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EFFECT OF (NH4)2SO4 ON RESISTANCE OF E.

COLI K12

Effect of Ammonium Sulfate on the Resistance of Escherichia coli K12 to Amoxicillin


Maya H. Patel
Freehold High School
Medical Sciences Learning Center: Proposal Paper

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

Effect of Ammonium Sulfate on the Resistance of E. coli K12 to Amoxicillin


Since the start of the modern antibiotic era, antibiotics have been used to treat infectious
diseases. Antibiotics such as penicillin target bacterial structures to inhibit growth. However,
researchers agree that antimicrobial resistance (AMR) is a result of the widespread use of
antibiotics (Aka & Haji, 2015). Infections caused by resistant bacteria pose a public health threat
because they are harder to treat, thus becoming more fatal towards a patient. (Aminov, 2010).
Resistant bacteria are harder to treat because they have a higher minimum inhibitory
concentration (MIC), which is the concentration of antibiotics needed to inhibit growth of the
bacteria after overnight incubation (Andrews, 2001). AMR is a natural phenomenon that will
always be prevalent, which is why research on AMR and its causes is necessary.
Antibiotics have also been used in the agriculture and livestock industries to prevent
diseases in animals (Cai, Fang, Wang, & Yu, 2014). As a result, antibiotics are released into the
environment with little regulation or prevention. This causes environmental stress and natural
selection to select for resistant bacteria (Carvalho, de Oliveria, Frazzon, Medeiros, & Van Der
Sand, 2015). For example, previous researchers studied the resistance of Gram negative isolates
from the Diluvio Stream and found that a significant number of isolates were resistant to
penicillins and cephalosporins, antibiotics used in both human and veterinary treatment
(Carvalho, et. al, 2015). Unfortunately, AMR can also be induced by non-antibiotic pollutants.
Other studies have found that bacteria exposed to heavy metal pollution and water disinfection
byproducts exhibited resistance against several antibiotics (Aleem & Malik, 2010; Dan, Gu,
Miao & Siyu, 2016). These pollutants induce the selection of resistant bacteria and play an
antibiotic resistance-role. Since a non-antibiotic pollutant can cause resistance, more
investigations on environmental AMR are needed.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

Inorganic nitrogen fertilizers such as ammonium sulfate are used in the agriculture
industry to enrich the soil. While nitrogen fertilizers can increase plant productivity, it can leach
from the soil and pollute surface water (Mengel, Johnson, and Vitosh, (1995); Blair, Coolon,
Herman, Jones & Todd, (2013); Stokstad, (2016)). Previous studies prove that long term
nitrogen enrichment alters the bacterial diversity of tallgrass prairie soil (Blair, et. al, 2013), but
it is unclear if nitrogen enrichment induces natural selection. This study plans to ease the
situation of AMR by seeing if ammonium sulfate fertilizers play an antibiotic-resistant role.
Since there is no way to stop the growth of AMR, the only solution is to recognize which
chemicals contribute to the problem, and reduce the use of these chemicals.
Therefore, the purpose of this present study is to examine the effect of ammonium sulfate
on the resistance of Escherichia coli K12 to amoxicillin. Susceptible E. coli K12 will be exposed
to high (up to MIC) and low (sub-MIC) concentrations of ammonium sulfate, and resistance will
be measured by observing the difference in MIC before and after treatment. The null hypothesis
is that ammonium sulfate has no effect on the resistance of E. coli K12 to amoxicillin. The
alternate hypothesis is if the prevalence of ammonium sulfate is related to the resistance of E.
coli K12 to amoxicillin, E. coli strains exposed to high and low concentrations will develop a
higher MIC.

Materials
The entity of this experiment is E. coli K12. Luria-Bertani (LB) agar will be needed to
grow the strains of E. coli. The dehydrated LB base is needed for this experiment because some
agar will have concentrations of ammonium sulfate. 500 grams (One container) of ammonium

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

sulfate is needed, and one container of amoxicillin is needed. Several inoculating loops will be
needed to transfer bacteria throughout the experiment. Finally, petri dishes and an incubator will
be needed.
Methods
E. coli K12 will be exposed to different concentrations of ammonium sulfate. These
methods are modeled after an experiment in where the researchers exposed E. coli K12 to
multiple cycles of various water disinfection products (Dan, et, al, 2016). Before the experiment
can take place, pretrials will be performed to determine the MIC of E. coli K12 for ammonium
sulfate and amoxicillin. To determine the MIC, the disk diffusion method will be used (Lorian
1980). After bacteria are plated on LB agar, disks containing concentrations of antibiotic or
ammonium sulfate will be placed in the center of the plate. After overnight incubation, the zones
of inhibition will be measured. These pretrials will be conducted through trial and error in order
to find the MIC for both chemicals. After these concentrations are determined, the high
concentration of ammonium sulfate will be one-half of the MIC, and the low concentration of
ammonium sulfate will be 10mg/L. (These concentrations are not final because the high, low,
and MIC concentrations are determined through trial and error.) E. coli K12 will be streaked on
Luria-Bertani agar plates and allowed to grow for 16 hours. One colony will be selected and
inoculated into a tube with 4.5mL of LB broth to be used as the isogenic seeds of the
experiment. The purpose of this is to provide an initial group of bacteria that have not been
exposed to any chemicals. 0.5 mL of isogenic E. coli and 4.5 of LB with one-half of the MIC of
ammonium sulfate will be incubated at 37C for 24 hours. Then, the 0.5 mL of E. coli will be
subcultured into 4.5 mL of LB with ammonium sulfate. 40 subcultures will be made, along with
a control of E. coli selected in only LB medium. Additionally, 10mg/L will be used for the low

