1. Bacterial Source Tracking in Stormwater
Calista Mills, Physical Scientist
USEPA, Gulf of Mexico Program
I want to start out with a disclaimer. My topic may lead you to believe that I am a microbiologist but I will highlight that I am a
Bacterial types (a) contain ribosomes (b) that are composed of a specific sequence of DNA/RNA bases (c), allowing them to be differentiated. The PhyloChip detects these differences, and thus is able to classify all types of DNA/RNA sequences in complex mixtures.

2. Bacterial Source Tracking in Stormwater
Calista Mills, Physical Scientist
USEPA, Gulf of Mexico Program
physical scientist – not a life scientist. So I am going to attempt to explain this as best as I can. Let me ask, who in the room has had some experience in microbial source tracking or microbiology in general? Awesome, thank you for volunteering to to help with any questions or clarity needed if I muck this up.

3. Gulf of Mexico Program
Water Quality Improvements
Coastal habitat & ecosystem enhancement, restoration &/or protection
Environmental Education & Outreach
Community Resilience
In case you haven’t heard of the Gulf of Mexico Program before we are focused on the health, productivity and restoration of the Gulf of Mexico and communities that rely on it.
Work in the gulf region and its watersheds through various partnering mechanisms.
Anticipating RFP to be released in June!
to sign up for the RFP list

4. Turkey Creek RARE Regional Applied Research Effort
use bacterial & viral fecal-indicators & landscape tools to identify & pinpoint pollution sources in an urban watershed
Bacterial source tracking is one component of a larger project. The Turkey Creek Project is funded as a regional applied research effort which is looking at five components:
nutrient concentrations in water,
presence of the pathogen indicator E. coli using IDEXX
DNA sequencing and taxonomic analysis of water using PhyloChip,
DNA sequencing and taxonomic analysis of sediment
presence and genotype of male-specific coliphages (FRNA)

5. Turkey Creek RARE Impaired - high fecal
Cooperate with state & local partners
Take quality assured data with local residents/students
Provide data for local Gulf decision makers
The Turkey Creek community is a low income ….. This project supplements existing monitoring programs with a fecal-source identification component.
Participation by the local community, students, residents and other partners imparts a sense of “ownership” between residents and their local waterways while providing water-quality data.
These data are shared with state and county stakeholders, Citizens’ Science partners, the Turkey Creek Steering Committee and EPA Regional Offices as they seek solutions to these contamination issues
Our hope is that what we learn from the project will be used to identify causes of impairment and demonstrate improvements after management actions such as LID, BMPs, and wastewater infrastructure upgrades take place
This information can also be used to help the State identify impairments at a lower cost.

6. This is the watershed we are working in and where our sites are located. Point sources include wastewater from subdivisions and trailer parks. Nonpoint sources include urban areas, wildlife, livestock, failing or aged septic systems. Water flows from Turkey Creek into Bernard Bayou, the back bay of Biloxi, then the Mississippi Sound and the Gulf of Mexico.

7. Turkey Creek RARE: E. coli
Sterile bottle sample collection
E. coli assays fluoresce under UV light
Add bacterial growth media (food) to 100 ml sample
Monitoring using the IDEXX Colilert test started in September of High E. coli levels occur often under both low and high-flow stream conditions. E. coli levels are higher after rain events. The RARE project is building on this monitoring. The Gulf Ecology Division at the EPAs Gulf Breeze Lab in Florida is the lead for the RARE project and we are offering field support.

8. Virology The plate shows viral plaques (red dots)
The next step is to determine sources of these fecal viruses
One component GED is looking at is viral fecal-indicators which are often indicative of the pollution source (human or animal)
Coliphage analyses: Approximately 1L of ambient water will be collected using the Nisskin, Van Dorn or a suitable water-collection device, and concentrated using the InnovaPrep concentrator, yielding less than 5 ml of concentrated environmental microbes. The concentrated water will be stored at 4 o C in RNase, DNase-free tubes within an hour following concentration and tested for viral plaques within 24 hr. The concentrated water will be analyzed for F+RNA coliphages using RT-PCR assays

