Characterization and Identification of Coagulase-negative, Heat-stable Deoxyribonuclease-positive Staphylococci (original) (raw)

1978, Journal of General Microbiology

Various characteristics of 13 coagulase-negative, weakly heat-stable deoxyribonucleasepositive staphylococci from human, veterinary and food sources were determined in an effort to identify them. Nine of the isolates were identified as coagulase-negative Staphylococcus aureus (2), Staphylococcus xylosus (2), Staphylococcus simulans (3), Staphylococcus capitis (1) and Staphylococcus sciuri subsp. lentus (1); the other four isolates, from food and veterinary sources, could not be identified as currently accepted or proposed species. Teichoic acid and peptidoglycan compositions were used as key taxonomic characteristics. The determination of heat-stable deoxyribonuclease activity can be useful to detect coagulase-negative S. aureus strains. However, this activity also appears to: be present in strains of other staphylococcal species. 276 J, L. GRAMOLI A N D €3. J. WILKINSON METHODS Taxonomic schemes. The schemes used for evaluation of the organisms were those of Baird-Parker (1974), which recognize S. aureus, S. epidermidis and S. saprophyticus plus the S. aureus biotype characteristics described by Hajek & MarShlek (1971). For further resolution, the coagulase-negative strains were evaluated according to the criteria of Kloos & Schliefer (1975a, b), Kloos et a/. (1976), Kloos, Schliefer & Smith (1976) and Schliefer & Kloos (1975), as recommended by the International Subcommittee (1976). Cultures. The test organisms, kindly provided by workers at several institutions, were from a variety of sources (Table 1). Isolates were maintained on BBL Brain Heart Infusion Agar (BHI agar) at 4 "C. Unless stated otherwise, all inocula were derived from overnight growth in 10 ml BBL Brain Heart Infusion Broth (BHI broth), and tests were performed at 37 "C. Cellular and coZonial morphology. Gram-stained preparations were examined under a light microscope for colour reaction, cell size and arrangement. Motility was determined with hanging drop preparations observed under a phase-contrast microscope. Colonial morphology was observed after growth on P agar for 5 d (Kloos, Tornabene & Schliefer, 1974). Respiratory system. Catalase production (Baird-Parker, 1963) and a modified benzidine test (Deibel & Evans, 1960) were determined simultaneously after overnight growth on 1 % (w/v) glucose agar. Oxidase was assayed as described by Kovacs (1956). Growth characteristics. Growth at 45 "C was determined after 24 and 48 h, and anaerobic growth in thioglycollate broth was determined according to Evans & Kloos (1972). Growth in the presence of lysozyme and salt tolerance were determined by the methods of Kloos et a/. (1974). Growth in crystal violet was determined by streaking cultures on Crystal Violet Agar (Difco) followed by examination after 24 and 48 h. Enzymic activities. Coagulase production was determined by the tube assay using fresh rabbit (Difco), porcine (gift of Geo. A. Hormel Co., Austin, Minnesota, U.S.A.) and human plasmas. Rabbit plasma was used as recommended by the manufacturer; porcine plasma was used undiluted. Human plasma was prepared from fresh pooled blood (gift of Donor Room, University of Minnesota Hospitals, Minneapolis, Minnesota, U.S.A.) collected in EDTA Vacutainers. Blood was centrifuged to remove the red cells, diluted 1 : 3 with distilled water, filter-sterilized and frozen in sterile test-tubes prior to use. The tube assay was performed by adding 0.25 ml BHI broth culture to 0.5 ml plasma in sterile, capped 12 x 75 mm tubes which were examined for clotting after 1,2, 3 , 4 and 24 h, and scored as 0 to 4 + (Turner & Schwartz, 1958). Clumping factor was determined with rabbit plasma (Difco) according to the manufacturer's instructions. Fibrinolysin was determined by a plate method (Hinton & Orr, 1957) using 15 % undiluted, filter-sterilized rabbit plasma incorporated into tempered 3HI agar. Soy Bean Trypsin Inhibitor (1 %, w/v; Sigma) was