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

1
Microbial Survival in the Environment with
Special Attention to Enteric and Respiratory
Pathogens

• Mark D. Sobsey
• ENVR 133

2
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

3
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
4
Some Physical Factors Influencing Microbe
Survival in the Environment
5
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 orcold
  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.)

6
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
7
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 lt1
  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.

8
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

9
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

10
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

Clumped interior microbes protected
Adsorbed partially protected
Embedded most protected
Dispersed least protected
Antimicrobial agent
11
Some Chemical Factors Influencing Microbe
Survival in the Environment
Effects
12
Chemicals and Nutrients Influence Microbial
Survival

• Antimicrobial chemicals
• Strong oxidants and acids
• Strong bases
• Ammonia antimicrobial at higher pH (gt8.0)
• 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

13
Some Biological Factors Influencing Microbe
Survival in the Environment
Effects
14
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

15
Factors Affecting Survival in Liquid

• Temperature
• Ionic Strength
• Chemical Constituents/Composition of Medium
• Microbial Antagonism
• Sorption Status
• Type of Microbe

16
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

17
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

18
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)

19
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 MgCa ions protect
• Generally very stable at ultra-cold temperatures,
  
• Some loss of infectivity occurs with freezing and
  thawing

20
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

21
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.

22
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 Waals forces
• Adsorption status greatly influences survival
• adsorbed microbes generally survive longer than
  unadsorbed microbes
• Protection and accumulation in sediments and soils

23
Colloid Particles and their Surface Electrical
Potentials
24
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

25
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

26
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

27
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

28
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

29
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

30
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

31
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)

32
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

33
Microbe Survival on Surfaces

• Adsorption State
• Air Water Interface
• Triple Phase Boundary
• Physical State
• Dispersed
• Aggregation
• Solids associated

34
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

35
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)

36
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

37

• 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.

High
38
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)

39
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)

40
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

41
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

42
Virus Survival in Fecal Waste

• Non-Enveloped Viruses
• In animal wastes, rotavirus survival to gt 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 lt2 days in lagoon and pit effluent
  (Schoenbaum, 1990)
• Swine Fever Virus survives up to 15 days in
  manure (Have, 1984)

43
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 lt1 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)

44
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