Re s i s ta n ce Ba s e d o n DRUG RECYCLING: COUNTIES ...1. Counties in Wyoming that do not donate...
1
1. Counties in Wyoming that do not donate antibiotics to the recycling program will have a higher rate of antibiotic resistant bacteria in wastewater. 2. There will be a more extensive antibiotic resistance profile, as determined by Kirby Bauer susceptibility testing, for isolates in counties that do not participate in the Wyoming Medication Donation Program compared to counties in Wyoming that do participate. To test our hypotheses, we provided containment to wastewater treatment plants, and they provided samples of influent water from the previously listed counties, Carbon, Niobrara, Sublette, Sheridan, Laramie, and Albany. Our research methods included: • Dilution and plating on enriched TSA plates 5 to non selectively or differentially culture growth • Replica Plating 4 from TSA agar to MacConkey/MUG agar, TSA+ ampicillin, TSA+ tetracycline and TSA+gentamycin • Kirby Bauer 5 with amoxicillin with clavulanic acid, azithromycin, ciprofloxacin, tetracycline, gentamycin, penicillin, cefazolin, and ampicillin Preliminary identification of isolates has been done through MALDI-TOF (Matrix- Assisted Laser Desorption Ionization - Time Of Flight), to confirm or deny the isolates as fecal coliforms, as well as to assist in analyzing Kirby Bauer Data. The counties that did not participate in the WMDP showed significantly higher ratios of resistant isolates per person. Niobrara, Carbon and Sublette Counties all had larger ratios of ampicillin resistance per person by an order of magnitude. The gentamicin ratios produced similar results: Laramie, Albany, and Sheridan Counties who participate in the program showed lower ratios of resistance per person. Niobrara, Sublette, and Sheridan counties produced the highest ratios of tetracycline resistance per person. These results indicate that counties in Wyoming that do not donate antibiotics to the recycling program have a higher rate of antibiotic resistant bacteria in wastewater than counties that do donate antibiotics, which supports our second hypothesis. Kirby Bauer data showed that the isolates that were resistant to one antibiotic were capable of multidrug resistance in many cases. Most isolates were completely resistant to the commonly prescribed antibiotic, penicillin. This is important because multidrug resistance of bacteria in wastewater is a higher threat than single drug resistant strains. We faced many challenges growing some of the isolates on Mueller-Hinton agar, and data for all of the isolates was not able to be obtained. The results from Kirby Bauer would be better analyzed if we had a complete set of data. Through these experiments, we have learned more about antibiotic resistance in wastewater, and our results align with the conclusions about the spreading of antibiotic resistance in wastewater that is outlined in the literature 2 . We determined that the three counties that participated in the WMDP had lower ratios of antibiotic resistance when compared to the three counties that do not participate. It should be noted that the extremely high ratio of bacterial resistant isolates per person found in Niobrara could be due to outside, confounding variables. These variables could include that this county is extremely agricultural, and high volumes of antibiotics are used in this industry 7 . This information could be used to spread awareness about medication donation programs and their positive impact, as well as increasing the funding for the Wyoming Medication Donation Program. • Special thanks to our community partners at the Wyoming Medication Donation Program, specifically Natasha Gallizzi • University of Wyoming Microbiology Program in the College of Agriculture and Natural Resources • UW Microbiology Prep-room Staff, especially John Willford and Holly Steinkraus • UW Learning Actively Mentoring Program • UW Bisha Lab, specifically Ella DeWolf and Brenna Lindsay • Wastewater Treatment Plant Employees at Albany, Carbon, Laramie, Niobrara, Sheridan and Sublette county Figure 4: Data collected and analyzed from replica plating presented in a graph for clearer comparison between counties and specific antibiotics. The green bars on the graph represent counties that do not participate in the WMDP, while the black bars are counties that do participate. Y axis has been segmented for clarity. 1. Centers for Disease Control and Prevention. Antibiotic/Antimicrobial Resistance.[Homepage on the Internet] c2017. [updated 2017 Aug 18; cited 2017 Dec 2]. Available from:https://www.cdc.gov/drugresistance/index.html 2. Berglund B, Fick J, and Lindgren, P.-E. Urban wastewater effluent increases antibiotic resistance gene concentrations in a receiving northern European river. Environ Toxicol Chem. 2015; 34: 192–196. 