- Email: [email protected]
Acne and propionibacterium acnes
Acne and Propionibacterium Acnes RICHARD A. BOJAR, PhD KEITH T. HOLLAND, PhD Abstract: The involvement of microorganisms in the development of acne has a long and checkered history. Just over 100 years ago, Propionibacterium acnes (then known as Bacillus acnes) was isolated from acne lesions, and it was suggested that P. acnes was involved in the pathology of the disease. The 1960s saw the use of antibiotics to treat acne, and the consequent clinical success combined with reductions in P. acnes gave new impetus to the debate. Over the past two decades, the inevitable emergence of antibiotic-resistant strains of P. acnes as a consequence of acne therapy not only has reopened the debate as to the role of P. acnes in acne, but also has created some serious health care implications.
T
he main obstacle to convincing the scientific community that P. acnes is a key factor in the development of acne is the difficulty in applying Koch’s postulates to the disease. Attempts to look for simple cause-and-effect relationships between P. acnes and acne have resulted in a plethora of anecdotal and circumstantial evidence, complicated by the fact that there is no suitable in vivo model and no therapeutic options that target P. acnes exclusively. Although efforts have been made to identify the virulence factors of P. acnes, consensus opinion on the initial changes in the pathology of an acne follicle has changed often. In particular, it remains a matter of debate as to whether comedone formation precedes or follows inflammation and whether the initial cellular infiltrate is neutrophilic or lymphocytic. Current scientific opinion generally views the cutaneous microflora as of secondary importance compared with local sebaceous gland activity, with significant microbial involvement occurring only after sebum production has increased and comedone formation has become established. In contrast, the pharmaceutical industry appears to view the process somewhat differently, because it continues to develop acne therapies with antimicrobial activity as their primary mode of action. Hopefully this article will go some way to reconcile these divergent views. At this point, the reader should be made aware of two issues with direct relevance to the discussion. First, there remains the possibility that other cutaneous microorganisms, such as staphylococci and Malassezia spp, may be involved in some cases of acne due to their close proximity to P. acnes in the pilosebaceous follicle. Second, P. acnes and other cutaneous microorganisms can From the Skin Research Centre, Division of Microbiology, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK. Address correspondence to R. A. Bojar, PhD, University of Leeds, Skin Research Centre, Division of Microbiology, Department of Biochemistry and Molecular Biology, Leeds LS2 9JT, UK. E-mail address: [email protected] © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
be isolated from both acne and normal skin. It is relatively easy to prove that a microorganism is the cause of a disease when it is cultivated from a normally sterile site and can be shown in vitro and in vivo to account for the pathology. However, it is far more difficult to assign a microorganism a pathogenic role when it is present in both normal and disease conditions. Modern views of pathogenicity (virulence) encompass the condition of the host (or target organ) and the diverse interactions between the host and the microorganism. This article discusses the biological features of cutaneous microorganisms, their location in the skin, their interaction with skin, the evidence implicating P. acnes in acne, and hypotheses on how P. acnes impinges on the pathology of acne.
Characteristics of Cutaneous Propionibacteria The cutaneous propionibacteria (P. acnes, P. avidum, P. granulosum, P. propionicum, and P. lymphophilum) are found as commensals on human skin and other keratinized epithelia. Over the years, they have been variously classified as Bacillus spp, Corynebacterium spp, anaerobic diphtheroids, and Propionibacterium spp, and this must be borne in mind when surveying the literature. They are gram-positive and nonmotile, and when first isolated exhibit a typical coryneform appearance under the microscope with irregular, short branching. Isolation is best performed at 35°C under anaerobic conditions on a growth medium supplemented with Tween 80, although propionobacteria are neither strictly anaerobic nor lipophilic. P. acnes and P. granulosum are commonly isolated from sebum-rich areas of the skin (eg, head, chest, and back) with reservoirs in the pilosebaceous follicle,1,2 whereas P. avidum is located mainly in the axillae.3,4 There is little information on P. propionicum and P. lymphophilum, and a sixth commensal strain formerly known as P. innocuum has recently been reclassified as Propioniferax innocua.5 0738-081X/04/$–see front matter doi:10.1016/j.clindermatol.2004.03.005
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The Microflora of Human Skin
Control of Microbial Colonization of the Skin
As a habitat for microorganisms, the human skin is open to contamination by microbial species from the environment, and yet it is not particularly suitable for colonization due to the environmental conditions resulting from its physical structure. Normal skin supports colonization by a limited number of resident microbial types, mainly gram-positive species able to tolerate the stresses associated with the physical environment. The stable microbial ecosystem is controlled partly by the physical environment (ie, pH, oxygen, ion, and growth substrate concentrations, ultraviolet radiation) and partly by synergistic and antagonistic interactions between the host and the resident cutaneous microflora and between microbial populations.6 Inhibitory substances, including bacteriocins, enzymes, and lowmolecular-weight inhibitors, are produced by cutaneous microorganisms7 and may help prevent colonization by pathogens.8 In addition to propionibacteria, resident microbial species come from the bacterial genera Staphylococcus, Micrococcus, Corynebacterium, and Acinetobacter; the yeast Malassezia; and a number of bacteriophage species. Although molecular techniques such as restriction fragment analysis of DNA and DNA/RNA base sequencing have been used successfully to classify resident skin microorganisms, routine identification continues to be based largely on phenotypic traits (eg, sugar fermentations, extracellular enzymes). The surprising stability of the resident microflora in response to changes in the skin environment suggests close coevolution of the skin microflora and the skin environment. In most adults, the surface of sebum-rich areas of skin and the pilosebaceous follicles support large, stable populations of propionibacteria, which are a benign presence and help maintain the ecosystem of healthy skin.9,10 They are also found in the mouth and gut, where again they possibly help stabilize complex microbial populations. Generally, they have a positive effect on human health by filling a niche that could otherwise be colonized by pathogenic microorganisms. However, when the host becomes compromised (through, eg, trauma, injury, or alterations in immune status), propionibacteria can display a pathogenic potential. Historically, most of the published work on the cutaneous propionibacteria has focused on P. acnes, due to the association with acne and its immunomodulatory effects. However, both P. granulosum and P. avidum have immunomodulatory properties, and P. granulosum has also been associated with acne. It is fair to say that despite investigations over many years, our understanding of the interactions between the cutaneous propionibacteria and the human host remains inadequate.
