Attenuation of Vibrio parahaemolyticus Virulence Factors by a Mixture of Natural Antimicrobials.

Reducing acute mortality in aquatic crustaceans using natural alternatives to antibiotics has become a necessity, firstly for its positive impact on the aquaculture industry and, secondly, because the extensive use of antibiotics may lead to increased levels of drug resistance in pathogenic microorganisms. This study aimed to investigate the effect of a mixture of natural antimicrobials on the in vitro and in vivo virulence abilities of Type VI secretion system (T6SS)-positive Vibrio parahaemolyticus (A3 and D4), strains known as having potentially harmful health consequences for aquatic crustaceans and consumers. Herein, we report that a natural antimicrobial mixture (A3009) was capable of significantly reducing the virulence of V. parahaemolyticus strains A3 and D4 in an in vitro infection model, using the fish cell line CHSE-214, an effect which correlates with the bacterial downregulation of hcp1 and hcp2 gene expression and with the ability of the antimicrobial to efficiently retain low cytotoxic levels (p < 0.001). We show for the first time that a natural antimicrobial is able to significantly reduce the mortality of shrimps in a challenge experiment and is able to significantly attenuate H2O2 release during infection (p < 0.001), indicating that it could harbor positive intestinal redox balance effects.

The Campylobacter jejuni Type VI Secretion System Enhances the Oxidative Stress Response and Host Colonization.

The role of the Type VI secretion system (T6SS) in Campylobacter jejuni is poorly understood despite an increasing prevalence of the T6SS in recent C. jejuni isolates in humans and chickens. The T6SS is a contractile secretion machinery capable of delivering effectors that can play a role in host colonization and niche establishment. During host colonization, C. jejuni is exposed to oxidative stress in the host gastrointestinal tract, and in other bacteria the T6SS has been linked with the oxidative stress response. In this study, comparisons of whole genome sequences of a novel human isolate 488 with previously sequenced strains revealed a single highly conserved T6SS cluster shared between strains isolated from humans and chickens. The presence of a functional T6SS in the 488 wild-type strain is indicated by expression of T6SS genes and secretion of the effector TssD. Increased expression of oxidative stress response genes katA, sodB, and ahpC, and increased oxidative stress resistance in 488 wild-type strain suggest T6SS is associated with oxidative stress response. The role of the T6SS in interactions with host cells is explored using in vitro and in vivo models, and the presence of the T6SS is shown to increase C. jejuni cytotoxicity in the Galleria mellonella infection model. In biologically relevant models, the T6SS enhances C. jejuni interactions with and invasion of chicken primary intestinal cells and enhances the ability of C. jejuni to colonize chickens. This study demonstrates that the C. jejuni T6SS provides defense against oxidative stress and enhances host colonization, and highlights the importance of the T6SS during in vivo survival of T6SS-positive C. jejuni strains.

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

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.

The in vitro and ex vivo effect of Auranta 3001 in preventing Cryptosporidium hominis and Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium is a major cause of diarrhea worldwide in both humans and farm animals with no completely effective treatment available at present. In this study, we assessed the inhibitory effect of different concentrations of Auranta 3001 (0.1, 0.5 and 1%), a novel natural feed supplement, on C. hominis and C. parvum invasion of human ileocecal adenocarcinoma (HCT-8), bovine primary cells and C. parvum invasion of HCT-8, bovine primary cells and bovine intestinal biopsies. The effect of the feed supplement on the production of pro-inflammatory cytokines IL-8 and INF-γ, the anti-inflammatory cytokine IL-10, the expression of CpSUB1 protease gene during infection was also assessed by quantitative PCR (q-PCR). Transepithelial electrical resistance (TEER) was employed to measure the integrity of tight junction dynamics of the culture models. RESULTS: Pre-treatment of intestinal cells or oocysts with the Auranta 3001 significantly reduced the invasiveness of C. hominis and C. parvum against HCT-8 and bovine primary cells in a dose dependent manner. The most pronounced reduction in the invasiveness of both parasites was observed when Auranta 3001 was present during infection. Levels of IL-8 were significantly reduced in both HCT-8 and bovine primary cells, while the levels of INF-γ and IL-10 showed opposite trends in the two cell lines during infection in the presence of Auranta 3001. CpSUB1 gene protease expression, which mediates infection, was significantly reduced suggesting that this enzyme is a possible target of Auranta 3001. CONCLUSIONS: Although, C. hominis and C. parvum use different invasion mechanisms to infect cells, the novel feed additive can significantly attenuate the entry of Cryptosporidium in HCT-8 cells, primary bovine cells and bovine intestinal biopsies and thus provide an alternative method to control cryptosporidiosis.