The

Essen(alness of Water
Blood in our veins approximates composi3on of sea water Concept of hydrophilic and hydrophobic nature of biological molecules These molecules determine shape of biological molecules and thus decide the specicity of all living processes
Essen(al for All living organisms 97% of the water is in the oceans Water covers 70% of the world

 We are a burgeoning human popula(on unable to move away from its waste

asparagus irriga(on

All Microbes Live in an Aqueous Environment

Ecology of aqua3c environments is complex Most aqua3c environments are teaming with life Microbes have evolved to live in:

Saturated salt solu3ons Below freezing to >110C Waters full of toxic substance , i.e. copper, cyanide, lead, silver, gasoline, oil, benzene, and many others

Terminology
Potable - (clean) water free of all objec(onable material, including pathogens, tastes, odors, colors, toxins, radioac3ve material, organisms, oils, gases, etc. Fresh non-salt or sea water Pollu(on anything that makes it Non-Potable Sewage the community waste or garbage that mother nature and we dump onto sewers or land

Typical Water Quality Standards

Drinking Water
No coliforms contamina3on acceptable

Recrea3onal water
200 fecal coliforms /100 ml

Fish and wildlife habitat

5000 fecal coliforms/100 ml

Shellsh
14 fecal coliforms/100 ml

Most Probable Number

10 ml, 1 ml and 0.1ml of water inoculated in lactose broth Coliforms iden3ed by gas produc3on Refer to tables and determine sta3s3cal range of number of coliforms Does not: Detect total number of bacteria Specic pathogens

Bacteria Found In Surface Water

Bacteria
Aeromonas Campylobacter jejuni

Disease/ infection
Enteritis Campilobacteriose

Symptoms
Very thin, blood- and mucus-containing diarrhea Flue, diarrhea, head- and stomachaches, fever, cramps and nausea Watery diarrhea, headaches, fever, homiletic uremia, kidney damage Nausea, stomachaches and watery diarrhea, sometimes fevers, headaches and vomiting Fevers Sickness, intestinal cramps, vomiting, diarrhea and sometimes light fevers Stomach aches, diarrhea and fevers, sometimes vomiting Heavy diarrhea

Escherichia coli

Urinary tract infections, neonatal meningitis, intestinal disease Plesiomonas-infection

Plesiomonas shigelloides

Typhus Salmonella

Typhoid fever Salmonellosis

Streptococcus

(Gastro) intestinal disease

Vibrio El Tor (freshwater)

(Light form of) Cholera

Pathogens of Most Concern on Fresh Produce

Salmonella Shigella Escherichia coli Campylobacter Yersinia entercoli7ca Staphylococcus aureus Clostridium species Bacillus cereus Vibrio species Vibrio species

Viruses (Hepa33s A, Norwalk) Parasites/Protozoa- (Giardia, Entamoeba, Toxoplasma, Sarccys7s, Isopora, Cryptosporidium, Eimeria, Cyclospora)

Waterborne Infec3ous Disease (U.S. 1997-1998)

Disease
Shigellosis

Agent Outbreaks Cases

Shigella sonnei 1 183

Giardiasis Giardia lambia 4 159 Cryptoporidiosis Cryptosporidium parvum 2 1432 Gastroenteri3s E. Coli 0157:H7 3 164 Acute Unknown 5 163 gastrointes3nal illness

Shigella

Other Important Water TransmiIed Organisms

Vibrio cholerae
Prevalent in U. S. in 1800s Currently common in Asia, Africa, La3n America Over 100,000 deaths and 2345 deaths in 2004 Transmihed through water, fresh vegetables and shellsh

Protozoa Found in Surface Water

Microrganism Disease Symptoms

Amoeba

Amoebic dysentery

Severe diarrhea, headache, abdominal pain, chills, fever; if not treated can cause liver abscess, bowel perforation and death

Cryptosporidium parvum

Cryptosporidiosis

Feeling of sickness, watery diarrhea, vomiting, lack of appetite

Giardia

Giardiasis

Diarrhea, abdominal cramps, flatulence, belching, fatigue Flu, swelling of lymph glands With pregnant women subtle abortion and brain infections

Toxoplasm gondii

Toxoplasmosis

Giardiasis and Cryptosporidiosis

Both are protozoans Transmission through water
(97% of all surface water carry cysts)

Resistant to chlorine, but can be ltered 1993 Milwaukee outbreak

(100,000)

Some Costly Cases

Cryptosporidium, 1993, Milwaukee, $55 million Pesteria piscicida, 1997, Chesapeake bay, $43 million 3700 beach closing in 1996 Mild case of diarrhea cost ~$280 for treatment and diagnosis

Life cycle of Cryptospoidium Transmission occurs mainly through Contaminated water.

Agricultural Water
Iden(fy source and distribu3on of water used Be aware of current and historical use of land Review exis3ng prac3ces and condi3ons to iden3fy poten(al sources of contamina(on. Maintain wells in good working condi(on Minimize contact of edible por(on of fresh produce with contaminated irriga(on water.

How are you applying the water?

Water Quality Evalua(on Log

Water Source
Open source, canal, Reservoir, pond, etc.

Irriga3on Pes3cide App. Hand Produce wash wash Y N Y N Y N Y N

Munciple water source Capped well, Annual test date Uncapped well, canal, reservoir, etc. Quarterly test date Municipal water source Quality report date

Y N

Y N

Y N

Y N

Public Health and Water Supply

Rou3ne monitoring of water quality using indicator organisms, indica3ng fecal contamina3on. To determine if fecal coliforms are from humans or other animals must test for fecal streptococci

Fecal coliform/fecal streptococci ra(os for humans and other animals Human Duck Sheep Chicken Pig Cow Turkey 4.4 0.6 0.4 0.4 0.4 0.2 0.1

Characteris3cs of a Useful Indicator

Useful for all water types Always present when pathogens are present Not present in the absence of the pathogen Correlated with degree of pollu3on More easily detectable than a pathogen Survive longer than the pathogen Not dangerous to work with

Bacterial-Indicator Organisms Common Groups

Coliforms Total coliforms Fecal coliforms Escherichia coli Streptococci fecal streptococci enterococci Spore Formers Clostridium perfringens

Indicator Organisms
General coliforms indicate water
in contact with plant or animal life (universally present)

Fecal coliforms mammal or bird

feces in water

Enterococcus bacteria (type of fecal

streptococci) feces from warm

blooded animals in water

These are not what generally make people sick

Problems With the Coliform Indicator Test

False Posi3ves

Enterobacter areogenes

False Nega3ves

Salmonella typhi

Some Factors Aec3ng Ra3o of Indicator Organisms to Pathogens

Feces from human popula3ons with higher infec3on rates are of greater concern All treatment methods and environmental condi3ons aect pathogens and indicators dierently
- Chlorinated water may have zero indicators and pathogens, but
loaded with viruses. - Pathogens can hide from treatment inside suspended solids.