Physical factors controlling colonization are mainly functions of the skin structure and include the number and size of follicles and glands, gland function (eg, sebum, sweat, apocrine), the flow of secretions (which determines nutrient availability and hydration levels), the integrity of barrier function, skin pH, and osmotic potential. Biochemical analysis of the skin surface environment has revealed a vast array of chemical compounds derived from sebum, sweat, and the metabolic activity of microorganisms on skin secretions. In normal skin, available soluble nutrients are mostly at low concentrations and are derived from sweat (vitamins, amino acids, and lactate) and sebum (lipids and amino acids). Antimicrobial compounds of the nonadaptive immune system may also influence the resident microbial population density and limit colonization by transient microorganisms. Three -defensins (HBD-1, -2, and -3) are produced by human keratinocytes;11 however, their role in limiting colonization by transient microorganisms is unclear, and their influence on the population density of the resident microflora is debatable. In contrast, dermicidin, which is secreted by mucosal cells of the sweat glands,12 has activity against both gram-positive and gram-negative bacteria under the pH and salt conditions of human sweat. Because there is no evidence that microorganisms colonize eccrine glands or ducts, dermicidin may play a role in maintaining sterility in eccrine follicles. The gram-positive cell wall of propionibacteria gives these bacteria high structural stability, making them resistant to drying, osmotic shock, and mechanical stress. These are important attributes, because the skin surface is relatively deficient in free water and subject to fluctuations in temperature, solar radiation, and salt/ ion concentrations. The density of resident microorganisms at any one site on the body varies widely among individuals and between sites on the same individual. The variation in colonization density and microbial diversity between skin sites depends largely on the number and density of sebaceous and sweat glands, but there is also wide variation within the population at any one site. This is well illustrated by the range in density of propionibacteria isolated from the face, an area rich in sebaceous glands. Skin surface counts within a population of 761 volunteers ranged from ⬍10 per cm2 of skin in some individuals to ⬎10,000,000 in others (Fig 1). The sebaceous-rich sites also support microbial growth within the follicles (Fig 2); again, there is huge variation in density within the population (Fig 3). Variation in colonization also extends to different follicles in the same individual, with up to 60% of pilosebaceous follicles from normal skin not being colonized.2 Such variation may be due to differences in follicle size and the rate of sebum secretion.13 There is also some evi-
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Figure 1. Population distribution of Propionibacterium spp. (mean, 4.829; SD, 1.392) and Micrococcaceae (mean, 4.527; SD, .858) isolated from the surface of the cheek using the Williamson and Kligman scrub wash (author’s data; n ⫽ 761). y surface Propionibacterium spp.; y surface Micrococcaceae.
Figure 3. Population distribution of Propionibacterium spp. (mean, 4.395; SD, 1.936) and Micrococcaceae (mean, 3.905; SD, 1.394) isolated from Permabond follicular biopsy specimens taken from the cheek (author’s data; n ⫽ 461). y follicular Propionibacterium spp.; y follicular Micrococcaceae.
dence suggesting spatial stratification of microbial populations within follicles, such that particular genera may inhabit distinct niches.14 However, the interactions between factors controlling the density and location of the cutaneous microflora remain to be defined. The physiological response and expressed phenotype of microorganisms depend largely on the concentration of available nutrients. The skin environment contains a wide range of polymers, including lipids, polysaccharides, and proteins, and the skin microflora
produces extracellular enzymes that degrade these polymers to release mostly end-products with direct nutritional value, with lipase and protease the most common enzymatic activities. Differences in nutrient availability at different skin sites over time will have a major influence on the phenotype of individual microbial species and will consequently determine the composition and density of the skin microflora. However, nutritional requirements are ultimately determined by genotypic traits, such as nutrient transport systems in the cytoplasmic membrane. All cutaneous microorganisms are chemoorganotrophs requiring organic nutrients and using oxidation of organic sources to produce energy and cell biomass. It may be assumed that to a large extent, molecules that are consistently present in the skin environment provide the minimal nutritional requirements to the resident microflora.
