THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 67 :283-284, 1976 Copyright © 1976 by The Williams & Wilkins Co.

Vol. 67, No.2 Print ed in U.S.A.

CONCISE REPORTS

A SEARCH FOR MYCOPLASMAS AND CHLAMYDIAE IN ACNE LESIONS

C. A. EVANS, M.D., PH .D., G. E. KENNY, PH .D. , SAN-PIN WANG, M.D. , AND E. E. LANGER, M.D.

Department of Microbiology and Immunology , Department of Pathobiology , and Division of Dermatology, Department of Medicine, University of Washington, Seattle, Washington, U. S. A .

No mycoplasmas and no chlamydiae were found in cultures of 75 uninflamed comedones, 72 papulopustular lesions, and 7 cystic lesions from 14 subjects with acne. Chlamydial serologic tests were negative for 11 subjects and showed antibodies in 3 subjects to 3 different antigenic types of trachoma agent. These results are viewed as substantial evidence against the possibility that either mycoplasmas or chlamydiae play an important part in the pathogenesis of acne.

According to a widely favored view of the patho­genesis of acne vulgaris, the acne bacillus (Propio­nibacterium acnes) and the free fatty acids it produces in sebum playa major part in the disease process [1-4]. This concept is not universally accepted as it leaves a number of questions un­solved [5,6]. In considering what microbial agents other than P. acnes might playa causal role in acne either in conjunction with P. acnes or independ­ently of it, we decided to investigate two major groups of microorganisms that have not previously been studied in relation to this disease, the chla­mydiae and the mycoplasmas.

MATERIALS AND METHODS

Fourteen volunteers, 9 male and 5 female, with acne , were studied. They ranged in age from 19 to 50 years , with a median age of 23.5 years. Thirteen were Caucasian and one was of Japanese descent. Most were students or employees of the University of Washington and none had received antibacterial therapy within the preceding 3 months according to their reports . Acne on their backs was graded by criteria similar to those of Pillsbury et al [7 ]. Seven subjects had Grade II disease, 6 had Grade III, and 1 had Grade IV acne.

All specimens were collected from the back. Contents of uninflamed lesions (open and closed comedones) were obtained with a sterile Schamberg comedone extractor without preliminary disinfection of skin. Seventy percent alcohol was briefly applied to the skin surface over inflamed lesions; the skin at the center of the lesion was

Manuscript received December 3, 1975; accepted for , ublication March 2, 1976. . ' This study was aided by the Poncin Scholarship Award :.'01-10-635500 and by the following grants from the ~ational Institutes of Health: AI 11915 and AI 10938 f ?rn the National Institute for Allergy and Infectious ;)}seases, EY 02219 from the National Eye Institute, and \ M 05250 from the National Institute of Arthritis, \1etabolism, and Digestive Diseases. . ~eprint requests to: Dr. C. A. Evans, Department of " ~lcrobiology and Immunology SC42, School of Medi­('me, University of Washington, Seattle, Washington :}8195.

nicked with a sterile scalpel and the contents were gently expressed with a comedone extractor. From each subject we prepared 2 pools: one contained material from 4 to 6 uninflamed lesions (open and closed comedones) , and the other contained material from a like number of papular and pustular lesions. Cystic lesions on 3 subjects were also tested. We tested the contents of 75 comedones and 72 papulopustular lesions. One of the latter pools con­tained material from 4 cystic lesions as well as 4 papulopustular lesions. Three other cystic lesions from 2 subjects were tested as 2 specimens, 1 from each subject.

As material was obtained from each lesion it was transferred to a Ten Broeck grinder and thoroughly ground in 0.4 ml of mycoplasma transport medium (see below). Because mycoplasmas are exceedingly sensitive to surface active agents, Triton X-100 was not used. The pooled specimen was dispensed into 4 equal parts to be tested for mycoplasmas, chlamydiae, and ordinary bacte­ria, respectively, with one portion frozen for storage.

283

Tests for Chlamydiae

All specimens for chlamydial isolation were kept frozen at -65°C before inoculation into cell culture. DEAE-dex­tran-pretreated He La 229 cells were used for isolation. Details of the procedure have been published [8]. The inoculated cells were examined for inclusion bodies after 72-hr incubation, and an additional passage was at­tempted. A specimen was considered negative if no inclusion bodies could be detected in the passage inocula­tion.