The ra3o of indictors to actual pathogens is not xed

Direct Tests For Pathogens

Involves selec3ve cul3va3on to large numbers
Time consuming Expensive Poten3ally dangerous to lab personnel

Molecular tests
Require tes3ng for each pathogen Expensive Require exper3se

Viral Sources of Waterborne Disease

Hepa33s A: inamma3on and necrosis of liver Norwalk-type virus: acute gastroenteri3s Rotaviruses: acute gastroenteri3s, especially in children Enteroviruses: many types aect intes3nes and upper respiratory tract Reoviruses: infects intes3nes and upper respiratory tract

Virus Detec3on
Very dicult and costly
Electron microscopy Immunoassays Cell cultures Reverse transcrip3on-polymerase chain reac3on (RT- PCR)

Chlorina3on of Water

The most commonly used sani(zer!

Methods of Treatment
Shock Chlorina3on (50-100 ppm, contact of at least 6
hours)

Con3nuous Chlorina3on for recurring bacterial

contamina3on problems a measurable amount of free residual chlorine

Chlorine Terms
Chlorine Dosage total added Chlorine Demand - inorganic Combined Residual Chlorine - organic Free Residual Chlorine

Chlorine Dosage

Chlorine Dosage
Chlorine Demand

Residual Chlorine

Chlorine Dosage

Inorganic

Free Residual Combined Chlorine Organic

Residual Chlorine

Chlorine Demand

Kill

Free Residual Chlorine

Chlorine remaining arer combining with organic maher Bacteria kill rate propor(onal to concentra(on of free residual
DPD, N,N-diethyl-p-phenylene-diamine

Bohom Line
Test your water as required and any3me you suspect a problem Work with your County Environmental Health Department Seek advise on interpre3ng the results what do they mean? If you ques3on the results, resample and retest

NEW

Water Microbiology as it Relates to Public Health

Human reservoir

Animal reservoirs

Wastewater

Groundwater

Surface water

Land surface

Aerosols

Domestic use

Crops

Aerosol

Shellfish

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.

2.2 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. Drinking water Filtration plant 11. Constructed wetland 12. Urban run-offs 13. Wastewater treatment 14. Industrial use 15. Industrial Re-use 16. Bore 17. Water table 18. River sediment 19. Mangrooves 20. Estuary 21. Recreational use

2.3 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

Recreation

Farming ac3vi3es

Forestry ac3vi3es

pump

stratification

C, N, S, O uxes & transforma3ons

Filtra3on & treatment

e Copper pip

Distribu3on system

INTERACTIVITY & INTERDEPENDENCY Ecology, Environmental & Public Health Microbiology Groups Regulatory Group Transparency Group

2.4 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

" " "

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 awarness: Flexible, transparent, achievable & realistic outcomes

" "

3. Bacterial Indicators of Water Pollution

3.1 What are bacterial indicators of pollution?

" Direct pathogen identification / isolation is impractical and / or impossible " lternate indirect indicator organism based inference is necessary: A niversally present in large nos. in warm blooded animal faeces u eadily detectable by simple methods r o not grow in natural waters d ersistence in water treatment regimes is similar to that for p pathogens

3.2 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 roportion of E. coli & coliforms as faeces leaves the body. P (Coliforms are are normal inhabitants of unpolluted soils & water). oliforms & waterborne disease outbreaks are not always linked & C 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 & recrea3onal 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 United States
a

Drinking 1-10 <10 0 0

Recreational

Thermotolerant coliforms Enterococci (recreational) Drinking Recreational 0 0 200c 35

Turbidtyb (NTU) <1-5 <1-5 0-4

<10,000d 200e <2,000d

1 (monthly)

< 1 out of the <40 monthly samples analysed or < 5% of the > 40 samples analysed monthly should be positive for coliforms b Nephlometric Turbidity Units 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

3.2 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) xample 1. US Envrion. Protection agency (USEPA, 1990): The water E 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. xample 2 EU Directive, 1998: E. coli, Enterococci & Coliforms 0 / 100ml. E Aesthetic parameters (color, conductivity, chloride, taste & ordour). The parameters should be taken in the context of health risk assessment.

3.3 Recent changes in coliform definition

Coliforms: Members of the family Enterobactericeae; produce acid & gas from lactose (24-48 h @ 362oC) Thermotolerant (fecal) coliforms: As above but were able to grow & ferment lactose at 44.50.2oC 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 -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 -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 Total Coliforms E. coli Enzyme / (substrate) -D-galactosidase (o-nitophenyl, 6-bromo-2-napthyl, 5-bromo-4-chloro-3-indolyl linked to -D-galactoside) SEE NEXT SLIDE -D-glucoronidase (5-bromo-4-chloro-3-indolyl, 4methylumbelliferyl linked to -D-glucoronide) SEE NEXT SLIDE -D-glucosidase (4-methylumbelliferyl, indoxyl- linked to -D-glucoroside)

Enterococci

Coliforms

E. coli E. aerogenes K. pneumoniae

all ferment

assay for

Lactose
If growth at at

Enzyme
named

35 oC Elevated temperature
of uses

-glucoronidase
detected with

Enzyme 44.5 oC
designate as named

MUG
designate as

-galactosidase E. coli
detected with

Fecal coliforms ONPG

designate as

Total coliforms

Tests for differentiating coliforms

4. 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) ommon commensals in warm blooded guts C 9 species (faecium, faecalis, durans, hirae dominate) 1 urvive longer & do not grow in the environment S n order of magnitude less than coliforms A ommercial test available C

2. Common Waterborne Pathogens

" are classified as members of domains Bacteria, Eucarya or virus. " they differ in: orphologies m rowth g hysiology & metabolism p ine genetic details f 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.

Crenarchaeota
Dinoflagellates Ciliates Green algae Plants Red algae

Animals

Brown algae Flagellates

Methanopyru

Korarchaeota

Evolution of Universal Ancestor (3.5 billion yrs) The Tree of Life - 16th November 2000

Thermotoga Thermodesulfobacteria Dictyoglomus Thermales Chrysiogen etes

Thermococcus

Aquifex

Bacterial pathogen
Sphingomonas Burkeholdaria E. E. E. E. coli coli coli coli 0157:H7 (hemorrhagic) (enteroinvasive) (enterpathogenic) (enteroitoxigenic)
P r o t e o b a c t e r i a

Phylum

Feces
H A

Urine
H A Potential

Disease

Enterobacterales + + + + + + + ? + + + + ? + + ? + ? -

Potential Strain dependent cramps, vomit, diarrhea, fever

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 Campylobacteria Arcobacter

Enterobacterales

Watery, bloody diarrhea Typhoid, enteric fever, abdominal pain Shigellosis (bacillary dysentery) Fish & crustaceans Cholera (Asiatic flu, Indian, El Tor) Legionellosis Potential

Enterobacterales Enterobacterales Vibrionales Legionellales Pseudomanadales Aeromoandales Desulovibrionales

Water diarrhea Stomach colitis (?)

