Dialogues in Health 1 (2022) 100063

Contents lists available at ScienceDirect

Dialogues in Health
journal homepage: www.elsevier.com/locate/dialog

Resistance on the rise: Assessment of antibiotic-resistant indicator organisms

in Shem Creek, Charleston, South Carolina
Brooke Emery a, Heather Fullerton b, Brian Bossak a,
⁎
a
Department of Health and Human Performance, College of Charleston, Charleston, SC, USA
b
Department of Biology, College of Charleston, Charleston, SC, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Shem Creek is a Charleston waterway well-known for numerous recreational activities such as paddle boarding, fish-
ARBs ing, and kayaking. However, Charleston WaterKeepers, a local organization, has consistently found high levels of
Public health coliform bacteria within Shem Creek. With antibiotic prescription rates surging and coastal flooding becoming an in-
Prevention creasing concern, antibiotic resistant bacteria (ARBs) have become both a public health and environmental risk. ARBs
Antibiotic resistance
can lead to the spread of antibiotic-resistant infections (ARIs) within populations. Precipitation influences bacterial
ARIs
concentrations in a body of water. Immediately after rainfall, the levels of bacteria rise tremendously. Runoff from live-
stock, sewage, and hospitals are known to contribute to the development of ARBs in bodies of water. Consistent water
testing is essential to preventing the risk and spread of ARIs and determining what additional factors contribute to the
development of ARBs in an aquatic environment. This pilot study found that precipitation was directly associated
with the levels of bacteria found within Shem Creek and validated that ARBs are present within local waterways in
Charleston, South Carolina.

1. Introduction [5]. Antibiotic resistance is a result of the transfer of genetic material be-
tween humans, animals, and the environment. Polluted environments are
Antibiotics are defined as substances that are produced by living organ- more likely to contain the genetic elements involved in the transfer of
isms and can prevent the growth of other organisms when in low concentra- antibiotic-resistant genes. The addition of fecal bacteria into the environ-
tions [1]. Recently, antibiotic resistance, the decrease in the effectiveness of ment further enhances the evolution of antibiotic-resistant genes (ARGs)
a specific antibiotic against bacteria, has become an urgent issue within the by providing genetic elements that specifically target ARGs [5]. Antibiotics
field of public health and environmental biology. Resistance genes in envi- are released into waterways from a variety of sources including human
ronmental bacteria have begun to transfer to human pathogens, contribut- waste, healthcare facilities, and the livestock industry [2,6].
ing to the increased prevalence of antibiotic-resistant bacteria (ARBs). In
addition to being a threat to natural ecosystems, ARBs are also a threat to 1.1. Antibiotic pollution and fecal coliforms
human health via ingested food or water [2]. The Environmental Protection
Agency (EPA) states that the two primary pathways of exposure to harmful Antibiotics can be transferred into the environment through the urine
water pollutants are through direct and accidental ingestion of water and and feces of both humans and domestic animals. Investigators have found
consumption of fish and shellfish from the polluted waterway [3]. This that coliform bacteria can be found in the feces of warm-blooded animals,
could include drinking water or water used for recreational purposes such the intestines of cold-blooded animals, in sediment, and on the surface of
as boating, fishing, and swimming. Annual global deaths for antibiotic- various plants [5]. A variety of intestinal bacteria are known sources of ge-
resistant infections (ARIs) are predicted to increase from 700,000 in 2014 netic elements that can accelerate the transfer of ARGs to pathogens in the
to approximately 10 million by 2050, costing $100 trillion [4]. High antibi- body [6]. Exposure levels from this mode of excretion depend on the
otic use is credited to many factors including over-prescription and ease of proportion of the population using a certain antibiotic at a given time,
accessibility through over-the-counter medication. Many bacterial species the dosage of the antibiotic, and the metabolism rate of the humans
began to develop antibiotic resistance abilities preceding human usage for and domesticated animals that make up that population [6]. Addition-
treatment of infectious disease. Additionally, every approved antibiotic ally, antibiotics in wastewater are not fully removed by water treatment
class has been met by resistance in at least one of the pathogens they target procedures which contributes to the presence of fecal bacteria in the

⁎ Corresponding author.
E-mail address: [email protected] (B. Bossak).

