Introduction to Microbial Source Tracking

Katharine G. Field Oregon State University

Fecal Pollution

• a continuing problem in coastal and fresh waters• causes economic losses, human health risks,

and environmental damage• standard methods of measuring fecal pollution

are slow, and do not distinguish its source

Potential Sources of Non-Point Source Fecal Pollution

Wildlife

Enteric viruses, Enteric viruses, bacteria, bacteria, parasitic protozoaparasitic protozoa……

E. coli E. coli 0157, 0157, SalmonellaSalmonella,,CryptosporidiumCryptosporidium……

Salmonella, Giardia, Cryptosporidium, Tularemia, Leptospira Salmonella, Giardia, Cryptosporidium, Tularemia, Leptospira ……

Storm drains, sewage plants, septic tanks, and agriculture

What’s Really Needed to Estimate Health Risk:

Either: direct monitoring of pathogens Either: direct monitoring of pathogens

Not practical as yetNot practical as yet

Or: some way of identifying the source of Or: some way of identifying the source of fecal pollution, and an accurate estimate of fecal pollution, and an accurate estimate of the health risks from different sourcesthe health risks from different sources

Fecal Indicator Bacteria (FIB)

•• E. coliE. coli, total and fecal coliforms, fecal , total and fecal coliforms, fecal enterococci (for marine waters), enterococci (for marine waters), ClostridiumClostridium

•• Permissible levels set by EPA based on Permissible levels set by EPA based on epidemiological studiesepidemiological studies

Indicator Bacteria: Limitations

1. Epidemiology studies were based on exposure to 1. Epidemiology studies were based on exposure to human fecal contamination, not animalhuman fecal contamination, not animal

•• We donWe don’’t know the risk from exposure to similar levels t know the risk from exposure to similar levels of animal fecal contaminationof animal fecal contamination

2. 2. E. coli and Enterococcus spp. can survive and proliferate in the environment.

•• Evidence suggests environmentally-adapted strains of indicators.

3. 3. E. coli and enterococci are not well correlated with many pathogens

Indicator Bacteria: Limitations

4. Standard 4. Standard E. coliE. coli and enterococci tests and enterococci tests dondon’’t distinguish the source of fecal t distinguish the source of fecal contaminationcontamination

•• Microbial Source Tracking (Fecal Source Microbial Source Tracking (Fecal Source Tracking) neededTracking) needed

Underlying assumptions:Underlying assumptions:

•• There are characteristics associated with feces There are characteristics associated with feces that identify the source (that identify the source (““hosthost--specific markersspecific markers””) )

•• These traits can be detected in waterThese traits can be detected in water

Often assumed (but not proven): Often assumed (but not proven):

•• survival of traits in water is proportionalsurvival of traits in water is proportional

•• quantitative detection estimates proportions of quantitative detection estimates proportions of each sourceeach source

Fecal source tracking: diagnosing the Fecal source tracking: diagnosing the source or sources of feces in watersource or sources of feces in water

Methods:Methods:

CultureCulture--based vs. culturebased vs. culture--independentindependent

LibraryLibrary--dependent vs. librarydependent vs. library--independentindependent

A A ““librarylibrary”” ((““host origin databasehost origin database””) is a ) is a collection of isolates from known sources of collection of isolates from known sources of feces, analyzed according to the method of feces, analyzed according to the method of fecal source identificationfecal source identification

FECAL SOURCE TRACKING:“LIBRARY-DEPENDENT” METHODS

• Culture a large number of bacterial isolates (E. coli, enterococcus) from different kinds of feces

• Use a screening method on the isolates

• Now culture bacterial isolates from contaminated water, screen them by the same method, and compare the screening profiles from water isolates to those from fecal isolates

PHENOTYPIC LIBRARY-DEPENDENT METHODS

Antibiotic resistance analysis (ARA, ARP, MAR, etc.)

Assumption: Humans, domestic animals, and wild animals have different exposure to antibiotics and thus their fecal bacteria show different resistance patterns

• Many problems with this assumption• Antibiotic resistance not stable • Method performed poorly in comparative tests with blind samples

• Fingerprinting methods that performed well in several comparative studies with blind samples: Ribotyping (Samadpour protocol: using 2 enzymes) and PFGE

• both can be effective methods of fecal source identification

GENOTYPIC LIBRARY-BASED METHODS: DNA Fingerprinting

Limitations to library-dependent methods:

• Slow and expensive

• Geographic variation: need a new “library” for each new area (or a VERY LARGE library)

• Some companies offering these methods don’t use the best protocols

• Some companies offering these methods claim to identify all isolates, but in reality, many to most isolates from the environment don’t match fecal isolates

Culture host-specific microbes:Phage: human vs. non-human only

• male-specific coliphages• Phages of Bacteriodes bacteria

Bacteria: • Bifidobacterium adolescentis, sorbitol-fermenting bifidobacteria for humans• Rhodococcus coprophilis for grazing animals

CULTURE-BASED, NON-LIBRARY-DEPENDENT METHODS

Old ratio method has been discredited:• ratio between fecal coliforms and fecalstreptococci• Although this ratio differs in fresh human and animal feces, the groups survive differently, so the ratio changes with time.

