European Journal of Clinical Pharmacology © by Springer-Verlag 1978
Europ. J. clin. Pharmacol. 14, 53-56 (1978)
Effect of Topical Application of Dexamethasone on Propionibacteria in the Pilosebaceous Duct M. Gloor, H. Funder, and M. Franke Universitfits-Hautklinik, Heidelberg, Federal Republic of Germany
Summary. A preparation of dexamethasone 0.02% in Eutanol G (n octyldodecanol) was applied once daily for three weeks, to the right side of the forehead of 25 male healthy test subjects. Eutanol G without dexamethasone was applied in the same way to the left side of the forehead. Before and at the end of the treatment period bacteria were removed from the pilo-sebaceous ducts by the method of Holland et al. [8]. Bacteria that grew under anaerobic conditions were evaluated quantitatively. Dexamethasone induced a significant increase in P. acnes, in all Propionibacteria and in the total bacterial count. The importance of these findings is discussed in relation to the development of steroid acne.
Key words: Dexamethasone, Eutanol G, skin bacteria, Propionibacteria, steroid acne.
In a previous paper we demonstrated that free fatty acids in the skin surface lipids increased at the expense of triglycerides after topical treatment with dexamethasone 21 sodium sulfobenzoate. This finding may be generalized to other corticosteroids [6]. The free fatty acids are mainly released by microbial lipases from the triglycerides of the sebaceous secretion. U n d e r in vivo conditions for the most part the lipases come from the Propionibacteria [1]. The increase in free fatty acids might be accounted for by an increase in bacteria or in lipase release from the bacteria or by activation of the lipases. The more pronounced lipolytic activity of P. granulosum means that an increase in P. granulosum at the expense of P. acnes could also be the cause of the increase in free fatty acids [22]. The previous investigations did not
clarify the mechanism of the increase in free fatty acids, nor did they demonstrate whether only lipolysis on the skin surface was intensified or lipolysis in the pilo-sebaceous duct, too, was affected. The present study was done to discover whether the enhancement of lipolysis brought about by the corticosteroids was caused by an increase in P. acnes or in P. granulosum in the pilosebaceous duct.
Material and Methods
a) Organization of the Tests 25 healthy male subjects, aged 17 to 24 years, were examined. Fortyeight hours before beginning the test and throughout the entire test period they avoided any contact with the facial skin, e. g. by washing the face, use of cosmetics etc. A preparation consisting of dexamethasone 0.02% 2 in Eutanol G 2 was applied once daily for three weeks, on the right side of the forehead. This solution was chosen since the vehicle guaranteed good solubility of the dexamethasone. At the same time Eutanol G, without the agent dexamethasone, was applied to the left side of the forehead. Neither the preparation containing the steroid nor that without it contained any preservative and both were used within 4 weeks after manufacture. Prior to treatment and at the end of the treatment period a quantitative investigation was performed on the bacterial flora in the pilosebaceous duct.
1 Manufacturer: Schering AG, Berlin, West Germany 2 2-octyldodecanol (Guerbet alcohol); manufacturer: HenkeI & Co., Dfisseldorf, West Germany
0031-6970/78/0014/0053/$01.00
54
b) Methods In order to collect bacteria from the pilosebaceous duct the glass stamp m e t h o d of Holland et al. [8] was employed. The m e t h o d is based on the principle that a drop of cyanoacrylate geP is applied to the skin and then a roughened glass sampling head is pressed on to the skin until the gel has b e c o m e firm. On withdrawing the glass stamp from the skin, parts of the hair follicles and pilo-sebaceous ducts remain attached. T h e bactericidal effect of the gel prevents superficial bacteria from being collected. T h e sampling head is always applied twice in succession to the same site. The bacterial counts are added together when a bacterial count per unit area of skin is to be determined. The initial investigation is always performed on the same area of the forehead and it is assumed that the bacterial count is almost identical on the contralateral area. T h e final investigation is always carried out on symmetrically situated areas on the two sides of the forehead. T h e glass stamps, to which parts of the follicles and pilo-sebaceous ducts adhere, are set into rotation under standardized conditions (for details see the original publication by Holland et al. [8]) in 12.5 ml reinforced clostridial medium (RCM) 4 with added 0 . 1 % T W E E N 805. Small glass beads were used to destroy the keratin, leading to release of bacteria. R C M 0.1 ml in dilutions from 10 ° to 10 .3 was spread on to reinforced clostridial agar 4 in the usual fashion. The cultures were incubated for 7 days at 37 °C, under anaerobic conditions, and then the bacteria were counted as usual. Aerobic cultures were deliberately not made since Propionibacteria were the main interest. Criteria for bacterial differentiation were the form and colour of colonies, as well as appearances in gram-stained smears. In this way Propionibacteria could be distinguished from gram-positive cocci and sarcines. Further differentiation of the cocci was not done, whereas in the Propionibacteria a distinction was made between P. aches and P. granutostun. T h e main criteria here were the results of the casein and phage susceptibility tests. T h e casein test is almost always positive with P. acnes and almost always negative with P. granulosum. As a rule, the phage susceptibility test is positive with P. acnes and negative with P. granulosum. It must be pointed out, however, that doubt has recently arisen as to the value of the phage susceptibility test, since not all P. aches strains react in the same way to all phages [9]. P. avidum, which 3 Permabound Contact Cement; manufacturer: Staident Laboratory, Staines, Middlesex, England 4 OxoidDeutschland, Wesel, West Germany 5 SigmaChemie, Neubiberg, West Germany
