Sampling and detection of microorganisms in the environment

1. Sampling and detection of microorganisms in the environment Gwy-Am Shin Department of Environmental and Occupational Health Sciences

2. Sampling

3. The challenges • Different microbe types • Different media types • Low numbers of pathogens in the environment

4. Infectious diseases • 1415 human pathogens (2001) • 217 viruses and prions • 538 bacteria and rickettsiae • 307 fungi • 66 protozoans • 287 helminths

5. Transmission of infectious disease • Person-to-person • Direct: person-to-person or animal-to-person • Indirect : droplet, fomites (toys), other vehicles • Environment • Airborne • Waterborne • Foodborne • Vectorborne

6. Low numbers of pathogens in the environment

7. Transmission of enteric pathogens

8. Low number of microbes in the environment • Need large volumes • Need to separate microbes from other materials

9. Steps in pathogen sampling in the environment • Concentration • Purification/Reconcentration • Analysis

10. Concentration in Individual media

11. Sampling microbes in water • Filtration is typically used for concentration • Several formats utilized: • Membrane, pleated capsule, cartridge, hollow fiber • Several types of media • cellulose ester, fiberglass, nylon, polycarbonate, diatomaceous earth, polypropylene, cotton, polysulfone, polyacrylonitrile, polyether sulfone

12. Filters to Recover and Concentrate Microbes from Liquids

13. Sampling microbes in air • Filters • Not recommended due to low sampling efficiency • Impingers • AGI sampler • Biosampler (SKC) sampler • Impactors • Anderson single and multistage sampler • Slit sampler • Rotary arm sampler • Centrifugal samplers • Cyclone sampler • Centrifugal sampler

14. Impingers

15. Impactors (I)

16. Impactors (II)

17. Centrifugal samplers

18. Sampling microbes from surfaces • Swabs • cotton, dacron, calcium alginate, sponge • Swipes/Wipes • cotton, nitrocellulose membranes, polyester bonded cloth, velvet or velveteen • Vacuum Filtration • Hepa bag vac, wet vac • Contact Plates and Paddles (RODAC) • 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

20. Purification/re-concentration

21. Purification/re-concentration • 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

22. Immunomagnetic Separation Y Antibody Bead Y Y Y Microbe

23. Immonomagnetic separation assay

24. Summary (Sampling) • Sampling methods are lagging behind detection methods • Speed isn’t everything • Negative results don’t necessarily mean target not there • There is a need to focus on the reliability and sensitivity of concentration methods • Difficulties with a single platform for any one media because of wide range of organisms and environmental conditions

25. Detection methods

26. Light microscope

27. Electron microscope

28. Sizes of microorganisms

29. Cultural methods (bacteria) Traditional approach • 1st step • pre-enrich and/or enrich using non-selective and then selective broth media • grow colonies on membrane filters • 2nd step • Transfer to differential and selective agars • Recover presumptive positive colonies • Biochemical, metabolic and other physiological testing • Serological or other immunochemical typing • Other characterization: phage typing, nucleic acid analyses, virulence tests

30. Enrichment Cultures • Observe for growth by turbidity, clearing, gas production, color change, etc. • Score as presence-absence (positive or negative) • (sometimes) Quantify using replicate and different volumes to compute a Most Probable Number Left: negative Right: positive (color change)

31. Cultural methods (bacteria) • Plating methods • Spread plating technique • Pour plating technique • Most Probable Number (MPN) technique

33. Different bacterial colonies on general media

35. Cultural methods (viruses and protozoa) • Animal infectivity assays • Mouse • Gerbils • Champagnes • Human • Cell-culture infectivity assays • Primary cell lines • Established cancer cell lines

37. Immunological methods

38. Antigen and antibody reaction

39. Immunological methods • Immunoprecipitation assays • Immunoblotting assays • Enzyme-Linked Immunosorbent assays (ELISA)

41. Nucleic acid-based methods

42. Structure of DNA

43. Nucleic-acid based methods • Gene probing • Southern and northern hybridization • Microarray • Polymerase Chain Reaction (PCR)

44. Gene probe detection

46. Real-Time PCR and Quantitative Fluorogenic Detection • Molecular beacon. Several 5' bases form base pairs with several 3' bases; reporter and quencher in close proximity. • If reporter is excited by light, its emission is absorbed by quencher & no fluorescence is detected. • Detection of PCR product by molecular beacon. • Beacon binds to PCR product and fluoresces when excited by the appropriate light. • [Fluorescence] proportional to [PCR product amplified]