Transcript Concentration and Purification of Airborne Microbes and Biotoxins

Microbial Indicator Concepts and Purposes
• The types of pathogens that can contaminate water, food,
air and other environmental media are diverse and there
are many different ones.
• Measuring all of these pathogens on a routine basis for
determining presence or absence or acceptable
concentration is not possible.
– Methods are not available to recover and measure some
of them,
– Methods are available for other pathogens, but they are
technically demanding, some are slow to produce results
and their costs are high.
• The alternative is to measure something other than a
pathogen that is indicative of contamination, predicts
pathogen presence and estimates human health risks.
What is Measured as Microbial Indicators and Why?
• Microbial indicators have been used for more than 100 years (since
late 1800s) to detect and quantify fecal contamination in water,
food and other samples
– Concerns were for bacteria causing water- and foodborne illness,
such as:
• Salmonella typhi: the cause of typhoid or enteric fever
• Vibrio cholerae: the cause of cholera
• Shigella dysenteriae and other Shigella species: dysentery
• Focus was and still is on detecting primarily human (or maybe
animal) fecal contamination as the source of these and other enteric
bacterial pathogens
• Detect fecal contamination by measuring:
– common enteric bacteria residing in the gut and shed fecally
– Chemicals associated with the gut or with anthropogenic fecal
contamination
– Something else associated with and predictive of fecal contamination
What is Measured as Microbial Indicators and Why?
• Microbial indicators also are used to indicate other conditions
unrelated to fecal contamination, such as :
– Food spoilage bacteria and molds
– Excessive microbial growth in water
• Causing appearance, taste and odor problems:
– “red water” from iron biofouling
– Blooms of algae and cyanobacteria (blue-green
algae)
» Some of the organisms harbor or release toxins
(“red tides”)
• Bacterial release from biological filters used in water
treatment
What is Measured as Microbial Indicators and Why?
• Airborne contamination:
– From wet buildings: molds and actinomycetes
– From industrial processes:
• bacterial endotoxins from cotton dust, solid waste and other
sources
• Microbial allergens from manufacturing processes (aerosols
and dusts)
– total airborne microbe concentrations
• In health care facilities
• In “clean room” manufacturing environments for electronics
and pharmaceuticals
• From composting operations
– Salivary bacteria from dentistry activities
Pathogen Detection and Monitoring
• Pathogen detection
– technically demanding,
– often tedious,
– slow to produce results,
– Often unreliable
– expensive.
• Done routinely in the health care field (clinical diagnostic
microbiology):
– often essential to patient treatment and care.
– provides national surveillance of infectious disease
epidemiology
Pathogen Analysis, Monitoring and Surveillance
• Until recently, rarely done for managing food quality
– Salmonella and E. coli O157:H7 are now monitored in meat and
poultry; Listeria monocytogenes monitoring also being done
• Rarely done for monitoring or managing water quality
– pathogen occurrence surveys and special studies:
• survey (18 months) for Giardia, Cryptosporidium and enteric
viruses in larger drinking water supplies using surface water
sources: ICR (Information Collection Regulation)
• survey for enteric viruses in ground water sources of drinking
water (data base for Ground Water Disinfection Rule)
– investigation of waterborne outbreaks and pilot/in-plant studies
– Pathogen monitoring sometimes done for biosolids (Class A)
• Salmonella, viable Ascaris ova, culturable enteric viruses
Sampling Considerations
What we want:
• Fast
• Sensitive
• Specific
• Easy to Perform
• Reliable (Accurate/Precise)
• Compatible with Downstream Detection
What do we have???
The Challenge of Environmental Sampling for Pathogens
• Variation in microbe type and distribution
• Low microbe numbers: need to concentrate them
• Non-random distribution and physical state of microbes
of interest: aggregated, particle-associated, embedded,
etc.
• Volume considerations
• Environmental factors may inhibit or interfere with
downstream detection
• Separate them from interfering and excess other
material
Detection of Pathogens in The
Environment
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Three main steps:
(1) recovery and concentration,
(2) purification and separation, and
(3) assay and characterization.
