Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Moos, Schleifer, and Smith 1976, 23& emend. Moos et al. 1996, S. sciuri subsp. curnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (7OOC) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. curnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnuticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. curnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).Staphylococcus sciuri is generally considered one of the most primitive Staphylococcus species; it is widely distributed in nature, is capable of growth on inorganic nitrogen salts as the sole source of nitrogen, and exhibits a wide range of biochemical activities (31-33). In the past 20 years since its original description (33), numerous laboratories have reported frequent isolation of this species from foods, farm animals, rodents, marsupials, marine mammals, and birds and occasional isolation from humans and their pets (1,2, 17,28,31,35,48,52,57,58).Most resident populations of S. sciuri are members of the normal cutaneous microflora of lower mammals. This species is rarely associated with infections. A preliminary investigation of a large collection of S. sciuri strains showed that a ribotyping method based on an analysis of genomic EcoRI fragments containing portions of the rRNA operons could be used to distinguish two major subgroups and additional strain variation within the species (10).In this study, we describe three subpopulations of S. sciuri which can be distinguished on the basis of their rib...

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...

Delimiting the genus. nov., are described on the basis of a phylogenetic analysis comparing 165 rRNA sequences, DNA-DNA liquid hybridization, DNA base composition, normalized ribotype patterns, macrorestriction pattern analysis and estimation of genome size using PFGE, cell wall composition, phenotypic characteristics and plasmid profiles. Compared with their closest relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 165 rRNA sequence similarities (934-9503 %), higher DNA G+C content (38-45 mol YO), absence of cell wall teichoic acids (with the possible exception of M. caseolyticus), unique ribotype pattern types and macrorestriction patterns, smaller genome size (approx. 1500-1800 kb) and generally larger Gram-stained cell size (14-205 pm in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus witulus and Staphylococcus lentus) by their oxidase activity. The four Macrococcus species can be distinguished from one another on the basis of DNA-DNA hybridization, ribotype pattern types, macrorestriction patterns and their phenotypic properties, including colony morphology, cell morphology, haemolysins, Staph Latex agglutination, acid production from a variety of carbohydrates, acetoin production, nitrate reduction, aesculin hydrolysis, and DNase and urease activities. The type species is M. equipercicus. The type strains of M. equipercicus, M. caseo/yticus, M. bovicus and M. carouselicus are ATCC 5183IT ( (Schleifer et al., 1982). These strains were isolated from the milk of cattle, but more recently several strains have been isolated from the abscesses of slaughtered lambs (De La Fuente et al., 1992) and one from the milk of goats (De Buyser et al., 1992).In a preliminary investigation (Ballard et al., 1995), we sampled the skin of 15 cattle, 25 goats, 14 horses, 10 ponies, 4 whales, 25 dolphins and meat products for the presence of Staphylococcus caseolyticus. This species was isolated from only three samples of raw beef and the skin of a pilot whale and so can still be thought of as a relatively uncommon species. Surprisingly, we discovered a group of three new species related to Staphylococcus caseolyticus living on the skin of cattle, horses and ponies. Phenotypic characterization. The following characteristics were determined as described previously Kloos et al., 1976; Webster et al., 1994;: Gram-stained cell morphology and cell arrangement, colony morphology and pigmentation, motility, anaerobic growth in thioglycollate semi-solid medium, catalase activity, acetylmethylcarbinol (acetoin) production, nitrate reduction, oxidase activity, pyrrolidonylarylamidase activity, aesculin hydrolysis, DNase activity, thermonuclease activity, ornithine decarboxylase activity, urease activity, staphylocoagulase activity, lysostaphin susceptibility, haemolysis of sheep, bovine and horse blood, and carbohydrate reactions. The presence of clumping factor and/or protein A was tested using the Staph Latex Kit ...

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 (...

When the hypervariable 16S-23S intergenic spacer regions found in prokaryotic ribosomal DNA (rDNA) are amplified from conserved adjacent sequences, homoduplex double-stranded DNA structures and heteroduplex structures containing substantial regions of single-stranded DNA are generated. The electrophoretic separation of these structures results in product profile patterns, which may be organized into highly correlated pattern groups of ribosomal spacer and heteroduplex polymorphism (RS/HP) types. In a test panel of 380 Salmonella strains that were analyzed by this procedure, 36 unique RS/HP types were observed. Of the 28 serovars in the test group, 21 showed single characteristic RS/HP types. The remaining seven serovars each contained multiple RS/HP types, which were also unique to individual serovars. Formation of heteroduplex structures with a substantially reduced electrophoretic mobility was observed in 29 of the 36 RS/HP pattern types. Because the mobility of these heteroduplex structures is sensitive to intergenic spacer sequence composition, the presence of these structures adds an additional diagnostic feature that is extremely useful in the differentiation of Salmonella serovars. The RS/HP types show sufficient diversity to be useful in the identification of many commonly observed Salmonella serovars. This analytical procedure is simple to perform and is well suited to rapid and inexpensive screening of large numbers of Salmonella strains.

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The primary structure of an elastase from the Antarctic fish Notothenia neglecta (NE) was elucidated by molecular cloning and cDNA sequence analysis. The cDNA of interest was isolated from a cDNA library obtained from Notothenia's pyloric caeca. The amino acid sequence identity with mammalian elastases ranges between 53 and 64%, but interestingly reaches 79% with one isoform (CEB) of two recently isolated cod elastases. The most interesting changes distinguishing the model of NE, predicted from the three dimentional structure of the native porcine elastase (PE), concern the catalytic crevice located in the inter-domains region. These features might be involved in the adaptation to cold of the Antarctic elastase.

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...