Listeria monocytogenes isolates from human sporadic and epidemic cases (n l 119) and from animal cases (n l 76) were characterized by automated ribotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) typing of the virulence genes actA and hly. This combination of typing methods differentiated 39 distinctive strains, each reflecting a unique combination of ribotypes, hly and actA alleles. Simpson's index of discrimination indicated a high discriminatory ability of ribotyping for both animal (0867) and human isolates (0857), which was further increased by the addition of hly and actA typing (0916 and 0904, respectively). Ribotype and hly allele data were further used to group isolates into three genetically distinct lineages. Each lineage is composed of several ribotype fragment subsets, each of which contains multiple ribotypes characterized by common ribotype fragments. To determine whether certain clones of L. monocytogenes show indications for unique pathogenic potential or host specificity, frequency distributions for five genetic characteristics (i.e. lineage, ribotype, ribotype fragment subset and hly and actA allele) were calculated for isolates from animal cases, human epidemic cases and human sporadic cases. Lineage III isolates were found less frequently in human cases (1 of 119 isolates) than in animal cases (8 of 76 isolates ; P l 0003). These results suggest the possibility of host specificity for non-primate mammals among lineage III strains. In addition, lineage I strains were found more frequently among human cases than among animal cases (P T 0001). Among the eight hly alleles observed, hly allele 1 was more common among human isolates as compared to animal isolates (P l 0002). We also identified one ribotype (DUP-1030) which was significantly more common among animal isolates (P l 0005) and one ribotype (DUP-1038 ; lineage I) which was significantly more common among human epidemic isolates as compared to human sporadic isolates (P T 0001). These findings confirm the presence of clonal groups of L. monocytogenes, which appear to be characterized by unique virulence or host specificity patterns. This study also establishes baseline data describing the genetic diversity of human and animal L. monocytogenes isolates which can be utilized in future surveillance programmes to track the emergence of new strains.

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inlA, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

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 (...

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