The Fine Structure of Diphtheroids of Erythrasma*

Vol. 45, No. 4

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

1967 by The Williams & Wilkins Co.

Printed in U.S.A.

THE FINE STRUCTURE OF DIPHTHEROIDS OF ERYTHRASMA* LEOPOLDO F. MONTES, M.D. AND S. H. BLACK, PH.D.

from the National Collection of Type Cultures

The causative organism of erythrasma has long been considered to be a fungus, variously named Micros pomm minutissimum, Nocardia minutissimo, Sporotrichum minutissimum, Micros paroides minutissimus, Oospora minutissima, and Discomyces minutissimus. Renewed interest in the etiology of this long-recognized (1) chronic infection of the stratum corneum has been stim-

(London), another was supplied by David Taplin, University of Miami (Florida), and one was cultured from one of our own patients. The medium of Sarknny et al. (5) was used for primary isolation and maintenance of the organisms; 20% (v/v)

fetal bovine serum, 78% (v/v) tissue culture medium No. 199, 2% (w/v) agar, and .05% (w/v)

tris (hydroxymethylaminomethane) were autoclaved together and used without adjustment of ulated by the clinical finding that erythrasma, pH or addition of antibiotics. Their typical counlike dermatophytic infections, fails to respond lonial morphology is pictured in Fig. 3. Bacteria to treatment with the antifungal agent, grisco- for electron microscopy were subcultured either in fulvin, but may be cured by erythromycin ad- nutrient broth (Difco) or on blood agnr plates. Electron microscopy. Skin biopsy specimens (6) ministered systemically (Fig. 1 and 2). That the from ten different patients, or bacteria harvested disease might be of bacterial origin was first sug- from the culture media, were fixed for 16 hr in gested by Lagana (2) who isolated a Car ynebac- either of two solutions: 1) 1% (w/v) OsO, in terium and a hemolytic Staphylococcus from veronal-acetate buffer, pH 6.0, supplemented with (w/v) CaCl, and 0.1% (w/v) tryptone (7); scrapings of the skin of patients with cry- 0.1% or 2) 1% (w/v) 0504 in veronal-acetate buffer, thrasma. More recently, Sarkany, Taplin and pH 7.4 (8). After fixation, the tissues or cells were Blank (3, 4) reported culturing, from scales of treated for 2 hr with 0.5% (w/v) uranyl acetate afflicted patients, a diphthcroid which, when in veronal-acetate buffer, pH 6.0, dehydrated inoculated onto the skin of human volunteers, through graded concentrations of acetone or ethanol, and embedded in a mixture of Epon and gave rise to clinical erytbrasma. The organism Araldito 506 (9) or in Maraglas (10). Sections has been described biochemically and has been were cut with a diamond knife on a Porter-Blum named Car ynebacterium minutissimum (5), the microtime and observed in an electron microscope type strain of which has been accepted by the (RCA EMU3F) with an accelerating voltage of National Collection of Type Cultures, London, 50 KY. England.

RESULTS

In view of the paucity of information available on the microanatomy of the coryncbacteria and as an adjunct to an ultrastructural study of the host-parasite relationship in erythrasma, we

Appearance of organisms in vivo. Sections of punch biopsies from patients displaying clinical crythrasma revealed bacterial cells located on the surface of the skin (Fig. 5 and 6) and in the superficial layers of the stratum corneum. An ultrastructural analysis of the host response to these infecting organisms was recently reported (11). The diphtheroids are seen as slightly elongated rods with rounded ends. Cells cut longitudinally appear broader at one end than at the other, thus conforming to the characteristic club-shaped appearance of whole cells seen with the light microscope (Fig.4). The organisms are seen to be surrounded by

have undertaken to describe the fine structure of Caryn.e bacterium minutissimum. MATERIALS AND METHODS

Cultures. Ten strains of Caryne bacterium minu-

tissimum, originally isolated from patients with crythrasma, were studied: eight were obtained * From the Departments of Dermatology and Microbiology, Baylor University College of Medi-

cine, Houston, Texas. Dr. Montes is currently in

the Department of Dermatology, University of Alabama Medical Center, Birmingham. This study was supported by a USPHS Research Career Development Award (No. I-K3-AI-31210-

extremely electron-dense material (Fig. 6).

Lilly and Company, Indianapolis, Indiana, and by a contract (No. DA-49-193-MD-2746) from the

Whether this electron-dense material is excreted by the organism or whether it may be of foreign origin (12) is not known.

Command Office of the Surgeon General. Received for publication June 10, 1966.

