Dr. Erin L. Young from the Utah Public Health Laboratory spoke a the Nanopore Community Meeting 2022. The ten-minute session was entitled “Long-read sequencing enables in-depth characterization of plasmids encoding KPC-2 in Citrobacter freundii outbreak. Young is a senior bioinformatician. Young explained that antimicrobial resistant (AMR) infections can be expensive and challenging to treat. Citrobacter freundii can carry genes conferring resistance on plasmids and… share them with other organisms. Their methods included sequencing 12 isolates of Citrobacter freundii with both Nanopore and Illumina sequencing. For Nanopore, they used the Ligation Sequencing v14 (SQK-LSK114 and Native Barcoding Expansion with MIN114 flow cells. They then sequenced on a GridION. Basecalling was performed with Guppy, and sequences were assembled with Unicycler. The hybrid assembly was annotated. Patient samples clustered except for one, which had KPC-3 while others had KPC-2. Using mmseq2, the gene positions were compared. The progressive mauve sequence compares plasmid sequences. The plasmids were similar but the KPC-2 containing plasmids of cluster 2 had a notable rearrangement. One patient seemed to have a very unique insertion, however, this region matches both cluster 1 and 2. Young emphasized that the sequencing of these plasmids from all patients provides a better understanding of the possibility of gene transfer event(s). This was a great application of sequencing of outbreak isolates to piece together potential transfer events.
