Skin microbiome and dysbiosis: Acne perspective

Introduction

Skin is the home for a vast variety of microorganisms, including archaea, bacteria, fungi, viruses and arthropods, which together make up what is known as skin microbiome^[1]. The skin microbiome majorly comprises Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%) and Bacteroidetes (6.3%)^[2]. The microbiome of epithelia and sebaceous follicles comprises Gram-positive bacteria such as Staphylococcus epidermidis and Cutibacterium acnes (C. acnes hereafter), and fungal species such as Malassezia. As skin commensals, C. acnes and S. epidermidis interact with the host, helping protect healthy skin from colonization by pathogens such as Staphylococcus aureus^[3], ^[4]. Any imbalance caused by internal/external factors in the microbiome composition is termed as dysbiosis. The dysbiosis factors mainly include genetic, hormonal changes/imbalances, western diet, overuse of cosmetics/antibiotics and infections^[5], ^[6]. Factors such as the western diet and overuse of antibiotics are also associated with changes in gut microbiome, which, in turn, has effects on skin microbiome via immune modulation, leading to skin ailments such as acne, eczema, psoriasis, rosacea, and keratosis pilaris^[7], ^[8], ^[9]. This white paper discusses skin microbiome and dysbiosis from the acne perspective.

Types of Acne

Acne (common term for Acne vulgaris) is a common inflammatory chronic skin problem, affecting the pilosebaceous units of hair follicles. About 85% of adolescents and young adults are estimated to have been affected by this disease. Acne is the second most common dermatological condition after dermatitis^[10]. Acne is mainly classified into four types based on the severity of lesions formed on the epidermis: (1) comedonal, (2) papular, (3) pustular and (4) nodules. The comedonal includes the non-inflammatory mild lesions commonly known as black heads and white heads, as shown in fig.1. Papular and pustular are inflammatory mild to moderate lesions, while nodules are the most severe ones^[11].

Fig. 1. Types of acne based on severity of lesions (Source)

Pathophysiology

The pathophysiology of acne mainly comprises four events:

• Over-secretion of sebum
• Proliferation and abnormal differentiation of keratinocytes in the hair follicle
• Bacterial colonization
• Host inflammatory response^[12].

These events are mostly interlinked, with each one likely to lead to the others.

For instance, sebum contains nutrients necessary for the bacterial growth and its over-secretion leads to bacterial colonization. Bacterial colonization is most evident by the presence of C. acnes, in numbers higher than usual, leading to imbalance i.e., dysbiosis. The balanced skin microbiome has a control over the proliferation of dwelling microbes as depicted in fig 2.

In healthy skin, C. acnes plays a beneficial role in the cutaneous microbiota of the pilosebaceous units. It limits the growth of pathogenic species, such as community-acquired methicillin-resistant S. aureus [13]. It also plays an important role in maintaining the acidic pH of the pilosebaceous follicle by hydrolyzing sebum triglycerides and secreting propionic acid^[14], ^[15]. The proliferation of C. acnes is regulated by the presence of S. epidermidis. Both the bacteria use glycerol as a shared carbon source for the production of different short-chain fatty acids (SCFAs), which act as antimicrobial agents against each other ^[16].

The dysbiosis in the C. acnes populations is caused by a set of events triggered due to puberty or hormone imbalances i.e., increased androgen levels that lead to change in the environment of sebocytes, such as pH changes and increased sebum secretion, resulting in the clogging of pores. The clogged pores create a hypoxic condition in the pilosebeceous units, resulting in a favorable environment for the unchecked growth of C. acnes^[17]. Furthermore, C. acnes stimulates the sebaceous glands and sebum synthesis via the corticotropin-releasing hormone (CRH)/ CRH receptor pathway, resulting in more lipogenesis and sebum production ^[18]. The gut microbiome dysbiosis, especially linked to acne, reveals the decreased levels of gut-beneficial bacteria such as Bifidobacterium and Lactobacillus^[19]. Gut dysbiosis is also linked to the alterations in sebum via Insulin-like growth factor-1 (IGF‐1), thereby triggering a shift in skin microbiome^[20].

C. acnes is involved in the modulation of the differentiation of keratinocytes, thereby resulting in local inflammation, which is regarded as the early stage of acne onset, i.e. formation of comedones, and further inflammation resulting in acne lesions^[21]. C. acnes extracts have been shown to be involved in the differentiation of keratinocytes by inducing b1, a3, a6s, aVb6 integrin expression and filaggrin expression in keratinocytes^[22].

