Xbai endonuclease

The genetic relationship among the CPKP isolates was determined by PFGE according to standardized protocol [50 (link)] with the XbaI endonuclease (New England Biolabs, Beverly, MA, USA) by using a CHEF-DR III apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA) for the separation of DNA fragments. XbaI-digested DNA from Salmonella enterica serotype Braenderup H9812 was used as a reference size standard, while PFGE patterns were digitally analyzed using the FPQuest (Bio-Rad Laboratories Pty Ltd., Hercules, CA, USA) software package.
PFGE profiles were compared using the Dice correlation coefficient with a maximum position tolerance of 1.5% and an optimization of 1.5%. Similarity clustering analysis was performed by using the Unweighted Pair Group Method using Averages (UPGMA), and a dendrogram was generated. Two PFGE profiles were classified as indistinguishable if the DNA fragment patterns matched each other completely, while clusters were selected using a cutoff at the 80% level of genetic similarity.

Representative O157 isolates were subjected to PFGE with XbaI endonuclease (New England BioLabs, Ipswich, MA, USA) using the CHEF Mapper system (Bio-Rad Laboratories, Hercules, CA, USA) as described previously [8 (link)]. The gel images were obtained using ethidium bromide stain. The electrophoretic patterns from PFGE were compared based on band position using FingerPrinting II software (Bio-Rad Laboratories, Hercules, CA, USA) and derived using the Dice coefficient with a maximum position tolerance of 1%. The strains were clustered using the unweighted pair group (UPGMA) method with arithmetic averages according to the manufacturer's instructions.

The evaluation of the genetic relationship between K. pneumoniae isolates was performed using PFGE and MLST. In the PFGE analysis, bacterial DNA was prepared and cleaved with 20 U XbaI endonuclease (New England Biolabs, Boston, MA, United States). Electrophoresis was performed in a 1.2% agarose gel (BMA Products, Rockland, ME, United States) prepared and ran in 0.5 × Tris-borate-EDTA buffer on a CHEF-DR III apparatus (Bio-Rad Laboratories, Richmond, CA, United States). The initial switch time was 1 s, the final switch time was 40 s, and the run time was 18 h at 6 V/cm. Gels were then stained in ethidium bromide, destained in distilled water, and photographed (GelDoc 2000; Bio-Rad). GelCompar II^® software (Applied Maths – bioMeriéux), with a Dice coefficient and a tolerance of 1.5, was used to determine the similarity and construct a dendrogram. MLST was performed by amplifying seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) on a selected number of ColR-CRKP isolates as previously described (Clinical & Laboratory Standards Institute [CLSI], 2021 ). The MLST website assigned the allele number and sequence type (ST)¹.