BEHAVIOR OF PATHOGENS IN THE

ENVIRONMENTM.S. Coyne, University of Kentucky

USDA-CSREES National Water Conference

February 5-9, 2006 San Antonio TX

The social, environmental, and scientific landscape for evaluating the risk of

pathogens and protecting against pathogens is changing.

Categories of Drivers, by Rank, Associated With Emerging and Re-emerging Human Pathogens

(Woolhouse and Gowtage-Sequeria. 2005)

*Rank1. Change in land use or agricultural practice2. Change in human demographics and society3. Poor population health (e.g. HIV, malnutrition)4. Hospitals and medical procedures5. Pathogen evolution (e.g. microbial drug resistance, increased

virulence)6. Contamination of food sources or water supplies7. International travel8. Failure of public health programs9. International trade10. Climate change

• Ranked by number of pathogen species associated with them. Fungi are associated with hospitalization and poor population health. Bacteria are associated with evolution, and contamination of food and water. Viruses are associated with international travel and change in land use and agriculture. Changing land use and agriculture are also associated with zoonoses.

Setting the Stage

• Significance of Pathogens in the Environment• Factors Affecting Pathogen Survival and

Transport• Behavior of Indicators in the Environment• Evidence for Pathogen Survival and Transport

in Soil and Water• Emerging Pathogen Issues

Significance of Pathogens in the Environment

• ~ 75 million cases of water- and food-borne diseases are reported annually (Jenkins and Bowman, 2004)

• Globally > 2 million persons die from drinking unsafe water (Kosek et al., 2003)

• Pathogens implicated in these diseases include:

Viruses (e.g. Avian Influenza, Hepatitis, Norwalk, Polio)Bacteria (e.g., Campylobacter, Clostridium, Salmonella)Fungi (e.g. Chytrids, Histoplasma, Aspergillus)Protozoa (e.g. Cryptosporidium, Cyclospora, Giardia)Helminths (e.g. Ascaria, Clonorchis)

Escherichia coli

Photograph courtesy of USDA-ARS

Cryptosporidium On the Intestinal Tract

(Source: Gardiner et al., 1988, An Atlas of Protozoan Parasites in Animal Tissues, USDA Agriculture Handbook No. 651.)

Ooocysts

Two Life Stages of Giardia

Cyst

TrophozoiteOriginal image by Arturo Gonzalez, CINVESTAV, Mexico (Source: Javier Ambrosio, UNAM, Mexico).

What Are Pathogens Sources? (Direct and Indirect)

Agricultural Runoff and Infiltration

CAFOs Septic SystemsSewage Lines Straight PipesUrban Runoff WildlifeBiosolids

Factors Affecting Pathogen Survival and Transport

• Extrinsic1. Chemical Environment – pH, aeration,

moisture, ionic strength2. Physical Environment – porosity, texture, temperature,

mineralogical properties3. Biological Environment – competition, predators, growth

rate4. Driving Forces – air and water

• Intrinsic1. Size2. Surface Chemistry: surface charge and hydrophobicity3. Motility4. Stage in life cycle5. Potential for biofilm formation

Adhesion is Complex and Multi-dimensional

• Primary Docking Stage

1. Chance meeting of conditioned surface andplanktonic viruses, bacteria, fungi, and protists

2. Reversible3. Influenced by: electrostatic and hydrophobic

interaction, temperature, hydrodynamic forces• Secondary Locking Phase

1. Binding between adhesions and surfaces2. Irreversible

a. Glycocalyx formation (bacteria)b. Significant inactivation (viruses)

3. Biofilm or microcolony formation commences4. Bacteria tend to become more hydrophobic and

stick better as growth rate increases

E. coli O157:H7

• Expresses multiple fimbrial and nonfimbrialadhesions with no role in pathogenicity

• Produces Cah, a surface-expressed protein causing autoaggregation and OmpA (outer membrane protein A), which facilitates binding

• Low surface hydrophobicity masked by extracellular polymers present in capsule

Classic Approach Has Not Been to Seek Specific Pathogens

• Why?Low DensityInfrequent OccurrenceCost of analysis

• Water quality monitoring is based on fecal indicator bacteria

Coliforms and Fecal ColiformsEnterococci

Useful Features of Coliforms and Fecal Coliforms

• Bacteria – very small (1-2 µm), prokaryotic, single-celled, proxies for bacterial pathogens