Classification and Identification
Figure 2. Gram Weigart–stained longitudinal section of a single pilosebaceous follicle, showing the multilobular sebaceous gland and the pilosebaceous duct colonized by gram-positive microorganisms (author’s data). Scale bar ⫽ 100 m.
Although much of the literature refers to P. acnes as an anaerobe, the cutaneous propionibacteria are not strictly anaerobic, and, although anaerobic conditions are used for primary isolation, all species will tolerate the presence of oxygen.15 After incubation on agar growth media, the colonies are characteristically domeshaped and beige to pink in color. Because of the limited range of species found on human skin, most isolates can be speciated using a limited number of biochemical tests (Table 1), and these tests continue to be used routinely in preference to more sophisticated techniques, such as bacteriophage and molecular typing techniques. Applying molecular typing initially proved difficult due to the lack of a suitable technique for lysing the robust cell wall of P. acnes. However,
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Table 1.
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The distribution and differentiation of human commensal propionibacteria
Distribution
Differentiation
Skin Eye Mouth Gut Aesculin hydrolysis Casein hydrolysis Catalase production Indole production Nitrate reduction
P. acnes
P. avidum
P. granulosum
P. propionicum
⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫻ ⻫ ⻫ ⻫ ⻫
⫹⫹⫹ ⫺ ⫺ ⫺ ⻫ ⻫ ⻫ ⫻ ⫻
⫹⫹⫹ ⫺ ⫹ ⫺ ⫻ ⫻ ⻫ ⫻ ⫻
⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫻ ⫻ ⫻ ⫻ ⻫
Distribution range: present at high density (⫹⫹⫹) to not present (⫺). Differentiation (biochemical test reactions): positive (⻫); negative (⫻).
progress has been possible in recent years due initially to the development of a specific lysis technique utilizing susceptibility to penicillin16 and subsequent developments in polymerase chain reaction and sequencing protocols for use with resiliant microorganisms such as mycobacteria. Consequently, the way is now open for a thorough analysis of cutaneous propionibacteria using established molecular typing methods.
Epidemiology The link between P. acnes and the development of acne is generally regarded as fact by most dermatologists and has by and large even become accepted by the general public through advertising for acne products. However, there is no formal proof linking P. acnes with acne, and the presence of propionibacteria is not a prerequisite for the development of acne.17 If P. acnes is involved in acne, whether its presence initiates certain features of the disease, such as inflammation, or whether it exacerbates inflammation once a lesion is formed remains unclear. Propionibacteria are known to inhabit distinct niches within the skin,14 including the pilosebaceous follicle, where they reside with Staphylococcus spp and Malassezia spp.2 However, colonization is by no means uniform. Although previous studies have demonstrated that some pilosebaceous follicles are effectively devoid of any viable microorganisms,17,18 applying molecular detection techniques to more accurately define the microbial community of the skin would be beneficial. Microbial involvement in acne is just one of several factors known to influence the progress of the disease, including chronic inflammation, ductal hypercornification, and excessive sebum production; these factors contribute differently to the overall disease in each individual. Therefore, elucidating a general mechanism for microbial involvement in acne has proven difficult. Most recent published research into the commensal propionibacteria has concentrated on case reports of opportunistic infections as a consequence of surgical procedures. However, the mechanisms that provoke
commensal cutaneous propionibacteria into a pathogenic role at other body sites have not been explained. Further confusion arises due to the widespread distribution of propionibacteria on the skin and the difficulty distinguishing between contamination and true colonization/infection.
Antibiotics and Propionibacteria The hypothesis that microorganisms are involved in acne is supported in part by the successful treatment of acne with antibiotics, such as erythromycin and clindamycin, and the reduced efficacy of these treatments when antibiotic-resistant propionibacteria are present.19 Although propionibacteria are susceptible to a range of other antibiotics, including penicillins and cephalosporins,20 the antibiotics of therapeutic value in acne are generally limited to lipid-soluble compounds. Extensive use of antibiotics to treat acne since the 1960s has led to the widespread occurrence of resistance in cutaneous propionibacteria,19 (although initial fears that antibiotic resistance, once acquired, would spread rapidly via transmissible genetic elements have been allayed, because resistance appears to result from specific mutations in 23S ribosomal RNA.21) Although this mechanism is ultimately self-limiting, current concerns regarding antibiotic resistance in the wider population and in pathogenic microorganisms confirms that the use of antibiotics to treat acne needs to be better controlled. Patient preference and compliance issues also dictate the prescription and use of both topical and oral antibiotics. The use of topical antibiotics will also be determined by additional factors, such as the site of acne. For example, topical antibiotics may be hard to apply to acne on the back. Although in some countries the incidence of resistant P. acnes exceeds 60%,22 the clinical relevance of this high incidence is unclear. The reasons for this are complex and include such factors as the anti-inflammatory effects of some antibiotics. To date, there are no adequate clinical studies defining the proportion of patients in which antibiotic-resistant P. acnes has a clinical effect. However, resistance should be
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suspected in patients who do not respond to antibiotic treatment, particularly tetracycline, doxycycline, and erythromycin. Antibiotic resistance in acne patients can by and large be overcome by the judicious use of nonantibiotic antimicrobial agents, such as benzoyl peroxide and azelaic acid.23 Ultimately, combination therapies containing an antibiotic and a nonantibiotic antimicrobial agent may prove to be the best approach for maintaining the efficacy of well-established treatment regimens.24 –27
Future Directions A key objective for research is to define in detail the interactions of P. acnes with human skin in both normal and acne states. Most likely, this will be achieved by developing living skin equivalent model systems colonized by P. acnes. Further progress will be possible once the genomic sequence of P. acnes has been elucidated; then the interaction between skin and P. acnes will be open to transcriptional analysis using DNA microarray technology.
