Microbial Survival in the Environment: with Special Attention to Enteric and

Respiratory Pathogens

Mark D. Sobsey

ENVR 133

Microbe Transmission Routes

• Direct Contact (Person to Person)• Indirect Contact

– Vector Transmission • Biological • Mechanical

– Vehicle Transmission• Respiratory transmission (by droplets or

aerosols)• Fecal-oral transmission (by ingestion of air-

borne contaminants, and contaminated foods or water

• Fomite transmission and self-inoculation after contact with fomitic surfaces

For microbes transmitted by respiratory and fecal-oral routes, transport and persistence in the environment is related to risk of host exposure, infection, and disease

Some Physical Factors Influencing Microbe Survival in the Environment

TEMPERATURE

• Greater Inactivation/death rates at higher temperatures• Lower survival rates at higher temperatures

– But, some microbes will grow or grow better at higher temperatures• Many microbes survive better at lower temperature

– Some bacteria experience “cold injury” or“cold shock” and cold inactivation

• Thermal inactivation differs between dry heat and moist heat– Dry heat is much less efficient than moist heat in inactivating microbes

• Some microbes survive very long times when frozen– Other microbes are destroyed by freezing

• Ice crystals impale them• Increased environmental temperatures can promotes pathogen spread

by insect vectors (mosquitoes, flies, etc.)

pH• Relative acidity or alkalinity• A measure of hydrogen ion (H+)

concentration• Scale:

– 1 (most acidic) to 14 (most alkaline or basic)

– pH 7 is neutral – Moving toward pH 1 the substance is

more acidic– Moving toward pH 14, the substance is

more alkaline.• Extreme pH inactivates microbes

– Chemically alters macromolecules– Disrupts enzyme and transport functions– Some enteric pathogens survive pH 3.0

(tolerate stomach acidity)– Some pathogens survive pH 11 and fewer

survive pH 12

Microbes are most stable in the environment and will grow in media (e.g., foods) in the mid pH range

Moisture Content or Water Activity• Drying or low moisture inactivates/kills some microbes

– Removing water content of some foods can preserve them– Most viruses rapidly inactivated in soil at <1% moisture; sme at a few%

• Moisture content of foods is measured as water activity, Aw.– Aw: ratio of the water vapor pressure of the substrate to the pressure of

pure water at the same temperature. – Vapor pressures is hard to calculate, so an alternative method is used to

measure Aw in food science:– Aw = moles of water ÷ (moles of water + moles of solute)– Pure water has a water activity of 1.00.– If 1 mole of a solute is added, then the solution has an Aw of 0.98. – Aw is measured on a scale of 0.00 to 1.00. – Most fresh foods have a water activity of 0.99. – Most spoilage microbes do not survive if an Aw below 0.91.

• some yeasts and molds that can survive at water activity of 0.61.

Physical Factors Influencing Survival, Continued• Ultraviolet radiation: about 330 to 200 nm

– Primary effects nucleic acids; absorbs the UV energy and is damaged• Sunlight:

– Ultraviolet radiation in sunlight inactivates microbes– Visible light is antimicrobial to some microbes

• Promotes growth of photosynthetic microbes• Ionizing radiation

– X-rays, gamma rays, beta-rays, alpha rays– Generally antimicrobial; bacterial spores relatively resistant– Main target of activity is nucleic acid– Effect is proportional to the size of the “target”

• Bigger targets easier to inactivate; a generalization; exceptions– Environmental activity of ionizing radiation in the biosphere is not highly

antimicrobial– Ionizing radiation is used in food preservation and sterilization

Atmospheric and Hydrostatic Pressure

• Most microbes survive typical atmospheric pressure• Some pathogens in the deep ocean are adapted to high

pressure levels (hydrostatic pressures): barophiles– Survive less well at low atmospheric pressures– Spores and (oo)cysts survive pressure extremes

• High hydrostatic pressure is being developed as a process to inactivate microbes in certain foods, such as shellfish– Several 100s of MPa of pressure for several minutes

inactivates viruses and bacteria in a time- and pressure-dependent manner

Role of Solids-Association in Microbial Survival • Microbes can be on or in other, usually larger particles

or they can be aggregated (clumped together)• Association of microbes with solids or particles and

microbial aggregation is generally protective• Microbes are shielded from environmental agents by

association with solids– Protection depends on type of solids-association– See diagrams, right

• Protection varies with particle composition– Organic particles: often highly protective

