MinION nanopore sequencing identifies the position and structure of bacterial antibiotic resistance determinants in a multidrug-resistant strain of enteroaggregative escherichia coli

The aim of this study was to use single-molecule, nanopore sequencing to explore the genomic environment of the resistance determinants in a multidrug-resistant (MDR) strain of enteroaggregative Escherichia coli serotype O51: H30, sequence type (ST) 38. Sequencing was performed on the MinION Flow cell MIN-106 R9.4. Nanopore raw FAST5 reads were base-called using Albacore v1.2.1, converted to FASTA and FASTQ formats using Poretools v0.6.0, and assembled using Unicycler v0.4.2, combining the long-read sequencing data with short-read data produced by Illumina sequencing. The genome was interrogated against an antimicrobial resistance (AMR) gene reference database using BLAST. The majority of the 12 AMR determinants identified were clustered together on the chromosome at three separate locations flanked by integrases and/or insertion elements [region 1 –catA, bla_OXA-1, aac(6′)-Ib-cr, tetA and bla_CTX-M-15; region 2 – dfrA1 and aadA1; region 3 – catA, bla_TEM-1, tetA and sul2]. AMR determinants located outside these three regions were a chromosomally encoded bla_CMY-16, mutations in gyrA and parC, and two plasmid-encoded AMR determinants, bla_OXA-181 and qnrS1 located on the same IncX3 plasmid. Long-read analysis of whole genome sequencing data identified mobile genetic elements on which AMR determinants were located and revealed the combination of different AMR determinants co-located on the same mobile element. These data contribute to a better understanding of the transmission of co-located AMR determinants in MDR E. coli causing gastrointestinal and extra-intestinal infections.