3. Wyoming Department of Health. Wyoming Medication Donation Program [homepage on the Internet]. C2016 [cited 2017 Dec 2]. Available from: https://health.wyo.gov/healthcarefin/medicationdonation/. 4. Gallizzi, Natasha. Wyoming Medication Donation Program. Pounds of Donations Listed by County Years 2015 & 2016; 2015 & 2016 (Unpublished report). 5. Paavilainen, T, Österblad, M, Leistevui, T, Huovinen P, Kotilainen, P. Screening for Antimicrobial Resistance in Normal Bacterial Flora of the Skin Using Replica Plating Method. European Journal of Clinical Microbiology & Infectious Diseases. 2000;19:956-959. 6. HardyDisc Antibiotic Susceptibility Test Instructions For Use. [homepage on the Internet]. c2015 [cited 2017 Dec 2]. Available from: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/HardyDiskASTProceduresandChart.pdf. 7. Census of Agriculture: County Profile, Niobrara County Wyoming. [homepage on the Internet]. C2012 [cited 2017 Dec 7]. Available from: https://www.agcensus.usda.gov/publications/2012/online_resources/county_profiles/wyoming/cp56027.pdf • The WMDP may play a crucial role in preventing antibiotic resistance in Wyoming wastewater. • In counties that participate in the WMDP there are less resistant bacterial isolates per person than in counties that do participate in the program. • This could have an influence on the future of the WMDP, and may be used to encourage the use of this program throughout the state. • The future of this research will be to continue examining the extent of resistance in wastewater around the state. • More samples will be examined through the same processes described in this research, and the experimental software MALDI-TOF will be used to identify each isolate. Bacterial antibiotic resistance is a rising problem in medicine, leaving many infections untreatable; the Centers for Disease Control estimates that 23,000 deaths have resulted from this growing issue 1 . Disposing of antibiotics incorrectly, such as through water systems, selects for bacterial mutants that are resistant to antibiotics. A 2015 study in the Berglund laboratories detailed that it is possible that many antibiotic resistance genes originate and are spread through wastewater due to the commingling of environmental bacteria, pathogenic bacteria and antibiotic waste 2 . In response to improper drug disposal, Wyoming implemented the Wyoming Medication Donation Program (WMDP). The WMDP is a program that collects medical waste from collection boxes around the state and either redistributes it or properly disposes of it through incineration as noted by the Wyoming Department of Health 3 . The role of the WMDP in preventing incorrect antibiotic disposal is detailed in Figure 1. Not only is this significant for human health, but the WMDP benefits the health of the environment by reducing water pollution. Twenty counties around the state participate in this donation program, while six do not 4 . Table 1 below includes preliminary data provided by WMDP that details the pounds of medication donated by each county in 2015 and 2016. The data presented in this table was crucial in establishing our experimental groups, three counties that do not participate in the WMDP, Sublette, Carbon and Niobrara, and three that do participate, Sheridan, Albany, and Laramie. While it seems obvious that participation in this program would reduce the level of antibiotic resistant bacteria in Wyoming’s wastewater, there was no data to support this positive impact. Figure 1: A schematic of the work that was done as well as the basics behind the antibiotic resistance that will be studied as a result of improper disposal of antibiotic waste. Table 1: The pounds donated per county in the years 2015 and 2016. Information for every county in Wyoming was supplied by the WMDP, included in the table above is only the counties relevant to this research. Name of County Pounds donated in 2015 Pounds donated in 2016 Albany 89.6 283.8 Carbon 0 0 Laramie 4381.9 3463.1 Niobrara 0 0 Sheridan 683.8 395.4 Sublette 0 0 Figure 2: The dilution and replica plating scheme that we used during the experiment. Steps 1-2 were done at each level of dilution. Steps 3-7 were only performed using the TSA plate from the level of dilution that produced a number of colonies between 30 and 300. Figure 3: The six images above show the results of replica plating. The first image is the growth from the plated water, diluted to the ratio written on the edge of the plate (ie. 1:2000). The second plate is the control TSA, the third is the MacConkey agar, fourth is TSA + ampicillin, fifth is TSA + tetracycline, and sixth is TSA + gentamycin INTRODUCTION METHODS RESULTS CONCLUSION DISCUSSION ACKNOWLEDGEMENTS RESOURCES Amox w/ Clauv Ciprofloxacin Tetracycline Gentamicin Penicillin Ampicillin Carbon Amp A S I S I R R Carbon Amp B S S S I R R Carbon Tet B S R R S R I Sheridan Amp