40 samples analysed monthly should be positive for coliforms

b Nephlometric Turbidity UnitsC > 90% are E.colid Compulsory limits, bathing is restricted if >20% samples over 14 day period are positivee 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 definitionColiforms: 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 definitionAcid & Gas from

lactoseAcid from

lactoseβ-galactosidase

Escherichia Escherichia Escherichia Klebsiella, Enterobacter, Citrobacter

Yersinia, Serratia, Hafnia, Pantoea, Kluyvera

Cedecea

Edwingella

Moellerella

Leclercia

Rahnella

YokenellaColiforms that can be present in the environment & in human faeces (bold ) and coliforms that are only environmental (bold & underlined)

Kit Manufacturers: IDEXX: Enterolert, Colisure, ColilertHach: m-ColiBlueBioControl: ColiCompleteChromocult: MerckGelman: MicroSure

Indicator group

Enzyme / (substrate)

Total Coliforms

β-D-galactosidase (o-nitophenyl, 6-bromo-2-napthyl, 5-bromo-4-chloro-3-indolyl linked to β-D-galactoside) SEE NEXT SLIDE

E. coli β-D-glucoronidase (5-bromo-4-chloro-3-indolyl, 4-methylumbelliferyl linked to β-D-glucoronide) SEE NEXT SLIDE

Enterococci β-D-glucosidase (4-methylumbelliferyl, indoxyl- linked to β-D-glucoroside)

Commercial kits based detection methods for microbial indicators

Coliforms E. coliE. aerogenes K. pneumoniae

Lactose

35 oC

Enzyme

β-galactosidase

ONPG

Total coliforms

Elevated temperature

44.5 oC

Fecal coliforms

Enzyme

β-glucoronidase

MUG

E. coli

If growth at

of

designate as

all ferment

at

uses

named

detected with

designate as

assay for

designate as

detected with

named

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.

1. 90% water related illness are microbial2. 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 deaths- heavy rainfall & inadequate treatment)

6. Northern Ireland: 2001 Cryptosporidium

7. Portugal: 2001 Cyanobacteria toxins reported

Information modified

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:Waterborne Pathogens: are classified as members of domains Bacteria, Eucarya or virus.

they differ in:morphologiesgrowth physiology & metabolismfine 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.

Flagellates

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Methanopyrus

Hal

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Evolution of Universal Ancestor (3.5 billion yrs)

Pla

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Animals

Gre

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Brown algae

Dip

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Fun

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Red

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Trichomonads

Cil

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lage

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Korarchaeota

EuryarchaeotaCrenarchaeota

EUKARYAEUKARYA (7) (7) ARCHAEAARCHAEA (3) (3) BACTERIA (21)BACTERIA (21)

Aq

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Chlamydia

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Actinobacte

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Verrucomicrobia

Acidobacteria

PlanctomycetesFibrobacter

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Spirochetes

Fusiforms

Proteo

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The Tree of Life - 16th November 2000

2. A list of bacterial waterborne pathogens2. A list of bacterial waterborne pathogensBacterial pathogen Phylum Feces Urine Disease

H A H A

Sphingomonas α Potential

Burkeholdaria β Potential

E. coli 0157:H7 (hemorrhagic)E. coli (enteroinvasive)E. coli (enterpathogenic)E. coli (enteroitoxigenic)

χ Enterobacterales

++++

+---

----

----

Strain dependent cramps, vomit, diarrhea, fever

Salmonellae speciesSalmonella enterica (serovar typhi)

χ Enterobacterales ++

+-

++

+-

Watery, bloody diarrheaTyphoid, enteric fever, abdominal pain

Shigella (S.flexneri, S. sonnei, S. dysenteriae, S. boydii)

χ Enterobacterales + - - - Shigellosis (bacillary dysentery)

Plesiomonas shigelloides χ Enterobacterales ? ? ? ? Fish & crustaceans

Vibrio cholera 01Vibrio cholera non-01

χ Vibrionales ++

--

--

--

Cholera (Asiatic flu, Indian, El Tor)

Legionella χ Legionellales - - - - LegionellosisPseudomonas χ Pseudomanadales Potential

Aermomonas hydrophila χ Aeromoandales + + - - Water diarrheaDesulfovibrio species δ Desulovibrionales Stomach colitis (?)

