The importance of this work is to identify which genes are induced or repressed depending on the drug tested. These bactrium are responsible for shigellosis, a bacterial infection of the intestine that results in bloody diarrhea and fever. Two antibiotic compounds were studied, rifampin (RP) and rifaximin (RX), both inhibiting RNA synthesis which inhibits growth and reproduction processes of the bacteria. Due to recently increasing resistance to antibiotics in shigella strains it is imperative to see which genes are being inhibited or induced with the current market antibiotics. The expression profiles of micro arrays will provide insight into the interaction between the drugs and bacteria. By inhibiting RNA synthesis, no new proteins will be produced, any proteins still present have yet to degrade.

#**The importance of this work is to identify which genes are induced or repressed depending on the drug tested. These bactrium are responsible for shigellosis, a bacterial infection of the intestine that results in bloody diarrhea and fever. Two antibiotic compounds were studied, rifampin (RP) and rifaximin (RX), both inhibiting RNA synthesis which inhibits growth and reproduction processes of the bacteria. Due to recently increasing resistance to antibiotics in shigella strains it is imperative to see which genes are being inhibited or induced with the current market antibiotics. The expression profiles of micro arrays will provide insight into the interaction between the drugs and bacteria. By inhibiting RNA synthesis, no new proteins will be produced, any proteins still present have yet to degrade.

Shigella flexneri 2a strain 301(Sf301) was used in the study. The drugs tested RP and RX were utilized at a concentration of 51.2 mg/mL and diluted with caMHB. The Minimal inhibitory concentrations of the two drugs for Sf301 were determined. Sf301 was taken from a 24 hour culture in caMHB and inserted into caMHB until reaching the desired optical density at OD600. Once the exponential growth phase was reached the drugs RP and RX were added to the cultures to yield final concentrations of 0xMIC, 0.25xMIC, 0.5xMIC, 1xMIC, 2xMIC, and 4xMIC. The drug treatment phase consists of cultures that had drugs added to create 0.5xMIC and 1xMIC with the solvent at 0.25%(v/v). The control cultures had the same final concentration but without the drugs added, solvent at 0.25%(v/v). After 10, 30, and 60 minutes samples were collected and washed for subsequent RNA isolation, each experiment was independently performed 3 times. The RNA was reverse transcribed to prepare a copy of the cDNA that was labeled with flourescent dyes. Cy3-dCTP dye for the control and Cy5-dCTP for the drug treated samples. The genome microarrays were created by purifying the amplified products of ORF-specific primer pairs with the MultiScreen-PCR plates. The products were readjusted to 100ng/microliter and spotted onto gamma amino propylsilan0coated GAPII slides by using Omni-Grid microarrayer. The protocol followed for the hybridization of cDNA to the DNA microarrays can be found http://www.ifr.ac.uk/safety/microarrays/protocols.html#Hybridisations. The slides were scanned by a GenePix 4100A scanner and quantified using GenePix Pro 6.0 software. Local background values were subtracted and the data set was filtered to exclude "not found" features from analysis. MIDAS sofware (http://www.tigr.org/software/tm4) was used to perform normalizations with flourescent signal ratios. The mean Cy5/Cy3 ratios of the gene were calculated to provide further analysis and yield if there were changes in the expression of a the gene. Significant change of expression is if the mean ratio is if there is anything greater than a twofold change eitehr way. qRT-PCR (Quantitative real-time PCR) performed on ABI 7000 instrument using Power SYBR Green Universal Master Mix. Primer Premier 5.0 software designated gene-specific primers. The manufactures recommendations were used for real time PCR. The amount of each gene present was measured relative to a standard transcript, 16S rRNA, and each cDNA was assayed in triplicate PCR reactions.

#**Shigella flexneri 2a strain 301(Sf301) was used in the study. The drugs tested RP and RX were utilized at a concentration of 51.2 mg/mL and diluted with caMHB. The Minimal inhibitory concentrations of the two drugs for Sf301 were determined. Sf301 was taken from a 24 hour culture in caMHB and inserted into caMHB until reaching the desired optical density at OD600. Once the exponential growth phase was reached the drugs RP and RX were added to the cultures to yield final concentrations of 0xMIC, 0.25xMIC, 0.5xMIC, 1xMIC, 2xMIC, and 4xMIC. The drug treatment phase consists of cultures that had drugs added to create 0.5xMIC and 1xMIC with the solvent at 0.25%(v/v). The control cultures had the same final concentration but without the drugs added, solvent at 0.25%(v/v). After 10, 30, and 60 minutes samples were collected and washed for subsequent RNA isolation, each experiment was independently performed 3 times. The RNA was reverse transcribed to prepare a copy of the cDNA that was labeled with flourescent dyes. Cy3-dCTP dye for the control and Cy5-dCTP for the drug treated samples. The genome microarrays were created by purifying the amplified products of ORF-specific primer pairs with the MultiScreen-PCR plates. The products were readjusted to 100ng/microliter and spotted onto gamma amino propylsilan0coated GAPII slides by using Omni-Grid microarrayer. The protocol followed for the hybridization of cDNA to the DNA microarrays can be found http://www.ifr.ac.uk/safety/microarrays/protocols.html#Hybridisations. The slides were scanned by a GenePix 4100A scanner and quantified using GenePix Pro 6.0 software. Local background values were subtracted and the data set was filtered to exclude "not found" features from analysis. MIDAS sofware (http://www.tigr.org/software/tm4) was used to perform normalizations with flourescent signal ratios. The mean Cy5/Cy3 ratios of the gene were calculated to provide further analysis and yield if there were changes in the expression of a the gene. Significant change of expression is if the mean ratio is if there is anything greater than a twofold change eitehr way. qRT-PCR (Quantitative real-time PCR) performed on ABI 7000 instrument using Power SYBR Green Universal Master Mix. Primer Premier 5.0 software designated gene-specific primers. The manufactures recommendations were used for real time PCR. The amount of each gene present was measured relative to a standard transcript, 16S rRNA, and each cDNA was assayed in triplicate PCR reactions.