LIPOLYTIC ACTIVITY OF RESIDENT FLORA OF THE SKIN: SOME OBSERVATIONS ON LIPASE ACTIVITY OF CORYNEBACTERIUM

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1 SKIN RESEARCH Dec Vol.10 No LIPOLYTIC ACTIVITY OF RESIDENT FLORA OF THE SKIN: SOME OBSERVATIONS ON LIPASE ACTIVITY OF CORYNEBACTERIUM ACNES AND STAPHYLOCOCCUS EPIDERMIDIS COMPARED WITH STAPHYLOCOCCUS AUREUS YASUO, ASADA*, M.D. Department of Dermatology, Kansai Medical School, Moriguchi, Osaka, Japan (Director: Prof. K. OHARA) INTRODUCTION It is generally recognized that facultative anaerobic corynebacterium acnes and aerobic and/or facultative anaerobic staphylococcus epidermidis (coagulase negative, mannitolfermentation negative) are considered to be apathogenic or less pathogenic, both survive saprophytically in the skin surface lipid film, in the epidermis and even in the superficial part of the pilosebaceous apparatus. These two kinds of bacteria constitute so called redident flora of the skin.4,5) On the other hand, staphylococcus aureus (coagulase positive, mannitol-fermentation positive) are considered to be pathogenic. They are usualy found in the lesions of pyodermas and rarely in the surface of the normal skin. *Associate professor, Department of Dermatology, Kansai Medical School. It is also accepted that free fatty acids on the skin surface are secondary split products of neutral fat (triglyceride) in sebum.11,12,26) One may assume that lipolysis of sebum starts after sebaceous cells have fallen apart and the amorphous mass has reached the sebaceous duct and continuously proceeded until or even after sebum reaches the skin surface. In the lipolytic process neutral fat in sebum is assumed to be split to free fatty acids by lipase originated from tissue cells and resident bacteria of the skin7,14,16,17,22,23) It is also observed that free fatty acids does not originate from bacterial lipids of resident bacteria.20) Reports have been made concerning lipolytic activity of various bacteria including staphylococci and corynebacterium acnes.3,5,6,8,14,22,24) Some state that there is distinct relation between lipolytic activity and virulence of staphylococcus.3,5,8,10,14) In the present study an attempt was made to pursue the lipolytic activity of bacteria which constitute resident flora of the skin, i.e., cory- 585

2 nebacterium acnes and staphylococcus epidermidis. Then a comparison was made between the lipolytic activity of staphylococcus epidermidis and that of staphylococcus aureus. MATERIALS AND METHODS The materials employed were as follows: (1) 29 strains of corynebacterium acnes isolated from the surface of the normal human skin and from lesions of acne vulgaris and 14 strains of corynebacterium acnes stored in the laboratory of the Department of Microbiology of Gifu University*, (2) 31 strains of staphylococcus epidermidis isolated from the normal skin surface and lesions of acne vulgaris, (3) 29 strains of staphylococcus aureus isolated from lesions of various pyodermas. Anaerobic culture for corynebacterium acnes was performed with Ueno's anaerobic jar method using steel wool19.25) (a modified method of Parker's iron wool method). (Fig. 1). FIGURE 1. Steel wool method for anaerobic culture For determination of lipase activity 4 substances were used, triacetin, tributyrin, tween 20 and tween 80. For triacetin test, two kinds of basic medium were prepared; (1) triacetin 10 ml B.T.B. 0.1 g heart infusion broth ad ml ph was adjusted at 6.8 with revelation of a green colour The medium was sterilized by filtration with Seitz- Filter. red (2) trypticase 20g natrium chloride 5g natrium sulfite 0.5g phenol red 0.017g agar 3.5g aqua ad ml ph was adjusted at 7.3 with revelation of a colour * These 14 strains are kindly presented from Dr. Ueno of the Department of Microbiology in Gifu University. 586