+ + + +

+ + + + +

+ -

Diarrhea Diarrhea Stomach ulcers Weil, swineherds, hemorrhagic Lung disease

Helicobacteria Duration of disease is between 1 to 42 days Leptospira Spirochaetes Mycobacteria aviumintracelllare Actinobacteria

Problems associated with bacterial identification

Phylum Cyanobacteria (blue green algae):

Information modified

" 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). " ALDI-TOF MS shows a large spectrum of oligopeptides & other poorly undertood M metabolities from cyanobacteria. " icrocystis exist as toxigenic organism in reservoirs & form blooms (summer to late M 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. Trichodesmium species

Nostoc punctiforme PCC 73102. "Anabaena cylindrica" str. NIES19 PCC 7122. Pseudoanabaena biceps PCC 7367. Lyngbya confervoides PCC 7419. 10%

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 toxinproducing illnesses, is in a state of flux. The previous identification by morphology & / or toxin production does not reflect the rRNA based molecular phylogeny.

Protozoa

Source Animal feces Nonfecal No No No ?

Disease

Entamoeba histolytica Giardia lamblia Cryptosporidium parvum Microsporidia: Enterocytozoon Septata Cyclospora cayatenensis Toxoplasma gondii Acanthamoeba Blantidium coli

Rare Yes Yes Yes

Amebiasis (dysentry, enetritis, colitis) Giardiasis (hikers disease) Cryptosporodiosis (cramp, vomit, fever, diarrhea) Cramp, vomit, fever, diarrhea

? Yes No Yes

? No Yes Yes Abdominal pain, bloody diarrhea

Virus Cytopathogenic human orphan (ECHO), polio, coxsackies Hepatitis A Virus (HAV) Hepatitis E Virus (HEV) Rotavirus A Rotavirus B Nowalk virus Snow mountain Astrovirus 100s of others (Developing new method to work with them?) Small small round structured virus (SSRV)

Group Entero

Faecal Source Human Yes Animal No

Disease Aseptic meningitis, infantile diarrhea, polio Infectious Hepatitis Acute gastroenteritis Acute gastroenteritis Acute gastroenteritis Uncertain

Hepatitis Rotavirus Calicivirus Astrovirus Picorna, Corona, parvo, picobirna, picotrirna

Yes Yes Yes Yes Yes Yes ?

No Pigs ? Yes Yes ? No ?

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

Table 2 Ecology / occurrence framework for waterborne pathogens Occurrence determinants:

Incidence, Lifecycle(s), Epidemiological data worldwide, reservoirs of agents (animal / human), geological distribution General, viable?, temperature (water pollution) Secondary hosts Biofilm

Detection:

Water-based vs water borne: Treatment barriers:

Source water quality Watershed management Treatment process configuration (driven by source water quality) Distribution concerns Treatment chain Distribution system Ingestion Dermal Inhalation

Microbial adaptation:

Pathways:

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

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 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
TOTAL DNA: ol%G+C M estric3on Paherns (RFLP, PFGE) R enome size G NA homology D DNA SEGMENTS: PCR based ngerprin3ng (ribotyping, ARDDRA, RAPD, AFLP, AP-PCR, rep-PCR) NA probes D NA sequencing D

RNA
rRNA sequencing MW RNA proles L

Plasmid DNA

DNA lectrophore3c paherns of total cellular or E cell envelope proteins (1D or 2D) ul3enzyme paherns (mul3locus enzyme M electrophoresis)

PROTEINS

mRNA

CHEMOTAXONOMIC MARKERS ellular fahy acids (FAME) C ycolic acids M olar lipids P uinones Q olyamines P ell wall compounds C xopolysaccharides E

EXPRESSED FEATURES orphology M hysiology (Biolog, API, ) P nzymololgy (APIzyme) E erology (monoclonal, polyclonal) S

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

Technique
Restric3on Fragment Length Polymorphism (RFLP) Low frequency restric3on fragment analysis (PFGE) Phage and bacteriocin typing Serological techniques Ribotyping DNA amplica3on (AFLP, AP-PCR, RAPD) Zymograms (mul3locus enzymes) Total cellular protein electrophore3c paherns DNA homology Mol% G+C DNA amplica3on (ARDRA) tDNA-PCR Chemotaxonomic markers Cellular fahy acid ngerprin3ng (FAME) rDNA / rRNA sequencing DNA probes DNA sequencing Highthrougput assays (Microarrays, Can3lever 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: F encoded by genes V heir presence makes the microbe pathogenic t ost E. coli in human/animals not pathogenic as VF genes are absent M quatic environment may be reservoir where virulence breed by A Plasmids/phage transmissision of VF (E. coli, Y.eneterocolitica & A. hydrophila) Viruses: Virus multiplication ost non-enveloped. Antigenic shift & drift in capsid proteins M Bacteria: almonella O (in LPS, endotoxin) & Vi (capsule) antigens S . coli may contain > 1 VFs: E -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:
uman species form animal species h nfectious forms from noninfectious forms i

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. unknown.

Microsporidia: 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.

4. Modern Techologies

a. Polymerase Chain Reaction (PCR)

Figure 1B

Figure 1A

Figure 1C

DNA Fundamentals

A short video clip to show the principle of PCR

2. What is PCR? DNA replication in a tube (in vitro). Xeroxing (copying) of DNA. 3. The Components of PCR The basic components of a PCR reaction are - one or more molecules of target DNA -two oligonucleotide primers - thermostable DNA polymerase - dNTPs 4. The Process of PCR Each PCR cycle requires three temperature steps to complete a round of DNA synthesis:

Cycle 1: The original DNA template will continue to be copied by the DNA polymerase until it stops or the process is interrupted by the start of the next cycle. Cycle 2: Amplicons of intermediate lengths produced Cycle 3: Amplicons of defined lengths will be produced. Cycle 4 onwards: Target sequence will be amplified exponentially The final number of copies of the target sequences is expressed as: (2n-2n)x where n = no of cycles, 2n = 1st product obtained after cycle 1 & 2nd products obtained after cycle 2 with undefined length and x= no. of copies of the original template

PCR target molecules accumulate as a function of cycle number with the exponential phase lasting for about 30 cycles under standard reactions conditions. The plateau phase results from limiting amounts of enzyme and reduced enzyme activity. The production of 1 billion copies of the specific targeted DNA from 1 template during the 30 cycles is theoretically possible but never practically achieved because of lack of 100% PCR efficiency. The products formed during the process could be mixtures of specific and non-specific products and these factors reduce PCR efficiency from the theoretical 100%.

