Waterborne Pathogens and State-of-art Detection Methods Dr Bharat Patel, Associate Professor in Molecular Microbiology &

1. Waterborne Pathogens and State-of-art Detection Methods Dr Bharat Patel, Associate Professor in Molecular Microbiology & Director, Clinical Microbiology PG Program, School of Biomolecular & Biomedical Sciences, Griffith University, Brisbane Australia

2. Section I. Indicators of Water Pollution

3. CONTENT • The Australian Cooperative Research Centres (CRC) • 1.1 Concept • 1.2 The FiveWater Related CRC • 1.3 CRC Water Quality & Treatment • 2. Introduction • 2.1 Microbes on our planet & their role • 2.2 Water as an environment • 2.3 Microbes & their role in water • 2.4 Why monitor water supplies? • 2.5 Ensuring the safety of drinking water. • 3. Bacterial Indicators of Pollution • 3.1 What are Bacterial indicators of pollution • 3.2 Total coliforms • 3.3 Changes in coliform definitions • 4. Alternatives to Total Coliforms

4. Section II. Risk Assessment Analysis Framework and Pathogens

5. CONTENT 1.Epidemiological data on some pathogens. 2.The current list of pathogens 3.How to monitor and assess the risk of pathogens?

6. SECTION III. Molecular Biology Databases and Tools

7. CONTENT Molecular Biology Bioinformatics Databases Online tools

8. SECTION IV. The Biology, Methods for Detection, Identification & Quantitation of Water-borne Pathogens

9. 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

10. Section I. Indicators of Water Pollution

11. 1. The Australian Cooperative Research Centres (CRC)

12. CRC Water Quality & Treatment CRC Wastewater Treatment CRC Freshwater ecology CRC Microelectronic engineering 1.1 The Concept of Cooperative Research Centres (CRC) in Australia • Participation by industry, universities, CSIRO and State Government bodies • $1 (in cash / in kind contributions) : $1 (cash from Fedral Government • Commercial focus • Skill acquisition and training for the industry. • Usually run by an independent board • 65 CRCs currently on the books ($320 million pa from participants to $140 million pa cash from Fedral Government). • CRCs may have synergies:

13. CRC for Coastal Zone, Estuaries and Waterway Management CRC for Waste Management and Pollution Control CRC for Freshwater Ecology CRC for Water Quality And treatment CRC for Catchment Hydrology 1.2 The Five Water Related CRCs

14. 1.3 The Cooperative Research Centre for Water Quality & Treatment • 2nd Round of Funding: 2001-2008 • Participants: 12 Research organisations (8 Universities), 16 Industry & 8 Associate partners • Key Objectives: • create a centre of excellence with the capability of pursuing world class research and training. • ensure that participants with their differing disciplines and backgrounds will interact effectively to optimise research outcomes. • increase the human skills base of the water supply industry and to train new post graduate students with specialist water quality skills. • commercialise Project Intellectual Property in such a manner as to ensure that the maximum benefit accrues to the Australian water industry, the Australian environment and the Australian economy generally

15. 2. INTRODUCTION

16. 2.1 Microbes on our planet & their roles 60% of the organisms are microbial (more microbes than human cells) Surive & thrive in virtually in all environments, often where no other “higher forms” of life exist. 1% have been characterised (24 kingdoms) & 99% remain uncharacterised (the tree of life has been generated using rRNA as chronometers) Efficient colonisers (rapid growth & doubling) Provide a service to the planet: Ecosystem servicing (biogeochemical cycle, flux) Biotechnology (vitamins, amino acids) Also produce harm: Directly as pathogens Indirectly producing byproducts (toxins) Simple morphology provides very little clues to their identities

17. NEW Water Microbiology as it Relates to Public Health Human reservoir Animal reservoirs Wastewater Land surface Groundwater Surface water Aerosols Domestic use Domestic use Crops Shellfish Recreation Aerosol 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.