• Readily culturable and ubiquitous in warm-blooded animals

• Capable of exponential growth in the right environment

Survival of Indicators in the Environment

• Longer survival in cool than warm temperatures

• Longer survival in clayey than sandy textured soil

• Longer survival in circumneutral than acid or alkaline pH

• Longer survival when moist than dry• Longer survival when shaded than exposed

to UV light

Influence of Temperature and Particle Size on Mortality (Howell et al., 1996)

Temperature and particle size are inversely related to E. coli survival in sediment but the effects are not interactive.

As particle size ↓ half life ↑ and as temperature↓ half life ↑.

Survival

• Greater transport when saturated than unsaturated

• Greater transport when flow is rapid than slow

• Greater transport when porous media is structured than unstructured

• Follow the water!

Transport of Indicators in the Environment

There is a statistically significant association between rainfall and disease in

the U.S.68% of waterborne diseases preceded by

precipitation exceeding the 80th

percentile.Largest reported outbreak of E. coli

O157:H7 in the U.S. occurred after a heavy rain that was linked to contaminated well

water in a NY fairground.(Curriero, F.C., J.A. Patz, J.B. Rose, and S. Lele. 2001. The association between extreme precipitation and waterborne disease outbreaks in the United States, 1948-1994. Am. J. Pub. Health. 91(8)1194-1199.)

Source: E.G. Beck et al. 2005. STATUS REPORT: Bacteria and othercontaminants in domestic water waters in the Jackson Purchase region. University of Kentucky Cooperative Extension Service ENRI-221.

Of 173 domestic wells sampled, 80%

contained some type of coliform

bacteria

The type of well affected the likelihood

of contamination

The concentration of fecal coliforms and fecal streptococci in leachate from beneath well structured and manured soils reflects the manure application rate.

The more waste you add – the more fecal bacteria (and pathogens?) you recover.

Is This a Good Assumption?

What Do We Know About Pathogen Transport and Survival in the Environment vs. What We

Think We Know?

Sedimentation velocity of Cryptosporidium oocysts and Giardia cysts attached to particles in biologically treated sewage effluent of different

size ranges.

(Source: Medema et al. 1998. Appl. Environ. Microbiol. 64:4460-4466)

The bigger they are –the faster they fall

Transport of Cryptosporidium parvum oocyststhrough overland flow in vegetative filter strips

(Source: Trask et al. 2004. J. Environ. Qual. 33:984-993.)

Cryptosporidium oocyst removal in filter strips

• 99.9% removal in filter strips of > 3m length and < 20% slope (Atwill et al., 2002)

• Log10 reductions of 1-3 per m of filter strip.

TransportFecal Coliform Infiltration Through Intact Soil

(McMurry et al., 1998)

Water and fecal coliforms move through just a

small portion of intact soils.

Leaching of E.coli O157:H7Gagliardi and Karns (2000)

Soil Core Soil Manure log CFU Amt RecoveredRecovered Amt Inoculated

No-till Clay loam + 5.4 31.0- 8.4 9.1

Silt loam + 8.8 20.5- 8.0 3.2

Sandy loam + 7.9 8.5- 7.2 0.6

Till Clay loam + 7.4 1.4- 8.1 4.4

Silt loam + 7.8 2.3- 8.2 3.8

Sandy loam + 7.9 2.1- 8.3 4.7

0.01

0.1

1

Sand 10mM KCl

Sand1mM KCl

Fe-coated

Al-coated

Porous Media

Stic

kin

g

Eff

icie

ncy

E.coliC. jejuni

(Redrawn from Bolster, C.H. and K.L. Cook. 2005. Personal communication)

Relative Sticking Efficiency of E. coli O157:H7 and C. jejuniin clean and coated sand ( initial loading rate 108 cells/mL)

0.0001

0.001

0.01

0.1

1

Sand 10 mMKCl

Sand 1 mM KCl

Fe-coated Al-coated

Porous media

Rel

ativ

e R

eco

very E.coli

C. jejuni

(Redrawn from Bolster, C.H. and K.L. Cook. 2005. Personal communication)

Relative Recovery of E. coli O157:H7 and C. jejuni in clean and coated sand ( initial loading rate 108 cells/mL)

Physical characteristics of spherical bacteriophages used as indicators

(Source: Dowd et al. 1998. Appl. Environ. Microbiol. 64:405-410.)