Aerosol Sampling
• Impactor
– Anderson single and multistage sampler
– Slit sampler
– Rotary arm sampler
• Impinger
– AGI sampler
– Biosampler (SKC) sampler
• Filters
– IOM/Button filter sampler
– Foam plug filter sampler
• Centrifugal
– Cyclone sampler
– Centrifugal sampler
• Precipitators
– Electrostatic precipitator
– Condensation trap
• Hybrid
Bioaerosol Sampling
John Scott Meschke
4225 Roosevelt Way NE, suite 2338
[email protected]
206-221-5470
Bioaerosols
• A collection of aerosolized biological particles (e.g.
microbes, by-products of living organisms) capable
of eliciting diseases that may be infectious, allergic,
or toxigenic with the conditions being acute or
chronic
• Size range 0.02–100 micrometers (typically 2-10
microns size range of most concern)
• Composition of the particles varies with source and
environmental conditions
• Particles can contain varying amounts of water
• Some are colloidal particles of soil, vegetation, other
material
• Viruses, bacteria and fungi (spores and hyphae) in
aerosols due to small size
• Many protozoa too large to remain airborne
Examples: Agents of Respiratory Infections
Viruses: influenza, measles (rubeola), chickenpox (herpes
varicella-zoster) and rhinoviruses (colds); Hantavirus
(from a rodent; mouse)
Bacteria: Legionella spp., tuberculosis and other
mycobacteria (Mycobacterium spp.), anthrax (Bacillus
anthracis), and brucellosis (Brucella spp.).
Fungi: diseases: histoplasmosis, cryptococcosis,
blastomycosis, coccidiodomycosis, and aspergillosis
Protozoans: Pneumocystis carinii pneumonia; prevalent in
immunodeficient hosts such as AIDS patients.
Acanthamoeba encephalitis; primary amebic
meningoencephalitis (PAM)
Reservoirs and Amplifiers of Airborne Microbes
Wide range, overall
Depends on the microbe
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humans,
animal,
soil
dust
water
air
Amplifiers:
• Places where microorganisms multiply or proliferate.
• Most reservoirs are potential amplifiers.
Airborne Microbes and their Reservoirs
Viruses:
• Mostly humans but some animals
• Some rodent viruses are significant: ex: Lassa Fever Virus and
Hantavirus.
Bacteria:
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Humans (TB & staphylococci),
other animals (brucella and anthrax),
water (Legionella)
soil (clostridia).
Fungi:
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soil and birds (Cryptococcus and Histoplasma)
dead plant material
wet surfaces (wood and other building materials)
indoor air (mycotic air pollution)
stagnant water for the opportunistic fungi (e.g., Aspergillus sp.).
Disseminators
• Devices causing microbes to enter airborne state or be
aerosolized; often the reservoir or amplifier.
• Any device able to produce droplets and aerosols:
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Humans and other animals: coughs and sneezes, esp.
Mechanical ventilation systems
Nebulizers and vaporizers
Toilets (by flushing)
Showers, whirlpools baths, Jacuzzi, etc.
Wet or moist, colonized surfaces (wet walls and other
structures in buildings)
– Environments that are dry and from which small particles can
become airborne by scouring or other mechanisms:
• Vacuuming or walking on carpets and rugs
• Excavation of contaminated soil
• Demolition of buildings
Bioaerosol Samplers
• Numerous sampler types
• Some adapted from dust or particle
samplers
• Some designed specifically for microbes
• Few specifically for non-microbial
bioaerosols (e.g. endotoxin), but generally
thought samplers used for microbe
collection are adaptable
Bioaerosol Samplers
• Gravitational samplers (e.g. settle plates)
– No special equipment only coated microscope
slide, agar plates, etc.
