Progress beyond the state of the art.
I here assessed HGT rates and associated changes in the gut microbiome under different sorts and levels of selection. Real-world investigations of this kind are scarce, and our work represents one of the few endeavors in this direction. To check HGT rates I used a newly developed method, OIL-PCR, and combined that with a phage treatment step to extend its applicability to settings where the HGT-agent is delivered through a donor-bacterium. In addition, I followed donor, plasmid and total bacterial numbers with qPCR. Where changes in the gut microbiome are currently only followed by marker gene sequencing, with relative abundance data as an outcome, I here used a combination of internal standard spike-ins and qPCR, obtaining absolute abundance data with its associated benefits for data analyses and interpretation. The availability of absolute data proved advantageous, enabling us to establish a correlation between plasmid transfer incidence and species abundance.
Next to antibiotic selection, I applied selective pressure through HMO supplementation, which provided the donor bacteria with an additional nutritional resource. To this end, I evaluated several carbohydrates and associated CAZymes for their specificity and created and tested an E. coli strain able to use this carbohydrate as the sole carbon source. I further tested the effects of this specific carbohydrate on the (mouse) gut microbiome, applying absolute abundance profiling, allowing an assessments of its safety for (mouse) health and gut microbiome composition, and the possibility to use this system for the creation of an exclusive metabolic niche.
Potential impact.
Little is known about the features that affect HGT rates within natural environments, such as the gut, nor the role of selection in that process. If these were well-understood, it would aid the development of predictive tools to guide health and environmental policies as well as current and novel GM modulation strategies.
For example, predicting the transferability of a gene/function and the role of selection in that process could help assessing the risk of spreading antibiotic or GMO genes within an animal microbiome. At this moment, we know herbicide-sprayed GMO consumption leads to the integration of herbicide resistance genes in animal GM and changes GM composition but it is currently not clear at which rates and conditions, nor whether certain taxa are preferentially affected. Such knowledge is however crucial to assess the safety of these compounds and organisms.
An increased insight into HGT rates of functions could also aid the development of next generation probiotics. Current probiotic strains often do not engraft and therefore fail to infer the health benefit. The creation of an exclusive metabolic niche would however stimulate strain engraftment and allow reversible engraftment by discontinuing the selective pressure. Furthermore, enhanced HGT mechanisms could adapt GM function in situ, circumventing the problems of current modulation strategies (e.g. prebiotics, probiotics or fecal material transplantation (FMT)). When these options fail, enhanced HGT mechanisms might be able to restore the functions lost through industrialization or disease.
Our results give crucial insights into the taxonomic breadth and dynamics of plasmid conjugation in a real-life system, elucidating the effect of species abundance and phylogenetic relationships, and therefore provide another step in the realization of these goals.
