Plasmid purification mini kit

Mueller-Hinton (MH) agar and broth were purchased from Thermo Fisher Scientific (East Grinstead, United Kingdom). Luria-Bertani (LB) broth, LB agar, ampicillin, amoxicillin, cefepime, CAZ, imipenem, meropenem, piperacillin, and tazobactam were obtained from Sigma-Aldrich (St. Louis, MO). Nitrocefin was purchased from Merck (Darmstadt, Germany). All strains used and constructed within this study are listed in Table 1. The characteristics of the clinical E. coli strain 50579417 harboring the OXA-48 plasmid (p50579417_3_OXA-48) have been described previously (61 (link), 62 (link)). The plasmid p50579417_3_OXA-48 was conjugated into rifampin-resistant E. coli TOP10 and subsequently isolated using a plasmid mini-purification kit (Qiagen, Germany). E. coli MG1655 (DA4201) was electroporated with p50579417_3_OXA-48 as published previously (63 (link)). Transformants positive for bla_OXA-48 were checked by PCR using REDTaq ready mix (Sigma-Aldrich, St. Louis, MO) and preOXA-48 primers (61 (link)).

Plasmid DNA was purified using a Plasmid mini purification kit (Qiagen Inc., CA, USA). The BAC-Tracker supercoiled DNA ladder (Epicentre Biotechnologies Inc., WI, USA) was used as a size marker for plasmid analysis. Plasmids were analyzed using the PCR-based replicon typing method to identify the plasmid type [15 (link)]. All detected replicon types were confirmed by sequencing.

After CAZ and CAZ-AVI exposure, plasmids were isolated using a plasmid mini-purification kit (Qiagen, Germany), and mutations within bla_OXA-48 were identified by Sanger sequencing (BigDye 3.1 technology; Applied Biosystems, CA) using preOXA-48 primers (61 (link)).
For a functional resistance profile, the native and mutated bla_OXA-48 genes were cloned in the pCR-blunt II-TOPO vector (Invitrogen, CA) and expressed in E. coli TOP10 (Invitrogen, CA). The PCR product was obtained by Phusion High Fidelity PCR mastermix with High Fidelity buffer (New England Biolabs, MA) and preOXA-48 primers (61 (link)). Transformants were selected on LB agar plates containing 50 or 100 mg/liter ampicillin. Insertion size was verified by Sanger sequencing using M13 forward (5′-GTAAAACGACGGCCAG-3′) and reverse (5′-CAGGAAACAGCTATGAC-3′) primers.

The pQE70 plasmid containing the genes of NColE7 and the Im7 immunity protein (a generous gift from prof. K.-F. Chak, Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei, Taiwan) served as a template for the amplification of DNA segments including the gene of the native and mutated NColE7. The primers applied in PCR are collected in Table S1. The obtained fragments were cloned into a pGEX-6P-1 vector (GE Healthcare BioSci.) within the EcoRI and XhoI cloning sites. The plasmids were transformed into E. coli DH10B cells and spread on an LB/Amp (10 μg/ml ampicillin) plates. The colonies (usually 1–3 colony/100 μl transformed cell) were cultivated in LB/Amp (10 μg/ml ampicillin) solution, which was then sedimented and the plasmids were purified with the QIAGEN Mini Plasmid Purification kit. The purified plasmids containing the gene of the mutant proteins were used as templates in a further PCR with the pGEX sequencing primers, and the products were sequenced. Inactive mutants were selected by E. coli DH10B cells. Since NColE7 itself is toxic for the cells, in native bacteria it is coexpressed with its cognate immunity protein (Im7) [24 (link), 25 (link)]. Here in the absence of Im7 gene the cloning procedure implied the selection of genes of nontoxic proteins.