Antimicrobial susceptibility testing will be performed for n antimicrobials and the sensitivity of isolates will be evaluated according to classification guidelines by the National Committee for Clinical Laboratory Standards (NCCLS) for or animals. The minimum inhibitory concentrations (MICs) will be determined according to standard protocols (Gilbert, White, & McDermott, 2007). Antimicrobial resistance will be defined using Clinical and Laboratory Standards Institute (CLSI) criteria.
Salmonella isolates purified from clinical cases shall be selected for whole genome sequencing (WGS) analysis. Salmonella genomic DNA will be purified using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA), and DNA concentrations will be measured with a Qubit™ fluorometer (ThermoScientific, MD). The DNA quality will be assessed on the Nanodrop Spectrophotometer (ThermoScientific, USA) and on a 1% agarose gel stained with ethidium bromide and visualized by UV illumination. The DNA libraries will be prepared from genomic DNA normalized to 0.2 ng/µL using the Nextera XT library Prep kit as per the preparation guide (Illumina Inc., San Diego, CA). The libraries will be normalized using bead-based procedure and pooled together at equal volume. Paired-end WGS will be performed using v3 chemistry at 2×250 cycles on the MiSeq platform (Illumina, San Diego, CA).
The FASTQ files will be trimmed and assembled de novo using CLC Genomics workbench 9.4 (Qiagen Bioinformatics, CA) and mapped to the Salmonella reference genome. The antibiotic resistance genes in the assembled Salmonella genomes will be identified by BLAST search against a local copy of the antibiotic resistance gene sequence data from ResFinder (Zankari et al., 2012) that uses BLAST for identification of acquired antimicrobial resistance genes in whole-genome data augmented with the Comprehensive Antibiotic Resistance Database CARD (Jia et al., 2017). As input, the ResFinder method can use both pre-assembled, complete or partial genomes, and short sequence reads (Zankari et al., 2012). The virulence genes in the genomes will be predicted using a similar approach. Salmonella virulence gene sequences will be extracted from Virulence Factor Database (Chen et al., 2005) and Salmonella genome assemblies will be searched against these sequences using BLAST with pre-set sequence length cut offs.
The Salmonella In Silico Typing Resource SISTR (Yoshida et al., 2016) shall be used to type and subtype the draft Salmonella genome assemblies obtained in this study. In addition to performing serovar prediction by genoserotyping, SISTR integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). The SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing a broader phylogenetic and epidemiological context.