Introduction

The development of antibiotics has been one of the most successful advances for modern human health (Martinez 2009). However, with the expansion of urban areas and anthropogenic activities such as development, recreation and resource use on the rise, antibiotics are being used more frequently in domestic, medical, and agriculture settings. Because antibiotics are not readily biodegradable and current water treatment facilities lack the ability to remove these drugs from waterways, the result is the evolution and persistence of antibiotic resistant bacteria in water sources (Mohanta et. al, 2014, Zheng et al., 2017). Aquatic sources of antibiotic resistant bacteria are of particular interest, because the spread of antibiotic resistance in natural ecosystems has been minimally studied (Pei et al., 2007). In addition, waterways provide an adequate distribution medium for antibiotic resistance in bacteria through nutrient availability and dispersal pathways (Ouyang et. al, 2015). Although antibiotic resistant bacteria are present naturally, their occurrence is heightened in clinical, industrial, and communal wastewater due to a constant low level of antibiotics present that allows for a heightened development and transfer of resistant genes (Czekalski et. al, 2012). Based on this information, there is strong evidence to suggest that increased population growth and the resulting impacts on surrounding ecosystems translates to increased antibiotic resistance levels above normal background resistant rates (Ouyang et al., 2015).

Understanding the link between urbanization and antibiotic resistance is important as human populations continue to increase and cities spread into new areas, potentially polluting ecosystems with antibiotics. In the next few decades, the population of Utah is expected to grow at more than double the national rate, reaching a projected 5.5 million people by 2065 (Perlich et al., 2017). The Salt Lake Valley maintains the most concentrated population accounting for 33% of Utah’s total population (Salt Lake City Population 2017). As populations increase, natural ecosystems are experiencing the impacts of rising urbanization on stream and river characteristics (Short et. al, 2005). The occurrence of antibiotic resistant bacteria threatens human health because the current advancement of new drug technology cannot keep up with the pace of antibiotic resistance (Storteboom et. al, 2010). Understanding the link between urbanization and antibiotic resistance is important, because as the human population continues to grow, increased pollutants--including antibiotics--will unmistakably make their way into our natural ecosystems. The combination of urbanized areas in the Salt Lake Valley located in close proximity to the less-urbanized mountainous areas/canyons of the Wasatch Front provide a strong urban and non-urban contrast for this ecological study.

Our study aimed to analyze the effects of urbanization on antibiotic resistance found in streams running through the Salt Lake Valley compared to less urbanized headwater sources. We plated samples from four Wasatch Front streams in antibiotic-treated nutrient agar and plates with no antibiotic treatment as a control using similar agar preparation and plating techniques as comparable studies (Esiobu et al., 2002). Visible growth of bacteria colonies indicated antibiotic resistance. The proportion of bacteria that survived the antibiotic treatment in comparison to the control treatment quantified the amount of antibiotic resistance observed.

Previous studies have focused on the effects of urbanization on freshwater ecosystems relating specifically to nutrient or chemical parameters, showing that urbanization increases levels of phosphorus, nitrogen, nitrate, potassium and ammonia, as well as presence of heavy metals or pesticides (Medeiros et. al, 2016). The current study will be the first to analyze antibiotic resistant bacteria presence and persistence in the Salt Lake Valley, and may reveal a positive relationship between urbanization and antibiotic resistance. Experiments conducted in China and Colorado support the hypothesis that urbanization translates to higher antibiotic resistance present in waterways (Ouyang et al. 2015, Storteboom et al. 2010). Due to the high levels of anthropogenic activity and urbanization in the Salt Lake Valley, we hypothesize that the sampling sites in urbanized sections of the streams will contain higher proportions of antibiotic resistant bacteria than the sampling sites in upstream, non-urban locations of the same streams The results of this study will increase the understanding of the relationship between urbanization and antibiotic resistance and inform local policies, water treatment and runoff management to address antibiotic resistance in the Salt Lake Valley.

Methods

Study area and Sampling sites

Our study was conducted using four of the major streams in the Wasatch Mountain Range, near Salt Lake City, Utah: Emigration Creek, Millcreek, Little Cottonwood Creek and Big Cottonwood Creek. Each stream originates with headwaters in a non-urbanized canyon area and flows down through the Salt Lake Valley where urbanization increases. Water samples were collected at accessible points upstream (nonurban) and downstream (urban) in each stream (Table 1). The nonurban upstream sampling locations were determined by accessibility and by approximate distance from the city center. The urban downstream sampling areas were collected from accessible green spaces located in urban areas in the Salt Lake Valley.