References 1. Puhvel SM, Reisner RM, Amirian DA. Quantification of bacteria in isolated pilosebaceous follicles in normal skin. J Invest Dermatol 1975;65:525–31. 2. Leeming JP, Holland KT, Cunliffe WJ. The microbial ecology of pilosebaceous units isolated from human skin. J Gen Microbiol 1984;130:803–7. 3. Leyden JJ, McGinley KJ, Mills OH, et al. Age-related changes in the resident bacterial flora of the human face. J Invest Dermatol 1975;65:379 –81. 4. McGinley KJ, Webster GF, Leyden JJ. Regional variations of cutaneous propionibacteria. Appl Environ Microbiol 1978;35:62–6. 5. Yokota A, Tamura T, Takeuchi M, et al. Transfer of Propionibacterium innocuum Pitcher and Collins 1991 to Propioniferax gen nov as Propioniferax innocua comb nov. Int J Syst Bacteriol 1994;44:579 –82. 6. Allaker RP, Noble WC. Microbial interactions on the skin. In: Noble WC, editor. The skin microflora and microbial skin disease. Cambridge, UK: Cambridge University Press, 1993:331–54. 7. Eady EA, Holland KT. Inhibitors produced by propionibacteria and their possible roles in the ecology of skin bacteria. Proc R Soc Edinburgh (Biol) 1980;79:193–9. 8. Holland KT, Cunliffe WJ, Roberts CD. The role of bacteria in acne vulgaris: a new approach. Clin Exp Dermatol 1978;3:253–7. 9. Holland KT, Ingham E, Cunliffe WJ. The microbiology of acne: a review. J Appl Bacteriol 1981;51:195–215. 10. Eady EA, Ingham E. Propionibacterium acnes: friend or foe? Rev Med Micro 1994;5:163–73. 11. Fulton C, Anderson GM, Zasloff M, et al. Expression of natural peptide antibiotics in human skin. Lancet 1997; 350:1750 –1.
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12. Schittek B, Hipfel R, Sauer B, et al. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol 2001;2:1133–7. 13. Kearney JN, Ingham E, Cunliffe WJ, et al. Correlations between human skin bacteria and skin lipids. Br J Dermatol 1984;110:593–9. 14. Kearney JN, Harnby D, Gowland G, et al. The follicular distribution and abundance of resident bacteria on human skin. J Gen Microbiol 1984;130:797–801. 15. Cove JH, Holland KT, Cunliffe WJ. Effects of oxygen concentration on biomass production, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture. J Gen Microbiol 1983;129:3327–34. 16. Tipper JL, Eady EA, Cove JH, et al. A method for lysing cutaneous propionibacteria and its use to clone antibiotic resistance determinants from Propionibacterium acnes. Br J Dermatol 1993;129:488 –9. 17. Leeming JP, Holland KT, Cunliffe WJ. The microbial colonization of inflamed acne vulgaris lesions. Br J Dermatol 1988;118:203–8. 18. Leeming JP, Holland KT, Cunliffe WJ. The pathological and ecological significance of microorganisms colonizing acne vulgaris comedones. J Med Microbiol 1985;20:11–6. 19. Eady EA, Cove JH, Holland KT, et al. Erythromycinresistant propionibacteria in antibiotic-treated acne patients: association with therapeutic failure. Br J Dermatol 1989;121:51–7. 20. Hall GS, Prattrippin K, Lowder CY, et al. Cidal activity of 3 cell-wall active agents against Propionibacterium acnes. Invest Ophthal Vis Sci 1993;34:1258. 21. Ross JI, Eady EA, Cove JH, et al. Clinical resistance to erythromycin and clindamycin in cutaneous propionibacteria isolated from acne patients is associated with mutations in 23S rRNA. Antimicrob Agents Chemother 1997; 41:1162–5. 22. Coates P, Vyakrnam S, Eady EA, et al. Prevalence of antibiotic-resistant propionibacteria on the skin of acne patients: 10-year surveillance data and snapshot distribution study. Br J Dermatol 2002;146:840 –8. 23. Bojar RA, Eady EA, Jones CE, et al. Inhibition of erythromycin-resistant propionibacteria on the skin of acne patients by topical erythromycin with and without zinc. Br J Dermatol 1994;130:329 –36. 24. Holland KT, Bojar RA, Cunliffe WJ, et al. The effect of zinc and erythromycin on the growth of erythromycin-resistant and erythromycin-sensitive isolates of Propionibacterium acnes: an in-vitro study. Br J Dermatol 1992;126: 505–9. 25. Eady EA, Jones CE, Tipper JL, et al. Antibiotic-resistant propionibacteria in acne: need for policies to modify antibiotic usage. BMJ 1993;306:555–6. 26. Bojar RA, Cunliffe WJ, Holland KT. The short-term treatment of acne vulgaris with benzoyl peroxide: effects on the surface and follicular cutaneous microflora. Br J Dermatol 1995;132:204 –8. 27. Eady EA, Bojar RA, Jones CE, et al. The effects of acne treatment with a combination of benzoyl peroxide and erythromycin on skin carriage of erythromycin-resistant propionibacteria. Br J Dermatol 1996;134:107–13.