http://dx.doi.org/10.1016/j.dialog.2022.100063
Received 21 June 2022; Received in revised form 19 October 2022; Accepted 23 October 2022
Available online 28 October 2022
2772-6533/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
B. Emery et al. Dialogues in Health 1 (2022) 100063

environment. Residues of consumed drugs, antibiotics, and stimulants 1.4. Antibiotic significance
are excreted in urine and feces from households and hospitals and
greatly contribute to the antibiotic compounds that are found in waste- The WHO splits antimicrobial classes into three different categories:
water [7]. The main pathway that leads to antibiotic resistance is the Critically Important, Highly Important, and Important. Categorization is de-
fecal-oral route in which people ingest or consume contaminated food termined based on three different criteria: Criterion 1: the specific antimi-
or water. crobial class is the only, or one of the few available treatments for a
severe bacterial infection; Criterion 2: the antimicrobial class is used to
1.2. Primary health care treat infections caused by bacteria that are from non-human sources or bac-
teria that may acquire resistance genes from non-human sources. Critically
In 2020, healthcare providers prescribed 201.9 million outpatient anti- important antimicrobials meet both criteria, highly important antimicro-
biotic prescriptions. Primary care physicians were responsible for 64.1 mil- bials meet either Criteria 1 or 2, and important microbes meet neither
lion of those outpatient prescriptions [8]. Specifically, South Carolina has criterion. Vancomycin, azithromycin, erythromycin, ampicillin, ciprofloxa-
an outpatient prescription rate of 700/1,000 people [8]. Urinary tract infec- cin, streptomycin, imipenem are categorized as critically important while
tions (UTIs) and upper respiratory tract infections (URTIs) are the top chloramphenicol, doxycycline, and tetracycline are highly important
reasons for prescribing antibiotics within primary health care [9]. As a re- [13]. Additionally, vancomycin-resistant Enterococci are considered a seri-
sult, these two infections are the most common ARIs in primary healthcare. ous threat by the CDC [16]. Ampicillin was used as our control antibiotic
Although most URTI cases are caused by viral infections that do not require because penicillins have historically been the most prescribed antibiotic
an antibiotic prescription, physicians often prescribe antibiotics to prevent in the United States. In 2020 alone, 23.2 million antibiotic prescriptions
a secondary bacterial infection [10]. Patient pressure towards antibiotic were for penicillins [8].
prescription is an additional factor that contributes to the over- Antibiotics are organized by prioritization factors. Prioritization factor 1
prescription of antibiotics in the primary healthcare setting. In a survey (P1) signifies that a large portion of the community is affected by diseases
that evaluated physician knowledge of antibiotics, their attitudes, expecta- that have limited antimicrobial treatment options. Prioritization factor 2
tions, and their prescription practices, 75.4% of respondents reported feel- (P2) signifies high usage of that specific antimicrobial class, which favors
ing pressured to provide an antibiotic prescription if their patient expects the development of resistance. Prioritization factor 3 (P3) antibiotics are
one [11]. used to treat infections where resistant bacteria are already known origi-
nate from non-human sources. Aminoglycosides (streptomycin) are P2
1.3. Anthropogenic sources of antibiotic pollution and P3. Carbapenems (imipenem) and other carbapenems are P1, and P2.
Glycopeptides (vancomycin) is P1, P2, and P3. Macrolides and ketolides
Internationally, the livestock industry accounts for over half of all anti- (azithromycin, erythromycin are P1, P2, and P3. Penicillins (ampicillin is
biotic usage. In 2013 alone, 131,109 tons of antibiotics were used. This P2. Quinolones and fluoroquinolones (ciprofloxacin) are P1, P2, P3.
number is projected to increase to more than 200,000 tons in 2030 [4]. An- Amphenicols (chloramphenicol) C2. Tetracyclines (doxycycline, tetracy-
tibiotics are used to prevent the spread of disease and as growth stimulants cline) is C1 [13].