M. Gloor et al.: Dexamethasone and Cutaneous Flora rarely occurs on the forehead, reacts in the phage susceptibility test in the same way as P. granulosum, and in the casein test in the same way as P. acnes. The results of the casein and phage susceptibility tests were identical in all cases, except one; the reliability of these tests must, therefore, be emphasized. For details of the technique of these methods of differentiation see Marples and McGinley [t4].
c) Statistical Evaluation Determinations of bacterial counts usually follow a logarithmic distribution. Therefore, the logarithmically transformed values were used for statistical analysis. T o permit logarithmic transformation to be done on zero isolation values, a factor of 1, related to 2 × 0.1 ml undiluted RCM, was added to each value. In the statistical evaluation the differences for log (bacterial count in 2 × 0.1 ml undiluted R C M + 1) between the right and the left side were calculated in the final investigation. A t the same time, a comparison was made of the results found in the initial and final investigations. The average differences were compared by the Wilcoxon matched paires signed rank test with the hypothetical mean value 0.1% was specified as the necessary level of significance. In order to draw any conclusion on the geometric mean count per square centimetre it was necessary to cab culate the geometric mean count per 2 × 0.1 ml undiluted R C M and to multiply this value by the factor of 80. It was not possible to carry out these calculations for P. granulosum since it could only be demonstrated in a few cases.
Results The geometric mean counts / cm 2 for P. acnes, all Propionibacteria combined and the total bacterial count in anaerobic culture are shown in Table 1. In the initial investigation P. granutosum was detected four times, and at the final investigation it was found twice on the side treated with the agent and three times on the side treated with the vehicle. The bacterial counts ranged between 80 to 80000 per cm 2. At the final investigation one result was obtained which suggested the presence of P. avidum on the side treated with the vehicle (casein + , phage suspectibility test --; bacterial count 96000/cm2). Sarcines were rarely detected. At the initial investigation they were seen twice, and in the final investigation four times on the side treated with the vehicle and five times on the side treated with the agent. Dexamethasone led to an increase in bacterial count for P. acnes, for all Propionibacteria and in the
M. Gloor et al.: D e x a m e t h a s o n e and CuI:aneous Flora
55
Table L Geometric m e a n count / cm 2 for P. aches, all Propionibacteria combined and the total bacterial count in anaerobic culture Geometric m e a n count / em 2
Before t r e a t m e n t A f t e r t r e a t m e n t with the vehicle A f t e r t r e a t m e n t with d e x a m e t h a s o n e
P. acnes
Propionibacteria (P. a c n e s + P. granulosum)
Total bacterial count (in anaerobic culture)
123 l 2705 11526
1834 3106 11669
5107 7563 36876
total bacterial count, regardless of whether the comparison was made between the side treated with the agent and the side treated with the vehicle, or between the initial value and the final value on the side treated with the agent (Table 1). All these differences were statistically significant, at the specified level of significance of 1%. This effect of dexamethasone was of quantitative importance. The apparent tendency towards an increase in bacterial count on the side treated with the vehicle, as compared with the initial investigation, could not be confirmed statistically. Detailed results from individual test subjects can be found in the thesis by Funder [3]. Discussion
Gloor and Mildenberger [6] showed that topical application of corticosteroids leads, as a rule, to increased free fatty acids in skin surface lipids. They were unable to show whether the increase in free fatty acids was caused by an increase in lipase-releasing bacteria and whether only lipolysis on the skin surface was influenced or if lipolysis in the pilo-sebaceous duct, too, was involved. From the present studies it is evident that topically applied dexamethasone leads to an increase in bacteria in the pilosebaceous duct, regardless of whether evaluation is made of P. acnes, of all the propionibacteria together or of the total bacterial count in anaerobic culture. The relatively low bacterial counts are striking, as compared to earlier investigations [5, 7] using the same method. This may have been caused by the fact that several of the test subjects were examined during a period of frosty weather. Studies by Duncan et al. [2] have indicated the possibility that environmental factors may influence the bacterial flora. For this reason, care should be taken in evaluating the increase in bacterial count during the treatment. In the statistical analysis, the most important comparison is that in the final investigation between the side treated with the vehicle and that treated with the agent, as this was not subject to any influence from environmental factors.