18. 2.2 Water as a Changable Heterogeneous Environment • 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

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20. 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

21. stratification Danger Donot enter Farming activities Recreation pump Forestry activities C, N, S, O fluxes & transformations Filtration & treatment Lead pipe Copper pipe Distribution system INTERACTIVITY & INTERDEPENDENCY Ecology, Environmental & Public Health Microbiology Groups Regulatory Group Transparency Group PVC pipe Biofilm development ?

22. 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

23. 2.5 Ensuring the safety of drinking water (management) • Primary assessment: Correct operation of water supply system • Verification: Proof that water is safe after supply. This includes monitoring for compliance. • Risk assessment: Maximum Acceptable Concentration (MAC). Should be zero but rarely technically & economically feasible. Compliance parameters • Compliance & risk assessment may be different for countries, states and applications. • Improved awarness: Flexible, transparent, achievable & realistic outcomes

24. 2.6 Ensuring the safety by monitoring & detection • Direct measurement of harmful agents • Microbes: Not usually undertaken. Difficult, expensive, time consuming & lack of technology. Risk -> Acute & short-lived • Chemicals: Usually undertaken. Technology exists. Risk -> Chronic exposure & delay between sampling, testing & acting on results is okay • Monitoring water quality barriers (catchment activities, filtration, disinfection) • Complete risk management system for health. Gaining popularity. • Currently used indicators of water quality • Inadequate, but will be used until “new” & “better” methods tried, tested & ratified. • Does not take into account emerging risks (microbes, chemicals). New risks, new ways.

25. 3. Bacterial Indicators of Water Pollution

26. 3.1 What are bacterial indicators of pollution? • Direct pathogen identification / isolation is impractical and / or impossible • Alternate indirect “indicator organism” based inference is necessary: • universally present in large nos. in warm blooded animal faeces • readily detectable by simple methods • do not grow in natural waters • persistence in water treatment regimes is similar to that for pathogens

27. 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 • 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

28. Table Bacteriological drinking water & recreational freshwater standards or guidelines Enterococci (recreational) 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 Units C > 90% are E.coli dCompulsory limits, bathing is restricted if >20% samples over 14 day period are positive e If 5 samples taken over 30 days are positive

29. 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) • 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.

30. 3.3 Recent changes in coliform definition Coliforms: Members of the family Enterobactericeae; produce acid & gas from lactose (24-48 h @ 36±2oC) Thermotolerant (fecal) coliforms: As above but were able to grow & ferment lactose at 44.5±0.2oC and include E. coli < Klebsiella, Enterobacter & Citrobacter (E. coli also produce indole from tryptophan). SEE “TESTS FOR DIFFERENTIATING COLIFORMS” SLIDE Report 71, 1994 Bacteriological Examination of Drinking water supplies: biochemical definition changed to “acid-only production from lactose” & therefore increased the numbers of species in the coliform category Enzymes: Lactose fermentation by the presence of -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.

31. Table showing coliform members by evolving definition Coliforms that can be present in the environment & in human faeces (bold ) and coliforms that are only environmental (bold & underlined)

32. Commercial kits based detection methods for microbial indicators • Kit Manufacturers: • IDEXX: Enterolert, Colisure, Colilert • Hach: m-ColiBlue • BioControl: ColiComplete • Chromocult: Merck • Gelman: MicroSure

33. Coliforms E. coli E. aerogenes K. pneumoniae assay for all ferment Lactose Enzyme at If growth at named 35 oC Elevated temperature -glucoronidase uses of detected with Enzyme 44.5 oC MUG named -galactosidase designate as designate as E. coli Fecal coliforms detected with ONPG designate as Total coliforms Tests for differentiating coliforms

34. 4. Alternatives to Coliforms as indicators of water pollution

35. 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

36. Section II. Risk Assessment Analysis Framework and Emerging Pathogens

37. 1.Epidemiology of some waterborne pathogens. 2.The current list of pathogens 3.How to monitor and assess the risk of pathogens?

38. 1. Epidemiology of some pathogens.

