INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 24,No. 1 January 1974, p. 79-101 Printed in U.S.A. Copyright 0 1974 International Association of Microbiological Societies Isolation and Characterization of Micrococci From Human Skin, Including Two New Species: Micrococcus ZyZae and Micrococcus kris tinae
WESLEY E. KLOOS, THOMAS G. TORNABENE, and KARL H. SCHLEIFER
Department of Genetics, North Carolina State University, Raleigh, North Carolina 2 760 7; Department of Microbiology, Colorado State University, Fort Collins, Colorado 80521; and Botanisches Institut der Universitat Miinchen, 8 Miinchen 19, Germany
Micrococci were commonly isolated from the skins of people living in various regions of the United States. Not all micrococci isolated in this investigation could be identified with the currently recognized species of Micrococcus, viz., M. luteus, M. varians, or M. roseus, and these micrococci therefore became the subject of further taxonomic study. As a result of this study, two new species are proposed: Micrococcus lylae and M. kristinae. The type strains of these species are ATCC 27566 and ATCC 27570, respectively. Numerous strains were isolated that were similar to M. sedentarius or M. nishinomiyaensis, species that were previously represented by only single strains. (ZoBell’s strain 541 [ ATCC 14392; CCM 3141 is designated herein as the type strain of M. sedentarius.) A few micrococci were left unclassified. A variety of morphological, physiological, biochemical, and genetic characters were examined for their use as taxonomic criteria, and key characters, many of which can be determined by simple laboratory procedures, were selected for species differentiation. The more sophisticated studies of aliphatic hydrocarbons and cell-wall peptidoglycans were also very useful in the taxonomy of the micrococci. The predominant micrococci found on human skin were M. luteus and M. varians.
The medical literature has, for many years, The purpose of this investigation was to made reference to a group of aerobic, sapro- classify species of micrococci found on human phytic bacteria known as Sarcina that have skin by using the most current taxonomic been commonly isolated from human skin ( 14, critieria. We also became involved with evaluat- 18, 28, 29, 33). Recent taxonomic studies have ing existing taxonomic criteria, exploring new indicated that most aerobic strains maintained characters, and estimating species variation in in various culture collections under the name of cutaneous populations. Sarcina belong to the species Micrococcus Some of the preliminary results of this study luteus (Schroeter 1872) Cohn 1872 or Micro- have been previously reported (W.E. Kloos and coccus varians Migula 1900 (19, 21, 22, 24, K. H. Schleifer, Abst. Annu. Meet. Amer. SOC. 40). In line with this, Marples et al. (30) Microbiol., 73rd, Miami Beach, p- 116, 1973). recently made reference to the species M. luteus in studies on the aerobic microflora of the human scalp. M. varians has not yet been MATERIALS AND METHODS reported in studies of human skin. However, it was not until 1973 (22) that a comprehensive Bacterial straips. Micrococci were isolated from the report on the taxonomic status of this species healthy skins of two groups of people. One group was was made available. Earlier reports were some- composed of 20 people living in Raleigh, N.C., who what confusing, and, with the lack of a clearly were sampled once each month for 6-13 months. The defined species status, cutaneous isolates may second group was composed of 115 people from 21 have been simply placed in Baird-Parker’s different regions of the United States who were Micrococcus subgroups together with other sampled once during the winter. These people were species (2). residents of the following areas: Seattle, Wash.; Fargo, N.D.; Orono, Me.; Durham, N.H.; Erie, Pa.; Cleveland, Paper no. 4198 of the Journal Series of the North Ohio; Somerville, N.J.; New Brunswick, N.J.; Tabor, Carolina State University Agricultural Experiment Iowa; Concordia, Kan.; Fort Collins, Colo.; Washing- Station, Raleigh, N.C. ton, D.C.; Richmond, Va.; Raleigh, N.C.; Pleasanton, 79 80 KLOOS, TORNABENE, AND SCHLEIFER INT. J. SYST. BACTERIOL.
Calif.; La Jolla, Calif.; Mesa, Ariz.; Austin, Tex.; San DNA base composition. The mol% of guanine plus Antonio, Tex.; Birmingham, Ala.; and Bartow, Fla. cytosine (G+C) in DNA was estimated by the thermal Samples from people of the Raleigh longitudinal denaturation method of Marmur and Doty (27). study were taken from two separate sites on the Genetic compatability with M. luteus. Transforma- forehead and one site from one cheek, one anterior tion of the M. luteus strain ATCC 27141 auxotrophs, nare, each axilla, each upper and lower arm, and each purE (ISU) and trpC23, was tested with donor DNA upper and lower leg. Samples were taken from similar from each strain. The transformation procedures used sites on people of the second group, with the were previously described by Kloos and Rose (20). exception that the anterior nare and axillae were Colony morphology and pigment. Cells from each omitted. These sites only occasionally produced culture were streaked for isolation on one-half micrococci and then usually in small numbers. portions of P agar plates and incubated for 5 days. At Representative strains of M. lylae, M. sedentarius, this time, the diameters of five widely isolated and M. kristinae are listed in Tables 1, 2, and 3, colonies were measured. Colonial characteristics, in- respectively. Additional strains that were analyzed for cluding profile, edge, surface texture, and pigment, only certain of the characters listed in these tables were observed daily throughout the incubation period. included M. lylae strains RS 14, RS 23, CPE 21, Cell morphology. Gram-stained smears were pre- JMWA 3, CS 11, HG 22, EDP 14, RW 13, RW 14, CR pared from 36-h-old cultures and examined for cell 13, KHND 24, KHND 25, KHND 2, BB 25, BB 13, morphology, staining properties, size, and aggregation DA 22, KAH 14, KAH 24, KAH 25, PH 11, PH 14, SD patterns. Several strains of each species were also 1, EF 23, and TF 14; M. sedentarius strains CPE 15, examined at 4-h incubation intervals over a 36-h SMCA 21, PH 1, RG 27, DBM 274, DBM 349, SM 93, period to check possible changes in cell morphology. and SM 15; and M. kristinae strains JB 11, RS 15, Motility. Motility was checked by using standard JMWA 11, LM 21, SG 11, MH 12, TB 12,DP26, KE procedures recommended for the Difco motility test 20, AB 2, MW 1, SR 13, and RG 1. medium (1). Procedures for isolating micrococci. Samples were Acetylmethylcarbinol production. The appearance obtained by using the following swab technique. of acetylmethylcarbinol was tested by the procedure Sterile cotton swabs were first moistened with a sterile of Coblentz (7). A loopful of a culture was suspended detergent containing 0.1% Triton X-100 (Packard) in in 5 ml of MR-VP broth. The inoculated broth tubes 0.075 M phosphate buffer, pH 7.9 (45). One swab was were shaken vigorously in a 34 C water bath for 48 h rubbed vigorously over each site (approximately 8 and then examined. cm’) on the forehead, cheek, apex of the axilla, or Nitrate reduction. Nitrate reduction was tested by hairy portions of the arms and legs. The anterior nare the sulfanilic acid and a-naphthylamine method and was sampled by swabbing the lining of the mucous confirmed with zinc dust (9). A loopful of a culture membrane. The duration of rubbing varied from 5 to was suspended in 5 ml of nitrate broth (Difco). The 15 s depending on the expected aerobic bacterial inoculated broth tubes were shaken vigorously in a 34 density for each region. Immediately after sampling, C water bath for 48 h and then examined. each swab was used to inoculate the entire surface of a Oxidase. Oxidase activity was determined on 18- to standard isolation agar plate (100-mm diameter). 