CULTURE INDEPENDENT METHODS:CHEMICAL METHODS

• caffeine, fecal sterols and stanols, bile acids, laundry brighteners, surfactants, and fragrances are used to diagnose human fecal contamination

• Their spread, persistence, and transport in water are not well correlated with FIB and microbial pathogens

• Some are removed by wastewater treatment, while others persist in sediments for years and may be resuspended

Culture-Independent Molecular Methodsfor Fecal Source Identification

• A paradigm change: Methods of molecular ecology applied to public health microbiology

• Methods: DNA extraction, PCR, gel electrophoresis

PCR = Polymerase Chain Reaction, a method of making millions of copies of a specified DNA sequence in a test tube

PCR is routinely used in medical and food safety testing. Today’s college graduates in biosciences have all learned it.

Culture-Independent Molecular Methodsfor Fecal Source Identification

• Assays include:- phage and viral pathogens (humanadenoviruses, enteroviruses, and polyoma virus; porcine teschoviruses and adenoviruses; and bovine/ovine adenoviruses and enteroviruses - virulence genes from E. coli, enterococci- 16S rDNA markers from fecal anaerobes

Plate Counts Direct Counts

Courtesy Mike Courtesy Mike RappeRappe, HIMB, HIMBSteve Steve GiovannoniGiovannoni, OSU, OSU

“The Great Plate Count Anomaly”: only 1 to 0.001% of microbes in the environment can be cultivateonly 1 to 0.001% of microbes in the environment can be cultivated.d.

Why use molecular methods, that don’t require culturing?

Culture bias: the ones that do grow are not necessarily representative of what’s in the natural population

• Using molecular methods to detect allows us to sample the entire population

• Molecular methods are faster

Using these tools, we developed:

• A method of fecal source detection that– is fast (less than 24 hours) and

inexpensive– doesn’t require culturing– doesn’t require a site-specific library

• based on extracting DNA from water samples and amplifying diagnostic gene sequences from uncultured Bacteroidales rRNA genes

Why Bacteroidales?

• found in both human and animal feces

• significant portion of the microbiota of the GI tract (more than E. coli or enterococci)

• only in animal body cavities, not in water

• anaerobic: limited survival and reproduction upon release

• species and strain differences among different animal hosts

1. Starting with fecal DNAs, look for unique host-species-specific Bacteroidales 16S rDNA markers

2. Show that markers can be recovered from fecally-contaminated natural waters

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Fragment Size (base pairs)

Rel

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nce sewage

effluent

cattle feceshuman feces

river water

bay water

3. Develop PCR primers from markers and test for specificity

humanfecal DNAs

cowfecal DNAs

Results: Specific primers for Results: Specific primers for ruminants and humansruminants and humans

Cow primers* specifically detect ruminant feces…

+control human

fecal DNAscow

fecal DNAs

*Geographically stable: *Geographically stable: Throughout the US, Hawaii, Europe, New Zealand, Australia, South Africa, Japan…

Human primers* specifically detect human feces.

Clone library analysis yielded more host-specific primers: PIG and HORSE

FECAL DNAsAMPLIFIEDwith PIGPRIMER

FECAL DNAsAMPLIFIEDwith HORSEPRIMER

But this approach doesn’t work well for closely-related hosts and host species that share fecal bacteria…

From L. K. Dick et al.,2005.From L. K. Dick et al.,2005.

Primer Sensitivity: 10 to 100 copies

Sensitivity assays:Sensitivity assays:((target = plasmid)

Target Target copy copy numbernumber

101011

101022

101033

101044

101055

101066

GenBacGenBac CF128 CF193CF128 CF193

We developed assays for human, ruminants, dogs, pigs, horses, elk…•Several others (birds) are being validated

• Other molecular assays– Toxin genes from E. coli (human, cow,

pig) (Olson group)

– Toxin gene from a human Enterococcusstrain (human) (Troy Scott, Joan Rose)

100-ml water sample

Filter

Extract DNA from filters PCR with

specific primers

Gel electrophoresis

Score results

E. coli and enterococci toxin

genes: enrich

Bacteroidales markers

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Is Source Tracking Quantitative?

Unanswered questions:• Do all markers have the same environmental survival?• Do markers have the same environmental survival as fecal indicator bacteria?• Do markers have the same environmental survival as pathogens?

Although it’s easy to measure markers quantitatively, we can’t interpret what the results mean until we can answer these questions…

Needed:

1. multiple markers for each species, utilizing a variety of targets.

2. Kits for source detection

Epidemiological studies needed to establish human health risks from fecal pollution from animal sources!

Conclusions:

1. No method of source tracking is ideal2. PCR of host-specific genetic markers is relatively rapid and

inexpensive, but lacks markers for certain species (wildlife)3. Library-dependent fingerprinting methods, like ribotyping and

PFGE, can identify many species, but take longer and cost more

4. Viral and chemical methods can document human fecal contamination

5. The best evidence supports taking a multi-tiered approach