39. Information modified 1. 90% water related illness are microbial 2. Canada (1974 – 1987): 32 waterborne outbreaks - Giardia:10, Norwalk & HAV: 5, 17 unknown origin. 2000: E. coli O157, 2001 Cryptosporidium. 3. USA (1993 – 1998): Cryptosporidium (Milwaukee, Las Vegas, Nevada) 2001: Microcystin & cylindrospermopsin found in Florida drinking water plant (5 times WHO guideline) 4. Europe (1980 –1990): Cryptosporidium (UK) 6. Vibrio cholera surveillance in India: 34 k (33 deaths) Flood related since July 2001 5. E. coli 0157 ->feces contaminated soil, to irrigation water, to food (Both E. coli 0157:H7 and VT6 gene strains isolated) Swaziland: 1992 (20k), Missouri: 1989, UK: several outbreaks reported, Wyoming: 1998, NY: 1999 (1k involved, 2 deaths, Campylobacter also implicated), Canada: 2001 (2K involved, 7 deaths- heavy rainfall & inadequate treatment) 6. Northern Ireland: 2001 Cryptosporidium 7. Portugal: 2001 Cyanobacteria toxins reported

40. 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.

41. 2. Common Waterborne Pathogens

42. Waterborne Pathogens: • are classified as members of domains Bacteria, Eucarya or virus. • they differ in: • morphologies • growth • physiology & metabolism • fine genetic details • Both classification & Identification is now increasingly based on their • molecular events & molecular details (see next figure). • The pathogens listed in the following tables have been detected in • water and / or in outbreaks. An attempt has been made to provide • their classification on the newly introduced molecular trend. • The biology of a number of the pathogens will be described and the possible targets sites for their identification highlighted.

43. EUKARYA (7)ARCHAEA (3)BACTERIA (21) Trichomonads Crenarchaeota Euryarchaeota Pyrodicitum Slime molds Fungi Red algae Green algae Ciliates Methanocococcus Dinoflagellates Plants Dictyoglomus Desulfurococcus Thermomicrobia Halophiles Animals Thermotoga Chrysiogenetes Thermodesulfobacteria Thermococcus Brown algae Proteobacteria Aquifex Deferribacter Thermales Nitrospira Flagellates Cyanobacteria Methanopyrus Firmicutes  Verrucomicrobia     Acidobacteria Microsporidia Korarchaeota Fibrobacter Planctomycetes Diplomonads Actinobacteria Chlamydia Evolution of Universal Ancestor (3.5 billion yrs) Fusiforms Spirochetes Bacteroides The Tree of Life - 16th November 2000

44. 2. A list of bacterial waterborne pathogens Proteobacteria Duration of disease is between 1 to 42 days

45. Information modified Problems associated with bacterial identification • Phylum Cyanobacteria (blue green algae): • Some 50 to 60 genera; some produce oligopeptide toxins& are of increasing concern (dermal, cytotoxin, mutantion causing and carcinogens). Lifelong exposure vs short term acute exposure • Toxins are produced by (a) nonribosomal peptide synthetase (NRPS) which have iterative catalytic domains. Overproduction of one or several sets up a catalytic reaction leading to production of the toxins. (b) Peptide kinase synthetase (PKS). • MALDI-TOF MS shows a large spectrum of oligopeptides & other poorly undertood metabolities from cyanobacteria. • Microcystis exist as toxigenic organism in reservoirs & form blooms (summer to late autumn) but reports of non-toxicogenic strains have been reported. • Some 60 toxins (collectively called Microcystin) are produced; these are thought to react with chlorine to produce other toxin bye-products • They have been traditionally classified on the basis of morphology & physiology which has created confusion. Based on 16S rRNA and DNA homology studies, the 23 species have now been identified as belonging to M. aeruginosa • Toxin production in strains vary based on growth conditions (in vivo and in situ) causing more confusion.

46. 10% Nostoc punctiforme PCC 73102. "Anabaena cylindrica" str. NIES19 PCC 7122. Pseudoanabaena biceps PCC 7367. Lyngbya confervoides PCC 7419. "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 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. 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. 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.

47. 2. A list of protozoal waterborne pathogens

48. 2. A List of viral waterborne pathogens

49. 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

50. 3. How to prioritise the list of pathogens for further studies? By using risk assessment analysis frame work