ENV H 452/542: Environmental and Occupational Health Microbiology II:

1. ENV H 452/542:Environmental and Occupational Health Microbiology II: Detection and Control of Environmentally Transmitted Microbial Hazards John Scott Meschke Office: 4225 Roosevelt Way NE, Suite 2338 Phone: 206-221-5470 Email: jmeschke@u.washington.edu Gwy-Am Shin Office: 4225 Roosevelt Way NE, Suite 2339 Phone: 206-543-9026 Email: gwyam@u.washington.edu

2. Course Description • This course will review environmental detection and control of pathogenic organisms. • The first half of the course will cover methods of sample collection, processing and target detection. • The second half of the course will examine methods of decontamination and disinfection, as well as other engineered controls of environmentally transmitted pathogens.

3. Course Objectives Students will gain a working knowledge of: • the methods used for sample collection, processing and target detection of environmentally transmitted pathogens, • methods of disinfection and decontamination of environmental media, and • practices and engineered controls for the containment of pathogens and prevention of their spread.

4. Text and References • Recommended Text: Environmental Microbiology(Maier, Pepper, and Gerba; Academic Press) • Readings will typically be assigned for each class session (20-25 pages; from text or will be distributed in class or on website) Reference Texts and Journals

5. Course Format • Lectures (typically) • 5 Minutes Questions • 30-40 Minutes Lecture Material • 5-10 Minutes Follow-up Questions • Student-Led Discussions • Division into 3 groups (based on CVs) • One group leads per session • Choose article (or series of articles) and come-up with list of discussion questions • Due 1 week before session

6. Grading Opportunities • On decimal scale: • A(3.9-4.0) Excellent work; typically >>90% of available points • D (0.9-1.1) very poor work; typically <66% of available points • Expected Graduate Average 3.7 or above • Expected Undergraduate Average 3.4 or above

7. Graduate Students CV (5%) Homework (10%) Midterm (25%) Class Participation (5%) Oral Exam (15%) Review Paper (15%) Final Exam (25%) Undergraduates CV (5%) Homework (20%) Midterm (30%) Class Participation (5%) Oral Exam (10%) Final Exam (30%) Grading Opportunities

8. CV • Each student will be required to provide a 1-2 page CV describing the student’s background and interests. • CVs will be due by the third class period.

9. Homework • Two (each worth 5 or 10% of overall grade for graduate and undergraduate students, respectively) • Chance to earn points back (1/2 points lost) • Must indicate why answer given is wrong (NOT why you missed it, i.e. I was thinking about something else) and Must indicate correct answer • Due one week after initially returned • Late homeworks may be penalized (10% for each class period late)

10. Midterm • Format: Short answer, Multiple choice, true/false explain, matching, fill-in the blank • Historically considered to be LONG • Chance to earn back points like on homework • Early or make-up exams offered only for emergency or prior arrangement • Format up to instructors

11. Participation • Contributing to classroom discussions • Asking questions that further instruction • Email response to instructor-posed questions • Peer review of papers

12. Review Paper • Graduate students ONLY; undergrads will assist with peer review • Topic relevant to course material • Work in teams of two • As long as necessary, but not to exceed 25 pages of double-spaced text; use ASM formatting • Follow posted deadlines

13. Oral Exam • Students will be required to make an appointment with the instructor on March 4th, 6th, or 9th. • Exams will consist of a relaxed discussion (~15 minutes) during which instructor will evaluate students grasp of course content. • Schedule by late February

14. Final Exam • March 18th, 2:30-4:20 • Similar format to midterm, may be problem solving questions • Open note, open book • NO opportunity to earn points back

15. Course Rules • Come to class, please try to let me know ahead of time if you can not make it. • Arrive on time • Turn in assignments on time • Come to class prepared (keep up with reading) • Be courteous (No newspapers, audible cell phones, PDAs, beepers) • Food and drinks are welcome (but keep it quiet) • Refrain from unnecessary talking • ASK QUESTIONS • Try to remain awake (at least no snoring please) • Let me know how I am doing (if I am moving too fast, not being clear, or otherwise not getting the message across, I need to know.)

16. Use of Indicator Microorganisms

17. Microbial Indicator Concepts and Purposes • The types of pathogens that can contaminate water, food, air and other environmental media are diverse and there are many different ones. • Measuring all of these pathogens on a routine basis for determining presence or absence or acceptable concentration is not possible. • Methods are not available to recover and measure some of them, • Methods are available for other pathogens, but they are technically demanding, some are slow to produce results and their costs are high. • The alternative is to measure something other than a pathogen that is indicative of contamination, predicts pathogen presence and estimates human health risks.