Table 1. Sample site locations, recorded using GPS latitude and longitude coordinates. Little Cottonwood upstream samples were taken from the lower part of Snowbird Ski Resort, downstream samples from Murray Park. Big Cottonwood upstream samples were taken from an accessible reach below Solitude Ski Resort up Big Cottonwood Canyon and downstream samples from Big Cottonwood Park. Millcreek upstream samples were collected at the winter gate and downstream samples from Evergreen Park. Emigration samples were collected upstream at Upper Kilyon Creek and downstream on the campus of Westminster University.

Site Name	Location
Little Cottonwood Creek Upstream	40°34′52″N 111°39′23″W
Little Cottonwood Creek Downstream	40˚39.508’N 111˚52.804’W
Big Cottonwood Creek Upstream	40˚38.720’N 111˚40.247’W
Big Cottonwood Creek Downstream	40.6799° N, 111.8577° W
Millcreek Upstream	40˚46.608’N 111˚42.555’W
Millcreek Downstream	40˚41.728’N 111˚49.618’W
Emigration Creek Upstream	40˚47.870’N 111˚42.602’W
Emigration Creek Downstream	40.7315° N, 111.8552° W

We collected three 50 mL water samples from pools located 100 m apart from each upstream and downstream site in 50 mL sterilized and labeled centrifuge tubes. The three samples were analyzed as separate collection sites. The water samples were collected from pools within 12 inches of the creek edge at all sample sites. Samples were collected between November 7, 2017 and November 16, 2017 on days with similar weather (sunny, mild conditions) at approximately the same time of afternoon to standardize collections. However, samples collected from upstream and downstream Emigration creek were collected in windy, warm and stormy weather on the same date. All water samples were transported to the laboratory within two hours of collecting, stored in a fridge, and plated within six hours of collection.

Plating Procedure

To determine the concentration of tetracycline to use for testing antibiotic resistance, we conducted a pilot study with three concentrations of tetracycline-treated plates (tetracycline hydrochloride T8032, 20 mg, Sigma Aldrich, Inc., Milwaukee, WI). These concentrations were obtained from previous studies (Duff 2017). Results from the pilot indicated that a concentration of 15 µg/mL tetracycline was sufficient for antibiotic treated plates due to no observed growth on any of the antibiotic containing pilot plates, and water samples were not diluted for the experiment. We used tetracycline because it is known to be a resilient antibiotic in the environment. Therefore, it lasts longer, spreads further, and accumulates to higher concentrations when released into the environment (Duff 2017). Tetracycline powder was stored at -20˚C until it was dissolved in 70% ethanol in corresponding proportions and filtered into ­­­­­­autoclaved Nutrient Agar Premix (Teknova, USA) using sterilized filters.

Approximately 10 µL of water from each sampling site was pipetted and spread on one control plate (nutrient agar, no tetracycline) and three 15 µg/mL tetracycline plates for a total of four plates (one control and three tetracycline treated plates) per sample site. Plates were placed in an incubator at 30°C and were monitored for growth every 24 hours for a 72 hour growth period, using pictures for analysis by ImageJ (Ouyjang et. al, 2015).

Increased Antibiotic Concentration Plating and Varied Media Plating Procedure

To learn more about what characterization of bacteria was antibiotic resistant and test the threshold of the resistance, we plated resistant bacteria on plates with higher tetracycline concentrations, as well as on Mannitol Salt Agar and Eosin Methylene Blue plates to characterize types of resistant bacteria present. Resistant bacteria was spread on new 15 µg/µL tetracycline-treated experimental plates and allowed to grow for 48 hours to maximize the abundance of resistant bacteria colonies present to conduct further tests on their resistance. Resistant bacteria were exposed to an increased antibiotic concentration of 30 µg/µL.

To characterize bacteria colonies as either gram negative and gram positive, we plated colonies on Mannitol Salt Agar (MSA) and Eosin Methylene Blue (EMB) media plates. We recorded and photographed growth patterns on all plates. These additional procedures were conducted to further describe and characterize antibiotic resistant bacteria, and to determine strength of resistance beyond minimal tetracycline concentrations, but colony forming units (CFUs) were not analyzed by ImageJ or statistically tested.

