Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic

Abstract

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, and automated ribotyping were compared for epidemiological typing of Clostridium botulinum type E using clinical and food isolates associated with four botulism outbreaks occurring in the Canadian Arctic. All type E strains previously untypeable by PFGE, even with the use of a formaldehyde fixation step, could be typed by the addition of 50 microM thiourea to the electrophoresis running buffer. Digestion with SmaI or XhoI followed by PFGE was used to link food and clinical isolates from four different type E botulism outbreaks and differentiate them from among 39 group II strains. Strain differentiation was unsuccessful with the automated ribotyping system, producing a single characteristic EcoRI fingerprint common to all group II strains. RAPD analysis of C. botulinum group II strains was not consistently reproducible with primer OPJ-6 or OPJ-13, apparently discriminating between epidemiologically related strains. A modified PFGE protocol was judged to be the most useful method for typing epidemiologically related C. botulinum type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination.

FIG. 1.

(A and B) PFGE patterns of SmaI digests of food and clinical isolates of C. botulinum type E originating from the Canadian Arctic run without (A) and with (B) 50 μM thiourea in the running buffer. Lanes 1, 8, and 15, low PFGE molecular weight markers; lanes 2 to 6, outbreak 3 strains; lanes 9 and 10, outbreak 4 strains; lane 11, epidemiologically unrelated clinical isolate with a PFGE pattern identical to that of strains in lanes 9 to 10; lanes 12 and 13, outbreak 1 strains; lanes 7 and 14, control type E strains typeable without thiourea. (C) PFGE analysis using SmaI digests of six C. botulinum type E strains analyzed with and without a formaldehyde fixation step. Lanes 1, 8, and 15, low PFGE molecular weight markers; lanes 2, 4, 6, 9, 11, and 13, strains fixed with formaldehyde; lanes 3, 5, 7, 10, 12, and 14, same strains without formaldehyde fixation. NF, no formaldehyde.

FIG. 2.

Dendrogram revealing the high level of genetic relatedness of the 11 epidemiologically related type E strains originating from four different outbreaks analyzed among 39 C. botulinum group II isolates and based on their XhoI PFGE patterns. The percentage of similarity among strains was determined using the Dice coefficient, and the clustering was performed by UPGMA.

FIG. 3.

Typical EcoRI ribopatterns of C. botulinum type E strains characterized with only two main lower fragments using an automated RiboPrinter system.

FIG. 4.

RAPD patterns of nine epidemiologically related and three epidemiologically unrelated type E strains of C. botulinum. Lanes 1, 8, and 15, DNA molecular weight marker VI; lanes 2 to 6, outbreak 3 strains; lanes 9 and 10, outbreak 4 strains; lanes 12 and 13, outbreak 1 strains; lanes 7, 11 and 14, epidemiologically unrelated type E strains.

FIG. 5.

Dendrogram showing the low genetic relatedness of 9 of the 11 epidemiologically related type E strains originating from four different outbreaks analyzed among 39 C. botulinum group II strains and based on their OPJ-13 RAPD patterns. The percentage of similarity among strains was determined using the Dice coefficient, and the clustering was performed by UPGMA.