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Title: Gene expression response of Salmonella enterica serotype Enteritidis phage type 8 to the subinhibitory concentrations of the plant-derived compounds,trans-cinnamaldehyde,and eugenol

Author
item KOLLANOOR JOHNY, ANUP - University Of Minnesota
item Frye, Jonathan
item Donoghue, Ann - Annie
item DONOGHUE, DAN - University Of Arkansas
item VENKITANARAYANAN, KUMAR - University Of Connecticut

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/6/2017
Publication Date: 9/26/2017
Citation: Kollanoor Johny, A., Frye, J.G., Donoghue, A.M., Donoghue, D., Venkitanarayanan, K. 2017. Gene expression response of Salmonella enterica serotype Enteritidis phage type 8 to the subinhibitory concentrations of the plant-derived compounds,trans-cinnamaldehyde,and eugenol. Frontiers in Microbiology. 8(1828):1-10. doi: 10.3389/fmicb.2017.01828.
DOI: https://doi.org/10.3389/fmicb.2017.01828

Interpretive Summary: Salmonella is often found in poultry and can be the cause of foodborne human infections resulting in gastroenteritis or enteric fever. Many strategies have been developed to reduce the level of Salmonella in poultry to prevent foodborne human infections. One of these is to inhibit the growth of Salmonella by adding natural plant extracts to chicken feed or water. Previous work found that trans-cinnamaldehyde (TC) and eugenol (EG) reduced colonization of chickens with S. Enteritidis PT8, a strain that has caused several outbreaks of human infections in the U.S. To understand how TC and EG reduced colonization of chickens, PT8 was grown in the laboratory with subinhibitory concentration of TC or EG added to the culture. The genes expressed by PT8 under these conditions were identified by extracting their RNA 30 min after TC or EG addition and comparing it to the RNA expressed before addition of the compounds. A Salmonella microarray assay was done to compare RNA expression with and without the plant compounds, and the genes induced or repressed were identified. TC and EG reduced the expression of genes required for motility, pathogenicity, invasion of intestinal cells, transport systems, and outer membrane proteins. Several metabolic and biosynthetic pathways in PT8 were also down-regulated during exposure to the plant compounds. Both TC and EG stimulated the expression of heat shock genes in PT8. The plant-derived compounds, TC and EG, exert antimicrobial effects on S. Enteritidis PT8 by multiple mechanisms, including suppression of bacterial virulence, cell membrane composition, transport systems, and energy metabolism required for growth in the host environment.

Technical Abstract: Background: Salmonella Enteritidis phage type 8 (PT8) is a major poultry-associated Salmonella strain implicated in foodborne outbreaks in the United States. We previously reported that two GRAS-status, plant-derived compounds, trans-cinnamaldehyde (TC) and eugenol (EG) significantly reduced S. Enteritidis colonization in broiler and layer chickens. To elucidate potential mechanisms by which TC and EG reduced PT8 colonization, a whole-genome microarray analysis of the bacterium treated with TC and EG was conducted. Results: S. Enteritidis PT8 was grown in Luria-Bertani broth at 37oC to an OD600 of ~0.5. Subinhibitory concentration (SICs; concentration that does not inhibit bacterial growth) of TC (0.01%; 0.75mM) or EG (0.04%; 2.46mM) were then added to the culture. S. Enteritidis PT8 RNA was extracted before and 30 min after TC or EG addition. Labeled cDNA from triplicate biological replicates was subsequently hybridized to a microarray of over 99% of S. Enteritidis PT4 genes, and the hybridization signals were quantified. Two plant compounds, TC and EG down-regulated (P<0.005) S. Enteritidis PT8 genes required for the expression of flagellar motility, regulation of Pathogenicity Island 1, invasion of intestinal epithelial cells, multiple transport systems and outer membrane proteins. Moreover, several metabolic and biosynthetic pathways in the pathogen were down-regulated during exposure to the plant compounds. Both TC and EG stimulated the expression of heat shock genes, such as dnaK, dnaJ, ibpB and ibpA in S. Enteritidis PT8 (P<0.005). The results obtained from microarray were validated using a quantitative real-time PCR. Conclusion: Plant-derived compounds, TC and EG, exert antimicrobial effects on S. Enteritidis PT8 by multiple mechanisms, including those associated with suppressing bacterial virulence, cell membrane composition, and transport systems.