Delimiting the genus Staphylococcus through description of Macrococcus caseolyticus gen. nov., comb. nov. and Macrococcus equipercicus sp. nov., and Macrococcus bovicus sp. no. and Macrococcus carouselicus sp. nov.

Four species of the newly proposed genus Macrococcus, namely macrococcus caseolyticus gen. nov., comb. nov. (formerly Staphylococcus caseolyticus Schleifer, Kilpper-Bälz, Fischer, Faller and Endl 1982, 19VP), Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. Macrococcus carouselicus sp. nov., are described on the basis of a phylogenetic analysis comparing 16S 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 closet relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 16S rRNA sequence similarities (93.4-95.3%), higher DNA G+C content (38-45 mol%), 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 (1.1-2.5% microns in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus vitulus and Staphylococcus lentus) by thier 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, Staphy 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. caseolyticus, M. bovicus and M. carouselicus are ATTCC 51831T (= DD 9350T) ATCC 13548T (= TDD 4508T) (Schleifer et al. 1982, ATCC 51825T (= DD 4516T) and ATCC 51828T (= DD 9348), respectively.

Pulsed-field gel electrophoresis of Staphylococcus hyicus and Staphylococcus chromogenes genomic DNA and its taxonomic, epidemiologic and ecologic applications in veterinary medicine.

One hundred and thirty-eight strains of Staphylococcus hyicus and 21 strains of S. chromogenes isolated from animals were analyzed by pulsed-field gel electrophoresis (PFGE) after restriction endonuclease Smal digestion of chromosomal DNA. Eighty-eight strains of S. hyicus from pigs with or without exudative epidermitis (EE) generated 16 to 26 fragments in the size range of < 1 to 485 kb, and yielded 39 different patterns. With regard to the strains from pigs with EE, PFGE patterns differed according to the country of origin. Outbreaks of EE occurring on four separate pig farms in Japan involved S. hyicus with different PFGE patterns. The PFGE patterns shown by S. hyicus strains from 4 kinds of animals were compared. Strains from pigs differed from those isolated from chickens (n = 45; 18 to 24 fragments of < 1 to 425 kb), cows (n = 3; 17 to 19 fragments of < 1 to 475 kb), and goats (n = 2; 16 or 17 fragments of < 1 to 1,125 kb). Also, each of the chicken, cow and goat strains had a host-specific fragment. The results suggest that PFGE analysis might be a useful marker for distinguishing ecovars within S. hyicus. In contrast, strains of S. chromogenes from pigs and cows generated 17 to 24 fragments ranging from < 1 to 545 kb. The PFGE patterns of S. chromogenes strains were more highly conserved than those of S. hyicus. S. chromogenes strains could be distinguished from S. hyicus strains by fragments within the range of 305 to 545 kb. The results indicate that PFGE analysis could be used to distinguish between S. hyicus and S. chromogenes. We conclude that PFGE analysis is a useful tool not only for species or strain identification but also for epidemiologic or ecologic studies of S. hyicus and S. chromogenes.

Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes.

Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus 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 (70 degrees C) 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. carnaticus 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. carnaticus 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. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).

An E. coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in host DNA degradation.

An E. coli B Tab strain, EM121, was isolated that restricts T4 denA (DNA endonuclease II) mutants at 37 degrees C and above, but is permissive for wild-type T4 at all temperatures examined. At 42 degrees C, other mutants affected in nucleic acid metabolism (T4 dexA, regA and uvsW strains) are also restricted. Genetic analysis revealed that one mutation (rpoB5081) in the RNA polymerase beta subunit gene is sufficient for restricting all denA mutants. rpoB5081, together with a second linked mutation, is also required for restricting the other T4 mutants, rpoB5081 (P806S), previously shown to increase transcription termination in E. coli K-12, causes delayed synthesis of T4 late proteins and reduced DNA synthesis in denA infections. Thus, T4 DNA synthesis and gene expression are impaired by the rpoB5081 beta subunit when degradation of host DNA is reduced. Because the restricted T4 mutants are not readily distinguished from wild-type phage under typical plating conditions, EM121 is an important host for screening and mapping T4 denA mutations.

Genomic DNA fingerprinting, using pulsed-field gel electrophoresis, of Staphylococcus intermedius isolated from dogs.

OBJECTIVES: To investigate the degree of polymorphism in the pulsed-field gel electrophoresis (PFGE) patterns of Staphylococcus intermedius and to assess the value of this typing method for discriminating strains. SAMPLE POPULATION: 52 S intermedius isolates from diseased and healthy dogs. PROCEDURE: Chromosomal DNA of S intermedius was digested with restriction endonuclease Sma I, and the fragments were separated by PFGE in a 1% agarose gel. RESULTS: Sma I cut the chromosomal DNA into 15 to 23 fragments ranging from about < 1 to 679 kb, and most of the detectable fragments were < 155 kb. Nine fragments, 115, 48, 33, 26, 16, 13, 10, 4, and < 1 kb, were shared by all or almost all (> 71%) of the strains examined. Of the 52 strains, each had a different pattern. S intermedius had a high degree of restriction fragment length polymorphism. The PFGE patterns obtained for S intermedius were stable and reproducible when the strains were tested in the different experiments. CONCLUSIONS: Genomic DNA fingerprinting by PFGE is an effective technique for discriminating S intermedius strains. The PFGE method appears to be a useful molecular marker for epidemiologic or ecologic studies of S intermedius.

Identification of the Staphylococcus sciuri species group with EcoRI fragments containing rRNA sequences and description of Staphylococcus vitulus sp. nov.

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 Staphylococcus 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).