Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most commonly isolated Salmonella serotypes from poultry (Centers for Disease Control and Prevention, 2010; Campioni et al., 2013; Gould et al., 2013), and is responsible for about one third of the reported human salmonellosis outbreaks in the United States (Gould et al., 2013). The pathogen poses a significant health concern for human health due to the contamination of poultry meat and eggs, which constitute the most common food products linked to human salmonellosis (Guard-Petter, 2001; Marcus et al., 2007). In chickens, the cecum is the most common site for Salmonella residency (Gantois et al., 2009; Kollanoor Johny et al., 2012a,b,c). 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 (Keller et al., 1995; Gantois et al., 2009). Therefore, reducing S. Enteritidis in the chicken intestinal tract would reduce contamination of poultry products, minimizing human health risk (Altekruse et al., 1993). 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 (reviewed in Kollanoor Johny et al., 2012a,b,c).

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 (Kollanoor Johny et al., 2008, 2010, 2012a,b,c). 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 (Upadhyaya et al., 2015). Trans-cinnamaldehyde, an aromatic aldehyde extracted from the bark of cinnamon (Cinnamomum zeylandicum), has well-known antimicrobial properties (Friedman et al., 2002, reviewed by Burt, 2004). Eugenol (EG), a compound obtained from clove (Eugenia caryophillis) oil, is also reported to be effective in killing pathogenic microorganisms (Suhr and Nielsen, 2003, reviewed by Burt, 2004). Both these compounds are classified as generally recognized as safe (GRAS) for use in foods by the United States Food and Drug Administration (USFDA) (TC–21CFR182.60; EG–21CFR582.60).

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) to a sub-inhibitory concentration of TC or EG using DNA microarrays. We sought to delineate the arsenal of genes affected by the two phytochemicals.

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. The major genes predicted to be differentially regulated in PT8 exposed to TC and EG based on the microarray data are provided in Table 1. The analysis identified 566 genes (9.7% genes on the array) with differentially expressed transcripts due to exposure to TC, and 483 genes (8.4% on the array) with differential expression as a result of EG exposure (M ± 1.5, P < 0.005; Tables 2, 3). TC significantly down-regulated 275 genes, whereas EG did so for 289 genes (P < 0.005). Many of the genes modulated are known to play a critical role in S. Enteritidis virulence in vivo based on previously published reports (Dhawi et al., 2011; Harvey et al., 2011) and are discussed here. Additional data on genes that are not discussed in depth in the manuscript can be found in the Supplementary Tables S1–S19.

Salmonella Enteritidis is a leading cause of foodborne salmonellosis worldwide. The increase in the number of outbreaks over the last few decades, where poultry and poultry products were implicated, has led to several pre- and post-harvest strategies to control the pathogen. We previously reported that supplementation of TC and EG in broilers (Kollanoor Johny et al., 2012a,b) and TC in layers (Upadhyaya et al., 2015) significantly reduced S. Enteritidis colonization in birds. We also observed that these compounds decreased Salmonella motility and invasion of avian epithelial cells in vitro (Kollanoor Johny et al., 2012b). Since analysis of transcriptional profiles of bacteria exposed to an antimicrobial molecule can yield new information regarding affected pathways and mechanisms of inhibition (Wilson et al., 1999; Gmuende et al., 2001), we conducted a microarray analysis of S. Enteritidis PT8 exposed to TC and EG. The goal was to delineate the genes affected by these plant-derived compounds, to gain insight into the molecular mechanism of how these compounds may cause reduced S. Enteritidis colonization in chickens.

A diverse repertoire of Salmonella virulence genes was down-regulated in response to treatment with TC and EG, including, critically, the genes involved in SPI-1 regulation. These genes are necessary for Salmonella uptake into non-phagocytic epithelial cells (McCormick, 2004). A direct control of SPI-1 genes is exerted by the activation by hilA-the major regulator of the island, resulting in the invasion of host tissue. Ellermeier and Slauch (2007) reported that other regulators, such as HilC and HilD work in conjunction with another regulator RtsA to express SPI-1. HilD, an AraC/XylS family member, binds directly to several sites within the hilA promoter and derepresses gene expression, which in turn results in the expression of other associated virulence genes. Both plant-derived compounds down-regulated the major regulators hilA, hilD, and hilC, although a greater effect was observed on hilC (Table 1, Supplementary Table S1). The T3SS encoded by SPI-1 is the pathogenic machinery to inject secretory proteins into host cells that help in the internalization of Salmonella (Kubori et al., 2000; Galan, 2001). The effector proteins are transported into the host cells with the help of three translocation proteins, sipB, sipC and sipD (Collazo and Galan, 1997; Galan, 2001). As revealed in the microarray data, TC down-regulated the effector protein genes (Supplementary Table S3).