in livestock and other animals used as sources of food. Twenty-five to The purpose of this study was to determine if confirmed high levels of
seventy-five percent of antibiotics given to animals are excreted into the en- bacteria have a direct relationship with the presence of ARBs. We hypothe-
vironment [12]. Antibiotics are consistently detected in the gastrointestinal sized that due to the high levels of enterococci found within Shem Creek by
environments of livestock, increasing the selective pressure for bacteria in Charleston WaterKeepers, we would find a higher-than-normal number of
livestock digestive systems to acquire antibiotic resistant genes (ARGs) ARBs as well. The information found through the completion of this exper-
and increase the abundance of resistant populations [4]. Several studies iment is an important contribution to data that shows national and interna-
have shown an increase in ARBs after the application of the manure from tional trends in antibiotic resistance.
animals treated with antibiotics is added to farmland as fertilizer [6]. Stud-
ies have shown high levels of tetracyclines, fluoroquinolones, macrolides, 1.5. Experiment structure
and sulfonamides in livestock manure [12]. Livestock farms are often lo-
cated near a water source for their animals. Due to this proximity, animal Little is known about the effect of other factors, such as tidal movement
wastes runoff contaminates surface waters with antibiotic residues and rainfall levels, on the prevalence of ARBs in recreational waters. This
and fecal coliforms [7]. An expert workshop with the Food Agricultural project will directly build upon the data that Charleston Waterkeepers has
Organization of the United Nations (FAO), the World Organization for been collecting since 2009 at one of their sampling locations within
Animal Health (OIE), and the World Health Organization recognized Charleston, South Carolina. Specifically, this project focused on the consis-
that ARBs were an international public and animal health concern tent high levels of fecal coliforms found by WaterKeepers at Shem Creek
that has been fueled using antibiotics in a variety of sectors. They em- Boat Launch. This project was testing the susceptibility of the bacteria
phasized that the types of antibiotics used within livestock were often found in Shem Creek to several commonly used or critically important an-
the same, or very closely related, to those used in human healthcare. tibiotics. Understanding the influence that tides and rainfall have on
There is evidence that human health is threatened by ARBs by increas- ARBs will help develop better methods for predicting high levels of bacteria
ing the prevalence of infections, increasing severity of infections, and and ARBs within recreational waters around Charleston. Increasing the
increased treatment failures [13]. Antibiotic resistance presents health awareness of increasing levels of bacteria will aid in preventing the spread
risks for farmworkers and communities around the United States in of ARIs within the surrounding communities. Additionally, environmental
the form of occupational hazards and potential food-borne illness [7]. information such as air temperature, water temperature, and water salinity
A study found that 80.5% of feces samples collected from healthy indi- were collected along with water samples. These factors are suspected to
viduals contained ARBs, with 98% of those bacteria being E. coli. The also contribute to the transmission of both resistant and non-resistant bac-
feces and intestinal flora of healthy individuals serve as reservoirs for terial strains in an environment [6].
multi-resistant bacteria strains. These bacteria enter the environment
along with treated sewage after being treated at wastewater treatments 2. Materials and methods
plants [14]. Poor water quality is associated with stormwater runoff of
human and animal feces and other bacterial sources into a waterway 2.1. Sampling
[14]. Another study found that E. coli strains from a sewage plant that
treated both municipal sewage and hospital waste had the highest prev- The first portion of this project consisted of water sampling and water
alence of ARBs [15]. filtration for isolation of bacteria. Water was collected from Shem Creek