C. acnes interacts with the host inflammatory factors such as Toll-like receptors (TLRs), antimicrobial peptides (AMPs), protease-activated receptors (PARs), and matrix metalloproteinase (MMP). It also upregulates the secretion of pro-inflammatory cytokines such as interleukins, including IL-1a, IL-1β, IL-6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GMCSF), through keratinocytes, sebocytes, and macrophages ^[23], ^[24]. The production of AMPs (LL-37, β-defensin 2), cytokines (IL-1α), and MMP is associated with the increased expression of the PAR-2 (G-protein-coupled receptor) in acne-affected skin^[25]. Further interaction between C. acnes and macrophages can induce IL-1β32, consequently activating the NLRP3-inflammasome pathway in antigen-presenting cells and myeloid cells^[26]. C. acnes has been shown to release extracellular vesicles (EVs), which induce cellular responses via TLR2 signal cascades. These EVs further induce IL-8 and GM-CSF and decrease epidermal keratin-10 and desmocollin, contributing to the development of acne lesions^[27]. Christie-Atkins-Munch-Petersen (CAMP) factor, a secretory virulence factor from S. aureus that was considered a main source of inflammation in acne vulgaris, is also found in genome of C. acnes ^[28]. The CAMP factor, when combined with acid sphingomyelinase (ASMase), which may be sourced from host or S. aureus, leads to acne lesions via cell lytic and hemolytic activites ^[29]. C. acnes also comprises enzymes that have lipase or ceramidase-like activities, leading to the formation of short chain fatty acids that are known to be pro-inflammatory factors. Further, the activity of ceramidases is linked to the impairment of skin barrier function^[30]. The different target activities of C. acnes in pathogenesis of acne are summarized in fig. 3.

The dysbiosis in case of C. acnes has been studied right up to the strain level using advanced genomic approaches and, together with morphological and biochemical approaches, it helps compare C. acnes strains belonging to the three main phylotypes (I, II and III). The more recent studies propose the reclassification into distinct subspecies: phylotype I as C. acnes subsp. acnes, phylotype II as C. acnes subsp. defendens and phylotype III as C. acnes subsp. elongatum^[31], ^[33]. Earlier studies have revealed the distinct microbiome profiles in patients with acne. Loss of C. acnes phylotype diversity in patients with severe inflammatory acne, and the predominant presence of a phylotype IA1, as compared to healthy controls, has been reported. With additional molecular typing methods, the SLST type A1 was found to be predominant in the acne group^[34], ^[35]. Type IA-2 (primarily ribotype 4 [RT4] and RT5) strains have also been associated with acne. The virulence of these strains was attributed to the presence of specific extra genomic elements encoding multiple virulence genes. A multi-locus sequence typing study has proposed that expression of dermatan-sulphate-binding proteins that contain putative phase/antigenic variation signatures by the type IA strains contributes to their role in the pathophysiology of acne and also sheds light on the recurrent nature of acne^[34].

Treatment

Conventional Therapies

Acne treatment generally involves mitigation of microbial load and inflammatory responses by using oral or topical drugs. Topical agents such as Sulphur, resorcinol, dapsone, benzoyl peroxide and salicylic acid have been very popular since ages, but their usage makes skin dry and irritable^[36]. Antimicrobials such as tetracyclines and macrolides have been considered to be most effective as they minimize the microbial load effectively^[37]. The key concerns with this treatment are collateral damage to the resident beneficial microflora such as S. epidermidis. There are several studies indicating the emergence of resistant pathogenic bacterial strains of P. acnes and S. aureus due to long term use of antimicrobials^[38]. Short-term use of antimicrobials though, effectively clears S. aureus during treatment periods, however, recolonization is typically seen within a month or two^[39], ^[40]. Use of oral antibiotics, which is also a common treatment option, could lead to the damage of beneficial bacteria of the gut, also leading to gastric disorders.^[41]. Fig.4, given below, outlines various therapies and the underlying side effects on the body.

Treatment with retinoid compounds is considered to address most of the aetiological factors of acne, as it shows significant reduction in sebum production, regulation of the number of comedones, lowering of C. acnes population while having anti-inflammatory properties^[42]. The anti–inflammatory effects are attributed to its effect on monocytes via two pathways, one involving TLR2/1 and the other involving CD14 expression^[43]. But they do pose significant adverse effects such as embryotoxicity in pregnant women^[44]. Hormonal therapy, mainly comprising of oral contraceptives and spironolactones, is the most common therapy, especially for women. Oral contraceptives have good efficacy in reducing both inflammatory and noninflammatory lesions. This treatment presents side-effects ranging from weight gain to cardiovascular disorders, mood swings to risk of both breast and ovarian cancers^[45]. Spiranolactones, though not FDA approved, have been widely used as adjuvant therapy for acne, alongside oral contraceptives or antibiotics. There are several debates on its usage as a single therapy, as it has side effects such as menstrual-related disturbances and moreover, experts warn about a theoretical future risk of fetal gender conversion in women of child -bearing age^[45].