Important previous studies of this problem have been published by Raab and Windisch [17-19]. They have shown that low doses of various corticosteroids can lead to metabolic enhancement of Candida albicans, Staph. aureus, Staph. albus and B. pyocyaneus. In the case of the first three bacteria a high concentration of corticosteroid causes inhibition of bacterial metabolism, but in B. pyocyaneus a high concentration of corticosteroid enhances its metabolism. These authors assumed that the latter was caused by metabolism of the corticosteroids by the bacterium. The studies were not concerned with Propionibacteria, but, in principle, they may also be applied to them. The present results were in agreement with their findings in so far as it is possible that only small amounts of dexamethasone penetrated the pilosebaceous duct. Our previous studies with antibiotics have shown that topically applied drugs may penetrate the pilosebaceous duct [5]. Many authors (reviewed in [15]) have assumed an influence of corticosteroids on infectious skin diseases. The present authors consider, however, that our findings are of only minor importance for this, since the general influence of corticosteroids on the host appears to be much more significant [20]. The present findings may be relevant in part to steroid acne. Steroid acne can only be compared with acne vulgaris in as far as comedo formation, but not pustule formation is concerned [16]. The results can only be discussed with regard to comedo formation. From experimental studies it seems probable that free fatty acids have a comedonogenic effect [4, 10, 12, 13]. They are formed in pilosebaceous ducts by splitting of triglycerides in the sebaceous secretion, aided by lipases which are mainly released by Propionibacteria [1, 21]. Propionibacteria also appear to have an additional influence on follicular hyperkeratosis in an as yet unclear manner [11]. A constitutional predisposition to develop follicular hyperkeratosis appears to be the prerequisite for a comedogenie effect. Plewig and Kligman [16] have shown that a constitutional predisposition towards acne is essential for the development of steroid ache. From the present
56 results it m a y b e a s s u m e d that the increase in b a c teria, especially in P. acnes, which is b r o u g h t a b o u t b y steroids, c a n i n d u c e c o m e d o f o r m a t i o n in s t e r o i d acne, as long as the p a t i e n t has the a p p r o p r i a t e c o n stitutional predisposition. This conclusion, based on the p r e s e n t results with d e x a m e t h a s o n e , m a y b e c o n s i d e r e d also to a p p l y to o t h e r corticosteroids.
