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 ConsiderationsWhat 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

• 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 Meschke4225 Roosevelt Way NE, suite 2338jmeschke@u.washington.edu206-221-5470

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, overallDepends on the microbe

– humans,– animal,– soil– dust– water– air

Amplifiers:• Places where microorganisms multiply or proliferate.• Most reservoirs are potential amplifiers.

Airborne Microbes and their ReservoirsViruses:• Mostly humans but some animals• Some rodent viruses are significant: ex: Lassa Fever Virus and

Hantavirus.Bacteria: • Humans (TB & staphylococci), • other animals (brucella and anthrax), • water (Legionella)• soil (clostridia).Fungi: • 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:

– 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:

– 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 75-1000 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

• 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

• 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

• 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)

– Recover them from this specific layer Flotation: microbe density < medium density

Isopycnic density gradient: microbe density = medium density at one depth

Immunomagnetic Separation

Y

Y

Y

Y

Bead

Antibody

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

: Virus Particle

: sCAR

Culture + media; :sCAR produced

(RT-) PCR

sCARpurification

: Blocking protein

Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)Pre-coated to provide available amine groups for covalent couplingof proteins or other ligands by glutaraldehyde-mediated coupling method

Covalent coupling to paramagnetic beads

Blocking post-coupling

Sample containing viruses

NA extraction