– Passive (non-volumetric), relies on collection
of particles by gravity settling
– Oversamples for larger particles
– Poor for collection in turbulent air; affected by
turbulent deposition or shadowing
Inertial Bioaerosol Samplers
• Allow collection of particles by size
selective sampling
• Includes impactors, sieves, stacked sieves
• Relies on particle tendency to deviate from
air flow streamlines due to inertia
• Particles deposited to solid or semi-solid
surface
Spore Traps
• E.g. Hirst, Burkhard, Air-o-cell,
Allergenco
• Initially designed for fungal spore
and pollen
• Sample at 10-20 Liters/minute
• Particles impacted on to coated
glass slide or adhesive tape
• Advantages: non-selective, direct
analysis after collection
• Disadvantages: may mask problem
species, does not assess viability
Impactors
• Similar to spore trap, but collection on slide or
agar plates
• Several designs tend to undersample smaller
particles; particle bounce can also be an issue
• Used at air flows of 10-30 Liters/minute
• Types:
– Single Stage or Multistage (e.g. Anderson)
– Rotary arm samplers (e.g. Rotorod, Mesosystems
BT550)
– Slit to agar samplers
– Sieve Samplers and Stacked Sieves (e.g. SAS)
Impactors
Impingers
• Air drawn through liquid (e.g. water, broth, mineral oil),
particles removed by impingement
• Allows dilution
• Problems with pass through, particle bounce, bubbling,
evaporation of liquid loss of viability
• Inlet efficiency decreased for particles above 10 microns
• Sampling rate 0.1-15 liters/minute (12.5 for AGI 30)
• Types:
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AGI
Biosampler
Shipe
Multistage
Impingers
Cyclones or Centrifugal Samplers
• Creation of vortex creating sufficient inertia to
trigger deposition of particles onto collection
surface; recovered in liquid (cyclone) or
semisolid medium (centrifugal)
• Allows dilution; high air sampling rates (up to 751000 LPM for cyclones, 40-100 LPM for
centrifugal samplers); small pressure drop
• Oversamples larger particles (can be used as
trap); poor collection below 5 micron
• Can be used in series or paired with other
samplers to overcome sampling bias (e.g.
Innovatek)
Large Volume Aerosol Samplers
• Biocapture BT 550 (Mesosystems)
– Rotary arm impactor, liquid collection
– 150L/min (~15 min)
• Bioguardian (Innovatek)
– Wet-walled multi cyclone, w/centrifugal impactor for
removal of large particles
– 100-1000L/min (1 min-12 hours)
• Spincon (Sceptor)
– Centrifugal wet concentrator, w/cyclonic
preseparation
– 450L/min (5 min-6 hours)
Aerosol Samplers
Non-Inertial Samplers
• E.g. Filtration, Electrostatic Precipitation,
thermal precipitators, and Condensation
traps
• Do not rely on inertia of particles for
operation, thus less reliant on particle size
(less particle size bias)
Filtration
• Simple equipment requirements
• Adaptable to personal sampling
• Less particle size bias (allows large and small
particle collection; dependent on inlet
size/shape)
• Continuous sampling over extended period
• Wide variety of sampling rates
• However, problems with desiccation leading to
reduced viability and difficulties with particle
recovery efficiencies
Filter Media
• Fiborous- mesh of material whose fibers are randomly
oriented (creating nominal pore size); depth filter
entrainment
– Glass fiber (works for proteinaceous bioaerosols)
• Membrane- a gel with interconnected pores of uniform
size (absolute pore size); depth filter entrainment
– Cellulose esters (commonly used for water and other liquids for
microbe concentration), PVC, PTFE, nylon, gelatin
• Flat disc or etched membranes- defined holes or pores
(absolute pore size); surface collection
– Silver, aluminum oxide, polycarbonate (most commonly filter
media for collection of microbes from air)
Filters
Electrostatic Precipitators
• Particles removed from air stream by electrical rather
than inertial forces
• Low pressure drop; low power; capable of large volume
sampling and high rates
• Draws air across high voltage field or corona discharge
inducing charge; surface collection
• Can be effective for very small particles, as well as larger
ones
• Problem with ozone production; loss of viability
• Examples– LVAS
– LEAP
Thermal Precipitation and
Condensation Traps
• Thermal precipitation
– Not commonly used
– Based on Thermophoretic motion
– Air passed between two plates (one heated and one