T
he main obstacle to convincing the scientific community that P. acnes is a key factor in the development of acne is the difficulty in applying Koch’s postulates to the disease. Attempts to look for simple cause-and-effect relationships between P. acnes and acne have resulted in a plethora of anecdotal and circumstantial evidence, complicated by the fact that there is no suitable in vivo model and no therapeutic options that target P. acnes exclusively. Although efforts have been made to identify the virulence factors of P. acnes, consensus opinion on the initial changes in the pathology of an acne follicle has changed often. In particular, it remains a matter of debate as to whether comedone formation precedes or follows inflammation and whether the initial cellular infiltrate is neutrophilic or lymphocytic. Current scientific opinion generally views the cutaneous microflora as of secondary importance compared with local sebaceous gland activity, with significant microbial involvement occurring only after sebum production has increased and comedone formation has become established. In contrast, the pharmaceutical industry appears to view the process somewhat differently, because it continues to develop acne therapies with antimicrobial activity as their primary mode of action. Hopefully this article will go some way to reconcile these divergent views. At this point, the reader should be made aware of two issues with direct relevance to the discussion. First, there remains the possibility that other cutaneous microorganisms, such as staphylococci and Malassezia spp, may be involved in some cases of acne due to their close proximity to P. acnes in the pilosebaceous follicle. Second, P. acnes and other cutaneous microorganisms can From the Skin Research Centre, Division of Microbiology, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK. Address correspondence to R. A. Bojar, PhD, University of Leeds, Skin Research Centre, Division of Microbiology, Department of Biochemistry and Molecular Biology, Leeds LS2 9JT, UK. E-mail address: [email protected] © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
be isolated from both acne and normal skin. It is relatively easy to prove that a microorganism is the cause of a disease when it is cultivated from a normally sterile site and can be shown in vitro and in vivo to account for the pathology. However, it is far more difficult to assign a microorganism a pathogenic role when it is present in both normal and disease conditions. Modern views of pathogenicity (virulence) encompass the condition of the host (or target organ) and the diverse interactions between the host and the microorganism. This article discusses the biological features of cutaneous microorganisms, their location in the skin, their interaction with skin, the evidence implicating P. acnes in acne, and hypotheses on how P. acnes impinges on the pathology of acne.
