Prep plasmid midi kit

Lipofectamine 3000 Reagent kit (Invitrogen) was used for plasmid transfection of mIMCD3 cells according to manufacturer’s protocol (3.75 μl of Lipofectamine 3000, 2.5 μg DNA and 5 μl of P3000 reagent in each well). siRNA transfections were also performed with Lipofectamine 3000 (Invitrogen) according to manufacturer’s protocol (0.75 μl of Lipofectamine 3000, 25 pmol siRNA in each well). Plasmids included pCGN (HA-tag, RRID:Addgene_53395), pCGN-HA-AURKA (gift from Olga Plotniknova), and pCGN-HA-AURKA KD (made using Q5 Site-Directed Mutagenesis Kit NEB.E0554S according to manufacturer’s protocol to contain the K162R variant described for pcDNA3.1-mRFP-AURKA K162R^{15 (link)}). All plasmids were prepared with Qiagen plasmid midiprep kits. siRNAs were All Stars negative control siRNA (SI03650318, Qiagen) and Mm_Aurka_1 Flexitube siRNA (SI00908803, Qiagen). Alisertib (10 mM in DMSO, S1133, SelleckChem) was added to media at 1 μM final concentration where indicated. 16 h after plasmid and siRNA transfections, fresh growth media was placed on mIMCD3 cells and wells harvested up to 24 h later.

Multilocus sequence typing (MLST) was conducted for identification of clonal correlation of the poxtA-positive S. haemolyticus (http://www.shaemolyticus.mlst.net Accessed on: 29 January 2021) [26 (link)]. Plasmid DNA from all poxtA-positive CoNS isolates was extracted using a Qiagen Prep Plasmid Midi Kit (Qiagen, Hilden, Germany) and transferred into a recipient S. aureus strain RN4220 by electroporation using Gene Pulser apparatus (Bio-Rad, Hercules, CA, United States) [27 (link)]. Electrotransformants were selected on brain heart infusion (BHI) agar containing 10 µg/mL of florfenicol. Electrotransformants were further confirmed for the presence of poxtA gene by PCR analysis. The successful electrotransformants were further screened for the presence of aadD, fexB, tet(L) and tet(M) genes by PCR.

To investigate the transferability of tet(X4)-bearing plasmid, conjugation assays were performed using streptomycin-resistance E. coli C600 as the recipient strain. Briefly, overnight cultures of donor and the recipient strains were 1:1 mixed and incubated at 37°C for 16 to 20 h. After incubation, 10-fold serial dilutions were mixed in sterile saline, and 100-μL samples were spread onto LB agar plates containing 4 mg/L tigecycline and 1,500 mg/L streptomycin. The tet(X4)-positive transconjugants were confirmed by PCR and ERIC-PCR (Versalovic et al., 1991 (link); Sun et al., 2019b (link)). Susceptibility of transconjugants was detected as mentioned previously. Plasmid analysis was performed using whole-genome sequence as described previously. Plasmid DNA was extracted using a Qiagen Prep Plasmid Midi Kit (Hilden, Germany).

Plasmid DNA from cfr-positive MRSA strains was extracted using a Qiagen Prep Plasmid Midi Kit (Qiagen, Hilden, Germany) and transferred into a recipient S. aureus strain RN4220 by electroporation using Gene Pulser apparatus (Bio-Rad, Hercules, CA, United States). Electrotransformants were selected on brain heart infusion (BHI) agar containing 8 μg/mL of florfenicol. The presence of cfr was further confirmed by PCR (Kehrenberg et al., 2009 (link)). To determine the location of cfr gene, DNA was separated by PFGE after treatment with S1 nuclease (Takara, Dalian, China) and plasmids carrying cfr were identified by Southern blot hybridization using a digoxigenin-labeled cfr probe (Roche, Mannheim, Germany) according to the manufacturer’s instruction.

E. coli C600 was used as the recipient for the conjugation experiment of MCR-producing Salmonella isolates. The transconjugants were selected on MacConkey agar containing colistin (2 mg/L) and streptomycin (2,000 mg/L), and finally confirmed by PCR and ERIC-PCR45 (link). Plasmids that are not transferable by conjugation were studied by transformation assay. Plasmid DNA was extracted using a QIAGEN Prep Plasmid Midi Kit. Purified plasmids were used in electroporation experiments with E. coli DH5α following the manufacturer’s instructions. Transformants were incubated at 37 °C for 1 h and were then selected on LB agar containing 2 mg/L colistin.