Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans characterized by fever, diarrhea, and abdominal cramps. In the human gut, Campylobacter adheres and invades the intestinal epithelium followed by cytolethal distending toxin mediated cell death, and enteritis. Reducing the attachment and invasion of Campylobacter to intestinal epithelium and expression of its virulence factors such as motility and cytolethal distending toxin (CDT) production could potentially reduce infection in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentration not inhibiting bacterial growth) of three GRAS (generally recognized as safe) status phytochemicals namely trans-cinnamaldehyde (TC; 0.005, 0.01%), carvacrol (CR; 0.001, 0.002%), and eugenol (EG; 0.005, 0.01%) in reducing the attachment, invasion, and translocation of C. jejuni on human intestinal epithelial cells (Caco-2). Additionally, the effect of these phytochemicals on Campylobacter motility and CDT production was studied using standard bioassays and gene expression analysis. All experiments had duplicate samples and were replicated three times on three strains (wild type S-8, NCTC 11168, 81–176) of C. jejuni. Data were analyzed using ANOVA with GraphPad ver. 6. Differences between the means were considered significantly different at P < 0.05. The majority of phytochemical treatments reduced C. jejuni adhesion, invasion, and translocation of Caco-2 cells (P < 0.05). In addition, the phytochemicals reduced pathogen motility and production of CDT in S-8 and NCTC 11168 (P < 0.05). Real-time quantitative PCR revealed that phytochemicals reduced the transcription of select C. jejuni genes critical for infection in humans (P < 0.05). Results suggest that TC, CR, and EG could potentially be used to control C. jejuni infection in humans.

Campylobacter jejuni is the leading cause of human foodborne illness globally, and is strongly linked with the consumption of contaminated poultry products. Several studies have shown that C. jejuni can form sanitizer tolerant biofilm leading to product contamination, however, limited research has been conducted to develop effective control strategies against C. jejuni biofilms. This study investigated the efficacy of three generally recognized as safe status phytochemicals namely, trans -cinnamaldehyde (TC), eugenol (EG), or carvacrol (CR) in inhibiting C. jejuni biofilm formation and inactivating mature biofilm on common food contact surfaces at 20 and 37°C. In addition, the effect of phytochemicals on biofilm architecture and expression of genes and proteins essential for biofilm formation was evaluated. For the inhibition study, C. jejuni was allowed to form biofilms either in the presence or absence of sub-inhibitory concentrations of TC (0.75 mM), EG (0.61 mM), or CR (0.13 mM) for 48 h and the biofilm formation was quantified at 24-h interval. For the inactivation study, C. jejuni biofilms developed at 20 or 37°C for 48 h were exposed to the phytochemicals for 1, 5, or 10 min and surviving C. jejuni in the biofilm were enumerated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surface at both temperatures ( P < 0.05). The highest dose of TC (75.64 mM), EG (60.9 mM) and CR (66.56 mM) inactivated (>7 log reduction) biofilm developed on steel (20°C) within 5 min. The genes encoding for motility systems ( flaA , flaB , and flgA ) were downregulated by all phytochemicals ( P < 0.05). The expression of stress response ( cosR , ahpC ) and cell surface modifying genes ( waaF ) was reduced by EG. LC-MS/MS based proteomic analysis revealed that TC, EG, and CR significantly downregulated the expression of NapA protein required for oxidative stress response. The expression of chaperone protein DnaK and bacterioferritin required for biofilm formation was reduced by TC and CR. Scanning electron microscopy revealed disruption of biofilm architecture and loss of extracellular polymeric substances after treatment. Results suggest that TC, EG, and CR could be used as a natural disinfectant for controlling C. jejuni biofilms in processing areas.

