Determining the Prevalence of Antibiotic Resistance in Inland,

Saltwater, and Freshwater Areas

Research Group: Ishanie Choudhury, Abigail Cabusao, Andrei Datol, Deontae Roberts
Faculty: Dr. Stacy Alvares, PhD, Biology Department, Bellevue College
Row   Inland Freshwater
Abstract Methods 1 Tetracycline concentration 3μg/ml 30μg/ml 3μg/ml 30μg/ml
This research study focused primarily on the prevalence of Soil was collected from four different sites (Mukilteo Beach, Lake        
antibiotic-resistant bacteria in the environment. Our group 2 Number of colonies per 356 308 196 54
Washington, Lake Meridian, and Westwood Highland hiking trail). “countable” plate
focused on two questions. The first one was if there would be These met the testing conditions our questions posed as they 3 Volume plated 0.2mL 0.2mL 0.2mL 0.2mL
a higher number of antibiotic-resistant bacterial colonies in represented saltwater, freshwater, and inland areas respectively.  
areas near water than in inland areas. The second question The soil was first diluted through serial dilution with a dilution factor 4 Dilution of plate 1/10 2
1/10 1/10 1/10
 
was whether areas near saltwater would have a higher number of 10 to ensure that at least one plate would have colonies that 5 Dilution factor 100 10 10 10
of antibiotic-resistant bacterial colonies than areas near were separated enough to count accurately. Each dilution was then  
freshwater. To answer these questions, soil samples were plated with MacConkey Agar in petri dishes. Five plates had no 6 Total tet R
CFUs per gram 1.78 x 10 5
1.54 x 10 4
9.8 x 10 3
2.7 x 10 3

soil
collected from sites that met the conditions described above. antibiotic (NA), three had tetracycline at a concentration of 3μg/ml  
Our team then tested these samples to assess the percent of (Tet3), and another three had tetracycline at a concentration of 7 Percent tetR CFUs 14.35% 1.24% 80.32% 22.12%
tetracycline-resistant bacterial cells present in each soil 30μg/ml (Tet30). The plates were then incubated at 28° C for 96  
Table 3. Total tetracycline resistant CFUs per gram soil counted in Tet3 and Tet30 plates from inland site and
sample. To determine the number of colony forming units hours. We then counted the colony forming units (CFUs) on the freshwater site. Percent of tetracycline resistant CFUs were then calculated for both sites.
(CFUs) as well as the percent of tetracycline resistant CFUs, plates that were “countable”, or had between 30 and 300 colonies.
the soil samples underwent serial dilution, plating, and After selecting the most “countable” plates from each category (NA,
incubation. We found that inland areas had a lower percentage Tet3, Tet30) of each plate set, we determined the percent of
of antibiotic resistance than areas near water. Additionally, we tetracycline-resistant bacterial cells present in each soil sample.
discovered that areas near freshwater had a higher percentage
of antibiotic resistance than areas near saltwater. Based on
these results, we can conclude that there are more antibiotic-
resistant bacteria in areas near freshwater than in saltwater or Results
inland areas.  Row   Saltwater Freshwater Inland Freshwater Figure 1, 2, and 3. NA plate, Tet3 plate, and Tet30 plate from saltwater site
1 Number of colonies per 620 1354 248 244
“countable” NA plate Discussion
Introduction 2 Volume plated 0.2mL 0.2mL 0.2mL 0.2mL We found that areas near water had a higher percentage of tetracycline
 
Microbes influence every single system on Earth. From populations of 3 Dilution of NA plate 1/103 1/10 1/103 1/10
resistant bacteria than inland areas at both concentrations of tetracycline.
microbes in oceans, to populations inside the human gut, microbes are   We also found that freshwater areas had a higher percentage of
absolutely necessary to our health and way of life. Antibiotics kill 4 Dilution factor 1000 10 1000 10 tetracycline resistant bacteria than saltwater areas at both concentrations of
  tetracycline. Although we expected to see more antibiotic resistance in
bacteria, and with the misuse and overuse of antibiotics, humans have 5 Total CFUs per gram 3.1 x 106 6.7 x 104 1.24 x 106 1.22 x 104 areas near water than in inland areas, we were surprised by the results to
bred the existence of superbugs. The term “superbug” is used to soil
  our other question. We did not expect freshwater areas to have more
describe bacteria that is resistant to nearly every antibiotic on Earth.
antibiotic resistance than saltwater areas. Another result that surprised us
“You can acquire drug-resistant bacteria in many different ways… from Table 1. Total CFUs per gram soil counted in NA plates from inland, freshwater, and saltwater sites.
was that inland areas had a slightly higher percentage of tetracycline
taking them when they aren't necessary, from eating meat treated with
resistant bacteria than saltwater areas. A possible explanation for these
antibiotics,.. Or from an antibiotic resistant infection spread from other Row   Saltwater Freshwater
results could be that saltwater is a harsher environment than freshwater as
people,” (Tishler, 2013). In this experiment, we measured the amounts 1 Tetracycline concentration 3μg/ml 30μg/ml 3μg/ml 30μg/ml
        salt can disrupt protein folding as well as other macromolecule processes.
of possible antibiotic resistance located from a sample site inland, to 2 Number of colonies per 416 736 440 170 Another explanation is that the ocean is a much larger area than a lake or a
samples from a saltwater site, and a freshwater site because “when “countable” plate hiking area, so the density of bacteria could differ. Some challenges we
people, our pets and livestock, take antibiotics, these substances and 3 Volume plated 0.2mL 0.2mL 0.2mL 0.2mL faced involved counting the CFUs in the soil samples. Some of our plates
possible resistant bacteria are passed via sewage and septic tanks into  
4 Dilution of plate 1/102 1/10 1/10 1/10
were overgrown or fungal, rendering them uncountable. While we did have
our water systems and the seas,” (Garrett, 2016). We hypothesized that   some countable plates, even those were somewhat difficult to count as
the inland site would have a less antibiotic resistance than sites near 5 Dilution factor 100 10 10 10 there were numerous colonies. This left room for human error. The data we
water and that the saltwater site would have more antibiotic resistance   collected can be applied to more than just soil. The environment can be
than the freshwater site. This study is important because the spread of 6 Total tetR CFUs per gram soil 2.08 X 105 3.68 x 104 2.2 x 104 8.5 x 103 exposed to antibiotics through a number of ways including feces, discarded
 
antibiotic resistance genes in the environment has a potential of unused prescriptions, and waterways. Our results imply that waterways are
7 Percent tetR CFUs 6.71% 1.19% 32.49% 12.56%
eliminating Earth’s microbiome. This threatens Earth’s oxygen levels,   a primary source of the spread of antibiotics. Perhaps this information will
makes viral infections harder to treat, and is detrimental to agriculture impact how farms, hospitals, and pharmaceutical companies go about the
production rates.  Table 2. Total tetracycline resistant CFUs per gram soil counted in Tet3 and Tet30 plates from saltwater site and disposal of antibiotics.
freshwater site. Percent of tetracycline resistant CFUs were then calculated for both sites.

References
Garrett, L. (2016, September 19). Antibiotic-Resistant Bacteria and the World’s Peril. Retrieved from https://blogs.scientificamerican.com/guest-
blog/antibiotic-resistant-bacteria-and-the-world-s-peril/
 
Tishler, L. W., MD. (2013, September 17). Drug-resistant bacteria a growing health problem. Retrieved from
https://www.health.harvard.edu/blog/drug-resistant-bacteria-a-growing-health-problem-201309176677