Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

Abstract

Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

The Natural History of Integrons

“…This can be inferred from the phylogeny of IntI protein sequences, which cluster according to the environment of origin rather than bacterial phylogeny. IntIs encoded by marine and soil/freshwater bacteria form the two distinct clades [7,10,16,25]. The same is true for attCs.…”

“…Recent metagenome studies indicate that integrons may indeed be even more diverse than indicated by surveys of sequenced genomes. Analysis of environmental samples has revealed that there are thousands of different integron 'classes' (discerned based on IntI amino acid homology) [25]. Similarly, the gene cassette repertoire of these environmental samples indicates that these elements are abundant and carry a diverse set of genes.…”

“…Collectively, they have acquired more than 130 different resistance genes [40], with more being continuously discovered [46][47][48][49]. Despite class 1 integrons being able to recognise and integrate a broad range of gene cassettes, there seems to be a preponderance of antimicrobial resistance genes out of the broader diversity of cassette functions that are associated with class 1 integrons [25]. This overrepresentation of resistance genes among class 1 integrons can best be explained by two factors: (1) Strong positive selection imposed by the human use of antimicrobials, and (2) their ability to confer resistance phenotypes in a wide range of hosts without needing to integrate into metabolic networks [50].…”

“…Class 1 integrons can now be detected on every continent, including Antarctica [25,43]. Such is their abundance that millions to billions of copies of class 1 integrons are now present in a single gram of faeces from humans and agricultural animals [51].…”

“…Environmental instability might provide opportunities for integron-carrying bacteria to invade specific niches and generate ecotypes. With improved sequencing technologies and methods for recovering integron sequences [25], we can investigate their ecological and evolutionary roles in natural environments more deeply.…”