3 The medium was sterlized by autoclaving and then added by sterilized triacetin to make 1% final concentration of triacetin. Spot inoculation with each bacterium was performed on each medium. After inoculation, each medium was incubated at 37 Ž for 48 or 72 hours under anaerobic or aerobic condition. Lipolytic activity was indicated by colour change of each medium due to a decrease of ph because of acetic acid produced from split of triacetin. When lipolytic activity was present, medium 1. changed from green to yellow and medium 2. changed from red to yellow. For tributyrin test, the basic medium was prepared as follows : hemin 10 ml yeast extract 3g pepton 5g agar 15g aqua ad. 1000ml ph was adjusted at 7.0. The medium was sterilized by autoclaving and cooled to 50 Ž, tributyrin was solved in a broth and shaken until complete suspension. This suspension was added to the medium of 50 Ž to make 1% final concentration of tributyrin. The medium was inoculated with spot inoculation of each bacterium and then incubated at 37 Ž for 48 or 72 hours. Lipolytic activity was indicated by the occurrence of a definite opaque zone around colonies by water solube lactic acid due to splitting of tributyrin (Fig. 2). For tween test, Sierras method") was used: The basic medium was prepared as follows: Difco bacto-pepton 10g natrium chloride 5g cacl. Ho 0.1g aqua ad ml Tween 20 and tween 80 are the esters of oleic acid (polyoxyethylene sorbitan mono-oleate) and of lauryl acid (polyoxyethylene sorbitar mono-laureate) respectively. Sterilized tween 20 and tween 80 are added o each basic medium to make 1% final concentration of each tween. Each tween medium was inoculated with spot inoculation of bacterium and incubated at 37 Ž for 48 or 72 hours. Lipolytic activity was indicated by the occurrence of a definite opaque zone around the colonies consisting of the calcium slat of fatty acid concerned (Fig.3). FIGURE 2 Tributyrin test: Opaque zone around a colony of corynebacterium acnes FIGURE 3 Tween 20 test: Opaque zone around a colony of staphylococcus aureus. (Only No. 124 is negative in tween test) 587

4 RESULTS The results of lipolytic activity tests of 43 strains of corynebacterium acnes and 60 strains of staphylococcus consisting of 31 strains of staphylococcus epidermidis and 29 strains of staphylococcus aureus are presented in Table 1, Figure 4 and Table 2, Figure 5, respectively. Relation between lipolytic activity and phage type of 14 strains of staphylococcus aureus performed by phage-typing are shown in Table 3. From the Table 1 and the Figure 4, it is found that positive lipolytic activity of corynebacrium acnes is 100% in triacetin test, 79-85% in tributyrin test, 93%-100% in tween 20 test and 28%-50% in tween 80 test, respectively. It was found that lipolytic activity of corynebacterium acnes was dominant in triacetin, and tween 20, but relatively weak in tween 80. It is also found that lipolytic activity of strains freshly isolated from lesions was higher than that of stored strains. Lipolytic activity of 31 strains of staphylococcus epidermidis is shown in Table 2 and in Figure 5. It iwas 97%-87% in triacetin test, 89% in tributyrin test, 58% in tween 20 test and tween 80 test, respectively. Triacetin and tributyrin showed a marked high positive reaction, while tween 20 and tween 80 showed a relatively low positive reaction. Regarding lipolytic activity of 29 strains of staphylococcus aureus showed a positive reaction 100% in triacetin test, 97% in tributyrin test, 90% in tween 20 test and 97% in tributyrin test, 90% in tween 20 test and 97% in tween test, respectively. High lipolytic activity was observed in staphylococcus aureus and staphylococcus epidermidis, the former much higher than the later. The relationship between lipolytic activity and phage type of 14 strains of staphylococcus TABLE 1 Lipolytic activity of corynebacterium acnes (43 strains) 588

