GENOMIC ANALYSIS OF CLOSTRIDIUM-BOTULINUM GROUP-II BY PULSED-FIELD GEL-ELECTROPHORESIS

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic anal yses of Clostridium botulinum (non-proteolytic) group II. DNA degradat ion problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ D NA isolation method was also assessed and gave indistinguishable resul ts. Genomic DNA from 21 strains of various geographical and temporal o rigins was digested with 15 rare-cutting restriction enzymes. Of these , ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patter ns, enabling strain differentiation. Twenty strains yielded PFGE patte rns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI r estriction fragments, the genome size of C. botulinum group II was est imated to be 3.6 to 4.1 Mb (mean +/- standard deviation = 3,890 +/- 17 0 kb), The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminati ng epidemiological method for studying C. botulinum group II at the mo lecular level.