THE JoURNAL oF INVESTIGATIVE DERMA'roLOGY

Copyright@ 1970 by The Williams & Wilkins Co. Vol. 54, No .

Printed in U.S. A .

FINE TRU TURE OF CORYNEBACTERI UM AGNES*

LEOPOLDO F. MONTES, M.D. AND WALTER H . WILBORX, PH .D.

ABSTRACT

An electron microscopic study was made of C. acnes grown in vitro and of unin­flamed open comedones in acne patients. Seven different strains grown anaerobically for 2.7 days, and comedones were fixed with the Ryter-Kellenberger procedure and cut into thin sections for electron microscopy. Floccular material attached to the cell wall, prominent mesosomes and more simple invaginations of the plasma membrane were characteristic features. The results showed C. acnes to be similar to other corynebac­teria studied previously with the electron microscope and to the orgamsms observed in lar{Te masses within comedones.

Corynebacterium acnes is one of the pre­dominant bacteria in normal human skin (1). Renewed interest in this organism has resulted in tudies to determine if it plays a role in the pathogenesis of acne vulgaris. C. acnes has been isolated from comedones and other types of acne lesion (2-6) and the biochemical char­acteristic of this bacterium have been defined (7, ) . Inj ction of C. acnes into steatocysto­mas re ulted in inflammation and rupture of the cysts (9) . imilar changes were produced by injection of living, but not dead, C. acnes into keratinous cy ts (10).

Immunologic techniques have revealed that serum antibody levels to C. acnes are higher in p r on with acne thnn in other individuals, nnd that th antibody J vel parallel the ' e­vcrity of acne (3). It has also been shown hat patient with ncne have increased serum

]eve] of compl ment fixincr antibodies to C. acn s ( 11) and a marked dermal hypernen i­tivit~· to th orgnni m (12).

Recently, Zierdt and W ertlake ( 13) reviewed studi de cribing C. acnes as a disease agent in

Thi Ludy wa upported b~· areer Dc,·elop­m nt ~ ward (LFM) 7-I\:3-.-\.I-31. 210-03, bv PHS ~rant DE 02110, and b)· equipment rrr::mts from Th Kr' g Foundation nnd )J a tional cience Foundation (GB-7489).

Th t hni ·al n istanc of Mi s Gwen Hamer and Mi s A. J. Narkate is gratefully nrlmmd­edg d.

Hequest for reprints should be addressed to L. F. Mont , M.D., Department of Derma tology.

niv r itv of lubama Medical enter, Birming­ham. Alabama 35233.

R ceiv d Augu t 2 . 1969; ace pted for publi­a tion o emb r 24. 1969.

*From th Department of Dermatology, Mi­cr biologY. and natom\·. niYer it:v of Aiabama M dicnl ent r. Birmingham .. ·\labama 35233.

33

a number of extracutaneous infections. They also examined cultures of C. acnes with the electron micro ·cope but did not describe its ultrastructur . Thu , we haYe studied the fine structure of cultured cells of C. acnes to pro­vide a basi for electron microscopic obser­vation of the ho. t-bacterium relationship in comedones and other acne le ions where ad­ditional types of microorganismn may occur (2, 6).

Fw. 1. Cell of C. acnes showing cell wall (cw) . pla ma m mbrane (pm) and an im·agination of the pla ma membrane (i). X 90.000.

Corynebacterium acnes

0 Fra. 2. Cell of C. acnes displaying a large central mesosome (m) and a closely related

volutin granule ( v) . X 132,000.

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340 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Fw. 3. Divi ling ·ell of C. acnes which ha r mained attach d to another cell (left side of pi ture) afL r apr 'iou divi ion. A large central mesosome (m) j ~ bown in the area wh r septum formation is taking place. Cell wall ( cw) and plasma membrane ( pm) are be­ginning to invaginate on oppo ite sides of the cell . Floccular material (f) adh rc to the ex­ternal surface of the cell. X 85 000 .

Corynebacterium acnes 341

lYIATERIAL AND METHODS

Observations were made on 7 strains of C. acnes . Four strains were provided by Dr. S. M. Puhvel (University of California, Los Angeles), one strain (6919) was obtained from the American Type Culture Collection, and two strains were i alated from two of our patient . Cultures were grown in the medium of Evans et al . ( 1) or in fluid thioglycollate medium (Baltimore Biological Laboratories) using an anaerobic jar with a Gas­pak envelope. After 48 hours to 7 day of growth, cells were harvested by centrifugation and fix:ed by the procedure of Ryter-Kellenberger (14) .