24-h-old cultures by a modification of the technique Inoculated plates were incubated at 34 C for 4 days of Kovacs (25) using N,Ndimethyl-p-phenyl- and then examined. If necessary, plates could be enediamine monohydrochloride. stored at 4 C for several weeks before the isolation of Salt tolerance. Growth at different NaCl concentra- strains. The above procedure was designed to produce tions of 0, 5, 7.5, and 10% in P agar was estimated by 100 to 700 colonies per plate for most samples. culture-streak development after incubation for 48 h. Isolation medium. The isolating medium (P agar) Streaks were made by lightly inoculating a lcm line (32) was nonselective and had the following composi- on the surface of an agar plate with a loopful of a tion: peptone (Difco), 10 g; yeast extract (Difco), 5 g; culture. Up to 16 streaks could be radially inoculated sodium chloride, 5 g; glucose, 1 g; agar (Difco), 15 g; on each plate. distilled water, 1,000 ml. P agar shipped to various Optimal growth temperature range. Growth on P collecting points was supplemented with the mold agar at different incubation temperatures between 15 inhibitor cy cloheximide (50 pg/ml). and 50 C was estimated by culture-streak development Culture conditions. P agar was used for the after incubation for 48 h. Streaks were prepared maintenance and propagation of all strains. Unless according to the procedure described above for salt otherwise noted, all cultures were maintained at 4 C tolerance determinations. and incubated at 34 C; inocula for tests were prepared Aerobic requirement. The aerobic requirement of from 24- to 48-h-old cultures. each strain was estimated by using the semisolid Catalase. A loopful of a culture was suspended in 2 thioglycolate medium described by Evans and Kloos ml of physiological saline. One milliliter of a 3% H, 0, (13). A loopful of a culture was suspended in 2 ml of solution was then added to the cell suspension, and physiological saline. A 0.1 -ml sample (approximately any bubbling was recorded. lo7 colony-forming units) of the cell suspension was Benzidhe test. The presence of cytochrome-con- then used to inoculate a tube containing 8 ml of fluid taining respiratory systems was determined by the (52 C) thioglycolate medium. Inoculated tubes were benzidine test, according to the procedure of Deibel allowed to solidify at room temperature and then were and Evans (1 1). incubated for 5 days. Growth characteristics were Deoxyribonucleic acid (DNA) isolation. The proce- noted at this time. dures for culturing strains for DNA extraction and Simmons citrate agar. Simmons citrate agar contains DNA isolation have been previously described (19). ammonium phosphate and sodium citrate as the sole VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 8 1 sources of nitrogen and carbon. The ability of strains plates were incubated for 48 h and then examined. to grow on this medium was estimated by culture- Lysozyme sensitivity was interpreted according to the streak development and change in the color indicator following spot inhibition scheme: + = susceptible, of the medium after incubation for 72 h. Streaks were complete growth inhibition; k = slightly resistant, prepared according to the procedure described above partial growth inhibition; - = resistant, no visible for salt-tolerance determinations. growth inhibition. Inorganic nitrogen agar. The ability of strains to The second procedure was more quantitative than grow on inorganic nitrogen was estimated by using a the spot technique described above and involved defined agar medium containing NH, H, PO, and determining the minimal inhibitory concentration NH4C1 as the only sources of nitrogen. The composi- (MIC) of lysozyme in P agar. A loopful of a saline cell tion of the medium was as follows: K,HPO,, 2 g; suspension was placed on each agar plate containing a NH4C1, 1 g; NH,H,PO,, 10 g; MgSO4*7H20;70mg; different concentration of lysozyme, as part of a FeSO, -7H,O, 1 mg; MnC12-4H,0, 0.5 mg; sodium two-fold dilution series. Up to 16 strains could be lactate, 5 g; Special Agar Noble (Difco), 15 g; tested on each plate. Plates were incubated for 48 h deionized water, 1,000 ml. The pH was adjusted to and then examined for evidence of culture growth. 7.0. Preparation of the medium required separate Lysostaphin susceptibility. The effect of lyso- autoclave sterilization of agar and other ingredients. staphin on strains was tested by placing a drop of a Sterile sodium lactate was added after the medium was sterile lysostaphin solution (200 pg/ml) on an inocu- autoclaved and mixed. lated agar overlay plate. Lysostaphin susceptibility was Cells from each culture were streaked for isolation tested on the same agar plate used in the above and incubated according to the same procedures procedure for testing lysozyme. Drops of each enzyme described above for determining colony morphology preparation were placed several centimeters apart to and pigment production. The relative percentage of prevent interactions. Lysostaphin susceptibility was growth on the inorganic nitrogen agar to that on P interpreted by using the same scheme described above agar was estimated by the ratio I/P X 100, where I is for lysozyme susceptibility tests. the average colony diameter on the inorganic nitrogen Antibiotic susceptibility. Two procedures were used agar and P is the average colony diameter on P agar. to test the effect of various antibiotics on strains. The The results of this test were later used as a basis for first procedure is recommended for routine laboratory nutritional studies on amino acid and vitamin require- screening and involved using sensitivity disks (Difco) ments. on inoculated agar overlay plates. The preparation of Carbohydrate reactions. The ability of strains to inocula and agar overlay plates and the incubation produce acid aerobically from various carbohydrates conditions followed procedures similar to those was detected by using an agar plate method. This described above for lysozyme susceptibility testing. method was more sensitive and expedient than the Six different lowconcentration disks could be tested standard broth method (41). Each carbohydrate agar on one plate without noticeable interference. was prepared by adding an appropriate sample of a The second procedure involved determining the sterile carbohydrate stock solution to a sterile Purple MIC of various antibiotics in P agar. MIC determina- Agar Base (Difco) medium. The final carbohydrate tions followed procedures similar to those used above concentration in the agar medium was 1%. Strains for lysozyme susceptibility testing. were streaked on the surface of agar plates according Aliphatic hydrocarbon analysis. Procedures for the to the same procedure described above for salt culturing of strains, cell extraction, column fractiona- tolerance. A total of eight streaks were inoculated on tion, and gas chromatography have been previously each plate. Plates were incubated and then examined described by Tornabene and co-workers (42-44). All at 1, 3, 5, and 7 days. Reactions were interpreted hydrocarbons were run on a stainless steel capillary according to the following scheme: ++ = strong acid column (26 m by 0.05 cm) coated with 10% Silicone reaction, wide zone (3 1-cm radius) of yellow indi- OV-17; temperature and gas flow rates varied. cator color surrounding the culture streak in 24 h; + = Cell-wall preparation and determination of the moderate acid reaction, narrow to medium zone of peptidoglycan type. Cell walls were prepared by yellow indicator color surrounding the culture streak disrupting a thick cell-wall suspension with glass beads in 24 to 72 h; f = weak acid reaction, bright yellow in a cell mill (Vibrogen-Zellmuhle, Buhler, Tubingen). indicator color under culture streak, but not extending Before disintegrating, the cell suspension was boiled into the surrounding medium within 72 h; - = no acid for 30 min to inactivate autolytic enzymes. The crude reaction, very faint to no yellow indicator color under cell-wall preparations were purified by treatment with the culture streak within 72 h. trypsin (37). Lysozyme susceptibility. Two procedures were used Procedures for determining the type of peptido- to test the effect of egg-white lysozyme on strains. glycan have been previously described (37, 39). The The first procedure is recommended for routine different types are abbreviated according to the laboratory screening and involved placing a drop of a proposal of Schleifer and Kandler (39). sterile lysozyme solution (400 pg/ml), with the aid of Chemicals. Egg-white lysozyme (muramidase) was a 2-ml syringe, on an inoculated agar overlay plate. purchased from Sigma Chemical Co., St. Louis, Mo.; The agar overlay was prepared by first adding 0.1 ml lysostaphin was from Schwartz/Mann, Orangeburg, N. of a saline cell suspension (approximately lo7 Y.; N,Ndimethyl-p-phenylenediamine monohydro- colony-forming units/ml) to a tube containing 3 ml of chloride was from Eastman Organic Chemicals, Ro- fluid, soft P agar (0.75% agar). The contents were chester, N.Y.; and a-naphthylamine was from Mathe- swirled immediately and then poured on the surface of son Coleman and Bell, Cincinnati, Ohio. Cyclo- a dry P agar plate. After the application of lysozyme, heximide and penicillin G were purchased from 82 KLOOS, TORNABENE, AND SCHLEIFER INT. J. SYST. BACTERIOL.
