INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1975, p. 62-79 Vol. 25, No. 1 Copyright 0 1975 International Association of Microbiological Societies Printed in U.S.A.

Isolation and Characterization of Staphylococci from Human Skin 11. Descriptions of Four New Species: warneri, Staphylococcus capitis, Staphylococcus horninis, and Staphylococcus simul ans

WESLEY E. KLOOS AND KARL H. SCHLEIFER Department of Genetics, North Carolina State University, Raleigh, North Carolina 27607, and Lehrstuhl fur Mikrobiologie, Universittit Mlinchen, 8 Munich 19, Germany

Staphylococci were isolated from the skins of people living in North Carolina and New Jersey and were studied in an attempt to resolve their natural relationships. As a result of this study, four new species are proposed in this paper: Staphylococcus warneri, S. capitis, S. hominis, and S. simulans. The type strains of these species are ATCC 27836, ATCC 27840, ATCC 27844, and ATCC 27848, respectively. The new species were established on the basis of a variety of morphological, physiological, biochemical, and antibiotic characters. Cell wall composition was particularly useful in resolving species and correlated well with other characters. Characteristic pigment production was useful in distinguishing several of the different species. A summary of the character variation found in the species and a scheme for the classification of human cutaneous staphylococci are included in this paper. The predominant staphylococci found on human skin were S. epidermidis and S. horninis.

A brief account of the classification of staphy- paper is concerned with the classification of four lococci and their occurrence on human skin is additional new species of human cutaneous presented in the accompanying paper (15).Most staphylococci; it also contains summary data on of the recent attempts at classifying cutaneous species character variation, occurrence of spe- staphylococci have utilized the Baird-Parker cies on human skin, and a classification scheme. scheme (2-4, 10, 11). This scheme involves the use of a small number of readily definable key MATERIALS AND METHODS characters to identify isolates at the species and Bacterial strains. Staphylococci were isolated subgroup levels. However, because of the small from the healthy skins of two groups of people. One number of characters used and the general lack group was composed of 20 people living in Raleigh, of information available on species character N.C., who were sampled once each month for 6 to 13 variation, the scheme is limited in accuracy and months. The second group was composed of 20 people living in New Jersey, who were sampled once during to the number of species and/or subspecies that the winter. Samples were taken from two separate it can resolve. sites on the forehead and one site from one cheek, one The specific purpose of our investigation was anterior and external nare, chin, each axilla, each to identify the species of cutaneous staphylo- upper and lower arm, and each upper and lower leg. cocci of humans. To attempt this, we reevalu- Randomly selected representative strains of the ated existing taxonomic criteria, explored the new species proposed in this paper are listed in Tables use of additional characters, and estimated 1, 2, 3, and 4, respectively. (Additional strains that species character variation in cutaneous popu- were analyzed for only certain of the characters listed lations. in the tables are too numerous to be cited here, but The results of studies on cutaneous strains of character information on these strains can be made available upon request to the authors.) Staphylococcus epidermidis (Winslow and Procedures for isolating staphylococci. Sampling Winslow) Evans 1916, S. saprophyticus Fair- techniques and the composition of the isolation me- brother 1940, and three new species, s. cohnii, dium have been described previously (6). S. haemolyticus, and S. xylosus, are presented Culture conditions. Culture conditions were simi- in the accompanying paper (15). The present lar to those previously described for the study of cutaneous micrococci (6). ' Paper no. 4401 of the Journal Series of the North Carolina Character determinations. Procedures for deter- Agricultural Experiment Station, Raleigh, N.C. 27607. mining deoxyribonucleic acid (DNA) base composi- 62 VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 63 tion, colony morphology and pigment, cell morphol- colonies or was a slight yellowish tint in the ogy, motility, aerobic and anaerobic growth in thio- center of colonies. Twenty percent of the strains glycolate, salt tolerance, growth temperature, cata- were unpigmented and had gray-white colonies. lase and benzidine activities, acetylmethylcarbinol Growth occurred in both the aerobic and production, nitrate reduction, carbohydrate reac- tions, and susceptibility to various antibiotics, lyso- anaerobic portions of the thioglycolate medium, zyme, and lysostaphin were similar to those described indicating a facultatively anaerobic capability. previously for micrococci (6).The minimal inhibitory Growth in the anaerobic portion was usually concentrations (MIC) of antibiotics and lysostaphin uniformly dense; however, 5% of the strains are as denoted throughout the text. demonstrated less growth, and 8% had only , hemolysis, deoxyribonuclease (DNase), individual colonies in the deeper, more anaero- phosphatase, and bacteriolytic activities were deter- bic portion of this medium. Fifteen selected mined by procedures described in the companion strains demonstrated fermentation of glucose paper (15). by lowering the pH of a yeast extract-glucose Procedures for making cell wall hydrolysates and determining peptidoglycan type, teichoic acid, config- broth from 6.8 to 4.7-5.0 after anaerobic incuba- uration of lactic acid, and the anaerobic fermentation tion. These strains produced nearly equal of glucose have also been described previously (16). amounts of D- and L-lactic acid from glucose. All strains grew well at NaCl concentrations RESULTS AND DISCUSSION up to 10%. Sixty-two percent of the strains grew poorly or failed to grow at a NaCl concentration Characterization of Staphylococcus of 15%. The optimal growth temperature range species. The characteristics of the S. aureus was 25 to 40 C. Eighty-four percent of the and other coagulase-positive strains isolated strains grew poorly or failed to grow at 15 C, but from human skin in this study were similar to 92% grew well at 45 C. those reported previously for various strains of All strains had either weak or moderate these organisms (3, 14, 16).Descriptions of four catalase activity and were positive for the new Staphylococcus species isolated from benzidine test. All failed to produce . human skin are as follows. Ninety-eight percent of the strains demon- (i) S. warneri sp. nov. (war. ner’i. M.L. gen. n. strated weak or no hemolysin activity, 95% warneri of Warner; named for Arthur Warner, demonstrated weak or no DNase activity, 74% Jr., from whom this organism was originally failed to reduce nitrates, and 78% failed to isolated.) Several strains tentatively identified demonstrate phosphatase activity; there was as members of this species were previously variable bacteriolytic activity. All produced designated S. epidernidis (CCM 2445; refer- acetylmethylcarbinol. ence 13) or s. pyogenes albus (ATCC 155; All strains produced acid aerobically from reference 16). Several strains, including SCH 5, glucose, fructose, sucrose, trehalose, and glyc- SCH 7, and SCH 13, were isolated by one of us erol. Seventy-eight percent of the strains pro- (K.H.S.) from dust and, based on their cell wall duced acid from maltose slowly, and 63% pro- composition and the configuration of the lactic duced acid from mannitol. Fifty percent of the acid they produced, were placed together with strains produced acid from ribose, and 24% the above-mentioned strains in Staphylococcus produced acid from galactose. Only 2% of the group I1 A3 (16). The following description of S. strains produced acid from mannose, and 11% warneri is based on a total of 38 strains, unless produced acid from lactose or turanose. All noted otherwise. failed to produce acid from rhamnose, xylose, Cells were gram-positive cocci, 0.5 to 1.2 pm arabinose, gentiobiose, cellobiose, melezitose, in diameter, nonmotile and nonsporeforming, xylitol, sorbitol, inositol, salicin, adonitol, dul- occurring predominantly in pairs and singly, citol, arabitol, erythritol, erythrose, raffinose, occasionally in tetrads. Only 12% of the strains melibiose, fucose, tagatose, lyxose, or sorbose. studied had approximately equal numbers of All strains were resistant to lysozyme and pairs and tetrads. were slightly resistant to lysostaphin (MIC, 200 Colonies on a P agar medium (6) were raised, pg/ml). All were susceptible to erythromycin usually had a slightly elevated center, and were (MIC, 0.4 to 1.6 pg/ml), tetracycline (MIC, 0.4 circular, entire, smooth, glistening, and opaque. to 0.8 pg/ml), and (MIC, 0.025 to 0.1 They were small and had a diameter of 3 to 5 pg/ml). Seventy-eight percent of the strains mm. The consistency of colonies or culture were susceptible to penicillin G (MIC, 0.012 to streaks was usually sticky. Eighty percent of 0.025 pg/ml), and 98% were susceptible or the strains were pigmented to some extent. slightly resistant to streptomycin (MIC, 6.2 to Pigment occurred as a characteristic bright 25 ,ug/ml). yellow-orange or yellow ring around the edge of Fifteen selected strains studied contained 64 KLOOS AND SCHLEIFER INT. J. SYST.BACTERIOL. peptidoglycan of the type L-LYs-G~Y~.~-~.~,L- staphylococci primarily on the basis of a combi- Sero.6-l.4.Their cell wall teichoic acid contained nation of its colony morphology, carbohydrate glycerol and glucose. reaction pattern, cell wall composition, lactic The guanine plus cytosine (G+C) content of acid configuration, and the lack of nitrate the DNA, as determined in six strains, was 34.7 reduction. There are also some significant dif- 0.2 mol%. ferences between S. warneri and certain other Some variable characters and the G+C con- species in the parameters of pigment pattern, tent of the DNA of some representative strains cell arrangement, anaerobic growth pattern in of S. warneri are given in Table 1. thioglycolate, growth at different NaCl concen- Strain ATCC 27836 (originally designated trations and temperature extremes, coagulase AW 25) is the type strain of S. warneri. A activity, hemolysis, acetylmethylcarbinol pro- description of this strain follows. duction, and phosphatase, DNase, and bacteri- Cells: Spheres, 0.8 to 1.0 pm in diameter, olytic activities. Several properties of's. warneri occurring ill pairs, tetrads, and singly. Non- overlap with those of S. huemolyticus, S. epi- motile and nonsporeforming. Gram positive. dermidis, and S. horninis, suggesting a rela- Agar colonies: Circular, entire, 3.0 to 3.5 mm tively close relationship between these species. in diameter, raised with slightly elevated ten- (ii) S. cupitis sp. nov. (ca'pi. tis. L. noun ca- ter, smooth with glistening surface, sticky con- put head; L. gen. noun capitis of the head; per- sistency. Originally opaque gray-white with yel- taining to that part of the human body where low-orange edge; subcultures have lost pigment cutaneous populations of this species are usu- and are now gray-white. ally the largest and most frequent.) Gordon (5) Catalase and benzidine tests are positive. in 1905 reported an unusual Staphylococcus Chemoorganotroph; metabolism is respira- that he considered to be different from the tory. commonly recognized Staphylococcus epider- Facultatively anaerobic. midis albus. Strains of this unusual type were Temperature relationships: Good growth at different from those of the above-mentioned 45 C, poor growth at 15 C. species in that they failed to produce acid from Growth on NaCl agar: Good growth with 10% maltose or lactose but produced acid from NaC1, no growth with 15%. mannitol. They also were predominately found Coagulases not produced. on the scalp as compared to the hands, face, or Phosphatase not produced. forearm. Although the few characters studied Acetylmethylcarbinol produced. by Gordon will not conclusively identify his Nitrates not reduced. isolate as belonging to S. capitis, nevertheless, Hemolysins: Weak hemolysis of bovine, they are consistent with those of S. capitis and, sheep, and human blood. hence, there is a reasonably good possibility DNase activity is weak. that he was observing this species. A later study Acid produced aerobically from glucose, fruc- by Marples et al. (9) on the aerobic microflora of tose, ribose, maltose, sucrose, trehalose, man- the scalp determined that the normal scalp nitoll, and glycerol. No acid from galactose, cocci are predominately micrococci of type 3. mannose, rhamnose, xylose, arabinose, lactose, However, according to Baird-Parker's scheme gentiobiose, cellobiose, melezitose, xylitol, sop- (3), the characteristics of type 3 micrococci are bitol, inositol, salicin, adonitol, dulcitol, arabi- not those of S. capitis. Hence, there is some tol, erythritol, erythrose, raffinose, melibiose, uncertainty concerning earlier reports of possi- fucose, tagatose, lyxose, or sorbose. ble encounters with this species. The following Antibiotic susceptibilities: Slightly resistant description of S. capitis is based on a total of 47 to lysostaphin (MIC, 200 pg/ml) and strep- strains, unless noted otherwise. tomycin (MIC, 12.5 pg/ml). Susceptible to peni- Cells were gram-positive cocci, 0.8 to 1.2 pm cillin G (MIC, 0.025 pg/ml), erythromycin in diameter, nonmotile and nonsporeforming, (MIC, 1.6 pg/ml), tetracycline (MIC, 0.8 pg/ occurring predominantly in pairs and tetrads. ml), and novobiocin (MIC, 0.025 pg/ml). Resist- Colonies on a P agar medium (6) were slightly ant to lysozyme. convex, circular, entire, smooth, slightly dull to Cell wall peptidoglycan: L-LYs-G~Y,.,, L- glistening, opaque and usually a distinctive Ser,.,. chalk white (unpigmented). They were very Cell wall teichoic acid composition: Glycerol small and had a diameter of 1 to 3 mm. and glucose. Growth occurred in both the aerobic and G-tC content of the DNA: 34.2 mol%. anaerobic portions of the thioglycolate medium, S. warneri can be distinguished from all other indicating.- a facult ativelv anaerobic caDa b ilitv. VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 65

