Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences.

Abstract

By using taxonomic characters derived from EcoRI restriction endonuclease digestion ofgenomic DNA and hybridization with a labeled rRNA operon from Escherichia coli, a polymorphic structure of Listeria monocytogenes, characterized by fragments with different frequencies of occurrence, was observed. This structure was expanded by creating predicted patterns through a recursive process ofobservation, expectation, prediction, and assessment of completeness. This process was applied, in turn, to normalized strain patterns, fragment bands, and positions of EcoRI recognition sites relative to rRNA regions. Analysis of 1346 strains provided observed patterns, fragment sizes, and their frequencies of occurrence in the patterns. Fragment size statistics led to the creation of unobserved combinations of bands, predicted pattern types. The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences. Each EcoRI site had a frequency of occurrence, and unobserved fragment sizes were postulated on the basis of knowing the restriction site locations. The result of the recursion process applied to the components of the strain data was an extended classification with observed and predicted members.Classification is the arrangement of strains into taxonomic groups on the basis of observed similarities. Bacteria have been classified into genera, species, and types with a variety of phenotypic characteristics to provide a basis for identification (1). Patterns of DNA restriction fragments containing portions of the rRNA operons provide another means of description and classification (2, 3).A bacterial genome contains numerous restriction enzyme recognition sites within and flanking the sequences that are highly conserved in related strains. By considering the mutational gains or losses of these sites as statistically independent events, we hypothesized that a species taxonomic structure incorporating all possible strain variation could consequently be defined. The conserved sequences and regional restriction sites inferred from our work in describing Listeria monocytogenes using EcoRI fragments containing sequences homologous to a rRNA operon from Escherichia coli (4) formed the basis of our analysis. Polymorphic fragments from different rRNA regions, each containing a given part of a given operon, could be combined into patterns, some of which would remain unobserved until the sample set became large enough to be truly representative of the natural population.By using matrix analysis on the data, the observed polymorphisms of the different rRNA regions were combined into patterns that have not yet been observed. The polymorphisms were also used to suggest the positions of EcoRI sites relative to an rRNA region. A maximum-likelihood model developed for use in this context predicted the pairings of restriction sites that led to the observed fragments and suggested pairings that could form additional sizes of rRNA sequence-containingThe publication costs of this article were defrayed in part by page ch...

Listeria monocytogenesIsolates from Foods and Humans Form Distinct but Overlapping Populations

“…EcoRI ribotyping was performed as previously described (5,15,17), by using the RiboPrinter Microbial Characterization system (Qualicon, Wilmington, Del.). Ribotype patterns were analyzed by using the RiboPrinter custom software, which normalizes fragment pattern data for band intensity and relative band size.…”

Ribotype Delineation and Description of Staphylococcus sciuri Subspecies and Their Potential as Reservoirs of Methicillin Resistance and Staphylolytic Enzyme Genes

“…The extracellular staphylolytic enzyme activity of selected strains was evaluated by visualizing zones of bacteriolysis following growth on a modified lysostaphin production medium (45) that contained heat-killed cells of S. aureus ATCC 6538P, as described previously (46), and by renaturing activity gel analysis Normalized ribotyping. Discrete and reproducible ribotype descriptions of strains resulted from a single method described previously in detail (11,24,59). Briefly, strains were lysed with staphylolytic enzymes and sodium dodecyl sulfate (SDS).…”

Characterization and Pathogenic Potential ofListeria monocytogenesIsolates from the Smoked Fish Industry

“…Wiedmann et al previously reported classification of L. monocytogenes isolates into three genetic lineages based on (i) ribotyping, a subtyping method based on restriction polymorphisms in the rRNA operon of prokaryotes (7,8,18); and (ii) allelic polymorphisms in the virulence genes hlyA, inlA, and actA (37). In their initial analysis, which included 20 human clinical L. monocytogenes isolates, all human epidemic isolates were classified into lineage I, while sporadic case isolates were classified into lineages I and II.…”