Campylobacteria ε + + - - DiarrheaArcobacter ε + + - - DiarrheaHelicobacteria ε + + - - Stomach ulcersLeptospira Spirochaetes - + - + Weil’, swineherd’s, hemorrhagicMycobacteria avium-intracelllare & other species

Actinobacteria + + - - Lung disease

Cyanobacteria (toxins) Cyanobacteria: taxonomy in flux

- - - - Mycrocystins (60), Cylindrospermopsin

Duration of disease is between 1 to 42 days

Proteobacteria

Problems associated with bacterial identificationPhylum 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.

10%

"Oscillatoria corallinae" str. CJ1 SAG8.92.

Gloeothece membranacea.Microcystis wesenbergii.

Chamaesiphon subglobosus PCC 7430.

"Calothrix desertica" PCC 7102.

Prochlorothrix hollandica.

Octopus Spring microbial mat DNA Yellow

Synechococcus elongatus.

Leptolyngbya boryanum PCC 73110.

Cylindrospermopsis raciborskii str. AWT205.

"Oscillatoria neglecta" str. M-82

Prochlorococcus marinus PCC 9511.

"Oscillatoria limnetica" str. MR1Cyanophora paradoxa (colorless flagellate alga) -- cyanelle.

Spirulina subsalsa str. M-223.

Phormidium "ectocarpi" str. N182.Phormidium minutum str. D5.

Microcystis holsatica.Microcystis elabens.

Phormidium mucicola str. M-221.Phormidium ambiguum str. M-71.

Gloeochaete wittrockiana str. SAG B 46.84 Glaucocystis nostochinearum str. SAG 45

"Plectonema boryanum" UTEX 485.Leptolyngbya foveolarum str. Komarek 1964/112.

"Oscillatoria rosea" str. M-220.Merismopedia glauca str. B1448-1.

Microcystis novacekii str. TAC20.Microcystis viridis.

Microcystis ichthyoblabe str. TAC48.Microcystis aeruginosa.

Prochloron didemni.Cyanobacterium stanieri PCC 7202.

Planktothrix rubescens str. BC-Pla 9303."Oscillatoria agardhii" str. CYA 18.

"Anabaena variabilis" IAM M-3.Nostoc muscorum PCC 7120.

Pseudoanabaena biceps PCC 7367.Lyngbya confervoides PCC 7419.

Nostoc punctiforme PCC 73102."Anabaena cylindrica" str. NIES19 PCC 7122.

Trichodesmium species

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 pathogens2. A list of protozoal waterborne pathogensProtozoa Source Disease

Animal feces

Non-fecal

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 ? Cramp, vomit, fever, diarrhea

Cyclospora cayatenensis

? ?

Toxoplasma gondii Yes No

Acanthamoeba No Yes

Blantidium coli Yes Yes Abdominal pain, bloody diarrhea

2. A List of viral waterborne pathogens2. A List of viral waterborne pathogensVirus Group Faecal Source Disease

Human AnimalCytopathogenic human orphan (ECHO), polio, coxsackies

Entero Yes No Aseptic meningitis, infantile diarrhea, polio

Hepatitis A Virus (HAV)Hepatitis E Virus (HEV)

Hepatitis YesYes

NoPigs ?

Infectious Hepatitis

Rotavirus ARotavirus B

Rotavirus YesYes

YesYes

Acute gastroenteritis

Nowalk virusSnow mountain

Calicivirus Yes ? Acute gastroenteritis

Astrovirus Astrovirus Yes No Acute gastroenteritis

100’s of others (Developing new method to work with them?) Small small round structured virus (SSRV)

Picorna, Corona, parvo,

picobirna, picotrirna

? ? Uncertain

Viruses:

Role of some human enteric & respiratory viruses (& some animal viruses) as waterborne pathogens has been well established

Most are nonenveloped (except corona & picobirna-viruses) – 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

2. Water transmission plausible? suspected? (see next slide)Fecal /oral, person-person, foodborne, waterborne)

NoYes

3. Water borne transmission demonstrated?

Yes NoRecognitionInvestigationReporting

Outbreak

YesHow many cases/outbreakUnder what circumstancesCommunicable / noncommunicableType source/treatment

Epidemiological studies

NoRecognitionInvestigationReporting

YesAttributable risk to water high or low due to type of water

NoMethodology problem

YesSeverity?Numbers of cases?In general or specific population?Secondary spread of disease?