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Growth in the anaerobic portion was usually of teichoic acid, and cell wall hydrolysates had uniforirnly dense; however, 11% of the strains to be concentrated for the detection of free demonstrated less growth, and 21% had only glycerol. Cell walls also contained additional individual colonies in the deeper, more anaero- glutamic acid. In partial acid hydrolysates, bic portion of this medium. Nineteen selected diglutamic acid was found, indicating that a strains of this species demonstrated the fermen- glutamic acid polymer is present. The addi- tation of glucose by lowering the pH of a yeast tional glutamic acid can be extracted with hot extract-glucose broth from 6.8 to 4.2-4.7 after trichloroacetic acid like the teichoic acid. anaerobic incubation. These strains also pro- The G+C content of the DNA, as determined duced predominately L-lactic acid and between in five strains, was 33.6 + 1.0 mol%. 5 and 25% D-lactic acid from glucose. Some variable characters and the G+C con- Ninety percent of the strains grew well at tent of the DNA of some representative strains NaCl concentrations up to 10%. All strains of S. capitis are given in Table 2. either grew poorly or failed to grow at a NaCl Strain ATCC 27840 (originally designated LK concentration of 15%.The optimal growth tem- 499) is the type strain of S. cupitis. A descrip- perature range was 30 to 40 C. All strains failed tion ofthis strain follows. to grow at 15 C but grew well at 45 C. Cells: Spheres, 0.9 to 1.2 pm in diameter, All strains had either weak or moderate occurring predominantly in tetrads and pairs. catalase activity and were positive for the Nonmotile and nonsporeforming. Gram posi- benzidine test. All failed to produce coagulases. tive. All strains demonstrated either weak or no Agar colonies: Circular, entire, 2 to 2.5 mm in hemolysin activity on bovine or sheep blood but diameter, slightly convex, smooth with glisten- usually produced good hemoly sin activity on ing surface. Opaque, chalk white. human blood. Strains had variable acetylmeth- Catalase and benzidine tests are positive. ylcarbinol production and had weak or no C hemoorganotroph; metabolism is respira- phosphatase activity. Eighty-three percent of tory. the strains reduced nitrates, and all strains Facultatively anaerobic. demonstrated weak DNase and moderate to Growth at extreme temperatures: Good strong b ac teriolytic activities. growth at 45 C, no growth at 15 C. By comparison with other staphylococci, S. Growth on NaCl agar: Good growth with 10% cupitis produced acid aerobically from only a NaC1, poor growth with 15%. few different carbohydrates. All strains pro- Coagulases not produced. duced acid from glucose, fructose, mannose, Phosphatase not produced. and ghrcerol. Ninety-one percent of the strains Acetylmethylcarbinol not produced. produced acid slowly from sucrose, and 94% Nitrates are reduced. produced acid slowly from mannitol. Only 2% of Hemolysins: No hemolysis of bovine and the strains produced acid from trehalose or sheep blood, but moderate hemolysis of human lactose. All failed to produce acid from galac- blood. tose, rhamnose, xylose, arabinose, ribose, mal- DNase activity is weak. tose, turanose, gentiobiose, cellobiose, melezi- Acid produced aerobically from glucose, fruc- tose, xylitol, sorbitol, arabitol, inositol, salicin, tose, mannose, sucrose, mannitol, and glycerol. adonitol, dulcitol, erythritol, erythrose, raf- No acid from galactose, rhamnose, xylose, arab- finose, melibiose, fucose, tagatose, lyxose, or inose, ribose, maltose, lactose, trehalose, turan- sorbose. ose, gentiobiose, cellobiose, melezitose, xylitol, All strains were resistant to lysozyme and sorbitol, inositol, salicin, adonitol, dulcitol, were slightly resistant to lysostaphin (MIC, 200 arabitol, erythritol, erythrose, raffinose, meli- pg/ml). All were susceptible to erythromycin biose, fucose, tagatose, lyxose, or sorbose. (MIC, 0.4 to 0.8 pg/ml), streptomycin (MIC, 1.6 Antibiotic susceptibilities: Slightly resistant to 6.2 pg/ml), and novobiocin (MIC, 0.05 to 0.1 to lysostaphin (MIC, 200 pg/ml); susceptible to pg/ml). Ninety-four percent of the strains were penicillin G (MIC, 0.05 pg/ml) , erythromycin susceptible to penicillin G (MIC, 0.025 to 0.05 (MIC, 0.4 pg/ml), streptomycin (MIC, 3.1 pg/ pglml), and 85% were susceptible to tetracy- ml), tetracycline (MIC, 0.4 pg/ml), and novobi- cline (MIC, 0.4 to 0.8 pg/ml). ocin (MIC, 0.05 pg/ml); resistant to lysozyme. Nineteen selected strains contained peptido- Cell wall peptidoglycan: ~-Lys-Gly~.1, L- glycan of the type ~-Lys-Gly~.~-~.~,L-Sero.e-1.2. Ser,.o. Their cell wall teichoic acid contained glycerol, Cell wall teichoic acid: Glycerol. and no neutral sugar or additional amino sugar G+C content of the DNA: 35.5 mol%. was detected. Cell walls contained low amounts S. cupitis can be distinguished from all other TABLE2. Variable characters of 20 strains of Staphylococcus capitis

