Bacterial strains
The 225 isolates used in this study were obtained from the Laboratoire de Biologie Moléculaire, d’épidémiologie et de surveillance des bactéries et virus transmissible par les aliments (LaBESTA)/Université Joseph KI-ZERBO, Burkina Faso. The strains were isolated from different sources and the serotype of each confirmed following the methodologies described in the International Organization for Standardization 6579 − 2017 (ISO 6579-1, 2017). The isolates were collected from clinical, veterinary, and food produce samples. Specifically, the breakdown was as follows; 28 from diarrheic patients; 105 from poultry feces, 22 from guinea fowl feces, 19 from poultry carcasses, 17 from eggs, 30 from fish and 4 were from sandwiches.
High-throughput molecular determination of Salmonella enterica serovars
We used the SMART method developed by Leader et al., 2009, with slight modification. Salmonella strains were streaked onto blood agar and incubated for 18–20 hrs at 36 °C. Then, one colony from each plate was cultured in 5 mL of Luria Bertani (LB) broth, (Difco™, Becton Dickinson and Company, Sparks, MD) and incubated for 18 hrs at 37 °C with shaking. The genomic DNA was then isolated from the overnight culture using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) and following the kit instructions for use. Once extractions were completed, the DNA was analyzed on the Nanodrop 2000 for DNA quality measuring the 260/280 nm. All DNA were then stored at -20 °C until ready for PCR and library preparation.
PCR amplification.
Each PCR mixture contained 12.5 µL of Immolase DNA polymerase 2X master mix (Bioline, Inc., Randolph, MA, USA), 2.5 µL of 10X primer master mix, 3 mM MgCl2, and 1 µL of extracted DNA with addition of nuclease free water to a final reaction volume of 25 µL. The cycling conditions used in the thermal cycler were 94 °C for 10 min; 25 cycles of 94 °C for 30 s, 57 °C for 90 s, and 72 °C for 30 s; 72 °C for 5 min; 15 cycles of 94 °C for 30 s, 68 °C for 90 s, and 72 °C for 30 s; and 72 °C for 5 min. For each run, the negative control was sterile water and positive controls were genomic DNA from Salmonella Typhimurium LT2, S. Typhi CT18, S. Enteritidis strains 21027 and 98104. The primers describe by Leader et al. (2009) were used in this study. The amplicon samples were then diluted and analysed on an ABI XXXX using capillary electrophoresis.
Genemapper software v3.5 (Applied Biosystems, Foster City, CA, USA) was used to analyze the sizes of resulting PCR products according to the protocol developed by Leader et al. (2009). Scoring was based upon the presence of a PCR product that corresponded to the predicted amplicon size, as detected in control reactions with DNA from S. Typhimurium, S. Typhi, and S. Enteritidis. Each PCR product detected was given a number (1 through 16) according to the size of the amplicon (Leader et al., 2009). The amplicons detected for each isolate were combined to create a SMART code that corresponds to serotypes previously screened by this method.
Whole genome sequencing of Salmonella strains
Extracted DNA was quantified using the Qubit double-strandedDNA high-sensitivity assay kit according to the manufacturer’s instructions (Life Technologies Corp., Carlsbad, CA, USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, san Diego, CA, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, Santa Clara, CA,USA) and quantified using a Qubit DNA HS assay kit in a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit (Illumina) with 500 and 300 cycles. The paired-end read length of 2 × 250 bp was used for 500 cycles and 2 × 150 bp for 300 cycles on the MiSeq platform (Illumina). The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequences were then assembled using the A5-miseq assembler (Coli et al., 2015), and the genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016).