Innovative Therapies

With increased awareness on skin microbiome and its role in immunity and overall skin health, there has been gradual shift in research towards safer, pathogen-targeting and microbiome-friendlier ways to treat skin ailments.

Probiotics

Oral probiotic therapy has been used since a long time as an adjuvant therapy, along with oral antibiotics, to replenish the lost gut flora. With emerging research on gut-brain-skin axis, the use of probiotics either in oral or topical therapies has been the focal point of several studies^[46]. Fig. 5 outlines a few probiotic therapies and their effect on acne pathophysiology in brief. Staphylococcus epidermidis, which is known to ferment glycerol and regulate the growth of C. acnes, is being considered as a probiotic therapy for acne. The glycerol fermented product is expected to result in a better outcome without harming other residential bacteria^[47]. A recent study has demonstrated the application of S. epidermidis encapsulated in polysulfone microtube array membranes (PSF MTAM), which has enhanced the glycerol fermentation activities of S. epidermidis^[48]. Oral supplementation with Lactobacillus paracasei NCC2461 was studied to assess its effect on skin integrity. Healthy volunteers underwent a capsaicin test to monitor skin sensitivity, while transepidermal water loss (TEWL) was utilized to measure the skin barrier function. The probiotic group showed improvement in both, skin sensitivity and skin barrier function, when compared to a control group^[49]. There are studies on its anti-inflammation activity carried out through the inhibition of the proliferation of CD-4 + T-cells and induction of the anti-inflammatory cytokines IL-10 and TGF-beta^[50].

An ex-vivo study utilizing Lactobacillus paracasei CNCM I-2116 demonstrated the inhibition of skin inflammation. The positive markers involved decrease in vasodilation, edema, mast cell degranulation, and TNF-alpha release, which were induced by substance P for the experimental purpose^[5]. In vivo studies utilizing an extract from Lactobacillus plantarum, showed a visible reduction in light acne lesions along with redness and effective reconstruction of skin barrier^[52].

In an in-vitro study, Streptococcus salivarius, which is a native commensal of the digestive system, was capable of inhibiting C. acnes growth via production of antimicrobial substances – Bacteriocin-like inhibitory substances (BLIS). Topical application of this organism was proposed as acne treatment, considering its efficacy also in anti-inflammation. They act by inhibiting proinflammatory cytokine IL-8 in epithelial cells and keratinocytes. They are found to modulate genes associated with epithelial layer adhesion and homeostasis. This way, they inhibit other bacteria growth without injuring the microbiome present in the skin^[53]. A phase-3 clinical trial was conducted with a topical preparation of Enterococcus faecalis SL-5 extract on patients with light to moderate acne vulgaris. The study resulted in significant reduction of inflammatory lesions in the treated group, indicating the inhibition of C. acnes and also anti-inflammation by decreasing the production of inflammatory mediators from the pathogen^[54].

Prebiotics

Konjac Glucomannan hydrolysates (GMH) are known for their prebiotic properties. They promote the growth of beneficial bacteria such as lactic acid bacteria, and inhibit the growth of pathogens, such as Escherichia coli or Listeria monocytogenes, when consumed orally^[55]. Their topical application was tested in volunteers suffering from acne, using spray formulation containing GMH at a concentration of 5% (w/v). Standard antibiotic therapy was used in comparison group. Acne severity index was measured before and during treatment. The results showed a visible improvement in the skin health after 40 days of treatment^[56].

The ability of konjac glucomannan hydrolysates (GMH) to inhibit C. acnes when used in combination with probiotics was studied in-vitro. The positive results from this study encourage a view for future use as therapeutic synbiotics alongside the selected probiotics for treating acne infections^[57]. Another study on combination of prebiotics and the probiotics has shown the combination to be contributing to the improved barrier function of skin. The main mechanism involved in it is the increased ceramide production^[58].

A study using oral prebiotics fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) in women with adult acne was conducted to evaluate the effect of prebiotics on metabolic factors such as glucose and lipid parameters. The study resulted in positive effects on glycemic and lipid metabolic parameters, which otherwise are known to play a vital role in sebum over secretion and initiating the acne flares^[59].