NoMedical treatment available1. Lab technique poor (sensitivity,

specificity, lack of use of good technology

2. Diagnosed but not reported (improve surveillance activities)

Table 1 Public health significance 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 hostsBiofilm

Treatment barriers:Source water qualityWatershed management Treatment process configuration (driven by source water quality)Distribution concerns

Microbial adaptation: Treatment chainDistribution system

Pathways:IngestionDermalInhalation

Table 2 Ecology / occurrence framework for waterborne pathogens

Table3 Treatment framework for waterborne pathogensOrganism properties & origins:

Physical: Size, surface properties, (charge, hydrophobicity, affinity for adsobtion), surface structure, settling rate, aggregration, spore-formationOxidant: Mechanism of actionOrigin: human, animal, naturally occurring

Disinfection kinetics:Disinfection sensitivity (chlorine/chloroamine, chlorine dioxide, ozone, advanced oxidation processes (AOP), UV, pottasium permanganate Synergistic / sequentialContact time

Organism survivability:Survival/transportInactivation/injury/culturabilitySurvival in sludges

Organism growth / regrowth:RegrowthGrowth 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 / replacementsMaintenance practices (flushing)

Water Quality Characteristics:ParticulatesChemical & physical (pH, temp, NOM, hardness, alkalinity)

Watershed management:Human activity (sewage inputs)Animal & environmental sources

Table3 Methods framework for waterborne pathogensObjectives:

Key criteria for relevant microorganismObjective for assay (qualitative vs quantitative)Potential for transferability

Evaluation:SensitivitySpecificityPositive predictive valuNegative predicitive valueRapidityThroughputCost

Validation by collaborative study:ReproducibilityAffordabilityPurpose of method

Standard method:Research training:

Training researchers in methodology approachesTraining technologist/analyst level

Criteria for defining potential risk posed by organisms:Source & level of sheddingSusceptible population & infectious dosePersistence & survivability in environmentSeverity of diseaseMode of transmissionPotential of secondary spreadTreatability of diseaseEcology 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

Molecular Biology

Bioinformatics

Databases

Online tools

Microbial Genomes

CONTENT

Molecular Biology DataBasesA. 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

Why study microbial genomes?

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

Microbial Genomes

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

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 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 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 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 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 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

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 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

pheR~25 kb

selC70 kb

pheV>170 kb

thrw~25 kb

E. coli K-12Chromosome

94 min

97 min

64 min

27 min

5.6 min

44 min

536

Strain #

CFT073

J96

535

82 min

leuX190 kb

metV60 kb

asnT 45 kb

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)?

E. coli genome studies:

Summary 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 Water-

borne Pathogens

CONTENT1. 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 SEGMENTS:• PCR based fingerprinting (ribotyping, ARDDRA, RAPD, AFLP, AP-PCR, rep-PCR)•DNA probes•DNA sequencing

TOTAL DNA:•Mol%G+C•Restriction Patterns (RFLP, PFGE)•Genome size•DNA homology

DNA

23S

16S

5S

tRN

A

DNA

Plasmid DNA

RNA

• rRNA sequencing

•LMW RNA profiles

mRNAPROTEINS

•Electrophoretic patterns of total cellular or cell envelope proteins (1D or 2D)

•Multienzyme patterns (multilocus enzyme electrophoresis)

CHEMOTAXONOMIC MARKERS

EXPRESSED FEATURES

•Cellular fatty acids (FAME)•Mycolic acids•Polar lipids•Quinones•Polyamines•Cell wall compounds•Exopolysaccharides

•Morphology•Physiology (Biolog, API, …)•Enzymololgy (APIzyme)•Serology (monoclonal, polyclonal)

DIFFERENT TARGETS FOR MICROBIAL IDENTIFICATION

Selection of Different Targets1. 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)

New

2. Biomolecule based Technology

Technique Family

GenusSp

ecies

The limits of resolution of various techniques in microbial identification

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)

Strain

3. The biology & detection methods of some pathogens

Virulence Factors (VF) of Water-borne PathogensVirulence 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 challengeBiology remains unstudied, biomarkers unavailableMethods have limitation & cannot differentiate:

•human species form animal species•infectious forms from noninfectious forms

Techniques such as Microscopy, PCR & RFLP of limited use for diagnosticsCharacteristics:

• 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: Ubiquitous parasite of insects, human & animals. Significance unknown.

Diagnostic Methods1. 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.