Drug Resistance in Bacteria:Assessinga Local Enemy

Maria Cilento*, Jeffrey Mercurio*, Thomas Powers, Andrew

Lippman,
Lisa Martinelli and Manuela Tripepi
MATERIALS & METHODS

INTRODUCTION
The ability of bacteria to become antibiotic-resistant is now a crucial
concern, not only to the Center for Disease Control (CDC), but also to
medication prescribers and those who consume antibiotics. With these
concerns, the PARE Program was developed at Yale University to create a
team in which high school students and undergraduate college students
would work together to focus on this matter. The PARE program aims to
focus on the understanding of antibiotic-resistant microbes throughout
geographic locations, which, in this experiment, were the Stockton
University campus and the Cedar Creek High School grounds. The
Students from Cedar Creek High School collected soil from different
locations around the school, and Stockton University undergraduates
collected soil samples around Stockton in order to help complete the
project. The PARE program allows the analysis of the amount of
tetracycline-resistant bacteria from different sites in order to locate
resistant populations.
Antibiotics are used in humans and animals in the medical field in
order to rid the body of specific harmful bacteria. Antibiotics that are
ingested by patients proceed to be flushed out of the digestive system in
the form of waste, and are released into the environment. This release of
antibiotics into the environment permeates into areas such as soil and
local water sources. As antibiotics are released into the environment,
there are negative effects on naturally occurring bacteria. Most bacteria
will die in the presence of excess antibiotic medication in the
environment, ultimately giving rise to the selection of bacteria that have
resistance to a particular antibiotic (i.e. tetracycline). Antibiotic resistance
is a heritable trait, therefore the prospering resistant population will
continue to grow as the antibiotic-sensitive population dies off. These
resistant mutants will begin to dominate the bacterial population and
thrive in the antibiotic environment. Usually, the development of
antibiotic-resistance in bacteria occurs within short distances of hospitals,
due to the significant amount of continuous drug runoff and disposal.
This translates into a public health issue: with greater populations of
antibiotic-resistant bacteria occupying areas in which people are
constantly present, the greater amount of people will be infected with this
new strain of antibiotic-resistant bacteria. The problem here is that once
infected with a resistant bacterium, scientists must then find a novel way
to rid their peoples bodies of it. Antibiotic-resistant populations can be

RESULTS

Figure 1 (right): This picture shows

one set of samples plated by a Stockton
undergraduate. The dilution pattern is
visible from left to right. Also notable is
the diminishing number of bacteria
colonies in plates with the higher
concentration of antibiotic. Different
types of colony morphologies are
visible, swarming bacteria (red arrow)
sometimes take over the plates, leaving
a smear that covers other colonies.
NA: no antibiotic. 3: 3g/ml of antibiotic. 30: 30g/ml of
antibiotic.

Fig: 2

Fig: 1

NA

NA # of colonies

3ug/mL # of
colonies

187

124

69

68

51

124

Fig: 3

*The residential area was the only

location to show true scientific value.
This could mean antibiotic resistance is
growing in the very same place that
Stockton students live.

Location

NA # of
colonies

3g/mL # of
colonies

30g/mL # of
colonies

% Resistant for
3g/mL

% Resistant for
30g/mL

L1: Soccer field

TM

58 (103)

21 (103)

N/A

N/A

L2: Freshmen
woods

47 (102)

132 (103)

117 (102)

1419%

6106%

L3: Across A&S

TM

Swarm

Swarm

Swarm

N/A

L4: Greenbriar Lot

TM

Swarm

Swarm

Swarm

N/A

L5: Residential
Area

268 (104)

224 (104)

23 (103)

83.60%

0.80%

L6: The Oasis

336 (102)

108 (103)

178 (104)

321%

5297%

L7: Outside USC

61 (102)

141 (103)

32 (103)

2311%

1359%

Table 2: Data collected after plating the Stockton samples were equally as
unyielding as with the 20 prior locations. Resistance frequency for L1 was
unable to be calculated due to all dilutions being too many (TM) to count.
L3 and L4 were excluded from the data set as well after clearly being
invaded by swarming colonies. This allowed the assessment of only 4 sets
of plates, with only 1 of these producing reliable scientific value and
significance.

30

Figure 2 (left): 20 samples were

collected by the Cedar Creek High
School students in a local perimeter of
the school (white building). Adjacent to
the school there is a housing
development that is partially under
construction (red oval). Our plan is to
test the same site at different times
over the year in order to observe any
impact human presence may have in
altering the prevalence of antibiotic
bacteria in this area.
30ug/mL # resistant
of

Location

CEDAR CREEK HIGH

SCHOOL

The ability of bacteria to become antibiotic-resistant is a concern, not

only to the Center for Disease Control (CDC), but also to those who simply
consume prescribed antibiotic medication.Infections that are resistant to
treatment with standard antibiotics are responsible for at least 23,000
U.S. deaths per year. Antibiotics are used in humans and animals in order
to rid the body of harmful bacteria. Antibiotics that are ingested by
patients destroy a targeted bacterium, and then proceed to be flushed
out of the digestive system in the form of waste, being released into the
environment.These antibiotic pollutants can then select for growth of
resistant microbes in the affected area. Individuals generally do not
become infected through handling of soil, but the prevalence of resistant
microbes in soil can be an indicator of environmental hot spots where
increased levels of antibiotic-resistant microbes have spawned due to the
presence of commercial antibiotics in the soils and waterways. This poster
shows the implementation of the PARE(Prevalence of AntibioticResistance in the Environment)program.This program, developed at Yale
University, aims to assess the prevalence of antibiotic resistance in the
environment. To develop a comprehensive understanding of the
prevalence of antibiotic-resistant microbes across a large geographic
range requires reporting at many different sites where all values are
determined using uniform methodology. Our group was selected to be one
of the ten institutions part of the 2015 pilot PARE program. We worked
together with students at the Cedar Creek High school and also analyzed
soil samples from the Stockton campus.PARE provides an opportunity for
undergraduate laboratory instructors to engage their students in an
authentic research experience with the aim of assessing the prevalence of
antibiotic resistance in the environment while also allowing students in
high school to take part in genuine data collection.