Image J Analysis and Statistical Analyses

Colony forming units (CFUs) were determined using ImageJ software and amount of antibiotic resistance was quantified by dividing CFUs formed on tetracycline treated plates by CFUs on control plates. Means, standard deviations, and percentages of all data were analyzed using both Excel 2010 and RStudio (Microsoft Office 2010, Microsoft, USA, R 3.1.0, The R foundation for Statistical Computing Vienna, Austria). To analyze the difference between upstream and downstream overall antibiotic resistance, a Wilcoxon rank sum test was performed after parametric assumptions were not met due to the result of a Shapiro-Wilk test. Individual upstream and downstream reaches were tested for difference in antibiotic resistance using the following tests: Little Cottonwood, Welch Two Sample t-test (normality found through a Shapiro-Wilk test), Big Cottonwood and Emigration, Wilcoxon rank sum test (non-normal based on a Shapiro-Wilk test). Millcreek samples had no bacterial growth on any tetracycline treated plates, therefore no statistical tests were run. These tests were chosen to test the difference between mean values of antibiotic resistance found in upstream and downstream reaches of Wasatch Front streams.

Results

We assessed the relationship between urbanization and antibiotic resistance in four Wasatch Front streams by analyzing the ratio of CFUs in tetracycline treated plates (15 µg/µL) compared to CFUs found on control plates, at non-urban (upstream) and urban (downstream) locations. All control plates exhibited extensive bacteria growth, and were used as a baseline of total bacteria found in each water sample. Antibiotic resistant CFUs were found on tetracycline treated plates from water samples in three of four streams (Little Cottonwood, Big Cottonwood, and Emigration creek), in both upstream and downstream reaches. No bacteria colonies were counted on tetracycline treated plates for upstream or downstream reaches of one creek (Millcreek).

Abundance of resistant bacteria

The mean percent resistance of bacteria in all downstream samples overall was 0.13% and the mean percent resistant of bacteria in all upstream samples overall was 0.23 %. To test for the difference between the means, a Shapiro-Wilk test was first performed on all data before statistical testing to assure that parametric assumptions were met. Antibiotic resistance in the upstream and downstream reaches of all combined stream sites was found to be non-parametric, so a Wilcoxon rank-sum test was performed. The mean percent resistance of bacteria between the upstream and downstream samples was found to be insignificant (p=0.64) (Figure 3). However, there was a significant amount of variance in the data due to some plates containing zero growth and some plates containing two to five antibiotic resistant bacteria colonies (upstream standard deviation 0.30, downstream standard deviation 0.19) (Figure 3).

Figure 3. Mean percent of resistant bacteria in upstream (non-urban) and downstream (urban) reaches of Wasatch Front streams. Boxes represent mean percent of antibiotic resistant bacteria in combined upstream sample sites and in downstream sample sites from the four streams analyzed. Error bars all extend to zero. Across the Wasatch Front, there is no significant difference between average upstream and downstream antibiotic resistant bacteria (p=0.64).

A similar result was found when comparing upstream and downstream resistant bacteria for each individual stream (Figure 4). Millcreek’s upstream and downstream percent resistance was found to be identical with no growth present on any of the experimental plates. Therefore, no statistical test was needed to prove against the difference between zero and zero (Figure 4). Big Cottonwood and Emigration stream bacterial growth was non-parametric data (based on a Shapiro-Wilk test), and so a Wilcoxon rank-sum test was performed for both data sets. Emigration creek’s upstream resistance was found to be 0.69% whereas the downstream resistance was found to be slightly lower at 0.51%, but the difference between resistance was not significant determined by the Wilcoxon rank sum test (p=0.077) (Figure 4). Big Cottonwood Creek upstream resistance was found to be 1.060% which was lower than the downstream resistance 1.180% , but the difference was found to be insignificant by the Wilcoxon rank sum test (p=0.18) (Figure 4). Little Cottonwood stream data was found to be normal through a Shapiro-Wilk test, so a Welch Two Sample t-test was performed. Little Cottonwood Creek upstream resistance was 0.91% which was higher than the downstream resistance of 0.21%. However, the difference was still found to be insignificant by a Welch Two Sample t-test (p=0.21) (Figure 4).

Figure 4. Mean percent of resistant bacteria in upstream (non-urban) and downstream (urban) reaches of each Wasatch Front stream. Boxes represent average of mean resistant bacteria in upstream sample sites and in downstream sample sites for each of the four streams analyzed. Error bars all extend to zero. There is no significant difference between the average antibiotic resistance in upstream and downstream reaches of Millcreek, Little Cottonwood (p=0.21), Big Cottonwood (p=0.18) and Emigration (p=0.07) streams (p>0.05 for all streams).