• Biofilms protect microbes in them• React with/consume antimicrobial chemicals

– Inorganic particles vary in protection• Opaque particles protect from UV/visible light• Inorganic particles do not always protect well

against chemical agents– Some inorganic particles are antimicrobial

• Silver, copper, other heavy metals/their oxides

Dispersed: least protected

Embedded: most protected

Adsorbed: partially protected

Clumped: interior microbes protected

: Antimicrobial agent

Some Chemical Factors Influencing Microbe Survival in the Environment

Effects

Chemicals and Nutrients Influence Microbial Survival• Antimicrobial chemicals

– Strong oxidants and acids– Strong bases– Ammonia: antimicrobial at higher pH (>8.0)– Sulfur dioxide and sulfites: used as food preservatives– Nitrates and nitrites: used as food preservatives– Enzymes:

• Proteases• Nucleases• Amylases (degrade carbohydrates)

– Ionic strength/dissolved solids/salts• High (or low) ionic strength can be anti-microbial

– Many microbes survive less in seawater than in freshwater– High salt (NaCl) and sugars are used to preserve foods

» Has a drying effect; cells shrink and die– Heavy metals:

• Mercury, lead, silver, cadmium, etc. are antimicrobial• Nutrients

– for growth and proliferation– Carbon, nitrogen, sulfur and other essential nutrients

Some Biological Factors Influencing Microbe Survival in the Environment

Effects

Biological Factors Influence Microbial Survival

• Chemical antagonistic activity by other microorganisms:– Proteolytic enzymes/proteases– Nucleases– Amylases– Antibiotics/antimicrobials: many produced naturally by

microbes– Oxidants/oxides– Fatty acids and esters; organic acids (acetic, lactic, etc.)

• Predation• Vectors• Reservoir animals

Factors Affecting Survival in Liquid

• Temperature

• Ionic Strength

• Chemical Constituents/Composition of Medium

• Microbial Antagonism

• Sorption Status

• Type of Microbe

Factors Affecting Survival in Aerosols

• Temperature• Relative Humidity• Moisture Content of Aerosol Particle• Composition of Suspending Medium• Sunlight Exposure• Air Quality (esp. “open air” factor)• Size of Aerosol Particle• Type of Microbe

Factors Affecting Survival on Surfaces

• Type of Microbe• Type of Surface• Relative Humidity• Moisture Content (Water Activity)• Temperature• Composition of Suspending Medium• Light Exposure• Presence of Antiviral Chemical or

Biological Agents

Effect of Virus Type on Survival

• Differences between virus families– Clear differences between enveloped and non-

enveloped viruses (Mahl, 1975)• Differences between virus genera

– Differences between Enteroviruses and Rhinoviruses• Differences between virus strains

– Survival of Influenza and Pseudorabies virus has been shown to be strain dependent (Platt, 1979; Mitchell, 1972)

• Differences associated with passage in different host cells– Ex. Yellow fever virus inactivation sensitivity at

intermediate RH increases with passage in HeLa cells (Hearn, 1965)

Microbe Survival in Liquid Media

• Temperature– Increased inactivation with increasing temperature– Most are inactivated rapidly (minutes) above 50oC– Some microbes are more thermotolerant than others

(e.g. Hepatitis A virus, bacterial/fungal spores, some helminth ova (ascarids)

• Most are inactivation more at higher temperatures• Chemical composition of media influences survival

– Protein/other organics & Mg&Ca ions protect– Generally very stable at ultra-cold temperatures,

• Some loss of infectivity occurs with freezing and thawing

Survival in Liquid Media

• pH– Direct effects on conformation of proteins and

other biomolecules– Indirect effects on adsorption and elution from

particles– pH range of stability is microbe-dependent

• Polio: 3.8 to 8.5 for maximum stability• Salt Content

– Variable effects on microbe survival– Affects microbe physiology (isotonic conditions),

adsorption and stability of biomolecules– Divalent cations (Mg2+) can increase thermo-

stability of viruses and bacteria• E,g., MHV, enteroviruses, HAV

Microbe Survival in Liquid Media

• Microbial Antagonism– Microflora influences microbe survival

• Metabolites: enzymes, VFAs, NH3 are antiviral

– Use of pathogen as a nutrient source

– Greater microbe survival documented in sterilized or pasteurized matrices, as compared to non-sterile matrices

– Phenomena demonstrated in sewage, fresh, estuarine, and marine waters, soils and sediments.