18. What is Measured as Microbial Indicators and Why? • Microbial indicators have been used for more than 100 years (since late 1800s) to detect and quantify fecal contamination in water, food and other samples • Concerns were for bacteria causing water- and foodborne illness, such as: • Salmonella typhi: the cause of typhoid or enteric fever • Vibrio cholerae: the cause of cholera • Shigella dysenteriae and other Shigella species: dysentery • Focus was and still is on detecting primarily human (or maybe animal) fecal contamination as the source of these and other enteric bacterial pathogens • Detect fecal contamination by measuring: • common enteric bacteria residing in the gut and shed fecally • Chemicals associated with the gut or with anthropogenic fecal contamination • Something else associated with and predictive of fecal contamination

19. What is Measured as Microbial Indicators and Why? • Microbial indicators also are used to indicate other conditions unrelated to fecal contamination, such as : • Food spoilage bacteria and molds • Excessive microbial growth in water • Causing appearance, taste and odor problems: • “red water” from iron biofouling • Blooms of algae and cyanobacteria (blue-green algae) • Some of the organisms harbor or release toxins (“red tides”) • Bacterial release from biological filters used in water treatment

20. What is Measured as Microbial Indicators and Why? • Airborne contamination: • From wet buildings: molds and actinomycetes • From industrial processes: • bacterial endotoxins from cotton dust, solid waste and other sources • Microbial allergens from manufacturing processes (aerosols and dusts) • total airborne microbe concentrations • In health care facilities • In “clean room” manufacturing environments for electronics and pharmaceuticals • From composting operations • Salivary bacteria from dentistry activities

21. Pathogen Detection and Monitoring • Pathogen detection • technically demanding, • often tedious, • slow to produce results, • Often unreliable • expensive. • Done routinely in the health care field (clinical diagnostic microbiology): • often essential to patient treatment and care. • provides national surveillance of infectious disease epidemiology

22. Pathogen Analysis, Monitoring and Surveillance • Until recently, rarely done for managing food quality • Salmonella and E. coli O157:H7 are now monitored in meat and poultry; Listeria monocytogenes monitoring also being done • Rarely done for monitoring or managing water quality • pathogen occurrence surveys and special studies: • survey (18 months) for Giardia, Cryptosporidium and enteric viruses in larger drinking water supplies using surface water sources: ICR (Information Collection Regulation) • survey for enteric viruses in ground water sources of drinking water (data base for Ground Water Disinfection Rule) • investigation of waterborne outbreaks and pilot/in-plant studies • Pathogen monitoring sometimes done for biosolids (Class A) • Salmonella, viable Ascaris ova, culturable enteric viruses

23. Microbial Indicators of Fecal Contamination • Traditional approach to protect/assess the "sanitary" quality of water (food) with respect to fecal contamination. • Quantify bacteria commonly present in intestines of warm blooded animals • high numbers • easy to measure • surrogates for pathogens • Developed when bacterial pathogens were recognized in late 1800s and early 1900s • Salmonella, Shigella, V. chloerae, etc.

24. Criteria for an Ideal Indicator of Fecal Contamination Applicable to all types of water (and other relevant samples). Present in feces, sewage and fecally contaminated samples when pathogens are present; numbers correlate with amount of fecal contamination; outnumber pathogens. No "aftergrowth" or "regrowth" in the environment. Survive/persist > than or = to pathogens. Easily detected/quantified by simple lab tests in a short time. Constant characteristics. Harmless to humans and other animals. Numbers in water (food, etc.) are associated with risks of enteric illness in consumers (dose-response relationship). Adapted from: Bonde, G.J. (1963) Bacterial Indicators of Water Pollution. Teknisk Forlag, Copenhagen; Bonde, G. J. (1966) Bacteriological Methods for Estimation of Water Pollution. Health Lab. Sci., 3: 124.

25. Microbial Indicators: No Ideal One • Bacteria are not always reliable indicators of all pathogens • Viruses and protozoa differ in size, response to environmental stressors and to treatment processes • No single indicator fulfills the criteria of an ideal fecal indicator • There is no ideal indicator, really • No single indicator is going to be suitable for all classes of pathogens • No single indicator will reliably predict pathogen health risks in all media and under all conditions

26. Introduction to Sampling of Environmental Media

27. Sampling Considerations What we want: • Fast • Sensitive • Specific • Easy to Perform • Reliable (Accurate/Precise) • Compatible with Downstream Detection What do we have???

