INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY Vol. 18, No. 3 July 1968 pp Copyright 1968, Iowa State University Press

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1 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY Vol. 18, No. 3 July 1968 pp Copyright 1968, Iowa State University Press THE NEOTYPE STRAIN FOR STAPHYLOCOCCUS EPIDERMIDIS(WINSL0W AND WINSLOW 1908) EVANS 1916 Rudolph Hugh and Madonna A. Ellis The George Washington University School of Medicine, Department of Microbiology, Washington, D. C. ABSTRACT. American Type Culture Collection (ATCC) strain has been proposed (Hugh and Ellis 1967) as the neotype strain for Staphylococcus epidermidis (Winslow and Winslow 1908) Evans The characteristics of this strain are presented and cornpared to the neotype of Staphylococcus aureus NCTC 8532 (ATCC 12600). ATCC has the characteristics which Winslow and Winslow attributed to the basionym, Albococcus epiderrnidis, and it conforms to the criteria proposed by the Subcommittee on Taxonomy of Staphylococcus and Micrococcus of the International Committee on Nomenclature of Bacteria, for the recognition of Staphylococcus epidermidis (1965). This paper constitutes the designation of ATCC as the neotype strain of Staphylococcus epidermidis under Rule 9d (1) Note 2(c) of the Bacteriological Code as approved at the 1967 Moscow Cong re s s. INTRODUCTION The Subcommittee on Taxonomy of Staphylococcus and Micrococcus of the International Committee on Nomenclature of Bacteria (ICNB), which met in Brno, Czechoslovakia, in 1964 proposed for recognition two species of staphylococci. They are Staphylococcus aureus Rosenbach 1884 and Staphylococcus epidermidis (Winslow and Winslow 1908) Evans A neotype for?. aureus was designated in 1958 by the Judicial Commission and the InternationalCommittee

2 232 INTERNATIONAL JOURNAL on Bacteriological Nomenclature (Opinion 17). Winslow and Winslow's strains of Albococcus epidermidis are no longer available for comparative studies and a neotype for this species has not been proposed. Hugh and Ellis (1967) proposed ATCC strain as the neotype for Staphylococcus epidermidis. This report describes ATCC in greater detail and serves to designate, according to the International Code of Nomenclature of Bacteria Rule 9d(l) Note 2(c), this strain as the neotype. The binary combination, Albococcus epidermidis, was first used and the species described by Winslow and Winslow (1908, 200) in their book "The Systematic Relationships of the Coccaceae." They credited Gordon (1905) with the first adequate description of this organism and recommended that the combination Staphylococcus epide rmidi s albu s be modified in accordance with the rules of nomenclature. The following proposal and description are found on pages of "The Systematic Relationships of the Coccaceae. I' "We propose, therefore, the name Alb. epidermidis (Gordon) for this type, characterizinst according to Gordon's description as follows: 2. ALB. EPIDERMIDIS (Gordon) Winslow. A parasitic coccus living normally on the surface animal body. Occurs singly, or in pairs, or irregular groups. Generally stains by Gram. Good to abundant, white surface growth. Moderate acid production in lactose, maltose, and glycerin media. Nitrates and neutral red reduced. Gelatin liquefied, somewhat slowly. Mannite not fermented. 'I It appears that Alice Evans (1916, 449) was the first to transfer Albococcus epidermidis to the genus Staphylococcus. Winslow and Winslow's description was emended to - include strains "for which the haemolytic action was considered negative... but possessed this function to a slight degree. The neotype strain should agree with Winslow and Winslow's description. Agreement with the emendation proposed by Evans and by the Subcommittee on the Taxonomy of Staphylococci and Micrococci would be desirable. The subcommittee recommends that staphylococcus be distinguished from micrococcus by the ability of the staphy- lococcus to grow and produce acid anaerobically in glucose medium. The subcommittee also recommends

