Ingenius bio imaging system

Five different ARG, namely bla_CTX, bla_TEM, bla_OXA, bla_SHV and sul1, were assessed in 40 randomly selected ESBL-producing Salmonella spp. isolates (10 from each matrix). The selected ARG were amplified by PCR using the primers listed in Table 1. Each reaction mixture consisted of 10 mL of SsoAdvanced Evergreen Supermix, 2 µL of each primer (reverse and forward), 5 µL of the DNA template and 3 µL of nuclease-free water, resulting in a final volume of 20 µL per reaction. The primers were prepared according to the manufacturer’s instructions (Inqaba Biotec, Pretoria, South Africa) to obtain stocks at working concentrations of each of the PCR primer. The CFX96 Touch™ real-time PCR detection system (Bio-Rad, South Africa) was used for PCR assays using the following conditions: initial denaturation at 98 °C for 2 min, followed by 40 cycles of amplification of denaturation 98 °C for 5 s, annealing for 30 s (60 °C for bla_CTX, 50 °C for bla_SHV, 58 °C for bla_OXA, 53 °C for bla_TEM and 65 °C for sul1 genes) and a primer extension at 72 °C for 5 s. Bio-Rad CFX Manager software (ver. 3.0) was used to acquire the generated data. The amplicon sizes were checked by running the amplicons on a 1% agarose gel (ThermoFisher, South Africa) stained with ethidium bromide (ThermoFisher, South Africa) and then visualised under a UV transilluminator (InGenius Bio Imaging System, Syngene, Cambridge, UK).

Cultured cells or brain tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2% Igepal CA-630 [NP-40], 0.25% sodium deoxycholate, 1 mM NaF, and 1 mM DTT supplemented with Mini Protease Inhibitor cocktail [Roche, Indianapolis, IN, USA; catalog no. 11873580001]). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (PALL, Port Washington, NY, USA; catalog no. 66485) or polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA; catalog no. 1620174) using the Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA; catalog no. 1704270). After blocking with 5% milk in Tris-buffered saline with Tween (TBST), primary antibodies were added to the blocking buffer. The membranes were then washed with TBST and incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies. The intensities of protein bands from the western blots were quantified using the Ingenius Bio Imaging system (Syngene, Frederick, MD, USA) and Gene Tools software. The signal intensities from phosphorylated and total proteins were normalized to respective tubulin levels, and the phosphorylated/total protein ratio was calculated.

The PCR amplification was performed on the extracted DNA samples using a combination of the universal 18 S rRNA gene forward primer 5′-GACGGGCGGTGTGTACA-3′ and the reverse primers 5′-CTGGTTGATCCTGCCAG-3′ or 5′-TGATCCTTCYGCAGGTTCAC-3′, respectively^{12 (link)}. These primers were used to amplify approximately 500 bp of the 18 S rRNA gene sequence. For the final reaction, each PCR reaction mixture (50 µL) contained DreamTaq^TM Green DNA polymerase (dNTPs and 4 mM MgCl₂), Master Mix (2×) (25 µL), nuclease-free water (22 µL), forward primer (1 µL) (0.2 µM) and reverse primer (1 µL) (0.2 µM) and metagenomic DNA (50–100 ng). The PCR cycling parameters used were as follows: initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 30 s and 72 °C for 1 min 30 s, with a 10 min extension at 72 °C, that was finally followed by rapid cooling to 4 °C. The PCR products were loaded into a 1% agarose gel and then visualised with an ultraviolet (UV) transilluminator (InGenius Bio Imaging System, Syngene, Cambridge, UK). The DNA was finally re-amplified with same sets of the two primers. The pooled samples were sequenced on the GS-FLX-Titanium series 454/Roche by Inqaba Biotechnology, South Africa.