Reservoirs of mobile antibiotic resistance genes in the gut microbiome of preterm infants

Opportunistic bacterial pathogens are increasingly acquiring resistance to antibiotics. One of the pathways by which bacteria acquire antibiotic resistance is through the horizontal transfer of resistance genes from other bacteria that inhabit the same ecosystem. Antibiotic resistance genes (ARGs) that spread via horizontal gene transfer (HGT) are of particular concern as they can rapidly disseminate through bacterial populations. Recent studies have highlighted that the complex microbial ecosystem of the human gut ('the gut microbiome') harbours a large diversity of ARGs. Notably, ARGs are not exclusively found in opportunistic pathogens. Several recent studies have determined that essentially harmless commensal bacteria in the human gut are the most prominent reservoirs of ARGs. These findings suggest that gut commensals may serve as a reservoir of antibiotic resistance genes that can be acquired by opportunistic pathogens by HGT. However, this hypothesis has so far not been formally tested.

In this study, we will determine the hosts of ARGs in the gut microbiome of preterm infants and quantify the ability of bacteria carrying ARGs to transfer these to opportunistic pathogens. The decision to target the gut microbiome of preterm infants in this study is based on the highly dynamic nature of their gut microbiome, which harbours both opportunistic pathogens (e.g. Escherichia coli and streptococci) and commensal bacteria (e.g. Bifidobacterium and non-toxigenic clostridia), thereby creating ample opportunities for HGT.

To address our overarching aim, we will first identify the bacteria that carry ARGs in the pre-term infant gut microbiome using a novel single-cell based method to link resistance genes with their bacterial hosts. We will then isolate these ARG-carrying bacteria and determine their genome sequence through a combination of two DNA sequencing technologies, specifically long-read Oxford Nanopore sequencing and short-read Illumina sequencing. This approach will lead to the complete assembly of the chromosome and independently replicating genetic elements (plasmids). We can thereby identify whether the antibiotic resistance genes are associated with genetic elements, like plasmids, that can be transferred between bacteria. Finally, we will quantify the efficiency by which commensal strains can transfer their ARGs to opportunistic pathogens. These experiments will be performed in bacterial growth media, but also using an innovative experimental set-up that mimics the conditions of the infant intestinal tract.

Taken together, this study will quantify the contribution of commensal bacteria to the spread of ARGs among pathogens. Our findings will be relevant to researchers that study AMR and/or the human microbiome and will contribute to the development of novel interventions to minimise the emergence and spread of antibiotic-resistant bacteria in preterm infants, who are at a particularly high risk for developing life-threatening bacterial infections.

A major factor contributing to the rapid spread of antimicrobial resistance (AMR) is the ability of antibiotic resistance genes (ARGs) to be transferred between bacteria in a process termed horizontal gene transfer (HGT). In this study we will test the hypothesis that commensal bacteria from the gut microbiome form a reservoir of ARGs and that these resistance genes can be acquired by opportunistic pathogens through HGT. This study will be performed using stool samples of preterm infants. The gut microbiome of the preterm infants comprises commensal bacteria and diverse opportunistic pathogens, creating ample opportunities for the HGT of ARGs.

We will first determine the microbial hosts of ARGs in the gut microbiome through a novel single-cell technique that results in a fusion PCR product that links an ARG with the 16S rRNA gene of its host. Sequencing of these amplicons will identify the bacteria that carry the targeted ARGs. We will then use this information to selectively culture and isolate the strains that carry resistance genes. Subsequently, the genomes of these isolates will be sequenced using a combination of short-read (Illumina) and long-read (Oxford Nanopore) sequencing, which will allow the complete assembly of the chromosomal and plasmid sequences of the strains that carry ARGs. Using the genomic data, we will be able to determine whether ARGs are associated with mobile genetic elements, which are most likely to be spreading through HGT. Finally, the ability of commensal strains to transfer their ARGs to opportunistic pathogens will be quantified in in vitro conjugation assays and in stool microcosm experiments, in which we will use shotgun metagenomics and novel bioinformatic tools to profile resistome dynamics.

This study addresses an important knowledge gap in the field of AMR research by quantifying the ability of opportunistic pathogens to acquire resistance genes from gut commensals through HGT.

In this project we will study the gut microbiome of pre-term infants to identify the pathways by which opportunistic pathogens can acquire antibiotic resistance genes from commensal bacteria. Both antimicrobial resistance (AMR) and the human microbiome are of considerable interest to industry and the general public. This proposal covers both these subjects, creating a number of opportunities to contribute to the economy and society.

According to the World Health Organisation, fifteen million babies are born prematurely every year. In low-income settings, half of the babies that are born at or below 32 weeks of gestation die, including due to a lack of basic care for infections. This project will contribute to the development of novel strategies that improve pre-term birth outcomes by reducing the likelihood of acquiring infections with drug-resistant bacteria.

This project will be of interest for industry in the infant nutrition field, as our studies will identify novel microbial hubs in the infant gut microbiome that facilitate the dissemination of antibiotic resistance genes. These findings can be translated to design new interventions to prevent the colonisation with antibiotic-resistant bacteria in the infant microbiome, e.g. by promoting the growth of bacteria that can outcompete the bacteria that harbour resistance genes. Industry engagement will be actively sought throughout the project, through existing links of the project team members with large multi-national companies that produce infant nutrition and smaller biotechnology companies that develop new approaches to modulate the microbiome to improve health.

The general public has become increasingly aware of the dangers posed by AMR, due, in part, to several campaigns that have been launched over the last few years. In addition, the human microbiome has become a topic of intense interest to the public. We therefore expect that our public engagement efforts, as described in this proposal, will find an audience that is eager to learn about the latest insights in AMR and the human microbiome and to discuss their own experiences and opinions with scientists. Indeed, it is very much the experience of the PI and Co-Is, on the basis of their ongoing public engagement efforts, that the general public is fascinated by research into these subjects. We therefore expect that the results of this project will be of considerable interest to news media, leading to broad dissemination of the project results.