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