Imported fish samples were purchased from different open markets. All fish samples during collection were placed in sterile polypropylene bag, placed in polystyrene box containing crushed ice and the temperatures was 4°C during transportation. The samples were transported to the laboratory and examined on the same day for the presence of Salmonella spp.
Salmonella strains were isolated from fish samples following the methodologies described in the International Organization for Standardization 6579-2017 (12). The fish samples were gently removed from coolers and processed in aseptic condition. The gills, intestines parts and skin parts were removed using sterile knifes. About 10 g of samples (fish gills, intestines parts and skin) were placed into a stomacher bag containing 90 mL of buffered peptone water (Liofilchem, Teramo, Italy) and homogenized using a stomacher (400 Circulator, Seward, London,UK) for 1 min and incubated for 24 h at 37 °C. From this non selective pre-enrichment, 0.1 mL were transferred into 10 mL of Rappaport-Vassiliadis broth (Oxoid, Basingstoke, England) and incubated for 24 h at 42°C. A loopful from the selective enrichment broth was streaked onto XLD (Oxoid, Basingstoke, England) agar and incubated for 24 h at 37 °C. Suspected colonies on selective agar plates were purified and bio-typed by using biochemical tests and API 20E strips (BioMerieux, Marcy l’Etoile, France).
Confirmed colonies were sent to the United States Department of Agriculture, Bacterial Epidemiology and Antimicrobial Resistance Research Unit for future analysis.
Antimicrobial susceptibility testing
The isolates were streaked onto Blood agar plates and incubated for 24 h at 37°C, and one colony from each plate was streaked onto new Blood agar plate for another 24h at 37°C. Minimum inhibitory concentrations (MIC, µg/mL) of all Salmonella isolates were determined by broth-microdilution using the Sensititre™ semi-automated antimicrobial susceptibility system (TREK Diagnostic Systems Inc., Cleveland, OH, USA) and the Sensititre™ Gram-Negative plate format, with plate code GN4F (Thermo, Fisher Scientific, USA), according to manufacturer’s directions. MICs of the isolates for the 24 antimicrobials were determined using colonies from the last 24h Blood agar plates, and each isolate was classified as resistant, intermediate, or susceptible to the antimicrobials tested using the breakpoints set by Clinical and Laboratory Standards Institute (CLSI) . Antimicrobials used breakpoints were as follows: Amikacin (≥ 64 µg mL-1); Piperacillin/tazobactam (≥ 128/4 µg mL-1); Tigecycline (≥ 1 µg mL-1); Ticarcillin/clavulanic acid (≥ 128/2 µg mL-1); Levofloxacin (≥ 2 µg mL-1); Nitrofurantoin (≥ 128 µg mL-1); Tetracycline (≥ 16 µg mL-1); Doripenem (≥ 4 µg mL-1); Minocycline (≥ 16 µg mL-1); Ertapenem (≥ 2 µg mL-1); Trimethoprim/sulfamethoxazole (≥ 4/76 µg mL-1); Imipenem (≥ 4 µg mL-1); Piperacillin (≥ 128 µg mL-1); Meropenem (≥ 4 µg mL-1); Gentamicin (≥ 16 µg mL-1); Cefazolin (≥ 8 µg mL-1); Tobramycin (≥ 16 µg mL-1); Ceftazidime (≥ 16 µg mL-1); Ampicillin/sulbactam (≥ 32/16 µg mL-1); Aztreonam (≥ 16 µg mL-1); Ampicillin (≥ 32 µg mL-1); Cefepime (≥ 32 µg mL-1); Ciprofloxacin (≥ 4 µg mL-1); Ceftriaxone (≥ 4 µg mL-1). For the analysis, isolates identified as intermediate were considered susceptible to the drug. E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213 were controls for determination of MIC.
For each isolate, a final inoculum of 5 x 105 CFU/ml was targeted. The panels were read after 18 h of incubation at 35°C.
Whole genome sequencing
Genomic DNA was isolated using the GenElute bacterial genomic DNAkit (Sigma-Aldrich, St. Louis, MO, USA) following instructions for Gram-negative bacteria, from 5 mL of overnight cultures grown in Luria-Bertani Broth, Miller (Difco™, Becton Dickinson and Company, Sparks, MD) at 37°C with shaking. The extracted DNA quality was read using NanoDrop 2000c spectrophotometer (Thermo, Fisher Scientific, USA). DNA was stored at -20°C prior to library preparation.
Extracted DNA was quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit according to the manufacturer’s instructions (Life Technologies, Inc., USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, USA) and quantified using a Qubit DNA HS assay kit in a Qubit fluorometer (Thermo, Fisher Scientific, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit with 500 and 300 cycles. The paired-end read length of 2 X 250 bp was used for 500 cycles and 2 X 150 bp for 300 cycles on the Illumina MiSeq platform. The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequence data were assembled using the A5-miseq assembler (14), and the genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (15).
The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession XXXXXX000000000. The version described in this paper is version XXXXXX010000000.
Identification of antibiotic resistance genes, chromosomal mutations, serotypes, MLST and plasmid from total genome sequence
Antibiotic resistance genes and chromosomal mutations were identified using ResFinder 3.2 (16). SeqSero 2 was used to determine the serotypes of salmonella strains from genome assembly data. MLST sequence type was identified using MLST database from the center of genomic epidemiology (17). The PlasmidFinder were used to detect plasmid from the strains (18).