B S I S S R R Sheridan Tet A I R R R R I Sheridan Gent A R I R R R R Niobrara Amp A I I R I R R Niobrara Amp B I R R S R R Niobrara Tet A S R R I R R Niobrara Tet B S R R S R R Sublette Amp B S S S S R R Albany Amp A I S S S R R Albany Amp B I S I S R R Albany Tet A S R S S R I Laramie Amp A R S R S R R Laramie Tet A R I R S R R Kirby Bauer Susceptibility Test HYPOTHESES Figure 5. The table above shows the results of Kirby Bauer Susceptibility testing 6 . S means that the isolate was susceptible, I stands for intermediate, and R means that the isolate was resistant to the antibiotic. The column on the left represents bacterial isolates from each county and which TSA+ antibiotic plate they were isolated on. Two antibiotics that were included in the Kirby Bauer test to account for the possibility that the isolates would be gram positive, are not included in this table due to the fact that all isolates grown were Gram negative, so those antibiotics were not applicable. All isolates with no growth, either in the Kirby Bauer Assay or from initial isolation, have also been removed from the table. DRUG RECYCLING: COUNTIES WITHOUT WYOMING MEDICATION DONATION PROGRAM SHOW MORE BACTERIAL ANTIBIOTIC RESISTANCE IN WASTEWATER PER PERSON Emily Armitage, Kathryn Moncrief, Natasha Radosevich, Rachel Watson & Gerard Andrews Al b a ny Car bon Lar amie Ni o brara Sheridan Sub l ett e 0.00000 0.00005 0.00025 0.00030 Counties Resistant Isolates per Person Resistance Based on Number of Colonies Per Person - Ampicillin Alb a n y C a rb o n Laram i e Nio br a r a Sherida n Sublette 0.00000 0.00001 0.00007 0.00008 Counties Resistant Isolates per Person Resistance Based on Number of Colonies Per Person - Tetracycline Al b an y C arb o n Lara m ie Niobrara Sheridan S ublette 0.000000 0.000002 0.000004 0.00004 0.00005 0.00006 Counties Resistant Isolates per Person Resistance Based on Number of Colonies Per Person - Gentamycin First trial runs of MALDI-TOF suggest that all isolated colonies tested were indeed Gram- negative bacteria either of the genera Klebsiella, Enterobacter, Escherichia or Citrobacter. REPLICA PLATING DATA KIRBY BAUER DATA MALDI-TOF DATA
Transcript of Re s i s ta n ce Ba s e d o n DRUG RECYCLING: COUNTIES ...1. Counties in Wyoming that do not donate...
1. Counties in Wyoming that do not donate antibiotics to the recycling program will have ahigher rate of antibiotic resistant bacteria in wastewater.
2. There will be a more extensive antibiotic resistance profile, as determined by Kirby Bauer
susceptibility testing, for isolates in counties that do not participate in the WyomingMedication Donation Program compared to counties in Wyoming that do participate.
To test our hypotheses, we provided containment to wastewater treatment plants,
and they provided samples of influent water from the previously listed counties, Carbon,Niobrara, Sublette, Sheridan, Laramie, and Albany.
Our research methods included:
• Dilution and plating on enriched TSA plates5 to non selectively or differentially
culture growth
• Replica Plating4 from TSA agar to MacConkey/MUG agar, TSA+ ampicillin, TSA+
tetracycline and TSA+gentamycin
• Kirby Bauer5 with amoxicillin with clavulanic acid, azithromycin, ciprofloxacin,
tetracycline, gentamycin, penicillin, cefazolin, and ampicillin
Preliminary identification of isolates has been done through MALDI-TOF (Matrix-
Assisted Laser Desorption Ionization - Time Of Flight), to confirm or deny the isolates asfecal coliforms, as well as to assist in analyzing Kirby Bauer Data.
The counties that did not participate in the WMDP showed significantly higher
ratios of resistant isolates per person. Niobrara, Carbon and Sublette Counties all had largerratios of ampicillin resistance per person by an order of magnitude. The gentamicin ratiosproduced similar results: Laramie, Albany, and Sheridan Counties who participate in theprogram showed lower ratios of resistance per person. Niobrara, Sublette, and Sheridancounties produced the highest ratios of tetracycline resistance per person. These results
indicate that counties in Wyoming that do not donate antibiotics to the recycling programhave a higher rate of antibiotic resistant bacteria in wastewater than counties that dodonate antibiotics, which supports our second hypothesis.
Kirby Bauer data showed that the isolates that were resistant to one antibiotic were
capable of multidrug resistance in many cases. Most isolates were completely resistant to the
commonly prescribed antibiotic, penicillin. This is important because multidrug resistance of
bacteria in wastewater is a higher threat than single drug resistant strains. We faced many
challenges growing some of the isolates on Mueller-Hinton agar, and data for all of theisolates was not able to be obtained. The results from Kirby Bauer would be better analyzedif we had a complete set of data.