Underneath the electron-dense material lies the cell wall, a tripartite structure consisting of

01) to L.F.M., by a research grant from the Eli

U.S. Army Medical Research and Development

342

v

Lt

I

'P

•1

4

-

-

•1

t—C I'

S

a -I

) I

•1 I

I

C

S

a

S.

•.

I

"I

*a' ••

.t-t. r S

S.

S

S

4

I

—

.

I —N

Fic. 1. Erythrasma involving the inguinocrural areas. The patient, a 62 year old Negro male, had the disease for 5 years before he was first seen in our hospital. He was also found to be a diabetic, with a fasting blood sugar level of 4.80 g %. FIG. 2. Photograph showing the same areas after 16 days of treatment with oral erythromyein estolate (Ilosone). The drug was given as follows: 250 mg q.i.d. for the first 7 days, 250 mg t.i.d. for another 4 days, and 250 mg b.i.d. during the last 5 days. No local treatment was used. Only residual hyperpigmentation is seen. The two dark spots mark the sites of biopsy specimen removal. Fie. 3. Typical colonial morphology of Corynebocterium minutissimum grown on the medium of Sarkany et al. (3). Fie. 4. Microscopic appearance of C. minulissimum stained by Gram's technic. Note the typical "diphtheroid" appearance of the cells.

344

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

'S

a 0

* k-—— 'S.

•4/

K4

4k

Js

Fie. 5. Section through the skin surface from a patient with erythrasma. The specimen was fixed by procedure 1. Note the volutin granule (V) in the cell at the lower left. Wisps of plasma membrane (arrows) are evident in the longitudinally-sliced cells. The nucleoplasm (N) of these cells shows compact arrangement. The superficial stratum corneum (SC) is shown to the left and bottom of the figure. X 48,298.

345

FINE STRUCTURE OF DIPHTHEROIDS

two dense bands, 35 A wide, separated by a light zone, 50 A wide (note inset of Fig. 6). The cell

viding cells (Fig. 11). Intracytoplasmic membranes are less-well-defined in cells fixed by pro-

wall adheres tightly to the underlying proto- cedurel (Fig. 13). plast, the limiting membrane of which is only ocThe cytoplasm of some cells contains irregcasionally well defined (arrows, Fig. 5). This ularly-shaped granules (Fig. 7) that are of explasma membrane, also a triple-layered structure traordinary electron-density, sometimes as large composed of two dense lines sandwiching a light as 0.1 in diameter; they are readily sublimed by band (total thickness about 75 A), is morpho- the electron beam. In some sections (Fig. 13 and

logically similar to the "unit membranes" (13)

15), the cytoplasm is seen to be packed with

that limit the cytoplasm of "higher" cells. The cytoplasm is characterized by the presence

dense granules, 120—150 A in diameter, that presumably are ribosomes. The nuclear region is another distinctive cytoplasmic feature of cells fixed by procedure 1. The

of volutin, irregularly-shaped, electron-dense granules (Fig. 5) that can easily be volatilized by electron bombardment (vacuole in cell pictured in Fig. 6). Also conspicuous in the cytoplasm is the nuclear region, a central area of lower electron-density than cytoplasm and filled with fine fibrils having a random arrangement, sometimes compact (Fig. 5) and sometimes sparse (Fig. 6). Cytoplasmic particles of ribosomal size or intracytoplasmie membranes are not well defined in these particular sections. Appearance of organisms in vitro. Figures 7—

nucleoplasm is not surrounded by a membrane and, compared to the cytoplasm, is seen to be of low electron-density. It contains thin fibrils (40—

50 A in diameter) which, at times, form whorls

(Fig. 7) but more usually make up a net-like mesh (Fig. 14). In some cells fixed by procedure 2, the fibers appear to run parallel to each other

or to be twisted one upon another (Fig. 16). In some sections, the nuclear material is not localized but apparently is dispersed among the gran-

16 demonstrate the appearance of sectioned ular elements of the cytoplasm (Fig. 15). Corynebacterium minutissimum grown in vitro. Surface-adhering material of high electron density is less conspicuous on cells fixed according