5. The Factors Affecting PCR I. Generalities: pipette water first, followed by the other ingredients. work on ice in order to minimise primers binding to the DNA template and to prevent functioning of the polymerase (even theoretically) prior to the first denaturing step. avoid aerosols while pipetting (or use aerosol-ressistant pipette tips) & work under laminar flow hoods. Be very accurate when dealing with small volumes. (Multiplex PCR of two different genomic DNA samples can be very susceptible to errors in pipetting). II Thermocyclers and PCR vials: The same PCR program will work slightly different on different thermocyclers (temperature and time profiles may differ) and therefore the PCR results using the same primer pair may vary. New PCR machine designs accommodate thin-walled 0.2 ml PCR vials (and/or 96 wells microtiter dishes). Contact between the metal and plastic is very good and aided by the downward pressure from the heated lid. Older machines accommodated 0.5 or 1.5 ml vials and the contact between the vials and the metal block is not always perfect because of slight differences in shape and wall thickness amongst manufacturers, often resulting in reduced or no amplification

II. Denaturing temperature and time:

50mM NaCl decreases the melting (denaturing) temperatures of 91-97oC (not 100 oC) 1 aq polymerase has a half-life of 30 min at 95oC. Denaturation temp as short as possible T 2-5 minutes initial denaturing step prior to the start of cycling is not necessary 1 min at 94oC (usually between 15 sec to 30 secs) avoids loss of Taq enzyme activity 7-28 bases. Longer primers 30-35 bp for multiplexing 1 both primers have a close melting temperature or Tm of within 5 oC. G+C content of 40-60% (Tms between 55-80oC are preferred). primer sequence with 1-2 GC pairs at the start and end improves priming efficiency three or more Cs or Gs at the 3'-ends of primers may promote mispriming Check for primer-primer interactions: 3'-ends of primers not complimentary Check for primer self-complementarity (ability to form 2o structures such as hairpins) primer sequences checked against DNA database (using BLAST programs) for uniqueness Useful ON-LINE programs for primer design can be found at the following URLs Primer0.5: http://trishul.sci.gu.edu.au/JaMBW/5/2/index.html WebPrimer:http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer Primer3: http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi alculate Tm of primer using rule of thumb 4(G + C) + 2(A + T)oC. C Temperature of annealing (Ta) should be about < 5oC below the lowest Tm of the pair of primers aq polymerase incorporates about 2000 nucleotides/minute at optimal temperature 72-78o C T Rule of thumb 1 min for a 1 kb product, 2 min for a two kb

III. Choosing and Primer Design:

IV. Primer Annealing Temperature:

V. Extension or Polymerisation:

Some Commercial thermostable DNA polymerases and their sources:

Deep Vent (Pyrococcus GB-D) Recombinant Vent (Thermococcus litoralis) Recombinant UlTma (Thermotoga maritime) Recombinant Tth (Thermus thermophilus) Recombinant Amplitaq (Thermus aquaticus) Recombinant Amplitaq Stoffel (Thermus aquaticus) Recombinant Hot Tub (Thermus flavus) Natural Pyrostase (Thermus flavus) Natural Tbr (Thermus brockianus) Natural Tfl (Thermus flavus) Natural Pfu (Pyrococcus furiosus) Natural Pwo (Pyrococcus wosei) Natural

Properties of some thermostable DNA polymerases:

VI. Reaction Volumes:

thin walled, 0.2 ml plastic vials for 96 well thermocyclers have heated lids (no oil). Reaction volumes (5, 25 or 100 ul) okay PCR product yield is higher in 5 L compared to 100 L volumes (product can be visualised) 100-500 nM each primer Purchased as 10-25 mM; 0.5-1ml primer is sufficient for 25-100 ml PCR reactions

VII. Primer Amount in PCR:

VIII. Template concentrations:

ithin limits, increasing primer and template concentration may improve the outcome of the W PCR reaction, and should be considered as a way to optimize PCR reactions Stock of 25 mM each stored as small aliquots (2-5 l) at -20o C. Centrifuge long term stored solutions as water condenses on the walls changing conc Dilute stocks in buffered water (10mM Tris pH 7.7-8.0) as acid pH hydrolysis dNTP to dNDP and dNMP

IX. Nucleotides (dNTP):

X. Relationship between MgCl2 and dNTP concentration:

00M dNTP each and 1.5mM MgCl2 is recommended with Taq polymerase, (Perkin Elmer 2 Cetus). Theoretically 6-6.5 g of DNA is synthesised from 25 l reaction. Besides magnesium bound by the dNTP and the DNA, Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets "trapped") whereas increases in magnesium concentration often have positive effects.

XI. Adjuvants in PCR Reactions:

Between 5-10% DMSO or glycerol promotes increase in PCR yield 0.8g/l BSA promotes better yield than DMSO or glycerol

Gel electrophoresis for detecting PCR products

Agarose Gels:
NuSieve agarose separates short products better than the regular agarose. More expensive but use less for the same gel strength as regular agarose. All regular agarose, irrespective of the brand, behave the same 600 bp separation: Run very fast (3-4 h for a 15-20 cm long 2-3% agarose gel). Bands are sharper

Non-denaturing PAA gels: Denaturing PAA gels:

6-10% PAA gels used for PCR products differing in only a few bp in length % PAA/7M urea sequencing gel is used to separate radiolabeled multiplex PCR products 6

Real Time detection of PCR products

o gels required. Recent method. Relies on the ability of a dye, SYBR Green, to N intercalate with double stranded amplicons produced during PCR, to produce fluorescence which is detected in a flurometer. (Dealt with in a subsequent section)

New

Distinguishing between PCR & Real Time PCR

PCR Primer design & annealing PCR cycling parameters 30 cycles Detection by Gel electrophoresis RealTime-PCR Primer Design & annealing Probe Design internal to PCR amplicons & Annealing PCR Cycling Parameters 30 cycles Detection of fluorescence every cycle (annealing and / or extension) Subsequent Gel Electrophoresis (if necessary)

b. RealTime-PCR

Real Time PCR

1. Introduction 2. General Principles & Concepts
A. What are Fluorescent dyes? B. What is Fluorescence Resonance Energy Transfer (FRET)? C. Some commonly used flurophores for labeling probes D. Quantitating Fluorescence E. Improving Fluorescence Signal Detection (new)

3. Instruments
A. LightCycler (Idaho Technologies ! Roche) B. Rotor-Gene (Corbett Research) C. iCycler (BioRad) D. Mx4000 Multiplex Quantitative PCR System (Stratagene) E. ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem) F. SmartCycler (Cephid)

4. Types of probes & design

A. DNA binding dyes B. Oligonucleotide Hybridisation Probes I. Hydrolysis Probes (TaqMan) II. Strand Displacement Probes A. Roche Dual probe B. Hair Pin Probes Molecular Beacon Sunrise UniPrimer Scorpion
Stem & Loop Duplex FRET Duplex

C. Peptide Nucleic Acid Probes (PNA)

5. Applications

1. Introduction

What is Real Time PCR?

Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.

WHAT IS THE DIFFERENCE BETWEEN PCR AND REALTIME PCR ?

" Fluorescence is measured every cycle " The signal is proportional to the amount of product " Observed in Real Time during PCR

WHAT CAN BE DONE WITH Real Time PCR?

Specific quantification of: DNA RNA Protein

2. General Principles & Concepts

2A. What are Fluorescent dyes?

New

When a population of fluorochrome molecules is excited by light of an appropriate wavelength, fluorescent light is emitted. The light intensity can be measured using a flurometer or by measuring a pixel-by-pixel digital image of the sample. In the later case, image analysis software, makes it possible to view, measure, render, and quantitate the resulting image. Excitation and Emission: Fluorodyes absorb light at one level (wavelength) & thereby boosts an electron to a higher energy shell (an unstable, excited state). The excited electron falls back to the ground state and the flurophore reemits light but at a second lower , longer wavelength. This shift makes it possible to separate excitation light from emission light with the use of optical filters. The wavelength (nm) where photon energy is most efficiently captured is defined as the Absorbancemax & the wavelength (nm) where light is most efficiently released is defined as the Emissionmax. The difference in absorbed & emitted wavelength = Stokes shift (). can be a large or small number depending on the loss of energy during fluorescence process.

2A. What are Fluorescent dyes?(contd)

New

The wavelegth range for which flurodyes absorb light is small (~ < 50nm) and light outside this range will not cause the molecule to fluoresce. 2. Linearity: Theintensity of the emitted fluorescent light is a linear function of the amount of fluorochrome present when the illuminating light has a constant wavelength and intensity (for example, using a controlled laser light source). The signal becomes nonlinear at very high fluorochrome concentrations. 3. Brightness: Fluorochromes differ in how much intensity they are capable of producing. This is important because a dull fluorochrome is a less sensitive probe than a bright fluorochrome. The brightness depends on two properties of the fluorochrome ts ability to absorb light (extinction coefficient). I he efficiency with which it converts absorbed light into emitted T fluorescent light (quantum efficiency). 4. Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks. Such fluorochromes are useful for analyzing changes in H+, Mg2+, or Ca2+ concentration & detecting lipids or double-stranded DNA. Photodestruction (photobleaching) of photosensitive dyes (eg fluorescein) is caused by intense light. Use antifade agents or lower the laser power

Fluorescent dyes have become the preferred method of detection for nucleic acids in Molecular Biology. " hey are used as single conjugated dyes to oligonucleotides for: T Automated fluorescent DNA sequencing, Fluorescent genotyping & Terminal Fragment Restriction Length Polymorphism (TFRLF) AND " As double or multiple conjugated dyes to oligonucleotides for simultaneous detection, identification and quantitative techniques in Real Time PCR (Molecular Beacons) based on the principle of Fluorescence Resonance Energy Transfer (FRET) or quenching.

Modified

2B. What is Fluorescence Resonance Energy Transfer (FRET)? FRET is a distance dependent interaction interaction between the excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon

2B. FRET (contd):

The Donor and Acceptor in close physical proximity (10 -100 Angstrom) can lead to FRET or Quenching
hv hv

(a) Physical proximity + hv (FRET +ve)

(b) No physical proximity + hv

Hybridization probes

(c) No hv

hv

hv

R R Q Q
(e) No Physical proximity + hv (Quenching released)

(d) Physical proximity + hv (Quenching)

TaqMan & Beacon Probes

2C. Some Commonly used flurophores for labeling probes

FLUORESCEIN TAMARA CY 5

494 / 518 nm
TET CY 3

650 / 690 nm
HEX

TEXAS RED

LC-RED

595 / 615 nm Consider the cost, ease of synthesis, proprietary, delivery

time etc

2D. Quantitating Fluorescence

A flurometer exploits the principles of fluorescence to quantitate fluorescent (dye) molecules in the following way: " A strong light source which produces light within a specific light range ( eg xenon arc lamp) is focused down to a tight beam. " he tight beam of light is sent through a filter which removes most T of the light outside of the target wavelength range for a particular fluorescent molecule. " he filtered light beam passes through the liquid target sample T striking some of the fluorescent molecules in the sample. " ight emitted from the fluorescent molecules that is traveling L orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range.

" he filtered light then strikes a photodetector or photomultiplier which T allows the instrument to give a relative measurement of the intensity of the emitted light. " luorescent molecules can be detected at concentrations below a level F visible to the subjective human eye & as fluorescence intensity vs. concentration is a linear relationship, dye concentrations can be determined with a good degree of accuracy

2E. Improving Fluorescence Signal Detection

A number of ways are available to improve detection and measurement of the emitted fluorescent signal. a. Elimination of the excited light from the collection pathway by several methods: Orienting the excitation light path so that the light does not shine into the collection pathway. Inserting optical filters into the collection pathway to reject the excitation wavelength. Delaying collection until after a pulse of excitation light has disappeared. b. The fluorescent signal can also be enhanced by increasing the dwell time or by scanning the sample multiple times and mathematically processing the signals to reduce random noise. Such methods are useful and practical for increasing the sensitivity at the low end. c. A band-pass optical filter can be used to reject broad-spectrum background emissions. This type of filter rejects wavelengths shorter and longer than the selected band, while allowing wavelengths in the selected wavelength range (centered around the fluorescent emissions of the sample) to pass through to the collection pathway.

Wide variety: Fluorochromes with a wide variety of characteristics are available, including fluorochromes that espond to pH or ion concentrations. R ocalize based on hydrophobic and hydrophilic interactions. L an be cross-linked to proteins, NA, lipids, or polysaccharides. C Commercial available: Fluorochromes are available crosslinked to many other molecules (eg fluorescently labeled monoclonal and polyclonal antibodies with a choice of fluorochrome, fluorescently labeled enzyme substrates, such as fluorescent chloramphenicol for chloramphenicol acetyl transferase (CAT) assays and fluorescein digalactoside for b-galactosidase assays (lacZ gene). Multiple-label possibility: A significant advantage of fluorescent labeling over other methods is the possibility of recording the fluorescence of two or more fluorochromes separately using optical filters and a fluorochrome separation algorithm. Thus, components can be labeled specifically and identified separately in the same sample or lane (EG Real Time PCR applications)

2F. Advantages of Fluorescence

(New)

Stability: The long shelf life compared to radiolabeled molecules. Fluoromonoclonal antibodies, oligonucleotide hybridization probes, and PCR primers can be stored for six months or more but antibodies labeled with 125I and 32P-labeled nucleotides and oligonucleotides become unusable in a month and a week respectively. Reagent batches can be standardized and used for extended periods in antigen localization, ELISAs, enzyme assays (such as CAT and kinase), PCR-based genetic typing assays (such as STR analyses), DNA sizing and quantitation, DNA sequencing, protein sizing and quantitation. Low hazard: Most fluorochromes are easy to handle, however, proper care should be observed (eg gloves) with DNA and RNA stains (mutagenic as they bind to these molecules). In contrast, lead or acrylic shields are required for handling radioactive materials and require special disposal protocols (eg shielded storage, long-term decay, or regulated land-fill disposal) Lower cost: The long shelf life and cheaper transportation and disposal costs for fluorochromes make fluorescent labeling, in many cases, less expensive than radiolabeling.

