Dig luminescent detection kit

The genomic location of the mcr-2 gene was verified by S1 nuclease digestion of genomic DNA followed by electrophoretic separation and Southern hybridization. A digoxigenin-labeled DNA probe (“DIG luminescent detection Kit”, Boehringer Mannheim GmbH, Mannheim) targeting a 567-bp PCR fragment specific for the mcr-2 gene using the aforementioned primers (Xavier et al., 2016 (link)) was used. In addition, we performed an in silico search of whole genome sequences with mlplasmids v. 1.0.0 (Arredondo-Alonso et al., 2018 (link)). To test whether the colistin resistance determinant was transferable, conjugation was performed by the broth filter mating method at 37°C using plasmid-free sodium azide resistant E. coli K12-J53 (J53 AziR) as recipient. Prior to the conjugation assays, all mcr-2-positive isolates were tested for their susceptibility to sodium azide. Transconjugants were selected on Endo agar plates containing 100 mg/L sodium azide and 2 mg/L colistin sulfate or containing 100 mg/L sodium azide and 4 mg/L colistin sulfate (Sigma-Aldrich, Germany, Karlsruhe, Germany). To confirm successful plasmid transfer, antimicrobial susceptibility testing of transconjugants, a PCR targeting the mcr-2 gene and plasmid profiling was performed as described above.

To determine the genomic location of carbapenemase genes, plasmid DNA, and I-CeuI digested whole-cell DNA were separated by agarose and PFGE, respectively, and analyzed by Southern blot hybridization. DNA probes were prepared using the PCR Dig Probe Synthesis Kit (Boehringer Mannheim GmbH, Germany) and consisted of a 1,486 bp PCR fragment specific for 16S rRNA genes (primers SK16R/SK16F), and an internal PCR fragment specific for the bla_OXA-48-like gene (743 bp; primers OXA-48A/B), respectively (Stolle et al., 2013 (link)). Detection of hybridized DNA molecules was done with the DIG Luminescent Detection Kit (Boehringer Mannheim GmbH, Mannheim, Germany).

Bacteria grown under the required growth conditions were pelleted and RNA was isolated using the SV total RNA purification kit (Promega) as described [34 (link)]. Total RNA (20 μg) was separated on MOPS agarose gels (1.2%), transferred by vacuum blotting for 1.5 h onto positively charged membranes (Whatman) in 10 x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate, pH7) using a semi-dry blotting system and UV cross-linked. Prehybridization, hybridization to DIG-labelled probes and membrane washing were conducted using the DIG Luminescent Detection Kit (Roche, Germany) according to the manufacturer's instructions. The DIG-labelled PCR fragments used as probes were produced by PCR using the DIG-PCR nucleotide mix (Roche, Germany) as described [34 (link)] with the following primer pairs: for the lcrF transcript—I214/I303, for the csrB and csrC transcripts—555/556 and 583/I82, for the rne transcript—IV529/IV530 and the pnp transcript—IV527/IV528 (see S2 Table).
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.