A Novel Natural Antimicrobial Can Reduce the in vitro and in vivo Pathogenicity of T6SS Positive Campylobacter jejuni and Campylobacter coli Chicken Isolates

Abstract

Human campylobacteriosis is considered one of the most common foodborne diseases worldwide with poultry identified as the main source of infection accounting for 50-80% of human cases. Highly virulent Campylobacter spp., positive for the Type VI secretion system (T6SS), which have an increased ability to adhere to and invade the host gastrointestinal epithelium are highly prevalent in poultry. Multidrug resistant strains of bacteria are rapidly evolving and therefore, new antimicrobials to supplement animal feed that are able to control Campylobacter species, are in great need. The work presented herein indicates that a novel phenolic antimicrobial, Auranta 3001, is able to reduce the adhesion and invasion of human intestinal epithelial cells (HCT-8) by two T6SS positive chicken isolates, C. jejuni RC039 (p < 0.05) and C. coli RC013 (p < 0.001). Exposure of C. jejuni RC039 and C. coli RC013 to Auranta 3001 downregulated the expression of hcp and cetB genes, known to be important in the functionality of T6SS. Furthermore, the reduced adhesion and invasion is associated with a significant decrease in bacterial motility of both isolates (p < 0.05-p < 0.001) in vitro. Most importantly our in vivo results show that Auranta 3001 is able to reduce cecum colonization levels from log 8 CFU/ml to log 2 CFU/ml for C. jejuni RC039 and from log 7 CFU/ml to log 2 CFU/ml for C. coli RC013. In conclusion, this novel antimicrobial is able to reduce the pathogenic properties of T6SS campylobacters in vitro and also to decrease colonization in vivo.

Keywords: Campylobacter coli; Campylobacter jejuni; HCT-8; attachment; gene expression; invasion.

FIGURE 1

The effect of Auranta 3001 on C. jejuni RC039 and C. coli RC013 motility. (A) shows the percentage decrease of C. jejuni RC039 motility and (B) of C. coli RC013 over control. Asterisks indicate significant differences (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). Error bars represent the standard deviation of means from three different experiments, each containing triplicate samples.

FIGURE 2

Adhesion and invasion of HCT-8 cells by C. jejuni RC039. The adherence (A) and invasion (B) of HCT-8 cells in the presence of Auranta 3001. Results are expressed as percentages of the initial inoculum. Asterisks indicate significant differences (Student’s t-test ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). Error bars represent the standard deviation of means from three different experiments, each containing triplicate samples.

FIGURE 3

Adhesion and invasion of HCT-8 cells by C. coli RC013. The adherence (A) and invasion (B) of HCT-8 cells in the presence of Auranta 3001. Results are expressed as percentages of the initial inoculum. Asterisks indicate significant differences (Student’s t-test ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). Error bars represent the standard deviation of means from three different experiments, each containing triplicate samples.

FIGURE 4

Alcian blue profiles of Campylobacter jejuni RC039 (A) and Campylobacter coli RC013 (B) capsule polysaccharides when co-cultured with HCT-8 cells pretreated with 0.1% Auranta 3001.

FIGURE 5

Effect of Auranta 3001 on C. jejuni RC039 and C. coli RC013 cetB (B) and hcp gene (A) expression after 3 h of exposure to 0.1 and 0.5% Auranta 3001. Asterisks indicate significant differences (Student’s t-test ^∗p < 0.05; ^∗∗p < 0.01). Error bars represent the standard deviation of means from three different experiments.

FIGURE 6

Transepithelial resistance of HCT-8 cells infected with C. jejuni RC039 and C. coli RC013 at 3 h postinfection using 0.5% Auranta during infection. Asterisk indicates significant difference (^∗∗P < 0.001) compared to uninfected cells. Error bars indicate standard deviations.