Calbiochem, Los Angeles, Calif. Streptomycin sulfate Micrococci could be further distinguished was kindly supplied by Eli Lilly and Co., Indianapolis, from staphylococci by comparing their lyso- Ind.; sodium methicillin was supplied by Bristol zyme and lysostaphin susceptibilities (17, 26) Laboratories, Syrapuse, N. Y.; and erythromycin base by using a simple and expedient spot test. was supplied by Abbott Laboratories, North Chicago, 111. Micrococci demonstrated a wide range of susceptibility to lysozyme, from very suscepti- ble to resistant, and were resistant to lyso- RESULTS AND DISCUSSION staphin. However, certain strains that were very susceptible to lysozyme occasionally showed Identification of the Micrococci. Preliminary evidence of a slight susceptibility to lyso- identification of skin isolates of micrococci staphin. Staphylococci were resistant to lyso- could, in most instances, be accomplished on zyme but were suceptible to lysostaphin by this the basis of colony morphology, growth rate, test. and pigment. Colonial characteristics became The G + C content of DNA from selected more distinguishable with age, and by 4 days of isolated strains of each Micrococcus species had incubation, the colonies were usually well a range of 66.0 to 72.1 mol% and was similar to developed. Micrococcus colonies were circular that reported by others for members of the and entire in smooth strains. They developed genus Micrococcus (22-24). much slower and were characteristically more Characterization of Micrococcus species. convex than Staphylococcus colonies com- Many of the characters that we chose to use in monly found on the same isolation plates. the classification of human cutaneous micro- However, a few uncommon strains,, produced cocci have been previously used in taxonomic colonies with an unusually lob convex profile studies of micrococci (19, 22-24, 3 1, 39). We that could be confused with that of certain also examined some additional characters, and staphylococci. Occasionally, colonies could be those which proved to be particularly usefulin confused with those of corynebacteria, Arthro- delineating species included colony morphol- bacter, or “Mycobacterium ” rhodochrous ogy, patterns of pigment development, nitrogen (kindly analyzed by Ruth E. Gordon, Rutgers requirements, some additional carbohydrate University, New Brunswick, N. J.). reactions, and antibiotic susceptibilities. Cells of micrococci appeared as typical Some attempt at strain identification was gram-positive or gram-variable cocci by light necessary for micrococci isolated from the same microscopy. Cell diameters ranged from 0.7 to person, where clonal populations appeared to 1.8 pm. Most species produced an abundance of be rather common. Strains were initially re- tetrads; however, some strains of certain species solved on the basis of differences in colony produced numerous diplococci. Cell morphol- morphology or pigment, or both. When avail- ogy did not change with culture age, and this able, at least 10 colonies of each suspected feature helped to distinguish suspected micro- strain were isolated, and their subcultures were cocci from coryneform bacteria, which demon- then examined for growth patterns in a strated cell elongation, e.g., in young cultures thioglycolate medium, carbohydrate reactions, (3,8, 15). growth on inorganic nitrogen agar, and anti- The micrococci were nonmotile, non-spore- biotic susceptibilities. In our analysis of strains, forming, and positive for catalase and benzidine it followed that an uncommon characteristic(s) tests (22-24). was considered to be a more accurate strain The growth of micrococci appeared in several marker than a common characteristic(s). Re- different patterns in the semisolid thioglycolate sults indicated that differences in colony medium. It usually occurred on or just beneath morphology or pigment were usually associated the agar surface or within an upper, aerobic, with differences in other characteristics, sug- green zone. One particular species produced gesting that our initial identification of strains tiny colonies in the lower anaerobic portion of on the isolation plates was reasonably accurate. this medium. By comparison, most strains of Descriptions of each Micrococcus species isolated staphylococci demonstrated good dif- isolated from human skin in this study are fuse growth or developing colonies in the presented in the following paragraphs. anaerobic portion. One human Staphylococcus (a) M. luteus (Shroeter 1872) Cohn 1872. species either failed to produce detectable Colonies were usually convex and uniformly growth or produced only a few small colonies pigmented; however, several uncommon strains in the anaerobic portion and might be occa- produced raised colonies with translucent, sionally confused with micrococci by this test depressed centers. The pigment of colonies (W. E. Kloos, K. H. Schleifer, and R. F. Smith, varied considerably but was usually different unpublished data). shades of yellow or cream white. Cream white VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 83
or unpigmented strains were more frequently resistant, to lysozyme. Most were slightly isolated from individuals living in northern resistant to methicillin and susceptible to states. streptomycin and erythromycin. All were sus- Most strains demonstrated good growth on ceptible to penicillin, novobiocin, tetracycline, inorganic nitrogen agar. Most of the auxo- chloramphenicol, neomycin, vancomycin, kana- t ro phic strains were stimulated by met hionine mycin, and polymyxin B. or cysteine and to a lesser extent by aromatic The hydrocarbon composition of six selected amino acids (J. W. Farrior and W. E. Kloos, strains was similar to that found in reference Abst. Annu. Meet. Amer. SOC.Microbiol., 73rd, strains of M. luteus (31, 43, 44). The major Miami Beach, p. 180, 1973). fractions included monounsaturated branched Most strains were susceptible to lysozyme, C27, C28, and C29 aliphatic hydrocarbons. susceptible to penicillin, erythromycin, strepto- Cell-wall peptidoglycans of seven selected mycin, and kanamycin, and slightly resistant to strains contained aspartic acid, lysine, glutamic methicillin. Approximately 1% of the strains acid, and alanine. The peptide patterns of were resistant to erythromycin, streptomycin, partial hydrolysates of M. lylae cell walls were or kanamycin. All were susceptible to novo- identical to those of cell walls of Streptococcus bio cin, tetracycline, chloramp henicol, neo- faeczum ( 16). Dinitrophenylation of the cell mycin, vancomycin, and polymyxin B. Other wall, however, indicated that, in contrast to cell characteristics were similar to those previously walls of S. faecium, a rather large amount of reported for reference strains of M. luteus (2, the a-amino groups of lysine residues was not 19, 24, 39,40). substituted. In some strains, the amount of (b) M. lylae sp. nov. (ly’lae. M.L. gen.n. lylae glutamic acid was significantly reduced. In of Lyla; named for Lyla Kloos, from whom this order to determine the amino acid sequence of organism was originally isolated.) Colonies were the peptide subunit, we first inhibited growing convex, entire, smooth, circular, and usually cells of strain ATCC 27566 with D-cycloserine unpigmented or cream white. Cells were gram- and isolated the nucleo tide-act ivat ed pep tid o- positive cocci and were 0.8 to 1.6 pm in glycan precursors (10). The amino acid se- diameter. They were nonmotile, non-spore- quence of the isolated tripeptide was: uridine- forming, and arranged predominantly in tetrads. 5 ’-diphosphat e-muramyl-L-alanyl-y-D-glutamyl- The optimal growth temperature range was 25 L-lysine. This proves that the normal peptide to 37 c. subunit was present in the peptidoglycan of Transformation of reference M. luteus auxo- these cell walls. The occurrence of unsubsti- trophs occurred at about 5% of homologous tuted a-amino groups of lysine in the intact values, suggesting considerable divergence in peptidoglycan may have been due to the action these two species, but, nevertheless, demon- of a powerful endopeptidase which splits the strating a significant genetic relationship. peptide bond between the y-carboxyl group of Growth occurred only in the aerobic portions glutamic acid and the a-amino group of lysine. of the thioglycolate medium, indicating a strict Strains containing a reduced amount of glu- aerobic requirement. All strains were positive tamic acid also contained a rather low amount for catalase and benzidine tests and were of L-alanine. These strains were also more oxidase positive. Most strains failed to produce susceptible to the action of lysozyme. By acetylmethylcarbinol and reduce nitrates. Most isolating peptidoglycan fragments of a lyso- grew well at all NaCl concentrations tested zyme lysate of cell walls of strain ATCC 27567 from 0 to 10%. by using Sephadex G50 and paper chromatogra- Strains grew poorly or failed to grow on phy, we found a rather high proportion of inorganic nitrogen agar and had an absolute u n su bstituted N-acetylglucosamine-N-acetyl- requirement for methionine (J. W. Farrior and muramic acid saccharides. This finding can W. E. Kloos, Abst. Annu. Meet. Amer. SOC. explain the rather low amount of D-glutamic Microbiol., 73rd, Miami Beach, p. 180, 1973). acid and L-alanine in the peptidoglycan. As in Except for one slow-growing strain, strains other strains of this group, the peptide linkage failed to grow on Simmons citrate agar. between D-glutamic acid and L-lysine is split, Most strains