IMPACT OF NOREPINEPHRINE ON THE GROWTH AND VIRULENCE OF CLOSTRIDIOIDES DIFFICILE
Clostridioides difficileinfection (CDI) is considered as an urgent threat to the publicby CDC, 2019.It causes life-threatening diarrhea and pseudomembranous colitis,mostly in those taking antibiotics or at the end of their antibiotic course.It is also notifiedas hospital-associated pathogensbecause one-third of the CDIhas occurredinthe health care center. Norepinephrine (NE) is a stress-associated neuroendocrine hormone released upon sympathetic stimulation to mediate stress.Gut walls are highly innervated by the sympathetic nervous system. During stress, elevated level of NE released in the GI tractcould influence bacterial overgrowth & translocation. It isalready known for its role in modulating the behavior of several bacterial pathogens suchas Staphylococcus, Escherichia coli, Salmonella, and Vibrio cholera. This study aims to evaluate the effect of NE treatment on the growth and virulence of C. difficile.Here, we studied the effect of NE on six different C. difficilestrains isolated from humans. To understandthe influence on growth, bacterial culture was treated (+/-)NE (5μM & 50 μM)during their log phase and recorded the density of the cell each time period for constructing the growth curve. In addition, after NE treatment, bacterial cells were taken for further analysis. For investigating the impact of NE on the virulence genes expression, a qPCR reaction was performed along with -RT / noRT control reactions for assessingthe RNA sample free from genomic DNA contamination. In the case of growth,higher growth was observed in VPI 10463at 6 hourtime pointonly,and in strain,NR 49277 significantly stimulated after 6 hoursand continued till 8 hours after treatmentwith50μM NE. In strain NR 49282, decreasedgrowth was observed at7-hourtime pointsafter 50 μM NEtreatment.But, there was no difference in cell density between control & 5μM NE treated bacterial culture in all strains.
Toxingenes(tcdA&tcdB)and flagellin gene(fliC),were upregulated in NR 49290, NR 49277 & VPI 10463strains in both concentrations of NE and down-regulated in NR 49282.In strain NR 32888, toxin genes were downregulated while treated with 5μM NEbut upregulated after 50μM NEtreatment, though fliC was downregulated in both concentrations. In strain NR 32891, tcdAwas downregulated,but tcdB& fliCwere upregulatedafter NE treatmentin both concentrations. Increased expression in pilin gene,pilA1in strain NR 49277, NR 49290, VPI 10463& NR 32891 in both concentrationswas observed. In addition, pilA3in NR 49277, VPI 10463& NR 32891 and PilA5in NR 49277 & NR 49290 showed an upregulation pattern while treated with both concentrations. Modulating this response, it is possible to reduce the pathogenicity of C. difficileduring medical care & antibiotic use.
- Master of Science
- Comparative Pathobiology
- West Lafayette