Campylobacter is one of the major foodborne pathogens that result in severe gastroenteritis in humans, primarily through consumption of contaminated poultry products. Chickens are the reservoir host of Campylobacter, where the pathogen colonizes the ceca, thereby leading to contamination of carcass during slaughter. A reduction in cecal colonization by Campylobacter would directly translate into reduced product contamination and risk of human infections. With increasing consumer demand for antibiotic free chickens, significant research is being conducted to discover natural, safe and economical antimicrobials that can effectively control Campylobacter colonization in birds. This study investigated the efficacy of in-feed supplementation of a phytophenolic compound, β-resorcylic acid (BR) for reducing Campylobacter colonization in broiler chickens. In two separate, replicate trials, day-old-chicks (Cobb500; n = 10 birds/treatment) were fed with BR (0, 0.25, 0.5, or 1%) in feed for a period of 14 days (n = 40/trial). Birds were challenged with a four-strain mixture of Campylobacter jejuni (∼106 CFU/ml; 250 μl/bird) on day 7 and cecal samples were collected on day 14 for enumerating surviving Campylobacter in cecal contents. In addition, the effect of BR on the critical colonization factors of Campylobacter (motility, epithelial cell attachment) was studied using phenotypic assay, cell culture, and real-time quantitative PCR. Supplementation of BR in poultry feed for 14 days at 0.5 and 1% reduced Campylobacter populations in cecal contents by ∼2.5 and 1.7 Log CFU/g, respectively (P < 0.05). No significant differences in feed intake and body weight gain were observed between control and treatment birds fed the compound (P > 0.05). Follow up mechanistic analysis revealed that sub-inhibitory concentration of BR significantly reduced Campylobacter motility, attachment to and invasion of Caco-2 cells. In addition, the expression of C. jejuni genes coding for motility (motA, motB, fliA) and attachment (jlpA, ciaB) was down-regulated as compared to controls (P < 0.05). These results suggest that BR could potentially be used as a feed additive to reduce Campylobacter colonization in broilers.

Human Campylobacter infections, a leading foodborne illness globally, has been linked with the high prevalence of this bacterium on raw retail chicken products. Reduction of Campylobacter counts on poultry products would greatly reduce the risk of subsequent infections in humans. To this end, this study investigated the potential of the phytophenolic compound β-resorcylic acid (BR) to reduce Campylobacter counts on postharvest poultry (chicken skin or meat). Four trials in total, two each on thigh skin or breast meat, were conducted in which chicken skin or meat samples (2 ± 0.1 g; 10 samples per treatment) were inoculated with 50 μL (∼10 CFU per sample) of a cocktail of four wild strains of C. jejuni. After 30 min of attachment, inoculated samples were dipped in a 0, 0.5, 1, or 2% BR solution for 30 s immediately followed by vigorously vortexing the samples in Butterfield's phosphate diluent and plating the supernatant for Campylobacter enumeration. In addition, the effect of BR on the color of skin and meat samples was studied. Moreover, the change in the expression of survival and virulence genes of C. jejuni exposed to BR was evaluated. Data were analyzed by the PROC MIXED procedure of SAS (P < 0.05; SAS Institute Inc., Cary, NC). All BR treatments significantly reduced Campylobacter populations on both chicken or meat samples by 1 to 3 log CFU/g compared with non-BR-treated washed controls. No significant difference in the lightness, redness, and yellowness of skin and meat samples was observed on exposure to BR wash (P > 0.05). Real-time PCR results revealed that BR treatment down-regulated expression of select genes coding for motility (motA, motB) and attachment (cadF, ciaB) in the majority of C. jejuni strains. Stress response genes (sodB, katA) were upregulated in C. jejuni S-8 (P < 0.05). Overall, our results suggest that BR could be effectively used as antimicrobial dip treatment during poultry processing for reducing Campylobacter on chicken carcasses.

Campylobacter jejuni is a major foodborne pathogen that causes gastroenteritis in humans. Chickens act as the reservoir host for C. jejuni , wherein the pathogen asymptomatically colonizes the ceca leading to contamination of carcasses during slaughter. The major colonization factors in C. jejuni include motility, intestinal epithelial attachment, acid/bile tolerance, and quorum sensing. Reducing the expression of the aforementioned factors could potentially reduce C. jejuni colonization in chickens. This study investigated the efficacy of subinhibitory concentration ( SIC ; compound concentration not inhibiting bacterial growth) of carvacrol in reducing the expression of C. jejuni colonization factors in vitro. Moreover, the effect of carvacrol on the expression of C. jejuni proteome was investigated using liquid chromatography-tandem mass spectrometry. The motility assay was conducted at 42°C, and the motility zone was measured after 24 h of incubation. For the adhesion assay, monolayers of primary chicken enterocytes (∼10 5 cells/well) were inoculated with C. jejuni (6 log cfu/well) either in the presence or absence of carvacrol, and the adhered C. jejuni were enumerated after 90 min of incubation at 42°C. The effect of carvacrol on C. jejuni quorum sensing and susceptibility to acid/bile stress was investigated using a bioluminescence assay and an acid–bile survival assay, respectively. The SIC (0.002%) of carvacrol reduced the motility of C. jejuni strains S-8 and NCTC 81-176 by ∼50 and 35%, respectively ( P < 0.05). Carvacrol inhibited C. jejuni S-8 and NCTC 81-176 adhesion to chicken enterocytes by ∼0.8 and 1.5 log cfu/mL, respectively ( P < 0.05). Moreover, carvacrol reduced autoinducer-2 activity and increased the susceptibility of C. jejuni to acid and bile in both the strains ( P < 0.05). Liquid chromatography-tandem mass spectrometry revealed that the SIC of carvacrol reduced the expression of selected C. jejuni colonization proteins critical for motility (methyl-accepting chemotaxis protein), adhesion (GroL), growth and metabolism (AspA, AcnB, Icd, Fba, Ppa, AnsA, Ldh, Eno, PurB-1), and anaerobic respiration (NapB, HydB, SdhA, NrfA) ( P < 0.05). Results suggest the mechanisms by which carvacrol could reduce C. jejuni colonization in chickens.