5 aureus showed that the lipolytic activity of two strains of phage type 71 indclued in phage group II isolated from lesions of impetigo was negative in tween tests but only one strain of phage type 71 isolated from carbuncle was positive in all tests. It was positive in another phage type in phage group II, i.e., type 3C/ 3B/55/71 (2 strains). It was positive in 3 strains of type 52/80 and 3 strains of 52/80/81 in group I and in 3 strains of untypable in all tests. It is interesting that strains of phage type 71 isolated from impetigo has a weak lipolytic activity while the other types of staphylococcus aureus have a hgh lipolytic activity. COMMENTS It is already believed that free fatty acids are secondary spilt products of triglycerides in sebum, consti- tuting an important component of the skin surface lipid film.11,12,13,16) It is also understood that corynebacterium acnes and staphylococcus epidermidis survive together in the skin surface lipid film and in the pilosebaceous apparatus and they are called resident flora of the skin. It is assumed that an over production of sebum in the pilosebaceous appratus results in a secondary of free fatty acids. Bacteriological studies of acne vulgaris show that corynebacterium acnes and staphylococcus epidermidis exist synergistically.1,2,9,17,21) As we already described, it is assumed that lipolysis of triglycerides in sebum starts after sebum is secreted from sebaceous gland and continues until or even after sebum reaches the skin surface. In this lipolytic process triglycerides in sebum is continuously dehydrolysed by lipase of tissue cells and of bacteria as follows : triglyceride diglyceride monoglyceride free fatty acid.1,12) The presence of lipase in tissue cells is demon- FIGURE 4 Lipolytic activity of corynebacterium acnes FIGURE 5 Lipolytic activity of staphylococcus epidermidis and staphylococcus aureus 589

6 TABLE 2 Lipolytic activity of Staphylococcus epidermidis and Staphylococcus aureus strated histochemically as nonspecific esterase activity of epithelial cells of the sebaceous duct.11,12) The presence of free fatty acids in the aseptic vernix caseosa of a newborne also evidences the presence of lipase activity in tissue cells.12) The presence of lipase activity of bacteria is reported by certain authors.1,3,5,6,14,18) Fatty substances used in testing lipolytic activity are represented by triacetin, tributyrin, tween 20, tween 40, tween 60 and tween 80. Gillepsie and co-workers5) and Jessen and co-workers8) emphasized egg-yolk test in testing lipolytic activity. There is an opinion that esters of long chain fatty acid (high fatty acid) are easily split by lipase than glyceride.18) Tweens are water soluble high fatty acid esters of polyoxyalkylene derivatives of sorbitan, while triacetin and tributyrin belong to triglycerides. Sierra18) described that there is some difference in lipolytic activity depending upon the kind of bacteria. For example, lipase of pseudomonas aeruginosa has lipolytic activity for high fatty acid ester but not for triglyceride, while bacillus megaterium does not split high fatty acid ester, although it is able to attack tributyrin as well as triactin. Sierra is also of an 590