Acid (aerobically) Anaerobic Anaerobic /___ Tetra- Cell wall Growth Acetyl- Nitrate from Penicillin Mol 70 thiogly- fermenta- L-Lact i c Hemol- Phos- I cyline glutamic acid Strain on 15% methyl- reduc- G MIC G+C colate tion of glu- acid (%) ysis p hat ase MIC (mol/mol NaCl agar carbinol tion (/Lg/mU growth@ cose (pH) - - (rdml) lysine)* su- vIan Ian Ser- Cly- crose -nose _.iitol ine cine ATCC 27840 35.5 + 4.6 89 - + - + + + 0.050 0.4 1.6 1.0 4.1 ATCC 27841 31.4 t 4.2 92 - + - + ++ t+ 0.050 50.0 1.4 1.0 3.5 ATCC 27842 + 90 + - + + + 50 .O 1.5 1.0 4.0 31.0 4.6 f 50.0 ATCC 27843 34.1 + 4.6 75 - + - + + ++ 0.025 0.4 1.8 1.2 4.1 RM 390 NDd * 4.5 87 - + - + + + 0.050 0.4 1.4 1.0 3.7 GS 14 ND + 4.6 89 f + f + + + 0.050 0.8 1.6 1.0 3.9 KES 14 ND + 4.4 81 - + * + + + 0.050 0.4 1.3 0.8 3.5 SE 21 ND + 4.7 90 * + - - + + 0.050 0.8 1.5 0.9 3.8 CK 17 ND * 4.4 91 - + + + 0.050 0.4 1.6 1.0 4.0 RK 23 ND + 4.5 90 f + + + 0.050 0.8 1.6 1.1 3.8 + KH 220 ND *C ND ND + 42 + 0.200 50.0 ND ND ND PM 378 35.8 + 4.4 90 f + ++ + 0.025 0.4 1.7 1.0 3.8 WK 11 ND *C 4.3 92 - + ++ ++ 0.050 0.4 1.6 1.1 3.9 WK 445 ND + 4.5 91 + t + ++ 0.050 0.4 1.7 1.1 4.3 DBM 213 ND t 4.3 92 * + + + 0.050 0.8 1.4 1.0 3.9 EK 11 ND + 4.7 82 - - + - 0.050 0.4 1 .a 1.1 3.8 HM 11 ND + 4.6 95 * + f + 0.050 0.4 1.6 BS 2 ND *C 4.7 90 + t + + 0.050 0.8 1.4 CE 2 ND + 4.7 87 f + i + 1.5 - + 0.050 0.4 SK 13 ND *C 4.3 88 - + I_++ 50.0 50.0 1.6 Symbols for anaerobic growth:I +, uniform dense; i, gradient of growth from dense to light down the tube; *C, gradient of growth plus individual colonies. * Moles of glutamic acid per mole of lysine is 1.0 in all other staphylococci. Reaction: ++,strong positive (carbohydrates only); +, positive; f, weak; -, negative. ND, Not determined. 68 KLOOS AND SCHLEIFER INT. J. SYST.BACTERIOL. staphylococci primarily on the basis of its small amount of oxygen present in the glucose me- and slightly convex, chalk-white colony, carbo- dium might result in the production of signifi- hydrate reaction pattern, cell wall composition, cant quantities of acid such as were observed. and lactic acid configuration. There are also The above-mentioned 18 strains could be subdi- some significant differences between S. cupitis vided into two groups on the basis of the type of and certain other species in the parameters of lactic acid isomers produced from glucose. One cell arrangement, anaerobic growth pattern in group contained eight strains that produced thioglycolate, growth at different NaCl concen- only D-lactic acid, and another group contained trations and temperature extremes, coagulase 10 strains that produced both L- and D-laCtiC activity, hemolysis, acetylmethylcarbinol pro- acid. The latter strains may contain, in addition duction, nitrate reduction, and phosphatase, to a D-laCtate dehydrogenase, an L-lactate dehy- DNase, and bacteriolytic activities. drogenase. Such differences were also noted in (iii) S. horninis sp. nov. (ho’mi.nis. L. noun S. aureus by Schleifer and Kocur (16). Most S. homo man; L. gen. noun hominis of man; uureus strains produce L- and D-lactic acid, but named for the host on whose skin this species is a few strains produce only L-lactic acid. Ninety- commonly found.) The following description of five percent of the strains grew well at NaCl S. horninis is based on a total of 240 strains, concentrations up to 7.5%, but 97% grew poorly unless noted otherwise. at 10% and failed to grow at 15%. The optimal Cells were gram-positive cocci, 1.0 to 1.5 pm growth temperature range was 28 to 40 C. in diameter, nonmotile and nonsporeforming, Seventy-two percent of the strains failed to grow occurring predominantly in tetrads with occa- at 15 C, and all strains grew well at 45 C. sional pairs. Thirty-eight percent of the strains All strains had either weak or moderate had approximately equal numbers of tetrads catalase activity and were positive for the and pairs. benzidine test. All failed to produce coagulases Colonies on a P agar medium (6)were raised and had weak or no hemolysin or DNase activi- to very slightly umbonate and, with age, usually ties. Eighty-three percent of the strains had no developed a wide beveled edge and elevated phosphatase activity, weak or no bacteriolytic center. The older colony profile appears to be activity, and reduced nitrates. Strains had species specific. Colonies were small, circular, variable acetylmethylcarbinol production. and usually entire, smooth dull, and opaque. All strains produced acid aerobically from Their diameter was 3 to 4 mm. Colony pigment glucose, fructose, maltose, sucrose, and glyc- was variable; 64% of the strains had yellow- erol. Ninety percent of the strains produced orange or yellowish pigment in the center of acid from trehalose, 81% produced acid from the colonies and 36% had gray-white (unpig- turanose, 60% produced acid from lactose, 52% mented) colonies. Older colonies ( 2 6 days) produced acid from galactose, and 51% pro- often showed concentric rings of lighter and duced acid from melezitose. Only 7% of the darker colors. strains produced acid from mannitol, and 10% Growth occurred in the aerobic portion and of the strains produced acid from mannose. All 84% of strains produced slight growth in the failed to produce acid from rhamnose, xylose, anaerobic portion of the thioglycolate medium, arabinose, ribose, gentiobiose, cellobiose, indicating a limited facultatively anaerobic xylitol, sorbitol, inositol, salicin, adonitol, dul- capability. Growth was much less intense in the citol, arabitol, erythritol, erythrose, raffinose, deeper, more anaerobic, portion of the medium melibiose, fucose, tagatose, lyxose, or sorbose. and was usually associated with the presence of All strains were resistant to lysozyme and a few very small individual colonies. Sixteen were slightly resistant to lysostaphin (MIC, 100 percent of the strains failed to produce any clear to 200 pg/ml). Seventy-seven percent of the signs of visible anaerobic growth. Eighteen strains were susceptible to penicillin G (MIC, selected strains demonstrated the fermentation 0.006 pglml), 97% were susceptible to strep- of glucose by lowering the pH of a yeast extract- tomycin (MIC, 1.6 to 6.2 pg/ml), 93% were glucose broth from 6.8 to 4.4-4.9 after anaerobic susceptible to erythromycin (MIC, 0.4 to 0.8 incubation. Considering the relatively poor an- pg/ml), 64% were susceptible to tetracycline aerobic growth of this species, it seemed some- (MIC, 0.4 pg/ml), and 99% were susceptible to what unusual to obtain acid from glucose under novobiocin (MIC, 0.1 to 0.2 pg/ml). A relatively anaerobic conditions. However, it should be large proportion of the strains (36%) was resist- mentioned here that this species rapidly pro- ant to tetracycline. duces very strong acid from glucose aerobically, Eighteen selected strains contained peptido- and even slight anaerobic growth or a slight glycan of the type ~-Lys-Gly~.~-~.~,L-Sero.6-1.3. VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 69