3 SYSTEMATIC BACTERIOLOGY 233 that?. aureus and 2. epidermidis be differentiated by the coagulase test and by anaerobic production of acid from mannitol. -- S. aureus coagulates plasma and produces acid anaerobically from mannitol in broth medium; 2. epidermidis neither coagulates plasma nor produces acid anaerobically from mannitol. MATERIALS AND METHODS Strains: Staphylococcus epidermidis ATCC (American Meat Institute Foundation (AMIF) strain Fussel, RH 2466) was isolated from the nose by Deibel. It was studied with other strains of?. epidermidis by Jones, Deibel, and Niven (1963) and was accessioned by ATCC on 8 April The neotype of Staphylococcus aureus, National Collection of Type Cultures (NCTC) 8532 (ATCC 12600, RH 2605) was used for comparative studies. These two strains were obtained from ATCC for the following study. Physiological Methods : Brain heart infusion (BHI) broth cultures, incubated for 18 to 24 hours, were iised as inocula for the various tests. All media were incubated at 30" C unless ofherwise indicated. Organisms from 24 hour infusion agar were stained by Gram's method. After 24 hours incubation at 30 C, infusion agar Petri plate cultures were stored at room temperature for a week; they were examined daily for pigmentation. Semisolid agar medium was used to detect motility. Acid production from carbohydrates and polyols was determined by the oxidative-fermentative principle of Hugh and Leifson (1953). Two basal media were compared: the medium recommended by the Subcommittee on the Taxonomy of Staphylococcus and Micrococcus of the ICNB (1965), and purple broth base (Difco 0227). One percent (w/v) glucose, mannitol, lactose, maltose, and glycerol was added to the basal media. The media were dispensed in 4 ml quantities in 13xl00mm tubes and autoclaved at 116 C for lominutes. Each staphylococcus strain was inoculated to two tubes of each medium. One of the duplicate tubes was immediately aseptically sealed with melted petrolatum. The cultures were incubated and examined daily until acid production was observed or for a maximum of thirty days. The tube method was used to test for coagulase. The test was performed by adding 0.5 ml of a 24 hour infusion broth

4 234 INTERNATIONAL JOURNAL culture to 0. 5 ml reconstituted, lyophilized rabbit plasma, followed by incubation for 18 hours at 37'C. Any degree of coagulation was interpreted as a positive test for coagulase. A strain was considered haemolytic if a zone developed around discrete colonies on 3. 5% defibrinated humen, rabbit, or sheep blood in heart infusion agar (Difco 0045). Sheep blood agar plates were incubated for 24 hours followed by refrigeration at 10 C for 24 hours to detect beta haemolysin, which is responsible for the hot-cold phenomenon (Glenny and Stevens 1935). Duplicate Petri plates of DNase test agar (Difco 0632) were heavily inoculated in 1 inch long streaks. After incubation for 24 hours and 48 hours, plates were flooded with. 1N HC1 to precipitate DNA remaining in the agar. Degradation of deoxyribonucleic acid was indicated by a clear zone around the growth (Jefbies, Holtman, and Guse 1957). A zone of clearing larger than 0.3 mm was interpreted as a positive test for the presence of extracellular deoxyribonuclease. Production of urease was estimated in Christensen urea medium (1946). Growth harvested from an infusion agar slant was transferred to urea slants and incubated at 30 C for 48 hours. Each strain was inoculated to Christensen medium without urea as a control. Swann (1954) 40% bile-aesculin agar was used to test the ability of staphylococci to hydrolyse aesculin and grow in the presence of bile. McConkey (1905) bile-salt neutral red lactose agar was used to test for neutral red reduction. The medium consists of 0. 5% sodium taurocholate, 2% peptone, 170 lactose, 0.0 1q0 neutral red, and agar. S. epidermidis growth is dark crimson and_s. aureus growthis yellow according to Andrews and Gordon (1907). The following methods were described by Hugh and Reese (1967): nitrate reduction, catalase production, indophenol oxida s e product ion, gelatin 1 iquef action, hydrogen sulfide production, malonate utilization, phenylalanine deamination, growth in potassium cyanide broth, indole production, methyl red test, acetyl-methyl- carbinol production, and citrate utilization. Utilization of 1- lysine, 1- arginine, and l-ornithine was determined in open and petrolatum sealed tubes of Mbller's medium. The basal medium, unfortified with these amino acids, was used as a control.

