Transcript Gwy-Am Shin
Sampling and detection of microorganisms in the environment Gwy-Am Shin Department of Environmental and Occupational Health Sciences Sampling The challenges • Different microbe types • Different media types • Low numbers of pathogens in the environment Infectious diseases • 1415 human pathogens (2001) – 217 viruses and prions – 538 bacteria and rickettsiae – 307 fungi – 66 protozoans – 287 helminths 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 Low numbers of pathogens in the environment Transmission of enteric pathogens Low number of microbes in the environment • Need large volumes • Need to separate microbes from other materials Steps in pathogen sampling in the environment • Concentration • Purification/Reconcentration • Analysis Concentration in Individual media 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 Filters to Recover and Concentrate Microbes from Liquids 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 Impingers Impactors (I) Impactors (II) Centrifugal samplers 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 Purification/re-concentration 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 Immunomagnetic Separation Antibody Y Bead Microbe Immonomagnetic separation assay 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 Detection methods Light microscope Electron microscope Sizes of microorganisms 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 Enrichment Cultures • Observe for growth by turbidity, clearing, gas production, color change, etc. • Score as presenceabsence (positive or negative) • (sometimes) Quantify using replicate and different volumes to compute a Most Probable Number Left: negative Right: positive (color change) Cultural methods (bacteria) • Plating methods – Spread plating technique – Pour plating technique • Most Probable Number (MPN) technique Different bacterial colonies on general media Cultural methods (viruses and protozoa) • Animal infectivity assays – Mouse – Gerbils – Champagnes – Human • Cell-culture infectivity assays – Primary cell lines – Established cancer cell lines Immunological methods Antigen and antibody reaction Immunological methods • Immunoprecipitation assays • Immunoblotting assays • Enzyme-Linked Immunosorbent assays (ELISA) Nucleic acid-based methods Structure of DNA Nucleic-acid based methods • Gene probing – Southern and northern hybridization – Microarray • Polymerase Chain Reaction (PCR) Gene probe detection 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]