Their cell wall teichoic acid contained glycerol Cell wall teichoic acid composition: Glycerol and, in addition, had 0.3 to 0.8 mol of glucosa- and glucosamine. mine per mol of glycerol. No neutral sugar was G+C content of the DNA: 33.7 mol%. found. This species produced only low levels of S. hominis can be distinguished from all other teichoic acids and, like S. capitis, required staphylococci primarily on the basis of a combi- concentration of the cell wall hydrolysates for nation of its colony morphology and pigment the detection of free glycerol. pattern, predominant tetrad cell arrangement, The G+ C content of the DNA, as determined poor or no detectable growth in thioglycolate, in five strains, was 33.0 & 1.0 mol%. low NaCl tolerance, and carbohydrate reaction Some variable characters and the G+C con- pattern. There are also some significant differ- tent of the DNA of some representative strains ences between s. hominis and certain other of S. hominis are given in Table 3. species in the parameters of cell wall composi- Strain ATCC 27844 (originally designated tion, lactic acid configuration, temperature ex- DM 122) is the type of strain of S. hominis. A tremes of growth, coagulase activity, hemolysis, description of this strain follows. acetylmethylcarbinol production, nitrate reduc- Cells: Spheres, 1.2 to 1.4 pm in diameter, tion, and phosphatase, DNase, and bacteri- occurring predominantly in tetrads and occa- olytic activities. Several of these properties sionally in pairs. Nonmotile and nonsporeform- overlap with those of S. epidermidis, S. he- ing. Gram positive. moly ticus, and S. warneri, suggesting a close Agar colonies: Circular, entire, 4 to 4.5 mm in relationship among these species. diameter, slightly umbonate with wide, beveled (iv) S. sirnulans sp. nov. (sim’u,lans. L. part. edge and elevated center, smooth dull surface. adj. simulans imitating; named for having simi- Yellow-orange pigment is produced in the ten- larities to certain coagulase-positive staphylo- ter of opaque colonies. cocci, including S. aureus.) The following de- Catalase and benzidine tests are positive. scription of S. simulans is based on a total of 13 C hemoorganotroph; metabolism is respira- strains. tory. Cells were gram-positive cocci, 0.8 to 1.5 pm Slightly facultatively anaerobic. in diameter, nonmotile and nonsporeforming, Growth at extreme temperatures: Good occurring predominantly in pairs and tetrads growth at 45 C, no growth at 15 C. and occasionally singly. Growth on NaCl agar: Good growth with 7.5% Colonies on a P agar medium (6) were slightly NaC1, slight growth with lo%, no growth with raised, circular, entire, smooth, slightly glisten- 15%. ing, translucent to slightly transparent, and Coagulases not produced. usually gray-white (unpigmented) . Twenty- Phosphatase not produced. three percent of the strains had a very slight tint Acetylmethylcarbinol produced weakly. of yellowish pigment. The colonies were rela- Nitrates are reduced. tively large and had a diameter of 5.0 to 7.5 mm. Hemolysins: No hemolysis of bovine, sheep, Growth occurred in both the aerobic and or human blood. anaerobic portions of the thioglycolate medium, DNase activity is weak. indicating a facultatively anaerobic capability. Acid produced aerobically from glucose, fruc- Growth in the anaerobic portion of the medium tose, galactose, maltose, lactose, sucrose, treha- was uniformly dense. This species demon- lose, turanose, and glycerol. No acid from man- strated the fermentation of glucose by lowering nose, rhamnose, xylose, arabinose, ribose, me- the pH of a yeast extract-glucose broth from 6.8 lezitose, mannitol, gentiobiose, cellobiose, to 4.7-5.0 after anaerobic incubation. The xylitol, sorbitol, inositol, salicin, adonitol, dul- strains could be subdivided into two groups on citol, arabitol, erythritol, erythrose, raffinose, the basis of the type of lactic acid isomers melibiose, fucose, tagatose, lyxose, or sorbose. produced from glucose. One group contained six Antibiotic susceptibilities: Slightly resistant strains that produced only L-lactic acid, and to lysostaphin (MIC, 100 pg/ml); susceptible to another group contained seven strains that penicillin G (MIC, 0.006 pg/ml), streptomycin produced both L- and D-lactic acid. The occur- (MIC, 6.2 pg/ml), erythromycin (MIC, 0.4 pg/ rence of strains producing L- and D-laCtiC acid or ml), and novobiocin (MIC, 0.1 pg/ml); resistant only L-lactic acid was also found in S. aureus to tetracycline (MIC, 100 pg/ml); resistant to (16). lysozyme. All strains grew well at NaCl concentrations Cell wall peptidoglycan: L-LYS-G~Y,.1, L- up to 10%. Seventy-seven percent of the strains Ser,.,. grew poorly at a NaCl concentration of 15%,and TABLE3. Variable characters of 20 strains of Staphylococcus hominis

