General results of the work done during the 2015 Summer fellowship are posted here.
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General results of the work done during the 2015 Summer fellowship are posted here.
UMD FIRE EP Stream 
Wei-Lee’s Summer Fellowship General Results
Examine:
Non point microbial source tracking
Design a time efficient protocol
Examine dog, deer, and E. Coli tracings
Methods:
Equal width increment sampling method
Location: Mill Creek River Watershed 3 points A, C, B
Bacterial plates: Colony Counts
Enterococci selective
DNA extraction with QUBIT quantification
PCR with dog, deer, and E. Coli
Quantified with QUBIT and checked with gel
Results:
Dog, Deer, Goose, and E. Coli are ran against samples
Observations:
Able to determine high amounts of E. Coli and Goose source.
Low or little Dog and Deer tracings
Able to determine that Source B had the starting source of E.Coli and Source A demonstrates that there are strong source of E.Coli but diluted as distance from B increased.
Able to determine that Source C had the highest starting source of Goose and Source A demonstrates that there are strong source of Goose but diluted as distance from C increased.
Conclusions:
Found a fast way to quantify different microbial source.
Need multiple trials to determine the precision
Need to calibrate control samples
Utilizing equal width increment sampling increases accuracy
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Sim’s Summer Fellowship Update
Overarching Research Summaries:
Overuse of antibiotics in many levels ranging from factory farms to prescriptions
Huge incline of resistant strains of bacteria
Bacteria evolve faster than medications can be made
Bacteria’s resistant illnesses’ dangers will be similar to pre-penicillin era
Few Key Resources:
“About Antimicrobial Resistance.” Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 08 Sept. 2015. Web.
URL: http://www.cdc.gov/drugresistance/about.html
Appelbaum, P. C. 2007. Microbiology of antibiotic resistance in Staphylococcus aureus. Clin. Infect. Dis. 45:S165-S170.
URL: http://cid.oxfordjournals.org/content/45/Supplement_3/S165.full
Friedman, Tom. “White House Announces National Strategy for Combating
Antibiotic-Resistant Bacteria.” Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 22 Sept. 2014. Web. 22 Sept. 2015.
URL: http://www.cdc.gov/media/dpk/2014/dpk-carb.html
Herper, Matthew. “Antibiotic Breakthroughs Fall Short.” Forbes.com. Forbes, 07 Feb. 2003. Web.
URL: http://www.forbes.com/2003/02/07/cx_mh_0207antibiotic.html
Ling LL et al. A new antibiotic kills pathogens without detectable resistance. Nature DOI.
URL: http://www.nature.com/nature/journal/v517/n7535/full/nature14098.html
Malachowa N., et al. 2011. Characterization of a Staphylococcus aureus surface virulence factor promoting resistance to oxidative killing and infectious endocarditis. Infect. Immun. 79:342–352.
URL : http://iai.asm.org/content/79/1/342.full
“NIAID-Funded Scientists Developing New Antibiotic To Fight Resistant Infections.” NIAID-Funded Scientists Developing New Antibiotic to Fight Resistant Infections. N.p., n.d. Web.
URL:http://www.niaid.nih.gov/topics/antimicrobialResistance/Pages /teixobactinFeature.aspx
Pottumarthy S, Fritsche TR, Jones RN., Evaluation of alternative disk diffusion methods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: validation report from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis 2005;51:57-62.
URL: http://www.sciencedirect.com/science/article/pii/S0732889304001580
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The Environmental Pathogens stream has various sub-streams with students focusing on different projects all related to the general theme of environmental pathogens. What was accomplished this summer can be divided into each of these sub-streams. The Dog and Dino projects had the most progress over the summer while the CONSERVE, Plant Pathogens, and Brackish vs. Fresh Water projects had modest progress, and the Fish project had the least progress. Details on individual project progression is provided below and are ordered as they were presented in the initial report about what the stream planned for the summer.
The CONSERVE project is still being developed. This summer, the students working on the project did background research on reclaimed water treatment, irrigation systems, and pathogen survival. They established a methodology for DNA extractions with collaboration from the Sapkota Lab and other labs doing analysis with CONSERVE. They made modifications to this protocol which was tested on non-CONSERVE research to determine a method which will produce the most effective DNA extractions: This included experimenting with enzyme cocktail compositions, final elution volumes, final elution processing, and water storage temperatures. While it was eventually determined that for the CONSERVE samples the protocols should be followed exactly as written in order to remain consistent within the larger project, the modified methods were effective for DNA extractions in other projects. The students also took preliminary samples from the Community Learning Garden to be processed for pathogen concentration. Bacteria such as E. coli, enterococcus, and enterobacter have been cultured from this water. DNA has been extracted and is ready to be processed for pathogen identification. In addition, paperwork for survey approval has been completed for a survey which will assess the public’s opinion on reclaimed water use.