Characteristics of Cutaneous Propionibacteria The cutaneous propionibacteria (P. acnes, P. avidum, P. granulosum, P. propionicum, and P. lymphophilum) are found as commensals on human skin and other keratinized epithelia. Over the years, they have been variously classified as Bacillus spp, Corynebacterium spp, anaerobic diphtheroids, and Propionibacterium spp, and this must be borne in mind when surveying the literature. They are gram-positive and nonmotile, and when first isolated exhibit a typical coryneform appearance under the microscope with irregular, short branching. Isolation is best performed at 35°C under anaerobic conditions on a growth medium supplemented with Tween 80, although propionobacteria are neither strictly anaerobic nor lipophilic. P. acnes and P. granulosum are commonly isolated from sebum-rich areas of the skin (eg, head, chest, and back) with reservoirs in the pilosebaceous follicle,1,2 whereas P. avidum is located mainly in the axillae.3,4 There is little information on P. propionicum and P. lymphophilum, and a sixth commensal strain formerly known as P. innocuum has recently been reclassified as Propioniferax innocua.5 0738-081X/04/$–see front matter doi:10.1016/j.clindermatol.2004.03.005
376 BOJAR AND HOLLAND
Clinics in Dermatology
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2004;22:375–379
The Microflora of Human Skin
Control of Microbial Colonization of the Skin
As a habitat for microorganisms, the human skin is open to contamination by microbial species from the environment, and yet it is not particularly suitable for colonization due to the environmental conditions resulting from its physical structure. Normal skin supports colonization by a limited number of resident microbial types, mainly gram-positive species able to tolerate the stresses associated with the physical environment. The stable microbial ecosystem is controlled partly by the physical environment (ie, pH, oxygen, ion, and growth substrate concentrations, ultraviolet radiation) and partly by synergistic and antagonistic interactions between the host and the resident cutaneous microflora and between microbial populations.6 Inhibitory substances, including bacteriocins, enzymes, and lowmolecular-weight inhibitors, are produced by cutaneous microorganisms7 and may help prevent colonization by pathogens.8 In addition to propionibacteria, resident microbial species come from the bacterial genera Staphylococcus, Micrococcus, Corynebacterium, and Acinetobacter; the yeast Malassezia; and a number of bacteriophage species. Although molecular techniques such as restriction fragment analysis of DNA and DNA/RNA base sequencing have been used successfully to classify resident skin microorganisms, routine identification continues to be based largely on phenotypic traits (eg, sugar fermentations, extracellular enzymes). The surprising stability of the resident microflora in response to changes in the skin environment suggests close coevolution of the skin microflora and the skin environment. In most adults, the surface of sebum-rich areas of skin and the pilosebaceous follicles support large, stable populations of propionibacteria, which are a benign presence and help maintain the ecosystem of healthy skin.9,10 They are also found in the mouth and gut, where again they possibly help stabilize complex microbial populations. Generally, they have a positive effect on human health by filling a niche that could otherwise be colonized by pathogenic microorganisms. However, when the host becomes compromised (through, eg, trauma, injury, or alterations in immune status), propionibacteria can display a pathogenic potential. Historically, most of the published work on the cutaneous propionibacteria has focused on P. acnes, due to the association with acne and its immunomodulatory effects. However, both P. granulosum and P. avidum have immunomodulatory properties, and P. granulosum has also been associated with acne. It is fair to say that despite investigations over many years, our understanding of the interactions between the cutaneous propionibacteria and the human host remains inadequate.
Physical factors controlling colonization are mainly functions of the skin structure and include the number and size of follicles and glands, gland function (eg, sebum, sweat, apocrine), the flow of secretions (which determines nutrient availability and hydration levels), the integrity of barrier function, skin pH, and osmotic potential. Biochemical analysis of the skin surface environment has revealed a vast array of chemical compounds derived from sebum, sweat, and the metabolic activity of microorganisms on skin secretions. In normal skin, available soluble nutrients are mostly at low concentrations and are derived from sweat (vitamins, amino acids, and lactate) and sebum (lipids and amino acids). Antimicrobial compounds of the nonadaptive immune system may also influence the resident microbial population density and limit colonization by transient microorganisms. Three -defensins (HBD-1, -2, and -3) are produced by human keratinocytes;11 however, their role in limiting colonization by transient microorganisms is unclear, and their influence on the population density of the resident microflora is debatable. In contrast, dermicidin, which is secreted by mucosal cells of the sweat glands,12 has activity against both gram-positive and gram-negative bacteria under the pH and salt conditions of human sweat. Because there is no evidence that microorganisms colonize eccrine glands or ducts, dermicidin may play a role in maintaining sterility in eccrine follicles. The gram-positive cell wall of propionibacteria gives these bacteria high structural stability, making them resistant to drying, osmotic shock, and mechanical stress. These are important attributes, because the skin surface is relatively deficient in free water and subject to fluctuations in temperature, solar radiation, and salt/ ion concentrations. The density of resident microorganisms at any one site on the body varies widely among individuals and between sites on the same individual. The variation in colonization density and microbial diversity between skin sites depends largely on the number and density of sebaceous and sweat glands, but there is also wide variation within the population at any one site. This is well illustrated by the range in density of propionibacteria isolated from the face, an area rich in sebaceous glands. Skin surface counts within a population of 761 volunteers ranged from ⬍10 per cm2 of skin in some individuals to ⬎10,000,000 in others (Fig 1). The sebaceous-rich sites also support microbial growth within the follicles (Fig 2); again, there is huge variation in density within the population (Fig 3). Variation in colonization also extends to different follicles in the same individual, with up to 60% of pilosebaceous follicles from normal skin not being colonized.2 Such variation may be due to differences in follicle size and the rate of sebum secretion.13 There is also some evi-
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2004;22:375–379
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Figure 1. Population distribution of Propionibacterium spp. (mean, 4.829; SD, 1.392) and Micrococcaceae (mean, 4.527; SD, .858) isolated from the surface of the cheek using the Williamson and Kligman scrub wash (author’s data; n ⫽ 761). y surface Propionibacterium spp.; y surface Micrococcaceae.