Acknowledgement. W e wish to t h a n k the " D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t " for their support. W e are grateful to Dr. K. T. H o l l a n d , D e p t . of M i c r o b i o l o g y , U n i v e r s i t y of Leeds, for familiarizing us with the bacteriological m e t h o d s a n d Dr. H.-J. J6rs, Ichthyol G e s e l l s c h a f t C o r d e s , H e r m a n n i & Co., H a m b u r g , for s u p p l y i n g the d e x a m e t h a s o n e p r e p a r a t i o n s . References 1. Cunliffe, W. J., Strangfeld, K., Holland, K. T., Roberts, C. D.: Lipolytic activity of microorganisms in acne vulgaris. Proc. Roy. Soc. Med. (Lond.) 68, 275-276 (1975) 2. Duncan, W. C., McBridge, M. E., Knox, J. M.: Bacterial flora, the role of environmental factors. J. invest. Dermatol. 53, 479-484 (1969) 3. Funder, H.: r21ber den EinflnB yon Dexamethason auf die Propionibakterien im Talgdriisenausfiihrungsgang bei topischer Applikation. Inaugural-Dissertation, Heidelberg 1978 4. Gloor, M., Habedank, W. D.: Zur Pathogenese der Acne vulgaris. Mtinch. med. Wschr. 118, 649-652 (1976) 5. Gloor, M., Kraft, H., Franke, M.: The effectiveness of topically applied antibiotics on anaerobic bacteria in the pilosebaceous duct. Dermatologica (Basel) 157, 96-104 (1978) 6. Gloor, M., Mildenberger, H.: On the influence of an external therapy with dexamethasone - 21 - sodium - m - sulfobenzoate on the amount of free fatty acids in the skin surface lipids. Arch. Dermatol. Res. 261, 33-38 (1978) 7. Holland, K. T., Cunliffe, W. J., Roberts, C. D.: Acne vulgaris: an investigation into the number of anaerobic diphteroids and members of the micrococcaceae in the normal and ache skin. Br. J. Dermatol. 96, 623-626 (1977) 8. Holland, K.T., Roberts, C. D., Cunliffe, W. J., Williams, M.: A technique for sampling microorganisms from the pilosebaceous duct. J. appl. Bact. 37, 289-296 (1974)
M. Gloor et al.: Dexamethasone and Cutaneous Flora 9. Jong, E. C., Ko, H. L., Pulverer, G.: Studies on bacteriophages of propionibacterium acnes. Med. Microbiol. Immunol. 161, 263-271 (1975) 10. Kanaar, P.: Follicular-keratogenic properties of fatty acids in the external ear canal of the rabbit. Dermatologica (Basel) 142, 14-22 (1971) 11. Kligman, A. M.: An overview on acne. J. invest. Dermatol. 62, 268-287 (1974) 12. Kligman, A.M., Wheatley, V.R., Mills, O.H.: Comedonogenicity of human sebum. Arch. Dermatol. 102, 267-275 (1970) 13. Lorincz, A., Krizek, H., Brown, S.: Follicular hyperkeratinisation induced in the rabbit ear by human skin surface lipids. 13th Int. Congr. Derm., Miinchen 2, p. 1016-1017. Berlin, Heidelberg, New York: Springer Verlag 1968 14. Marples, R. R., McGinley, K.J.: Corynebacterium acnes and other anaerobic diphteroids from human skin. J. med. Microbiol. 7, 349-357 (1974) 15. Noble, W. C., Somerville, D. A.: Microbiology of human skin. London, Philadelphia, Toronto: W. B. Saunders 1974 16. Plewig, G., Kligman, A.M.: Induction of acne by topical steroids. Arch. Dermatol. Res. 247, 29-52 (1973) 17. Raab, W.: Probleme der lokalen Corticosteroidbehandlung. Heidelberg: Hiithig Verlag 1971 18. Raab, W., Windisch, J.: Bakterienstoffwechsel und Corticosteroide. Arch. klin. exp. Dermatol. 233, 363-375 (1969) 19. Raab, W., Windisch, J.: Der Einfluf3von Corticosteroiden auf den Stoffwechsel yon Hefepilzen. Arch. klin. exp. Dermatol. 236, 422--427 (1970) 20. Raft, M.J., Werner, A.S.: Staphylococcal infection in chick embryos: The effect of hydrocortisone on intra-allantoic infection. J. infect. Dis. llg, 4-24 (1968) 21. Shalita, A.: Genesis of free fatty acids. J. invest. Dermatol. 62, 332-335 (1974) 22. Whiteside, J. A., Voss, J. G.: Incidence and lipolytic activity of propionibacterium acnes (corynebacterium acnes group I) and p. granulosum (c. acnes group II) in acne and in normal skin. J. invest. Dermatol. 60, 94-97 (1973) Received: March 3, 1978 accepted in revised form: April 28, 1978
Prof. Dr. M. Gloor Universitgts-Hautklinik VogstraBe 2 D-6900 Heidelberg Federal Republic of Germany
Europ. J. clin. Pharmacol. 14, 53-56 (1978)
Effect of Topical Application of Dexamethasone on Propionibacteria in the Pilosebaceous Duct M. Gloor, H. Funder, and M. Franke Universitfits-Hautklinik, Heidelberg, Federal Republic of Germany
Summary. A preparation of dexamethasone 0.02% in Eutanol G (n octyldodecanol) was applied once daily for three weeks, to the right side of the forehead of 25 male healthy test subjects. Eutanol G without dexamethasone was applied in the same way to the left side of the forehead. Before and at the end of the treatment period bacteria were removed from the pilo-sebaceous ducts by the method of Holland et al. [8]. Bacteria that grew under anaerobic conditions were evaluated quantitatively. Dexamethasone induced a significant increase in P. acnes, in all Propionibacteria and in the total bacterial count. The importance of these findings is discussed in relation to the development of steroid acne.
