Instagene

Serotype was determined using the CDC standard protocol for the molecular determination of Salmonella serotype (CDC, 2009 ). Briefly, DNA from a pure culture was isolated using Instagene (BioRad). Multiplex PCR was set up using Qiagen HotStar Master Mix (Qiagen), 1 ul of DNA and thermocycled under the following conditions: 95°C, 15 min; 30 cycles of 94°C for 30 s, 48°C for 90 s, 72°C for 90 s; then 72°C for 10 min. DNA from the PCR reactions was then hybridized to the beads with specific O- and H-Ag probes before addition of strepavidin-R-phycoerythrin (Invitrogen div. Life Technologies, Grand Island, NY). After incubation the samples are read using the Bio-Plex instrument (BioRad). Positives are determined based on the ratio of signal to noise using a no template DNA negative control. Serotype is determined based on which antigens are positive for each sample. Traditional serotyping was performed on isolates where neither PFGE analysis nor molecular serotyping could infer a serotype. Traditional serotyping was performed at FDA's Center for Veterinary Medicine, Laurel, MD.

The dried blood spots were sent to the Malaria Research Centre, University of Malaysia, Sarawak, Malaysia, to identify the Plasmodium species through molecular analysis. The DNA was extracted from the dried blood spots using InstaGene (Bio-Rad Laboratories, USA), as described previously [46 (link)]. The DNA samples were initially analyzed through a nested polymerase chain reaction (PCR) with genus-specific primers for detecting Plasmodium, as described previously [47 (link)]. The Plasmodium-positive samples were then analyzed through a nested PCR with species-specific primers for P. knowlesi, P. coatney, P. cynomolgi, P. inui, and P. fieldi parasites, as described previously [48 (link)].

For the molecular study, the genomic DNA from ESBL-producing Salmonella spp. was extracted using the InstaGene™ matrix (Bio-Rad, Johannesburg, South Africa) according to the manufacturer’s instructions as detailed by Raseala et al. [23 (link)].
For the invA amplicon sequencing analysis, all suspected ESBL-producing Salmonella spp. isolates grown on ESBL-supplemented CHROMagar™ Salmonella Plus plates were pooled together per sample source and transferred into a DNase-free Eppendorf tube. These bacterial cells were disrupted in a 2 mL microfuge tube containing 1.5 mL of 1 × PBS and 2% Tween 20 (Sigma Aldrich, South Africa) using a Disruptor Genie^® Vortex mixer (Scientific Industries Inc., NY, USA). Each microfuge tube containing bacterial cells was placed under centrifugation at 10,000 × rpm for 5 min, and the resulting bacterial pellet was used to extract genomic DNA using a ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

DNA was extracted from blood spots on filter papers with the use of InstaGene (Bio-Rad Laboratories, USA) as described previously [40 (link)]. The DNA samples were first examined by nested PCR assays based on the small subunit ribosomal RNA genes of Plasmodium with the aid of genus-specific primers (rPLU1 and rPLU5 in nest 1 amplification, and rPLU3 and rPLU4 in nest 2) as described previously [41 (link)]. Plasmodium-positive samples were then examined by nested PCR assays using species-specific primers to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi, as described previously [42 (link)]. The products of the PCR amplification were analysed by gel electrophoresis in 2.7% agarose gels and were stained by Sybersafe before being observed under UV light.

The dried blood spots were sent to the Malaria Research Centre, University of Malaysia, Sarawak, Malaysia, to identify the Plasmodium species through molecular analysis. The DNA was extracted from the dried blood spots using InstaGene (Bio-Rad Laboratories, USA), as described previously [46 (link)]. The DNA samples were initially analyzed through a nested polymerase chain reaction (PCR) with genus-specific primers for detecting Plasmodium, as described previously [47 (link)]. The Plasmodium-positive samples were then analyzed through a nested PCR with species-specific primers for P. knowlesi, P. coatney, P. cynomolgi, P. inui, and P. fieldi parasites, as described previously [48 (link)].

For the isolation of thermophilic Campylobacter spp., 25 g of pooled rectal fecal samples were diluted 1/10 in Preston broth, homogenized and incubated for 18±2 h at 42 ℃ for enrichment. Suspensions (0.1 mL) were then subcultured onto a Chromogenic-Campylobacter Selective Agar (CASA^® Agar, Biomerieux) and incubated at 42 ℃ in a microaerobic atmosphere (5% O₂, 10% CO₂, 85% N₂) for 48–72 h. To confirm the presumptive Campylobacter and identify the species present in the pool of feces, DNA was extracted from a loopful of bacterial culture (InstaGene, BioRad, CA, USA) and screened for the presence of C. jejuni and C. coli in a multiplex real-time PCR (TaqMan^®Campylobacter Multiplex assay, ThermoFisher Diagnostics). Individual colonies were then tested using the same multiplex real-time PCR to confirm their identity and were stored for further characterization.

Fresh colonies of isolates were grown from cryovial beads following two consecutive streaks on MRS agar. The DNA was extracted using InstaGene (Bio-Rad, Hemel Hempstead, UK), according to the manufacturer’s instructions, and stored at −20 °C.