Dorothy Jones, R. H. Deibel and C. F. Niven
J. Bacteriol. 1963, 85(1):62.

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0 to 7. Illinois Received for publication 25 July 1962 In the classification of the family MicroEvans. epidermidis includes a heterogeneous collection of mass-forming cocci sharing the following characters: (i) ability to grow anaerobically in a standardized medium containing glucose. NIVEN. S. F. The physiological characteristics considered to be of primary import are: (i) ability to grow anaerobically in a defined complex medium with glucose or pyruvate as the energy source. In experiments concerned with the anaerobic utilization of the various substances as energy sources.2. The University of Chicago. coagulase-negative. 5 g. the basal medium consisted of Tryptone (Difco). 62 The present paper attempts to provide a more definitive characterization of the species S. (iii) ability to ferment serine as an energy source. K2HPO4. R. H. Carbon dioxide production from glucose was determined qualitatively by the method of Williams and Campbell (1951). 5 g. and (iii) inability to ferment mannitol anaerobically. hyicus [a pathogenic. broth cultures were placed in a 6-liter desiccator and flushed twice with hydrogen. Anaerobic requirement for uracil was determined in the casein hydrolysate medium of Gretler et al. Most of the physiological tests have been described previously (Evans and Niven. and deoxyribonucleic acid hydrolysis determined by the method of Di Salvo (1958). 1 liter. DEIBEL. Sompalinsky. . (ii) inability to produce coagulase. Although S. 5 g. AND C. Bacteriol. H. where the genus Staphylococcus includes two species. 1953). coccaceae. I Journal paper 234. In addition. JR. These authors suggested that the facultative species capable of growing anaerobically in a standardized medium containing glucose be placed in the genus Staphylococcus. MATERIALS AND METHODS Source of strains.asm. epidermidis. energy source. Chicago. DEIBEL. 10 g. Downloaded from http://jb. This distinction between Micrococcus and Staphylococcus has been incorporated into Bergey's Manual (Breed. The majority of the strains employed in this study were isolated from food products. 1963. and compared with other staphylococci. Murray. homogeneous species. Copenhagen. 1950). yeast extract (Difco). and Smith. 1957). hyicus was obtained from D. and from a variety of clinical sources. catalase-positive coccus isolated by Sompalinsky (1950. Chicago. Biotin requirement was determined in Biotin Assay Medium (Difco). (iv) the requirement for biotin and uracil (under anaerobic conditions) in a semisynthetic medium. aureus and S. AND C. pH 7. American Meat Institute Foundation. For anaerobiosis. Uracil was added to give a final concentration of 1 mg/100 ml of medium. 2014 by guest ABSTRACT JONES. the taxonomic position of Al. 85:62-67. NaCl. J. Bradford. and The Department of Microbiology. aureus is a well-defined. The egg yolk opacity test was detected on Colbeck EY agar plates (Difco). and those species incapable of anaerobic growth in the medium be assigned to the genus Mlicrococcus.-The species Staphylococcus epidermidis is defined more precisely. JR. epidermidis. and (v) inability to reduce nitrate beyond nitrite. and shown by him to be the etiological agent in an outbreak of contagious impetigo of swine] was investigated and compared with S. Physiological methods. NIVEN. (ii) inability to produce coagulase. Identity of Staphylococcus epidermidis. Division of Bacteriology. Institut de Serologie Veterinaire de l'Etat. American Meat Institute on September 22. (1955). S.IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS' DOROTHY JONES. and Niven (1955) proposed the separation of the genus Staphylococcus from Micrococcus on the basis of their growth relationship to oxygen. R. DOROTHY (American Meat Institute Foundation. One strain of Al. The final gas phase consisted of a mixture of 90% hydrogen and 10% carbon dioxide.). Growth was estimated by determining the optical density of cultures in a Spectronic 20 spectrophotometer at a wavelength setting of 600 m.. 10 g. I11. F. epidermidis. distilled water.