Key words: Dexamethasone, Eutanol G, skin bacteria, Propionibacteria, steroid acne.
In a previous paper we demonstrated that free fatty acids in the skin surface lipids increased at the expense of triglycerides after topical treatment with dexamethasone 21 sodium sulfobenzoate. This finding may be generalized to other corticosteroids [6]. The free fatty acids are mainly released by microbial lipases from the triglycerides of the sebaceous secretion. U n d e r in vivo conditions for the most part the lipases come from the Propionibacteria [1]. The increase in free fatty acids might be accounted for by an increase in bacteria or in lipase release from the bacteria or by activation of the lipases. The more pronounced lipolytic activity of P. granulosum means that an increase in P. granulosum at the expense of P. acnes could also be the cause of the increase in free fatty acids [22]. The previous investigations did not
clarify the mechanism of the increase in free fatty acids, nor did they demonstrate whether only lipolysis on the skin surface was intensified or lipolysis in the pilo-sebaceous duct, too, was affected. The present study was done to discover whether the enhancement of lipolysis brought about by the corticosteroids was caused by an increase in P. acnes or in P. granulosum in the pilosebaceous duct.
Material and Methods
a) Organization of the Tests 25 healthy male subjects, aged 17 to 24 years, were examined. Fortyeight hours before beginning the test and throughout the entire test period they avoided any contact with the facial skin, e. g. by washing the face, use of cosmetics etc. A preparation consisting of dexamethasone 0.02% 2 in Eutanol G 2 was applied once daily for three weeks, on the right side of the forehead. This solution was chosen since the vehicle guaranteed good solubility of the dexamethasone. At the same time Eutanol G, without the agent dexamethasone, was applied to the left side of the forehead. Neither the preparation containing the steroid nor that without it contained any preservative and both were used within 4 weeks after manufacture. Prior to treatment and at the end of the treatment period a quantitative investigation was performed on the bacterial flora in the pilosebaceous duct.
1 Manufacturer: Schering AG, Berlin, West Germany 2 2-octyldodecanol (Guerbet alcohol); manufacturer: HenkeI & Co., Dfisseldorf, West Germany
0031-6970/78/0014/0053/$01.00
54
b) Methods In order to collect bacteria from the pilosebaceous duct the glass stamp m e t h o d of Holland et al. [8] was employed. The m e t h o d is based on the principle that a drop of cyanoacrylate geP is applied to the skin and then a roughened glass sampling head is pressed on to the skin until the gel has b e c o m e firm. On withdrawing the glass stamp from the skin, parts of the hair follicles and pilo-sebaceous ducts remain attached. T h e bactericidal effect of the gel prevents superficial bacteria from being collected. T h e sampling head is always applied twice in succession to the same site. The bacterial counts are added together when a bacterial count per unit area of skin is to be determined. The initial investigation is always performed on the same area of the forehead and it is assumed that the bacterial count is almost identical on the contralateral area. T h e final investigation is always carried out on symmetrically situated areas on the two sides of the forehead. T h e glass stamps, to which parts of the follicles and pilo-sebaceous ducts adhere, are set into rotation under standardized conditions (for details see the original publication by Holland et al. [8]) in 12.5 ml reinforced clostridial medium (RCM) 4 with added 0 . 1 % T W E E N 805. Small glass beads were used to destroy the keratin, leading to release of bacteria. R C M 0.1 ml in dilutions from 10 ° to 10 .3 was spread on to reinforced clostridial agar 4 in the usual fashion. The cultures were incubated for 7 days at 37 °C, under anaerobic conditions, and then the bacteria were counted as usual. Aerobic cultures were deliberately not made since Propionibacteria were the main interest. Criteria for bacterial differentiation were the form and colour of colonies, as well as appearances in gram-stained smears. In this way Propionibacteria could be distinguished from gram-positive cocci and sarcines. Further differentiation of the cocci was not done, whereas in the Propionibacteria a distinction was made between P. aches and P. granutostun. T h e main criteria here were the results of the casein and phage susceptibility tests. T h e casein test is almost always positive with P. acnes and almost always negative with P. granulosum. As a rule, the phage susceptibility test is positive with P. acnes and negative with P. granulosum. It must be pointed out, however, that doubt has recently arisen as to the value of the phage susceptibility test, since not all P. aches strains react in the same way to all phages [9]. P. avidum, which 3 Permabound Contact Cement; manufacturer: Staident Laboratory, Staines, Middlesex, England 4 OxoidDeutschland, Wesel, West Germany 5 SigmaChemie, Neubiberg, West Germany
