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

Authors: JOHNY, ANUP KOLLANOOR - University Of Minnesota; Frye, Jonathan; Donoghue, Ann - Annie; DONOGHUE, DAN - University Of Arkansas; PORWOLLIK, STEFFEN - University Of California; MCCLELLAND, MICHAEL - University Of California; 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: Johny, A., Frye, J.G., Donoghue, A.M., Donoghue, D.J., Porwollik, S., Mcclelland, M., Venkitanarayanan, K. 2017. Gene expression response of Salmonella enterica Serotype Enteritidis phage type 8 to subinhibitory concentrations of the plant-derived compounds trans-cinnamaldehyde and eugenol. Frontiers in Microbiology. 8:1828. https://doi.org/10.3389/fmicb.2017.01828.
DOI: https://doi.org/10.3389/fmicb.2017.01828

Interpretive Summary: Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most commonly isolated Salmonella serotypes from poultry, and is responsible for about one third of the reported human salmonellosis outbreaks in the United States. In chickens, the cecum is the most common site for Salmonella residency. The cecal colonization by the pathogen results in fecal shedding, invasion of reproductive organs, contamination of egg shells and yolks, and carcass contamination during slaughter. Therefore, reducing S. Enteritidis in the chicken intestinal tract would reduce contamination of poultry products, minimizing human health risk. Thus, intervention strategies for controlling S. Enteritidis colonization in chickens are critical for improving the microbiological safety of poultry-derived foods. Since a multitude of sources can transmit S. Enteritidis to chickens at farms, a variety of pre-harvest approaches, especially in-feed supplementation of antimicrobials, has been explored for reducing the pathogen persistence in poultry We previously reported that two phytophenolics, namely trans-cinnamaldehyde (TC) and eugenol (EG), were effective in reducing S. Enteritidis in vitro and in broiler chickens. Further, we observed that in-feed supplementation of TC in layers significantly reduced S. Enteritidis colonization in the internal organs of birds and egg-borne transmission of the bacterium. In light of our previous findings, the objective of this study was to analyze the genome-wide response of S. Enteritidis Phage Type 8 (or PT8, a major poultry-associated Salmonella strain implicated in foodborne outbreaks in the United States) to a sub-inhibitory concentration of TC or EG using DNA microarrays. The DNA microarray analysis revealed that about 10% of genes in the S. Enteritidis PT8 genome were significantly modulated after exposure to TC and EG. Many of the genes modulated are known to play a critical role in S. Enteritidis virulence in vivo based on previously published reports. In conclusion, the plant-derived compounds TC and EG exert antimicrobial effects on S. Enteritidis PT8 by affecting multiple genes, including those associated with virulence, colonization, cell membrane composition, and transport systems.

Technical Abstract: 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 plant-derived compounds generally recognized as safe (GRAS), trans-cinnamaldehyde (TC), and eugenol (EG), significantly reduced S. Enteritidis colonization in broiler and layer chickens. To elucidate potential PT8 genes affected by TC and EG during colonization, a whole-genome microarray analysis of the bacterium treated with TC and EG was conducted. S. Enteritidis PT8 was grown in Luria-Bertani broth at 37'C to an OD600 of ~0.5. Subinhibitory concentrations (SICs; concentration that does not inhibit bacterial growth) of TC (0.01%; 0.75 mM) or EG (0.04%; 2.46 mM) were then added to the culture. S. Enteritidis PT8 RNA was extracted before and 30 min after TC or EG addition. Labeled cDNA from three replicate experiments was subsequently hybridized to a microarray of over 99% of S. Enteritidis PT4 genes, and the hybridization signals were quantified. The plant-derived compounds down-regulated (P < 0.005) expression of S. Enteritidis PT8 genes involved in flagellar motility, regulation of the Salmonella Pathogenicity Island 1, C and EG also suppressed transcription of genes encoding multiple transport systems and outer membrane proteins. Moreover, several metabolic and biosynthetic pathways in the pathogen were down-regulated during exposure to the plant-derived compounds. Both TC and EG stimulated the transcription 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. The plant-derived compounds TC and EG exert antimicrobial effects on S. Enteritidis PT8 by affecting multiple genes, including those associated with virulence, colonization, cell membrane composition, and transport systems.