NEERS/SNECAFS Joint Meeting, May 9, 2003

Project Author: Kristen Whiting-Grant, Maine Sea Grant Cayce Dalton*, AmeriCorps/Maine Conservation Corps

Fred Dillon*, U Southern Maine-Muskie School for Public Service Steve Jones, Jackson Estuarine Lab, U New Hampshire

Michele Dionne, Wells National Estuarine Research Reserve* presenters

Microbial Source Tracking in Two Southern Maine Watersheds

Funded by a grant from:Cooperative Institute forCoastal and EstuarineEnvironmental Technology (CICEET)

In cooperation with partners from:

Project Partners

What is Bacterial Pollution?

Indicates presence of fecal matter in water.

Risk of illness from water contact.

Grounds for shellfish bed closure.

Sources of Bacterial Pollution? • Unmanaged livestock/pet waste• Leaking sewer pipes/storm overflows• Wildlife (incl. mammals and birds)• Malfunctioning septic systems

• Inexpensive surrogate for fecal pathogens.

• Countable, not just presence/absence.• Regulatory standards for shellfish

areas and recreational waters.

Importance of Bacteria as Water Quality Indicators

What is Microbial Source Tracking?

• ID strains of indicator bacteria or virus

• Phenotypic or Genotypic methods

• Unknown strains from environment compared to strains found in host animals

• Close matches are a basis for source identification

• Experimental technique, gaining attention

EXPERIME

NTAL!

Why Use MST?• Addresses biggest weakness of conventional bacterial

tests: not source specific.

• Knowing sources means corrective measures focused, saving public resources and reducing frustration.

• Example: Expensive sewer extension in Wells, Maine, did not significantly reduce fecal coliform levels in Little River Estuary.

Deer Scat Water Sample

Study Area

Local Need: History of fecal contamination in So. ME results

in closed clam flats. Mirrors state & national issue.

Wells Beach enjoys significant tourism (despite shark).Bacteria = public health issue.

MST Step 1: Intensive Water Sampling

Sample freshwater tributaries of estuary during winter clamming season.

Half of sampling during post-storm, snowmelt or high flow conditions.

Test is membrane filtration using mTEC + urea for E. coli.

MST Step 2: Collect Fecal Samples For Reference Library

E. coli obtained from about 10 fecal samples.

Includes 3 human (septic, sewage, direct fecal).

Includes other probable sources: dog, deer, cow, raccoon, etc.

MST Step 3: Select and Save BacteriaSamples with high E. coli are identified.

10 bacteria are isolated on TSA and refrigerated.

Isolates transported to Jackson Estuarine Lab for ribotyping within about two weeks.

All water samples tested for E. coli

Over ten months, 390 water samples were collected and filtered.

From samples with high E. coli, bacteria are isolated

Isolates were made from 136 of 390 water samples.(Ten bacterial isolates per water sample.)

From isolates, a few representative samples are ribotyped

Genetic analysis conducted on 27 of 390 water samples.We counted 22,856 bacterial colonies. 159 were ribotyped.

Source Species DatabaseSpecies Species

ME ME & NH ME ME & NHPets 6 17 Livestock/chickens 46Cat -- 2 Cow -- 30Dog 6 15 Horse -- 14Humans 40 86 Chicken -- 2Human 10 14 Birds 2 48Septage 17 17 Cormorant -- 13Wastewater 13 55 Duck -- 4Wildlife 23 116 Goose -- 19Coyote 10 15 Grouse 2 2Deer 3 41 Pigeon -- 2Grey Fox 3 3 Robin -- 3Muskrat -- 3 Seagull -- 5Raccoon 4 28Red Fox 3 26 TOTAL 75 317

159 bacterial colonies from streams/estuary ribotyped.Compared to 75 bacterial colonies from watershed.Compared to 317 bacterial colonies from ME-NH region.

Results of MST in Maine:

MST on small subset of samples.

Detailed view of geographic distribution of bacteria.

Ribotyping:Lab Procedures

• DNA extracted & purified.• DNA digested w/restriction enzyme.• DNA separated via gel electrophoresis.• DNA denatured & blotted onto membrane. • Hybridization with E. coli rRNA DNA probe.• DNA exposed to a chemiluminescent

substrate & digitally imaged. • Image enhanced & optimized in computer.

Ribotyping:Data Analysis

• DNA patterns are analyzed by cluster analysis and by computing a similarity coefficient.

• Source species identification for sample patterns based on degree of matching to source species patterns.

Matching ribotype gel banding patterns:

Unknown

Which of these patterns is most similar to the unknown?Duck

Human

Seagull

Human

Unknown

Human

Human

Duck

Seagull

Human = 93% similarity

Researcher Sets Similarity Criteria

Researcher must decide what tolerance and percent similarity qualify as a match:

Higher standard means greater certaintybut potentially too few matches.

Of 159 ribotyped isolates:at >80%, 112 had source-IDat >85%, 70 would have source-IDat >90%, 30 would have source-ID at >95%, 10 would have source-ID

Results of Ribotyping in Webhannet Watershed: Local Source Library

Largest single source: Human (18% of 53% known)Largest category: Wildlife (24% of 53% known)

Birds 0%*

Livestock 0%*

HUMAN 18%

Pets 11%

Wildlife 24%

Unknown 47%

* not in library

Results of Ribotyping in Webhannet Watershed: Regional Source Library

Largest single source: Human (18% of 70% known)Largest category: Wildlife (29% of 70% known)

HUMAN 18%

Birds 3%Livestock 11%

Wildlife 29%

Pets 9%

Unknown 30%

CONCLUSIONS

• Webhannet report being drafted now. Awaiting steering committee input re: management ideas.

• Results provide data in place of speculation.

• As more regional MST is conducted, library size should expand and % unknown diminish.

• Perhaps most useful when one or two major sources are suspected and impact is severe.

• Driven by need and interest, the basic science underlying MST is being researched further.

More info at web site: www.umseagrant-mst.org