Ez seq service

cDNA encoding SMPD3 and SMPD4 were kindly donated by Dr. Hannun [15 (link)] and Dr. Mangini, respectively [12 (link)]. 3xHA-miniTurbo-NLS_pCDNA3 was a gift from Alice Ting (Addgene plasmid # 107172; http://n2t.net/addgene:107172 (accessed on 31 October 2018); RRID:Addgene_107172). Ref. [16 (link)]. All steps of the cloning process were performed according to the protocol provided by the manufacturer. To enable cloning, restriction enzyme recognition sites were introduced into the SMPD3 and SMPD4 cDNA using overhanging primers in a 2-step PCR (Appendix B Table A2). Vectors were digested using enzymatic restriction digestion, dephosphorylated with Shrimp Alkaline Phosphatase (New England Biolabs, Ipswich, MA, USA), and ligated to purified PCR products using T4 DNA ligase (NEB). Plasmids were introduced into One Shot Stbl3 chemically competent E. coli (Thermo Fisher Scientific Inc., Waltham, MA, USA). For miniprep, a colony was inoculated and grown in Luria–Bertani medium with 50 µg/µL of ampicillin, and plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands). For midiprep, DNA was isolated using the NucleoBond Xtra Midi kit (Bioké, Leiden, The Netherlands). DNA fragments amplified with PCR were sequenced by the Macrogen EZ-Seq service (Macrogen, Amsterdam, The Netherlands).

Chromosomal DNA from L. plantarum wild-type strains and their derivatives were obtained with the Microbial DNA extraction kit (Cabru, Milan, Italy) according to the manufacturer’s instructions. Quantity and quality of genomic DNA were assessed using a BioTek Eon spectrophotometer (BioTek, Winooski, VT, United States) and by visualization on 0.8% agarose gel. The primers RFNFpl (CAGCGCCTTGTTTGAT) and RFNR (TGGCCGTCTTTGACTA) (Macrogen, Madrid, Spain) were used to amplify a 649-bp fragment including the rib regulatory region. The PCR were carried out in a 25-μL volume reaction containing 20 ng of genomic DNA, 5 μL of 5× HotStar HiFidelity PCR Buffer, 0.2 nm of each primer, and 2.5 U/μL of the HotStar HiFidelity DNA Polymerase (Qiagen, Hilden, Germany). The thermal profile of the PCR was as follows: 95°C for 5 min, 35 cycles of 95°C for 30 s, 53°C for 45 s, 72°C for 75 s, and a final extension at 72°C for 7 min. Clean-up was performed by using a QIAquick PCR purification kit (Qiagen), and quantification and purity of the amplicons were determined spectrophotometrically and by visualization on 1.2% agarose gel. Sequencing was performed according to the EZ-seq service (Macrogen). Multiple sequence alignments of the RFN were performed using the Clustal Omega program¹.

To determinate the clonal dissemination of ESBL-producing E. coli, a sequence type analysis was performed following the scheme described by Wirth et al. [24 (link)] and seven housekeeping genes were amplified and sequenced for each isolate (adk, fumC, gyr, icd, mdh, purA and recA). DNA extraction was performed with a DNeasy^® Blood & Tissue kit (Qiagen, Barcelona, Spain) using a pretreatment protocol for Gram-Negative Bacteria. For amplification, 3 μL of DNA extract was mixed with 5 μL of buffer 10× (Bioline, London, UK), 5 μL of dNTPs (Bioline), 1.5 μL of MgCl₂ 50 mM (Bioline), 2 μL of each primer Sigma-Aldrich, Steinheim, Germany) and 1.5 U of Inmolase™ DNA polymerase (Bioline) in a final volume of 50 μL. The amplification conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 1 min at 95 °C, 1 min at 60 °C (mdh, gyrB, and recA) or 1 min at 64 °C (fumC, icd, purA and adk), 2 min at 72 °C, and a final elongation step of 5 min at 72 °C. Sequence reactions were performed by EZ-Seq Service (Macrogen Europe) and sequences data were imported into the E. coli MLST database website [25 ] to determine MLST type. These data were analyzed using BioNumerics v7.5 software (Applied Maths, Sint-Martens-Latem, Belgium).