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Boat Landing twice a day for two weeks, once during an incoming tide and United States according to the CDC [16]. If an organism was resistant or in-
once during an outgoing tide. Sampling times were based off the South Car- termediately resistant to three or more of these antibiotics, then they were
olina’s Department of Health and Environmental Control’s (SCDHEC) Tide also tested, using the same protocol, for resistance against streptomycin,
Tables for June 2021. Sampling was conducted at the Shem Creek boat tetracycline, and chloramphenicol. Resistance was determined by measur-
landing during incoming and outgoing tides to determine the total number ing the diameter of the zones of inhibition (ZI) to the nearest millimeter.
of fecal coliforms present (see Supplemental Table #1 for exact sampling National Committee for Clinical Laboratory Standards (NCCLS) guidelines
times). Subsequently, stock cultures were prepared and tested for antibiotic were used to determine the resistance of the organisms to vancomycin,
resistance to six commonly used antibiotics using the Mueller-Hinton anti- azithromycin, and doxycycline. Ampicillin, tetracycline, ciprofloxacin,
biotic disk diffusion assay method. Along with collection of water samples, streptomycin, and chloramphenicol susceptibility was measured using
environmental factors including water temperature and salinity were mea- methodology from published studies [34–36]. Zone of Inhibition values
sured using a YSIProplus (YSI, Yellow Springs, Ohio). The height of the can be seen in Supplemental Table #2. Organisms were determined to be
tides and the air temperature for Cooper River Entrance were recorded at either resistant (R), intermediately resistant (IR), or susceptible (S) to the
time of sampling using data from [17]. Two water samples were collected antibiotics. If an organism was resistant or intermediately resistant to
in sterile collection tubes, placed directly onto ice, and transported back three or more antibiotics, it was considered a multi-resistant bacterium
to the lab within thirty minutes of collection. Samples were collected as (MRB) and sent off to be sequenced and identified. Supplemental
far down the dock as possible and submerged until the entire collection Table #2 shows the ZIs for each antibiotic.
tube was filled. Once in the lab, vacuum filtration was used to filter the
water samples. Sample water was filtered in volumes of 1mL, 2mL, 5mL, 2.4. Isolate identification
or 10mL to calculate the total fecal coliforms on MacConkey agar. The
5mL and 10mL samples were consistently too many to count (TMTC), Isolate sequences were inputted in MEGA11 to align the sequences and
while 1mL and 2mL were within the countable range of 20-80 colonies. cut them down to a maximum of 750 nucleotides. Once the sequences were
Agar plates with filters were incubated for 18-24 hours at 37°C. After the shortened and aligned, they were entered into the U.S. National Library of
incubation period, the number of bacterial colonies was counted and Medicine’s National Center for Biotechnology Information Basic Local
recorded for each volume. Total coliforms per 100mL of water was then Alignment Search Tool (NCBI BLAST) for identification [21]. The two
calculated using the formula [(# of colonies x 100)/(sample volume)]. NCBI nearest species are listed in Supplemental Table #3. Isolates were
classified by Silva High Quality Ribosomal RNA Databases to determine
2.2. Bacterial colony isolation the family of the bacteria [22].

