FEMS Microbiology Letters 3 (1978) 51-56 © Copyright Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press 51 RELATIONSHIPS AMONG SOME COAGULASE-NEGATIVE STAPHYLOCOCCI BASED UPON DEOXYRIBONUCLEIC ACID RE-ASSOCIATION G. PULVERER, M. MORDARSKI, A. TKACZ, K. SZYBA, P. HECZKO and M. GOODFELLOW Institute of Hygiene, University of Cologne, West Germany, Institute of lmmunology and Experimental Therapy, Wrocfaw, Poland, Institute of Microbiology, MedicalAcademy, Krak6w, Polandand Department of Microbiology, University of Newcastle, England Received 9 November 1977 1. Introduction Although aerobic, catalase-positive, coagulase- negative staphylococci are an increasing source of human infection [1,2] there is little agreement on their classification and identification [3]. Schleifer and Kocur [4] proposed a classification based upon wall structure and lactic acid metabolism and, in sub- sequent studies, recognized nine species using additional criteria [5,6]. In contrast, the ICSB Subcommittee on the Taxonomy of Staphylococci and Micrococci [7] only recognize Staphylococcus aureus, S. epidermidis and S. saprophyticus. DNA re-association assays have proved invaluable in establishing the relationships between closely related bacteria whose classification is confused and complex [8-10]. The aim of the present study was to determine the relationships between representatives of the nine coagulase-negative taxa recognized by Kloos and Schleifer [11]. 2. Materials and Methods 2.1. Test strains and growth conditions Details of the strains and their sources are given in Table 1. All strains were maintained routinely at 4°C on nutrient agar supplemented with glucose (2% w/v). For DNA extraction, organisms were grown in submerged culture on a reciprocal shaker (160 strokes per minute) for 8 h at 37°C in the medium described above (minus agar). Flasks of 750 ml were inoculated with cells from the log phase of batch culture and organisms harvested at the late log phase. Strains which showed resistance to lysis had penicillin (1 to 10 units/ml) added to the culture medium 2 h before harvesting. Cultures were checked for purity, harvested by centrifugation, washed in 0.15 M saline-0.1 M ethylenediaminotetraacetate (EDTA) pH 8.0, and stored at -20°C. 2.2. Extraction of DNA Harvested organisms (1 g wet weight) were sus- pended in 1 ml buffer (0.03 M Tris-HC1, 0.1 M EDTA, pH 8.0) containing 5 mg lysozyme and 1 mg pronase and incubated for 18 h at 37"C. After lysis, sodium dodecyl sulphate (2% w/v) was added and the suspension heated at 60°C for 30 to 60 min. Lyso- zyme resistant strains, cultivated in the presence of penicillin, were treated with staphylolysin (5 mg/g; Schwartz Mann, Dickinson Co., Orangeburg, U.S.A.) for 2 h at 37°C prior to lysozyme treatment. The DNA was isolated using a phenol method [12] adapted from Saito and Miura [13]. The purity of DNA preparations was assessed on the basis of the follow- ing: absorbance at E26o/2so and E2ao/2so; reaction for protein with Folin reagent, and diffusion tests with concanavalin A for polysacchaddes [14]. In addition to the routine orcinol test for ribonucleic acid, the samples were checked using the more sen- sitive method described by Chov [15]. The purified DNA was stored at 50C in 0.1 X SSC (SSC : 0.15 M NaC1, 0.015 M trisodium citrate) containing a few drops of chloroform. Downloaded from https://academic.oup.com/femsle/article-abstract/3/1/51/594882 by guest on 30 May 2020