Discovery of the fourth mobile sulfonamide resistance gene

Abstract

Background: Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.

Results: Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (> 1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe.

Conclusions: Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

Keywords: Bioprospecting; Environment; Evolution; Metagenomics; Pharmaceutical; Resistome.

Fig. 1

Predicted functions of open reading frames separated by samples and primers. The results are based on known homologues in the CARD database

Fig. 2

Schematic diagram of the different arrangements of sul4 recovered by amplicon sequencing of integron datasets: a RS_PETL, Hyderabad, India. The read begins with an attI site, as the integron-integrase gene was not amplified by the primers. Alignment of 1.10 Kbp of the gene cassettes to the read in b RS_Pune, Pune and RS_PETL, Hyderabad, India. The first gene cassette is a hypothetical protein (WP_019224580.1). Primers used to further confirm the context found in RS_Pune and RS_PETL are indicated by arrows. The rest of the arrangements are contigs that resulted from assembling different shotgun metagenomics datasets. c mgm4622354.3 (MG-RAST ID) collected from Kolkata, India. d mgm4510219.3 collected from Malaysia. e mgm4709385.3 collected from Sheffield, UK. f ERR1414268 (EBI ID) collected from Käppala sewage treetment plant, Sweden. Downstream of ISCR20 are hypothetical proteins (WP_076836759.1 and WP_011927925.1) g mgm4537907.3 collected from Hong Kong and h mgm4714564.3 collected from Beijing, China. Due to the lower read coverage, contigs covering the entire sul4 gene were not recovered in the f–h metagenomes

Fig. 3

Collapsed phylogenetic tree of Sul4 and known chromosomal dihydropteroate synthase proteins from the NCBI RefSeq database excluding plasmid-born genes, along with proteins encoded by mobile sulfonamide resistance genes sul1, sul2, and sul3 from the CARD database. Branches are annotated with the accession number and the identified species name, with the phyla in bold. Collapsed clades are distinguished by red edges and the size of the blue bubbles corresponds to the number of proteins in the collapsed clade. The full version of the tree is available as an Additional file 9 in Newick format

Fig. 4

Map of primer pairs used to amplify partial class 1 integrons