Figure 3. Population distribution of Propionibacterium spp. (mean, 4.395; SD, 1.936) and Micrococcaceae (mean, 3.905; SD, 1.394) isolated from Permabond follicular biopsy specimens taken from the cheek (author’s data; n ⫽ 461). y follicular Propionibacterium spp.; y follicular Micrococcaceae.
dence suggesting spatial stratification of microbial populations within follicles, such that particular genera may inhabit distinct niches.14 However, the interactions between factors controlling the density and location of the cutaneous microflora remain to be defined. The physiological response and expressed phenotype of microorganisms depend largely on the concentration of available nutrients. The skin environment contains a wide range of polymers, including lipids, polysaccharides, and proteins, and the skin microflora
produces extracellular enzymes that degrade these polymers to release mostly end-products with direct nutritional value, with lipase and protease the most common enzymatic activities. Differences in nutrient availability at different skin sites over time will have a major influence on the phenotype of individual microbial species and will consequently determine the composition and density of the skin microflora. However, nutritional requirements are ultimately determined by genotypic traits, such as nutrient transport systems in the cytoplasmic membrane. All cutaneous microorganisms are chemoorganotrophs requiring organic nutrients and using oxidation of organic sources to produce energy and cell biomass. It may be assumed that to a large extent, molecules that are consistently present in the skin environment provide the minimal nutritional requirements to the resident microflora.
Classification and Identification
Figure 2. Gram Weigart–stained longitudinal section of a single pilosebaceous follicle, showing the multilobular sebaceous gland and the pilosebaceous duct colonized by gram-positive microorganisms (author’s data). Scale bar ⫽ 100 m.
Although much of the literature refers to P. acnes as an anaerobe, the cutaneous propionibacteria are not strictly anaerobic, and, although anaerobic conditions are used for primary isolation, all species will tolerate the presence of oxygen.15 After incubation on agar growth media, the colonies are characteristically domeshaped and beige to pink in color. Because of the limited range of species found on human skin, most isolates can be speciated using a limited number of biochemical tests (Table 1), and these tests continue to be used routinely in preference to more sophisticated techniques, such as bacteriophage and molecular typing techniques. Applying molecular typing initially proved difficult due to the lack of a suitable technique for lysing the robust cell wall of P. acnes. However,
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Table 1.
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The distribution and differentiation of human commensal propionibacteria
Distribution
Differentiation
Skin Eye Mouth Gut Aesculin hydrolysis Casein hydrolysis Catalase production Indole production Nitrate reduction
P. acnes
P. avidum
P. granulosum
P. propionicum
⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫻ ⻫ ⻫ ⻫ ⻫
⫹⫹⫹ ⫺ ⫺ ⫺ ⻫ ⻫ ⻫ ⫻ ⫻
⫹⫹⫹ ⫺ ⫹ ⫺ ⫻ ⫻ ⻫ ⫻ ⫻
⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫻ ⫻ ⫻ ⫻ ⻫
Distribution range: present at high density (⫹⫹⫹) to not present (⫺). Differentiation (biochemical test reactions): positive (⻫); negative (⫻).
progress has been possible in recent years due initially to the development of a specific lysis technique utilizing susceptibility to penicillin16 and subsequent developments in polymerase chain reaction and sequencing protocols for use with resiliant microorganisms such as mycobacteria. Consequently, the way is now open for a thorough analysis of cutaneous propionibacteria using established molecular typing methods.
Epidemiology The link between P. acnes and the development of acne is generally regarded as fact by most dermatologists and has by and large even become accepted by the general public through advertising for acne products. However, there is no formal proof linking P. acnes with acne, and the presence of propionibacteria is not a prerequisite for the development of acne.17 If P. acnes is involved in acne, whether its presence initiates certain features of the disease, such as inflammation, or whether it exacerbates inflammation once a lesion is formed remains unclear. Propionibacteria are known to inhabit distinct niches within the skin,14 including the pilosebaceous follicle, where they reside with Staphylococcus spp and Malassezia spp.2 However, colonization is by no means uniform. Although previous studies have demonstrated that some pilosebaceous follicles are effectively devoid of any viable microorganisms,17,18 applying molecular detection techniques to more accurately define the microbial community of the skin would be beneficial. Microbial involvement in acne is just one of several factors known to influence the progress of the disease, including chronic inflammation, ductal hypercornification, and excessive sebum production; these factors contribute differently to the overall disease in each individual. Therefore, elucidating a general mechanism for microbial involvement in acne has proven difficult. Most recent published research into the commensal propionibacteria has concentrated on case reports of opportunistic infections as a consequence of surgical procedures. However, the mechanisms that provoke
commensal cutaneous propionibacteria into a pathogenic role at other body sites have not been explained. Further confusion arises due to the widespread distribution of propionibacteria on the skin and the difficulty distinguishing between contamination and true colonization/infection.
