Deciphering the Mechanisms of Silver Resistance in Escherichia coli.

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Fall 2018


Background: Silver has been used medicinally since 750 AD. It’s widespread use as an antimicrobial agent in medical and health settings has made the menacing threat of resistance more possible in the near future. While there is little known about the mechanisms of silver resistance, our previous work shows mutations within specific genes that may contribute to resistance in Escherichia coli. These genes include cusS which controls silver homeostasis through expression of the CusCFBA efflux pump, ompR which is required for porin synthesis, purL which is involved in purine synthesis, and rpoB the RNA polymerase beta-subunit. Question: This study focuses on evaluating cusCFBA efflux pump expression in Escherichia coli strains that are either silver resistant or harbor single cusS mutations. Hypothesis: Both resistant and single mutant strains harboring cusS mutations will exhibit an increase in expression of genes associated with the cusCFBA efflux pump operon. Method: We began by growing 10mL cultures of up both silver resistant and single cusS mutant strains of E. coli acquired from our previous work in presence and absence of silver. RNA was then extracted from each sample using the Monarch RNA extraction kit from NEB® and quantified using a Quantifulor® from Promega. Each sample was then diluted to a final concentration of ~300ng/uL. RT-PCR was then used to evaluate gene expression of target genes. We first looked at the housekeeping gene cysG to ensure normalized expression across samples, then we evaluated cusC expression to assess expression of the efflux pump. Results: Assessment of our cysG data showed equal expression across all samples indicating its validity as a control for this experiment. cusC expression show up regulation in some of our cell lines and a decrease in expression in others. Future direction: We will now evaluate expression of other genes in the operon as well as porin expression through looking at ompC and ompF also using RT-PCR. Broader Impact: This study will help us to understand the mechanisms of silver resistance before resistance becomes widespread in nature, in an effort to have measures in place as it becomes more prominent.

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