2F. Advantages of Fluorescence (contd)

(New)

3. Real Time PCR Instruments

General Description of Instruments 1. PCR cycler: 1. 96 well format, 8 tube format, capillary (glass) 2. Air or block heater 3. Temeperature ramp, temperature gradient 2. Fluorescence emission & detection : 1. Fluorometer 2. CCD camera 3. Excitation source: xenon, halogen, laser 3. Fluorescent Dye Labeling of: 1. Oligonucleotides 2. Peptide Nucleic acids (PNA) 4. Near Infra Red Dyes: 1. Available but no commercial labeling service available

Xenon Arc lamp (250-1000 continuous) Glass capillaries + Air (not metal block) = rapid

Idaho LightCycler

The Lightcycler performs PCR in small-volume glass capillary tubes, contained within a rotor-like carousel, that are heated and cooled in an airstream. The carousel is rotated past a blue light-emitting diode, and fluorescence is read by three photodetection diodes with different wavelength filters that allow the use of spectrally distinct fluorescent probes. Assays based on DNA-binding dyes, hydrolysis probes, molecular beacons and dual hybridisation probes are possible. Up to 32 reactions are typically carried out in 520 l volumes and PCR is completed in less than 20 min. The fluorescence readings taken at every cycle of the PCR reaction are displayed immediately after each measurement, allowing amplification runs to be terminated or extended, as appropriate, during individual runs.

Dual Light source Excitation: 470 & 530 32 x 0.2 ml plastic tubes Air heated, centrifugal mixing

Rotor-Gene from Corbett Research

Real Time Detection

1a. Excitation filters 1b. Emission filters Tungsten halogen light source Microplate format Cycler
(350 - 1000nm continuous)

iCycler from BioRad

Biorad Instruments have recently launched an optical module that fits their standard thermal cycler and transforms it into a real-time RT-PCR system. This instrument is capable of generating and detecting a wider range of excitation frequencies than either the ABI 7700 or the Lightcycler. At present, it can monitor up to four different fluorescent reporters at any one time and can be used for any one of of the alternative fluorescent RT-PCR strategies. Furthermore, unlike the ABI 7700 which scans its 96 samples sequentially, this instrument can scan up to 96 samples simultaneously, with a sampling frequency that can be defined by the user.

Quartz tungsten halogen lamp (excitation range of 350 to 750nm) 96 well plates Fast cycling (90 min) CCD camera for capturing fluorescence

Mx4000 Multiplex Quantitative PCR System from Stratagene

The ABI Prism 7700 (Perkin-ElmerApplied Biosystems) contains a built-in thermal cycler with 96-well positions, and is able to detect fluorescence between 500 nm and 660 nm. Fluorescence is induced during the PCR by distributing laser light to all 96 samples contained in thin-walled reaction tubes via a multiplexed array of optical fibres. The resulting fluorescent emission returns via the fibres and is directed to a spectrograph with a charge-coupled device (CCD) camera. Because each well is irradiated sequentially, the dimensions of the CCD array can be used for spectral resolution of the fluorescent light. This instrument can be used for assays based on DNA-binding dyes, molecular beacons and hydrolysis probes.

4. Probe types & Design

A. Fluorescent DNA Binding Dyes

B. Oligonucleotide Hybridisation Probes

I. Hydrolysis Probes (TaqMan)

TaqMan Probe
Forward Primer

Probe

This strand is not shown below

Reverse Primer

R
Forward Primer

Forward Primer

Q
Probe

The TaqMan probe binds to ssDNA at a combined annealing and elongation step. It is degraded by the polymerase which releases the reporter dye (R) from the quencher (Q).

New

Designing TaqMan Probes

TaqMan Probe Design: Keep the G-C content in the 3080% range. Avoid runs of an identical nucleotide especially Guanine Do not put Gs on the 5' end. Select the strand that gives the probe more Cs than Gs. For single-probe assays, Tm should be 6870 C Primer Design: Choose the primers after designing the probe. Design the primers as close as possible to the probe without overlapping the probe. Keep the G-C content in the 3080% range. Avoid runs of an identical nucleotide especially Guanine The Tm should be 5860 C. The five nucleotides at the 3' end should have no more than two G and/or C bases.
NOTE: Applied Biosystems provide Primer Express for design of primers and probes in real time with Real Time Quantitative PCR systems (7700, 5700)

B. Oligonucleotide Hybridisation Probes

II. Strand Displacement Probes

A. Roche Dual probe

Microbe using identification16S rRNA genes

Adjacent probes

New

Designing Dual Adjacent Hybridisation Probes

(i) Identify useful Primer / probe regions (use any of the Primer software) (ii) Check primer / probe specificity using Fasta against Genbank database (iii) Check Tm using http://alces.med.umn.edu/rawtm.html (iv) Check propenisity of probe to self anneal using Oligo Selection Program (v) Probe Tms should be near equal and 5-10C greater than primer Tms (vi) The 3 end of the upstream probe should be labeled by fluorescein, which serves as the donor in the FRET and blocks extension from the probe (vii) the 5 end of the downstream probe should be labeled by Cy5, which serves as the acceptor in the FRET, and the 3 end of the probe should be phosphorylated to block extension the probes should be separated by one base (viii) the probes should be placed on one strand near a primer on the opposite strand.

Designing rRNA gene directed fluorroprobes for detection & identification of Campylobacter & Arcobacter by Real Time PCR
Forward PCR primer

FITC probe

Cy5 probe

Reverse PCR primer

2 1

Figure 1: Continuous monitoring of fluorescence during PCR in which DNA templates from A. butzleri ATCC (--), C. jejuni ATCC (-+-) and A. skirrowii ATCC (--) show a significant increase in fluorescence emission whereas templates from E. coli (--), C. upsaliensis (--) and C. hyointestinalis (-o-) show only a marginal increase when compared to no DNA template control (--) which shows no increase. Template DNA was prepared using the rapid boiling method Figure 2. Derivative melting curves (-dF/dT) determined by the dissociation of fluorogenic adjacent hybridisation probes from the target amplicons enables discrimination of A. butzleri ATCC (Tm 68 oC), A. skirrowii (Tm 64 oC), C. jejuni ATCC and C. coli (Tm \ 66 oC) from each other. E. coli, C. upsaliensis, and C. hyointestinalis and template DNA produce no Tm.

