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

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 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 Y Y Y Y Bead Antibody Microbe

Immonomagnetic separation assay

Summary (Sampling) Sampling methods are lagging behind detection methods Difficulties with a single platform for any one media because of wide range of organisms and environmental conditions 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

Detection methods

Light microscope

Electron microscope

Sizes of microorganisms

Cultural methods (bacteria) Traditional approach 1 st step –pre-enrich and/or enrich using non-selective and then selective broth media –grow colonies on membrane filters 2 nd 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 presence- absence (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

Nucleic acid-based methods

Antigen and antibody reaction

Structure of DNA

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

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]