Peptido- glycan :mol/mo Acid (aerobically) from of glu- Teichoic Anaerobic Bac- Inaerobic L- Ni- Peni- Erythro. Tetra- tamic acid (mol Zolony ferments He- Acetyl- Deoxy- terio- Mol ?6 thiogly- Lactic trate :illin G mycin cycline acid) of gluco- Strain of moly- met hy 1- ribonu- lytic Pig- colate acid meduc. MIC MIC MIC saminel G+C menta sis :arbinol clease activ- - - - - (%) tion rglml) (rglml) (rglml) mol of ity W aJ aJ % 8 glycerol) - 0) E +-'0 xm .- 2 .d uh 3 z s mE 5 - __ - - - ATCC 27844 33.7 YO f 4.5 0 f + ++ ++ + 0.006 0.4 100.0 0.8 4.1 0.5 ATCC 27845 32.3 YO fC 4.5 0 f + f ++ + 0.006 0.4 100.0 0.6 4.3 0.5 ATCC 27846 NDd - fC 4.6 0 + f f - + 0.006 0.4 0.4 1 .o 3.9 0.3 ATCC 27847 29.9 fC 4.9 50 - + - + + 6.2 0.4 50.0 1.1 3.8 0.4 WK52 ND -C 4.5 0 f + ++ + + 0.006 0.4 0.4 0.9 3.9 0.8 PM 375 ND - -C 4.4 43 - + ++ + + 0.006 0.4 0.4 1.3 3.6 ND SM 67 33.5 YO- - 4.6 38 f f ++ ++ + 0.006 0.8 0.4 1.2 3.8 0.5 JL 248 ND YO -C 4.6 0 + ++ ++ - 6.2 400.0 100.0 1 .o 4.0 0.7 TW 52 ND Y f 4.5 49 + ++ ++ + 0.006 0.4 200.0 1.3 3.6 0.5 TW 139 ND Y f 4.4 49 + ++ ++ + 12.5 0.4 0.4 0.9 4.1 0.8 GH 149 ND YO - 4.5 0 + - ++ + 0.006 0.4 0.4 1.2 3.8 0.5 KH 211 ND Y fC 4.5 0 - ++ ++ + 25.0 0.8 0.4 0.7 4.4 0.3 AW 178 35.8 Y f 4.4 42 + ++++ - 6.2 8.4 100.0 0.8 4.1 0.4 RM 122 Y - - - ND - fC- 4.5 51 - + ++ + 0.006 0.8 0.4 0.9 4.3 0.4 MCS 13 ND 4.5 0 f f f + + 0.050 200.0 100.0 1.1 3.3 0.5 GS 16 ND Y! 37 - f 4.5 f + + ++ + 0.050 0.8 200.0 1.3 3.5 0.4 DGS 13 ND 47 + 4.5 f f + ++ ++ 0.006 400.0 0.4 1.1 3.6 0.8 BS 14 ND 45 - 0.006 200.0 4.6 f + ++ + 100.0 0.6 3.9 0.3 LK 47 ND ND ND + f ++ + 0.006 0.4 0.4 ND ND ND MK 314 ND ND ND + -++ ++ -+ 12.5 0.4 0.4 -ND -ND ND a Symbols for colony pigment: YO, yellow-orange center; Y, yellowish center; -, gray-white (unpigmented). Symbols for anaerobic growth: f, gradient of growth from moderate to light down the tube; +C, gradient of growth plus individual colonies; -C, individual colonies only (usually <50); -, no 2detectable growth. Reaction: ++, strong positive (carbohydrates only); +, positive; f, weak; -, negative. ND. Not determined.

- TABLE4. Variable characters of 13 strains of Staphylococcus simulans

I I I 1 1 I

Anaerobic Acid (aerobically) from Peni- fermenta- L-Lactic Acetyl- Nitrate Phos- Bacteri- Erythro- Tetra- Growth cillin G Strain Mol tion of acid :r:t methyl- reduc- pha- olytic mycin cycline 1G+C NaCl at l5 MIC MIC glucose ('1 carbinol tion tase activity agar ' (PW (ccdml) (ccg/mU Galac- Man- Ri- Malt- Tre- Ser- Gly- lltose nose bose ose halosf ine cine -a - ATCC 27848 4.7 100 + 3.1 0.8 200.0 0.2 4.9 ATCC 27849 4.7 63 i ++ 0.012 0.4 0.4 0.1 5.7 ATCC 27850 4.8 60 + ++ 0.025 400.0 200.0 0 6.0 ATCC 27851 4.8 100 - -+*++ 1.6 0.8 400.0 0.1 4.7 AW232 4.8 100 ~ 0.012 0.4 100.0 0 1-++* - ++ 0.2 5.6 SM 128 4.8 64 f I-++-+ ++ 0.025 0.8 0.8 0 5.3 SM 81 4.9 100 - '-+ + - ++ + 25.0 400.0 0.4 0 5.1 KL 299 4.8 69 + + - ++ ++ 0.012 0.4 0.4 0 5.0 KL 104 4.9 70 f 1--+--+++ 0.012 400.0 200.0 0 4.8 MAW 222 37.2 4.8 75 f - ++ - - - + 0.025 0.8 0.8 0.1 5.2 HK 12 ND 4.9 -+-f ++ ~ 25.0 0.8 0.4 0.2 4.8 DW 196 4.8 *-**++ * 12.5 0.8 200.0 0.1 5.4 CE 13 ND 5.0 -++--+ ++ 0.800 0.4 6.2 0 5.0

Reaction: ++, strong positive (carbohydrates only); +, positive; +, weak; -, negative. ND, Not determined. 72 KLOOS AND SCHLEIFER INT.J. SYST.BACTERIOL.

23% failed to grow at this concentration. The phatase activity. Ninety-two percent of the optimal growth temperature range was 25 to strains reduced nitrates or demonstrated mod- 40 C. Eighty-five percent of the strains grew erate to good bacteriolytic activity. All had well at 15 C, and all grew well at 45 C. weak to moderate DNase activity. All strains had weak or moderate catalase All strains produced acid aerobically from activity and were positive for the benzidine test. glucose, fructose, lactose, sucrose, and glycerol. All failed to produce coagulases. All strains Seventy-seven percent of the strains produced demonstrated weak or no hemolysin activity on acid from mannose and trehalose, and 92% bovine or sheep blood but produced weak to produced acid from mannitol. Fifty-four per- good hemolysin activity on human blood. Sev- cent of the strains produced acid from ribose. enty-seven percent of the strains failed to pro- Sixty-nine percent of the strains failed to pro- duce acetylmet hylcarbinol or had weak phos- duce acid from maltose; 31% produced only

TABLE5. Morphological characters of Staphylococcus species

~ ~ Colony Colony pigment Colony profile

Total Colony CI no. of Species diametera strains (mm) T studied

- S. aureus ...... 46 7.1 0.1 100 100 S. simulans ...... 13 6.5 * 0.2 100 100 15 s. xylosus ...... 102 7.0 * 0.1 5 12 8 71 17 12 68 32 S. cohnii ...... 42 5.9 i 0.2 21 21 53 7 10 83 95 5 S. saprophyticus . . 83 6.5 * 0.1 18 11 29 20 80 98 2 S. haenolyticus . , . 117 5.4 * 0.1 13 32 23 13 87 92 8 14 S. warneri ...... 38 3.8 mo.1 20 22 78 98 2 12 S. horninis ...... 240 3.7 * 0.05 36 18 82 91 9 61 38 S. epidermidis .... 204 3.1 * 0.04 99 1 2 17 S. capitis ...... 47 2.4 * 0.07 1 96 I100 1100 100 14 70

iand s %. Percentages of strains possessing the character jicated.