Campylobacter jejuni infection in humans is strongly associated with the consumption of contaminated poultry products. With increasing consumer demand for minimally processed and natural product, there is a need for novel intervention strategies for controlling C. jejuni. Antimicrobial coatings are increasingly being used for preventing food contamination due to their efficacy and continuous protection of product. This study investigated the efficacy of pectin and chitosan coating fortified with eugenol to reduce C. jejuni on chicken wingettes. Pectin, chitosan, and eugenol are generally recognized as safe status compounds derived from berries, crustaceans, and cloves respectively. Each wingette was inoculated with a mixture of 4 wild-type strains of C. jejuni (approximately 107 CFU/sample) and randomly assigned to controls, pectin (3%), chitosan (2%), eugenol (0.5, 1, or 2%), or their combinations. Following 1 min of coating, wingettes were air-dried, vacuum sealed, and sampled on 0, 1, 3, 5, and 7 d of refrigerated storage for C. jejuni and aerobic counts (n = 5 wingettes/treatment/d). In addition, the effect of treatments on wingette color and expression of C. jejuni survival/virulence genes was evaluated. All 3 doses of eugenol or chitosan significantly reduced C. jejuni and aerobic bacteria from 0 d through 7 d. Incorporation of 2% eugenol in chitosan improved coating efficiency and reduced C. jejuni counts by approximately 3 Log CFU/sample at the end of 7 d of storage (P < 0.05). Similarly, the antimicrobial efficacy of pectin was improved by 2% eugenol and the coating reduced C. jejuni by approximately 2 Log CFU/sample at 7 d of storage. Chitosan coating with 2% eugenol also showed greater reductions of total aerobic counts as compared to individual treatments of eugenol and chitosan. No significant difference in the color of chicken wingettes was observed between treatments. Exposure of C. jejuni to eugenol, chitosan, or combination significantly modulated select genes encoding for motility, quorum sensing, and stress response. Results demonstrate the potential of pectin or chitosan coating fortified with eugenol as a postharvest intervention against C. jejuni contamination on poultry products.

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

The efficacy of the natural plant‐derived compound, eugenol (EG), as an antimicrobial wash treatment to reduce Campylobacter jejuni in postharvest poultry was investigated. The antimicrobial efficacy of EG was studied as a suspension, emulsion, or nanoemulsion treatment (two trials each). In each trial, chicken skin samples were inoculated with C. jejuni (∼7.2 Log CFU/sample), washed with treatments (0, 0.125, 0.25, 0.5, 1, or 2% EG corresponds to 0, 7.61, 15.22, 30.45, 60.90, or 121.8 mM, respectively) for 1 min, drip dried for 2 min, and then processed at 0, 8, and 24 hr of refrigerated storage (n = 5 samples/treatment/time point). All doses of the EG suspension consistently reduced C. jejuni counts with the greatest reduction (>2.0 Log CFU/sample) for the 2% dose when compared with controls (p < .05). EG emulsions or nanoemulsions did not provide any additional reduction in C. jejuni when compared to EG suspension. Our results suggest that EG could be an effective postharvest intervention strategy for reducing C. jejuni contamination on poultry products. Practical Applications Campylobacter jejuni, a leading cause of foodborne illness in humans, is strongly associated with the consumption of contaminated poultry products. Interventions reducing C. jejuni contamination in poultry would reduce the risk of subsequent human infections. In this study, the antimicrobial efficacy of eugenol was studied in three different delivery systems; suspension, emulsion, or nanoemulsion. Our results demonstrated that eugenol was effective in reducing C. jejuni counts on chicken skin and can be used as a potential strategy to reduce Campylobacter on poultry products.