7 TABLE 3 The relationship between phage-type and lipolytic activity opinion that tweens are better than glycerides in testing bacterial lipase activity. The lipolytic activity of staphylococci is reported by Sierra18), Delmotte,3) Pospigil14) Gillepsie and co-worker,5) Jessen and co-workers8) etc. in tween test or egg-yolk test. Delmotte, PospisSil, Gillepsie and Jessen reported that there is distinct correlationship between lipolytic activity and virulence of staphylococci, i.e., coagulase positive strains (staphylococcus aureus) have higher lipolytic activity than that of coagulase negative strains (staphylococcus epidermidis). Delmotte estimats the lipolytic activity of 56 strains of staphylococcus aureus using tween 20, tween 40, tween 60 and tween 80 and observs the activity in 80%-92% of these strains. Pospisil also reports the lipolytic activity of 258 strains of staphylococcus aureus and 42 strains of staphylococcus epidermidis on the Sierra's tween method observing lipolytic activity in 60% of staphylococcus aureus strains and 47% of staphylococcus epidermidis strains. Jessen reports that there is almost a complete correspondence between egg-yolk reaction and Sierras tween reaction in lipolytic activity test of staphylococcus. Few reports seem to have been made of lipolytic activity of corynebacterium acnes. Ueno24) reports a positive lipolytic activity of this bacterium for triacetin and tributyrin. Scheimann reports that by the suppreses bacterial flora with the antimicrobial agent indirectly suppresses bacterial lipase activity, resulting in the decreased formation of free fatty acids. Kirshbaum and Kligman9) reports that when suspension of corynebacterium acnes are injected into closed epithelial cysts, which theoretically contain no free fatty acids, marked inflammation with rupture of the cyst occurs. One may assume that when the organisms have been injected into sebumcontaining cyst, there is a consequent formation of irritant substances such as free fatty acids. In the present results, staphylococcus epidermidis and staphylococcus aureus showed a high lipolytic activity in triacetin test and tributyrin test but in tween test staphylococcus epidermidis showed a low lipolytic activity while staphylococcus aureus showed a high activity. Generally, staphylococcus aureus showed a higher lipolytic activity than staphylococcus epidermidis. Presently, corynebacterium acnes showed a very high lipolytic activity in triacetin test and in tributyrin test, while it showed a very low activity in tween 80 test and a high activity in tween 20 test. Thus the patterns of lipolytic activity of corynebacterium acnes and staphylococcus epidermidis in the 4 fatty substances used were found quite similar. A few reports have been made of relations between phage type and lipolytic activity of staphylococcus aureus. Jessen and co-workers state that 13 strains of phage type 71 isolated from impetigo are all nega- 591

8 tive in egg-yolk test but 30 strains of phage type 71 isolated from pypdermas other than impetigo are positive in 93% (28/30). They also report that 8 strains of phage group II except type 71 isolated from impetigo were egg-yolk positive in 89% (7/8), and 75 strains of phage group I isolated from boils are eggyolk positive in 92% (69/75). In the present results with triacetin, tributyrin, tween 20 and tween 80, 6 strains of phage group I are all positive for all the four fatty substances employed and 5 strains of phage group II are all positive for triacetin (100%) but are positive for tributyrin in 80%, for tween 20 and for tween 80 in 60% respectively. In 5 strains of phage group II, 2 strains of type 71 isolated form impetigo are positive for triacetin in 100%, for tributyrin in 66% but are all negative for tween. 20 and tween 80, while one strain of phage type 71 isolated from carbuncle and other two strains of phage type 3C/3B/55/71 are all positive for all four fatty substances. SUMMARY ipolytic activity of 43 strains of corynebacterium acnes and 29 strains of staphylococcus epidermidis and 31 strains of staphylococcus aureus, using L triacetin, tributyrin, and Sierras tween method, was reported. Activity of corynebacterium acnes showed high per centage of positive reaction for triacetin, tributyrin and tween 20 but low per centage for tween 80. Lipolytic activity of staphylococcus epidermidis was high for triacetin, tributyrin but relatively low for tweens test showing similar lipolytic pattern of corynebacterium acnes. Staphylococcus aureus showed higher lipolytic activity than staphylococcus epidermidis. The relation between phage type and lipolytic activity of staphylococcus aureus showed that strains of phage type 71 from impetigo were all negative for tween 20 and tween 80, while other types were positive in very high per centage. REFERENCES 1. ASADA, Y.,: The relation between lipolysis of sebum and resident bacteria of the skin. Hifu, (Skin research) 9: , 1967 (in Japanese). 2. ASADA, Y.: The bacteriology of acne vulgaris. Hifuka no Rinsho (J. Clinical derm.). 9: , 1967 (in Japanese). 3. DELMOTTE, A.: L'activite lipolytique microbienne decelee par la methode de Sierra avec reference speciale au m. pyogenes var aureus. Leeuwenhoek. Amsterdam. 24: , EVANS, C. E.: Bacterial flora of the normal skin. J. invest. derm., 15: GILLEPSIE, W. A., and ALDER, V. G.: Production of opacity in egg-yolk media by coagulase-positive staphylococci, J. path. & bact., 64: , HORACEK, J., and Pospisil, K.: Lipazy stafylokokku kozniho povrechu. Bratislavaske. Lekarske. Listy, 45: , HERRMANN, F.: Acne-Probleme. Fortschritte d. prakt. Derm. u. Venerol., Band 5: , JESSEN, O., FABER, V., ROSENDAL, K. and ERIKSEN, K. R.: Some properties of staphylococcus aureus, possibly related to pathogencity. Part I: A study of 446 strains from different types of human infection. Part II: In vitro properties and origin of the infecting strains correlated to mortality in 190 patients with staphylococcus aureus bacteriemia. Acta path. et microbiol. scandin., 47: , KIRSCHBAUM, J. 0. and KLIGMAN, A. M.: The pathogenic role of corynebacterium acnes in acne vulgaris, Arch. derm., 88: , NOBLE, C. W.: Virulence and the biochemical characters of staphylococci. J. path. bact. 91: , NICOLAIDES, N. and WELLS, G. C.: On the biogenesis of the free fatty acid in human 592