Other Virulence Factor genes

Forward PCR primer

Designing virulence gene directed fluorroprobes for detection, identification & differentiation of Campylobacter coli & Campylobacter jejuni from other species by Real Time PCR

Cy5 probe

Reverse PCR primer

Figure-1- Real time detection Real time detection of hippuricase gene for Campylobacter jejuni (--), Campylobacter coli (--), Campyloacter hyointestinalis (-),Campylobacter upsaliansis(--),E. coli (--) and Negative control (-+-) (No template Figure 2: Melting temperature of hippuricase gene for Campylobacter species

Quantitation of gene copy numbers

B. Oligonucleotide Hybridisation Probes

II. Strand Displacement Probes

B. Hair Pin Probes Molecular Beacon Sunrise UniPrimer Scorpion Stem & Loop Duplex FRET Duplex

MOLECULAR BEACONS Molecular Beacons are hairpin structures composed of a nucleotide base paired stem and a target specific nucleotide loop. The loop consists of target specific nucleotide (probe) sequences The stem is formed by annealing of complementary nucleotide bases of the probe sequence. A fluorescent moiety (reporter)is attached to one end of the arm and a non-fluorescent quenching moiety is attached to the other arm. The stem keeps both the moieties in close proximity so that fluorescence is quenched

(Loop)

Stem

5 3

3 5

5 5 3

Q
5

Denaturation

3 5

5 3

Primer molecular beacon annealing

3 5

Extension
5 3

Operation of Molecular Beacon (MB): MB is non-fluorescent due to close proximity of the non-fluorescent quencer (Q) and the fluorescent Reporter. However when the probe denatures and the loop anneals to the target sequence of the amplicon, a conformational reorganization occurs separating the quencher from the fluorophore and thereby producing fluorescence which is proportional to the amplicons produced during PCR

SUNRISE UNIPRIMER PROBE

Similar to Molecular Beacon except that the stem contains a poly A (15 mer) tai. This tail is complimenatry to the polyT tail of one f the primers.

AAAAAAAAAAAAAAA

Q
PolyA Tail

Primer with polyT tail

Sunrise Probe with polyA tail binds to the primer polyT tail at annealing.

Q
hv

Q
AAAAAAAAAAAAAA

The Sunrise probe changes conformation during denaturation & quenching by DABCYL is removed allowing FITC to fluoresce

Sunrise UniPrimer Probe is a modification of Molecular Beacon

The template & probe denature The primer is part of the Scorpion probe

Scorpion stem-loop format

Primer, stopper to prevent read PCR through, probe sequence, fluorophore & quencher (detection system).

The primer is extended

The primer binds to the target

The probe binds to the complimentary sequence of the DNA

Duplex Scorpion Format

Similar to the Ste-loop Scorpion except the probe sequence is part of the stem. There is no loop in this case.

FRET Duplex Scorpions with 3 different versions of the quencher oligonucleotides

In general, design complexty: Dual adjacent > Taqman > Stem Loop Ideally, MBs should hybridise at their annealing temps (fluorescent) & free MBs should be closed (nonfluorescent) Use Oligo4.0 or percent GC rule to calculate that the loop sequence length (usually 15-30 nucleotides) is such that it dissociates from its target at temperatures 7-10 oC higher then the annealing temp of the PCR. Add two complimentary arms on either side of the loop probe sequence. (usually 5-7 nucleotides; 5 GC rich stems melt between 55 & 60, 6 between 60 & 65 and 7 between 65 & 70). In order that it remains closed in the absence of the target, the length & GC content should be 7-10 oC higher then the annealing temp of the PCR. The melting temperature of the stem cannot be predicted by the GC rule as the stems forms by an intramolecular hybridisation event There should be no in between conformational changes, ie should always be the intended hairpin structure an d nothing in between. Commercial program available for making MB probes. ..\..\My Documents\My Pictures\BD100Tour.exe

Designing Stem Loop Molecular Beacon (MB) Probes

New

Probe Detection of the different fluorescent probes during Real Time PCR

, Dual probe

SOME REALTIME-PCR APPLICATIONS

" imultaneous identification, detection and quantitation S of pathogenes in human/food/water/animal samples " ene expression analysis (splicing variants for example) G " ingle Nucleotide Polymorphism (SNP) analysis S " rotein expression analysis P " hromosome aberrations C

Application: Bacterial Pathogen detection isteria monocytogenes L ampylobacter jejuni group C rcobacter group A eptospira group L Application: Bacterial Non-Pathogen detection hermoanaerobacter species T aloramator species C ervidobacterium species F

Advantages of Adjacent Probe Technique with Real Time PCR (Idaho -> Roche):
1. Rapid requiring < 30 mins in a Light Cycler 2. rRNA and / or rRNA genes can be used = flexible 3. Simultaneous detection, identification & quantitation 4. PCR primer design + probe design = extremely specific assays possible 5. Different flurodyes available. Multiplexing possible 6. Population dynamics in an ecosystem can be followed 7. Forms a powerful tool when used in conjunction with rRNA sequencing & FISH

c. Pulse Field Gel Electrophoresis (PFGE)

 agarose gel electrophoresis is a fundamental technique in molecular biology but is generally unable to resolve fragments greater than 20 kilobases in size (whole microbial genomes are usually greater than 1000 kilobases in size) PFGE (pulsed field gel electrophoresis) is a adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments) essential technique for estimating the sizes of whole genomes/chromosomes prior to sequencing and is necessary for preparing large DNA fragments for large insert DNA cloning and analysis of subsequent clones also a commonly used and extremely powerful tool for genotyping and epidemiology studies for pathogenic microorganisms

Pulsed Field Gel Electrophoresis (PFGE)

Principle of PFGE
two factors influence DNA migration rates through conventional gels - charge differences between DNA fragments - molecular sieve effect of DNA pores DNA fragments normally travel through agarose pores as spherical coils, fragments greater than 20 kb in size form extended coils and therefore are not subjected to the molecular sieve effect the charge effect is countered by the proportionally increased friction applied to the molecules and therefore fragments greater than 20 kb do not resolve PFGE works by periodically altering the electric field orientation the large extended coil DNA fragments are forced to change orientation and size dependent separation is reestablished because the time taken for the DNA to reorient is size dependent

Principle of PFGE

Principle of PFGE
the most important factor in PFGE resolution is switching time, longer switching times generally lead to increased size of DNA fragments which can be resolved switching times are optimised for the expected size of the DNA being run on the PFGE gel switch time ramping increases the region of the gel in which DNA separation is linear with respect to size a number of different apparatus have been developed in order to generate this switching in electric fields however most commonly used in modern laboratories are FIGE (Field Inversion Gel Electrophoresis) and CHEF (ContourClamped Homogenous Electrophoresis)