1 cimen were dehydrated in acetone and em­bedded in Araldite (15). Several uninflamed comedone were removed with a comedo ex­tractor from the back and face of the t\\·o patient and fixed as described for the cultures. Thin ec­tion of the pla tic embedded cultures and come­dones ~ere cut on an ultramicro tome and placed on copper grids coated with a carbonized collodion supporting film. Sections stained n·ith lead citrate (16) or without lead taining were reviewed with a Philips EM 200 or a Philips E I 300 electron microscope at 40-100 kv.

RE SULTS

Appearance of C. acnes m Cultures

Although the cells of each strain differed in size and hape, they po ses ed many ultra-tructural imilarities. In sections, bacteria

were rounded, oval or elongated. Rounded cells were 0.3-0.6 f.L in diameter. The lange t cell were 3-4 p. in length and about 0.5 f.L in width. Cells from strain ATCC 6919 were t rikingly more rounded than cells from the

other strain . Cells of each strain showed floccular material adherent to the cell surface and beneath this material was the cell wall. In most cell these two envelope had a similar electron density and were so closely apposed that they could not be easily differentiated from each other (Figs. 1-3).

The pla rna membrane was in contact with the cell wall. It was somewhat wavy and com­J:O~ d of two electron den e layers separated by an electron lucent layer (Fig~. 1-7). Me o orne~ , produced by whorled invagina­tions of the plasma membrane, were promi­nent and clearly showed the trilaminar char­acteri-tic of the plasma membrane. Meso-orne lay near and sometime within the

nucleoplasm or were centrally located and re­lated to the septum of dividina cells (Figs. 2, 3, 7). In addition to meso orne , more simple

Fro. 4. Another dividing cell of C. acnes. X 77,000.

intracytoplasmic invagination of the pla rna membrane were frequently seen (Fig. 1).

The cytoplasm of C. acnes was dense and amorpbou . Ribosomes were difficult to detect in most cells because of the dense cytoplasm, but they wer sometime. visible in s ctions stained with lead citrate (Fig. 6). Area of low electron density, possibly containing lipid, were pre ent in the cytopla. m. In most cells, polypho phate (volutin) granule were not ob~ served but some were occa ionally seen in close relation to mesosomes (Fig. 2).

The nuclear region was usually fairly well circum cribed in one area of the cell. How~

342 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 5. These dividing cells of C. acnes display different stages of septum formation. Cell division is occurring simultaneously in several different planes. Note the di per ed arrange­ment of the nuclear material (n). X 90,000.

ever, in some instances, it was dispersed and its typical confluent structure was seen in many portions of the cell. Nucleoplasm char­acteristically showed areas of low electron density filled with DNA filaments (Figs. 4, 5, 9).

Cell division was common and daughter cells often remained attached after completion of

septum formation (Fig . 3, 9). Dividing cells often displayed several septa indicating the line of division could occur in more than one plane (Fig. 5).

Appearance of Organisms in Comedones

Loncritudinal ection through uninfl.amed comedone (Ficr~. , 10) bowed many free

Corynebacterium acnes 343

bacteria associated with cornified cells or embedded in an amorphous material of moder­ate electron density believed to repre ent ebum. In those areas of the comedone

where bacteria were present in laro-e number , the ultrastructural similarities of the organi ms were impressive, suggesting the presence of one species. The resemblance of these bacteria to cultured cells of C. acnes was al o impres ive. Examples of these similarities are illustrated in Figures 7-10.

DISCUSSIO

Zierdt and W ertlake ( 13) described three dif­ferent morphologic types in cell of C. acnes: a spherical form, a pleomorphic type, and a cornynebacterium form which they called the definitive C. acnes. Althouo-h our study was not de igned to confirm that three types exist, orne evidence wa obtained to support this

view. This possibility will be important to consider when studying acne lesion which may include cocci in addition to C. acnes .