5 SYSTEMATIC BACTERIOLOGY 235 RESULTS Staphylococcus epidermidis ATCC has the following characteristics. Morphology: Gram-positive cocci, u in diameter, occurring singly, in pairs, or in clusters. It is asporogenous and nonmotile. Physiology: This facultative organism produces a dense turbidity in neutral peptone broth in hours at temperatures of 22"C, 37"C, and 42 C. Colonies on infusion agar are white, entire, smooth, and glistening. It grows well in neutral BHI broth containing 10% sodium chloride. There is no growth in Mbller potassium cyanide broth. Comparable results were obtained in oxidative-fermentative studies using the JAMS Subcommittee medium and purple broth medium. However, indicator color change in purple broth medium could be detected within 24 hours whereas a similar color change in committee medium required between 24 and 48 hours of incubation. ATCC grew and produced acid in open and sealed tubes of media containing 1% glucose, lactose, and maltose. There was good growth in the open tubes of mannitol and glycerol media, but no growth in the duplicate sealed tubes containing these substrates. Acid was produced from glycerol in the open tube, but not from mannitol. Catalase, nitratase, gelatinase, urease, and acetylmethyl-carbinol were produced; neutral red was reduced. The organism grew and produced arginine dihydrolase in open and sealed tubes of Mbller's medium containing arginine. Methyl red indicator, when added to a 5-day buffered glucose peptone broth culture, turned orange rather than red or yellow. The organism grew in the presence of 4070 bile. Milk was coagulated after 11 days, and growth occurred on potato after two days, C oagula s e, extra c ellula r deoxy r ibonu c le a s e, nit r ita s e, indole, indophenol oxidase, hydrogen sulfide, phenylalanine deaminase, and lysine and ornithine decarboxylases were not produced. Citrate and malonate were not utilized. Aesculin was not hydrolyzed. The guanine plus cytosine ratio was 34.6 moles percent. Enterotoxins A, By C, and D were not serologically detectable. ATCC was not lysed by the following Staphylococcus aureus phages: 3B/3C/6/7/29/42B/42E/44-3/ m / 7 m 1/83/187/UC18. Biotin is required by this strain for aerobic and anaerobic growth; uracil is also re-

6 2 36 INTERNATIONAL JOURNAL quired for anaerobic growth according to Jones, Deibel, and Niven ( 19 63). The characteristics of the neotype of S. epidermidis and of the neotype of S. aureus are compared in Table Table 1. Characteristics of the neotype of Staphylococcus epidermldls ATCC and of the = o w o f Staphylococcus aureus ATCC Glucose open sealed d-mannitol open.sealed Lactose open sealed naltose open sealed Glycerol open sealed Growth in BHI at 22O, 31, and 42 C Catalase Indophenol oxldase Coagulase Lip och rome Extracellular deovyrlbonuclease Baemolysls of rabbit, sheep, and human erythrocytes Nltratase Nitri ease Reduction of neutral red Gelatin hydrol ysls Urease, Chrlstensen Indol e Methyl red Acetyl-methyl-corbinol Cltrate, Simmons Phenylalanine deamlnase Malonate Hydrogen sulfide production, K1 i gler 1-lysine decarboxylase 1-arglnlne dlhydrolase 1-ornithine decarboxylase Growth on 40% bile agar Hydrolysis of aesculln Growth In 107. sodium chlorlde broth Coagulation of milk Growth on potato Enterotoxlns A, B, C, and D Guanine plus cytosine ratio Phage lysls pattern ** S. eplde rmidis NG 2 2 NG orange % 2. aureus i -k NG i U K * ** KEY: Posltlve test; subscripts Indicate number of days required to become positive - Negative test NG No growth WK Weak reaction, slowly produced * Sllvestrl, L. G. and L. R. Htll, ** Phage set: 3~/3C/6/1/29/42B/42E/44A/47/53/54/75/77/00/01/03/~0l/UC~~. ** Phane pattern: 6/1/42B142~/41/53/54~15/11~~1/~3~UC18.