Students working on the Fish project completed background research on how water quality affects the health of aquatic wildlife, how to dissect a fish for the purpose of identifying sickness and infection, and previous studies regarding Anacostia River fish. Mostly, they are looking for water contamination and infection, and indicators of illness inside of fish organs for signs of major sickness due to the poor water quality. Examples include redness inside of stomach indicating possible infection, and degradation of intestinal cilia. Possible water collection sites were compiled and visited. Paperwork is being completed for submission to board for approval to do dissections.
The stream had two potential collaborations being developed at the beginning of the summer. Risk Communication and Resilience stream contacted us about collaborating with them on a project to track how a potential bioterrorist agent (a bacterium) would move through the water system by using an analog bacterium. RCR has already done most of the background research and suggested a common soil bacteria to use as an analog, but because the analog bacteria is a common soil bacteria, we are currently working out a way to label the bacteria to properly track it. There are restrictions on releasing modified organisms into the environment, so we need to determine how we could properly track which bacteria is “ours”
while we monitor how it moves through a water system.
Additionally, the Autonomous Unmanned Systems stream contacted us about developing a robot for water collection. During the course of their work this summer, they were able to develop a prototype robot which we will begin testing in the fall. This prototype was engineered specifically for the Fish project, but could be useful for other collection sites that are difficult to access.
This summer the Dino project saw significant progress. The sampling sites of the dinoflagellate project expanded throughout the UMD campus and Dinoflagellate PCR’s were also run on water samples collected outside of college park. The majority of positive samples come from large basins, reservoirs, ponds, and puddles. Stagnant water shows the best results. Some of the notable positive results include a puddle outside of Centreville Hall, the basin by the Xfinity Center Parking Garage, and another basin near the parking lot in front of the Xfinity Center. Some of the sites tested are displayed below with stars on the location: We then successfully topo-cloned the DNA into a vector and are ready to submit it for sequencing for the first time in the environmental pathogens lab. We have also designed a model method/protocol for other students to follow so in the upcoming semester we can send out large quantities of samples for sequencing and help the Delwiche Lab study the phylogeny of these organisms.
Over the past three months individuals working on the Dog Park study have been able to identify fecal derived dog feces, E. Coli, enterococcus and salmonella in the water adjacent to various parks. Testing for quantitative amounts of these pathogens have yet to be conducted, but the quality of these contaminants are under investigation. This has caused us to successfully broaden our water sample sites, and compare ordinances put into place. A survey has been submitted to question random individuals at the site, to see if knowledge of what’s in the water could affect their habits and treatment of the environment. Methodology for DNA extractions have been established and refined from protocol used by the Sapkota Lab and the CONSERVE project. Preliminary samples taken from various sites have shown that water processing is best when done within a two week period in order to have enough viable DNA to do various polymerase chain reactions (PCR).
In addition to the projects initially described at the beginning of the summer, the EP stream also developed some new projects during the course of the fellowship. These two projects are the Plant Pathogens project and the Fresh vs. Brackish Water E. coli project. The Plant Pathogens project was created this summer as a way to study the transmission of plant disease through water. Studies have shown that plant pathogens including bacteria, fungi, oomycetes, nematodes, and viruses can be transmitted through water. Because of this we designed a diagnostics test to identify plant pathogens, particularly the geni of Phytophthora and Pythium. These two oomycetes are some of the most prevalent plant pathogens (Phytophthora gets its notoriety for being responsible for the Irish Potato Famine) and are known to transmit reproductive spores through water. The diagnostics test will allow the environmental pathogens stream to study the transmission of these pathogens through various water sources and potentially aid the CONSERVE study. We have successfully tested a positive control and gained results from water samples as well as created a foundation for further plant pathological studies and species identification within water systems.
The second project in development is a comparison of E. coli isolated from fresh verses brackish water. Many authorities consider the quantity of bacteria a greater threat than the extent of their virulent properties. With this in mind, a project has been established to compare the virulence gene profiles of brackish versus freshwater E. coli strains local to the Chesapeake Bay. To begin, background research was conducted on common E. coli virulence genes and pathotype mechanisms. Initial samples of brackish and freshwater E. coli strains were then isolated from the Chesapeake Bay from three locations. DNA extractions were performed on these samples, followed by a PCR to identify bacterial pathogens and examine potential microbial sources, as well as to determine the presence of virulent genes known to induce diarrheagenic effects. Evidence of enterobacteria, campylobacter, V. cholera and V. vulnificus bacteria further verify that these E. coli strains have originated from fecal contamination. Potential sources of this bacteria were identified as goose or muskrat feces. Additionally, samples were cultured on selective media and both E. coli and Enterococcus ssp. were isolated, although the amount was
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