Body fluids extraction column based kit

The represented samples were collected based on the minimum required sample for the interpretation. The isolates were sub-cultured on 5% sheep blood agar, and incubated for 24 h at 37 °C. DNA extraction was carried out with the QIAGEN body fluids extraction (column-based) kit. All isolates were screened for CTX-M 14 (Cefotaximase-Munich), CTX-M 15, NDM (New Delhi metallo-ß-lactamase), VIM (Verona Integron Encoded Metallo-Beta-Lactamase producing), IPM (imipenem), CMY (cephamycin), DHA (Docosahexaenoic acid), OXA-48 (Amber class D beta-lactamase), and KPC (Klebsiella pneumonia carbapenemase), using multiplex PCR, as described by Rotor-Gene real-time PCR (Qiagen Instrument AG: 5 Flex HRM) with the probe-based AMR multiplex PCR kit (Streck ARM-D kit beta-lactamases), via the previously described protocols by the manufacturers. Verification of the kit was conducted with positive and negative control cultures (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC BAA 1705, Klebsiella pneumoniae ATCC BAA 1706, Klebsiella pneumoniae ATCC 70063, Enterobacter cloaciae NCTC 13406, Klebsiella pneumoniae ATCC BAA 2146).

Molecular detection of antimicrobial resistant genes for all the isolates from humans (n = 200), poultry (n = 140), and aquaculture (n = 60) were carried out at the MRI. The represented samples were collected based on the cost of the kit and minimum required sample for interpretation. The isolates were sub-cultured on 5% sheep blood agar, and incubated for 24 h at 37 °C. DNA extraction was performed using the QIAGEN body fluids extraction (column-based) kit. The extracted DNA was screened for CTX-M 14, CTX-M 15, NDM, VIM, IPM, CMY, DHA, OXA-48, and KPC, using multiplex PCR, as described by Rotor-Gene real-time PCR (Qiagen Instrument AG: 5 Flex HRM) with the probe-based AMR multiplex PCR kit (Streck ARM-D kit beta-lactamases), as described by manufacturers’ protocols.