cooled); particles collected on cooler plate
• Condensation trap
– Relies on manipulation of relative humidity
– Bioaerosol used as condensation nuclei
– Particles collected by settling
Recovery from Air
• Factors that will affect the recovery of microbes
from air samples:
– Sampling Rate
– Environmental Factors may reduce sampling
efficiency (e.g. Swirling winds)
– Sampling Time
– Organism Type and Distribution
– Particle Size and Distribution
– Target of detection method to be utilized
– Sampler Choice
• Collection efficiency
• Recovery efficiency
• Particle Size Bias
Recovery from Air
• Factors that will affect the recovery of microbes
from air samples:
– Sampling Rate and Sampling Time (sampled volume)
– Concentration factor
– Environmental Factors may reduce sampling
efficiency (e.g. Swirling winds)
– Organism Type and Distribution (need for replication)
– Target of detection method to be utilized
– Sampler Choice
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Collection efficiency (d50)
Retention efficiency
Recovery efficiency
Particle Size Bias
Loss of viability
– Sampler Calibration
Collection Efficiency: Calm Air
Collection Efficiency: Calm Air
Collection Efficiency: Calm Air
Collection Efficiency: Flowing Air
Sample Line Losses
• To minimize make as short as possible,
minimize angles
Separation and Purification
Separation and Purification Methods
• Purification, separation and secondary
concentration of target microbes in primary
sample or sample concentrate
– Separate target microbes from other particles
and from solutes
– Reduce sample size (further concentrate)
Separation/Purification Methods
• Variety of physical, chemical and
immunochemical methods:
– Sedimentation and flotation (primarily
parasites)
– Precipitation (viruses)
– Filtration (all classes)
– Immunomagnetic separation or IMS (all
classes)
– Flow cytometry (bacteria and parasites);
an analysis, too
Secondary Concentration and
Purification
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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
Chemical Precipitation Methods
• Viruses: precipitate with polyethylene glycol
or aluminum hydroxide
– resuspend PEG precipitate in aqueous buffer
– dissolve aluminum floc in dilute acid solution
– both have been used as second-step
concentration and purification methods
• Parasites: precipitate with calcium
carbonate
– dissolve precipitate in dilute sulfamic acid
Other Recovery and Concentration Methods
• Minerals, such as iron oxide and talc; used to
adsorb viruses
• Synthetic resins: ion exchange and
adsorbent
• Other granular media: glass beads and sand
Less widely used; less reliable, cumbersome;
uncertain elution, desorption, exchange
efficiencies
Initial Recovery and Concentration of
Pathogens
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Flotation centrifugation
– Layer or suspend samples or microbes in
medium of density greater than microbe
density; centrifuge; microbes float to surface;
recover them from top layer
Isopycnic or buoyant density gradient
centrifugation
– Layer or suspend samples or microbes in a
medium with varying density with depth but
having a density = to the microbe at one
depth.
– Microbes migrate to the depth having their
density (isopycnic)
Flotation: microbe
– Recover them from this specific layer
density < medium
density
Isopycnic density
gradient: microbe
density = medium
density at one
depth
Immunomagnetic Separation
Antibody
Y
Bead
Microbe
Virus Capture Plus RT-PCR to Detect Infectious
Viruses - The sCAR System
• The cell receptor gene for Coxsackieviruses and
Adenoviruses has been cloned and expressed,
producing a soluble protein receptor, sCAR
• Expressed, purified and bound sCAR to solid
phases to capture infectious Coxsackieviruses
from environmental samples
– The nucleic acid of the sCAR-captured viruses is RT-PCR
amplified for detection and quantitation
Application of sCAR with Para-Magnetic Beads for
Virus Particle Capture and then RT-PCR
sCAR
purification
Covalent coupling
to paramagnetic beads
Culture + media;
:sCAR produced
Blocking
post-coupling
: sCAR
: Virus Particle
: Blocking protein
Sample
containing
viruses
(RT-) PCR
NA
extraction
Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)
Pre-coated to provide available amine groups for covalent coupling
of proteins or other ligands by glutaraldehyde-mediated coupling method