0022-202X/80/7406-0437$02.00/ 0 THE JOURNAL OF I NVESTIGATIVE DERMATOLOGY , 74:437-439, 1980 Copyright © 1980 by The Williams & Wilkins Co.

Vol. 74, No.6 Pn:nted in U.S .A.

In Vitro Effects of Skin Lipid Extracts on Skin Bacteria in Relation to Age and Acne Changes

MARIA BASTA, M.Sc., JANusz WILBURG, M.D., AND PIOTR B. HEczKo, M.D.

Department of Bacteriology, Institute of Microbiology, Medical Academy, Krakow, Poland

Skin lipid extracts of 185 persons of different age were tested in vitro against single representatives of skin bacteria: Staphylococcus aureus, S. epidermidis, Propi­onibacterium acnes, P. granulosum and Corynebacterium sp- in order to find differences in their inhibitory effects. Percentages of the extracts inhibiting the first 4 bacteria were nearly the same (about 20%) while corynebacteria were inhibited by only 3 extracts. Extracts showing these properties were isolated mainly from adolescents with acne changes, young adults, about 20 yr old, and from children 8-10 yr old, but not from elderly people. The age-related differences in growth inhibition may be re­lated to known differences in relative composition of the sebum constituents according to age.

Forty-two extracts of acne patients and healthy adults and adolescents showed an inverse effect in vitro: they enhanced bacterial growth. Growth enhancement was demonstrated only for P. granulosum.

Skin lipids possess a n ability to inhibit growth of different bacteria both in vivo and in vitro [1-3]. It has been demon­strated that a removal of fatty substances from a given region of the human skin increases survival rate of bacteria artificially applied to this skin surface [ 4]. The in vivo inhibitory effect of the skin lipids is attributed only to gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes) and to Candida albicans but not the gram-negative rods like Esch­erichia coli or Pseudomonas aeruginosa. This activity is re­lated to the in vitro inhibitory properties of extracts of the skin lipids exerted against the same organisms. Antibacterial prop­erties of the skin lipid extracts seem to be similar to inhibitory effects of some free fatty acids on different microorganisms [3,5-7]. However, the inhibitory properties of the fatty acids are not restricted to S . aureus and S. pvogenes since they have been demonstrated, although at different degree [8-10], for all resident bacteria of the human skin. Recently, Aly et al [11,12] have shown that skin lipid extracts of different individuals differ in their abilities to suppress the growth of S. aureus. The inhibitory properties of the skin lipids are correlated with lower counts of the total skin flora of a given individual. Numbers of diphtheroids in comparison to staphylococci and micrococci on the skin of these persons are significantly increased.

It is our purpose in this communication to show that the in vitro inhibitory properties of the skin lipid extracts are extended over resident bacteria of the human skin and that these indi­vidual characteristic of the sebum is related to the age and some skin conditions of the studied persons.

Manuscript received August 30, 1979; accepted for publication De­cember 18, 1979.

These investigations were partially supported by grant No. 05-339-C from the Center for Disease Control, U.S. Public Health Service, Atlanta, GA.

Reprint requests to: Dr. P . B. Heczko, Institute of Microbiology, M edical Academy, 18 Czysta Street, 31-121 Krakow, Poland.

Abbreviations: cfu: colony forming units MH: Mueller-Hinton agar TSB: Tryptic Soy Broth

MATERIALS AND METHODS

Subjects

Altogether 185 persons of both sexes were studied. This group consisted of 22 children 8-10 yr old, 13 adolescents 15-17 yr old, without acne symptoms, 94 adolescents of the same age with distinct acne lesions, 36 young adults 20-22 yr old and 20 persons over 70 yrs old. All acne patients were tested before any local or systemic use of antibiotics. Only patients with moderate uncomplicated acne were selected for the study.

Bacteria

Five test microorganisms isolated from healthy human skin were used: S. aureus, S. epidermidis, Propionibacterium a.cnes, P. granu­losum and lipophilic Corynebacterium sp. Their taxonomic position was proven on the base of tests given in a last edition of Bergey's Manual [13]. The bacteria were preserved on agar slan ts before testing.