Antibiotics and Propionibacteria The hypothesis that microorganisms are involved in acne is supported in part by the successful treatment of acne with antibiotics, such as erythromycin and clindamycin, and the reduced efficacy of these treatments when antibiotic-resistant propionibacteria are present.19 Although propionibacteria are susceptible to a range of other antibiotics, including penicillins and cephalosporins,20 the antibiotics of therapeutic value in acne are generally limited to lipid-soluble compounds. Extensive use of antibiotics to treat acne since the 1960s has led to the widespread occurrence of resistance in cutaneous propionibacteria,19 (although initial fears that antibiotic resistance, once acquired, would spread rapidly via transmissible genetic elements have been allayed, because resistance appears to result from specific mutations in 23S ribosomal RNA.21) Although this mechanism is ultimately self-limiting, current concerns regarding antibiotic resistance in the wider population and in pathogenic microorganisms confirms that the use of antibiotics to treat acne needs to be better controlled. Patient preference and compliance issues also dictate the prescription and use of both topical and oral antibiotics. The use of topical antibiotics will also be determined by additional factors, such as the site of acne. For example, topical antibiotics may be hard to apply to acne on the back. Although in some countries the incidence of resistant P. acnes exceeds 60%,22 the clinical relevance of this high incidence is unclear. The reasons for this are complex and include such factors as the anti-inflammatory effects of some antibiotics. To date, there are no adequate clinical studies defining the proportion of patients in which antibiotic-resistant P. acnes has a clinical effect. However, resistance should be
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suspected in patients who do not respond to antibiotic treatment, particularly tetracycline, doxycycline, and erythromycin. Antibiotic resistance in acne patients can by and large be overcome by the judicious use of nonantibiotic antimicrobial agents, such as benzoyl peroxide and azelaic acid.23 Ultimately, combination therapies containing an antibiotic and a nonantibiotic antimicrobial agent may prove to be the best approach for maintaining the efficacy of well-established treatment regimens.24 –27
Future Directions A key objective for research is to define in detail the interactions of P. acnes with human skin in both normal and acne states. Most likely, this will be achieved by developing living skin equivalent model systems colonized by P. acnes. Further progress will be possible once the genomic sequence of P. acnes has been elucidated; then the interaction between skin and P. acnes will be open to transcriptional analysis using DNA microarray technology.
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12. Schittek B, Hipfel R, Sauer B, et al. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol 2001;2:1133–7. 13. Kearney JN, Ingham E, Cunliffe WJ, et al. Correlations between human skin bacteria and skin lipids. Br J Dermatol 1984;110:593–9. 14. Kearney JN, Harnby D, Gowland G, et al. The follicular distribution and abundance of resident bacteria on human skin. J Gen Microbiol 1984;130:797–801. 15. Cove JH, Holland KT, Cunliffe WJ. Effects of oxygen concentration on biomass production, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture. J Gen Microbiol 1983;129:3327–34. 16. Tipper JL, Eady EA, Cove JH, et al. A method for lysing cutaneous propionibacteria and its use to clone antibiotic resistance determinants from Propionibacterium acnes. Br J Dermatol 1993;129:488 –9. 17. Leeming JP, Holland KT, Cunliffe WJ. The microbial colonization of inflamed acne vulgaris lesions. Br J Dermatol 1988;118:203–8. 18. Leeming JP, Holland KT, Cunliffe WJ. The pathological and ecological significance of microorganisms colonizing acne vulgaris comedones. J Med Microbiol 1985;20:11–6. 19. Eady EA, Cove JH, Holland KT, et al. Erythromycinresistant propionibacteria in antibiotic-treated acne patients: association with therapeutic failure. Br J Dermatol 1989;121:51–7. 20. Hall GS, Prattrippin K, Lowder CY, et al. Cidal activity of 3 cell-wall active agents against Propionibacterium acnes. Invest Ophthal Vis Sci 1993;34:1258. 21. Ross JI, Eady EA, Cove JH, et al. Clinical resistance to erythromycin and clindamycin in cutaneous propionibacteria isolated from acne patients is associated with mutations in 23S rRNA. Antimicrob Agents Chemother 1997; 41:1162–5. 22. Coates P, Vyakrnam S, Eady EA, et al. Prevalence of antibiotic-resistant propionibacteria on the skin of acne patients: 10-year surveillance data and snapshot distribution study. Br J Dermatol 2002;146:840 –8. 23. Bojar RA, Eady EA, Jones CE, et al. Inhibition of erythromycin-resistant propionibacteria on the skin of acne patients by topical erythromycin with and without zinc. Br J Dermatol 1994;130:329 –36. 24. Holland KT, Bojar RA, Cunliffe WJ, et al. The effect of zinc and erythromycin on the growth of erythromycin-resistant and erythromycin-sensitive isolates of Propionibacterium acnes: an in-vitro study. Br J Dermatol 1992;126: 505–9. 25. Eady EA, Jones CE, Tipper JL, et al. Antibiotic-resistant propionibacteria in acne: need for policies to modify antibiotic usage. BMJ 1993;306:555–6. 26. Bojar RA, Cunliffe WJ, Holland KT. The short-term treatment of acne vulgaris with benzoyl peroxide: effects on the surface and follicular cutaneous microflora. Br J Dermatol 1995;132:204 –8. 27. Eady EA, Bojar RA, Jones CE, et al. The effects of acne treatment with a combination of benzoyl peroxide and erythromycin on skin carriage of erythromycin-resistant propionibacteria. Br J Dermatol 1996;134:107–13.