failed to produce detectable acid and thus the action of common N-acetylmura- from glucose, fructose, mannose, maltose, or mic acid-L-alanine amidases may release the sucrose. All failed to produce acid from dipeptide L-Ala-D-Glu which is bound to galactose, rhamnose, xylose, ribose, lactose, muramic acid. The rather high amount of glycerol, sorbit 01, arabinose, adonitol, dulcitol, muramic acid residues found which were not raffinose, melibiose, or mannitol. substituted by a peptide subunit also supports Approximately one-half of the strains were this contention. The peptidoglycan type, how- resistant, and the other half were slightly ever, is not changed by the action of cell-wall 84 KLOOS, TORNABENE, AND SCHLEIFER INT. J. SYST. BACTERIOL. lytic enzymes and can be written for all strains Simmons citrate agar: No growth. as L-Lys-Asp . Acetylmethylcarbinol: None produced. The G + C content of the DNA, as Acid from carbohydrates: None from galac- determined in six strains, is 68.6 f 0.9 mol%. ' tose, rhamnose, xylose, ribose, arabinose, lac- Some variable characters and the G + C tose, glycerol, sorbitol, adonitol, dulcitol, raf- content of DNA in representative strains of M. finose, melibiose, or mannitol. Zylae are shown in Table 1. Antibiotic susceptibilities: Susceptible to Strain ATCC 27566 (originally designated JL novobiocin, tetracycline, chloramphenicol, neo- 178) is the type strain of M. ZyZae. A mycin, vancomycin, kanamycin, and poly- description of this strain follows. myxin B. Spheres, 0.9 to 1.2 pm in diameter, occurring Other characters and the G + (2 content of predominantly in tetrads, occasionally in pairs. the DNA of this strain are shown in Table 1. Nonmotile. Non-sporeforming. Gram positive. M. ZyZae can be distinguished from the closely Cell-wall peptidoglycan: L-Lys-Asp. related species M. Zuteus by lysozyme suscepti- Aliphatic hydrocarbons: Predominantly br-A- bility, genetic compatibility, and the type of C27, br-A-C28, and br-A-C29. cell-wall peptidoglycan. There are also some Agar colonies: Circular, entire, 4 to 5 mm in differences between these species in the param- diameter, convex, smooth with glistening sur- eters of pigmentation, nitrogen requirements, f ace. U np ig m en t ed. nitrate reduction, acid from maltose and suc- Chemoorganotrophic; metabolism respira- rose, and susceptibility to penicillin and methi- tory. cillin. Several of these parameters overlap Strictly aerobic. slightly with those of M. varians. Genetic compatibility with M. Zuteus: Per- (c) M. sedentarius ZoBell and Upham 1944, centage of homologous transformation of purE 260. The original description of this species was = 2.0; trpC23 = 3.7. reported by ZoBell and Upham (47) and was
TABLE 1. Variable characters of 20 strains of Micrococcus lylae - - ..,
c 4cid from .^ 2 M 1 5
Yz E c G.2 -i G 3 O .2an -3 3 2 % 2- ss€s -3 2- 3 M- G+C gs: 2 3E1) 3 az Strain (%I) 3.5 23 3 -8 -za - ATCC 27566 67.1 3 + > 1,600 6.2 0.05 0.2 1.6 ATCC 27567 ND~ 13 f 6.2 0.8 0.01 0.0: 0.8 ATCC 27568 72.2 6 5 6.2 0.8 0.02 1.6 0.4 ATCC 27569 ND 2 + > 1,600 6.2 0.05 0.2 1.6 RM 324 67.7 3 + 6.2 0.2 0.0 1 0.1 0.8 TW 226 70.5 1 + > 1,600 6.2 0.05 0.2 1.6 WK 312 67.0 2 + > 1,600 6.2 0.05 0.2 1.6 HK 62 ND 1 + > 1,600 6.2 0.05 0.2 1.6 SL 166 ND <1 + 25 6.2 0.1 0.1 3.1 DM 5 ND <1 + 3.1 0.8 0.05 0.1 6.2 MAW 332 ND 8 + 6.2 1.6 0.02 0.1 0.8 MAW 262 ND 2 + 12.5 6.2 0.1 0.2 0.8 MK 312 67.3 2 + > 1,600 6.2 0.05 0.2 1.6 DW 154 ND 1 + > 1,600 6.2 0.05 0.2 1.6 SM 144 ND <1 f 6.2 1.6 0.02 0.8 3.1 SM 249 ND 2 + > 1,600 0.2 0.1 0.1 0.8 RK 1 ND <1 * 25 0.2 0.02 0.1 15 CK 1 ND <1 * > 1,600 6.2 0.05 0.2 1.6 EK 5 ND <1 f > 1,600 6.2 0.02 0.1 1.6 + AS 1 -ND <1 - 3.1 -0.4 0.01 0.1 6.2 a LY, light yellow; YW, yellow-white; CW, cream white; PW, pink-white; -, unpigmented. Calculated from the ratio I/P X 100 (see text). Reaction: +, positive; ?, weak; -, negative. ND, not determined. VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 85 based on one strain, designated 541 by ZoBell, Most strains were susceptible to lysozyme. that was isolated from a slide submerged in sea All were resistant to methicillin and penicillin, water. This strain has since been deposited in and this property appears to be species specific. the American Type Culture Collection and the Most were susceptible to erythromycin; how- Czechoslovak Collection of Microorganisms, ever, 28% of the strains were resistant or where it is referred to as ATCC 14392 and CCM slightly resistant to this antibiotic. Resistant 3 14, respectively. strains were isolated from individuals living in During the course of this study, we isolated a several different regions of the United States number of strains of M. sedentarius from and did not appear to be localized in any human skin. Since numerous strains were specific area. Hence, it would appear that available for analysis, we are able to provide a erythromycin resistance is a relatively common more comprehensive estimate of the parameters characteristic of this species. Most strains were of this species. suceptible to chloramphenicol and kanamycin. Colonies were, convex to pulvinate, entire, Ten percent of the strains were resistant to circular, and usually smooth, and developed chloramphenicol or kanamycin. All were sus- rather slowly. Colonies were commonly cream cept ib le to streptomycin, novo biocin , tetra- white or deep buttercup yellow. Many strains cycline, neomycin, vancomycin, and polymyxin produced a brownish exopigment. Cells were B. gram-positive or gram-variable cocci which were The major aliphatic hydrocarbons of 10 usually embedded in a gram-negative, slime-like selected strains of this species had significantly layer. The cell diameter was 0.8 to 1.1 pm. longer carbon chains than those found in other Cells were nonmotile and non-sporeforming and species. They were predominantly monoun- were arranged predominantly in tetrads or saturated branched C30, C3 1, and C32 aliphatic tetrads in cubical packets. The optimal growth hydrocarbons. A few strains also had major temperature range was 28 to 36 C. This was fractions of monounsaturated branched C28 or significantly higher than the 20 to 25 C range C33 and/or normal C32 aliphatic hydrocarbons. originally reported for the ZoBell and Upham Cell walls of this species were distinguished strains (47). by a rather large amount of glutamic acid per Transformation of reference M. luteus auxo- mol of peptide subunit. A part of the glutamic trophs occurred at about 0.1 to 0.8% of the acid, however, does not belong to the peptido- homologous value. This presumed transforma- glycan since it can be easily extracted with tion is approaching the lower limit of our test trichloroacetic acid at 60 C after treatment for to detect recombination (0.05 to 0.1%) but, 6 h. Therefore, we used trichloroacetic acid- nevertheless, appears to be significant. The extracted cell walls for further studies. The cell results would suggest a distant but detectable walls of all nine strains tested contained lysine, genetic relationship between M. sedentarius and glutamic acid, and alanine with about 3 mol of M. luteus. glutamic acid per mol of lysine. The peptide Growth occurred only in an upper, aerobic, patterns of the partial acid hydrolysates of green zone of the thioglycolate medium, indi- these cell walls were identical and indicated cating a strict aerobic requirement. All strains that, besides the normal peptide subunit (Mur- were positive for catalase and benzidine tests L-Ala-y-D-Glu-L-Ly s-D-Ala), a y-glu tamy l-glu- and failed to produce acetylmethylcarbinol. tamic acid peptide occurs in the interpeptide Most failed to reduce nitrates, were oxidase bridge. The occurrence of N-terminal glutamic negative, and grew well at NaCl concentrations acid confirms the finding that the interpeptide up to 7.5 to 10%. bridge consists of glutamic acid residues. In All strains were unable to grow on inorganic addition to N-terminal glutamic acid, we also nitrogen agar and had an absolute requirement found N-terminal L-alanine. The position of for methionine (J. W. Farrior and W. E. Kloos, this alanine has not yet been identified. In one Abst. Annu. Meet. Amer. SOC.Microbiol., 73rd, strain, ATCC 27575, a higher amount of Miami Beach, p. 180, 1973). Most strains also alanine occurs in the peptidoglycan than in that required tyrosine, arginine, valine, lysine, leu- of the other strains. This high amount of cine, and pantothenic acid. Strains failed to alanine is matched by the presence of penta- grow on Simmons citrate agar. peptide subunits (Mur-L-Ala-y-D-Glu-L-Lys-D- Most strains failed to produce detectable acid Ala-D-Ala), and this was proved by the isolation from glucose and lactose. All failed to produce of a D-alanyl-D-alanine peptide from partial acid from fructose, galactose, mannose, rham- acid hydrolysates of cell walls. Although it is nose, xylose, ribose, maltose, sucrose, glycerol, not yet possible to give the exact primary