... 1+ referred to as "denitrifying" staphylococci.. epidermidis group. hyicus demon. Esculin . 1957). + Ml. 1two denitrifying strains. further incubation this curd was digested slowly.. hyicus was devoid of strated weaker enzyme activity than S.. used.......... 2014 by guest TABLE 1.. All the years....... Lactose ..VOL.. + physiological characterization. Strains (53) of coagulase-negative... one of the denitrifying staphyand sucrose..... 1+.. sorbitol. 1953) stressed inability to ferment maltose strains included in this study produced opacity in his description of M....Peptonization. and upon Maltose .. Although M. 1lower final pH value than the S. However.. 1produce acetoin........ epidermidis within Hydrolysis of deoxyribonucleate. be bic) .......... epidermidis inulin. arabinose.. aureus... frucwith S... + 1±....... aureus exhibited a weak and delayed (48 to 72 hr) strains and M. 4tion of these strains from the typical S....5 bic fermentation of mannitol is usually associated * All strains produced acid from glucose... 14. although all of the S. 2lococcus strains H60 and H92) reduced nitrate beyond nitrite... hyicus would be termed more 45 C.. 85.. 43 grew epidermidis hyicus staphylo(15) (1) cocci (2) under these conditions... StaphyMannitol (aero+ lococcus strains H60 and H92 will. it has been observed that the dismutation aureus of S....... melibiose.. Mannitol (anaerobic) .. reaction.. were selected for a more complete Coagulase . 1+ purposes.. Organism and no..... activity... In recent its reaction was decidedly positive.... 14+ All of the strains utilized glucose as a source of energy under anaerobic conditions (Table 1). This characteristic merits separa. and were classified tentatively as S... Sompalinsky (1950.. Egg yolk opacity ...... 1appropriately Staphylococcus hyicus. and M. therefore. 13+. Of the on September 22. litmus milk.. II+ 4strains of S. strains fermented this substrate... the S.... Hydrolysis for purposes of presentation.. 14+........ In contrast.. + This reaction in litmus milk has not been reported Trehalose . 7No distinct fermentation pattern was detected Melezitose . 11+. 1957). generic name associated with this species will be Sodium hippurate.. or by Mll. aureus were included for comparative Pigmentation + Yellow... 11+.. Anaerobic utilization of pyruvate. and fermented glucose to a CO2 cose + from glucose.. epidermidis (Breed et al. 12Three of the strains (Al. 1+. epidermidis strains.. 13previously for S... + 1+. In the following discussion... of strains tested gram-positive cocci were screened for their ability Physiological MicroDenitriStaphyloto utilize glucose as a source of energy under characteristic coccus coccus fying anaerobic conditions.... 9+.. In addition.. 4rennetlike curd in litmus milk (Difco).positive reaction.... 5. 2+. hyicus evidenced appreciable deoxyribonuclease activity..7 5... salicin.. 3+. glucose. Anaerobic glucose 17 of these strains. 1+.. hyicus formed a Galactose . aureus.. 13in the S. 1Gelatin . Maltose was not fermented by the denitrifying staphylococci.. None . hyicus. Although the anaero........... epidermidis (Breed et al. hyicus and StaphyArginine ...... hyicus..2-5........... the more common Starch .. Six or more Nitrate reduction.Final pH. together with the 1 strain of utilization ... Only S... dextrin. Physiological characteristics of the RESULTS strains tested General physiological characterization.... 1963 IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS 63 Downloaded from http://jb...asm....4-4... 14+. epidermidis Fermentation* (acid) strains. raffinose.. the Mannose .... all of these strains failed to Acetoin from glu.. 1+. Growth 10 C . 24 hr.. lococci fermented this alcohol. hyicus... 61+... The majority of the S. and M. epidermidis...... None of the strains evidenced tose...... M.+ Consequently. Glycerol ..... 8+... cellobiose..... nor acid production from xylose. catalase-positive..