Flagellar motility and chemotaxis are the two critical features by which bacteria survive under a wide variety of environments (Soutourina and Bertin, 2003), including the intestinal lumen and ceca. Flagellar motility is also required for colonization by Salmonella (Ciacci-Woolwine et al., 1998), and is considered a virulence factor in the bacterium (Josenhans and Suerbaum, 2002). In the regulation of flagella, the flhDC master operon plays a key role (Kutsukake et al., 1990; Soutourina and Bertin, 2003). Among other genes, the master operon is necessary for transcriptional activation of the fliA gene, encoding σ²⁸, a sigma factor associated with the transcription of late flagellar genes, such as fliC (filament structural protein). It was interesting to note that that these associated genes were all down-regulated by EG (Table 1). Apart from the flhCD-regulated genes (early flagellar operon), the middle and late operon genes, such as flgKLMN, fliZ, motAB, and cheAWYZ, were also down-regulated by EG (Table 3, Supplementary Table S5). In contrast, TC did not exert strong effects on these genes.

Porins are outer membrane aqueous channels used in the diffusion of various compounds across the membrane that can also aid in adapting the bacteria to various conditions and confer antibacterial drug resistance (Gil et al., 2009). In the current study, TC and EG down-regulated porins, such as ompW, ompC, ompS1, and nmpC by 2-fold. Recently, Gil et al. (2009) reported that ompW expression in Salmonella was activated in response to oxidative stress. Both plant-derived compounds down-regulated ompW by 4.3- and 2.7-fold, respectively. Methner et al. (2004) reported that ompC deletion mutants of S. Enteritidis resulted in decreased virulence and colonization in chickens in conjunction with deletions of rpoS or phoP. Our results revealed that ompC was down-regulated by 2.8-fold by both compounds (Table 1). Porin ompS1, down-regulated by TC and EG in our study, is regulated by the EnvZ/OmpR two-component signal transduction system (Florez-Valdez et al., 2003). Rodriguez-Morales et al. (2006) reported that ompS1 is required for virulence in S. Typhimurium in mice.

It was recently reported that during colonization in the gut Salmonella makes use of a wide range of carbon compounds, including D- glucose, melibiose, L-ascorbate, and other unusual compounds, such as sialic acid, ethanolamine, and propanediol resulting from the degradation of host mucosa (Dhawi et al., 2011). We observed that TC and EG down-regulated some of the glucose metabolism genes (agp, pykA), and TC reduced the transcription of melABR genes (Supplementary Table S8). Moreover, both plant-derived compounds were found to apparently reduce the bacterial capacity to utilize propanediol and ethanolamine as an energy source or tetrathionate as a terminal electron acceptor (Table 1), suggesting that TC and EG may interrupt the pathways critical for Salmonella survival and successful competition with the host microflora. During colonization of the host intestine, Salmonella can use energy and nutrient sources unavailable to commensal bacteria, including propanediol and ethanolamine. These compounds cannot be used for energy without a terminal electron acceptor, and the host flora mainly consists of mostly fermentative bacteria unable to perform this electron transport. Salmonella, however, is uniquely capable of using these compounds by employing tetrathionate as a terminal electron acceptor. When tetrathionate is used as an electron acceptor, it is reduced to thiosulfate, which is then regenerated via oxidation by the reactive oxygen compounds released by the host in response to invasion by Salmonella. Salmonella can therefore outcompete the host flora, resulting in inflammatory diarrhea, with Salmonella cells as major bacterial component of the excreta thus accomplishing transmission to the next host organism.