sorbitol, arabinose, adonitol, dulcitol, raffinose, structure of the peptidoglycan of this species, it melibiose, or mannitol. is rather clear from the quantitative amino acid 86 KLOOS, TORNABENE, AND SCHLEIFER INT. J. SYST. BACTERIOL. composition and the peptide patterns of the uncertain. Mur-L-Ala-y-D-Glu-L-Lys-D-Ala sub- partial acid hydrolysates that all strains belong unit, y-glutamyl-glutamic acid peptide bridge, to the same peptidoglycan type. It is interesting and N-terminal L-Ala and Glu. that most of the interpeptide bridges contain Aliphatic hydrocarbons: Predominantly brAA- y-glutamyl-glutamic acid, as in the case of some C3 1, br-A-C32, and n-A-C32. coryneform bacteria (4). However, in the Agar colonies: Circular, entire, 3 to 3.5 mm coryneform bacteria, meso-diaminopimelic acid in diameter, pulvinate, and smooth with a dull occurs in position 3 of the peptide subunit, surface. Cream white pigment. whereas in M. sedentarius strains, L-lysine is Chemoorganotrophic; metabolism respira- occupying this position. The above situation tory. may indicate a distant relationship of M. Strictly aerobic. sedentarius to certain coryneform bacteria. Genetic compatibility with M. Zuteus: Per- The G + C content of the DNA, as centage of homologous transformation of purE determined in six strains, is 67.7 k 0.6 mol%. = 0.2; trpC23 = 0.4. Some variable characters and the G + C content of the DNA in representative strains of Simmons citrate agar: No growth. M. sedentarius are shown in Table 2. Inorganic nitrogen agar: No growth. ZoBell’s strain 541 (ATCC 14392; CCM 314) Acetylmethylcarbinol: None produced. is here designated as the type strain of M. Acid from carbohydrates: None from fruc- sedentarius. A description of this strain is tose, galactose, mannose, rhamnose, xylose, presented below. ribose, maltose, lactose, sucrose, glycerol, sorbi- Spheres, 0.8 to 1.O pm in diameter, occurring tol, arabinose, adonit 01, dulcit 01, raffinose, predominantly in tetrads. Nonmotile. Non- melibiose, or mannitol. sporeforming. Gram positive. Antibiotic susceptibilities: Susceptible to Cell-wall peptidoglycan: Primary structure streptomycin, novobiocin, tetracycline, chlor-
TABLE -2. Variable characters of 20 strains of Micrococcus sedentarius Acid from
% 5 G + (1 Strain (%) iz6 CCM 314 69.3b cw + 1.6 200 25 6.2 ATCC 27573 NDe BY f 6.2 100 12.5 12.5 ATCC 27574 67.4 cw + 0.4 400 25 3.1 ATCC 27575 ND cw f 0.4 800 50 0.1 MK 136 66.0 CY f 1.6 800 50 0.05 PM 451 ND cw + 0.4 8 00 25 0.02 DM 51 67.2 BY + 3.1 200 25 0.1 DM 140 ND cw + 0.4 400 25 0.05 TW 93 ND cw + 25 400 25 25 DBM 420 70.6 cw + 1.6 400 25 0.1 SM 326 67.3 BY f 1.6 200 25 0.2 SM 328 ND BY 2 1.6 200 25 6.2 TM 61 ND cw + 3.1 400 12.5 0.05 KH 234 ND cw + 1.6 400 25 0.05 AW 68 ND cw + 25 400 25 6.2 CM 259 ND cw + 6.2 400 25 0.1 RM 327 ND cw + 3.1 400 25 0.05 BB 4 ND cw + 1.6 800 25 0.05 JB 1 ND CW + 12.5 400 25 3.1 KS 13 ND YW + -1.6 800 25 0.05 a BY,buttercup yellow; CY,cream yellow; YW,yellow-white; CW,cream white. G + C content of strain CCM 314 was determined by Rosypal et al. (36). Reaction: + = positive; +_ = weak; - = negative. ZoBell and Upham (47) reported the production of acid from glucose (and maltose) for the original strain. ND, not determined. VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 87 amphenicol, neomy cin, vancom ycin , kana- required cysteine and either thiamine alone or mycin, and polymyxin B. thiamine and pantothenic acid (J. W. Farrior Other characteristics and the G+C content of and W. E. Kloos, Abstr. Annu. Meet. Amer. the DNA of this strain are shown in Table 2. SOC. Microbiol., 73rd, Miami Beach, p- 180, M. sederztarius can be distinguished from 1973). Eisenberg and Evans (12) have previ- other species of Micrococcus by penicillin and ously reported the stimulation of some refer- methicillin resistance, nitrogen requirements, ence strains of M. roseus by thiamine. genetic compatibility , aliphatic hydrocarbons, Most strains produced acid from rhamnose, peptidoglycan, and, to a lesser extent, by xylose, and sorbitol. The production of acid exopigment. from rhamnose appears to be species specific. (d) M. varians Migula 1900. Colonies of M. Strains were either resistant or slightly varians were usually difficult to distinguish resistant to lysozyme, very susceptible to M. Zuteus. from those of However, most strains methicillin and penicillin, and susceptible to produced colonies with a convex profile lower erythromycin, streptomycin, novobiocin, tetra- M. Zuteus, than that of colonies of strains of cycline, chloramphenicol, neomycin, vanco- and young colonies showed a distinctly lighter mycin, kanamycin, and polymyxin B. pigment near the colony edge. Some uncom- mon strains developed an ornate matted or Other characteristics were similar to those wrinkled surface that appears to be species previously reported for reference strains of M. specific. The pigment of the colonies varied roseux (23). considerably, but was usually different shades (f) M. kristinae sp. nov. (kris’ti”’nae. M. L. of yellow. Colonies or culture streaks were gen.n. kristinae of Kristin; named for Kristin usually sticky in consistency. Holding, from whom this organism was origi- Most strains failed to produce acetylmethyl- nally is0 late d .) carbinol. Colonies were convex to umbonate, entire or Most strains grew poorly or failed to grow on crenate, smooth or rough, and circular. Young, inorganic nitrogen agar. Most of the auxo- 2- to 3-day-old colonies were very distinctive trophic strains were stimulated by methionine and could be readily identified by their small or cysteine and thiamine (J. W. Farrior and W. size, high convex profile, and pale cream to pale E. Kloos, Abst. Annu. Meet. Amer. SOC. orange pigment. By 4 days of incubation, the Microbiol., 73rd, Miami Beach, p. 180, 1973). pigment became intensified in the center of the All strains were resistant to lysozyme, very colony, and the colony profile appeared less susceptible to methicillin and penicillin, and convex and more umbonate. The internal susceptible to novobiocin, tetracycline, chlor- structure of colonies in most strains was amphenicol, neomycin, vancomycin, kana- granular, and cells were very difficult to mycin, and polymyxin B. Most were sensitive disperse in aqueous solutions or media. Cells to erythromycin and streptomycin. As with M. were gram-positive cocci and were 0.7 to 1.1 sederztarius, a significant proportion of strains pm in diameter. They were nonmotile and (18%) was resistant or slightly resistant to non-sporeforming and were arranged in tetrads erythromycin. Eight percent of the strains were that were usually found in large, adherent resist ant to streptomycin. clusters. The optimal growth temperature range Other characteristics were similar to those was 26 to 38 C. previously reported for reference strains of M. We were unable to detect transformation of varians (22). M. luteus reference auxotrophs by DNA prepa- (e) M. ruseus Flugge, 1886. All strains rations from strains of M. kristinae. produced smooth, glistening colonies that were Growth occurred both in an upper, aerobic, different shades of pastel or orange-red. The green zone and as tiny colonies in the anaerobic profile of the colonies, like that in M. varians, portion of the thioglycolate medium. The was usually low convex. Also, pigment ap- appearance of colonies deep in the thio- peared lighter near the edges of young colonies. glycolate tube would suggest that this species The colonies of some strains had a glistening can grow anaerobically to a limited extent and, wet appearance, and these could be confused therefore, is slightly facultatively anaerobic. All with similarly pigmented strains of Corynebac- strains were positive for catalase and benzidine terium equi and LLMycobacteriurn” rhodo- tests and were oxidase positive, and they chrous. produced acetylmethylcarbinol. Most strains Most strains produced low levels of acetyl- failed to reduce nitrates and grew well at all met hylcarbinol. NaCl concentrations tested from 0 to 10%; Most strains were unable to grow on inor- strains grew very poorly at an NaCl concentra- ganic nitrogen agar. The auxotrophic strains tion of 15%. 88 VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 89
TABLE 3. Variable characters of 20 strains of Micrococcus kristinae
Acid from .L f! 9 c3 E: -..d .--n 3 .Y E Strain G + C (%) ha5%
ATCC 27570 66.8 PO 2 f ++ + 0.2 0.05 ATCC 2757 1 66.2 PO 3 + ++ + 0.2 0.05 ATCC 27572 66.5 PC