Bacteriophage Isoelectric Diameter (nm) Lipid Host point content bacterium

MS2 3.9 24 0 Escherichia coliATCC 15597

PRD1 4.2 63 16 Salmonellatyphimurium LT-2

Qß 5.3 24 0 Escherichia coliATCC 17853

FX174 6.6 27 0 Escherichia coliATCC 13706

PM2 7.3 60 13 Alteromonas espejiaATCC 2725

MS2, PRD1, Qß , FX174, and PM2 have differing isoelectricpoints (pI 3.9, 4.2, 5.3, 6.6,and 7.3 respectively).

Transport of indicator viruses

through sand columns.

(Source: Dowd et al. 1998.Appl. Environ. Microbiol. 64:405-410.)

Size > 60 nm means flow characteristics rather than surface characteristics dominate

Extraction and Transport of Phage from Biosolids(Source: Chetochine et al. 2006. Appl. Environ. Microbiol. 72:665-671)

MS-2 Extracted from 7% Class B Biosolids

MS-2 leached through sand

93% of all coliphage

Extracted in 1st two

extractions

Pathogen Survival Media

Pathogen Feces Soil WaterCampylobacter 7-40 d – 2-9 dClostridium perfringens – – –Cryptosporidium 30-400 d 160-500 d –

parvumEscherichia coli 50-600 d 40 d 50-90 d

O157:H7Giardia lamblia > 60 d 1-38 d –Listeria – 10 d >14 d

monocytogenesMycobacterium 56 d – 175 d

paratuberculosisSalmonella 50 d > 180 d > 70 dYersinia 10 d – > 100 d

enterocolitica

Pathogen vs. NonpathogenMortality in Soil (Mubiru et al.,

2000)

Survival was most directly affected by relative clay content.

Survival Soil type but not strain made a difference in mortality.

Decline of Salmonella, Listeria, Campylobacter, and E. coli O157 in cattle wastes. Solid wastes (A and C) and liquid wastes (B and D) were obtained from beef cattle (A and B) and dairy cattle (C and D).

(Source: Hutchison et al. 2005. Appl. Environ. Microbiol. 71: 691-696.)

Survival of E. coli and Pathogens in Various Manure Treatment Systems

(Source: Grewal et al. 2006 Appl. Environ. Microbiol. 72:565-574)

E. coli

0

1

2

3

0 3 7 14 28 56

Days

# o

f S

am

ple

s

Po

sit

ive f

or

Cu

ltu

res

Compost - Sawdust

Compost -Straw

Stack - Sawdust

Stack- Straw

Lagoon

Salmonella

0

1

2

3

0 3 7 14 28 56

Days

# o

f S

am

ple

s

Po

sit

ive f

or

Cu

ltu

res

Compost - Sawdust

Compost -Straw

Stack - Sawdust

Stack- Straw

Lagoon

Listeria

0

1

2

3

0 3 7 14 28 56

Days

# o

f S

amp

les

Po

siti

ve f

or

Cu

ltu

res

Compost - Sawdust

Compost -Straw

Stack - Sawdust

Stack- Straw

Lagoon

Mycobacterium

0

1

2

3

0 3 7 14 28 56

Days

# o

f S

amp

les

Po

siti

ve f

or

Cu

ltu

res

Compost - Sawdust

Compost -Straw

Stack - Sawdust

Stack- Straw

Lagoon

*

*

DNA still detectable at 175 d

Two Examples of Emerging Pathogen Issues

• Novel vectors – Mollusks and Protists• Antibiotic Resistance – Enhanced Survival• Global Changes – Increasing range of

Plasmodium, Amphibian Die Off • New Agricultural Activity – Flukes and

Aquatic Food (Fish and Shrimp farms)• Changing Demographics – Clostridium

difficile• Leisure Exposure (Listeria in pets)