TABLE6. Physiological characters of Staphylococcus species

Growth on: Growth at: Anaerobic growth in thioglycolate semisolid medium 10% NaCl 15% NaCl 15 C Total no. agar agar Species of strains Gradi- Scat- Scat- studied Uni- ent: tered tered Noni +a i - formly dense colo- colo- + + + *- dense to nies nies light > 50 <50 ------S. aureus ...... 46 986 2 96 4 40 60 60 36 4 100 S. simulans ...... 13 100 100 77 23 85 15 100 s. xyl0.sus ...... 102 68 11 14 4 3 100 38 48 14 100 19 19 62 S. cohnii ...... 42 24 47 17 7 5 100 21 64 15 100 76 17 7 S. saprophyticus ...... 83 59 30 9 1 1 100 30 70 100 38 36 26 S. haemolyticus ...... 117 20 8 66 4 2 100 15 45 40 43 57 100 S. warneri ...... 38 87 58 100 8 54 38 8 76 16 92 8 S. horninis ...... 240 10 44 30 16 97 3 97 28 72 100 S. epidermidis ...... 204 98 11 16 79 4 96 21 79 100 S. capitis ...... 47 68 11 21 90 10 51 49 LOO 100 - - - - - OGrowth: +, good; *, weak; -, none. Percentages of strains possessing the character indicated. VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 73

weak acid from this carbohydrate. Eight per- The G+C content of the DNA, as determined cent of the strains produced weak acid from in six strains, was 35.6 f 0.6 mol%. galactose. All failed to produce acid from rham- Some variable characters and the G+C con- nose, xylose, arabinose, turanose, gentiobiose, tent of the DNA of some representative strains cellobiose, melezitose, xylitol, sorbitol, inositol, of S. simulans are given in Table 4. salicin, adonitol, dulcitol, arabitol, erythritol, Strain ATCC 27848 (originally designated erythrose, raffinose, melibiose, fucose, tagatose, MK 148) is the type strain of S. simulans. A lyxose, or sorbose. description of this strain follows. All strains were resistant to lysozyme and Cells: Spheres, 0.8 to 1.0 pm in diameter, were susceptible to lysostaphin (MIC. 12.5 to 25 occurring predominantly in pairs and tetrads pg/ml). All were susceptible to streptomycin and occasionally singly. Nonmotile and non- (MIC, 1.6 to 3.1 pg/ml) and novobiocin (MIC, sporeforming. Gram positive. 0.1 to 0.2 pg/ml). Fifty-four percent of the Agar colonies: Circular, entire, 6.5 to 7.0 mm strains were susceptible to penicillin G (MIC, in diameter, slightly raised, smooth with 0.012 pg/ml), and 77% were susceptible to slightly glistening surface. Translucent to erythromycin (MIC. 0.4 to 0.8 pg/ml). Forty- slightly transparent and gray-white (unpig- six percent of the strains were susceptible to mented). tetracycline (MIC, 0.4 pg/ml). This species Catalase and benzidine tests are positive. contained peptidoglycan of the type L-LYS- Chemoorganotroph; metabolism is respira- Gly,-,. The cell wall teichoic acid contained tory. glycerol and galactosamine. Facultatively anaerobic.

TABLE7. Biochemical characters of Staphylococcus species I Bacterio- I I Hemolysis methyl- Nitrate Phospha- Deoxyribo- Total no. Coagulase lytic ~~~~~1 reduction tase nuclease Species of strains 1 1 activity production studied I I

+a - ++

s. aureus ...... 46 100b 100 S.simulans ...... 13 100 s. xylosus ...... 102 100 g S. cohnii ...... 42 100 S. saprophyticus ...... 83 100 S. haemolyticus ...... 117 100 S. warneri ...... 38 100 10 12 S. hominis ...... 240 100 <1 16 83 S. epidermidis ...... 204 100 89 6 S. capitis...... 47 100 100

“Reactions: +, positive; +, weak; -, negative. ’Percentages of strains giving the reaction indicated.

TABLE8. Carbohydrate reactions of Staphylococcus species: monosaccharides under aerobic conditions I I

D(+) 1 B-D(-) 1 D(+) D(+) D(+) Total no. Glucose Fructose Galactose Mannose Xylose krabinoseL(+) Ribose 1 D(-) Species of strains -7 I studied ++, ++=+ ++ + - ++, ~ +

S.aureus ...... 46 1OOb 100 100 89 S. simulans ...... 13 100 100 8 92 77 s. rylosus ...... 102 56 38 6 95 5 62 29 9 95 S. cohnii ...... 42 86 12 2 100 10 90 51 100 100 S. saprophyticus ...... 83 85 14 1 92 5 3 8 92 100 S. haemolyticus ...... 117 98 2 24 36 40 29 24 47 3 100 S. warneri ...... 38 100 79 21 5 19 76 2 100 S. hominis ...... 240 100 100 33 19 48 S. epidermidis ...... 204 100 100 80 5 15 S. capitis...... 47 100 79 21 100

a Reactions: + +, strong acid; +, moderate acid; *, weak acid; -, negative. ’Percentages of strains giving reaction indicated. 74 KLOOS AND SCHLEIFER INT. J. SYST. BACTERIOL. I I1 I

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I

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...... VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. II. 75

Growth at extreme temperature: Good weak hemolysis of bovine blood, good hemolysis growth at 15 and 45 C. of human blood. Growth on NaCl agar: Good growth with 10% DNase activity weak. NaC1, no growth with 15%. Acid produced aerobically from glucose, fruc- Coagulases not produced. tose, ribose, maltose (very weak), lactose, su- Phosphatase activity weak. crose, trehalose, mannitol, and glycerol. No acid Acetylmethylcarbinol not produced. from galactose, mannose, rhamnose, xylose, Nitrates are reduced. arabinose, turanose, gentiobiose, cellobiose, Hemolysins: No hemolysis of sheep blood, melezitose, xylitol, sorbitol, inositol, salicin, adonitol , dulci tol, arabitol, erythritol, ery- t hrose, raffinose, melibiose, fucose, t agatose, lyxose, or sorbose. Antibiotic susceptibilities: Susceptible to ly- Total Level of susceptibility" sostaphin (MIC, 25 pglml), streptomycin (MIC, no. of Species 3.1 pglml), erythromycin (MIC, 0.8 pg/ml), and strains 12.5 25 50 100 200 novobiocin (MIC, 0.1 pglml); slightly resistant studied (+) (+) (+I (+I (*I to penicillin G (MIC, 3.1 pglml); resistant to S. aureus ...... 51 10 82 8 tetracycline (MIC, 200 pg/ml); resistant to S. simulans ...... 13 23 77 lysozyme. s. xylosus ...... 101 27 67 6 Cell wall peptidoglycan: L-LYs-G~Y,., S. cohnii...... 42 100 , L S. saprophyticus .... 83 28 72 Ser,,. 2, S. haemolyticus ..... 116 34 66 Cell wall teichoic acid composition: Glycerol S. warneri ...... 38 8 92 and galac tosamine. S. hominis ...... 178 41 59 S. epidermidis ...... 152 68 32 G+C content of the DNA: 34.0 mol%. S. capitis ...... 48 8 92 S. simulans can be distinguished from all other staphylococci primarily on the basis of a combination of its colony morphology, lack of acetylmethylcarbinol production, carbohydrate reaction pattern, and cell wall composition. There are also some significant differences be-