The floccular material attached to the cell wall was similar to that observed in C. minutis­simum (17) and C. avis (1 ) . In C. avis (18) the adherence of daughter cells following clivi-ion was attributed to the cohe~ive prop­

erties of the superficial floccular rna terial. Hard (1 ) bowed that this material i~ a lipid and Jolly (19) suggested that it may be associated with pathoo-enicity. In orne of our previou studies of C. minutissimU?n, a ma­terial of similar appearance wa more abun­dant in vivo (erythrasma) t han in cultured cells. Electron microo-raphs (17 ) depicted the floccular material a contributino- to the ad­herence of diphtheroids to the skin. I t remains to be een if t his i al o the case with C. acnes in human skin.

Prominent mesosomes, such as tho e seen in this study, have been observed previously in bacteria present in comedones (20) . Mesosomes in B. subtilis and B. licheniformis have been associated with eptation and wall-forming capacity. L forms of these organism~, which lack mesosome , are unable to septate and make cell walls. In contrast, their bacillary forms contain meso omes and possess this ca­pacity (21). -The possibility of the spherical type of C. acnes repre enting an L form was di~cu sed by Zierdt and Wertlake (13) . L

Fw. 6. Ribo omes (r) m cell of C. acnes. X 98,000 .

forms were not seen in our cultures but it will be important to determine whether these occur in skin.

The infrequency of polypho phate o-ranules neemed to characterize six of the seven strains. It should be remembered, however, that in certain other bacteria (22), including C. diphtheriae (23), t he polyphosphate content i a function of the growth phase, beino­lowest in the exponential phase and highest in older culture . More strain. need to be studied before the ability of C. acnes to produce polyphosphate granules can be ascertained. Polyphosphate granules are very prominent in C. minutissimum ( 17), the only other member of the cutaneous flora studied in vitro and in vivo thus far with the electron microscope.

Results of this investigation have revealed triking similarities between many of the bac­

teria fo und in uninflamed comedones and cells of C. acnes grown in vitro . It is note­worthy that a specific immunofluorescent tech­nique has shown a larger number of C. acnes in uninfl.amed comedone (24). Although ul­trastructural criteria for recognition of C. acnes cannot yet be definitely established, it

0

Fw . 7-10. Compari on of two cells of cultured C. acnes (Figs. 7 & 9) with two ba teria in com dones (Figs. & 10). Cell wall and plasma membrane are somewhat better outlined in bacteri of comedones (Figs. 8 & 10) but the resemblance of all the cells i triking. X 65 000 (Fig. 7); X 82 000 (Fig. 8); X 62,000 (Fig. 9); X 74,000 (Fig. 10).

344

Corynebacterium acnes 345

seems certain that knowledge gained from this study will provide a useful background for in vivo identification and future observa­tions on the relationship of this bacterium to normal and pathologic skin.

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14. Ryter, A. and Kellenberger E.: tude au microscope electronique d pla rna con­tenant de lacide desoxyribonucl ique. Z. N aturforsch., 13b: 597, 195 .

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17. Montes, L. F. and Black, . H.: The fine tructure of diphth roid of erythrasma. J.

Invest. Derm., 48: 342 1967. 18. H ard, G. C.: Electron microscopic xamina­

tion of Corynebacl riwn ovis. J. Bact., 97: 1480, 1969.

19. Jolly, R. D.: Some obs rvation on urface lipids of virulent and att nuat d trains of Corynebacterium ovis. J. Appl. Bact riol., 29 : 189, 1966.

20. Montes, L. F. and Wilborn, W . H.: Location of bacterial skin flora. Brit. J. Derm., 81: 23, 1969.

21. Ryter, A. and Landman, 0. E.: El ctron mi­croscope study of th relationship b tween me osome los and table L tate (or proto­plast state) in Bacillus subtilis. J. Bact., 88: 457, 1964.

22. Harold, F. M.: Inorganic polyphosphates in biology: structure metabolism and function. Bact. Rev., 80: 772. 1966.

23. Kawata, T.: Electron microscopy of the fine structure of Coryn bacterium diphtheriae, with special refer nee to th intracytoplasmic membrane ystem. Jap. J. Microbial., 5: 442, 1961.

24. Imamura S., Pochi, P . E.. Struu J. S. and McCabcs, W. R.: The localization and dis­tribution of Corynebacterium acnes and its antigens in normal skin and in lesions of acne vulgari . J. Invest. erm., 53: 143, 1969.