Copper-induced microbiota shifts and its effect on pig-gut colonisation by sil and sopE encoding Salmonella

The gut microbiota plays a crucial role in the exclusion of bacterial pathogens such as Salmonella from animal hosts. Pig farmers are particularly interested in excluding Salmonella from herds because this pathogen compromises productivity, impacts animal welfare and presents a risk to the human population by gaining entry into the food chain. Maximising productivity in pig production while maintaining a healthy and safe product requires a complete understanding of the interaction of Salmonella with the pig including the gut microbiota. Central to this project are the consequences of diet-induced variation in the microbiota composition and the genetics of Salmonella on this interaction.

The need for a greater understanding is extremely timely since the emergence of a variant of Salmonella Typhimurium (monophasic S. Typhimurium ST34) primarily in pig populations has spread globally and now accounts for over half of all S. Typhimurium infections in people in the UK, and over 1 in 10 of all Salmonella infections. Since the virtual eradication of Salmonella Enteritidis from layer hens and breeding flocks in the UK through biosecurity and vaccination regimens, ST34 is now the single most common cause of Salmonella infection resulting from UK livestock. Coincident with the emergence of ST34 has been the widespread use of copper supplementation in the diet of pigs to decrease infections during weaning, and as a growth promotor during fattening. Its perceived importance as a growth promotor has increased since antibiotics were banned for such use in 2005.

The ST34 strain is genetically unique in being more resistant to copper and having acquired a toxin called SopE. Published and preliminary data suggest that both of these factors have the potential to affect the ability of Salmonella to exploit the gut of pigs on a diet supplemented with therapeutic levels of copper. Genomic epidemiology of ST34 strains in the UK suggests that ST34 strains with increased resistance to copper and SopE have a fitness advantage over those that have either lost or never acquired the associated genes. Furthermore, that different components of the microbiota have been implicated in colonisation resistance depending on the genetics of the Salmonella, including the presence of the sopE gene.

There is a pressing need therefore to not only understand the changes in the composition of the pig gut microbiota that result from a copper supplemented diet, but also the impact that this has on colonisation resistance to distinct genetic variants of Salmonella. This timely information will lead to evidence-based decisions on farm practices that maximise productivity, animal welfare and food safety.

The project aims to address these gaps in knowledge by characterising the changes in the gut bacteria of pigs that occurs as a result of supplementation of feed with elevated levels of copper. This will identify potential mediators of colonisation resistance to Salmonella. The effect of changes in the gut microbiota on colonisation by Salmonella will be investigated using an in vitro gut model and with experimental infections of groups of pigs in a containment laboratory. Finally, in order to further study bacteria of the gut that mediate gut colonisation resistance to Salmonella we will culture gut microbiota bacteria and investigate their direct and indirect interaction with Salmonella in vitro, and through collaboration in follow-on experiments using the mouse model of infection.

The project will generate a large amount of data of interest to researchers addressing related questions in the pig industry such as feed conversion (productivity) and biomedicine where the pig is used as a model to study human disease. Resources made freely available will include a large number of genome sequences and a collection of cultured bacteria of the pig gut complementing those recently made available in the pig intestinal bacteria collection (PiBAC).

We will test the hypothesis that copper (Cu)-induced gut microbiota changes in the pig provides a niche exploited by S. Typhimurium ST34 in a sil and sopE gene-dependent mechanism.

The effect on gut bacterial communities of supplementation of pig feed with therapeutic concentrations of Cu will be determined in 120 pigs on a working farm. Piglets will be weaned on a high protein diet supplemented with either 10ppm (nutritional) or 150ppm (therapeutic) Cu and the microbiota defined by metataxonomic analysis using shotgun metagenome sequencing. Computational analysis will be used to identify species and strains that differ in the two feed groups. The role of the sil and sopE genes in colonisation of the pig gut will be determined using an in vitro gut model and experimental infections of pigs. ST34 strains in which sil or sopE or both are deleted will be constructed and assessed for their ability to grow in the presence of in vitro microbiota cultures established from faecal samples collected from pigs on Cu nutritional or Cu therapeutic diets. The same ST34 strains will be tested by experimental infection of pigs from each diet.

Together, these experiments will reveal whether the sil and sopE genes increase colonisation of the pig gut and whether this is dependent on microbiota changes associated with a diet with therapeutic concentrations of copper. Gut microbiota whose composition is altered in response to therapeutic concentrations of copper will be isolated from faecal samples of pigs, and potential direct or indirect agonistic and antagonistic properties assessed by co-culture in vitro or with organic phase extracts of spent media.

In a follow-on study, and through an ongoing collaboration with the Baumler lab (UC Davis), we will test the hypothesis that specific microbiota isolates affect colonisation of Salmonella in the murine model of infection using precision microbiota editing methodology.