Progress 01/01/16 to 12/31/17
Outputs
Target Audience:Target audiences: 1) Undergraduate and graduate students through formal and informal research experiences in the laboratory. 2) Ag animal producers through published results in peer-reviewed journals and presentations at local, national and international meetings. 3) Ag policy makers and legislators through increased awareness of the impact of plasmid curing agents on antibiotic resistance in animals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student working on this project had the opportunity to learn about the ecology of plasmids in bacterial cells and how these factors influence the overall evolution of bacterial populations. This student performed PCR and DNA sequence based analyses to evaluate the impact of curing agents on plasmid abundance and distribution. The student also performed experiments without curing agents to understand the baseline nature of plasmids over time. This work will be highlighted in a manuscript of which the student will be the lead author. How have the results been disseminated to communities of interest?We are currently generating a manuscript describing our results and plan to submit to a peer-reviewed journal within the next two months. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Over the course of the project period, several experiments were conducted with R-plasmid containing Klebsiella and E. coli strains. Results from these experiments generated novel findings on the importance of plasmid-encoded toxin-antitoxin systems and other mechanisms by which these factors maintain themselves within host cells. We made significant discoveries regarding general rates of plasmid acquisition and loss both under laboratory conditions (in vitro) and in murine models (in vivo). A maunscript describing these results is being prepared for submission to a peer-reviewed journal. In general, most of the R-plasmids that were evaluated in our study contained factors (e.g. toxin-antitoxin systems) that largely overcame the curing effects of menthol and SDS. While these compounds were able to cure "simple" plasmids at rates exceeding the basal (random) curing rate, they were unable to cure R-plasmids that had evolved complex mechanisms to maintain themselves inside host bacterial cells. However, we learned enough about these plasmids to hypothesize which fators are important and so future work can focus on disrupting these genes either before curing agents are used or in conjunction with curing agents. One potential approach would be to use pathogen-specific phage to deliver CRISPR-mediated nucleases that disable toxin-antitoxin machinery. The added pressure for curing from menthol and SDS might then lead to the loss of R-plasmids in these populations. While not all of the project objectives were reached, a great deal of information and novel understanding came from this project and we look forward to publishing our results in the next few months.
Publications
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Target audiences reached by the past year's efforts: 1) Undergraduate and graduate students through formal and informal research experiences in the laboratory. 2) Ag researchers through local presentations at local presentations of research results. Changes/Problems:We were excited to discover that plasmid content in our preliminary mouse experiments changed significantly over time even in the absence of exposure to plasmid curing agents. We were curious to know wether this happens in bacteria in people and have taken the opportunity to quantify plasmid content in E. coli isolated from fecal samples of several volunteers. This work has revealed many interesting observations that we continue to follow up on. Although this aspect of the project was unanticipated, we believe it is significant to the field and will advance the current understanding of plasmid mediated microbiome dynamics. However, this interesting sidebar will not detract from our ability to accomplish the goals of the proposed research and we are still on track to complete the proposed research by the end of the project period (12/31/2017). What opportunities for training and professional development has the project provided?Training - This project has provided an excellent opportunity for my graduate student to learn a number of molecular biology techniques. My lab manager and I have been able to instruct this student in plasmid preparations, PCR primer design, and murine modeling. Professional - I have increased my knowledge through individual study of the effects of plasmids on colonization of the murine gut. We think that our research will add significantly to this area and potentially lead to novel understanding of these genetic factors. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1) Quantify plasmid loss under varying levels of plasmid curing agents (menthol and SDS), temperature (room temperature and mammalian and avian body temperature), and bacterial density (low and high). All experiments to date have been at a high bacterial density and we will be conducting replicate experiments at lower density soon. 2) Co-culture R-plasmid carrying strains against R-plasmid cured counterparts to quantify differences in relative fitness. These exeriments have been started and are beginning to show interesting results. We anticipate completing this protion of the project in the next month and may be able to publish our findings if our initial observations are supported. 3) Colonize germ-free mice with R-plasmid carrying strains of Enterobacteriaceae and treat with plasmid curing agents (menthol and SDS). 4) Quantify loss of R-plasmids in Enterobacteriaceae from mouse fecal pellets. We will begin exposing resistant E. coli colonized mice to menthol and SDS and comparing the rate of R-plasmid loss to the rate that occurs spontaneously (i.e. without exposure to curing agents). We anticipate that these experiments will be completed by the end of this coming summer (2017). 5) Remove plasmid curing agents and monitor bacterial population dynamics to understand the longer-term effects of plasmid curing agents. These experiments will be conducted beginning in June 2017. We have begun to increase our breeding colony of mice to have an adequate of age- and sex-matched animals to accomplish this objective.
Impacts
What was accomplished under these goals?
Specific objectives and accomplishments: 1) Quantify plasmid loss under varying levels of plasmid curing agents (menthol and SDS), temperature (room temperature and mammalian and avian body temperature), and bacterial density (low and high). We have tested the effects of menthol and SDS on ESBL-producing E. coli isolates at different temperatures. We have found variable effects upon repeat exposure to these agents, suggesting that chance events may be influencing our results. All experiments to date have been at a high bacterial density and we will be conducting replicate experiments at lower density soon. 2) Co-culture R-plasmid carrying strains against R-plasmid cured counterparts to quantify differences in relative fitness. We have begun to quantify fitness differences between isolates that have different plamsid content. Not enough experiments have been done to draw solid conclusions, but it appears that some isolates are more fit in the absence of antibiotic resistance plasmids. More results will help to clarify the outcome of this objective. 3) Colonize germ-free mice with R-plasmid carrying strains of Enterobacteriaceae and treat with plasmid curing agents (menthol and SDS). 4) Quantify loss of R-plasmids in Enterobacteriaceae from mouse fecal pellets. These two objectives are experimentally linked and are being pursued at the same time. Mice have been colonized with resistance E. coli and monitored for 12 weeks to evaluate colonization dynamics and the rate at which R-plasmids are lost simply by chance. We have shown that E. coli colonize GF mice rapidly and maintain a high level of abundance. We have also transplanted human stool samples into GF mice (i.e. humanized mice) and have shown that E. coli in this complex bacterial community are stable over a long period of time. We are now ready to begin exposing resistant E. coli colonized mice with the plasmid curing agents and comparing the rate of R-plasmid loss. 5) Remove plasmid curing agents and monitor bacterial population dynamics to understand the longer-term effects of plasmid curing agents. We have not yet conducted these experiments, but plan to begin in a few weeks time.
Publications
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