Through these experiments, we have learned more about antibiotic resistance inwastewater, and our results align with the conclusions about the spreading of antibioticresistance in wastewater that is outlined in the literature2. We determined that the threecounties that participated in the WMDP had lower ratios of antibiotic resistance whencompared to the three counties that do not participate. It should be noted that the
extremely high ratio of bacterial resistant isolates per person found in Niobrara could bedue to outside, confounding variables. These variables could include that this county isextremely agricultural, and high volumes of antibiotics are used in this industry7. Thisinformation could be used to spread awareness about medication donation programs andtheir positive impact, as well as increasing the funding for the Wyoming Medication
Donation Program.
• Special thanks to our community partners at the Wyoming Medication Donation Program,
specifically Natasha Gallizzi
• University of Wyoming Microbiology Program in the College of Agriculture and Natural
Resources
• UW Microbiology Prep-room Staff, especially John Willford and Holly Steinkraus
• UW Learning Actively Mentoring Program
• UW Bisha Lab, specifically Ella DeWolf and Brenna Lindsay
• Wastewater Treatment Plant Employees at Albany, Carbon, Laramie, Niobrara, Sheridan and
Sublette county
Figure 4: Data collected and analyzed
from replica plating presented in a graphfor clearer comparison between countiesand specific antibiotics. The green barson the graph represent counties that donot participate in the WMDP, while the
black bars are counties that doparticipate. Y axis has been segmentedfor clarity.
1. Centers for Disease Control and Prevention. Antibiotic/Antimicrobial Resistance.[Homepage on the Internet] c2017. [updated 2017 Aug18; cited 2017 Dec 2]. Available from:https://www.cdc.gov/drugresistance/index.html
2. Berglund B, Fick J, and Lindgren, P.-E. Urban wastewater effluent increases antibiotic resistance gene concentrations in a receivingnorthern European river. Environ Toxicol Chem. 2015; 34: 192–196.
3. Wyoming Department of Health. Wyoming Medication Donation Program [homepage on the Internet]. C2016 [cited 2017 Dec 2]. Availablefrom: https://health.wyo.gov/healthcarefin/medicationdonation/.
4. Gallizzi, Natasha. Wyoming Medication Donation Program. Pounds of Donations Listed by County Years 2015 & 2016; 2015 & 2016(Unpublished report).
5. Paavilainen, T, Österblad, M, Leistevui, T, Huovinen P, Kotilainen, P. Screening for Antimicrobial Resistance in Normal Bacterial Flora ofthe Skin Using Replica Plating Method. European Journal of Clinical Microbiology & Infectious Diseases. 2000;19:956-959.
6. HardyDisc Antibiotic Susceptibility Test Instructions For Use. [homepage on the Internet]. c2015 [cited 2017 Dec 2]. Available from:https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/HardyDiskASTProceduresandChart.pdf.
7. Census of Agriculture: County Profile, Niobrara County Wyoming. [homepage on the Internet]. C2012 [cited 2017 Dec 7]. Available from:https://www.agcensus.usda.gov/publications/2012/online_resources/county_profiles/wyoming/cp56027.pdf
• The WMDP may play a crucial role in preventing antibiotic resistance in Wyoming
wastewater.
• In counties that participate in the WMDP there are less resistant bacterial isolates perperson than in counties that do participate in the program.
• This could have an influence on the future of the WMDP, and may be used to encouragethe use of this program throughout the state.
• The future of this research will be to continue examining the extent of resistance in
wastewater around the state.
• More samples will be examined through the same processes described in this research,and the experimental software MALDI-TOF will be used to identify each isolate.
Bacterial antibiotic resistance is a rising problem in medicine, leaving many
infections untreatable; the Centers for Disease Control estimates that 23,000 deaths haveresulted from this growing issue1. Disposing of antibiotics incorrectly, such as through watersystems, selects for bacterial mutants that are resistant to antibiotics. A 2015 study in theBerglund laboratories detailed that it is possible that many antibiotic resistance genesoriginate and are spread through wastewater due to the commingling of environmental
bacteria, pathogenic bacteria and antibiotic waste2.
In response to improper drug disposal, Wyoming implemented the WyomingMedication Donation Program (WMDP). The WMDP is a program that collects medicalwaste from collection boxes around the state and either redistributes it or properly disposes
of it through incineration as noted by the Wyoming Department of Health3. The role of theWMDP in preventing incorrect antibiotic disposal is detailed in Figure 1. Not only is thissignificant for human health, but the WMDP benefits the health of the environment byreducing water pollution.
