From amplicons to metagenomes: Long read sequencing the environment

First to the lecturn in the Environmental metagenomics breakout session was Dr. Rob James from the Wellington lab at the University of Warwick, who kicked off by explaining how complex environmental matrices, such as soil, sediment, and excreta are home to diverse microbial communities and that long-read sequencing of such communities can provide a wealth of information on the selective drivers of antimicrobial resistance in the environment. However, he pointed out that working with environmental samples can be challenging. For example, while harsh DNA extraction methods may provide complete microbial representation, they also result in DNA fragmentation, which is suboptimal for generating complete closed genome assemblies. On the other hand, gentler extraction methodologies may preserve the integrity of the DNA but microbial representation is often incomplete.

Rob revealed that there are a number nanopore sequencing projects underway at the University of Warwick, including the study of a novel endophytic fungi that obtained funding after an initial pilot study undertaken using the MinION RevD Flow Cell provided to delegates at last year’s London Calling.

In the first study presented, the team at Warwick used nanopore sequencing to characterise the plasmids associated with two E. coli ST131 isolates originating from a river sample, obtained downstream of a waste water treatment plant (WWTP). These strains had previously been identified as resistant to the antibiotic cefotaxime; however, attempts to resolve the plasmid composition of these strains using a short-read sequencing approach failed due to highly repetitive nature of the plasmids. To overcome these challenges, the team applied long-read nanopore sequencing to identify the antimicrobial resistance (AMR) gene composition and provide a scaffold for annotation. However, Rob stated that they ‘actually got considerably further than that’. To start with, they isolated the plasmids from their hosts by conjugating into the E. coli lab strain ET12567. Using antibiotic selection, they isolated the single plasmids of interest which carried the blaCTX-M variants 15 and 27 genes conferring resistance to the antibiotic cefotaxime. They then used lambda exonuclease and exonuclease I to degrade genomic DNA associated with E. coli. Using nanopore sequencing, the team was able to fully assemble (using Canu) and map the antibiotic resistance genes in two plasmids; p48 and p61, which were isolated from two different ST131 samples. For p48, they discovered that almost two thirds of the genome is dedicated to conjugation and pili construction. This is a classic example of a conjugative plasmid carrying a selective advantage for blaCTX-M-15. Conversely, for the p61 plasmid, the team couldn’t find any evidence for a transfer region. Rob suggested that this plasmid had lost its ability to conjugate due to genome streamlining and host-plasmid co-evolution ensuring retention of the plasmid through vertical transmission. Interestingly, the plasmid contains a multiple drug resistance region on a class I integron or as Rob likes to call it ‘a jumping gene cassette of death’. It also containes a tox-antitox system, which ensures that any daughter cells that don’t inherit the plasmid through vertical transmission are not viable. Summarising this study, Rob stated that it is a great example of divergent selection in the environment in the presence of antibiotics.