Microbial Indicator Concepts and Purposes

1. 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.

2. 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

3. 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

4. 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

5. 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

6. 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

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

8. 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

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

10. 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

11. Bioaerosol Sampling John Scott Meschke 4225 Roosevelt Way NE, suite 2338 jmeschke@u.washington.edu 206-221-5470

12. Bioaerosols • A collection of aerosolized biological particles (e.g. microbes, by-products of living organisms) capable of eliciting diseases that may be infectious, allergic, or toxigenic with the conditions being acute or chronic • Size range 0.02–100 micrometers (typically 2-10 microns size range of most concern) • Composition of the particles varies with source and environmental conditions • Particles can contain varying amounts of water • Some are colloidal particles of soil, vegetation, other material • Viruses, bacteria and fungi (spores and hyphae) in aerosols due to small size • Many protozoa too large to remain airborne

13. Examples: Agents of Respiratory Infections Viruses: influenza, measles (rubeola), chickenpox (herpes varicella‑zoster) and rhinoviruses (colds); Hantavirus (from a rodent; mouse) Bacteria: Legionella spp., tuberculosis and other mycobacteria (Mycobacterium spp.), anthrax (Bacillus anthracis), and brucellosis (Brucella spp.). Fungi: diseases: histoplasmosis, cryptococcosis, blastomycosis, coccidiodomycosis, and aspergillosis Protozoans: Pneumocystis carinii pneumonia; prevalent in immunodeficient hosts such as AIDS patients. Acanthamoeba encephalitis; primary amebic meningoencephalitis (PAM)

16. Reservoirs and Amplifiers of Airborne Microbes Wide range, overall Depends on the microbe • humans, • animal, • soil • dust • water • air Amplifiers: • Places where microorganisms multiply or proliferate. • Most reservoirs are potential amplifiers.

17. Airborne Microbes and their Reservoirs Viruses: • Mostly humans but some animals • Some rodent viruses are significant: ex: Lassa Fever Virus and Hantavirus. Bacteria: • Humans (TB & staphylococci), • other animals (brucella and anthrax), • water (Legionella) • soil (clostridia). Fungi: • soil and birds (Cryptococcus and Histoplasma) • dead plant material • wet surfaces (wood and other building materials) • indoor air (mycotic air pollution) • stagnant water for the opportunistic fungi (e.g., Aspergillus sp.).

18. Disseminators • Devices causing microbes to enter airborne state or be aerosolized; often the reservoir or amplifier. • Any device able to produce droplets and aerosols: • Humans and other animals: coughs and sneezes, esp. • Mechanical ventilation systems • Nebulizers and vaporizers • Toilets (by flushing) • Showers, whirlpools baths, Jacuzzi, etc. • Wet or moist, colonized surfaces (wet walls and other structures in buildings) • Environments that are dry and from which small particles can become airborne by scouring or other mechanisms: • Vacuuming or walking on carpets and rugs • Excavation of contaminated soil • Demolition of buildings

19. Bioaerosol Samplers • Numerous sampler types • Some adapted from dust or particle samplers • Some designed specifically for microbes • Few specifically for non-microbial bioaerosols (e.g. endotoxin), but generally thought samplers used for microbe collection are adaptable

20. Bioaerosol Samplers • Gravitational samplers (e.g. settle plates) • No special equipment only coated microscope slide, agar plates, etc. • Passive (non-volumetric), relies on collection of particles by gravity settling • Oversamples for larger particles • Poor for collection in turbulent air; affected by turbulent deposition or shadowing

21. Inertial Bioaerosol Samplers • Allow collection of particles by size selective sampling • Includes impactors, sieves, stacked sieves • Relies on particle tendency to deviate from air flow streamlines due to inertia • Particles deposited to solid or semi-solid surface

22. Spore Traps • E.g. Hirst, Burkhard, Air-o-cell, Allergenco • Initially designed for fungal spore and pollen • Sample at 10-20 Liters/minute • Particles impacted on to coated glass slide or adhesive tape • Advantages: non-selective, direct analysis after collection • Disadvantages: may mask problem species, does not assess viability

23. Impactors • Similar to spore trap, but collection on slide or agar plates • Several designs tend to undersample smaller particles; particle bounce can also be an issue • Used at air flows of 10-30 Liters/minute • Types: • Single Stage or Multistage (e.g. Anderson) • Rotary arm samplers (e.g. Rotorod, Mesosystems BT550) • Slit to agar samplers • Sieve Samplers and Stacked Sieves (e.g. SAS)