All strains grew poorly on inorganic nitrogen M. kristinae was generally more active in the agar. Preliminary nutritional studies have indi- production of acids from carbohydrates than cated some multiple amino acid and vitamin were the other species studied. All strains requirements (J. W. Farrior and W. E. Kloos, produced acid from glucose, fructose, mannose, Abst. Annu. Meet. Amer. SOC.Microbiol., 73rd, sucrose, and glycerol. Most produced acid from Miami Beach, p. 180, 1973). Most strains were maltose and sorbitol, and some produced acid stimulated by the combination of leucine, from galactose and lactose. The strong produc- lysine, valine, tyrosine, and niacin. Some were tion of acid from mannose was specific for this stimulated by met hio nine, leu cin e, aspar tic species. All strains failed to produce acid from acid, or thiamine, in addition to the other rhamnose, xylose, ribose, arabinose, adonitol, metabolites. Those that were not stimulated by dulcitol, raffinose, melibiose, or mannitol. methionine were slightly inhibited by cysteine. All strains were resistant to lysozyme and Except for one slow-growing strain, strains susceptible to methicillin, penicillin, erythro- failed to grow on Simmons citrate agar. mycin, streptomycin, novobiocin, tetracycline,
FIG. 1. Colonies of Micrococcus species isolated from human skin. Colonies were 5 days old. The “seed” inoculum was first incubated at 34 C for 3 days and then allowed to remain at 25 C for 2 days. A, M. luteus; top row, fall rows read kftto right), DM25.5, GH210, TW 285, PM 187; middle row, TW I, PM231, HK 255. MA W 72; bottom row, PM 184, PM 167, DM 21 7, TW 240. B, M. lylae; top row, RM 324, ATCC 27566 (JLl78); bottom row, MAW 262, ATCC 27568 (TW 265). C, M. varians; top row, MAW 64, LK 132, RG 24; middle row, PM 180, TM 1 75, KL 15; bottom row, TM 206, EK 11, PM 25. D, M. kristinae; top row, A W 220, A TCC 275 70 (PM 129); bottom row, ATCC 27571 (KH 189), JM 8. E, M. roseus; top row, JL 208, KH 6, CM 160; bottom row, CJ 23, HK 270, RM 337. F, M. sedentarius; top row, ATCC27574 (PM450),ATCC27575 (GH252/,MK 136; bottom row, ATCC 27573 (DM lo), DM 1. G, M. nishinomiyaensis; top row JL 205, A W 112,; bottom row, GH 223, SK 3. H, strains of uncertain taxonomic status; top row, WK 132, LK 335, CM 9; middle row, SM 239, KR 13, KE 11; bottom row, VN 6. X 2.5. Ph)h)Oh,NO OPOWWOh,
+I
Ph,
cc. r woowh,oro rnOO\DPOro NA Vlr Buttercup yellow I h, Chrome yellow m4 c h,
w LL h, oh, Light yellow -1
rcL N+OP yellow-white
unpigm ented wl Pink-white
c r A r Orange
cream-white edge Pastel red I
CI 0 Orange-red I Smooth glistening
Smooth dull
h, h, Vlr Stippled dull Rough Ornate matted Tetrads and packets Tetrads, < 10% diploco cci
+ PW N OW 0-
VI wlr Diplococci, 10- 2 w 00 - 40%tetrads Diplococci, Percent homologous c \o transformation Growth on Growth on 4P of M. luteus Growth on inorganic Simmons Acetylmethyl- ATCC 27 14 1 10% nitrogen ci t ra t e carbinol Nitrate Oxidase Total (R and SR) 1 NaCl agara agara productiona reduction' activitya no. of _- - strains purE trpC23 + 2 - - + f- ++- Species __ - M. luteus 402 8122 7922 64 18 18 100 2 6 92 75 20 5 M. lylae 21 521 521 14 86 90 10 5 85 75 25 M. sedentarius 24 0.3 * 0.2 0.5 + 0.2 100 100 4 96 4 96 M. varians 106 <0.1 0.1 100 6 23 71 100 60 74 11 15 1 25 74 M. roseus 20 <0.1 <0.05 5 95 15 75 15 10 35 65 M. kristinae 24 TABLE 6. Carbohydrate reactionsof Micrococcusspecies: monosaccharidesa I Total I D-Glucose D-Galact ose D-Mannose 1 L-Rhamnose D-Xylose N no. of ~ - ~ Species strains ++b + ++++- ++ +-++_- + ~ __ ~ M. luteus 375 <1 8 92 <1 99 100 7% M, lylae 38 3 5 92 8 92 100 5 M. sedentarius 26 4 19 77 100 100 M. varians 111 26 70 4 96 4 20 80 100 38 M. roseus 20 25 65 10 85 15 100 --$40 M. kristinae 30 100 100 10 90 100 M. nishinomi- 50 30 6 64 4 96 24 36 40 2 100 yaensis - - Data are given as percentage of strains. Reactions: ++, strong acid; +, moderate acid;+, weak acid; -, negative. Reactions were performed under aerobic'conditions. 92 KLOOS, TORNABENE, AND SCHLEIFER IN. J. SYST. BACTERIOL. TABLE 7. Carbohydrate reactions of Micrococcus species: disaccharides and sugar alcoholsa Disaccharides Sugar alcohols Total Maltose Lactose Sucrose Glycerol Sorbitol no. of - - - - Species strains ++ + + ++ ~ - - ~ M. luteus 375 14 65 100 100 M. lylae 38 90 100 100 M. sedentarius 26 4 100 100 100 M. varians 111 16 6 24 100 100 M. roseus 20 30 60 25 M. kristinae 30 80 13 10 100 7 M. nishinomi- 50 100 100 712 26 62 100 100 yaensis - - - - a Reactions and data are expressed as in Table 6. TABLE 8. Lysozyme susceptibility of Micrococcus species Level of susceptibility' Total no. of 0.1- 0.4- 1.6- 6.2- 25- 100- 400- Species strains 0.2 (+) 0.8 (+) 3.1 (+) 12.5 (2) 50 (2) 200 (?) 800 (-) 21,600 (-) 14 25 51 10 <1 M. lylae 42 9 24 19 48 M. sedentarius 25 28 40 24 8 M. varians 110 5 21 74 M. roseus 20 10 50 20 20 M. kristinae 24 100 M. niihinomi- 33 21 9 12 58 y aensis ' Expressed as MIC (numbers) and spot inhibition (symbols in parentheses). MICs are expressed as micrograms per milliliter. Spot inhibition is expressed as: +, complete inhibition; f, partial inhibition; -, no inhibition. Data are given as percentage of strains. chloramphenicol, neomycin, vancomycin, kana- The G+C content of the DNA, as determined mycin, and polymyxin B. in six strains, is 66.8 f 0.2 mol%. Six of the seven selected strains had pre- Some variable characters and the G+C con- dominantly monounsaturated branched C28 tent of the DNA in representative strains of M. and C29 aliphatic hydrocarbons. One strain had kristinae are shown in Table 3. predominantly C29 and C30 aliphatic hydro- Strain ATCC 27570 (originally designated carbons. These patterns are- unique to this PM 129) is the type strain of M. kristinae. A species. description of this strain follows. The amino acid composition of hydrolysates Spheres, 0.7 to 1.O ym in diameter, occurring of purified cell walls of M. kristinae is rather in tetrads and tetrad clusters. Nonmotile. similar to that of M. varians and M. roseus Non-sporeforming. Gram-positive. strains. Comparison of the peptide patterns of Cell wall peptidoglycan: L-Lys-L-Ala3-4. partial acid hydrolysates of cell walls of M. Aliphatic hydrocarbons: Predominantly br- kristinae strains with that of M. roseus or M. &C28 and br-A-C29. varians indicated that they contain the same Agar colonies: Circular and slightly crenate, 2 peptidoglycan type, viz., L-Lys-L-Ala34. to 2.5 mm in diameter, slightly umbonate, Further studies have shown that cell walls of slightly rough surface, granular structure. Pale M. kristinae contain additional glucosamine as a orange pigment in colony center becoming constituent of the cell-wall polysaccharide. M. lighter toward the edge. roseus and M. varians do not have the addi- Che moorganot ro phic ; metabolism respira- tional glucosamine but rather contain other, as tory. yet unidentified, amino sugars in their cell-wall Slightly fa cult atively anaerobic . polysaccharide. Genetic compatibility with M. luteus: None VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 93 TABLE 9. Methicillin susceptibility of Micrococcus species Level of susceptibilitya ~ 0.006- 0.025- 6.2- Total 0.012 0.05 12.5 400-800 Species strains (20-18) (17-15) (14-12) (11-8) (74) (3-0) (0) M. luteus 321 <1 91 M. lylae 40 60 M. sedentarius 26 81 M. varians 102 29 48 M. roseus 20 10 90 M. kristinae 23 M. nishinomi- 37 yaensis a Data are expressed as MIC and disk zone inhibition (numbers in parentheses). MICs are expressed as micrograms per milliliter. Disc zone inhibition represents the width (in millimeters) of the zone of antibiotic inhibition, measured from the edge of a 5-pg methicillin sensitivity disk (Difco). TABLE 10. Penicillin G susceptibility of Micrococcus species ~~ I Level of susceptibilitya I 0.025- Total no. 0.05 0.1-0.2 0.4-0.8 1.6-3.1 6.2-1 2.5 25-50 Species of strains (15-1 2) (1 1-8) (7; (0) (0) I M. luteus 43 1 21 70 1 M. lylae 31 74 7 M. sedentarius 26 19 81 M. varians 106 21 33 26 17 31 M. roseus 20 90 10 I M. kristinae 25 8 76 16 M. nishinomi- 39 69 1 31 ya en sis I Levels of susceptibility and data are expressed as in Table 9, except that a 2-unit penicillin G sensitivity disk (Difco) was used for disk zone inhibition determinations. detectable, i.e., <0.1% homologous transforma- growth. M. kristinae appears to be somewhat tion. related to M. roseus and M. varians and also Simmons citrate agar: No growth. possibly to a halophilic strain designated M. Acetylmethylcarbinol produced. haEobius (34). Acid from carbohydrates: Acid from glucose, (g) M. nishinorniyaensis Oda 1935. Colonies fructose, mannose, sucrose, and glycerol. No of this species are easily distinguished from the acid from rhamnose, xylose, ribose, arabinose, colonies of other Micrococcus species, but adonitol, dulcitol, raffinose, melibiose, or man- occasionally they can be confused with those of nitol. certain coryneform bacteria. Most strains pro- Antibiotic susceptibilities: Resistant to lyso- duced colonies that were small, smooth, and zyme (MIC > 1,600 pg/ml). Susceptible to convex to slightly umbonate with a bright erythromycin, st rep t omycin, novo biocin, tetra- orange pigment. Some strains produced an cycline, chloramphenicol, neomycin, vanco- orange exopigment. mycin, kanamycin, and polymyxin B. Strains either grew poorly or failed to grow Other characters and the G+C content of the on inorganic nitrogen agar. Most