Gene Expression Analysis of the AI-2-Controlled Genes and Biofilm Formation-Related Genes of the Antibiotic-Resistant Escherichia coli at Different Growth Stages
Gene Expression Analysis of the AI-2-Controlled Genes and Biofilm Formation-Related Genes of the Antibiotic-Resistant Escherichia coli at Different Growth Stages Baho S.1 and Samarasinghe S.2 1,2 De Montfort University/Leicester/LC/United Kingdom Background- Antibiotic resistance along with biofilm formation represents current major issues faced by healthcare institutions worldwide. Autoinducer-2 (AI-2), which is coded by luxS, is one of the quorum sensing signals that acquired great attention recently for its role in many bacterial activities through inducing many genes’ regulation. Most studies focus on seeking for new antibiotics to treat biofilms but very little focus on the bacterial communication signals and gene expression study at specific stages of bacterial growth. In our work, we are trying to analyze gene expression at different biofilm growth stages in antibiotic resistant Escherichia coli CTX-M-15. This might give new information about biofilm formation mechanism and the role of quorum sensing and its relation to antibiotic resistance, which in turn, might give new insights into using new therapeutic alternatives other than the traditional antibiotic. Methods The amount of biofilm formation at different time points (6, 12, 24 & 48 hours) was measured in E. coli CTX-M-15 using Tissue Culture Plate Assay (TCP). 6 wells tissue culture plates were used in this study using AB broth medium, incubated under static conditions at 37°C, and OD570 values for crystal violet absorbance rate represented biofilm amount. RNA extraction was performed for the growing strain in AB broth at the above mentioned periods of incubation plus a 0-time incubation to be the calibrator, followed by cDNA reaction. qPCR analysis was performed for the selected 7 AI-2 controlled genes plus luxS, 21 biofilm formation-related genes and CTX-M-15 resistance gene. Results Biofilm formation amount was the highest at the 6 hr time of incubation, then started to decrease by time. The expression of 2 out of 21 biofilm formation-related genes (fimA and fimC) showed the highest upregulation at 6 hr time of incubation when 0 time was chosen as the calibrator, while 10 genes at 12 hr time of incubation and 6 genes at 24 and 48 hr time of incubation were upregulated when 6 hr time was chosen as the calibrator. All the 7 AI-2 controlled genes showed their highest upregulation at the 24 hr time of incubation along with luxS and CTX-M-15 resistance gene. Conclusion qPCR and TCP analysis demonstrates that biofilm formation-related genes in comparison with biofilm amount measured by TCP can be utilized as a model to study the up- and down- regulation of such genes. Also, it shows that AI-2 has a profound effect during biofilm formation stages, and antibiotic resistance might be controlled by AI-2 as the resistance gene upregulated along with other AI-2 controlled genes
Citation : Baho, S and Samarasinghe, S. (2018) Gene Expression Analysis of the AI-2-Controlled Genes and Biofilm Formation-Related Genes of the Antibiotic-Resistant Escherichia coli at Different Growth Stages. ASM Microbe 2018, Atlanta, June 2018.
Research Group : Infectious Disease Research Group
Research Institute : Leicester Institute for Pharmaceutical Innovation - From Molecules to Practice (LIPI)
Peer Reviewed : Yes