CHEF Electric Field 1 + + + + + + + Switch Time Electric Field 2 +

Preparation of DNA for PFGE

ideally a genomic DNA preparation that contains a high proportion of completely or almost completely intact genome copies would be suitable for PFGE conventional means of DNA preparation are unsuitable for PFGE as mechanical shearing and low-level nuclease activity will result in fragmented DNA with an average size much smaller than an entire microbial genome (usually less than 200 kb in size) the solution to this is to prepare genomic DNA from whole cells in a semisolid matrix (ie. agarose) that eliminates mechanical shearing a very high concentration of EDTA is also used at all times in order to eliminate all nuclease activity

Preparation of DNA for PFGE

1) intact cells are mixed with molten LMT agarose and set in a mold forming agarose plugs 2) enzymes and detergents diffuse into the plugs and lyse cells 3) proteinase K diffuses into plugs and digests proteins 4) if necessary restriction digests are performed in plugs (extensive washing or PMSF treatment is required to remove proteinase K activity) 5) plugs are loaded directly onto PFGE and run

Preparation of DNA for PFGE

for restriction digests, conventional enzymes are unsuitable as they cut frequently on an entire genome sequence producing DNA fragments that are far too small rare cutter restriction endonucleases cut genomic DNA with far less frequency than conventional restriction enzymes such as HindIII, BamHI etc. many rare cutter REs have 6-bp (or longer) recognition sites eg. NotI GCGGCCGC in many cases the frequency of cutting is highly species dependent eg. BamHI will cut far less frequently on a low GC% genome when compared to a intermediate or high GC content genome suitable rare cutter enzymes therefore have to be determined experimentally for each new species being studied

d. New High Throughput Methods

a. DNA MicroArrays

DNA Microarray
a completely annotated microbial genome sequence, whilst a powerful scientific tool, still doesnt provide all of the information needed to understand the complete biology of an organism as it essentially a static picture of the genome for truly complete characterisation, the dynamic nature of gene expression within a microbial cell needs to be determined microarray technology allows whole organism gene expression to be investigated PCR products of every gene from a complete genome sequence are bound in a high density array on a glass slide these arrays are probed with fluorescently labelled cDNA prepared from whole RNA under specific environmental conditions the level of cDNA for each ORF is then quantified using high resolution image scanners

DNA MICROARRAYS TECHNIQUES: The development based on bioinformatics knowledge genes & genomes; High throughput & can analysise complex gene expression profiles There are different formats for DNA high density microarrays:
DNA arrays (Stanford University development 1999): 0.5 5kb c ligonucleotide synthesised (Genechip) arrays (Affymetrix, 1998): O 20-25 base ligomers / PNA, in situ or spotted O An example of how a microarray is made by in situ synthesis approach is shown as a movie. ..\..\My Documents\GeneChip.mov

STEPS IN THE DNA ARRAY TECHNIQUE: 1. Probe Selection - cDNA / oligo with known identify: Small oligos, cDNA, chromosome 2. Chip Fabrication Putting probes on the chip: photolithography, pipette, drop-touch, piezoelectric (inkjet), electric 3. Target fluroscently labeled sample: RNA (mRNA) to cDNA 4. Assay: Hybridisation, ligase, base addition, electric, electrophoresis, fluocytometry, PCRDIRECT, TaqMan 5. Readout: Flurorescence 6. Informatics: Robotic controls, image processing, DBMA, WWW, bioinformatics EXAMPLES: BioMerieux is developing for a water company a 4 h fecal indicator test using Affymetrix technology 2 has 400k oligonucleotide probes, small volumes of sample required limits the usefulness 1cm TaqMan type: Leptospira for WHO regional reference laboratory, Campylobacter & Arcobacter for QHSS, Brisbane.

An example of Microarray hybridisation

a microarray containing 97% of the predicted ORFs from Mycobacterium tuberculosis was used to investigate the response to the antituberculosis drug isoniazid (INH) INH was found to induce several genes related to outer lipid envelope biosynthesis consistent with the drugs physiological mode of action a number of additional genes were also induced which may provide potential drug targets in the future

INH untreated - green INH treated - red Overlay

Yellow = Red + Green (no change in expression) Green (untreated controls) ie expressed without INH treatment Red = expressed as a result of INH treatment

The effect of Isoniazid (INH) on the gene expression of Mycobacterium tuberculosis using DNA microarray technique

The Future of DNA Microarrays

New

1. Studies on the mechanism of toxicity of drugs to humans:

INH is very safe, but like any medicine it can sometimes cause side effects. (yellowish skin, dark urine, vomiting, loss of appetite, nausea, changes in eyesight, unexplained fever, unexplained fatigue & stomach cramps). Human DNA arrays can be used to investigate the mechanism of toxicity on human which may not be possible using animal models.

2. Studies on cyanobacterial toxins extracted from nature blooms:

A human DNA array can be used for testing water which has been contaminated by cyanobacterial blooms before and after treatment. This will provide useful information on exposure levels (time & concentration).

3. The role of uncultured viruses on different human cell lines:

This will provide a rapid method for identifying the most susceptible cell lines that can be used to isolate the offending pathogen.

Cantilever arrays

What are cantilever arrays? Cantilever arrays are produced by microfabrication in silicon using dryand wet-ething techniques. The cantilevers are 500 m long, 100 m wide and about 1 m thick. The spring constant is 20 milliNewton per meter, resulting in a resonance frequency of about 4 kHz. The reproducibility of the resonance frequency from cantilever to cantilever within the array is better than 2%. Owing to their high flexibility, such cantilevers are appropriate for measuring tiny changes in surface stress. For such applications as mass determination, we have designed special cantilevers with a thickness of about 8 m, producing a resonance frequency of around 50 kHz.

Cantilevers are used for imaging in scanning force microscopy but is now being tested as a nanotech sensor. A thin flexible beam made of silicon coated with a sensor layer serves as a chemical sensor. Eight cantilevers aligned in a row form a nanomechanical cantilever sensor array, which can detect small amounts of analytes via very specific reactions. The analyte can also be characterized via its diffusion properties through the coating, e.g. a polymer layer.

Nanomechanical cantilever array sensor If analyte molecules dock on the surface of the cantilevers the surface stress at the interface changes, causing the cantilever to bend.The amount of bending is quantitated.

DNA hybridisation cantilever array The binding of two complementary single stranded oligonucleotides can be observed in a setup of two cantilevers, each functionalized with a different synthetic oligonucleotide (red and blue). If the complementary oligonucleotide (green) is injected, it binds preferably to the red oligomer, but not to the blue one. This hybridization process involves bending of the cantilever due to steric and charging effects. If the complementary sequence to the blue strand is injected, the second cantilever bends.

Thank you for your attention