auxotrophic DNA of this strain are shown in Table 3. strains required cysteine or methionine and M. kristinae can be distinguished from other niacin, and were further stimulated by trypto- species by colony morphology and pigment, phan, valine, aspartic acid, glutamic acid, acid production from glycerol, sorbitol, and proline, and lysine (J. W. Farrior and W. E. mannose, nitrogen requirements, aliphatic hy- Kloos, Abst. Annu. Meet. Amer. SOC.Micro- drocarbon production, and slight anaerobic biol., 73rd, Miami Beach, p. 180, 1973). TABLE 11. Relative mol 74 compositions of the aliphatic hydrocarbons of Micrococcus species Aliphatic hydrocarbonsa Representative br - br- br- br- br- br- br- n- br- br- n- br- br- br- n- br- br- Species strainsb AC2 1 AC22 AC23 AC24 AC25 AC26 AX27 AC27 AC28 A-C29 lC29 AC30 AC3 1 AC32 AC3 2 AT33 AC34 M. luteus ATCC 272 0.7 0.6 8.1 4.7 25.4 11.7 48.8 ATCC 381 5.8 5.3 22.8 23.1 43.0 ATCC 382 0.9 0.4 8.8 2.4 4aQ 5.0 - ATCC 540 Tr 1.o 2.9 26.7 8.9 26.5 25.7 2.8 5.5 ATCC 4698 3.2 1.4 18.0 7.3 m ATCC 27141 2.0 2.7 21.3 14.3 ELL FD 533 4.4 1.1 15.9 0.6 14.5 61.3 2.2 BKM B963 4.8 28.9 8.7 36A 4.8 rn M. lylae ATCC 27566 2.2 3 .O 16.1 21.8 56.7 0.2 ATCC 27567 2.3 2.5 11.5 12.6 37.3 1.9 13.8 17.9 0.3 0.04 ATCC 27569 1.7 2.4 9.4 17.5 69.0 RM 324 0.8 1.8 16.7 24.5 56.2 0.02 TW 226 5.7 23.9 21.5 48.9 WK 312 2.1 10.7 8.9 78.3 M. seden- CCM 314 5 .O 9.1 21. 24. 24.9 14.4 Tr tarius ATCC 27573 14.8 6 .O 33.7 14.3 21.3 5.1 4.8 ATCC 27574 3.5 -14.0 28.4 26.6 15.7 11.8 Tr ATCC 27575 2.9 1.9 22.1 41.2- 26.9 5 .O DM 51 0.6 6.6 3 -0 -49.0 14.5 -18.0 7.1 1.2 DM 140 Tr 0.3 1.o 0.9 4.1 23.5 4.8 41,e- m 8.0, MK 136 34.4 30.6 15.7 DBM 420 3.2 -20.1 42.3 27.9 6.5 Tr SM 326 9.3 2.3 13.5 29.0 29.9 10.3 5.7 TW 93 1.8 5.1 5.1 12.6 -29.7 29.6 15.0 1.1 M. varians ATCC 401 0.3 1.5 6.7 20.5 21.2 44.0 2.6 3.2 ATCC 533 5.4 13.0 40.9 14.8 I_- 25.9 ATCC 9341 4.6 21.6 13.4 54.4 6 .O CCM 268 4.9 10.2 26.0 30.4 0.6 14.6 10.9 2.4 CCM 884 6.8 9.0 35.7 --20.0 5.5 20.2 0.8 2.0 CM 217 12.7 4.3 3.4 -41.2 -16.0 22.4 - M. roseus ATCC 412 2.1 4.6 21.9 51.6 8.9 10.9 ATCC 416 2.8 4.7 25.9- 48.4 9.5 8 .O 0.3 0.4 ATCC 516 2.1 4.2 30.8 -50.1 8.8 4.0 KH 6 2.7 3 .O 38.6 40.3 12.2 3.2 RM 337 1.8 4.6 30.4 --53.4 8 .O 1.8 VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 95 Many strains produced weak to moderate acid from glucose and galactose; of 50 strains, one strain produced weak acid from mannose, and two strains produced weak acid from fructose. Most strains were resistant to lysozyme, slightly resistant to methicillin, and susceptible to penicillin, erythromycin, and streptomycin. One strain was slightly resistant to erythro- mycin, and one was resistant to streptomycin. All strains were susceptible to novobiocin and neomycin. The susceptibility to novobiocin was slightly less than that found in other species. Only two of 24 strains tested contained detectable quantities of hydrocarbons. Mono- unsaturated branched C22, C23, C25, C26, and C27 aliphatic hydrocarbons were predominant in these strains. The quantities of hydrocarbons in these strains were similar to those found in the other species, i.e., within a range of 0.7 to 35% of the total lipids. The occurrence of long-chain, branched hydrocarbon isomers in microorganisms had previously been limited to the genus Micrococcus (31, 35, 42-44; T. G. Tornabene, Ph.D. thesis; D. Takemoto and T. G. Tornabene, unpublished data). However, we have recently found significant quantities of long-chain, branched hydrocarbons in an Arthrobacter strain (CCM 1647), in Corynebac- x 2 terium sp. ATCC 21188, and in strain CCM 2 135, designated M. conglomeratus, that con- tains meso-diaminopimelic acid in its cell wall and may be related to coryneform bacteria (4). The major hydrocarbons present were monoun- saturated branched C22, C23, C25, C27, and C29 aliphatic hydrocarbons. Several other strains of Arthrobacter and other coryneform bacteria that we examined failed to demon- strate aliphatic hydrocarbons. We feel that the rather unique occurrence of long-chain, 1 0 branched hydrocarbon isomers in micrococci CI and certain coryneform bacteria and the close resemblance of certain of their peptidoglycans (except for the absence of meso-diaminopimelic v)/N acid in micrococci) (5, 39), G+C content of their DNA (46), colony morphology, pigment, and certain physiological characteristics (4, 8) suggest the possibility of a taxonomic relation- ship between the genus Micrococcus and the Corynebacteriaceae. The sporadic occurrence of aliphatic hydrocarbons in strains of M. nishinomiyaensis may be suggesting a distant relationship of this species to certain coryne- form bacteria. Other characteristics of M. nishinomiyaensis and the designation of a type strain are being reported separately as part of a collaborative study with M. Kocur, Czechoslovak Collection of Microorganisms, University J. E. Purkyne, Brno, Czechoslovakia (manuscript submitted for publication) . TABLE 12. Molar ratio of amino acids in purified cell wallsof Micrococcus species andpeptidoglycan type Molar ratio of amino acids Glutamic acid Representative 01 Img Glu tamic Aspar tic Speciesa strains of(0 cell wall) Lysine acid Alanine GI ycine Serine acid Peptidoglycan type M. luteus CCM 169 0.50 1.o 1.o 1.98 1 .o L-Lys-peptide subunit M. lylae ATCC 27566 0.48 1.o 1 .oo 1.33 1.01 L-Lys-Asp ATCC 27567 0.19 1.o 0.42 1.39 1.oo ATCC 27568 0.1 1 1.o 0.34 1.34 1.02 ATCC 27569 0.41 1.o 1.oo 1.30 0.99 RM 324 0.24 1.o 0.5 1 1.29 1.oo TW 226 0.40 1.o 1.oo 1.26 1.oo WK 312 0.34 1.0 1.oo 1.32 1.01 M. sedentarius CCM 314 1.35 1.0 2.92 2.08 Complete primary structure ATCC 27573 1.64 1.o 2.98 2.08 uncertain (see text) ATCC 27574 1.32 1 .o 2.86 2.04 Mur-L-Alay-D-Glu-L-Ly s- ATCC 27575 1.35 1 .o 2.56 2.29 D-Ala,-, subunit DM 51 1.59 1.o 3.00 2.09 y-Glytamyl-glutamic acid DM 140 1.35 1.o 2.89 2.08 pep tide bridge MK 136 1.49 1.o 2.99 2.09 N-terminal L-Ala and Glu DBM 420 1.51 1.o 2.75 2.02 TW 93 1.57 1.o 2.76 2.00 M. varians CCM 418 0.48 0.98 1.o 5.1 1 L-Lys-L-Ala, -4 M. roseus CCM 679 0.42 1.o 1 .o 4.98 L-Lys-L-Ala, -4 M. kristinae ATCC 27570 0.40 0.93 1 .o 4.89 L-Lys-L-Ala, -4 ATCC 27572 0.37 0.96 1.0 4.85 AW 220 0.35 0.95 1 .o 4.80 MK 322 0.38 0.98 1.o 5 -00 LK 146 0.42 0.95 1 .o 5.08 DU 95 0.4 1 0.96 1 .o 4.87 M. nishinomi- CCM 2140 0.87 1 .o 2.0 1.91 , 1.78 L-Lys-L-Ser, -D-Glu yaensis a Amino acid composition of cell walls and peptidoglycan type of strains of M. luteus, M. varians,M. roseus, and M. nishinomiyaensis have been previously reported by Schleifer and Kandler (38, 39) and are used as references for species comparisons. TABLE 13. Abbreviated schemefor the classification of human cutaneous micrococcia - Acid from 0 0 c0 ?, nit. c a 0 N 4 .3 % c, 8 0 a h 1 c( w0 2 Species 8 i5 n B M. luteus -L>$ Y> 30-1 25 ->+,-1 - > +-, - - Se SR S C27, C28, L-Lys-pep- cw, - C29 tide sub- unit M. lylae L>$ ZW, 1-12 ->r ->+,I! +, * ->? 3R, R 3-SR S C27, C28, L-LYs-AsP - > YH C29 M. seden- L-s zw> 1.1-0.8 - - ->A S-SR R R C30, C31, Primary Q tarius BY C32, C33 structure 4 uncertain (see text) +- I->+,- + R L-Lys-L- M. varians L-s Y GO.1 -9 ->+ vs-s vs-s C25, C26, C27 Ah, -4 M. roseus L-s PR 3 <0.1 k>- I->+,- ->+ I-, f > SR-R vs vs C24, C25 L-Lys-L- OR Ala, -4 M. kris- S PO <0.1 + - > %,i +, * ++ R S S C28, C29 L-Ly S-L- tinae Ah, -4 M. nishi- S 0 <0.1 ->: +, +, - +, 1 - > +, SR-R SR SR C22, C23, L-Lys-L- nomi- C25, C26 Ser, - yaensis C27 D-Glu usually - - absent Characters were selected on the basis of their ease of distinction and wide differences (270%) in frequencies between two or more species. See Tables 4 through 12 for details of characters. A single listed symbol denotes a character frequency of about 95 to 100%. The notation > denotes a frequency greater than; a comma between symbols denotes nearly equal frequency. L, large; S, small. (See Table 4 for colony diameter.) BY, buttercup yellow; Y,yellow; YW,yellow-white; CW, cream white; -, unpigmented; PR,pastel red; OR, orange-red; PO,pale orange with cream edge; 0, orange. Reaction: ++, strong positive;+, positive; +,weak; -, negative. VS, very susceptible; S, susceptible; SR,slightly resistant; R, resistant. (See Tables 8,9, and 10 for data on levels of susceptibility.) TABLE 14. Characters of strains of uncertain taxonomic status' n Acid from ---3 1? 3 M -.-M ...M3 .m3 ...3. z 2 Y 3 8 3 0 3.