Harvey et al. (2011) reported that within the lumen of chicks, the degradation of 1, 2-propanediol occurs at a much higher rate, requiring cobalamin synthesis. The pdu operon required for propanediol utilization works in conjunction with cob and cbi operons involved in vitamin B12 synthesis. Our results revealed that TC and EG significantly down-regulated pdu and cbi operons (Table 1). It was previously observed that Salmonella could utilize ethanolamine, which is derived from host cells and membranes, as a sole source of carbon, nitrogen, and energy (Garsin, 2010). Catabolism of ethanolamine is under the control of the eut operon that primarily consists of 17 clustered genes (Kofoid et al., 1999) involved in the conversion of the compound to acetyl-CoA for energy (Harvey et al., 2011). The plant compound TC was found to down-regulate these genes (Supplementary Table S14). An anaerobic environment may exist in the chicken cecum (Harvey et al., 2011), and oxygen may not be present as a terminal electron acceptor. In that case, other electron acceptors are required, and ethanolamine and 1, 2-propanediol could be utilized in that function by Salmonella in the cecum (Price-Carter et al., 2001). Tetrathionate may be present in the cecum of chicks due to the presence of yolk sac, which is rich in sulfur. Tetrathionate is reduced to thiosulphide and H₂S, involving proteins encoded by ttr and phs (Harvey et al., 2011). Our results indicated that TC down-regulated the transcription of ttr genes (Supplementary Table S12), whereas both plant-derived compounds reduced phsABC transcription (Supplementary Table S18). Overall, TC and EG may disrupt Salmonella's ability to use a unique energy source, possibly reducing its ability to compete successfully with the host's microflora.

Some heat shock genes and genes conferring antibiotic resistance were up-regulated by TC and EG. While the mar (multiple antibiotic resistance) locus genes were up-regulated by both TC and EG, the efflux pump genes acrAB were only up-regulated by EG. The MarA protein positively regulates transcription of acrAB, and it was therefore unexpected that the latter genes were not up-regulated after exposure to TC. However, the transcriptional repressor marR was also found to be up-regulated by both TC and EG, potentially indicating a compensatory mechanism on reducing marAB-related resistance. Heat shock proteins dnaJ, dnaK, ibpA, ibpB, and htpG were found to be up-regulated by both plant-derived compounds (P < 0.005). However, clpPX-mediated heat shock protection mechanisms were least affected (Supplementary Table S19).

Some limitations of this study include the use of nutrient broth for the gene expression studies as opposed to growth within a characterized chicken-derived intestinal cell line, although broth cultures have been previously used for similar experiments (Cao et al., 2011; Jessica et al., 2013; Upadhyay et al., 2013). To our knowledge, immortalized chicken intestinal cell lines are unavailable, and developing and maintaining a primary chicken intestinal cell line has its challenges, including the high variability in confluency across different propagations. In a separate study, we conducted invasion assays using the only available permanent avian cancer cell line (Budgerigar Abdominal Tumor Cells; BATCs) and found that these two phytophenolics significantly reduced Salmonella invasion without affecting the viability of cells (Kollanoor Johny et al., 2012b). However, being a permanent cell line obtained from a different avian species (parakeets), a direct correlation of these results to a chicken-derived cell environment is not guaranteed. However, the selected phytophenolics reduced the cecal colonization of S. Enteritidis in broiler chickens in at least 5 independent in vivo experiments (Kollanoor Johny et al., 2012a,b), lending support to the notion that TC and EG directly affect S. Enteritidis virulence genes. However, this hypothesis needs to be further validated.

Genes required for S. Enteritidis PT8 virulence and colonization, including those conferring flagella-associated motility, those enabling invasion of epithelial cells, genes encoding T3SSs, genes regulating the synthesis of effector proteins delivered by T3SS, and genes encoding other surface virulence structures and OMPs were found to be down-regulated after exposure to subinhibitory concentrations of TC or EG. Moreover, the exposure to both phytochemicals brought about transcriptional changes of S. Enteritidis PT8 genes involved in the degradation of carbon compounds, those related to amino acid-, carbohydrate-, and lipid-transport, those associated with secondary metabolism, and genes encoding the biosynthesis of molybdopterin and vitamin B12. The genes regulating pathways associated with tetrathionate reduction, propanediol, and ethanolamine utilization, and H₂S production were also affected. Finally, a few genes regulating antibiotic resistance and heat shock were up-regulated by the plant-derived compounds, including transcriptional activators and repressors, with as yet undefined phenotypic net results.

AK carried out microarray experiments, RT-qPCR assays, involved in the study design and wrote the manuscript. JF trained AK to conduct microarray analysis, preliminary interpretation of statistical analysis and corrected the manuscript. AD and DD participated in the design of the study and corrected the manuscript. SP and MM designed and constructed the microarrays, interpreted the hybridization results and performed statistical analysis of the data. KV conceived and designed the study and corrected the manuscript. All authors read and approved the manuscript.