9. Microbial Source Tracking
DNA from target animals or bacteria is released into the water.
Sample is collected and DNA is extracted.
Specific sequences are amplified and identified.
Source determination is made.
Microbial fecal- indicators in water and sediments
Biofilm, water or sediments
MST is a more useful tool than culturing or coliform because most fecal bacteria are unable to survive outside of the host so by targeting pieces of DNA instead of the whole organism, more accurate determinations can be made.
Microbes from the water column and from the sediment will be sequenced to determine if the fecal contamination is being re-suspended from the sediments or is primarily in the water column

10. Phylogenetic Similarity
Turkey Creek sediments were collected this past summer and fall, 2016 and preliminary data suggest that the microbial community is 80% different at the Rippy station than the other communities in the same watershed

11. MST: qPCR EPA qPCR Methods One genetic sequence per method
$375 per sample per species
qPCR methods target either a host specific bacteria, or a host specific DNA sequence that is shed through fecal waste. Very expensive to outsource at $375 per sample per species.
2 Existing methods – Enterococci (1609.1/1611.1)
2 Draft methods – Bacteroidales and E.coli (Draft B/C)
6 New MST methods in development – (2)Human/(2)Cow/Dog/Pig
Available in R7 (and other regions) once approved
Available Commercially – EPA Developed and licensed to Source Molecular

12. MST: Phylochip Phylogenetic Microarray
developed by Gary Anderson at UC Berkeley
profile over 60,000 bacterial taxa in one test
The EPA is currently evaluating the PhyloChip microarray as a Next Generation Microbial Source Tracking technology which could be used along side existing MST technologies to further our understanding of bacterial communities, water health, and fecal identification.
The PhyloChip is a Phylogenetic Microarray developed by Gary Andersen of University of California Berkeley.
The chip is based on the Green Genes 16S ribosomal DNA library.
It is capable of detecting microbial species that are present even at very low abundance
Reproducible for multiple replicates and comparable with other experiments
Images: LBNL

13. MST: PhyloChip qPCR on a large scale
Over 1 million genetic probe sequences including controls
Developed using 80 fecal reference libraries
$500 per sample, batches of 10
Phylochip utilizes the same basic concepts as qPCR MST by amplifying DNA and using an intensity to determine concentration. The phylochip uses 1.1 million distinct genetic sequences to identify 60,000 distinct bacterial signatures in one test.
Image: LBNL

14. Unique 25 base DNA probe designed for each species from database
Fluorescent antibody bound to probes with match
There are 6.5 million locations on each GeneChip allowing multiple tests to be conducted on a single glass surface. Each DNA probe consists of unique 25 base sequence designed for species from the reference database being used. DNA from a sample are tagged with fluorescence. When the sample is applied to the chip, DNA find their match on the chip (“hybridization”); With a laser scanner you can identify which microbes are present.
Multiple DNA sequences or probes are used on the PhyloChip and each probe is paired with a single-base mismatch control probe to check performance characteristics of each sample.
Images: LBNL

15. Bird
Horse
Cow
Pig
Dog/Cat
Human
The Phylochip can profile waste of various animals based on reference libraries.
Common sequences (PhyloChip probes) can be used to train predictive models.
80 Fecal reference libraries were used in development of the Phylochip.
Human Sewage, Dogs/Cats, Birds, Cows, Horses, Deer/Elk, Pigs, Seals/Sea Lions, Rats, Raccoons
Images: LBNL

16. Bacterial Animal Sources
Fecal reference libraries were used to create generic “Waste Profiles”. Each source creates a specific fingerprint that can be used to track and quantify it’s contribution. PhyloChip measures these unique fingerprints making it a powerful tool for pollution detection and monitoring
Images: EPA R7

17. Hybridization intensity
Generating a profile
Bacterial species (probes)
Hybridization intensity
Source fingerprint
When contaminants are unknown it is important to develop a reference database of contaminated samples to train a predictive model for detection unknowns.
Machine learning algorithms used for predictive modeling to discriminate sources
Resulting bacterial profile shown as change against baseline. (Sample comparison)
Image: LBNL