Soil samples were transferred into a sterile collection tube without being touched by the
collectors. A smart phone was used to capture the longitude and latitude values at each
location. Data was recorded on the collection data sheet. Tubes of samples were labeled
with the date, time collected, and name of collector. These samples were obtained from
both Cedar Creek High School students and Stockton University undergraduates. The
samples collected by the high school students were then transported to Stockton
University, where serial dilutions were made for the different soils on varying amounts of
antibiotic plates. 1g of soil was mixed with 9mL of sterile water and vortexed to create a
suspension. Serial dilutions were created by transferring 100L of this water-soil
suspension to 900L sterile water in a microfuge tube labeled 1/10 2. These steps were
repeated for the following dilutions by using 100L of the 1/10 2 dilution to create the 1/103
dilution and 100L of the 1/103 dilution to create the 1/104 dilution. One nutrient agar plate,
one 3g/mL-tetracycline plate and one 30g/mL-tetracycline plate were labeled for each
dilution. 100L of each dilution was plated for each different type of plate. Plates were then
wrapped in parafilm and allowed to incubate at 28C for 72 hours. The serial dilutions and
plating were completed by Stockton University undergraduates. CFUs (colony forming
units) were then counted by Stockton Undergraduates and pictures were taken of all plates.
Numbers found here were used to calculate the frequency of tetracycline resistance in soil
samples taken at different locations. Numbers were uploaded to a main database in order
to compile all results. The total cell count for each plate was found by putting the [total
number of CFUs that grew on each antibiotic plate] over [the dilution factor] [0.1mL of
plated solution]. The resistance frequency was found by putting the [total number of cells
calculated on the antibiotic plates] over [the total number of cells calculated for the NA
plates] per dilution factor.

Figure 3: 7 samples
were collected around
the Stockton University
academic
and
residential
campuses.
Soil was collected by
undergraduate
students. Locations 1
through 7 are labeled
by blue pins and white
text.
Locations
are
listed in Table 2 below.

STOCKTON
UNIVERSITY

ABSTRACT

% Resistant for 3ug/mL % Resistant for 30ug/mL

colonies Note: The 20 separate locations in this data set were not

indicated on this poster to avoid cluster on the map.

62.40%

15.6%

75.0%

13.0%

176

35

140.0%

28.0%

96

66

68

68.75%

70.8%

Swarm

Swarm

Swarm

N/A

N/A

239

91

12

38.0%

5.0%

384

105

140

10.10%

134.6%

TM

Swarm

Swarm

N/A

N/A

21

11

13

52.0%

61.9%

10

177

165

67

93.0%

37.0%

11

84

63

14

7500%

1666.0%

12

320

277

205

85.65%

64.0%

13

372

108

35

28.80%

0.9%

14

300

152

132

50.70%

400.0%

15

36

118

41

32.78%

11.4%

16

420

296

120

14.20%

576.9%

17

112

180

174

1607%

1553.0%

18

268

155

47

57.84%

N/A

19

312

34

10.80%

1.6%

20

528

253

174

48.0%

48.0%

Table 1: Data obtained by Stockton undergraduate students after plating the samples
collected around Cedar Cedar Creek High School. Some students were unable to count
(swarm) or calculate (N/A) due to mistakes in dilutions and resulting overgrowth.
Dilution mistakes also account for some of the frequencies above reaching over 100% of
the population, which is of course not possible. The yellow-highlighted rows in the table
represent samples that were successfully plated, diluted, and calculated for using the
correct procedure. It is seen that resistance frequencies reaching over 15% could possibly
be inhabiting these local areas.

CONCLUSIONS & FUTURE DIRECTIONS

The PARE program allows for screening of antibiotic resistant bacteria in soil, while
also allowing undergraduate students to take part in genuine scientific data
collection and also engaging high school students.
Our group was one of ten chosen to be part of the PARE pilot program. We decided
to study the tetracycline resistance present within Stocktons campus and around
the Cedar Creek High school. Our aim is to repeat sampling at the same sites over
time in order to gain accurate measures and check the difference in antibiotic
resistance bacteria intermittently.
Of particular interest is the location around Cedar Creek that will experience an
increasing amount of human activity as the housing development approaches
completion. We hope to track the impact of human residency on the presence of
antibiotic-resistant bacteria on this site. We are planning to repeat
theassessmentin spring of 2016.
During plating, it is possible that other bacteria will invade the plates, sometimes
creating a characteristic pattern on the agar. Swarming colonies made much data
collection impossible, sometimes by taking over an entire plate. This accounts for
some error in accuracy due to the loss of colony counts for these plates.
Being the first time we implemented the PARE program, we also experienced some
inevitable difficulty with some of the project (i.e. swarming bacteria). Issues that
need
to
be
addressed
also
include
technique
training
regarding
dilutiontechniquesand plating techniques. These are areas that we will adjust as
we implement the program in further years, in order to create a better experience
for all students involved.
In the future, selection and genotyping of the bacteria isolated through the PARE
program will allow us to gain an even clearer picture of which populations of
Acknowledgments
bacteria are becoming more antibiotic-resistant.
We thank Dr. Carol Bascom-Slack, from Yale University, for her assistance and for providing the resources used to
work on this experiment.
As well as Dr. Karen Hogan, at the University of Pennsylvania for providing lab space and guidance.
* These authors contributed equally to this work