- FI E 3 2.3 2 3 Possibly G+C M 5 ." related Strain (%I 3 & Peptidoglycan species ~- 3 ~~ WK 132 69.6 >1,600 6.2 0.05 ND~ L-Lys-L-Ala, -% M. lylae or M. luteus CM 9 ND > 1,600 1.6 0.8 ND L-Lys-L-Ala, -4 M. varians VN 6e 67.2 > 1,600 0.05 -0.003 C24, C25, L-Lys-L-Ala, -4 M. varians C26, C27 KR 13 ND 800 0.01 0.003 ND ND M. roseus KE 11 ND 800 0.006 0.001 ND ND Same as above RG 21 ND 800 0.01 0.003 ND ND Same as above LK 335 67.5 25 50 3.1 None ND M. nishinomi- yaensis SM 239 35.5 3.1 0.01 0.001 ND ND Genus unknown a All strains were gram-positive cocci arranged in diplococci and tetrads, nonmotile, non-sporeforming, positive for catalase and benzidine tests, strictly aerobic, failed to grow or grew poorly on inorganic nitrogen agar, failed to produce acid from lactose, maltose, or D-mannose, were susceptible to novobiocin (except LK 335, slightly resistant), erythromycin (except CM 9, slightly resistant; SM 239, resistant), streptomycin (except VN 6, resistant), and neomycin, and resistant to 2 lysostaphin. Characters which were different from those found in possibly related species are underlined. YW, yellow-white; OW, orange-white; PO, pink-orange; R, red; R/O, red and orange sectors; YO, yellow-orange; P, pink. Reaction; +, positive; +, weak; -, negative. ND, not determined. Strain VN 6 and several other similar strains were isolated in 1965 from the skins of Vietnamese people and from air in Qui Nhon, South Vietnam. 9 W b 9 0rE VOL. 24,1974 CHARACTERIZATION OF MICROCOCCUS SP. 99 TABLE 15. Frequency of occurrence of Micrococcus species on human skin Skin sites occupied by species Species of total micrococci (%Ib present per site (%)' Per Per Per Individuals Per individual individual individual with species individual with species with genus with species Species (%IQ (K and sjz) (X and sx) (K and %) (R and sx) M. luteus 90 462 3 51 k 3 55t 3 58 2 3 M. lylae 33 7+ 1 23 + 3 7+1 20 f 3 M. sedentarius 13 4+ 1 26 f 8 2 % 0.8 13+5 M. varians 75 245 2 322 2 23 % 3 30+ 3 M. roseus 15 2 2 0.5 155 3 2 t 0.7 13 + 5 M. kris tina e 25 62 1 23 * 4 6k2 24 f 4 M. nishinomi- 28 6f 1 22 f 4 5+1 1724 yaensis a A total of 115 individuals was sampled. Members of the genus Micrococcus were present in 96% of the individuals. Mean number of skin sites sampled and analyzed per individual was 1 1 f 1. ' Mean number of total Micrococcus colonies isolated per site per individual was 29 + 10. The number of colonies isolated from each site represents an estimated 23- 5 1% of the total Micrococcus colony-forming units present. Character variation in Micrococcus species. teristics in M. luteus and M. varians compared Summary data of the characters found in the with that observed in the United States (W. E. various Micrococcus species isolated from hu- Kloos and W. C. Noble, unpublished data). man skin are shown in the following figure and Also, we might expect to find some differences tables: colony morphology and pigment, Fig. 1 with micrococci isolated from the skins of and Table 4; cell arrangements, Table 4; genetic other mammals. Recent studies have shown compatibility with M. luteus, growth on inor- that strains of M. varians isolated from several ganic nitrogen, Simmons citrate, and NaCl different species of rodents living in the Dismal agars, ace t y lmet hy lcarbinol production , nitrate Swamp area in North Carolina were distinctly reduction, and oxidase activity, Table 5 ; carbo- different from human strains in having a bright hydrate reactions, Tables 6 and 7; lysozyme chrome-yellow or orange pigment, and all susceptibility, Table 8; methicillin susceptibil- strains produced acid from xylose (R. J. ity, Table 9; penicillin susceptibility, Table 10; Zimmerman and W. E. Kloos, unpublished aliphatic hydrocarbon composition, Table 1 1; data). cell-wall amino acid composition and pep- Characterization of strains of uncertain tidoglycans, Table 12. Several characters that taxonomic status. Several types of rare strains were useful in strain identification but not were isolated that did not appear to belong to particularly useful for resolving species have any of the above-mentioned seven species. They been omitted from the summary tables. In differed from these species by at least one or addition to outlining species character varia- more key characteristics. Since most types were tion, these tables are useful for making com- represented by only one or a few strains, we parisons of the newly proposed species with will not attempt to provide species names to previously recognized species. An abbreviated these types but will merely refer to them by scheme showing a minimal number of key strain designation. A summary of their distin- characters used for the classification of cutane- guishing characters is shown in Table 14. Their ous Micrococcus species is presented in Table colonies are shown in Fig. 1. One strain, 13. designated SM 239, possesses many of the It should be pointed out that species charac- morphological and biochemical characters that ter variation found in this study may be are uniquely typical of micrococci but has a somewhat different when strains are obtained G+C content that is entirely too low. It, from people living in other geographical areas. therefore, cannot be considered as a member of For example, in examining micrococci from the the genus Micrococcus, but it also does not skins of people living in London, England, we appear to be a member of Staphylococcus. have found significant differences in the fre- Occurrence of Micrococcus species on human quency of certain colony morphological charac- skin. A summary of the occurrence and 100 KLOOS, TORNABENE, AND SCHLEIFER INT. J. SYST. BACTERIOL. distribution of Micrococcus species on humans 1957. Bergey’s manual of determinative bacteriol- is shown in Table 15. As can be seen from the ogy, 7th ed. The Williams & Wilkins Co., data, M. luteus and M. varians were the Baltimore. predominant species found on human skin in 7. Coblentz, L. M. 1943. Rapid detection of the this study. Some differences were noted in the production of acetylmethylcarbinol. Amer. J. Pub. Health 33:815-817. relative occurrence of certain species on indi- 8. Conn, H. J., and I. Dimmick. 1947. Soil bacteria viduals from different regions of the United similar in morphology to Mycobacterium and States, and these observations will be reported Corynebacterium. J. Bacteriol. 54: 29 1-303. elsewhere in another communication. 9. Cowan, S. T., and K. J. Steel. 1965. Manual for the identification of medical bacteria. Cambridge University Press. ACKNOWLEDGMENTS 10. Cziharz, B., K. H. Schleifer, and 0. Kandler. 1971. A new type of peptide subunit in the We are greatly indebted to Margaret Musselwhite for murein of Arthrobacter strain 539. Biochemistry her excellent technical assistance and maintenance of 10: 3574-3578. the many cultures used in this study. We are also 11. Deibel, R. H., and J. B. Evans. 1960. Modified grateful to E. Hagner and B. Weyrich for their capable benzidine test for the detection of cytochrome- technical assistance. We are thankful for the assistance containing respiratory systems in microorganisms. provided by Judy Mullins (Washington), David J. Bacteriol. 79:356-360. Berryhill and Jon Lindgren (North Dakota), Nancy 12. Eisenberg, R. C., and J. B. Evans. 1963. Energy Richardson (Maine), William Chesbro and Rita Fur- and nitrogen requirements of Micrococcus roseus. man (New Hampshire), Walter Dobrogosz (Pennsyl- Can. J. Microbiol. 9:633-642. vania and Ohio), Susan Kloos and Wesley L. Kloos 13. Evans, J. B., and W. E. Kloos. 1972. Use of shake (New Jersey), Sandra Gregory (Iowa), Gwendolyn cultures in a semisolid thioglycolate medium for Fletcher (Kansas), Jackie Warner (Washington, D.C.), differentiating staphylococci from micrococci. Gerald Hieronymus (Virginia), Willard Blevins (Ala- Appl. Microbiol. 23: 326-33 1. bama), Claudia Juhl and Alan Proctor (California), 14. Glass, M. 1973. Sarcina species on the skin of the Elaine Anderson (Arizona), Sandra Thurlow (Colo- human forearm. Trans. St. John’s Hosp. Derma- rado), James Higbee and Myrtle Wing (Texas), and tol. SOC.59:56-60. Lyle Dodd (Florida) in collecting skin samples for this 15. Goodfellow, M., A. Fleming, and M. J. Sackin. study. We also thank the many persons that provided 1972. Numerical classification of “Mycobacte- us with their skin flora. rium” rhodochrous and Runyon’s Group IV This research was supported in part by Public mycobacteria. Int. J. Syst. Bacteriol. 22:81-98. Health Service research grant A1 08255 from the 16. Kandler, O., K. H. Schleifer, and R. Dandl. 1968. Institute of Allergy and Infectious Diseases. Funds Differentiation of Streptococcus faecalis An- were provided by the North Carolina Agricultural drewes and Horder and Streptococcus faecium Experiment Station for the color plate, which was Orla-Jensen based on the amino acid composition photographed by Ralph Mills. of their murein. J. Bacteriol. 96: 1935-1939. 17. Klesius, P. H., and V. T. Schuhardt. 1968. Use of lysostaphin in the isolation of highly polymerized REPRINT REQUESTS deoxyribonucleic acid and in the taxonomy of aerobic Micrococcaceae. J. Bacteriol. 95: 739-743. Address requests for reprints to: Dr. Wesley E. 18. Kligman, A. M. 1965. The bacteriology of normal Kloos, Department of Genetics, North Carolina State skin, p. 13-31. In H. I. Maibach and G. University, Raleigh, N.C. 27607. Hildick-Smith (ed.), Skin bacteria and their role in infection. McGraw-Hill Book Co., New York. LITERATURE CITED 19. Kloos, W. E. 1969. 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