Sampling and detection of microorganisms in the environment

Gwy-Am ShinDepartment 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• 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 presence-absence (positive or negative)

• (sometimes) Quantify using replicate and different volumes to compute a Most Probable Number

Left: negativeRight: 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]