Overview
How do students generate data for our surveillance maps? Scroll down to learn.
Student Modules
Brief Overview
Schematic Diagram of the PARE modules. Each can be completed in about 2-3 undergraduate laboratory class periods.
Students collect environmental samples of soil or water from their local environment based on their hypotheses of which areas might harbor high levels of resistance. Students then return back to their labs and perform classic microbiological or molecular biology techniques. Microbiology techniques include serial dilution and plating, colony counting and colony-forming unit (CFU) calculations. Molecular biology techniques include DNA extraction, PCR and gel electrophoresis. Students report their findings in the PARE Global Database which displays as spatial data on publicly accessible maps so students can examine their results and compare to other samples around the world. Below is a synopsis of the data-generating modules, but a collection of skill-building laboratory activities is also available. Click on the Modules tab to find them.
Environmental Field Work & Sampling
STEP 1: Students form a hypothesis about a collection site with high expected resistance. Given what they've learned about antibiotics in the environment, they select a publicly accessible location they believe will have high antibiotic resistance in soil or water samples.
STEP 2: Students collect samples from the local environment and upload sample location to the PARE Global Database. Students in teams of 4 head into the field to collect soil or water samples from publicly accessible sites. They upload their sample location and various attributes about the site using a standardized Sample Collection form.
STEP 3: Sample Upload Verification Students visit the the interactive spatial map displaying all soil and water samples to verify that their sample point correctly uploaded to the database.
Below is the sample collection form used by students in the field.
This is the sample collection survey created with Survey123 that students fill out in the field.
Photo: Dr. Teddie Rahube’s students at Botswana International University of Science and Technology
PARE Student Soil & Water Samples
Below is an Online web map showcasing the 2,400+ samples collected by PARE students globally.
The web map showcases soil and water samples. PARE students have been collecting soil samples since 2014 and the the program has recently expanded into water sampling as of spring 2024. Click on a point to learn about the students' sample sites.
PARE Soil and Water Samples Web Map - data collected by students participating in PARE.
Microbiology Part 1
Determining Percent Resistant Bacteria
Students learn how to determine the percentage of bacteria in their sample that are resistant to tetracycline antibiotic.
Tetracycline was selected for this surveillance project, in part, because it is inexpensive, easy to use, and it has a long history of use and study in agriculture ( Daghrir and Drogui, 2013 ; Zhou et al., 2017 )
Step 1: Students return to their labs and perform classic microbiological techniques starting with serial dilutions of their environmental sample.
Step 2: Students then plate their dilutions on two plate sets as follows:
- 5 - No Antibiotic plates
- 3 - Low Dose Tetracycline (3ug/ml)
- 3 - High Dose Tetracycline (30ug/ml)
In total, there are 22 plates per soil or water sample.
Step 3: Students incubate their samples
Schematic showing how student dilute and plate their samples.
Microbiology Part 2
Determining Percent Resistant Bacteria
Step 4: After incubating the samples, at least 2 students from each group analyze the growth on the plate and count the colonies.
Instructional Video - Counting Colonies
Step 5: Students then calculate the number of tetracycline-resistant colony forming units (CFUs) and percent resistance at 3ug/ml and 30ug/ml doses of tetracycline.
Definition - Colony-forming units (CFUs) are a unit of measurement used in microbiology to estimate the number of viable microbial cells in a sample that can multiply under controlled conditions
Step 6: Students enter their results into the PARE Global Database. A unique sample ID joins this new data to their original soil or water collection site. Results are automatically curated and displayed on a publicly accessible map.
Photos: 1) Undergraduate teaching assistant Serenity Beaumont at Tufts University's Lab Science Investigations summer course for high school students directed by Dr. Revati Masilamani and 2) a video clip displaying the CFU Survey123 the students fill out.
Tetracycline Resistance CFU Results
Below is a swipe map showing the results of students' analysis calculating the average Tetracycline 3ug/ml colonies vs Tetracycline 30ug/ml colonies ranging from zero to 100% resistance. Darker colors indicate higher levels of resistance per gram of soil. There are 1801 samples that passed curation tests and are displayed in the map. Swipe to see the difference in resistance between 3ug/ml and 30ug/ml.
ArcGIS Online Histogram screenshot showing the distribution of sample CFU results with 3ug/ml of Tetracycline and 30ug/ml of Tetracycle resistance. In the instant application, students can interact with the graph as well.
Note: Not all students plate serial dilutions of soil to calculate CFUs. This module is typically completed by university level students but not high school students, for biosafety reasons. The data shown on this map have undergone rigorous data curation processes which were built into the survey to eliminate incorrect data entered by students.
Molecular Techniques
Determining if resistance genes are present
Students return to the lab to complete polymerase chain reaction (PCR) testing to amplify DNA sequences and identify which resistance genes are present in their sample.
Polymerase chain reaction (PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment. Source: https://www.genome.gov/
STEP 1: Extract total genomic DNA from soil or water sample
Step 2: Use PCR to amplify antibiotic resistance genes
Step 3: Visualize using gel electrophoresis. Students check to see if the following genes are present: tetA, tetB, tetM, tetO, mcr1, ndm1, or other.
Step 4: Students enter their PCR results in a another survey form (see the survey on the right), which joins the new data back to their GPS sample location and displays it in an publicly accessible map.
Antibiotic Resistance Genes Detected
ArcGIS Online Pie Chart screenshot showing the breakdown of genes detected. The colors in the pie chart match the map symbology. In the instant application, students can interact with the graph as well.
Below is a web map showing the results of students' PCR analysis determining which genes were detected in their sample. Out of the samples that detected antibiotic resistance genes, 47.1% detected TetM alone, whille 20.2% detected TetM and TetB, and 1.7% detected TetM along with another unlisted gene.
Note: Not all classrooms calculate PCR data. This module is a favorite of high school teachers due to low biosafety concerns.
PCR sample results that passed curation steps
Thank you to Carolyn Talmadge, the Tufts Data Lab (Research Technology) and Andrew Boylan (Tufts University Senior) for support with GIS, ESRI software, and spatial analyses.