Medium-colored, dark pink, dark purple, and metallic green colonies

were subcultured on EMB plates, while colonies that were lighter than 2.5. Statistical analysis
the medium, yellow, tan, or translucent were subcultured on hektown
enteric (HE) agar plates for 18-24 hours at 37°C. Approximately five Statistical analysis of study data was performed using SPSS software.
different-colored colonies were selected for isolation per water sample. In Correlations were assessed to determine the relationship between average
total, two-hundred and twelve total colonies were isolated on either EMB colony-forming units (CFUs) and precipitation. Three correlations were
or HE plates. Approximately five different-colored colonies were performed: Kendall’s tau, Spearman’s Rho, and Pearson correlations. A
selected for isolation per water sample. EMB agar is formulated for the Microsoft Excel spreadsheet was created to include average CFUs, precipita-
isolation of fecal coliforms due to two dyes: Eoin Y and methylene tion levels, tidal movements for each sampling time. The average CFUs and
blue. These dyes are inhibitory for gram-positive organisms and indi- precipitation levels from this document were entered into SPSS and each
cate a low pH when lactose is fermented [18]. HE agar is formulated correlation was conducted separately using bivariate correlation settings.
to differentiate Salmonella and Shigella organisms from other gram- A Pearson correlation measure the relationship between two different
negative rods. Gram-positive organisms are inhibited by bile salts, variables and ranges between -1.00 and +1.00. For a Pearson correlation
bromthymol blue, and acid fuchsin [19]. Ferric ammonium citrate to be valid, the data must be normally distributed. Once normal distribution
causes a black precipitate to form when hydrogen sulfide is produced has been confirmed, data analysis can continue. Experimental data was
[19]. Escherichia coli (E. coli) can be differentiated from other lactose confirmed to fit a normal distribution by using the Kolmogorov-Smirnov
fermenters based on its metallic-green sheen when grown on EMB (KS) test of Normality via Social Science Statistic’s webpage [23].
[18]. Therefore, each different colored colony suggested different levels Two-tailed was selected if the relationship between the two variables was
of lactose fermentation and a different species. unknown and one-tail was selected if the relationship between the two var-
iables was predicted to be either positive or negative [24]. A value of 0.00
2.3. Subculturing and antibiotic susceptibility testing means that there is no correlation between the two variables. A value closer
to -1.00 signifies an inverse relationship and a value closer to +1.00
One-hundred and thirteen colonies were isolated onto EMB agar and signifies a direct relationship between the two variables. Significance
ninety-nine colonies were isolated onto HE agar. Thirty-four (24.3%) HE level values were calculated simultaneously. A significance value below
samples and forty-two (37.2%) EMB samples grew on their designated 0.05 is considered statistically significant [25].
plates and were tested for antibiotic resistance using methodology based
off the Kirby-Bauer Disk Diffusion Susceptibility Test Protocol described 3. Results
by [20]. Mueller-Hinton plates were prepared using dehydrated media ac-
cording to manufacturer’s instructions. A sterile cotton swab was immersed 3.1. Colony-forming units
in a bacterial colony resuspension set at a standard density for inoculation
of the MH agar plates. The antibiotic discs were placed on each labeled If every volume for a specific sample was TMTC, a maximum value of
MH plate using a Self-Tamping Sensi-Disc Dispenser. 12,000 was given as the average CFUs for that sample time. The highest av-
The six antibiotics used to test susceptibility in this experiment were erage CFUs for the incoming samples was 12,000 and the lowest was 1,060.
doxycycline, imipenem, ampicillin, azithromycin, ciprofloxacin, and van- For the outgoing samples, the highest average CFU count was 12,000 and
comycin. These antibiotics were chosen based on factors such as high pre- the lowest was 536.67. On the fifteenth day, there was an average CFUs
scription levels, antibiotic class, and threat severity of antibiotic-resistant value of 12,000, the highest out of all the incoming samples. For the outgo-
organisms based on data from the Center for Disease Control. Azithromycin ing samples, the fifteenth day had the second highest average CFUs value
and doxycycline are included in the top five prescribed antibiotics in the (5366.67) compared to Day 3 which was 12,000.

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B. Emery et al. Dialogues in Health 1 (2022) 100063