9 skin surface fat. J. invest. derm., 29: , NICOLAIDES, N.: Human skin surface lipids: origin, composition and possible function. Advances in Biology of the Skin: Chapter XI, , 1963, Pergamon Press. 13. NICOLAIDES, N., KELLUM, R. E. and WOOLY, P. V.: The structures of the free unsaturated fatty acids of human skin surface fat, Arch. Bioch. Biophysics, 105: , POSPIIL, L.: Quntitative Lipasebestimmung der aus verschiedenen Dermatosen isolierten Staphylokokken. Derm. Wschr., 48: , PILLSBURY, D. M.: Foundamental cutaneous bacteriology. Dermatology: Saunders, 1956, ROTHMAN, S.: Physiology and Biochemistry of the Skin. Chicago, University of Chicago Press, 1954, STRAUSS, J. S. and POCHI, P. E.: Newer views of skin diseases. Ed., Yaffe, H. S., Little, Brown Comp., Boston, 1966, SIERRA, G.: A simple method for the detection of lipolytic activity of micro-organisms and some observations on the fatty substrates. Leeuwenhoek. Amsterdam. 23, 15-22, SUZUKI, S. and UENO, K.: The culture technic for anaerobic bacteria. Modern media 11: , 1965, (in Japanese). 20. SAITO, K. and ASADA, Y.: Fatty acid composition of skin surface lipids and resident bacterial lipids. Biochm. Biophys. Acta, 137: , SHEHADEN, N. H. and KLIGMAN, A. M.: The bacteriology of acne. Arch. derm., 88: , STEIGLEDER, G. K. and ROTTCHER, K. H.: Die Fahigkeit der Hautoberflache zur Esterspaltung und Esterbildung II, Ober Pilze, Hefen und Bakterien als Trager von Esterasen. Arch. klin. u. exp. Derm., 209: , SCHEIMANN, L. G., KNOX, G., SHER, D. and ROTHMAN, S.: The role of bacteria in the formation of free fatty acids on the human skin surface. J. Invest. derm., 94: , UENO, K.: Studies of anaerobic bacteria as resident flora and some observations of their pathogenicity: Abstract of symposium of clinical bacteriology of non-spolulating anaerobic bacteria, 1968, , (in Japanese). 25. UENO, K.: The practice of anaerobic culture by the method of steel wool. Media circle, 57: , 1964, (in Japanese). 26. WHEATLEY, V. R.: Possible clinical application of study of the cutaneous lipid. Bull. N.Y. Acad. Med., 4: ,