1. An Enzyme Too Far Intraspecific Competition and the Fitness costs of β -lactamase in E. coli Justin Dalton IntroductionMethodsConclusions Surviving isn't easy. In order to succeed, any organism must choose where best to devote its resources to survive in its environment. But what if that environment changes? How will it fare competing with members of its species perfectly at home in this new world? Could traits that an organism has spent generations developing become a useless drain on resources overnight? Bacteria have spent countless generations developing biochemical defenses to combat our rampant use of antibiotics (Riley and Wertz 2002). One of the most common mechanisms of resistance requires bacteria to make the enzyme β-lactamase. When threatened, the enzyme is worth having, but what if the antibiotic isn’t present? Every enzyme a bacterium makes represents resources it could have used to grow and reproduce. In the absence of antibiotics, B-lactamase becomes unnecessary, and a constant drain on resources. Is the burden of producing β-lactamase enough to affect competition? Could an antibiotic resistant bacterium find itself out competed entirely by its non-resistant brethren in an environment free of antibiotics, its genetic information lost? Ampicillin Sensitive Ampicillin Resistant Competition Isolated Growth of Wild Type Isolated Growth of Transformed Combined Growth of Wild Type and Transformed Samples at One Hour Intervals Viable cell count by standard plate count Proportion viable by Live/Dead fluorometric staining Total biomass by BCA protein assay Objectives Determine the outcome of competition between wild type and ampicillin resistant E. coli. Predict long term effects of competition on population through ecological modeling. References Fig. 1 Total population of resistant and non-resistant bacteria measured each hour by standard plate count of a 10 5 dilution. Non-linear fit shown in red for resistant population and blue for non-resistant. Fig. 4 Percent of cells viable measured each hour by ratio of fluorescence at 530nm and 630nm. Live and dead cells stained by propium iodide and STYO-9 stain respectively. Fig. 3 Fluorescent staining of culture with propium iodide and STYO-9 dyes. Live cells appear in green and dead cells appear in red. Fig. 2 Live biomass of resistant and non-resistant populations measured each hour by BCA protein assay. Non-linear fit shown in red for resistant population and blue for non resistant. Measurements based on three replicate samples. Fig. 5 Projected outcome based on founding populations. Resulting isocline indicates competitive exclusion of resistant strain. Fig. 6 Projected population over ecological time by the Lotka-Volterra model. Competitive exclusion of resistant strain occurs within twenty generations β-lactamase production has a significant effect on competition between E. coli. In the absence of ampicillin, resistant bacteria are outcompeted by ampicillin sensitive bacteria within twenty generations. This outcome is supported by the ecological model based on the two populations. Increased resource use and reduced cell viability in the resistant population contribute to this outcome. Resistant and non-resistant populations reduce average cell size in response to competition. Competition in the wild may contribute to keeping antibiotic resistant bacteria in check. Antibiotics present in the environment reverse the outcome of competition seen in this study, thus contributing to the current epidemic of increased antibiotic resistance in wild bacteria. Marciano, David C., Karkouti, Omid Y., and Palzkill, Timothy. 2007. A Fitness Cost Associated With the Antibiotic Resistance Enzyme SME-1 β-Lactamase. Genetics. 176:2381-2392 Martinez, Jose L., Baquero, Fernando, and Andersson, Dan I. 2007. Predicting antibiotic resistance. Annual Review of Microbiology. 5:12:958-965 Gelder, Leen De, Ponciano, José M., Abdo, Zaid, Joyce, Paul, Forney, Larry J., and Top, Eva M.. 2004. Combining Mathematical Models and Statistical Methods to Understand and Predict the Dynamics of Antibiotic-Sensitive Mutants in a Population of Resistant Bacteria During Experimental Evolution. Genetics. 168:1131-1144 Acknowledgments I would like to thank Dr. Mathur for her constant help and advice throughout this project. Her support and encouragement made senior thesis one of my most memorable experiences here at York. Results