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

concentration.. After 40 cycles of both high and low concentrations, the cultures will be serially
diluted on LB agar and allowed to grow for 16 hours at 37C. 30-40 clones will be selected,
along with the control culture by streaking them onto LB agar for 5-6 hours at 37C. The MICs
of E. coli will be determined against amoxicillin, and will be determined as the concentration of
antibiotics that inhibits 90% of growth in LB.
Results
A two sample t test will be used to compare the mean MIC of each treatment group vs its
control group. The treatment groups are the high concentrations of ammonium sulfate and the
low concentrations of ammonium sulfate. The two sample t test will determine if the difference
in MICs is statistically significant. If the difference is statistically significant, it will indicate
selection for resistance. To do this test, calculate the test statistic:

Using a standard table of t-values, see if the p-value is less than 0.05. If it is less than 0.05, it is
statistically significant.

Discussion
The results of this study will be evaluated in light of the hypotheses. If the results show a
significant change in MIC, ammonium sulfate increases the resistance of E. coli K12.
Limitations include that this experiment only applies to E. coli and amoxicillin. This
research does not apply to all different types of bacteria and antibiotics. Further research can
show the effects of ammonium sulfate on other types of resistance and bacteria. Another

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

limitation is that this experiment is a simulation of the environment, and isolates from the
environment will not be studied. Future research can study bacteria from the environment as
opposed to bacteria continuously exposed to ammonium sulfate.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

References
Aka, S. T., & Haji, S. H. (2015). Sub-MIC of antibiotics induced biofilm formation of
Pseudomonas aeruginosa in the presence of chlorhexidine. Brazilian Journal of Microbiology,
46(1), 149-154. http://doi.org/10.1590/S1517-838246120140218
Aminov, R. I. (2010). A Brief History of the Antibiotic Era: Lessons Learned and
Challenges for the Future. Frontiers in Microbiology, 1, 134.
http://doi.org/10.3389/fmicb.2010.00134
Andrews, J. M. (2001). Determination of minimum inhibitory concentrations [Abstract].
Journal of Antimicrobial Chemotherapy, 49(6), 1049-1049. doi:10.1093/jac/dkf083
Coolon, J. D., Jones, K, L., Todd, T, C., Blair, J, M., & Herman, M, A. (2013). LongTerm Nitrogen Amendment Alters the Diversity and Assemblage of Soil Bacterial Communities
in Tallgrass Prairie. PLoS ONE, 8(6), e67884. http://doi.org/10.1371/journal.pone,0067884
Dan, L., Siyu, Z., Miao, H., & Gu, A. Z. (2016). Water Disinfection Byproducts Induce
Antibiotic Resistance-Role of Environmental Pollutants in Resistance Phenomena.
Environmental Science & Technology, 50(6), 3193-3201. doi:10.1021/acs.est.5b05113
Fang, H., Wang, H., Cai, L., & Yu, Y. (2014, December 16). Prevalence of Antibiotic
Resistance Genes and Bacterial Pathogens in Long-Term Manured Greenhouse Soils As
Revealed by Metagenomic Survey. Environmental Science & Technology Environ. Sci. Technol.,
49(2), 1095-1104. doi: 10.1021/es504157v
Lorian, V., M.D. (1980). Antibiotics in laboratory medicine (3rd ed.). Baltimore, MD:
Williams & Wilkins.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

Malik A., & Aleem, A. (2010). Incidence of metal and antibiotic resistance in
Pseudomonas spp. from the river water, agricultural soil irrigated with wastewater and
groundwater. Environmental Monitoring and Assessment Environ Monit Assess, 178(1-4), 293308. doi: 10.1007/s10661-010-1690-2
Stokstad, E. (2016). The Nitrogen Fix. Science, 353(6305), 1225-1227.
Vitosh, M. L., J. W. Johnson, and D. B. Mengel. 1995. Selecting forms of nitrogen
fertilizer. Extension Bulletin E-2567 (July), Michigan State University Extension, East Lansing,
MI.

EFFECT OF (NH4)2SO4 ON RESISTANCE OF E. COLI K12

Appendix A
Hypothesis draft: If the prevalence of ammonium sulfate is related to the resistance of E. coli
K12 to amoxicillin, E. coli strains exposed to high and low concentrations will develop a
higher MIC.
Independent variable:
Concentrations of ammonium
sulfate applied to E. coli K12

Background questions:
How do nitrogen fertilizers benefit the soil?
How do I determine the concentrations to use?
What bacteria should be used?
How many cycles of incubation should be run?

Dependent variable:
Quantitative:
MIC of E. coli K12 after
treatment

Constants

Qualitative:
If the ammonium sulfate
visible inhibited growth on
the petri dish
Experimental group: E. coli
exposed to ammonium sulfate
Control group: E. coli on an
agar plate without any other
chemical

Amount of agar the bacteria is plated on


The antibiotic that is used to test resistance
Incubation time
Number of incubation cycles

Experimental group 1:
High concentration of
ammonium sulfate (Half of
the MIC value)

Experimental group 2:
Low concentration of
ammonium sulfate (10 mg/L)

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