DISCUSSION

Our electron microscopic observations of thin-

to procedure 1 (Fig. 7) than on cells infecting sectioned skin from patients with erythrasma esskin (Fig. 5 and 6) and is virtually absent in cells tablish the presence of "diphtheroids" on and in fixed by procedure 2 (Fig. 8—11). The tripartite the horny layer of the stratum corneum. Confirstructure of the cell wall is apparent in cells fixed mation that these diphtheroids are the etiologic by both procedures. agent of erythrasma, as suggested by Sarkany The plasma membrane is poorly defined in et al. (3—5), was not sought in these investigamost of these cells. However, there is apparent, in tions, but it seems significant that frequently cells fixed by procedure 2, a clear zone between only diphtheroids were found in and on the the cell wall and the protoplast that is compa- stratum corneum from patches of erythrasma rable in thickness (25—30 A) to the electron light and that frequently only diphtheroids could be band of a typical unit membrane and is consid- cultivated. ered to represent the plasma membrane. That Not surprisingly, those organisms cultured the dark bands are not generally visible is per- from afflicted patients were, as indicated above,

haps attributable to their having a density similar to organisms inhabiting the stratum similar to that of adjacent structures. Readily corneum. Indeed, they were found to resemble apparent in cells fixed by procedure 2 are intra- closely Corynebacteriu,n diphtheriae (15) and cytoplasmic membranes. These membranes are C. fasciens (12), the only other members of the seen to vary in shape and complexity from genus Corynebacterium which, to our knowledge, simple closed, concentric rings (Fig. 9 and 11) have been studied in ultrathin sections under the to open semi-circular or horse-shoe-shaped for- electron microscope. Typically, the features of mations (Fig. 8 and 10). These "mesosomes" these reported organisms, and those pictured (14) are usually observed in close proximity to, above, have been: 1) a triple-layered cell wall, and in occasional cases (Fig. 10) to be continuous a rather more complex structure than is comwith, the plasma membrane; they are commonly

present at regions of septum formation in di-

monly seen in other Gram-positive bacteria (16) 2) the presence of electron-dense volutin granules

lr,9. at

t.

t: 7 .sc

1( N

LptI' Fra. 6. Corynebacterium minutissimum observed on the surface of the skin in a patch of erythrasma. The specimen was fixed by procedure 1. Note the heavy layer of electrondense material adhering to the surface of the organism as well as to the horny layer of the stratum corneum (SC). The tripartite structure of the cell wall is readily apparent. The inclusion (V) in the cytoplasm is all that remains from electron bombardment of a volutin granule. The nucleoplasm (N) displays a typical sparse arrangement of fibrils. X 87,480. The inset shows at higher magnification details of the surface structures. The cell wall (CW) is clearly seen, while the plasma membrane (PM) is barely resolved. X 174,960. 346

- F'?.

A

'It-

FINE STRUCTURE OF DIPHTHEROIDS

Fie. 7. Section through C. minutis.simum from a broth culture incubated overnight. The cells were fixed according to procedure I. Particularly striking are the triple-layered cell wall (CW), the volutin granules (V), and the electron-light nucleoplasm (N). Several rounded areas (M) of low density in the cytoplasm are probably mesosomes. X 75,600.

347

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

348

a-

A.

-

•.h C#

I Pie. 8—11. C. mznutisszrnum grown 7 days in broth culture nnd fixed according to procedure 2. Pie. 8. General appearance of week-old cells. The clear zone between the cell wall and the underlying protoplast is considered to be the middle layer of the plasma membrane. Notable in these cells are the intracytoplasmic membranes. X 48,600. Fm. 9. The intracytoplasmic membranes show a characteristic concentric ring arrangement. >< 77,800.

Fm. 10. The mesosome in this dividing cell appears to be continuous with the plasma

membrane (PM). >< 104,976.

Fm. 11. Another dividing cell displaying a large mesosome proximal to the invaginating

membrane and wall. 1< 121,824.

that can be caused to sublime under the electron

closely approximating that for a variety of

beam (17); and 3) elaborate intracytoplasmic extensions of the plasma membrane generally

corynebacterial species (19) all lend support to the establishment of the organism of erythrasma

termed mesosomes (14).

as a Corynebocterium.

The accumulated facts: 1) that the organism

That Corynebacterium minutissimum is in-

constantly associated with erythrasma resembles, volved in the etiology of erythrasma seems cerin thin-section, the corynebacteria that have thus tain. But the question of whether C. minutisfar been investigated; 2) that the organism is re- sf mum is the sale etiologic agent, as claimed by lated biochemically to a great variety of diph- Sarkany et at. (3—5), or whether this diphtheroid

thcroids studied (18) and 3) that the DNA ex- must occur symbiotically with yet another "untracted from the organism displays a base ratio cultivable" organism to produce the disease, as

Fic. 12—14. C. minutissimum from a broth culture incubated overnight and fixed accord-

ing to procedure 1.