.. 2014 by guest of pyruvate is an energy-yielding process in which lipoic acid plays a key role (Gunsalus... CB8 ... The chemical nature of this viscous material will be the subject of a future publication...... OB14 . S... citrate..... All of the S....... Results determined after 24 hr of incubation at 37 C..asm. Characteristically..... and Niven (1958) and Deibel and Niven (1960) reported that the anaerobic utilization of pyruvate.. 1954). and.... serine... citrate......... growth with pyruvate... No slime was detected when the strains were cultured with other energy sources.... and M. it is assumed that a deamination (serine dehydrase) takes place with the No.. and serine as the energy source required an exogenous source of lipoic acid.. the majority of the S... The utilization of L-serine as an energy source by S. BACTERIOL.... whereas none of the aerobic Micrococcus strains was capable of growing under these conditions (Table 2).. 41 S. Deibel.. The entire Staphylococcus-Micrococcus collection was tested for the ability to utilize pyruvate as a source of energy under anaerobic conditions... 1 Micrococcus hyicus + + and one S.. although they did evidence a significant growth response with pyruvate.... 170 .. Quite often this material interfered with the optical-density determination of the growth response. Typical growth responses (optical density X 100) of staphylococci with pyruvate and glucose as anaerobic energy sources* Organism and strain Staphylococcus epidermidis Fussell . 1960) as energy sources can be used to distinguish Streptococcus faecalis from Streptococcus faecium....... epidermidis strain were able to utilize Micrococcus sp 10 this amino acid as a source of energy (Table 4). Anaerobic utilization of glucose and mechanistic aspects of serine degradation were pyruvate by various Staphylococcus strains* not investigated... the denitrifying staphylococci. 1962). A high incidence of slime production was ob- J. 70 80 110 119 97 107 122 113 122 140 130 122 37 110 90 77 * The basal medium is described under Materials and Methods.... In contrast....... Worthy of mention are the relative growth responses of the various groups to pyruvate and glucose (Table 3). DEIBEL.. cultested tures evidencing growth with serine produced large quantities of ammonia. S.. .. AND NIVEN 64 TABLE 3.....64 .. of strains Glucose Pyvtr Organism formation of pyruvate.... * Malate... For comparative purposes..... malate............. denitrifying staphylococci.. aureus 43 .. aureus. Unlike Streptococcus faecalis (Deibel et al.. aureus strains were unable to derive energy from this amino acid.. no exogenous lipoic acid requirement could be demonstrated with the on September 22......... 3RB2 ...... B5B ... + + Staphylococcus epidermidis.... ascorbate. Results determined after 24 glycerol were also tested for their ability to serve hr of incubation at 37 C. epidermidis... Although the TABLE 2.... Deibel (1962) reported slime production by S. epidermidis strains when tested in a casein hydrolysate.. S.. as energy sources for the staphylococci under vate aureus .... hyicus. epidermidis paralleled closely the utilization of pyruvate (Table 4).. faecalis utilized these substrates..... gluconate.......... served when the S. M.. and The basal medium is described under Materials and Methods. the growth response with glucose was determined. furthermore... hyicus exhibited equal or usually higher optical densities with pyruvate as the energy source. S. Downloaded from http://jb..... H92 ........... semisynthetic medium (Deibel. More recently.. NU13B .. only cocci 2 + + one of the two denitrifying strains.. Denitrifying staphylococci H60 ........ faecalis in a pyruvate medium........ 10 + + Although the vast majority of the strains emDenitrifying staphyloployed in this study hydrolyzed arginine..... the S.JONES. aureus strains demonstrated a superior growth response with glucose as the energy source.. Substrate Pyruvate Glucose 119 116 122 135 90 .... citrate. Micrococcus hyicus ... 1958). epidermidis.......... Evans............ and M.. epidermidis strains were cultured with pyruvate as the energy source. hyicus strains utilized pyruvate and glucose.... and arginine (Deibel.. However... .