Increased Resistance and Diversity of Resistant Bacteria

Bacteria from Little Cottonwood upstream and Emigration downstream samples successfully responded to the increased concentration of tetracycline and grew on plates of 30 µg/µL tetracycline. Little Cottonwood Upstream and Emigration downstream resistant bacteria samples were analyzed further on MSA and EMB media plates. Little Cottonwood and Emigration antibiotic resistant colonies grew on the EMB plates and therefore were characterized as gram negative bacteria, because they produced variable dark purple and black growth. These samples did not produce a brilliant green growth and consequently were determined to not be E.coli. The second media, MSA, was only responsive to the Emigration stream samples. The Emigration downstream water sample was also characterized to contain antibiotic resistant gram positive bacteria. Because Emigration downstream samples contained both resistant gram positive and gram negative bacteria, we determined that there were multiple colonies of resistant bacteria isolated from these samples. Little Cottonwood stream samples were unable to ferment the mannitol. Therefore, they were not determined to contain gram positive bacteria, only gram negative bacteria. These results were not statistically analyzed, only noted and suggested in terms of future studies.

Discussion

The importance of studying antibiotic resistance in aquatic pathways is crucial as humans consume antibiotics increasingly and release antibiotic excess into ecosystems. Antibiotic resistance that develops in aquatic ecosystems may transfer to the human environment because of exposure and subsequent genetic variation (Martinez 2009). Once introduced to humans, the development and persistence of antibiotic resistant bacteria becomes a threat to human health (Van Boeckel 2014). Bacteria that develop resistance to antibiotics compromise the success of the initial treatment (Davies and Davies, 2010). Understanding the consequences and causes of antibiotic resistance is important to ensure the success of antibiotics as a successful treatment.

Recent research has linked the effects of urbanization to increased antibiotic resistance. Because water quality is a growing concern and urbanization is expanding in the Salt Lake Valley, assessing the presence of antibiotic resistant bacteria in Wasatch Front water sources is important (Ouyang et. al, 2015). Antibiotic release into waterways may come from a variety of sources, including municipal wastewater, agriculture or medical settings. Previous studies have found antibiotic contamination and the presence of antibiotic resistant bacteria in post-wastewater treatment runoff (Watkinson et al. 2009, Batt et al. 2006, Le-Minh et al., 2010). If wastewater treatment processes fail to remove antibiotics from aquatic pathways, following the distribution of these drugs and resulting antibiotic resistance becomes crucial.

Our study aimed to analyze the effects of urbanization along the Wasatch Front on antibiotic resistance, comparing upstream (nonurban) and downstream (urban) water samples for colony growth on tetracycline treated plates. Antibiotic resistant colonies grew on tetracycline treated plates from upstream and downstream water samples in Little Cottonwood, Big Cottonwood and Emigration streams. We did not detect antibiotic resistance in either upstream or downstream samples from Millcreek stream. Based on two-sample statistical tests, the null hypothesis was not rejected for all Wasatch front streams in total as well as individual streams, which means there was no significant difference between antibiotic resistance in upstream or downstream water samples. Therefore, we found that urbanization has no significant effect on the presence of antibiotic resistant bacteria. Compared to the results of similar studies, our results are worth further contemplation.

Sample site differences in our study may account for differing results found in other studies. Water flowing from the top of Little Cottonwood, Big Cottonwood, Millcreek and Emigration canyons and down through the Salt Lake Valley does not come in contact with agricultural wastewater, a common source of antibiotic use and release. Hospitals and other medical institutions often release antibiotics in high concentrations, and little attention was given to this source in our study (Watkinson et al. 2009). Additionally, Little Cottonwood and Big Cottonwood streams go through a water treatment plant near the mouth of the canyon, on the urbanization boundary before water is released through the Salt Lake Valley. Both of these canyons also have a history of mineral extraction and currently have high traffic due to outdoor recreation use, which could contribute to the occurrence of antibiotic resistance found in upstream reaches. Finally, bacteria may develop antibiotic resistance in unperturbed ecosystems, and with no selective pressure will not persist, which may explain why antibiotic resistance was detected in small amounts both upstream and downstream (Mohanta 2014). These parameters can explain why no significant difference was found between urban and nonurban antibiotic resistance in our study. This may warrant a more holistic approach to assessing antibiotic resistance in Wasatch Front Streams in the future. Studies could focus on the history of how these areas were used and length of residential exposure, to show why antibiotic resistant bacteria are just as prevalent in downstream and upstream reaches in the waterways that were measured. Additionally, determining sources of the largest contributors of antibiotic waste would also be useful to direct focus on specific waste management programs.

