Genomic Analysis of tet(X)-Positive Isolates

Genomic DNAs of the tet(X)-positive isolates were extracted using the Puregene yeast/bact. kit B (Qiagen, Gaithersburg, MD) according to the instructions of the manufacturer and were sequenced by using the HiSeq 4000 system (Illumina, San Diego, United States). The isolates were taxonomically assigned using GTDB-Tk (version 1.3.0) with the Genome Taxonomy Database (release 95) (34 (link)). The sequence similarities of tet(X)-harboring plasmids were analyzed using BRIG version 0.95 (35 (link)). Representative strains with various genetic contexts of tet(X) genes were selected to be further sequenced using the PromethION platform (Nanopore, Oxford, UK). Hybrid assembly of short-read and long-read sequencing data was performed using Unicycler version 0.4.8 (36 (link)).
Phylogenetic analysis was performed using Parsnp version 1.2 (37 (link)), and the numbers of single-nucleotide polymorphisms (SNPs) among the core genomes were determined by using MEGA X (38 (link)). Functional annotation was performed using the RAST server (39 (link)). Antibiotic resistance genes (ARGs) were identified using ResFinder 4.0 (40 (link)) and CARD (https://card.mcmaster.ca/) with a threshold of nucleotide identity of >90% and coverage of >90%. Synteny analysis was performed using Easyfig (41 (link)).