TABLEll. Antibiotic susceptibilities of Staphylococcus species

Penicillin G Novobiocin Erythromycin Tetracycline

Level of sus- Level sus- Level of susceptibility* Level of susceptibilit! of Total 1 ceptibility ceptibility no. of I - Species strains studied" 8"o ? y 0 82 "9, zz&s" 2-4- ST1- 3 g& G+ 3AzF 40 Jq 72 d~o~ddd~o5%?8~4.;) 0szz T+c 3- - S. aureus ...... 51 45' 20 8 27 96 96 4 100 S. simulans ...... 13 46 8 23 23 100 77 23 46 8 46 s. xylosus ...... 101 2 26 47 25 62 26 12 80 2 18 S. cohnii ...... 42 2 91 7 62 17 21 84 14 2 S. saprophyticus ... 83 95 5 1 82 7 11 93 4 3 S. haernolyticus .... 116 49 4 <1 25 21 98 95 5 90 <1 9 S. warneri ...... 100 S. hominis ...... 64 36 S. epidermidis ..... 152 <1 83 17 S. capitis ...... 48 - 85 -15 a Strains were isolated from persons who were not exposed to antibiotics for at least 3 months prior to col- lecting. It would be expected that the above base-level frequencies might shift toward higher frequencies of re- sistant strains during or shortly after antibiotic exposure (8). bExpressed as MIC (numbers) and disk zone inhibition (numbers in parentheses). MICs are expressed as micrograms per milliliter. Disk zone inhibition represents the width (in millimeters) of the zone of antibiotic inhibition, measured from the edge of a 2-U penicillin G, 5-pg novobiocin, 2-pg erythromycin, and 30-pg tetracycline sensitivity disk (Difco), respectively. 'Data are given as percentages of strains. TABLE12. Scheme for the classification of human cutaneous staphylococcia

Cell I'erminal Colony mange 4naerobic pH in Growth Coagu- Hemol. Colony profile Lactic Acety!- Nitrate ribonu-D~xy- diam- ment growth in yeast on 10% ;rowth Growth lase ysis Phos- Species and light acid methyl- reduc- eter be- thiogly- extract- NaCl 3t 15 C at 45 C (pref. (pref. phatase transmis- isomer(s~ rarbinol tion clease domi- colated glucose agar rabbit) bovine1 (mm) sion* nantY medium

S. aureus ...... 6-8 RT (PS) 4.3-4.6 DL,(L) + + + + S. simulans ...... , , 5-7 RT V 4.7-5.0 DL,L + + - (-) s. xylosus ...... 5-9 (RST) PS 4.9-5.6 L> D + (-) - f, - S. cohnii ...... 5-7 (CO) PS 5.3-6.0 L,DL + (+) - +** S. saprophyticus . . . . 5-8 (LCO) PS 5.0-5.4 DL + V - (+) S. haemolyticus . . , . . 4-8 (LCO) V 4.9-5.5 D + + - +. f S. warneri ...... 3-5 (CO) V 4.7-5.0 DL + + + S. hominis ...... 3-4 (CO) T 4.4-4.9 DL,D f + V S. epidermidis ...... 2-4 RT V 4.3-4.6 L (*) + - + S. capitis ...... 1-3 co (T) 4.2-4.7 L>D + + - V -- - Acid (aerobically)from ResistanceI to I Cell-wall composition Peptidoglycan I Teichoic acid (moVmol of (components) 1 -I----glutamic acid) Glutamic acid polymer Species Q (glutamic acid 3 2 1.3 mol/mol lysine) : Q E! .3 R -z 5 d S. aureus ...... + + 5.0-6.0 S.simulans . . . . , , , . . + (+) 4.7-6.0 s. xylosus . . , ...... + + 4.9-5.7 S.cohnii ...... + + 4.94.0 S. saprophyticus . . . . + + 4.1-5.1 S. haemolyticus . . . . . +,- + 0.9-1.4 3.3-4.0 S. warneri ...... + + 0.6-1.4 3.3-4.4 S.hominis ...... + t 0.6-1.3 3.3-4.3 S. epidermidis . . . :. . + - 0.7-1.5 4.0-5.0 S. capitis ...... -+ - 0.8-1.2 3.5-4.3 a Characters were selected based on their ease of distinction and wide differences (270%) in their type frequencies between two or more species. A single listed symbol denotes a type frequency of 90 to 100%;parentheses around symbols denote a type frequency of 70 to 89%; two symbols are listed for a character when either type is in frequency below 70'70, but together equal 80 to 100%. Symbols for characters (unless noted otherwise):+, positive; i, weak; -, negative: V, variable (+, f, and -). Symbols for colony profile and light transmission: RT, raised (flat) and translucent; RST. raised (flat) and slightly translucent; CO, convex and opaque: LCO. low convex and opaque. Symbols for cell arrangement: PS, pairs and single cells; T, tetrads; V, variable (pairs, singles, and tetrads common). Symbols for anaerobic growth: +. dense uniform; *. gradient of growth from dense to light down tube; +C, large individual colonies; c. small individualcolonies to absence of visible growth: V, variable (dense uniform, gradient growth, and individual colonies). 'Without exposure. 'ND, Not determined. (All of the strains were not tested. Some of the strains of S. xylosus and S. saprophyticus tested contained glucose. and most of the strains of S.saprophyticus tested contained glucosamine.) VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 77 tween S. simulans and certain other species in Species colony morphology and pigment pat- the parameters of anaerobic growth in thio- terns are illustrated in Fig. 1. These characters glycolate, lactic acid configuration, growth at were very useful in making the preliminary different NaCl concentrations and temperature species identification of strains on the original extremes, coagulase activity, hemolysis, acetyl- isolation plates. Species having very distinctive methylcarbinol production, nitrate reduction, colony characteristics include S. hominis, S. and phosphatase, DNase, and bacteriolytic ac- warneri, S. epidermidis, and S. capitis. It was tivities. Several of the properties of S. sirnulans somewhat difficult to differentiate a certain few overlap with those of five unusual coagulase- strains of S. xylosus, S. saprophyticus, S. positive strains isolated in this study that cohnii, and S. haemolyticus from one another appear to be similar to strains previously de- using these characters alone. scribed by Reeder and Ekstedt (14)and Oeding It should be pointed out that the species (12). There is also an overlap of several proper- character variation observed in this study, ties with those of human strains of s. aureus. It through very similar for the strains isolated from would appear then that S. sirnulans is closely cities in North Carolina and New Jersey, may related to the coagulase-positive staphylococci. be somewhat different with strains obtained Character variation in Staphylococcus from certain other geographical areas. For ex- species. Summary data of the morphological, ample, in this study we did not recover any of physiological, biochemical, and antibiotic char- the violet-pigmented strains of S. epiderrnidis acters found in the various Staphylococcus found by Marples (7) on people living in Phila- species isolated from human skin are shown as delphia, Pa., and recently found by one of us follows: colony morphology and pigment and (W.E.K.) and W. C. Noble (unpublished data) cell arrangements, Table 5; anaerobic growth on people living in London, England. Strains in thioglycolate, growth on 10% and 15% NaCl originating from animals other than man may agar, and growth at 15 and 45 C, Table 6; also show somewhat different character varia- coagulas e , he mol y sis , acety lmet hy lcarbinol tion from the human strains. production, nitrate reduction, phosphatase, Occurrence of Staphylococcus species on DNase, and bacteriolytic activities, Table 7; human skin. A summary of the occurrence and carbohydrate reactions, Tables 8 and 9; lyso- distribution of Staphylococcus species on hu- staphin susceptibility, Table 10; and antibiotic mans is shown in Table 13. As can be seen from susceptibilities, Table 11. A summary scheme the data, some species show preferences for showing the key characters used for the classifi- certain regions of the cutaneous habitat. S. cation of cutaneous Staphylococcus species is epiderrnidis was a predominant species in all presented in Table 12. This information is regions tested, including the head, nares, axil- based on species described in the companion lae, arms, and legs. S. hominis was a predomi- paper (15) and this one. nant species on the head, axillae, arms, and