Further identification tests revealed that Little Cottonwood stream has some gram-negative antibiotic resistant colonies of bacteria. We also found that colonies from Little Cottonwood samples were resistant above the 15 µg/mL threshold, and are resistant to at least 30 µg/mL of tetracycline. This information supports that bacteria found in Little Cottonwood stream is resistant to tetracycline above minimum antibiotic resistance standards (Esiobu et al. 2010). However, colonies grown on additional testing plates were from water samples upstream, and additionally from an area designated as a watershed, with heavier restrictions placed on contaminants and area use. This may indicate an additional source of antibiotics coming from upstream Little Cottonwood Canyon.

We also tested Emigration stream CFUs beyond original tetracycline treatments, and identified both gram-positive and gram-negative resistant strains of bacteria. Bacteria from Emigration stream was additionally resistant to a higher concentration of 30 µg/mL tetracycline, above minimum antibiotic resistance standards. Because both gram-negative and gram-positive strains were identified, we can determine that more than one type of bacteria has developed antibiotic resistance in Emigration stream. The implications of these findings are that antibiotic resistance in Emigration stream is not limited to one strain of bacteria, and may be spreading.

Although no significance was found relating urbanization and antibiotic resistance in Wasatch Front streams, the results of this study still yield important implications. Our study found antibiotic resistant strains of bacteria in both upstream and downstream reaches of three out of four streams sampled, which carry health consequences if these strains persist and expand. Even if antibiotic resistant bacteria detected were not selected for due to the presence of antibiotics, the predicted increase in Salt Lake Valley population and urbanization may introduce antibiotics to aquatic systems and advance antibiotic resistance (Perlich et al., 2017). Additionally, because we found multiple strains of antibiotic resistant bacteria in Emigration stream, special attention should be paid to monitoring the release of antibiotics and water treatment processes. The downstream waterways we tested were physically and chemically treated by water treatment plants to remove contaminants, but we did not observe a significant reduction in antibiotic resistance post-treatment.

Subsequent studies may analyze water samples with agricultural runoff for antibiotic resistance, and go into more depth with the comparison of upstream and downstream reaches of Wasatch Front streams by testing more locations. Testing for multiple resistance by plating water samples on multiple types of antibiotic plates may reveal the extent of antibiotic resistance in the Salt Lake Valley, as well as testing resistant strains against higher concentrations of antibiotics. Utilizing different methods to identify antibiotic resistant bacteria, such as different plating methods or PCR may help target specific sources of antibiotic release. Keeping antibiotic use and release in check may help slow the development of antibiotic resistant bacteria, and prolong the use of this important treatment in human disease.

The rejection of our hypothesis brings up additional questions as to why antibiotic resistance was detected equally in urbanized and non-urban reaches of Wasatch Front streams. This could indicate additional factors causing antibiotic resistance apart from urbanization. Additionally, antibiotic resistance may develop as a mutation in bacteria without selection imposed by antibiotics, therefore our methods may have picked up on antibiotic resistant bacteria occurring naturally (Mohanta 2014). If this is the case, other urbanizing areas may have strains of antibiotic resistant bacteria without the selective pressure from presence of antibiotics, and if development continues without concern for release of antibiotics into ecosystems, these few strains may multiply and flourish. Additional limitations to our study include the short experimental time period, the temporal restraints of when the samples were collected, the small replicates of stream samples collected, the small replicates of plates for stream samples, the location of where the samples were collected, and the analysis of plate bacterial count.

Studying the relationship between antibiotic release into ecosystems and the development of antibiotic resistant bacteria is important on a global perspective. As developing countries gain access to antibiotics and use them frequently, concern over antibiotics released into aquatic pathways due to incomplete water treatment processes is important to consider. Bacteria can develop antibiotic resistance without the presence of antibiotics but if introduced to antibiotics, selection for resistance will be prominent. Globally, Salt Lake City can be used as a model for others locations conducting urbanization studies due to its highly urbanized areas and its close proximity to the Wasatch Mountains. It’s possible that human technology will be unable to keep up with the evolution and spread of antibiotic resistance, and the loss of this important treatment will become a global health concern.

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Environmental Science / Biology Student

Salt Lake City, Utah

Rosanise enjoys many outdoor activities including climbing, skiing, and hiking. During this research project she enjoyed the field aspect as well as compiling and analyzing the data she obtained. Producing an entire research manuscript was also a highlight of this research project for Rosanise.

Hannah is an identical twin and immerses herself in veterinary medicine and wildlife ecology. She also loves climbing, running and biking. She enjoyed conducting this research because of the applicability of the project to the Salt Lake Valley and appreciated the chance to compare rural and urban variation in reaches of each stream and resistance of the bacteria. The most challenging part of this research for her was not having a baseline of the antibiotic resistance previously conducted in the Wasatch area to which to compare results.