Microbe Survival in Liquid Media

• Adsorption– Several possible mechanisms:

• Ionic attractions and repulsions • covalent reactions (with active chemicals) • hydrogen bonding • hydrophobic interactions • double layer interactions• van der Waal’s forces

– Adsorption status greatly influences survival• adsorbed microbes generally survive longer than

unadsorbed microbes• Protection and accumulation in sediments and

soils

Colloid Particles and their Surface Electrical Potentials

Colloidal Particles and their Charge Properties

• Colloids: small charged, suspended particles– Most microbes are colloids

• Particle surface is charged– strongly bound layer of opposite charged

counterions; the Stern layer– Positive ions are attracted by a negative

colloid and vice-versa• Stern layer: layer of actual particle and its

immediately bound counter ions.• Beyond Stern layer: diffuse layer of ions that

move with the particle when it is in motion• Zeta potential : potential at the shear plane;

the layer of bound ions moving with the particle

Aqueous Liquids on Hydrophilic and Hydrophobic Surfaces

• Water is polar and hydrophilic

• Droplets spread out on hydrophilic (polar) surfaces

• Droplets form a round bead on hydrophobic (non-polar) surfaces

• Contact angle: angle describing the interaction of a water droplet with a surface

• Influence microbe survival on surfaces

MicrobeSurvival in Liquid Media

• Organic Matter

– In liquid media, organic matter increases microbe survival

• Increased oxidant demand protects from oxidation

• If an enzyme substrate, protects from enzymatic attack

• Can coat to protect microbe particles

– In soils, organic matter has variable effects on microbes

• Possible competition for adsorption sites

• May coat or protect microbe particles

• Bacteria may grow of organics are nutrients

Microbe Survival in Liquid Media

• Antimicrobial Chemicals– Ionic and non-ionic detergents, particularly for

enveloped viruses and some bacteria– Ammonia is virucidal; ammonium ion is not– Germicides (chlorine, ozone, etc.)

• Light– Direct microbicidal activity below wavelengths of

370 nm– Indirect antimicrobial activity:

• stimulation of microflora growth• triggering formation of reactive oxidants• activation of photoreactive chemicals

Microbe (Virus) Survival in Aerosols

• Relative Humidity and Moisture Content– Viruses with lipid survive better at lower relative

humidity– Viruses with little or no lipid content survive better at

higher relative humidity– Viral inactivation or retention of infectivity may be a

function of stabilization (drying of aerosol) and of rehumidification of aerosol particle upon collection

– Effect of relative humidity on virus survival may be influenced by temperature effects

Microbe Survival in Aerosols• Temperature

– Survival decreases with increased temperature

• Suspending Media– composition influences microbe stability– effect is microbe dependent

• Salts stabilize some viruses (e.g. Poliovirus)• Removal of salts stabilize other viruses (e.g. Langat, Semiliki

Forest virus)• Proteinacious material and organic matter may have similar

mixed effects, depending on microbe type• Polyhydroxy compounds stabilize some virus types (e.g.

Influenza) but have no effect on other viruses

Microbes Survival in Aerosols

• Oxygen and Air Ions– Oxygen has little direct effect on most viruses but may

influence bacteria– But, oxygenation may be synergistic with higher

temperature and sunlight to inactivate microbes– The “Open Air Factor” has been shown to have

virucidal activity• Poorly characterized chemical agents in open air

that reduce virus survival compared to clean laboratory air

• May be reaction products of ozone and olefins

Microbe Survival in Aerosols

• Light– Virucidal activity of UV light is a greater in air

than in liquid media– Photosensitivity is virus type-dependent and

may be related to the envelope• Non-enveloped viruses (Poliovirus, Adenoviruses

and FMDV) are more resistant to UV light than enveloped viruses (vaccinia, herpes simplex, influenza, and Newcastle disease virus) (Jenson, 1964; Donaldson 1975; Applyard, 1967)

Microbe Survival in Aerosols

• Aerosol Particle Size– Airborne microbes may be more rapidly

inactivated in smaller aerosol particles than larger ones (some studies)

– Other studies observed no effect of particle size on virus survival

• Aerosol Collection Method– Abrupt rehydration of virus particles and other

microbes upon collection may lead to their inactivation– Prehumidification may improve recovery of infectious

virus– Effect is virus type-dependent

Microbe Survival on Surfaces

• Adsorption State– Air Water Interface– Triple Phase Boundary

• Physical State– Dispersed– Aggregation– Solids associated

Microbe Survival on Surfaces

• Relative humidity– Similar effects as seen in aerosols; effects are

microbe type dependent

• Moisture Content– In soils moisture content directly related to

microbe survival• Dessication• Enhanced predation

Microbe Survival on Surfaces

• Temperature– Effects as observed in liquid media and

aerosols– Interaction between relative humidity and

temperature pronounced on surfaces for certain virus types (e.g. Polio, Herpes Simplex), less important for others (e.g. Vaccinia) (Edward, 1941)

Microbe Survival on Surfaces

• Suspending Media– Effects similar to effects on survival in aerosols

• Presence of fecal material• Presence of salts

• Type of Surface– Little effect by non-porous surfaces on most viruses

• important for some virus types (Herpes simplex)– Effects more pronounce for porous surfaces (e.g.

fabrics: cotton, synthetics and wool• Light

– Effects similar to those in aerosols and liquids

Microbe type: Resistance to chemical disinfectants:• Vegetative bacteria: Salmonella, coliforms, etc.:

low• Enteric viruses: coliphages, HAV, Noroviruses:

moderate• Bacterial Spores• Fungal Spores• Protozoan (oo)cysts, spores, helminth ova, etc.