Twenty counties around the state participate in this donation program, while six do
not4. Table 1 below includes preliminary data provided by WMDP that details the pounds ofmedication donated by each county in 2015 and 2016.
The data presented in this table was crucial in establishing our experimental groups,three counties that do not participate in the WMDP, Sublette, Carbon and Niobrara, and
three that do participate, Sheridan, Albany, and Laramie. While it seems obvious thatparticipation in this program would reduce the level of antibiotic resistant bacteria inWyoming’s wastewater, there was no data to support this positive impact.
Figure 1: A schematic of the work that was done as well as the basics behind the
antibiotic resistance that will be studied as a result of improper disposal of antibioticwaste.
Table 1: The pounds donated per county in the years 2015 and 2016.
Information for every county in Wyoming was supplied by the WMDP,included in the table above is only the counties relevant to this research.
Name of County Pounds donated in 2015 Pounds donated in 2016
Albany 89.6 283.8
Carbon 0 0
Laramie 4381.9 3463.1
Niobrara 0 0
Sheridan 683.8 395.4
Sublette 0 0
Figure 2: The dilution and replica plating scheme that we used during the
experiment. Steps 1-2 were done at each level of dilution. Steps 3-7 were onlyperformed using the TSA plate from the level of dilution that produced a numberof colonies between 30 and 300.
Figure 3: The six images above show the results of replica plating. The first image is the
growth from the plated water, diluted to the ratio written on the edge of the plate (ie. 1:2000). The second plate is the control TSA, the third is the MacConkey agar, fourth is TSA + ampicillin, fifth is TSA + tetracycline, and sixth is TSA + gentamycin
INTRODUCTION METHODS RESULTS
CONCLUSION
DISCUSSION
ACKNOWLEDGEMENTS
RESOURCES
Amox w/ ClauvCiprofloxacin Tetracycline Gentamicin Penicillin Ampicillin
Carbon Amp A S I S I R R
Carbon Amp B S S S I R R
Carbon Tet B S R R S R I
Sheridan Amp B S I S S R R
Sheridan Tet A I R R R R I
Sheridan Gent A R I R R R R
Niobrara Amp A I I R I R R
Niobrara Amp B I R R S R R
Niobrara Tet A S R R I R R
Niobrara Tet B S R R S R R
Sublette Amp B S S S S R R
Albany Amp A I S S S R R
Albany Amp B I S I S R R
Albany Tet A S R S S R I
Laramie Amp A R S R S R R
Laramie Tet A R I R S R R
Kirby Bauer Susceptibility Test
HYPOTHESES
Figure 5. The table above shows the results of Kirby Bauer Susceptibility testing6. S means
that the isolate was susceptible, I stands for intermediate, and R means that the isolate wasresistant to the antibiotic. The column on the left represents bacterial isolates from eachcounty and which TSA+ antibiotic plate they were isolated on. Two antibiotics that wereincluded in the Kirby Bauer test to account for the possibility that the isolates would begram positive, are not included in this table due to the fact that all isolates grown were
Gram negative, so those antibiotics were not applicable. All isolates with no growth, eitherin the Kirby Bauer Assay or from initial isolation, have also been removed from the table.
DRUG RECYCLING: COUNTIES WITHOUT WYOMING MEDICATION DONATION
PROGRAM SHOW MORE BACTERIAL ANTIBIOTIC RESISTANCE IN
WASTEWATER PER PERSONEmily Armitage, Kathryn Moncrief, Natasha Radosevich, Rachel Watson & Gerard Andrews
Albany
Carbon
Laramie
Niobrara
Sheridan
Sublette
0.00000
0.00005
0.00025
0.00030
Counties
Re
sis
tan
t Is
ola
tes p
er P
erso
n
Resistance Based on Number of Colonies
Per Person - Ampicillin
Albany
Carbon
Laramie
Niobrara
Sheridan
Sublette
0.00000
0.00001
0.00007
0.00008
Counties
Re
sis
tan
t Is
ola
tes p
er
Pe
rso
n
Resistance Based on Number of Colonies
Per Person - Tetracycline
Albany
Carbon
Laramie
Niobrara
Sheridan
Sublette
0.000000
0.000002
0.0000040.00004
0.00005
0.00006
Counties
Re
sis
tan
t Is
ola
tes p
er P
er
so
n
Resistance Based on Number of Colonies
Per Person - Gentamycin
First trial runs of MALDI-TOF suggest that all isolated colonies tested were indeed Gram-
negative bacteria either of the genera Klebsiella, Enterobacter, Escherichia or Citrobacter.
REPLICA PLATING DATA
KIRBY BAUER DATA
MALDI-TOF DATA
