To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species.Strains of Listeria monocytogenes are classified into the taxon based on genotypic and phenotypic similarities (1, 2). A general method for classification and identification of strains by using DNA restriction fragments containing portions of rRNA operons has been described (3, 4). This method has been applied to the genus Listeria (5) and to L. monocytogenes (6), demonstrating its utility for classifying, identifying, and typing strains.We have described a standard method for species description by using conserved sets of species-specific rRNA gene restriction endonuclease-derived fragments (7). In the present study, > 1000 strains ofL. monocytogenes were characterized by using EcoRI fragments containing sequences complementary to an rRNA operon from Escherichia coli. The pattern structure of the species was described in detail by the use of fixed electrophoretic conditions, fragment standards, electronic imaging, and software for mobility normalization. In addition, we introduced the use of continuous-scale relative intensity in recording patterns from -9000 strains of -200 species. The L. monocytogenes patterns were arranged into the taxonomic structure by the use of squared correlation values (8) and visual assessment. We assigned letter names to the rRNA regions, each containing a given part of a given operon, and differentiated strains by restriction fragment length polymorphisms ofThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adverti...

Strains of a new species, Staphylococcus vitulus, were isolated from food and a variety of mammals. This species was recognized on the basis of the results of an analysis of genomic EcoRI fragments containing portions of the rRNA operons. The patterns of hybridized fragments obtained from strains belonging to the new taxon were sorted into a distinguishable cluster and were distinct from the StaphyroCoccus lentus and Staphylococcus sciuri patterns. The results of DNA-DNA hybridization reactions demonstrated that strains in this cluster were more closely related to S. lentus and S. sciuri than to other Staphylococcus species and yet were significantly different. While these strains had some of the phenotypic characteristics of the S. sciuri species group, the newly recognized taxon could be distinguished by its very small colonies on P agar, absence of alkaline phosphatase activity, and lack of acid production from L-arabinose, maltose, N-acetylglucosamine, D-mannose, and raffinose. The type strain of the new species is strain DD 756 (= ATCC 51145).A general method for distinguishing bacterial species by using restriction fragments containing portions of their rRNA operons has been described previously (12,26,31). This method has been applied to the genus Staphylococcus (8,9,29) and recently was recommended as a way to distinguish a newly described staphylococcus from previously described taxa (7).In this study, the electrophoretic patterns of restriction fragments labeled by hybridization with an rRNA operon from Eschen'chia coli were used to characterize organisms belonging to the Staphylococcus sciuri species group. When the patterns were sorted on the basis of similarity by using correlation values, clusters of strains identified as S. sciuri and Staphylococcus lentus were formed. We also distinguished another cluster of novobiocin-resistant, oxidase-positive staphylococci. This third taxon and its relationship to the S. sciuri species group are described in this paper. MATERIALS AND METHODSBacterial strains. In this study, strains were identified by their DuPont numbers. Table 1 shows the strains which we studied, other designations of some strains, the species or subspecies t o which each strain belongs, and the source of each strain.Characteristic determinations. The following characteristics were determined as previously described (18,19,21,22): colony morphology and pigment, motility, anaerobic growth in t hioglycolate broth, cat alase activity, acetylme t hylcarbinol (acetoin) production, nitrate reduction, tube coagulase activity, clumping factor, hemolysis of bovine blood, carbohydrate reactions, and susceptibility to various antibiotics. Clumping factor and protein A were detected with a Staph Latex kit (Remel, Lenexa, Kans.). The oxidase test was performed by using a Microdase disk (Remel) (10). Pyrrolidonyl arylamidase activity was determined by using the Pyr broth and Pyr reagent of Carr-Scarborough Microbiologicals (Stone Mountain, Ga.) for identification of group A streptococci and enterococci (...

Ribotyping is a molecular method for the characterization, identification, and typing of bacterial isolates that has value in epidemiological studies. To demonstrate the utility of this technique for typing of Listeria monocytogenes, four outbreaks of epizootic listeriosis in ruminants were investigated through coordinated detection and characterization methods utilizing classical microbiology and nucleic acid-based techniques. L. monocytogenes strains isolated from clinical samples and the silage consumed by the affected animals were ribotyped to establish the causal relationship between feed and the disease outbreak. For all but one outbreak, we were able to isolate L. monocytogenes strains represented by the same ribotype from both clinical and silage samples. Additional L. monocytogenes strains with ribotypes different from those of the respective clinical samples were isolated from all silage samples. This indicates that a diverse population of L. monocytogenes strains exists in farm environments, of which some may be more likely than others to cause disease.

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...