Antimicrobial peptides

The ability of bacteriocins to inhibit C. acnes was reported in some studies on bacteriocin- secreting organisms such as Lactococcus sp. HY 449, Streptococcus, Enterococcus faecalis SL-5 or Lactobacillus plantarum. The studies showed the reduction of the inflammatory lesions caused by acne bacterium, suggesting the bacteriocins as therapy for acne^[60]. A study was conducted to examine the effectiveness of bacteriocin AS-48, alone and in combination with host antimicrobial enzyme lysozyme, against C. acnes. Lysozyme constitutes the innate immune system to treat conditions produced by various pathogens. Microscopy and bioassay techniques indicated this combination to be effective against both free cells and biofilms of C. acnes. Topical usage of these substances was recommended due to the absence of any cytotoxicity in the cell lines tested^[61].

Nisin J is a novel nisin variant bacteriocin of Staphylococcus capitis APC 2923, resident of the human skin microbiota. Nisin J exhibits antimicrobial activity against a variety of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and C. acnes. The genetic variation of this nisin J from the known nisin variants is that it is lacking the nisin-regulatory gene nisRK, as well as the nisin-immunity gene nisI. Nisin J is comparatively longer with 35 amino acids, making it difficult for the pathogens to develop resistance against the antimicrobial^[62].

Phage therapy

Specific bacteriophages that are intracellular parasites dwelling within C. acnes were isolated from sebaceous follicles of healthy individuals. These isolated phages were identified to be highly homogeneous, showing limited genetic diversity. The tested phages had ideal features such as lytic lifecycle, lack of lysogeny-related genes, and the presence of endolysin-encoding genes that could be leveraged for therapy of acne^[63]. An in-vitro study utilizing C. acnes phages formulated into a topical preparation showed effective eradication of C. acnes bacteria, and the formulation preserved the bacteriophage for at least 90 days when stored at 4 degrees Celsius^[64]. The phage therapy is targeted and it poses less risk to non-targeted residential microbiome. The resistance to phages can be addressed by genetic engineering of effective bacteriophages. Further, the presence of endolysins ensures bacterial cell lysis, and resistance is not yet reported for endolysins^[65].

Nitric oxide

The beneficial role of nitric oxide in improving the blood flow, wound healing and immune building is widely known. The endogenous production (NO) is carried out by bacteria such as Nitrosomonas, which are now also being used in the cosmetics industry^[66]. The compounds releasing nitric oxide, such as berdazimer sodium- SB 204, are currently in phase-3 trials for treating acne. They could be targeting bacteria via nitrosative and oxidative mechanisms, thereby lowering the risk of resistance development^[67], ^[68].

NO-nanoparticles were studied, and the results confirm the prevention of C. acnes-induced inflammation by both, clearing the organism and inhibiting microbial stimulation of the innate immune response. The mechanism by which NO-np regulates the inflammatory factor IL-1β secretion is by inhibiting the gene expression of caspase-1 and IL-1β ^[69].

Vaccine

In preliminary investigations, a vaccine against C. acnes-virulence factors such as the Christie-Atkins-Munch-Petersen (CAMP) factor was found to be effective in reducing the production of pro-inflammatory cytokines IL-8 and IL-1β^[70].

Collaborations

• Bayer and Azitra Inc. have announced collaboration for the identification and characterization of skin microbiome bacteria. The partnership will leverage Azitra’s proprietary panel of Staphylococcus epidermidis strains to identify potential candidates for the treatment of adverse skin conditions and diseases^[71].
• Royal DSM and Belgian Life Sciences start-up S-Biomedic have signed a commercial agreement to develop and commercialize a skin care active that uses probiotic technology to treat acne^[72].
• S-Biomedic earlier collaborated with Beiersdorf to complete the development of its acne treatment and commercialize it ^[73].
• Cosmetics giant L’Oréal has collaborated with microbial genomics company uBiome to tackle challenges in microbiome research^[74].
• Unilever ventures has extended its financial support to Gallinée, a French player in the area of probiotic cosmetics ^[75].
• Symrise has collaborated with a bioengineering company, Probi to produce Lactobacillus-based skin care ingredients^[76].

Commercial Products

Product: Mother Dirt AO+ mist

Company: AO Biome

Product description: Clinically researched to restore clarity & balance to the human skin in 4 weeks, this spray comprises Nitrosomonas eutropha (ammonia-oxidizing bacteria), a peacekeeper that once existed on our skin but was cleaned away with modern hygiene and lifestyles. Removing the ammonia from the skin helps keep the pH balanced and the production of nitrite and nitric oxide helps maintain balance across other micro-organisms. It also promotes a calming and soothing effect on the skin.