M. Gloor et al.: Dexamethasone and Cutaneous Flora rarely occurs on the forehead, reacts in the phage susceptibility test in the same way as P. granulosum, and in the casein test in the same way as P. acnes. The results of the casein and phage susceptibility tests were identical in all cases, except one; the reliability of these tests must, therefore, be emphasized. For details of the technique of these methods of differentiation see Marples and McGinley [t4].
c) Statistical Evaluation Determinations of bacterial counts usually follow a logarithmic distribution. Therefore, the logarithmically transformed values were used for statistical analysis. T o permit logarithmic transformation to be done on zero isolation values, a factor of 1, related to 2 × 0.1 ml undiluted RCM, was added to each value. In the statistical evaluation the differences for log (bacterial count in 2 × 0.1 ml undiluted R C M + 1) between the right and the left side were calculated in the final investigation. A t the same time, a comparison was made of the results found in the initial and final investigations. The average differences were compared by the Wilcoxon matched paires signed rank test with the hypothetical mean value 0.1% was specified as the necessary level of significance. In order to draw any conclusion on the geometric mean count per square centimetre it was necessary to cab culate the geometric mean count per 2 × 0.1 ml undiluted R C M and to multiply this value by the factor of 80. It was not possible to carry out these calculations for P. granulosum since it could only be demonstrated in a few cases.
Results The geometric mean counts / cm 2 for P. acnes, all Propionibacteria combined and the total bacterial count in anaerobic culture are shown in Table 1. In the initial investigation P. granutosum was detected four times, and at the final investigation it was found twice on the side treated with the agent and three times on the side treated with the vehicle. The bacterial counts ranged between 80 to 80000 per cm 2. At the final investigation one result was obtained which suggested the presence of P. avidum on the side treated with the vehicle (casein + , phage suspectibility test --; bacterial count 96000/cm2). Sarcines were rarely detected. At the initial investigation they were seen twice, and in the final investigation four times on the side treated with the vehicle and five times on the side treated with the agent. Dexamethasone led to an increase in bacterial count for P. acnes, for all Propionibacteria and in the
M. Gloor et al.: D e x a m e t h a s o n e and CuI:aneous Flora
55
Table L Geometric m e a n count / cm 2 for P. aches, all Propionibacteria combined and the total bacterial count in anaerobic culture Geometric m e a n count / em 2
Before t r e a t m e n t A f t e r t r e a t m e n t with the vehicle A f t e r t r e a t m e n t with d e x a m e t h a s o n e
P. acnes
Propionibacteria (P. a c n e s + P. granulosum)
Total bacterial count (in anaerobic culture)
123 l 2705 11526
1834 3106 11669
5107 7563 36876
total bacterial count, regardless of whether the comparison was made between the side treated with the agent and the side treated with the vehicle, or between the initial value and the final value on the side treated with the agent (Table 1). All these differences were statistically significant, at the specified level of significance of 1%. This effect of dexamethasone was of quantitative importance. The apparent tendency towards an increase in bacterial count on the side treated with the vehicle, as compared with the initial investigation, could not be confirmed statistically. Detailed results from individual test subjects can be found in the thesis by Funder [3]. Discussion
Gloor and Mildenberger [6] showed that topical application of corticosteroids leads, as a rule, to increased free fatty acids in skin surface lipids. They were unable to show whether the increase in free fatty acids was caused by an increase in lipase-releasing bacteria and whether only lipolysis on the skin surface was influenced or if lipolysis in the pilo-sebaceous duct, too, was involved. From the present studies it is evident that topically applied dexamethasone leads to an increase in bacteria in the pilosebaceous duct, regardless of whether evaluation is made of P. acnes, of all the propionibacteria together or of the total bacterial count in anaerobic culture. The relatively low bacterial counts are striking, as compared to earlier investigations [5, 7] using the same method. This may have been caused by the fact that several of the test subjects were examined during a period of frosty weather. Studies by Duncan et al. [2] have indicated the possibility that environmental factors may influence the bacterial flora. For this reason, care should be taken in evaluating the increase in bacterial count during the treatment. In the statistical analysis, the most important comparison is that in the final investigation between the side treated with the vehicle and that treated with the agent, as this was not subject to any influence from environmental factors.