(Redrawn from Sproston et al. 2006. Appl. Environ. Microbiol. 72:144-149)

SourceContaminated fecesLand-applied manure

Carriageof E. coli

O157 By SlugsDeroceras reticulatum

E. coli Contaminatedvegetables

A. Adsorption by contact

B. Ingestion

C. Transfer by contact

E. Excretion in feces

0.21% of nativeslugs

14 d persistence

21 d persistence

Persistence of Mycobacterium in Protists(Source: Mura et al., 2006. Appl. Environ. Microbiol. 71:854-859.)

Log # of Mycobacterium

detected by qRT-PCR

7

6

5

4

Weeks0 5 10 15 20 25 30

Persistence of Glycopeptide Resistant Enterococci (GRE) Since the Termination of

Avoparcin(Source: Sørum et al. 2006. Appl. Environ. Microbiol. 72:516-521)

% GRE Positive

Sample Poultry Farmers

1995 Avoparcin Use Banned

1998 100 282000 82 322001 96 252002 83 292002-03 72 26

Despite the absence of selective pressure, resistance persists

Questions?

Amphibian Mortality

• Batrachochytrium dendrobatidis – a chytridomycete fungus causing chytridomycosis in amphibians

• Mass die-offs and extensive species declines on 4 continents (Stuart et al., 2004)

References• Atwill, E.R., L. Hou, B.M. Karle, T. Harter, K.W. Tate, and R.A. Dahlgren.

2002. Transport of oocysts through vegetative buffer strips and estimated filtration efficiency. Appl. Environ. Microbiol. 68(11):5517-5527.

• Chetochine, A.J. et al. 2006. Leaching of phage from class B biosolids and potential transport through soil. Appl. Environ. Microbiol. 72(1):665-674

• Dowd,S.E., S. D. Pillai, S. Wang, and M. Y. Corapcioglu. 1998. Delineating the specific influence of virus isoelectric point and size on virus adsorption and transport through sandy soils. Appl Environ Microbiol. 64: 405-410.

• Grewal, S.K. et al. 2006. Persistence of Mycobacterium avium subsp. tuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure. Appl. Environ. Microbiol. 72(1):565-574.

• Hutchison, M.L., L. D. Walters, T. Moore, D. J. I. Thomas, and S. M. Avery. 2005. Fate of pathogens present in livestock wastes spread onto fescue plots. Appl. Environ. Microbiol. 71(2): 691-696.

• Jenkins, M.B. and D.D. Bowman. 2004. Viability of pathogens in the environment. p. 75-79. In W.L. Hargrove (ed.) Workshop Report:Pathogens in the Environment. Kansas Center for Agriculture Resources and the Environment, Kansas State University, Manhattan, KS.

References• Kosek, M. et al. 2003. The global burden of diarrhoeal disease as

estimated from studies published between 1992 and 2000. Bull. WHO 81:197-204.

• Medema,G.J., F. M. Schets, P. F. M. Teunis, and A. H. Havelaar.1998. Sedimentation of free and attached Cryptosporidium oocysts and Giardiacysts in water. Appl. Environ. Microbiology.64:4460-4466.

• Mura, M. et al. 2006. Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga. Appl. Environ. Microbiol. 72:854-859.

• Sørum , M. et al. Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3-8 years after the nam on Avoparcin. Appl. Environ. Microbiol. 72(1):516-521.

• Sproston, E.L. et al. 2006. Slugs: Potential novel vectors for Escherichia coli O157. Appl. Environ. Microbiol. 72(1):144-149.

• Stuart et al. 2004. Status and trends of amphibian declines and extinctions worldwide. Science. 306:1783-1786.

• Torres, A.G. et al. 2005. Differential binding of Escherichia col O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures. Appl. Environ. Microbiol. 71(12):800-8015.

• Trask, J.R. P.K. Kalita, M.S. Kuhlenschmidt, R.D. Smith, and T.L. Funk. 2004. Overland and near-surface transport of Cryptosporidium parvumfrom vegetated and nonvegetated surfaces. J. Environ. Qual. 33:984-993.