FIG. 12. Multiple layers in the septum of this divided cell are particularly well defined.

X 102,976.

FIG. 13. Another dividing cell in which the ribosomes (R) are especially outstanding.

Some intracytoplasmic membranes (arrows) are also evident. >< 92,340.

FIG. 14. The electron-light nucleoplasm (N) shows fibrils that appear to form a net-like

mesh. X 102,976.

350

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 15. Section through cells of C. minutissimum from broth culture and fixed according to procedure 1. The nucleoplasm (N) is not localized but, rather, appears to be interspersed among the cytoplasmic elements, including ribosomes (R). X 97,200.

FINE STRUCTURE OF DIPHTHEROIDS

351

2. Lagana, I.: Contribution to the study of the

pathogenic agent of erythrasma and its therapy (in Greek). Acta Microbiol. Hellen., 5: 69, 1960.

3. Sarkany, I., Taplin, D. and Blank, H.: Ery-

thrasma—common bacterial infection of the skin. J. A. M. A., 177:

130, 1961.

4. Sarkany, I., Taplin, D. and Blank, H.: Incidence and bacteriology of erythrasma. Arch. Derm. (Chicago), 85: 578, 1962.

5. Sarkany, I., Taplin, D. and Blank, H.: Organism causing erythrasma. Lancet, 7250: 304, 1962.

6. Montes, L., Owens, D. and Knox, J.: Applica-

tion of the Ryter-Kellenberger fixation method to electron microscopic study of bac-

teria on the skin surface. J. Invest. Derm., 45: 93,

1965.

7. Ryter, A. and Kellenberger, E.: Etude au microscope electronique de plasmas contenant de l'acide desoxyribonucleique. Z. Naturforsch., iSB: 597, 1958.

8. Palade, C.: A study of fixation for electron microscopy. J. Exp. Med., 95: 285, 1952.

9. Mollenhauer, H.: Plastic embedding mixtures for use in electron microscopy. Stain Techn., 39:

111, 1964.

V. and Freeman, J.: Technical modifications in Maraglas em-

10. Spurlock, B., Kattine,

bedding. J. Cell Biol., 17: 203, 1962. 11. Montes, L., McBride, M., Johnson, W., Owens, FIG. 16. C.

minutissimum fixed according to

procedure 2. The nucleoplasm (N) is characterized by fine fibrillar threads arranged in parallel arrays. >< 129,600.

D. and Knox, J.: Ultrastructural study of the host-bacterium relationship in erythrasma. J. Bact., 90: 1489, 1965.

12. Glauert, A.: The fine structure of bacteria. Brit. Med. Bull., 18: 245, 1962.

13. Robertson, J.: Ia The Structure and Function suggested by Meyer-Rohn and Meinhof (20), remains to he answered. SUMMARY

1. The fine structure of Corynebacterium mmutissimum, the causative organism of crythrasma, has been studied. 2. Distinctive features of these cells include a

tripartite cell wall, elaborate intracytoplasmic extensions of the plasma membrane, and electrondense volutin inclusions.

of Subcellular Components. Biochemical So-

cietv Symposia. No. 16. Cambridge, Cambridge University Press, 1959.

14. Fitz-James, P.: Participation of the cytoplasmic membrane in the growth arid spore formation of bacilli. J. Biophys. Biochem. Cytol., 8: 507, 1960.

15. Kawata, T.: Electron microscopy of the fine structure of Coryne bacterium diphtheriae, with special reference to the intracytoplasmic membrane system. Jap. J. Microbiol., 5: 443, 1961.

16. Salton, M. R. J.: The Bacterial Cell Wall. Amsterdam, Elsevier Publishing Company, 1964.

3. The diphtheroids seen in the stratum cor- 17. Vanderwinkel, E. and Murray, R. C. E.: Organelles intracytoplasmiques bactdriens et neum of biopsy specimens taken from patients site d'activité oxydo-rSductrice. J. Ultrastruct. Res., 7: 185, 1962. with erythrasma were no different from those cultured in the laboratory. REFERENCES 1. Burchart, M.: Uber eine bei Chloasma vorkommende Pilzform. Med. Zeit., 2: 141, 1859.

18. McBride, M. and Montes, L.: Unpublished observations. . 19. Mandel, M.: Personal communication.

20. Meyer-Rohn, J. and Meinhof, W.: Zur Frage

der bakteriellen Genese des Erythrasma. Arch. KIm. Exp. Derm., 222: 57, 1965.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.