Evolution of Antibiotic Resistance in Bumblebee Gut Symbionts Público

Abstract

Exposure to antibiotics can often cause adverse effects on beneficial organisms, such as bumblebees, in cropping systems. It is vital to address this issue, as bee pollination plays a major role in the global economy. Field studies have indicated that bee-associated microbes can develop antibiotic resistance. However, it is still unclear how bumblebees acquire antibiotic- resistant genes within their gut microbiome, and how readily these mutations could arise in specific symbionts. Various environmental conditions within the gut, such as biofilm formation, could impact the ability of symbionts to gain antibiotic resistance, yet it is unclear whether biofilm formation plays an important role in facilitating this process. By using an evolution passaging assay with increasing antibiotic concentrations on static and shaking plates, we explored the potential for inducing antibiotic resistance in a strain of the bumblebee gut symbiont Gilliamella via de novo mutation. Furthermore, we explored whether biofilm formation could impact the evolution of resistance in Gilliamella. Previous literature has indicated frequent presence of antibiotic-resistance genes in bee symbionts Gilliamella and Snodgrassella in natural populations. Thus, I hypothesized that Gilliamella can gain antibiotic resistance through de novo mutations. Cultures grown in a planktonic environment are found to have the potential to develop antibiotic resistance at higher concentrations, thus I hypothesized that strains grown in shaking conditions can have a higher resistance to antibiotics. We found that antibiotic resistance in the bumblebee symbiont Gilliamella could be evolved via passaging and the state, either biofilm or planktonic, in which Gilliamella grew did not impact the evolution of resistance. However, we found that the state did impact the symbiont’s growth rate. Further experiments will allow us to better understand and manage antibiotic resistance within the bee community. Moreover, the design of these experiments can be a tool to study how antibiotic resistance affects the functions of bee core symbionts and their impacts on bee behavior. Microbial interactions could further impact a symbiont's ability to evolve antibiotic resistance. In future studies, community-level evolution assays should be conducted to further explore symbionts' ability to evolve in a community setting.

Table of Contents

TERMINOLOGY .............................................................................................................................. 1 

INTRODUCTION ............................................................................................................................ 2 

METHODS ...................................................................................................................................... 4

Figure 1 .......................................................................................................................................... 4

Strains Description........................................................................................................................... 5 

Table 1............................................................................................................................................ 5

1. Minimum Inhibitory Concentration (MIC) Assay for Ancestral Susceptible Strains ............................. 5

2. Crystal Violet Assay ....................................................................................................................... 5

3. Evolution of Antibiotic Resistance via Sequential Passaging ............................................................. 5

4.1. MIC Assay for Putatively Evolved strains - Ability to Maintain Resistance ....................................... 6

4.2. Strain Sequencing ....................................................................................................................... 7

5. Disc Diffusion Assay - Ability to Maintain Resistance in a Different Environmental Condition ............. 7

Statistical Analyses ........................................................................................................................... 8 

RESULTS ........................................................................................................................................ 10

1. MIC Assay for Susceptible Ancestral Strains.....................................................................................10

Figure 2 .......................................................................................................................................... 10

2. Crystal Violet Assay ....................................................................................................................... 11

Figure 3 ........................................................................................................................................... 11 

Figure 4 ........................................................................................................................................... 12

3.1. Evolution of Antibiotic Resistance via Sequential Passaging: Two-Fold Increments ......................... 13 

Figure 5 ........................................................................................................................................... 13

3.2. Evolution of Antibiotic Resistance via Sequential Passaging: 3 ppm Increments .............................. 14 

Figure 6 ........................................................................................................................................... 14 

Table 2............................................................................................................................................. 14 

Figure 7 ........................................................................................................................................... 15 

Table 3............................................................................................................................................. 16 

Table 4............................................................................................................................................. 16

4. MIC Assay of Putatively Evolved Strains .......................................................................................... 17 

Figure 8 .......................................................................................................................................... 17 

Table 5............................................................................................................................................ 18 

Table 6............................................................................................................................................ 18

5. Disc Diffusion Assay- Ability to maintain resistance in different environmental conditions ................ 19

Figure 9 .......................................................................................................................................... 19 

DISCUSSION ................................................................................................................................... 20

Initial MIC determination ................................................................................................................. 20

Evolution of Resistance .................................................................................................................... 20

Shortcomings .................................................................................................................................. 23

Future Directions ............................................................................................................................ 23

Big Picture ...................................................................................................................................... 23

REFERENCES.................................................................................................................................. 25

APPENDIX...................................................................................................................................... 29

Table A1......................................................................................................................................... 29 

Table A2......................................................................................................................................... 29 

A3. Material Transfer Agreement................................................................................................... 32 

Figure A1 ....................................................................................................................................... 32 

Figure A2 ....................................................................................................................................... 33 

Table A3......................................................................................................................................... 33 

Table A4......................................................................................................................................... 33 

Table A5......................................................................................................................................... 34 

Figure A3 ....................................................................................................................................... 34 

Figure A4 ....................................................................................................................................... 34 

Figure A5 ....................................................................................................................................... 35 

Figure A6 ....................................................................................................................................... 35

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Microbiology Biology, Evolution and Development Biology, Ecology
Palavra-chave	Symbionts Antibiotic resistance Bumblebee Gut microbiome Evolution
Committee Chair / Thesis Advisor	Gerardo, Nicole , Emory University
Committee Members	Vazquez-Prokopec, Gonzalo , Emory University Vega, Nic , Emory University

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