TABLE13. Occurrence of Staphylococcus species on human skin

Mean 7%of skin sites occupied by Mean % of species of total staphylococci (CFU) % of 40 specieshndividual recoveredsite per individual" individ- Species uals with Head Nares Axillae Arms Legs Head' Nares Axillae species 4b 2 2 4 4 1.6 x 104~ 7.8 x lo6 1.0 x 10'

S. aureus ...... 52 12 43 4 11 9 5 30 0.02 S. simulans ...... 12 32 1 72 1 0.1 0.05 s. xylosus ...... 42 15 5 5 11 8 4 0.06 0.2 S. cohnii ...... 35 17 15 6 18 19 3 0.3 0.2 S. saprophyticus ... 70 31 12 17 28 32 3 0.1 0.3 S. haemolyticus .... 78 21 5 8 28 26 4 0.3 0.9 S. warneri ...... 52 15 12 0 99 1 0.3 0 S. epidermidis ..... 100 87 78 92 82 62 46 63 64 S. hominis ...... 100 69 77 22 5 34 S. capitis ...... 65 27 17 11 0.8 0.3

* The number of colonies isolated from each site to determine the percentage of species present represents an estimated 22 f 1% of the total staphylococcus colony-forming units (CFU) recoverable. Number of sites per individual. Average number of staphylococci per site per individual. 78 KLOOS AND SCHLEIFER INT. J. SYST.BACTERIOL. VOL.25, 1975 STAPHYLOCOCCI FROM HUMAN SKIN. 11. 79 legs, and to a lesser extent in the nares. S. anaerobic glucose utilization tests in Staphylococcus- cupitis was a predominant species on the head Micrococcus identification. Int. J. Syst. Bacteriol. 24:l-5. and to a lesser extent on the arms. S. uureus, as 5. Gordon, M. H. 1905. Report on bacterial test whereby expected from a considerable number of earlier particles shed from the skin may be detected in the air. reports, was often a predominant species in the Annu. Rep. Med. Officer Local Gov. Board (London) nares. 5:387-409. 6. Kloos, W. E., T. G. Tornabene, and K. H. Schleifer. 1974. Isolation and characterization of micrococci from ACKNOWLEDGMENTS human skin, including two new species: Micrococcus We are greatly indebted to Margaret Musselwhite for her lylae and Micrococcus kristinae. Int. J. Syst. Bacteriol. excellent technical advice and assistance throughout this 24~79-101. study. We are also grateful to E. Hagner, I. Pomper, and B. 7. Marples, R. R. 1969. Violagabriellae variant of Staphylo- Popp for their capable assistance. We are thankful for the coccus epidermidis on normal human skin. J. Bacte- help provided by Merrill Emmett for maintaining and ship- riol. 100:47-50. ping cultures, Susan Kloos and Wesley L. Kloos for assisting 8. Marples, R. R., and A. M. Kligman. 1971. Ecological in collecting skin samples from persons living in New Jersey, effects of oral antibiotics on the microflora of human and Renee Johnson for assembling and typing tabular data skin. Arch. Dermatol. 103: 148-153. and assisting in preparing the manuscript. We also thank the 9. Marples, R. R., A. M. Kligman, L. R. Lantis. and D. T. many people who provided us with samples of their skin flora. Downing. 1970.The role of the aerobic microflora in the This research was supported by Public Health Service genesis of fatty acids in human surface lipids. J. Invest. research grant A1 08255 from the Institute of Allergy and Dermatol. 55:173-178. Infectious Diseases and the Deutsche Forschungsgemein- 10. Mitchell, R. G., V. G. Alder, and K. Rosendal. 1974. The schaft. Funds were provided by the North Carolina Agricul- classification of coagulase-negative Micrococcaceae tural Experiment Station for the color plate, which was from human and animal sources. J. Med. Microbiol. photographed by Ralph Mills and printed by Prolab, Raleigh, 7:131-135. N.C. 11. Noble, W. C. 1969. Skin carriage of the Micrococcaceae. J. Clin. Pathol. 22:249-253. 12. Oeding, P. 1973. Wall teichoic acids in animal Staphylo- REPRINT REQUESTS coccus aureus strains determined by precipitation. Acta Address reprint requests to: Dr. Wesley E. Kloos, Depart- Pathol. Microbiol. Scand. Sect. B 81:327-336. ment of Genetics, North Carolina State University, Raleigh, 13. Perry, J. J. 1969. Isolation of Staphylococcus epidermidis N.C. 27607. from Tobacco. Appl. Microbiol. 17:647. 14. Reeder, W.J., and R. D. Ekstedt. 1973. Unique teichoic LITERATURE CITED acid isolated from the cell walls of a strain of Staphylo- coccus aureus. Infect. Immunity 7586-588. 1. Auletta, A. E., and E. R. Kennedy. 1966. Deoxyribonu- 15. Schleifer, K. H., and W. E. Kloos. 1975. Isolation and cleic acid base composition of some members of the characterization of staphylococci from human skin. I. Micrococcaceae. J. Bacteriol. 92:28-34. Amended descriptions of Staphylococcus epidermidis 2. Baird-Parker, A. C. 1963. A classification of micrococci and Staphylococcus saprophyticus and descriptions of and staphylococci based on physiological and biochem- three new species: Staphylococcus cohnii, Staphylococ- ical tests. J. Gen. Microbiol. 30:409-427. cus haemolyticus, and Staphylococcus xylosus. Int. J. 3. Baird-Parker, A. C. 1965. The classification of staphylo- Syst. Bacteriol. 25:50-61. cocci and micrococci from world-wide sources. J. Gen. 16. Schleifer, K. H.,and M. Kocur. 1973. Classification of Microbiol. 38:363-387. staphylococci based on chemical and biochemical 4. Davis, G. H. G., and B. Hoyling. 1974. Observations on properties. Arch. Mikrobiol. 93:65-85.

FIG. 1. Colonies of Staphylococcus species isolated from human skin. The “seed” inoculum was incubated at 34 C for 5 days (unless otherwise noted). (All rows read from left to right.) (A)S. aureus: top, GH 38; middle row, KL 207, JL 68, KES 1; bottom row, GH 39, MA W306,HK 91 (Staphylococcus sp. related tolor a variety of S. aureus). (3)S. simulans: ATCC 27848 (MK Z#), ATCC 27849 (AW 215), ATCC 27850 (KL 240). (C) S. xylosus: top row, A W 124, DM 37, A W244, GH 30; middle row, DSM 20266 (KL162), KH 168, TM 76, RM349; bottom row, DBM 194, GH 9 (Staphylococcus sp. related tolor variety of S. xylosus). (D)S. cohnii: DM 224, DSM 20260 (GH 137),MK 247. (E)S. saprophyticus: top row, TW111, KL 20, DM 100; bottom row, SE 11, GH l%, SM 118. (F)S. haemolyticus: top row, DM 70, TW268, GS 1, MK 67; bottom row, KH 99, DSM2026.3 (SM 131), RM 419. (G)S. warneri: top row,MAW314, ATCC 27838 (MK 155),ATCC 27836 (AW25); bottom row, ATCC 27837 (RM 130),ATCC 27839 (DW 138), DM 229. (HI) S. hominis (incubated 5 days): top row,ATCC 27844 (DM 122), GH 180, LK 89, JM 16; bottom row, ATCC 27845 (KL 2431, BS 14, ATCC 27847 (KES 131, ATCC 27846 (MK 129). (H2)S. hominis (incubated 7days):colony identification same as above as in (Hl).(I) S. epidermidis: top row, KL 275, MK 214, A W269; bottom row, RK 13, JRM 1, KH 141, (4 S. capitis: ATCC 27840 (LK 499), ATCC 27842 (MCS 11), ATCC 27841 (DM 267).