org/ on September 22. S. the composition of these 37 C. includes aerobic uracil for anaerobic growth (Table 5). Hill (1959). and Cowan (1951) placed the entire group in the one genus. Shaw et al. The anaerobic growth response to glucose was was added to the basal medium. in an exhaustive investigation of strain of S.14+. i. using the method of Evans et al. of Gretler the results (1955). glucose. studied utilized these compounds. The literature reflects this DenitriMicroStaphylounsatisfactory condition in the plethora of generic stpyincoccus S. Arginine * The basal medium is described under Materials afermentans were too heterogeneous a collection and Methods. bic) the central question of whether this group of + + + _ microorganisms should be included in one genus Biotin or separated into two genera still is not resolved * Readings made after 24 hr of incubation at completely. Confirming as a source of utilization the anaerobic pyruvate all of the coagulase-negative staphylococci rewith obtained results the of energy paralleled quired the vitamin biotin for growth (Table 5). and ascorbate. 1963 TABLE 4. Stitt. The additional a sharp differentiaturbidimetrically. agreed with the conclusions of Shaw et al. -. this be effected tion could et al.. In addition. Hill preferred to incubation at 37 C. (1955). entire group as a linear series. Serine + 3+ 6. under Materials and Methods. 1+. The semisynthetic media are described genera remains in a fluid state. Nutritional studies.aureus) and the acetoin-producing strains (S. 85. malate. none of the other organisms the little-studied meat Micrococcus-Staphylococcus group. and S. Biotin and uracil (anaerobic) requirements anaerobic strains of the family Micrococcaceae is of staphylococci* far from satisfactory. afermentans. of modification uracil in combination with guanine and adenine.(1951) regarding S. Staphylococcus. aureus which utilized citrate. 1951) in the genus some strains of S. epidermidis strains required defined by Evans et al. In addition. and strains which he could not anaerobic conditions. Results determined after 24 hr of to warrant species status. 2014 by guest oc .but was of the opinion that S. M. epider(15) midis Micrococcus hyicus (1) Denitrifying staphylococci (2) 65 staphylococci at one extreme and the thermoduric saprophytes at the other. saprophyticus. (1955). additional. no requirement. aureus and S. a primary separation of denitrifying strains manifested no uracil requiretypes was effected by employing a physiological or when uracil. (1955). unlike the genus Micrococcus as group. and thus emphasized the validity of this DISCUSSION primary differentiation of physiological types in The classification of the aerobic and facultative TABLE 5. reAbd-el-Malek and Gibson (1948) regarded the quirement. aureus.+ 1+. strated an absolute requirement for uracil by saprophyticus. the pathogenic undetermined requirement. S. With the exception of one roseus.asm. These authors recognized five species: S. In the present study. hyicus did not grow the method of Evans et al. Although some + 14+. aureus when grown under Staphylococcus. placed the coagulase-positive strains (S. estimated requirements of this strain were not investigated. Utilization of serine and arginine as energy sources by staphylococci* Substrate Sta phylococcus aureus (9) S. this genus as proundertaken with the Staphylococcus-Micrococcus posed by Pohja.IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS VOL. in manner. 14. Shaw. S.+ progress has been made in the direction of a more Uracil (anaerorational and universally acceptable classification. lactis. + + + + 1957). ment. Pyruvate (anaerobic) Downloaded from http://jb. 1. roseus. Richardson (1936) demon. Symbols: +. using the Adansonian method of classification (Sneath. Pohja (1960). saprophyticus. This requirement has never classify in this way were assigned to the genus been exploited fully in any taxonomic study Micrococcus. epidercoccus tan ce (s t subs nitaphylmidis1der sustaceaureus (15) hyicus and specific epithets that have been and still TesTes (1) COCCi2 the be termed what can are employed to classify Micrococcus-Staphylococcus group. reclassify Staphylococcus roseus as Micrococcus anaerobic conditions. The two and facultative anaerobic organisms. Consequently. 93% of the S. 1. 1. lactis and S. Moreover.

The two denitrifying strains reacted similarily.. Lipoic acid requirement for anaerobic utilization of pyruvate as an energy source by Streptococcus faecalis. In this study. produced acetoin neither from glucose (confirming the findings of Sompalinsky) nor from pyruvate. S. Downloaded from http://jb. U. DEIBEL. 1960.. 1948. 1957. Lipoic acid and the fermentation of serine and malate by Streptococcus faecalis. NIVEN.. DEIBEL. R. AND N. epidermidis on the basis of other criteria. B. ACKNOWLEDGMENTS The authors wish to extend appreciation to P. DEIBEL. however. Although the results obtained with the deni- J. On the basis of its ability to utilize glucose and pyruvate anaerobically. 1960. R. H. R. epidermidis. G. SMITH. Studies in the bacteriology of milk. 1959). p. EVANS. BREED. failed to grow anaerobically when provided with glucose or pyruvate as the energy source. DEIBEL. MURRAY. two strains of S. Cowan. St. aureus employed in this study. the denitrifying staphylococci and M. All the strains of S. JR. Proc. which is presumably deaminated to pyruvate. The preliminary results obtained with these strains indicate that they may differ sufficiently from other staphylococci to merit species rank. for the use of his laboratory in the isolation of the clinical strains employed in this study. may also be employed to define S.. p. DEIBEL. Y. H. . James Clinical Laboratories. Thesis. II. 1948. a case can be made for the reclassification of M.. 15:249-260. In the mouse. 