Serotonin modulates Campylobacter jejuni physiology and invitro interaction with the gut epithelium.

Microbial endocrinology, which is the study of neurochemical-based host-microbe interaction, has demonstrated that neurochemicals affect bacterial pathogenicity. A variety of neurochemicals, including norepinephrine, were shown to enhance intestinal epithelial colonization by Campylobacter jejuni. Yet, little is known whether serotonin, an abundant neurochemical produced in the gut, affects the physiology of C. jejuni and its interaction with the host gut epithelium. Considering the avian gut produces serotonin and serves as a major reservoir of C. jejuni, we sought to investigate whether serotonin can affect C. jejuni physiology and gut epithelial colonization in vitro. We first determined the biogeographical distribution of serotonin concentrations in the serosa, mucosa, as well as the luminal contents of the broiler chicken ileum, cecum, and colon. Serotonin concentrations were greater (P < 0.05) in the mucosa and serosa compared to the luminal content in each gut region examined. Among the ileum, colon, and cecum, the colon was found to contain the greatest concentrations of serotonin. We then investigated whether serotonin may effect changes in C. jejuni growth and motility in vitro. The C. jejuni used in this study was previously isolated from the broiler chicken ceca. Serotonin at concentrations of 1mM or below did not elicit changes in growth (P > 0.05) or motility (P > 0.05) of C. jejuni. Next, we utilized liquid chromatography tandem mass spectrometry to investigate whether serotonin affected the proteome of C. jejuni. Serotonin caused (P < 0.05) the downregulation of a protein (CJJ81176_1037) previously identified to be essential in C. jejuni colonization. Based on our findings, we evaluated whether serotonin would cause a functional change in C. jejuni adhesion and invasion of the HT29MTX-E12 colonic epithelial cell line. Serotonin was found to cause a reduction in adhesion (P < 0.05) but not invasion (P > 0.05). Together, we have identified a potential role for serotonin in modulating C. jejuni colonization in the gut in vitro. Further studies are required to understand the practical implications of these findings for the control of C. jejuni enteric colonization in vivo.

Japanese quail (Coturnix japonica) as a novel model to study the relationship between the avian microbiome and microbial endocrinology-based host-microbe interactions.

BACKGROUND: Microbial endocrinology, which is the study of neuroendocrine-based interkingdom signaling, provides a causal mechanistic framework for understanding the bi-directional crosstalk between the host and microbiome, especially as regards the effect of stress on health and disease. The importance of the cecal microbiome in avian health is well-recognized, yet little is understood regarding the mechanisms underpinning the avian host-microbiome relationship. Neuroendocrine plasticity of avian tissues that are focal points of host-microbiome interaction, such as the gut and lung, has likewise received limited attention. Avian in vivo models that enable the study of the neuroendocrine dynamic between host and microbiome are needed. As such, we utilized Japanese quail (Coturnix japonica) that diverge in corticosterone response to stress to examine the relationship between stress-related neurochemical concentrations at sites of host-microbe interaction, such as the gut, and the cecal microbiome. RESULTS: Our results demonstrate that birds which contrast in corticosterone response to stress show profound separation in cecal microbial community structure as well as exhibit differences in tissue neurochemical concentrations and structural morphologies of the gut. Changes in neurochemicals known to be affected by the microbiome were also identified in tissues outside of the gut, suggesting a potential relationship in birds between the cecal microbiome and overall avian physiology. CONCLUSIONS: The present study provides the first evidence that the structure of the avian cecal microbial community is shaped by selection pressure on the bird for neuroendocrine response to stress. Identification of unique region-dependent neurochemical changes in the intestinal tract following stress highlights environmental stressors as potential drivers of microbial endocrinology-based mechanisms of avian host-microbiome dialogue. Together, these results demonstrate that tissue neurochemical concentrations in the avian gut may be related to the cecal microbiome and reveal the Japanese quail as a novel avian model in which to further examine the mechanisms underpinning these relationships. Video abstract.

Draft Genome Sequences of Campylobacter jejuni Strains Isolated from Poultry.

Four wild-type Campylobacter jejuni strains isolated from the cecal contents of broiler chickens were sequenced. The average genome size was 1,622,170 bp, with 1,667 to 1,761 coding sequences and 47 to 51 RNAs. Multiple genes encoding motility, intestinal colonization, toxin production, stress tolerance, and multidrug resistance were present in all the strains.

Trans-Cinnamaldehyde, Eugenol and Carvacrol Reduce Campylobacter jejuni Biofilms and Modulate Expression of Select Genes and Proteins.

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.

Edible Coatings Fortified With Carvacrol Reduce Campylobacter jejuni on Chicken Wingettes and Modulate Expression of Select Virulence Genes.

Campylobacter jejuni, a leading cause of foodborne disease in humans, associate primarily with consumption of contaminated poultry and poultry products. Intervention strategies aimed at reducing C. jejuni contamination on poultry products could significantly reduce C. jejuni infection in humans. This study evaluated the efficacy of gum arabic (GA) and chitosan (CH) fortified with carvacrol (CR) as an antimicrobial coating treatment for reducing C. jejuni on chicken wingettes. Aforementioned compounds are generally recognized as safe status compounds obtained from gum arabic tree, crustaceans and oregano oil respectively. A total of four separate trials were conducted in which wingettes were randomly assigned to baseline, saline control (wingettes washed with saline), GA (10%), CH (2%), CR (0.25, 0.5, or 1%) or their combinations. Each wingette was inoculated with a cocktail of four wild-type strains of C. jejuni (∼7.5 log10 cfu/sample). Following 1 min of coating in aforementioned treatments, wingettes were air dried (1 h) and sampled at 0, 1, 3, 5, and 7 days of refrigerated storage for C. jejuni and total aerobic counts (n = 5 wingettes/treatment/day). In addition, the effect of treatments on wingette color was measured using a Minolta colorimeter. Furthermore, the effect of treatments on the expression of C. jejuni survival/virulence genes was evaluated using real-time quantitative PCR. Results showed that all three doses of CR, CH or GA-based coating fortified with CR reduced C. jejuni from day 0 through 7 by up to 3.0 log10 cfu/sample (P < 0.05). The antimicrobial efficacy of GA was improved by CR and the coatings reduced C. jejuni by ∼1 to 2 log10 cfu/sample at day 7. Moreover, CH + CR coatings reduced total aerobic counts when compared with non-coated samples for a majority of the storage times. No significant difference in the color of chicken wingettes was observed between treatments. Exposure of pathogen to sublethal concentrations of CR, CH or combination significantly modulated select genes encoding for energy taxis (cetB), motility (motA), binding (cadF), and attachment (jlpA). The results suggest that GA or CH-based coating with CR could potentially be used as a natural antimicrobial to control C. jejuni in postharvest poultry products.

Trans-Cinnamaldehyde, Carvacrol, and Eugenol Reduce Campylobacter jejuni Colonization Factors and Expression of Virulence Genes in Vitro.

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.

Application of ß-Resorcylic Acid as Potential Antimicrobial Feed Additive to Reduce Campylobacter Colonization in Broiler Chickens.

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, ß-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.