Salmonella typhimurium

Three different datasets were used for evaluation in the present study, comprising selected Salmonella Montevideo [17] (link), Staphylococcus aureus CC398 [5] , and Salmonella Typhimurium DT104 [18] (link) from previous studies.
For S. Montevideo 12 closely related outbreak strains where sequenced once by US Food and Drug Administration using Roche Genome sequencer FLX system, Illumina MiSeq and Life Technologies Ion Torrent and made publicly available (Table S1), although only the MiSeq data was used in the original study [16] (link). The raw data were downloaded from the Sequence Read Archive (SRA). For Staphylococcus aureus CC398, the completely sequenced and annotated strain SO385 (AM990992.1) as well as four additional strains were selected from a previously published study [5] and sequenced twice using both MiSeq and Ion Torrent. HiSeq was used in the original study for sequencing. All the strains except for the reference strain were chosen from the same clade, named IIa1i in the original study. The strains are not epidemiologically related but have all been isolated from Danish Pigs and are shown to be closely related in the original study. For S. Typhimurium DT104 the reference strain NCTC 13348 (HF937208.1) and an additional three isolates from the same outbreak [18] (link) were sequenced twice on both MiSeq and Ion Torrent.
Genomic DNA (gDNA) was purified from the isolates using the Easy-DNA extraction kit (Invitrogen) and DNA concentrations determined using the Qubit dsDNA BR Assay Kit (Invitrogen). The isolates were sequenced twice on the MiSeq platform (Illumina) and Ion Torrent PGM (Life Technologies).
For Ion Torrent the isolates were sequenced following the manufacturer’s protocols for 200 bp gDNA fragment library preparation (Ion Xpress Plus gDNA and Amplicon Library 96 Preparation), template preparation (Ion OneTouch System), and sequencing (Ion PGM 200 Sequencing kit) using the 316 chip. For MiSeq the isolates chromosomal DNA of the isolates was used to create genomic libraries using the Nextera XT DNA sample preparation kit (Illumina, cat. No. FC-131-1024) and sequenced using v2, 2×250 bp chemistry on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA).

The bacterial species tested in this study (Table 1) were obtained from the Food Microbiology Laboratory at Auburn University (USA). Salmonella Typhimurium and S. Enteritidis were incubated in 20 ml of Trypticase Soy Broth (TSB, Difco Laboratories Inc., USA) for 16 h at 37°C. After cultivation, each bacterial culture was washed 3 times with phosphate-buffered saline (PBS, pH 7.2, Sigma-Aldrich Co., USA) by centrifugation at 5,000 ×g for 5 min. The precipitated cells were resuspended in PBS and each bacterial concentration was determined using a preconstructed standard curve. A Salmonella cocktail was prepared by mixing equal amounts of S. Typhimurium and S. Enteritidis. Other bacterial species (Table 1), except Listeria spp., were cultured in TSB whereas two strains of Listeria were cultured in TSB containing 0.6% yeast extract (TSBYE) for 16 h at 37ºC. After cultivation, each bacterial culture was washed and centrifuged for preparing a bacterial suspension according to the abovementioned procedures.

Salmonella typhimurium ATCC 14028 and Lactobacillus rhamnosus GG ATCC 53103 were obtained from the Institute of Animal Nutrition, Northeast Agricultural University (Harbin, Heilongjiang, China). Luria-Bertani (LB) medium was used to activate and incubate S.T at 37 ℃ in a shaker incubator until the logarithmic growth phase. The bacterial cells were collected by centrifugation at 1000 × g for the subsequent experiments. The crude EPSs were separated and purified according to described procedures with slight modifications [18 (link)]. In brief, LGG was cultured at 37 ℃ for 36 h in skim milk-modified medium supplemented with 10% glucose. And then heated in a boiling water bath for 10 min to denature the proteins and inactivate the enzymes. Cells and proteins were removed by centrifugation at 10,000 × g and 4 ℃ for 15 min. After filtering to obtain the supernatant, trichloroacetic acid was added to a final concentration of 4% (w/v) for 12 h. The supernatant was obtained by centrifugation, 3 times the volume of precooled ethanol was added to the supernatant, and the mixture was incubated at 4 ℃ for 12 h and then centrifuged to obtain the precipitate. The precipitate was dissolved in deionized water, dialyzed using a dialysis bag (molecular weight cutoff of 8000–14,000 Da) for 72 h and then lyophilized in a freeze-dryer. The content of EPSs was analyzed using the phenol sulfuric acid method.

We measured how DNA fragments that provide antibiotic resistance to E. coli influence susceptibility in Shigella sonnei HNCMB 25021, K. pneumoniae NCTC 9131 and Salmonella enterica subsp. enterica serovar Typhimurium str. LT2. For this purpose, we used a representative set of 13 plasmids that were isolated in our antibiotic selection screens. For each strain, the provided resistance levels (that is, the MIC) were measured with a standard 12-step microdilution method in 96-well plates, and the MIC fold change was determined by comparing them to the MIC of the corresponding empty vector harbouring control strains. MICs were determined on the basis of cell growth (OD₆₀₀) after 24 h incubation (37 °C, 180 r.p.m.).