Pig Production Without the Use of Antibiotics - Impact on the Pig Resistome and Microbiome

Antibiotic resistance is currently one of the biggest threats to human health. Pig farms are known to be reservoirs for horizontally transferrable resistance genes and multi-drug resistant pathogens. In Denmark the total amount of antibiotics used in pig rearing is around double that used for human consumption. To meet growing consumer demand, Danish Crown launched an initiative in pig farming called Raised Without Antibiotics (RWA). These RWA pigs receive ear tags at birth that can be removed should they require antibiotic treatment for bacterial infections. If pigs receive antibiotic treatment they are reared alongside the RWA pigs.

Since both the pigs’ microbiome and their resistome are altered by administered antibiotics, the tagging of individual pigs makes these commercial operational farms suitable for case control studies. Such studies do not appear to be available in existing literature.

The objectives of this PhD thesis are to study the following: 1) the effect of antibiotic treatment on the pig microbiome and resistome and their dynamic development over time; 2) the effect of antibiotics on mobile genetic elements and the potential of resistance gene transference; and 3) the potential transference of antibiotic resistance between untreated pigs and pigs treated with antibiotics. In this project we studied the faecal microbiome and resistome from pigs with the following three study designs: 1) a cohort study without intervention at an RWA farm that followed the pigs from birth to slaughter to cover the temporal effect that antibiotics have on the microbiome and resistome of the pigs with no intervention; 2) an endpoint study at the slaughterhouse of RWA pigs from six different farms to determine if there is a general trend in microbiome and resistome patterns before reaching the consumer; 3) a separation cohort study with minor interventions at one RWA farm where pigs were separated into three treatment pens to investigate if the separation of the treated and untreated pigs has diverging effects on the pigs’ microbiome and resistome.

By utilizing 16S rRNA sequencing for microbiome studies, high throughput qPCR for resistome studies and Hi-C metagenomics for connecting the two, this project has extended the current knowledge of microbiome and resistome dynamics in pigs raised without antibiotics versus pigs treated with antibiotics. In the first study design, we found that the microbiome and resistome of treated and untreated pigs diverge after treatment but converges again before slaughter. These results were substantiated by the fact that OUA and non-OUA pigs are indistinguishable at slaughter. To investigate if this convergence was a result of mixing treated and untreated pigs, the separation study was done. The study showed that the microbiomes in the mixed pen converged, while they did not when treated and untreated RWA pigs were separated. The resistomes were found to be transferred from treated pigs to untreated pigs in mixed pens, resulting significantly different resistomes but indistinguishable levels of resistance gene abundance at the end of the separation study.

In conclusion, the RWA pigs had a lower abundance of antibiotic resistance genes for at least 10 weeks compared to treated pigs. We suggest that the reason that the microbiomes of treated and untreated pigs seem to converge over time in RWA farms is due to the mixing of the RWA and pigs treated with antibiotics. We showed that mixing the RWA pigs with pigs treated with antibiotics resulted in a higher abundance of antibiotic resistance genes in the RWA pigs microbiomes compared to the RWA pigs that were separated from pigs treated with antibiotics. The objective of RWA farming is to reduce the amount of antibiotics used in pig farming. This thesis suggests that a significant additional benefit could be obtained by separating the treated and untreated RWA pigs at the RWA farms as this would lower the amount of antibiotic-resistant bacteria in the RWA pigs.