– Cryptosporidium parvum oocysts– Giardia lamblia cysts

– Ascaris lumbricoides ova– Acid-fast bacteria: Mycobacterium spp.

Least

Most

High

Virus Survival in Drinking Water

• Non-Enveloped Viruses– Poliovirus survives in sterile water for nearly 300 days

(Schwartzbrod, 1975)– Astroviruses survive up to 90 days in dechlorinated

drinking water (Abad, 1997)

• Enveloped Viruses– Herpes Simplex Virus survives in distilled water for up

to 1 day and in tap water for up to 4 hours (Nerurkar, 1983)

– Psuedorabies virus survives in chlorinated tap water for less than one day (Schoenbaum, 1990)

Virus Survival in Fresh Water

• Non-Enveloped Viruses– Coxsackie virus survives up to 30 days in

stream water with a 2 log10 reduction

– From epidemiologic evidence Noroviruses survive at least 4 months in fresh water (Kukkula, 1999)

Virus Survival in Soil/Groundwater

• Non-Enveloped Viruses– groundwater velocity models: survival in groundwater

calculated to be up to 200 days (Vaughn, 1983)– Echovirus survival in sandy soil: 170 days in seeded

studies (Bagdasaryan, 1964)– Seeded studies: Poliovirus, Norwalk, and MS2

detected in soil suspensions up to 70 weeks by RT-PCR; Infectious Polio was still detected in Groundwater after 70 weeks (Meschke, 2001)

• Enveloped Viruses– Pseudorabies virus persists in well water up to 7 days

Virus Survival in Sea/Brackish Water

• Non-Enveloped Viruses– Coxsackie A9 virus survives in marine waters for up

to 30 days with ≤2 log10 reduction (Nasser, 2003)• Similar survival of HAV, FCV, and Polio (Callahan,

1995; Kadoi, 2001; and Wait, 2001)• Enveloped Viruses

– Bovine Diarrhea Virus survives in various water types 6 to 24 days (Pagnini, 1984)

– Viral Hemorrhagic Septicemia virus persisted up to 40 hours in filtered Seawater with 50% reduction; survival up to 36 days in cell culture medium-amended seawater

Virus Survival in Fecal Waste

• Non-Enveloped Viruses– In animal wastes, rotavirus survival to > 6 months, 1 log10

reduction (Pesaro, 1995)– FMDV persist up to 100 days in a fecal slurry (Bartley, 2002)– Poliovirus persists up to 180 days in sand saturated with

septic liquor (Yeager, 1979)• Enveloped Viruses

– In animal wastes, herpes virus reduced 1 log10 in less than 1 week (Pesaro, 1995)

– Pseudo rabies virus survives up to 2 weeks in swine urine; <2 days in lagoon and pit effluent (Schoenbaum, 1990)

– Swine Fever Virus survives up to 15 days in manure (Have, 1984)

Virus Survival in Aerosols

• Non-Enveloped Viruses– Rotavirus SA11 survives up to 223 hours, depending

on humidity (Sattar, 1984)– Rhinovirus persists up to 24 hours with <1 log10

reduction• Enveloped Viruses

– Vaccinia, VEE, and Influenza virus survive in aerosols for up to 23 hours (Harper, 1961)

– New Castle Disease Virus persists 4-16 hours (Hugh-Jones, 1973)

– Human Coronavirus (229E) persists at 50% humidity with up to 20% infectious at 6 days (Ijaz, 1985)

Virus Survival on Surfaces• Non-Enveloped Viruses

– Poliovirus survives up to 20 weeks on wool blanket fabric (Dixon, 1966)

– HAV recovered from stainless steel surfaces after 96 hours; and from plastic surfaces after 1 month (Mbithi, 1991)

– Rotavirus persists for up to 10 days (Sattar, 1986)• Enveloped Viruses

– Influenza persists for several weeks on dust, cotton sheets, and glass slides (Edward, 1941)

– RSV was reduced by 2 log10 after 24 hours (Kingston, 1968)– Parainfluenza virus persists up to 12 days on plastic

surfaces (Parkinson, 1983)– Human Coronavirus persists up to 6 hours with 1-2 log10

reduction