Stanislav Rosypal

Identification of bacteriophage types and their carriage in Staphylococcus aureus.

Summary.Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified.

Genomic Variability of Staphylococcus aureus and the Other Coagulase-Positive Staphylococcus Species Estimated by Macrorestriction Analysis Using Pulsed-Field Gel Electrophoresis

The genomic DNAs of 95 culture collection and hospital Staphylococcus aureus subsp. aureus strains of various origins, as well as the genomic DNAs of other coagulase-positive Staphylococcus species, were cleaved with restriction endonuclease SmaI and subjected to pulsed-field gel electrophoresis. The levels of similarity of the SmaI restriction patterns of the S. aureus subsp. aureus strains varied from 30 to 100%, which is considered characteristic of this species; thus, these organisms belonged to the same species restriction group. Within this range of similarity values 13 S. aureus intraspecies restriction groups were identified, and each group consisted of strains whose levels of similarity ranged from 65 to 100%. S. aureus subsp. aureus CCM 885T (T = type strain) belonged to the major intraspecies restriction group that comprised 39% of the S. aureus strains which we studied. The strains of the other coagulase-positive staphylococci, including Staphylococcus aureus subsp. anaerobius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus schleiferi subsp. coagulans, clustered with their type strains in separate restriction groups. S. aureus subsp. aureus exhibited almost no similarity to these species. We found 44-kb SmaI fragments in all of the S. aureus subsp. aureus and S. aureus subsp. anaerobius strains studied, and these fragments are considered characteristic of the species S. aureus. The high level of homology of these fragments was confirmed by the results of DNA hybridization experiments in which we used representatives of individual intraspecies restriction groups. Of the other staphylococci studied, only Staphylococcus epidermidis and one strain of S. hyicus contained these fragments. However, the levels of homology between these fragments and the fragments of S. aureus were found to be very low.

Molecular typing of exfoliative toxin-producing Staphylococcus aureus strains involved in epidermolytic infections

Genotyping of sixteen exfoliative toxin-producing (ET-positive) strains of Staphylococcus aureus isolated in maternity units of two distant hospitals was accomplished by PFGE, ribotyping, PCR ribotyping, and prophage carriage. Three strains secreted combined ETA + ETB, and the remaining produced ETA and enterotoxin C, or TSST-1. The comparison of various genomic profiles resulted in the identification of nine genotypes. The presence of one prevailing genotype was demonstrated in each hospital. Evidence was given that the outbreak-related ET-positive strains causing the skin disease pemphigus neonatorum disseminated in both the hospitals did not originate from a single source or a common ancestor.

Complex genomic and phenotypic characterization of the related species Staphylococcus carnosus and Staphylococcus piscifermentans

On the basis of numerical analysis of 100 phenotypic features, the strains of two species, Staphylococcus carnosus and Staphylococcus piscifermentans, were differentiated into two separate phenons corresponding with the macrorestriction patterns of their genomic DNA, as well as with the results of ribotyping and PCR amplification of enterobacterial repetitive intergenic consensus sequences. One of the S. carnosus strains, the F-2 strain, was shown to be marginal, exhibiting the lowest genomic and phenotypic similarity to the S. carnosus type strain DSM 20501T. Two of the strains studied (strains S. carnosus SK 06 and S. piscifermentans SK 05) were phenotypically convergent, forming a separate phenon. They were phenotypically similar, even though the genomic DNA of one of them was homologous with that of the S. carnosus type strain, whereas that of the other was homologous with the genomic DNA of the S. piscifermentans type strain. In such cases, fingerprinting methods (particularly macrorestriction analysis and ribotyping) served as important correctives, as they allow phenotypically convergent strains to be distinguished on the basis of their genomic profiles. The results of this paper support the proposal for the new species Staphylococcus condimenti as well as the new subspecies Staphylococcus carnosus subsp. utilis.

Deoxyribonucleic acid base composition of some micrococci and sarcinae

AbstractThe strains designated in this paper asMicrococcus lysodeikticus, M. sodonensis, M. flavus, Sarcina flava, S. pelagia, S. variabilis, S. marginata, S. subflava, S. citrea, S. lutea andStaphylococcus afermentans have similar DNA base compositions. The mole % GC (guanine plus cytosine) contents in DNA of these strains ranged from 71.8 to 73.3 as calculated from the denaturation temperature (Tm). They may be, therefore, closely related. However, at variance with Kocur and Martinec (1962) they do not seem to be identical withMicrococcus luteus (Schroeter 1872) Cohn 1872, because the neotype culture of the latter species has a different content of guanine and cytosine in its DNA (GC=66.3%). Sarcina aurantiaca, Micrococcus dentrificans andM. luteus have a similar DNA base composition. However, they are not identical as they differ from each other in several physiological characters.In the strains designated asStaphylococcus roseus andSarcina erythromyxa the content of GC varies within the range 72–72.8%. These species do not differ from each other physiologically. They form a pink pigment, reduce nitrates, do not hydrolyze casein and gelatin, and do not produce urease. They seem, therefore, to be identical, which confirms the conclusion of Kocur and Martinec (1962) who designated them asMicrococcus roseus Flügge 1886. Micrococcus conglomeratus differs significantly in DNA base composition from almost all strains of the groupM. lysodeikticus—Staphylococcus afermentans, also fromMicrococcus luteus, M. roseus andM. denitrificans. It differs fromSarcina aurantiaca only physiologically.

Deoxyribonucleic acid base composition and taxonomy of violet-pigmented cocci

The content of guanine and cytosine in DNA of the violet-pigmented micrococci designated asStaphylococcus flavocyaneus andMicrococcus flavocyaneus varies within the range of 70.8 – 72.0%. These species have similar deoxyribonucleic acid base compositions and do not differ physiologically and morphologically: they both produce yellow and violet pigments, hydrolyse gelatin and casein, reduce nitrates and do not form lipase. Therefore we consider them in accordance with Kocur and Martinec (1962, 1963) identical. They do not, however, seem to be identical withMicrococcus luteus (Schroeter, 1872) Cohn 1872 because the content of guanine and cytosine in DNA of the neotype culture of this species was found to be 66.3%.Micrococcus luteus differs from the violet pigmented micrococci also physiologically. It does not produce violet pigment, does not hydrolyse gelatin and casein and does not produce urease. For the violet pigmented strainMicrococcus cyaneus the use of the original designation is recommended:Micrococcus cyaneus (Schroeter) Cohn 1872, as it differs from the other violet cocci not only physiologically — it does not produce yellow pigment, oxidises mannitol, dulcitol and sorbitol, produces lipase and does not hydrolyse casein — but also in its DNA base composition.