Important previous studies of this problem have been published by Raab and Windisch [17-19]. They have shown that low doses of various corticosteroids can lead to metabolic enhancement of Candida albicans, Staph. aureus, Staph. albus and B. pyocyaneus. In the case of the first three bacteria a high concentration of corticosteroid causes inhibition of bacterial metabolism, but in B. pyocyaneus a high concentration of corticosteroid enhances its metabolism. These authors assumed that the latter was caused by metabolism of the corticosteroids by the bacterium. The studies were not concerned with Propionibacteria, but, in principle, they may also be applied to them. The present results were in agreement with their findings in so far as it is possible that only small amounts of dexamethasone penetrated the pilosebaceous duct. Our previous studies with antibiotics have shown that topically applied drugs may penetrate the pilosebaceous duct [5]. Many authors (reviewed in [15]) have assumed an influence of corticosteroids on infectious skin diseases. The present authors consider, however, that our findings are of only minor importance for this, since the general influence of corticosteroids on the host appears to be much more significant [20]. The present findings may be relevant in part to steroid acne. Steroid acne can only be compared with acne vulgaris in as far as comedo formation, but not pustule formation is concerned [16]. The results can only be discussed with regard to comedo formation. From experimental studies it seems probable that free fatty acids have a comedonogenic effect [4, 10, 12, 13]. They are formed in pilosebaceous ducts by splitting of triglycerides in the sebaceous secretion, aided by lipases which are mainly released by Propionibacteria [1, 21]. Propionibacteria also appear to have an additional influence on follicular hyperkeratosis in an as yet unclear manner [11]. A constitutional predisposition to develop follicular hyperkeratosis appears to be the prerequisite for a comedogenie effect. Plewig and Kligman [16] have shown that a constitutional predisposition towards acne is essential for the development of steroid ache. From the present
56 results it m a y b e a s s u m e d that the increase in b a c teria, especially in P. acnes, which is b r o u g h t a b o u t b y steroids, c a n i n d u c e c o m e d o f o r m a t i o n in s t e r o i d acne, as long as the p a t i e n t has the a p p r o p r i a t e c o n stitutional predisposition. This conclusion, based on the p r e s e n t results with d e x a m e t h a s o n e , m a y b e c o n s i d e r e d also to a p p l y to o t h e r corticosteroids.