7 SYSTEMATIC BACTERIOLOGY 2 37 DISC USSION Numerous freshly isolated clinical strains and reference strains of staphylococci were studied in search of a suitable strain which could be used as a neotype for S. epidermidis. ATCC was found to be a suitable candtdate. Strain ATCC has the characteristics which Winslow and Winslow attributed to the species Albococcus epidermidis. The organism is nonhaemolytic in accordaxe with Evans' emendation of the description of Albococcus epidermidis. It does not produce coagulase, nor does it produce acid anaerobically from mannitol; thus it meets the emendations of the description of 2. epidermidis proposed by the Subcommittee on Taxonomy of Staphylococcus and Micrococcus. Strain ATCC is established, according to Rule 9d( 1) Note 2(c) of the International Code of Nomenclature of Bacteria, as the neotype of Staphylococcus epidermidis (Winslow and Winslow 1908) Evans 1916; it also serves as the neotype for Albococcus epidermidis. ACKNOWLEDGMENTS The authors are indebted to E. R. Kennedy of Catholic University, Washington, D. C., for determining the guanine plus cytosine ratio; to E.P. Casman of the Food and Drug Administration, Washington, D. C., for testing for enterotoxins A, B, C, and D; and to C. Zierdt of the National Institutes of Health, Bethesda, Maryland, for determining the lysotypes and for supplying numerous clinical strains. REFERENCES Andrewes, F. W. and M.H. Gordon Report on the biological characters of the staphylococci pathogenic for man. 35th Ann. Rept. of the LOC. Gov. Board Containing Rept. Med. Off. for the Years Supple, Baird-Parker, A. C Subcommittee on Taxonomy of Staphylococci and Micrococci Minutes of fir st Meeting (5th-6th October, 1964). Intl. Bull. Bact. Noman. Tax. 15: Subcommittee on Taxonomy of Staphylococci and Micrococci. Recommendations, Intl. Bull. Bact. Nomen. Tax. 15:

8 2 38 INTERNATIONAL JOURNAL Buchanan, R. E Conservation of the generic name Staphylococcus Ro senbach, designation of Staphylococcus aureus Rosenbach as the nomenclatural type of the genus Staphylococcus Rosenbach, and the designation of a neotype culture of Staphylococcus aureus Rosenbach. Opinion 17. Intl. Bull. Bact. Nomen. Tax. 8:138. Christensen, W. B Urea decompositionas a means of differentiating Proteus, and paracolon cultures from each other and from Salmonella and Shigella types. J. Bact. 52: Evans, A. C The bacteria of milk freshly drawn from normal udders. J. Inf. Dis. 18: Glenny, A. T. and M. F. Stevens Staphylococcus toxins and antitoxins. J. Path. and Bact. 40: Gordon, M.H Report of some charactzs by which various Streptococci and Staphylococci may be differentiated and identified. 33rd Ann. Rept. LOC. Gov. Board Containing the Rept. Med. Off. for the Years Supple Hugh, R. and E. Leifson The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J. Bact. - 66: and M.A. Ellis The neotype strain for Staphylococcus epidermidis (Winslow and Winslow 1908) Evans Bact. Proc. her. SOC. Microbiol. Abstracts of the 67th meeting, New York, p. 62. and R. Reese Designation of the type strain for Bacterium anitratum Schaub and Hauber Intl. J. System. Bact. 17: Jeffries, C. D., D. FFHoltman and D. G. Guse Rapid method for determining the activity of microorganisms on nucleic acids. J. Bact. 73: Jones, D., R.H. Deibel and7. F. Niven, Jr Identity of Staphylococcus epidermidis. J. Bact. 85: MacConkey, A Lactose-fermenting bgcteria in faeces. J. Hyg. 5: Rosenbach, F. J Mikro-organismen bei den Wund- Infections-Krankheiten des Menschen. J. F. Bergmann. Weisbaden. pp Silvestri, L. G. and L. R. Hill Agreement between deoxyribonucleic acid base composition and taxonomic classification of gram-positive cocci: J. Bact. - 90:

9 SYSTEMATIC BACTERIOLOGY 2 39 Swann, A The use of a bile-aesculin medium and of Maxted' s technique of Lancefield grouping in the identification of enterococci (Group D Streptococci). J. Clin. Path. 7~ Winslow,-C. E. A. and A. R. Winslow The Systematic Relationships of the Coccaceae. John Wiley and Sons, New York. pp