Collection of Sebum Extracts

Skin lipids were extracted by ethyl ether from about 450 cm2 area of skin of interscapular region by use of sterile, previously deg:t·eased, cotton balls which were then shaken for 10 min in tubes containing .20 ml of the same solvent. After this the balls were pressed against tube walls and discarded and then solution fil tered through sintered glass filter to remove desquamated cells and concentrated with nitrogen to a volume of 1 ml. The residue was stored at -20°C under nitrogen until tested.

Inhibitory Effects of Sebum Extracts

The agar diffusion method was followed as described by Aly et al [11,12]. Test bacteria, in appropriate concentrations in Tryptic Soy Broth (TSB) (Difco) estimated by nephelometry (S. aureus and S . epidermidis about 5 x 104 colony forming units (cfu) / ml, P. acnes and P. granulosum about 5 X .107 cfu / ml and aerobic corynebacteria about 1 X 106 cfu/ml) , were flooded over surface of Mueller-Hinton (MH) agar (Oxoid) for staphylococci or MH agar with 1% glucose for aerobic and anaerobic corynebacteria. Seeded plates were well dried at a room temperature and then wells of 6 mm diameter were bored in the agar. Concentrated lipid extracts were added to the wells in a volume of 20 JLl and the plates were incubated at 37°C, aerobically for 24 lu, in the case of staphylococci and aerobic diphtheroids, and anaerobically for 5 days, in the case of propionibacteria. Inhibition of growth of the tested strains was demonstrated by zones of a clear agar surrounding the wells without visible bacterial growth while enhancement as clear-cut zones of abundant and confluent growth of the bacteria markedly exceeding that found elsewhere on the same plates. The identity of the bacteria isolated from zones of the enhanced growth with incoulated ones were checked on the base of biochemical pattern and resistance to several antibiotics. In all cases the same P. granulosum strain was found . Diameters of zones of growth inhibition or enhancement were then measured. They varied from 7 to 24 mm for aerobic bacteria and from 7 to over 30 mm for propionibacteria. Only the zones exceeding 10 mm of diameter were regarded as positive tests. Controls consisted of seeded plates with wells filled with the solvent only and sterile plates with wells filled with the extracts.

RESULTS

Effects of the lipid extracts tested on different test bacteria are shown in Table I. About 20% of all extracts caused inhibition of growth of both staphylococcal strains and propionibacteria, while the aerobic diphtheroid was inhibited by only 16% of extracts. About 22% of the extracts enhanced growth of P. granulosum test strain.

437

438 BASTA, WILBURG, AND HECZKO

The enhancement of the bacterial growth around wells filled with extracts was visible only in some plates inoculated with P. granulosum; this effect was found in 1 extract from a healthy adolescent, 18 from acne patients, and 23 from adults. On the other hand no extract of children and elderly individuals was active in this respect.

Numbers of extracts showing inhibitory properties against staphylococci and propionibacteria dirfered depending on the group of the individuals tested (Table II).

Extracts taken from healthy adolescents were not inhibitory for all bacteria tested except for one extract which inhibited staphylococci and propionibacteria.

Basically the same results were found for the extracts of elderly people; only 2 of them inhibited multiplication of exclu­sively P. acnes.

No distinct differences were found between extracts of chil­dren, acne patients and healthy adults in their frequency of growth inhibition of S. epidermidis, P. acnes, and Corynebac­terium sp. However, differences were encountered with respect to P. granulosum and S. aureus.

Fow- skin lipids samples from children suppressed the growth of S . epidermidis, 7 of P. acnes and 10 of P. granulosum. Growth of S. aureus and Corynebacterium species was unaf­fected. One-fow-th of skin lipid extracts of adolescents with acne and nearly a half of those of children showed growth inhibition of P. granulosum while only 2 extracts of adults were inhibitory for this bacterium.

More than one-fifth of extracts of lipids isolated from the skin of acne patients and adults inhibited S . aureus, others, with one exception, were inactive against this organism.

DISCUSSION

Our results of testing of antibacterial effect of the skin lipid extracts done on a greater group of individuals than reported

TABLE I. In vitro effect of 185 skin lipid extracts on different bacteria

Effect Test organism on s. P. Coryne· bacterial s. P.

growth aureu.s epider· acnes granu · bact. midis losum. sp.