Acknowledgement. W e wish to t h a n k the " D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t " for their support. W e are grateful to Dr. K. T. H o l l a n d , D e p t . of M i c r o b i o l o g y , U n i v e r s i t y of Leeds, for familiarizing us with the bacteriological m e t h o d s a n d Dr. H.-J. J6rs, Ichthyol G e s e l l s c h a f t C o r d e s , H e r m a n n i & Co., H a m b u r g , for s u p p l y i n g the d e x a m e t h a s o n e p r e p a r a t i o n s . References 1. Cunliffe, W. J., Strangfeld, K., Holland, K. T., Roberts, C. D.: Lipolytic activity of microorganisms in acne vulgaris. Proc. Roy. Soc. Med. (Lond.) 68, 275-276 (1975) 2. Duncan, W. C., McBridge, M. E., Knox, J. M.: Bacterial flora, the role of environmental factors. J. invest. Dermatol. 53, 479-484 (1969) 3. Funder, H.: r21ber den EinflnB yon Dexamethason auf die Propionibakterien im Talgdriisenausfiihrungsgang bei topischer Applikation. Inaugural-Dissertation, Heidelberg 1978 4. Gloor, M., Habedank, W. D.: Zur Pathogenese der Acne vulgaris. Mtinch. med. Wschr. 118, 649-652 (1976) 5. Gloor, M., Kraft, H., Franke, M.: The effectiveness of topically applied antibiotics on anaerobic bacteria in the pilosebaceous duct. Dermatologica (Basel) 157, 96-104 (1978) 6. Gloor, M., Mildenberger, H.: On the influence of an external therapy with dexamethasone - 21 - sodium - m - sulfobenzoate on the amount of free fatty acids in the skin surface lipids. Arch. Dermatol. Res. 261, 33-38 (1978) 7. Holland, K. T., Cunliffe, W. J., Roberts, C. D.: Acne vulgaris: an investigation into the number of anaerobic diphteroids and members of the micrococcaceae in the normal and ache skin. Br. J. Dermatol. 96, 623-626 (1977) 8. Holland, K.T., Roberts, C. D., Cunliffe, W. J., Williams, M.: A technique for sampling microorganisms from the pilosebaceous duct. J. appl. Bact. 37, 289-296 (1974)
M. Gloor et al.: Dexamethasone and Cutaneous Flora 9. Jong, E. C., Ko, H. L., Pulverer, G.: Studies on bacteriophages of propionibacterium acnes. Med. Microbiol. Immunol. 161, 263-271 (1975) 10. Kanaar, P.: Follicular-keratogenic properties of fatty acids in the external ear canal of the rabbit. Dermatologica (Basel) 142, 14-22 (1971) 11. Kligman, A. M.: An overview on acne. J. invest. Dermatol. 62, 268-287 (1974) 12. Kligman, A.M., Wheatley, V.R., Mills, O.H.: Comedonogenicity of human sebum. Arch. Dermatol. 102, 267-275 (1970) 13. Lorincz, A., Krizek, H., Brown, S.: Follicular hyperkeratinisation induced in the rabbit ear by human skin surface lipids. 13th Int. Congr. Derm., Miinchen 2, p. 1016-1017. Berlin, Heidelberg, New York: Springer Verlag 1968 14. Marples, R. R., McGinley, K.J.: Corynebacterium acnes and other anaerobic diphteroids from human skin. J. med. Microbiol. 7, 349-357 (1974) 15. Noble, W. C., Somerville, D. A.: Microbiology of human skin. London, Philadelphia, Toronto: W. B. Saunders 1974 16. Plewig, G., Kligman, A.M.: Induction of acne by topical steroids. Arch. Dermatol. Res. 247, 29-52 (1973) 17. Raab, W.: Probleme der lokalen Corticosteroidbehandlung. Heidelberg: Hiithig Verlag 1971 18. Raab, W., Windisch, J.: Bakterienstoffwechsel und Corticosteroide. Arch. klin. exp. Dermatol. 233, 363-375 (1969) 19. Raab, W., Windisch, J.: Der Einfluf3von Corticosteroiden auf den Stoffwechsel yon Hefepilzen. Arch. klin. exp. Dermatol. 236, 422--427 (1970) 20. Raft, M.J., Werner, A.S.: Staphylococcal infection in chick embryos: The effect of hydrocortisone on intra-allantoic infection. J. infect. Dis. llg, 4-24 (1968) 21. Shalita, A.: Genesis of free fatty acids. J. invest. Dermatol. 62, 332-335 (1974) 22. Whiteside, J. A., Voss, J. G.: Incidence and lipolytic activity of propionibacterium acnes (corynebacterium acnes group I) and p. granulosum (c. acnes group II) in acne and in normal skin. J. invest. Dermatol. 60, 94-97 (1973) Received: March 3, 1978 accepted in revised form: April 28, 1978
Prof. Dr. M. Gloor Universitgts-Hautklinik VogstraBe 2 D-6900 Heidelberg Federal Republic of Germany