18. Profile of entire community
Extract DNA/RNA
from filtered sample
Amplify with PCR
ml of water sent through a Polycarbonate membrane filter, 47mm, 0.45μm within 6 hours of collection, stored frozen (- 80 o C) and shipped to LBNL for analysis using the PhyloChip.
Analyze composition
Profile of entire community
Images: LBNL

19. Performance evaluation
Blinded study: 41 different methods, 12 fecal sources, 64 unknowns
100% correct identification of all dominant sources with PhyloChip
PhyloChip identified all sources, including sources with no specific markers available (e.g. deer, chicken)
Source
True positive rate
Number false positives
Chicken
100
Cow 75 1
Deer
Dog
Goose 2
Gull 83
Horse
Human stool
Pig
Pigeon
Septage
Sewage 93
In a blinded study of 41 different methods, 12 fecal sources, 64 unknowns the phylochip correctly identified 100% of all dominant sources.
Phylochip was the only method that identified all sources.
Cao et al Water Research 47:
Images: LBNL

20. Case Study *
Horseshoe Cove
Baker Beach
Both beaches exceed water quality limits for fecal indicator bacteria
Two beaches on the San Francisco Bay that suffer from E.coli levels above the action limit. Opposite sides of the Golden Gate Bridge.
Images: LBNL

21. Case Study Fecal signal from different sources
Using the phylochip they were able to determine that one beach was being impacted by a nesting seagull population while the other beach was impacted by a human wastewater discharge.
Images: LBNL

22. The future for you PhyloChip can be tailored for specific analysis
The goal of this evaluation is to develop a chip that costs under $100 per sample.
Regional sewage libraries for hot topic animals
Ask for next gen MST – OECA will give next gen dollars to run samples in your region.
One cool thing about the phylochip is that it can be tailored to what you need or are interested in. The EPA evaluation period will conclude in November At the conclusion of this contract, a bulk evaluation of the data will be conducted in order to make recommendations for the creation of an EPA specific microarray. This microarray will be tailored to meet all regional needs as well as meeting a lower price point.

23. Troy Pierce, EPA Gulf of Mexico Program, pierce.troy@epa.gov
Can incorporate EPA MST methods that are already based on 16s gene, as well as methods based on host specific genes by mixing primers.
Available in inter-agency agreement with LBNL
Several funding initiatives in the works to begin trials of PhyloChip. Not just CAFO activities, but water quality, soil health, community characterization, Harmful Algal Bloom research and identification, etc. Ultimately trying to custom tailor an EPA chip that will be ran here in Region 7 for all regions. Eventual uses could include Freshwater, Saltwater, Soil, and Air samples.
Method 1615 – Enterovirus and Norovirus
Chips are currently $500 for each sample, and are processed in batches of 10. The contract only allows purchases in increments of 10 in order to keep the price point low.
The goal of this evaluation is to develop a chip that costs under $100 per sample.
Multiple DNA oligonucleotide probes target taxa specific regions of the 16S rRNA gene to identify virtually all known bacteria and archaea.  This gene is used to create RNA copies of itself that are one of the two main building blocks of the ribosome complex, which is essential for making proteins in the cell (Figure 2).  The advantage of using this gene is that it is universally present in all microbes and small sequence variations within the gene can be used as a “bar-code” for bacteria and archaea identification.  Multiple DNA probes on the PhyloChip are used to identify sequence variation in the 16S rRNA gene and each probe is paired with a single-base mismatch control probe to minimize the effect of non-specific hybridization.  The current Generation 3 (G3) PhyloChip contains a total of 1,100,000 different DNA probes targeting.
Contact: Calista Mills, EPA Gulf of Mexico Program,
Troy Pierce, EPA Gulf of Mexico Program,

24. qPCR Microarray qPCR highly sensitive.
qPCR can be tailored to very specific targets.
One DNA sequence
$375 per sample
Microarray generates massive amounts of data.
Microarray can produce results in more diverse samples.
over 60,000 bacterial taxa
$500 per sample
Both useful tools that compliment each other.