3.2. Precipitation each of the sequenced samples. Fourteen of the twenty samples (70.0%)
were a part of the Pseudomonadaceae family, four (20.0%) belonged
Throughout the two-week sampling period, there was a total of 4.02 to the Enterobacteriaceae family, one (5.00%) belonged to the
inches of precipitation. The highest levels of precipitation occurred on the Xanthomonadaceae family, and two (10.0%) isolates were labeled as Unclas-
fifteenth day of sampling (1.67 inches), and there were six days that expe- sified by Silva. Therefore, it is concluded that Pseudomonas species showed
rienced no rainfall (0 inches). Precipitation was most directly related to the highest rate of resistance to the selected antibiotics. Pseudomonas
average CFUs when there was a lag of 0 days. This means that the levels protegens strain CHA0 was the only sample that was IR/R to six different an-
of bacteria within the samples was most influenced by the rain that had tibiotics. Twenty-five percent of the Pseudomonas isolates were IR/R to
occurred within the same day of sample collection. Fig. 1 shows the corre- three antibiotics, 50.0% were resistant to four antibiotics, and 16.7%
lation between the number of average CFUs and the level of precipitation in were resistant to five antibiotics.
inches. According to the lines of best fit, there is a positive correlation Of the nineteen multi-resistant isolates, there were three samples that
between rainfall and average CFUs during incoming tides, but there is a we IR/R to five antibiotics: Pseudomonas protegens strain CHA0,
negative correlation between rainfall and average CFUs during outgoing Stenotrophomonas maltophilia strain IAM, and Pseudomonas aeruginosa strain
tides. The highest level of rain experienced was on Day #13. Precipitation DSM 50071. The remaining samples were IR/R to four or less antibiotics
levels were determined using NOAA’s Climate Data Online Search [26]. and can be read about in detail in Supplemental Table #3.
The relationship between the average CFUs for both sampling times of
each day and the daily precipitation is shown by Fig. 2. Additionally, 3.5. Association between precipitation and CFU concentration
Supplemental Table #1 summarizes the precipitation and CFU data for
the sampling period. Within two of the three correlations calculated using SPSS, statistically
significant values were calculated. The statistical correlation examined
3.3. Antibiotic susceptibility and resistance were Pearson’s correlation due to the normal distribution of the data. As
seen in Table 1, the Pearson correlation coefficient between average CFUs
Of seventy-six isolates tested for antibiotic resistance, two isolates and precipitation was 0.449, which was indicated as significant by SPSS.
(2.63%) were resistant to zero antibiotics, twenty isolates (26.3%) were re- The significance level calculated by SPSS was p=0.008 between the two
sistant to one antibiotic, and thirty-two (42.1%) isolates were resistant to variables.
two antibiotics. One isolate (1.32%) was found to be R/IR to six antibiotics,
three isolates (3.95%) were R/IR to five antibiotics, nine isolates (11.8%) 4. Discussion
were R/IR to four antibiotics, and seven isolates (9.21%) were R/IR to
three antibiotics. All isolates were susceptible to CIP 5. All the isolates Fecal coliform levels have been measured within Charleston, South Car-
were also susceptible to IPM 10 except for three isolates. Approximately olina throughout the last decade; however, antibiotic resistance has not
28.9% of tested isolates were IR/R to AZM 15 and 3.95% to D 30. At the been a focal point. This data sets forth a procedure that can be used to con-
conclusion of the testing, twenty MRBs were identified. MRBs are defined tinue testing Shem Creek and other popular recreational waters for the
as a sample that is intermediately resistant (IR) and/or resistant (R) to presence of ARBs. There is little data for ARB levels within any waterways
three or more antibiotics and show significant threat to public health. in Charleston and this data can serve as the basis of new programs that work
Two of the samples were found to be duplicate isolates, resulting in only to monitor the presence and development of dangerous ARBs. A British
nineteen samples being tested with additional antibiotics. Of the nineteen study examined fecal swabs from surfers who were considered more likely
isolates that were tested with the three additional antibiotics due to their to ingest seawater than non-surfers and were found to be more at risk of car-
multi-resistance properties, approximately 63.2% were IR/R to C30, rying cephalosporin-resistant E. coli. Another study found that recreational
10.5% to S10, and 0% to TE 30. swimming may be a risk factor for UTIs and other bacterial infections [6].
These results emphasize the needs for extensive testing for ARBs and fecal
3.4. Species identification coliforms of both fresh-water environments as well as other recreational
bodies of water, such as beaches, that may experience runoff from the sur-
Supplemental Table #3 presents the sequence number, family, NCBI rounding environment. In a recent study, ciprofloxacin was found in sew-
two nearest identifications, and the specific antibiotic susceptibility of age sludge at a concentration of one milligram per kilogram. However,

Fig. 1. Linear correlation of precipitation and average CFUs with line of best fit

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Fig. 2. Average daily CFUs relating to daily precipitation

infections in a healthcare setting may be caused by a variety of things in-

Table 1 cluding contaminations of medications, intravenous fluids, and medical
SPSS statistical analysis. equipment. All but one of sample was a gram-negative organism. Addition-
Correlations ally, gram-negative organisms are known to be a cause of catheter-related
Precipitation CFUs
BSIs associated with contaminated fluids [28].

