Genomic DNA of the isolates was extracted using a G-spin Genomic DNA extraction kit (iNtRON, Korea) according to the manufacturer’s instructions. Multilocus sequence typing (MLST) of seven selected housekeeping loci (adk, atpA, ddl, gdh, gyd, purK, and pstS) with polymerase chain reaction (PCR) amplification was used for genotyping of VREF isolates [20 (link)]. The e-BURST algorithm was used to analyze the relatedness of each VREF isolate ST [21 (link)]. The presence of virulence genes esp and hyl was detected by PCR [22 (link), 23 (link)], and confirmed by sequencing [24 (link)].
In cases in which both blood and rectal VREF isolates showed an identical ST, pulsed field gel electrophoresis (PFGE) was conducted to determine the clonal association. For PFGE, bacterial DNA was digested with the Sma I restriction enzyme (TaKaRa Bio Inc., Shiga, Japan) and separated by electrophoresis using a CHEF DR II system (Bio-Rad Laboratories, Hercules, CA, USA). The PFGE patterns were analyzed using Gel Compar II software (Applied Maths, Kortrijk, Belgium). Potential clonal relatedness was determined at a ≥ 80% level of similarity [25 (link)].
In cases in which both blood and rectal VREF isolates showed an identical ST, pulsed field gel electrophoresis (PFGE) was conducted to determine the clonal association. For PFGE, bacterial DNA was digested with the Sma I restriction enzyme (TaKaRa Bio Inc., Shiga, Japan) and separated by electrophoresis using a CHEF DR II system (Bio-Rad Laboratories, Hercules, CA, USA). The PFGE patterns were analyzed using Gel Compar II software (Applied Maths, Kortrijk, Belgium). Potential clonal relatedness was determined at a ≥ 80% level of similarity [25 (link)].
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