28. The Challenge of Environmental Sampling for Pathogens • Variation in microbe type and distribution • Low microbe numbers: need to concentrate them • Non-random distribution and physical state of microbes of interest: aggregated, particle-associated, embedded, etc. • Volume considerations • Environmental factors may inhibit or interfere with downstream detection • Separate them from interfering and excess other material

29. Detection of Pathogens in The Environment • Three main steps: • (1) recovery and concentration, • (2) purification and separation, and • (3) assay and characterization.

30. Pathogen Detection Techniques Targets: • ATP • Nucleic Acid • PCR methods • Microarray methods (fluorometric, electrochemical) • Protein/Lipid • Immunological methods • Mass Spectrometry methods • Whole Organism • Microscopy • Culture

31. Viruses Rotaviruses Noroviruses Adenoviruses Enteroviruses Poxviruses

32. Bacteria Bacillus Francisella Escherichia Yersinia Vibrio Salmonella Shigella

33. Protozoans Cryptosporidium Giardia and Cryptosporidium Entamoeba Microsporidia Cyclospora

34. Viruses: smallest (0.02-0.3 µm diameter); simplest: nucleic acid + protein coat (+ lipoprotein envelope) Bacteria: 0.5-2.0 µm diameter; prokaryotes; cellular; simple internal organization Protozoa: most >2 µm- 2 mm; eucaryotic; uni-cellular; flexible cell membrane; no cell wall; wide range of sizes and shapes; hardy cysts

35. Water Concentration • Distribution of pathogens in water necessitates sampling of large volumes of water (1-1000s of liters) • Filtration is typically used for concentration • Several formats utilized: • Membrane filter, pleated capsule, cartridge, hollowfiber • Several types of media • cellulose ester, fiberglass, nylon, polycarbonate, diatomaceous earth, polypropylene, cotton, polysulfone, polyacrylonitrile, polyether sulfone

36. Filters to Recover and Concentrate Microbes from Liquids

37. Types of Filtration • Size Exclusion/Retention • Adsorption/Elution

38. Surface Sampling • Current Methods (5-90% recoveries, generally poorly characterized) • Swabs (better for gram negatives?) • Cotton • Dacron • Calcium Alginate (may inhibit PCR and be toxic to cell culture) • Sponge (Polyurethane and Cellulose) • Swipes/Wipes • Cotton • Nitrocellulose membranes • Polyester bonded cloth • Velvet or Velveteen • Vacuum Filtration • Hepa bag vac • Wet Vac • Rinse/Elute • Contact Plates and Paddles (RODAC) (better for gram positives?) • New Methods • Adhesive Strips and Paddles • Scraping/Aspiration Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999; Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003

40. Aerosol Sampling • Impactor • Anderson single and multistage sampler • Slit sampler • Rotary arm sampler • Impinger • AGI sampler • Biosampler (SKC) sampler • Filters • IOM/Button filter sampler • Foam plug filter sampler • Centrifugal • Cyclone sampler • Centrifugal sampler • Precipitators • Electrostatic precipitator • Condensation trap • Hybrid

41. Impingers

42. Impactors

43. Filters

44. Large Volume Aerosol Samplers • Biocapture BT 550 (Mesosystems) • Rotary arm impactor, liquid collection • 150L/min (~15 min) • Bioguardian (Innovatek) • Wet-walled multi cyclone, w/centrifugal impactor for removal of large particles • 100-1000L/min (1 min-12 hours) • Spincon (Sceptor) • Centrifugal wet concentrator, w/cyclonic preseparation • 450L/min (5 min-6 hours)

45. Aerosol Samplers

46. Separation and Purification Methods • Purification, separation and concentration of target microbes in primary sample or sample concentrate • Separate target microbes from other particles and from solutes • Reduce sample size (further concentrate)

47. Separation/Purification Methods • Variety of physical, chemical and immunochemical methods: • Sedimentation and flotation (primarily parasites) • Precipitation (viruses) • Filtration (all classes) • Immunomagnetic separation or IMS (all classes) • Flow cytometry (bacteria and parasites); an analysis, too

48. Secondary Concentration and Purification • PEG (polyethylene glycol) • Organic Flocculation • IMS (Immunomagnetic separation) • Ligand capture • BEaDs (Biodetection Enabling Device) • Capillary Electrophoresis • Microfluidics • Nucleic Acid Extraction • Spin Column Chromatography • Floatation • Sedimentation • Enrichment