24. Impactors

26. Impingers • Air drawn through liquid (e.g. water, broth, mineral oil), particles removed by impingement • Allows dilution • Problems with pass through, particle bounce, bubbling, evaporation of liquid loss of viability • Inlet efficiency decreased for particles above 10 microns • Sampling rate 0.1-15 liters/minute (12.5 for AGI 30) • Types: • AGI • Biosampler • Shipe • Multistage

28. Impingers

29. Cyclones or Centrifugal Samplers • Creation of vortex creating sufficient inertia to trigger deposition of particles onto collection surface; recovered in liquid (cyclone) or semisolid medium (centrifugal) • Allows dilution; high air sampling rates (up to 75-1000 LPM for cyclones, 40-100 LPM for centrifugal samplers); small pressure drop • Oversamples larger particles (can be used as trap); poor collection below 5 micron • Can be used in series or paired with other samplers to overcome sampling bias (e.g. Innovatek)

31. 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)

32. Aerosol Samplers

33. Non-Inertial Samplers • E.g. Filtration, Electrostatic Precipitation, thermal precipitators, and Condensation traps • Do not rely on inertia of particles for operation, thus less reliant on particle size (less particle size bias)

34. Filtration • Simple equipment requirements • Adaptable to personal sampling • Less particle size bias (allows large and small particle collection; dependent on inlet size/shape) • Continuous sampling over extended period • Wide variety of sampling rates • However, problems with desiccation leading to reduced viability and difficulties with particle recovery efficiencies

35. Filter Media • Fiborous- mesh of material whose fibers are randomly oriented (creating nominal pore size); depth filter entrainment • Glass fiber (works for proteinaceous bioaerosols) • Membrane- a gel with interconnected pores of uniform size (absolute pore size); depth filter entrainment • Cellulose esters (commonly used for water and other liquids for microbe concentration), PVC, PTFE, nylon, gelatin • Flat disc or etched membranes- defined holes or pores (absolute pore size); surface collection • Silver, aluminum oxide, polycarbonate (most commonly filter media for collection of microbes from air)

36. Filters

37. Electrostatic Precipitators • Particles removed from air stream by electrical rather than inertial forces • Low pressure drop; low power; capable of large volume sampling and high rates • Draws air across high voltage field or corona discharge inducing charge; surface collection • Can be effective for very small particles, as well as larger ones • Problem with ozone production; loss of viability • Examples- • LVAS • LEAP

38. Thermal Precipitation and Condensation Traps • Thermal precipitation • Not commonly used • Based on Thermophoretic motion • Air passed between two plates (one heated and one cooled); particles collected on cooler plate • Condensation trap • Relies on manipulation of relative humidity • Bioaerosol used as condensation nuclei • Particles collected by settling

39. Recovery from Air • Factors that will affect the recovery of microbes from air samples: • Sampling Rate • Environmental Factors may reduce sampling efficiency (e.g. Swirling winds) • Sampling Time • Organism Type and Distribution • Particle Size and Distribution • Target of detection method to be utilized • Sampler Choice • Collection efficiency • Recovery efficiency • Particle Size Bias

40. Recovery from Air • Factors that will affect the recovery of microbes from air samples: • Sampling Rate and Sampling Time (sampled volume) • Concentration factor • Environmental Factors may reduce sampling efficiency (e.g. Swirling winds) • Organism Type and Distribution (need for replication) • Target of detection method to be utilized • Sampler Choice • Collection efficiency (d50) • Retention efficiency • Recovery efficiency • Particle Size Bias • Loss of viability • Sampler Calibration

41. Collection Efficiency: Calm Air

42. Collection Efficiency: Calm Air

43. Collection Efficiency: Calm Air

44. Collection Efficiency: Flowing Air

45. Sample Line Losses • To minimize make as short as possible, minimize angles

46. Separation and Purification

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

48. 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

49. 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

50. Chemical Precipitation Methods • Viruses: precipitate with polyethylene glycol or aluminum hydroxide • resuspend PEG precipitate in aqueous buffer • dissolve aluminum floc in dilute acid solution • both have been used as second-step concentration and purification methods • Parasites: precipitate with calcium carbonate • dissolve precipitate in dilute sulfamic acid