Patient serum was tested with the microimmuno­fluorescence method against Trachoma-lymphogranu­loma venereum (LGV) representative antigens. Only re­actions observed at 1:8 or higher dilutions of serum were considered positive. Type-specific antibody was determined by the pattern of the reactions [9 J .

Tests for Mycoplasmas

Specimens to be cultured for mycoplasmas were emul­sified in transport medium (trypticase soy broth with 0.5% bovine albumin) and inoculated onto E agar and Mes agar (0.05 ml each) and E broth (0.1 ml). Media and methods are described in [10 J. The E agar plate was incubated in an atmosphere of 2.5% CO 2 in air at 37°C, the E broth at 37°C in a sealed tube, and the Mes agar

284 EVANS ET AL

plate was incubated anaerobically in the GasPak (BBL) system at 37°C. Cultures were examined at 3, 6, and 21 days. The E broth was subcultured at 6 days and 21 days to E agar and Mes agar and incubated as above.

RESULTS

No chlamydiae were isolated. All 30 specimens derived from 14 subjects were negative. In tests for trachoma antibody, the serum of 11 subjects was negative. The other 3 subjects showed antibody, each to one of 3 different antigenic types of trachoma agent-CJ, B, and DE.

No mycoplasmas were isolated even though the medium and conditions were sufficient to recover most known mycoplasma species [10].

DISCUSSION

Our fail ure to recover chlamydiae in cell cultures along with the mostly negative results in serologic tests for trachoma antibodies constitutes persua­sive evidence that no members of this group of infectious agents play an essential role in acne. If chlamydiae were present, their requirements for growth in vitro and their antigenic make-up must have been markedly different from those of the broad group of agents encompassed by the designa­tion Chlamydia trachomatis.

The known species of mycoplasma exhibit great diversity in their requirements for in vitro cultiva­tion as well as in their disease-producing capabili­ties. The methods used in the present studies should have been adequate to detect either aerobic or anaerobic species unless they were more highly sensitive to exposure to oxygen than other species that infect man, or their growth requirements were different from those of the known mycoplasmas. The acne lesion seems, on an a priori basis, an unlikely habitat for these membrane-bound orga­nisms because organisms of this group tolerate

Vol. 67, No.2

excess fatty acids poorly [11,12] even though some mycoplasmas have a specific requirement for fatty acids.

We deeply appreciate Dr. George F. Odland's encour­agement and his assistance in facilitating this study, and the expert technical assistance of Ms. Sidney Hallam.

REFERENCES

1. Strauss JS, Pochi PE: Intracutaneous injection of sebum and comedones. Arch Dermatol 92:443-456, 1965

2. Kellum RE: Acne vulgaris-studies in pathogenesis: relative irritancy of free fatty acids from C2 to C 16.

Arch Dermatol 97:722-726, 1968 3. Kligman AM, Katz AG: Pathogenesis of acne vul­

garis. I. Comedogenic properties of human sebum in external ear canal of the rabbit. Arch Dermatol 98:53-57, 1968

4. Freinkel RK: Pathogenesis of acne vulgaris. N Engl J Med 280:1161-1163, 1969

5. Voss JG: Acne vulgaris and free fatty acids . Arch Dermatol 109:894-898, 1974

6. Fulton JE, Weeks JG, McCarty L: The inability of bacterial lipase inhibitor to control acne vulgaris (abstr). J Invest Dermatol 64:281-282, 1975

7. Pillsbury DM, Shelley WB, Kligman AM: Dermatol­ogy . Philadelphia, Saunders, 1956, pp 806-810

8. Kuo CC, Wang SP, Wentworth BB, Grayston JT: Primary isolation of TRIC organisms in the HeLa 229 cells treated with DEAE-dextran. J Infect Dis 125:665-668, 1972

9. Wang SP, Grayston JT: Human serology in Chlamydia trachomatis infection with microim­munofluorescence. J Infect Dis 130:388-397, 1974

10. Kenny GE: Mycoplasma, in Manual of Clinical Microbiology. Edited by EH Lennette, EH Spauld­ing, JP Truant. Washington, DC, American Society of Microbiology, 1974, pp 333-337

11. Rodwell AW: Nutrition and metabolism of Myco­plasma mycoides var mycoides. Ann NY Acad Sci 79:499-507, 1960

12. Razin S, Rottem S: Fatty acid requirements of Mycoplasma laidlawii . J Gen Microbiol 33:459-470, 1963