Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics

Abstract

Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.

Figure 1. Protocol for using IPCR to evaluate the horizontal gene transfer potential of ARGs in the environment.

After sample collection (1) and total DNA extraction (2), the DNA is digested with restriction enzymes and resulting fragments are self-ligated into circular DNA molecules (3). DNA flanking the ARG is amplified with IPCR using ARG targeting primers (4). The amplicons are sequenced using long read sequencing with PacBio SMRT cell technology and the ARG associated MGEs are identified (5).

Figure 2. Schematic representation of an IPCR amplicon resulting from digestion, self-circularization and amplification of DNA flanking a resistance gene.

Mobile genetic element is digested with restriction enzymes and self-ligated into a circular molecule (1) and amplified (2). The IPCR amplicon contains the up- and downstream regions surrounding the target ARG, which have MGE associated genes (mge1-5) and co-selected ARGs (co1 and co2). The digestion and ligation site separates the up-and downstream regions of the ARG in the IPCR amplicon.

Figure 3. Mobile genetic elements containing sul1 resistance genes identified from IPCR consensus sequences.

The restriction and ligation site, where the MGE sequence is discontinuous, is marked with an arrow. The genes and open reading frames are oriented as in Fig. 2 in relation to the MGE sequence.

Figure 4. tetM resistance gene containing mobile genetic elements identified from IPCR consensus sequences.

The restriction and ligation site, where the MGE sequence is discontinuous, is marked with an arrow. The genes and open reading frames are oriented as in Fig. 2 in relation to the MGE sequence.