Automated Ribotyping Using Different Enzymes To Improve Discrimination ofListeria monocytogenesIsolates, with a Particular Focus on Serotype 4b Strains

Abstract

To develop improved automated subtyping approaches for Listeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymes EcoRI, PvuII, and XhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination ‫؍‬ 0.518). A combination of PvuII and XhoI showed the highest discriminatory ability (index of discrimination ‫؍‬ 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.

Multi-Virulence-Locus Sequence Typing Identifies Single Nucleotide Polymorphisms Which Differentiate Epidemic Clones and Outbreak Strains ofListeria monocytogenes

“…Early multilocus enzyme electrophoresis (MEE)-based subtyping (3) and restriction enzyme analysis (57) showed that strains from many different outbreaks were closely related even though those outbreaks were geographically and temporally distinct. These findings were also supported by data generated using ribotyping (12), virulence gene polymorphism analysis (59), and pulsed-field gel electrophoresis (PFGE) (4,5,28,31). Kathariou (30,31) subsequently compared those studies and defined four epidemic clones of L. monocytogenes (epidemic clone I [ECI], ECIa, ECII, and ECIII).…”

“…1). The isolates from the coleslaw outbreak (Canada, 1981) and the two soft cheese outbreaks (California, 1985, andSwitzerland, 1983 (12). However, both MEE and ribotyping have relatively low discriminatory power.…”

Listeria monocytogenes Serotype Identification by PCR

“…Because of the importance of L. monocytogenes epidemiology to human health, a number of discriminatory subtyping methods have been described for this organism (2,6,9,15,16,19,21,22,27,31). Although pulsed-field gel electrophoresis is the most commonly employed subtyping technique, new subtyping technologies are constantly introduced and tested in hopes of increasing the resolution, speed, and reproducibility of L. monocytogenes subtyping.…”

Recent Advances in the Study of the Genotypic Diversity and Ecology of Listeria monocytogenes

“…Over the last decade, a vast number of reports of inexpensive and rapid methods to type Listeria spp. have been published 8,20,22,62,66) . The overall goal has been to develop methods that are more discriminatory than existing serotyping and phage-typing methods.…”