Precipitation Pearson Correlation 1 0.449⁎⁎

4.1.1. Pseudomonas putida
Sig. (1-tailed) 0.008
N 28 28 Pseudomonas putida has been found to serve as an exchange platform
CFUs Pearson Correlation 0.449⁎⁎ 1 for antibiotic-resistant genes (ARGs) and is considered a serious threat for
Sig. (1-tailed) 0.008 antibiotics resistant infections in hospital settings. This species of bacteria
N 28 28
harbors metallo-beta-lactamase genes that can increase the likelihood of
* Significant correlation. resistance to beta-lactams such as carbapenems. This specific class of
drugs are considered “last-resort” drugs for critically ill patients. In a hospi-
tal setting, P. putida often originates from contaminated fluid and hospital
ciprofloxacin-sensitive strains remained common [6]. This remained true wastewater systems [29].
for the samples collected from Shem Creek as every sample tested showed
susceptibility to ciprofloxacin. 4.1.2. Stenotrophomonas maltophilia
Stenotrophomonas maltophilia is a known cause of hospital-acquired
4.1. Public health and environmental significance of ARBs infections that occurs mostly in immunocompromised individuals.
S. maltophilia is considered an environmental bacterium, but its sources
The following bacteria were isolated from the samples and are known to and reservoirs are not always naturally available [30]. Transmission from
have an impact on the environment and/or public health: Pseudomonas person-to-person is infrequent outside of the healthcare setting. Therefore,
putida strain NBRC 14164, Stenotrophomonas maltophilia strain IAM hospital environments often act as the reservoir for the development and
12423, Yokenella regensburgei strain CIP 105435, Klebsiella oxytoca strain growth of this specific bacteria. The majority of S. maltophilia strains are
JCM 1665, and Enterobacter bugandensis strain 247BMC. The genus Pseudo- characterized by their resistance to antibiotics in the carbapenem class
monas is one of the most diverse and significant groups of bacteria. Specif- which is supported by the data shown above. Isolate 9 showed resistance
ically, the bacteria in this genus can withstand various forms of stress to both carbapenems tested in this procedure (IPM 10, VA 5). S. maltophilia
including physical, chemical, and antibacterial substances [13]. Pseudomo- is often recovered from wet environments such as sink traps, bottled water,
nas species have been prioritized by the WHO as bacteria that requires the rivers, and wells. It has also been found in a variety of foods and animals
development of new, effective drugs [16]. Pseudomonas species are urgent such as frogs, lizards, raw cow’s milk [30]. This supports the prediction
public health threats for four reasons: the produce a variety of beta- that runoff from both livestock and hospital environments may influence
lactamases that promote resistance to certain antibiotics; they act as the development of ARBs in Shem Creek.
pathogens that infect those who are immunocompromised; and they are
associated with sepsis, wound infections, UTIs, and some forms of pneumo- 4.1.3. Yokenella regensburgei
nia [13] The overexpression of efflux pump systems contributes to the de- Isolate 18 was identified as Yokenella regensburgei. Y. regensburgei is the
velopment of resistance to a wide range of drugs in Pseudomonas species sole species of Yokenella genus. It has previously been isolated from the
[27]. Supplemental Table #3 presents the sequence number, family, NCBI upper respiratory tract, wounds, knee fluid, urine, sputum, and feces
nearest identification, and antibiotic susceptibility of each of the sequenced [31]. Considering that Y. regensburgei is a known fecal coliform, it is possi-
samples. Outbreaks of Enterobacter and Klebsiella infections in a healthcare ble that this bacteria was transferred into Shem Creek due to leakage in
setting may be caused by a variety of things including contaminations of sewage systems or runoff from flooding and precipitation. It is also found
medications, intravenous fluids, and medical equipment [28]. Fourteen in intestinal tracts of insects and insect-feeding animals such as bats
percent of inpatient bloodstream infections (BSIs) were caused by gram- which could also explain its presence in the sampling waters. Infections
negative bacteria according to the National Nosocomial Infections Surveil- with Y. regenburgei are rare, but are possible and unpredictable. A study test-
lance System from 1992 to 1999. Outbreaks of Enterobacter and Klebsiella ing the susceptibility patterns of Y. regensburgei strains found that all strains

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B. Emery et al. Dialogues in Health 1 (2022) 100063

were susceptible to various beta-lactams, all aminoglycosides (including 5. Conclusion