1958. 163-164. saprophyticus (NCTC 7292 and 7612) obtained from S. required uracil.S. Richardson (1936) investigated six strains of S. hyicus as a Staphylococcus. Bacteriol. Its pathogenicity was established previously in pigs by Sompalinsky (1950. one strain. epidermidis. The University of Chicago. R. R. D. Moreover. Staphylococcus hyicus differs sufficiently from S. R. epidermidis to remain outside this species. aureus. the observation that some of the facultative anaerobic strains reduced nitrate beyond nitrite further aided in delineating the remaining group. Colindale. Bergey's manual of determinative bacteriology. LITERATURE CITED ABD-EL-MALEK. pathogenic Staphylococcus. 1953) and confirmed in this laboratory in mice. Hill. The staphylococci and micrococci of milk. 1962. The inability of S. The anaerobic requirement for uracil may also prove to be of taxonomic value. 114. M. Bacteriol. Fagan. Utilization of pyruvate as an energy source for Streptococcus faecalis. NIVEN. 1951. F.. epidermidis as defined by Breed et al. 164. Chicago. Ill. AND C. Arginine as an energy source for the growth of Streptococcus faecalis. H. J. the nature of which was not determined. epidermidis. Proc. London... hyicus either exhibited no such requirement or else required an additional growth supplement. These results indicate the necessity for further studies with this species prior to determining its exact taxonomic position. classified by us as S. The fermentation of serine.66 JONES. AND NIVEN trifying staphylococci warrant separation of these microorganisms from S. although it may well be incorporated in the description of S. GIBSON. Bacteriol. necrotic skin lesions develop after 3 to 5 days when the organism is injected subcutaneously. their exact taxonomic position must await further study. (1957) appears to be synonymous with S.. Public Health Service. The Williams & Wilkins Co. BACTE:RIOL. Proc. Dairy Res. AND C. However. JR. p. hyicus. Central Public Health Laboratory. However. T. Acetoin production has long been considered an important characteristic within this group of microorganisms (Abd-el-Malek and Gibson. This work was supported in part by grant E-1951 from the National Institutes of Health. epidermidis on September 22. This organism possesses a unique set of characteristics in that it is a coagulase-negative.asm. S. and four grew anaerobically when only uracil was added. J. and all but one of the strains of S. AND T. H. but all these strains produced acetoin from pyruvate. 7th ed. E. saprophyticus as described by Hill (1959). Chicago Heights. aureus to ferment pyruvate with facility detracts from the possible use of this character to define this species. F. 2014 by guest the Micrococcus-Staphylococcus group. produced acid from glucose but failed to produce acetoin. Baltimore. Shaw et al. Although the property of pathogenicity alone may not contribute to species status. The other strains grew only if hydrolyzed nucleic acid was added..

1955. n. D. 1957. GRETLER.VOL. The application of computors to taxonomy. Vitamin nutrition of the staphylococci with special reference to their biotin requirements. S. STITT. SOMPALINSKY. Serumlab. 20: 277-283. 13: 715-722. R. J. AND L. Food Technol. SHAW. 85. 1936. M. J. 1959. J. 30:2184-2190. Intern Bull. HILL. COWAN. F. 9:191-196.. JR. Tech. NIVEN. H. CAMPBELL. WILLIAMS. EVANS. J. Med. on September 22. vol. L. SNEATH. 70:44-49. 1953. Fennica. AND C.. The nutrition of Staphylococcus aureus. 59:545-550. Schweiz. sp. B. 0.. F. Bacteriol. Microbiol. J. Nomen. C. 17: 201-266. The detection of heterofermentation by lactic acid bacteria. J. I. POHJA. B. Biochem. Gen. 1963 IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS of staphylococci. Gen. Downloaded from http://jb. 96. F. A. p. Vet. P. AND S. D. SOMPALINSKY. C. Statens. J. Gen. 1951. M.. MUCCIOLO. Bull. JR. AND C. J. Taxon. JR. J. C. NIVEN. B. L. Agral. Federation Proc. De l'impetigo contagiosa suis et du Micrococcus hyicus. Bacteriol. RICHARDSON. Medd. W. T. 2014 by guest SALVO. AND C. P. Tierheilk. G. J. 5:306.. BRADFORD. W. B.asm. Arch. 1951. 1950. Comments concerning the taxonomy of the genera Micrococcus and Staphylococcus. J. 5: 61-66. EVANS. Acta. Oxidative transfer reactions of lipoic acid. 95:302-309.. EVANS. L. Micrococci in fermented meat products. M. Impetigo contagiosa suis. 5:1010-1023. Staphylococci and their classification. Microbiol. Necessity for uracil in anaerobic growth. 280-283. 1958. A. Microbiol. Bacteriol. NIVEN. A comparative study of known food poisoning staphylococci and related varieties. 1954. 1960. The Adansonian classification Di 67 . Deoxyribonuclease and coagulase activity of micrococci. 1955. GUNSALUS.