JOURNAL OF BACTERIOLOGY, Nov., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 5Printed in U.S.A.

Ultrastructural Study of the Host-BacteriumRelationship in Erythrasma

LEOPOLDO F. MONTES, MOLLIE E. McBRIDE, WILHELM P. JOHNSON, DONALD W. OWENS,AND JOHN M. KNOX

Department of Dermatology, Baylor University College of Medicine, Houston, Texas

Received for publication 21 June 1965

Erythrasma, for many years considered asuperficial fungal infection, has recently beenrecognized as a bacterial disease. This was firstsuggested in 19C0 by Lagana (Acta Microbiol.Hellen. 5:69, 19C0) following isolation of a diph-theroid from lesions of erythrasma. Sarkany,Taplin, and Blank (J. Invest. Dermatol. 37:283,

4 4

0~~~~~~~~~~-

FIG. 1. Superficial stratum corneum inerythrasma.Numerous diphtheroids are seen. "acCallum-Goodpasture stain for bacteria in tissue. X 1,500.

1961) described the consistent isolation of agram-positive diphtheroid from lesions of cry-thrasma. This observation, subsequently confirmedby others (Munro-Ashman, Wells, and Clayton,Brit. J. Dermatol. 75:401, 1963), led to thebiochemical characterization of tHis diphtheroid,Corynebacteriuanmintissimim, the type strainof which was recently accel)ted by the NationalCollection of Type Cultures, London, England(Sarkany, Taplin, and Blank, Lancet 2:304,1962).

The localization by Sarkany et al. of thisorganism within cornified cells suggested to usthat skin from erythrasma could be used for astudy of a human host-bacterium relationshipat the ultrastructural level. Also, the frequencyof erythrasma, and the accessibility of the lesions

FIG. 2. Cross section of the superficial hyper-keratotic sratum corneum in erythrasma. Bacteriaare seen in the wide intercellular spaces (IS) andwithin cornified cells (C); the skin suirface is at thetop of the picture. X 5,700.

to multiple and innocuous biopsies, seemed tooffer a good l)ossibility of success for an in-vestigation of this nature.The 10 adult patients (5 men and 5 women)

used in this study fulfilled the diagnostic criteriafor erythrasma (clinical ap)earance, red fluo-rescence under Wood's light, positive culturesfor C. minutissimum, clinical and bacteriologicalresponse to systemic erythromycin). Negativemycological studies revealed that these subjectshad no superimposed fungal infection on the

1489

J. BACTERIOL.

FIG. 3. Skin surface in erythrasma. Several diph-theroids are seen. One (1) is penetrating the stratumcorneunm (SC); another (2) shows an electron-densevolutin granuile (V). X 14,000.

FIG. 5. Bacterium within a cell of the stratumcorneum. The cell wall (CW), the plasma membrane(PM), and an electron-dense cytoplasmic granule(V) (volutin?) are seen. The intercellular spaces(IS) of the stratum corneum are also shown. An areaof decreased electron density in the stratum corneumis apparent (arrows). X 31,000.

FIG. 4. Bacter-ium penetrating a corniftedI cellf ruom

an intercellular space (IS). Atrrows indicate thepoints at which the boundaries of this cornified cellare broken. 7'he fibrillar keratin (K) an(d the bac-terial cell wall (CW) are seen. X 33,700.

patches of erythrasma. The isolation mediumdescribed by Sarkany et al. (Arch. Dermatol.83:578, 1962) was employed for the bacterio-logical cultures.

Several skin specimens, 2 mm in diameter,were obtained in a manner previously described(Montes, Owens, and Knox, Experientia 20:672,1964) from the axilla or the crural region of eachlpatient. Immediately after removal, the speci-mens were fixed according to the Ryter-Kellen-berger technique (Z. Naturforsch. 13b:597, 1958).Embedding was l)erformed in a mixture of Eponand Araldite (Mollenhauer, Stain Technol.39:111, 1964). A Porter-Blum ultramicrotomeequipped with a diamond knife was used to pre-pare the sections. These were cut perpendicularlyto the skin surface. An RCA EMU3F electronmicroscole with an accelerating voltage of 50 kvwas utilized for study of the material. From eachpatient, one specimen was fixed in formalin andembedded in paraffin; 4-,u sections were stainedwith hematoxylin and eosin, periodic acid-Schiff,and the MacCallum-Goodpasture stain forbacteria in tissue (Mallory, Pathological Tech-nique, W. B. Saunders Co., Philadelphia, 1942).These sections were used for histologiccal ob-servations (Fig. 1-7).Under the electron microscope numerous

bacteria were seen at different levels of the stra-tum corneum: proliferating over the skin surface(Fig. 3); lying freely between the superficialcornified cells (Fig. 2); penetrating these cells

1490 NOTES

VOL. 90, 1965

FIG. 6. Another bacterium observed within the stratum corneum. Bacterial cell wall (CW) andmesosomes (M) are shown. The intercellular spaces (IS) and the fibrillar keratin (k) are seen in thestratum corneum. X 37,000.

FIG. 7. Details of the cell wall (CW) and plasma membrane (PM) of same bacterium as shown inFig. 6. X 135,000.

from the intercellular spaces (Fig. 4) or, lessfrequently, directly from the skin surface (Fig. 3);and intracellularly within the keratinized cells(Fig. 2, 5, 6). Whereas most organisms observedon the skin surface were characterized by ahomogeneous fine structure, bacteria withinthe stratum corneum were quite pleomorphic.Furthermore, dividing organisms were morecommon on the surface than within the stratumcorneum.The stratum corneum itself was hyperkera-

totic, with the superficial layers widely separatedand the cell boundaries disrupted at the sites ofbacterial penetration (Fig. 4). In the keratinizedcells, cytoplasmic areas of decreased electrondensity, frequently observed around intracellularbacteria, were suggestive of a keratolytic proc-ess (Fig. 5). Hopefully, studies presently in

progress will help to interpret the precise natureof this change. Electron micrographs of the fullstratum corneum thickness have shown thatbacteria in erythrasma can penetrate as deeplyas one-half the thickness of that layer. Thisobservation was confirmed under the light micro-scope by a study of sections stained with theMacCallum-Goodpasture method for bacteriain tissue. Observation of sections stained withperiodic acid-Schiff showed no fungal elementsin the stratum corneum of these patients.

This investigation was supported by a researchgrant from the Eli Lilly Co., Indianapolis, Ind.

S. H. Black, G. F. Odland, E. Kellenberger, D.Taplin, and R. P. Williams helped in the inter-pretation of the results. Nedra Morelanid, SylviaMcCrevey, and Gordon Adams gave valuable tech-nical assistance.

NOTES 1491