streptomycin), chloramphenicol, quinolones (ciprofloxacin), and tetracy-
clines (tetracycline, doxycycline). Strains were found to be either resistant The prevalence of ARBs in Shem Creek suggest that high levels of fecal
or intermediately resistant to erythromycin and azithromycin [31]. How- coliforms are associated with an increased presence of ARBs. It can be con-
ever, isolate 18 only showed resistance to VA 5, AZM 15, and AM 10. cluded that areas with high antibiotic usage rates may have high prevalence
This may be because Y. regensburgei was the second species listed on NCBI of ARBs in their waterways due to runoff from a variety of sources such as
BLAST [21]. livestock facilities, hospitals, and sewage treatment plants. It can also be
concluded that precipitation has a direct relationship with the number of
4.1.4. Klebsiella oxytoca average CFUs due to the positive relationship between average CFUs and
Isolate 4 was identified as Klebsiella oxytoca. K. oxytoca is a known cause precipitation. As precipitation increases, the amount of runoff that enters
of community and hospital-acquired infections that resemble those caused waterways also increases. In frequently-flooding areas, increased precipita-
by Klebsiella pneumonia. The most common regions of the body that tion may lead to sewer overflow and an influx of sewage and waste into
K. oxytoca are found are the gastrointestinal and respiratory tract. Resistant local waterways. It is important to increase the frequency of water testing
K. oxytoca outbreaks have been seen largely in hospital environments. Envi- in frequently used and recreational waterways so that community leaders
ronmental reservoirs have been found to play a role in infection with can develop strategies to prevent the spread of antibiotic-resistant bacterial
K. oxytoca [32]. infections within their populations. These findings will allow community
members to become increasingly aware of the health risks of ARBs and em-
4.1.5. Enterobacter bugandensis phasize the negative effects of over-prescribing antibiotics on both the envi-
Isolate 17 and 18 both identified as Enterobacter bugandensis. ronment and public health. Specifically for Charleston, it is important to
E. bugandensis is known to be associated with neonate sepsis and dangerous monitor the antibiotic prescription rate as well as local precipitation levels.
bloodstream infections. Other enterobacter infections can include bacter- As of 2020, South Carolina had the 9th highest antibiotic prescription rate in
emia, lower respiratory tract infections, skin and soft-tissue infections, the United States at 700 prescriptions per 1000 persons [8]. According to
UTIs, septic arthritis, osteomyelitis, and more. This species can survive on the results of this study, South Carolina’s high prescription rates, and
skin, on dry surfaces, and in contaminated fluids, making it common in a Charleston’s susceptibility to flooding, Charleston waterways are at risk
hospital setting. E. bugandensis is known to show resistance to ampicillin, for increased levels of ARBs due to antibiotic runoff from local hospitals,
amoxicillin/clavulanic acid, ciprofloxacin, tetracycline, and various other residential and commercial sewage, and farmland. The methodology of
antibiotics [33]. In this experiment, Isolate 17 and 18 showed resistance this project has never been utilized in Charleston before the completion
to VA 5, AZM 15, and AM 10. of this study and can be used to enhance the efforts to decrease the levels
of antibiotic runoff into the environment and raise awareness to the health
4.2. Statistical correlation of average CFUs and precipitation risks of ARBs and ARIs. If more funds were available for use, this study
would be expanded to other waterways around Charleston to more accu-
A Pearson correlation was utilized due to the normality of the data. Nor- rately determine the effect that geographical location and environmental
mal distribution fit was determined using the KS test of Normality via Social surroundings have on the levels of ARBs.
Science Statistic’s webpage [23]. The correlation coefficient between aver-
age CFUs and precipitation for the Pearson correlation was 0.499, which Funding
was marked as significant by SPSS. Since this value approaches +1.00, a
positive relationship can be concluded between the two variables and a di- This work was supported by the College of Charleston’s Undergraduate
rect relationship can be confirmed between average CFUs and precipita- Research and Creative Activities Program [SU 2021-11].
tion. The significance value for the Pearson correlation was 0.008 as seen
in Table #1.
Declaration of Competing Interest
4.3. Limitations
The authors declare that they have no known competing financial inter-
This study only determined ARB levels at a single location. A more accu-
ests or personal relationships that could have appeared to influence the
rate understanding of the causes and influential factors of the development
work reported in this paper.
of ARBs in waterways could be gathered through the testing of multiple
sites. The prevalence of specific antibiotics in the water at the sampling
Appendix A. Supplementary data
location was not determined which also limits our understanding as to
why certain antibiotics showed more or less resistance compared to others
Supplementary data to this article can be found online at https://doi.
tested. The subculturing methods used resulted in only a specific type of
org/10.1016/j.dialog.2022.100063.
resistant bacteria. By utilizing a more inclusive subculturing method, it
would be possible to determine the different types of ARBs present in that
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