Xbai restriction enzyme

PFGE fingerprinting was performed using the XbaI restriction enzyme (ThermoScientific, Waltham, MA, USA), 1% SeaKem agarose gel, and the universal laboratory standard Salmonella enterica subsp. enterica serovar Braenderup (ATCC® BAA664™) according to the standard PulseNet protocol (http://www.pulsenetinternational.org). Electrophoresis was performed using the Bio-Rad laboratories CHEF DR-III system (Bio-Rad Laboratories, Bio-Rad Laboratories Inc., Hercules, CA, USA) with a run time of 12 h and switch time of 5–40 s (https://www.cdc.gov/pulsenet/). Gels were stained with ethidium bromide. For isolates showing identical pulsotypes or that were untypable by XbaI, PFGE was repeated using the secondary enzyme AvrI. PFGE profiles were analyzed with the BioNumerics software version 7.6.1 (Applied Maths, Sint-Martens-Latem, Belgium), with profiles assigned as different pulsotypes if three or more bands were different between the two of them. Pulsotypes were clustered based on the BioNumerics software analysis through dice correlation coefficients with an optimization of 1.5% and tolerance of 1.5%.

In the event that at least 2 household members were colonized in the same home, the genetic relatedness of isolates from hemodialysis patients, household contacts, and pets was determined using pulsed-field gel electrophoresis (PFGE), which was performed using the XbaI restriction enzyme (Thermo Scientific, Waltham, MA, USA) [15 (link)]. The cluster analysis was performed using BioNumerics software version 6.0, and dendrograms were generated by the unweighted pair group method using average linkages. A similarity cutoff of 80% was used to define genetically related strains.
Additionally, multiple-locus sequence typing (MLST) was performed on a representative sample of the isolates, according to the group generated by PFGE [16 (link)]. The sequence type and the clonal complex were assigned using the web database (https://pubmlst.org/). Finally, all Escherichia coli isolates were typified using the Clermont method to establish the phylogroups that represented the highest clinical risk in colonized people. For this, quadruplex PCR and 2 simple PCRs were performed to recognize the 7 defined phylogroups (A, B1, B2, C, D, E, and F) according to the previously described protocol [17 (link)].

TALENs targeting exon5 of Spink5 gene were designed using TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/) [43 (link),44 (link)], assembled using the Golden Gate Cloning system[43 (link)], and cloned into the ELD-KKR backbone plasmid as described previously [45 (link)]. DNA binding domains of TALENs specific for the desired target site within Spink5 gene (Fig 1B) consisted of following repeats: NI-HD-HD-NN-HD-NI-NN-NG-NI-NN-NI-NG-NN-NG-NN-NI-NI-HD-NG (5´ TALEN-Spink5) and HD-NG-NN-HD-NG-NG-NG-NI-NG-NI-NN-NN-NN-NG-NI-HD-NG-HD-NI-HD (3´ TALEN-Spink5). Both TALEN plasmids were used for production of TALEN encoding mRNA as described previously [46 (link)]. 5 μl of TALEN mRNA (with total RNA concentration of 40 ng/μl) was mixed with 100 μM of targeting single-stranded oligonucleotide (Sigma-Aldrich; Fig 1B) and the final solution was microinjected into C57BL6/N-derived zygotes. Genomic DNA isolated from tail biopsies of newborn mice were screened by PCR (primers F1: 5´-CCTGTCTCTGCCTTCAGACC-3´ and R1: 5´-GGCTGTGGTAACTGTCCAAAA-3´) and subsequent RFLP analysis using XbaI restriction enzyme (Thermo-Scientific).