Inhibition 37" 2o.o• 38 20.5 40 21.6 35 18.9 3 1.6 Enhancement 0 0 0 42 22.8 0 No change 148 80.0 147 79.5 145 78.4 108 58.3 182 98.4

"Number and 6percentage of extracts.

TABLE II. Inhibitory effects of sllin lipid extracts isolated from different age groups on test microorganisms

Group of

person (age)

T est organism

Children (8-10)

Healthy adolescents

(15-17) Adolescents with acne

(15-17) Young adults

(20-22) Elderly people

(over 70)

Total

Number of

persons

22

13

94

36

20

185

s. S. aureus epider·

midis

4

28 24

8 9

0 0

37 38

"Corynebacterium sp. (lipophilic) .

P. acnes

7

1

21

9

2

40

6 Numbers of extracts causing inhibition of growth.

P. granu· losunt

10

22

2

0

35

Coryne· bac. sp."

0

0

2

1

0

3

Vol. 74, No. 6

before [11,12] confirmed that skin lipids of a proportion of normal individuals possess an ability to suppress the in mtro growth of S . aureus. However, this effect of the lipids is not restricted to this organism but it involves also other groups of microorganisms residing on the normal skin surface. The mech­anism of growth inhibition of the skin bacteria by lipids of some persons remains still obscme in spite of several studies [12,14]. It is possible that some fatty substances, most probably free fatty acids, present in different concentrations in these ~km lipids due to different metabolism of the host are respons1b!e for their antibacterial effect. We have demonstrated that skm bacteria are susceptible to different degrees to only several fatty acids from a wide variety present in the sebum [8-10].

The screening method applied by us and also by Aly et al [11,12] possesses several limitations and therefore it limits a quantitative assay. It depends strongly on the concentration of the sebum constituents which may be different due to individual rate of sebum production [15] or sampling procedw-e. The former factor is most probably partially responsible for differ­ences in antibacterial activity found by us between lipids ob­tained from persons of different age groups. It has been dem­onstrated that the general sebum excretion rate reaches the highest values in adolescents and young adults [16,17]. Also acne patients regularly show increased sebum production and some changes in composition especially in seborrhoic regions of the skin, which have been sampled in our studies fl·om [18-21].

Several fatty acids contained in sebum were found by us to be inhibitory for different skin bacteria [7-9] including S. au­reus and Streptococcus pyogenes [10]. AU of these findings may explain in part more n·equent inhibitory activity of the sebum extracts obtained from adults and acne patients. On the other hand the extracts of elderly people were generally noninhibitory for the bacteria tested. This may reflect some senile changes in the skin lipids metabolism since it corresponds to distinct changes in skin flora [22,23] including an increased rate of skin carriage of S. aureus [24 ].

Unfortunately these preliminary investigations were done on single representatives of the skin flora and therefore no more precise conclusions on inhibition of different bacteria by the extracts can be given now. However, it is interesting that no differences were found in susceptibility of P. acnes and P. granulosum to the extracts of acne patients in spite of different density of their populations on skin in relation to acne changes [25] and also of their differential susceptibility to fatty acids, especially to unsaturated ones [9].

Enhancement of growth of P. granulosum by extracts, iso­lated only from acne patients and young adults was not en­countered· by the previous investigators. However, this may be caused by special requirements of the strain used for some substances present only in these extracts. This interesting phe­nomenon needs for further investigation since it corresponds to increased density of P. granulosum populations on the skin of young individuals [23,25].

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June 1980

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13. Buchanan RE, Gibbons NE: Bergey's Manual of Determinative Bacteriology, 8th ed. Baltimore, Williams & Wilkins, 1974

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16. Cotterill JA, Cunliffe WJ , WiHiamson B. Bulusu L: Age and sex

EFFECTS OF SKIN LIPID EXTRACTS ON BACTERIA 439

variation in skin surface lipid composition and sebum excretion rate. Br J Dermatol 87:333- 340, 1972

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Dermatopathology Symposium New York City

This year 's annual symposium on dermatopathology at N ew York U niversity School of M edicine is entitled " Histologic Diagnosis of Neoplastic Skin Diseases: A M ethod by Pattern Analysis." The dates a re October 16-19, 1980. Address all inquiries to: A. B ernard Ackerman, M .D ., Dermatopathology Section, 530 First Ave., NYC 10016, 212-340-7250.