The human gut microbiome as a reservoir of antibiotic resistance

Aims:
A. To link antibiotic resistance genes (ARGs) in the human gut microbiome to their bacterial hosts
B. To assess the ability of ARGs in the human gut microbiome to spread to opportunistic pathogens.

Background
The human gut harbours a complex microbial community ('the gut microbiome'), that contributes to human health and disease. Previous work by the group of Prof. Van Schaik and others have revealed that the gut microbiome comprises a large number of antibiotic resistance genes (ARGs).1,2 Current methodologies to study the gut microbiome, like metagenomic shotgun sequencing or high-throughput quantitative PCRs, do not allow for the identification of the bacteria and mobile genetic elements that carry ARGs.3 The extent by which ARGs can spread between members of the gut microbiome, including to gut-dwelling opportunistic pathogens like Escherichia coli and the enterococci, also remains unclear.

Hypothesis
Most of the ARGs in the gut microbiome will be harboured by anaerobic gut commensals. These ARGs may be transferred to both Gram-positive and Gram-negative opportunistic pathogens.

Experimental Methods and Research Plan
1. Bacterial reservoirs of ARGs in the gut microbiome
In this project, we will determine the linkage of ARGs with their bacterial hosts in human stool samples. We will use faeces from paediatric and adult patients and healthy individuals (n = 5 for each, 15 samples in total). The stools are available through collaborators and can be shared for research purpose in compliance with current study protocols and ethical regulations. The faecal samples will first be screened for the presence of the important resistance genes blaNDM, blaKPC, mcr-1, vanA and vanB by quantitative PCR, and, if found to be positive, linkage between resistance genes and their bacterial hosts will be determined by a recently developed method, termed epicPCR for Emulsion, Paired Isolation and Concatenation PCR.4

2. Transferability of resistance genes to opportunistic pathogens
Once the identity of the bacteria carrying ARGs has been established, these bacteria will be cultured from the relevant samples using a variety of enrichment methods.
After their isolation, the genomes of the isolated bacteria will be sequenced, using a combination of short- and long-read sequencing, resulting in complete assemblies of the chromosome and plasmids. The annotated genome sequences will be used to determine whether resistance genes are associated with mobile genetic elements, specifically plasmids and conjugative elements, or are carried on the chromosome, which would minimize their potential for horizontal transfer.5
Upon completion of the genomic characterization of the resistance-gene-carrying bacteria, we will perform conjugation assays to assess the ability of these bacteria to serve as donors of resistance genes to the opportunistic pathogens Escherichia coli and Enterococcus faecium. Transfer of ARGs will be quantified by assays on laboratory media, and in human faecal suspensions.

Expected Outcomes and Impact
This project will lead to important insights into the dynamics of ARGs in the gut microbiome. Due to the role of the microbiome in a variety of complex diseases and conditions (including inflammatory bowel disease, colon cancer, and obesity) and the widespread use of antibiotics, which is the main driver of the emergence of antibiotic resistance, it is of considerable importance to study the dynamics of the reservoir of ARGs in the gut microbiome. This information can be used for the development of therapies that use antibiotics or other antimicrobials to target specific groups of bacteria in the gut microbiome, while minimizing the acquisition of ARG, leading to drug resistance.
The PhD student will acquire expertise in state-of-the-art methods in aerobic and anaerobic microbiology and molecular biology, specifically in the fast-moving area of the human gut microbiome. In addition, the student will be