9.1.4 Cutaneous Microbiology

Microbes of the skin belong to the microbial ecosystem of the body.

The terms microbiom and microbiota are used interchangeably, however, there are differences:

Cutaneous microbiota: all different micro-organisms as a population living on the skin. Refers to the taxonomy. Subcategories i.e. arm pit, feet, groin, nose etc.

Cutaneous microbiome: all micro-organisms and their genetic material on the skin. Refers to the bacteria first and their genes secondary.

Metagenome: these are the genes of cutaneous microbes in a specific environment. Refers to collective functions of microbial genes.

Metatranscriptome: transcriptomes induced by microbiota.

The cutaneous microbiome encompasses the microbes that live in and on the skin, including bacteria, fungi and viruses. Archeae and mites (acari group) do not play primarily a significant role. Like many aspects in the development of infant physiology, the cutaneous microbiome is dynamic, continuously evolving and diversifying with age, environment (height, latitude, temperature, clothes, skin moisture, pH, sebum), personal hygiene, different forms of stress, nutritional status, culture, exposure to skin irritating or protecting agents, UV- light, antibiotics and/or genetic and epigenetic factors (barrier function, innate and adaptive immune system).

At time of delivery, the neonatal skin is ad hoc exposed to a new environment outside the uterus with a microbe rich world.

The mode of delivery determines the newborn′s cutaneous microbiome composition. Bacteria on the skin of newborns delivered vaginally are similar to their mothers′ vaginal flora, containing predominantly Lactobacilla. The skin of newborns delivered via cesarean section is colonized by bacteria most similar to those on their mothers′ skin, in particular Staphylococci, Streptococci, Corynebacteria and Cutibacteria. In contrast to the microbiome of adults, the early skin microbiome does not differ significantly in composition based on anatomic location.

(figure 1)

Between the 3rd week up to the 3rd month of life, infants begin to develop the anatomic site‐ specific bacterial profiles similar to adults. Colonization takes place in interfollicular, intrafollicular and acrosyringeal areas. Around the 6th week of life, infant skin and nares become colonized by Staphylococcus and Corynebacterium quite similar to adults but still differs significantly from the adult skin microbiome until late adolescence. Malassezia colonization increases with age, becoming adult‐like by 1 month of life. The strain sequence identity of Malassezia colonizing neonates and their mothers are very similar, suggesting transmission from mother to infant. During the time of infancy, Firmicutes (specifically genera Staphylococcus and Streptococcus) are the predominant bacteria on the skin, followed by bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. In adults, Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are the predominant phyla, and Corynebacterium, Cutibacteria, and Streptococous and Staphylococcus are the predominant genera.

An average healthy human body possesses 10x14 microorganisms – 10-fold the number of human cells.

A single square centimetre of the human skin can contain up to one billion microorganisms. These diverse communities of bacteria, fungi, mites and viruses can provide protection against disease, but can also exacerbate skin lesions, promote disease and delay wound healing. Microbial diversity is the degree of hererogeneity, the more the healthier for the host.

It is now known that according to localisation and temporal diversity of a healthy, adult skin microbiome from 20 different skin sites 19 bacterial phyla were detected, but the most prominent sequences were assigned to four phyla: Actinobacteria (52%), Firmicutes (24%), Proteobacteria (17%), and Bacteroidetes (7%). Within these phyla Cutibacteria and Staphylococci species dominate sebaceous areas (glabella, alar area, external auditory canal, neck, sternum and back). In moist sites, Corynebacteria species predominate (nare, axilla, antecubital and popliteal fossa, interdigital spaces, inguinal and gluteal creases and umbilicus). Staphylococcal species were also identified here. In dry skin sites, such as the volar forearm, hypothenar, palms and buttocks, a mixed population of bacteria resides (figure 2).

Depending on commensal or pathogenic microbes living on the skin, the local and systemic immune response is different. Whereas commensal microbes live in homeostatic balance with the host, a disruption occurs when pathogenic microbes come to the scene or those are overgrowing normally suppressed by the host cells (epidermal and dermal compartments) or commensal microbe defense mechanisms.

Therefore, an important challenge faced by the host`s skin immune system is to distinguish between beneficial and pathogenic microbes, which can share very similar molecular patterns that are first recognized by the innate immune system (such as lipopolysaccharide, peptidogycans, lipoproteins and flagellin). Discrimination between acquired microbes during delivery and infancy may be a feature of the developing adaptive immune system, which can recognize discrete molecular sequences, and, depending on the development of the immune system respond with both pro- and anti-inflammatory mediators and activation of cells depending on the nature of the antigen. For example, S. epidermis induces AMPs such as beta defensins 2 and 3 enhancing the host response to S. aureus, activates mast cell-mediated antiviral reactions, can suppress inflammatory reactions during wound healing, and stimulates resident cutaneous T-cells for maturation. Stress can modulate the composition of the microbiome in general. On the skin, the function of Staph. epidermidis is disturbed and can in turn influence the Staph.aureus nasal colonization and can reduce the lipoteichoic acid production by Staph. epidermidis which in turn influences via miR 143 TLR-2 production and growth of C.acnes. An infection by intracellular pathogens initiates the development of T helper 1 (TH1) cells, whereas extracellular pathogens induce the differentiation of TH2 and TH17 subsets (atopic dermatitis, acne). These are proinflammatory cells which coordinate many aspects of the innate and adaptive immune response to clear microbial invaders.

At the beginning of life, in the late in utero status and after delivery, the vernix caseosa is a key player in the development of early cutaneous innate immunity. It contains LL-37 and lysozyme, two antimicrobial substances that work synergistically, as well as lactoferrin, alpha‐defensins, and other antimicrobial peptides. Vernix appears to selectively inhibit some bacteria (Klebsiella, Bacillus megaterium, Listeria monocytogenes, Group B Streptococcus, and Candida albicans), but not Pseudomonas aeruginosa, coagulase‐negative Staphylococcus, or Serratia marcescens. This selective inhibition may be mediated, in part, by the role of vernix in the development of the acid mantle.

First of all, the understanding of the differences in the microbiome in healthy children and in healthy adolescents as well as in adults and in the aged population is necessary for changes that occur in a disease. The unique structure of the skin of newborns is influenced by and influences itself cutaneous microbial colonization vice versa (skin barrier function and genetic failures of skin structures, vaginal or cesarean delivery) and a possible settlement by non-commensal microbes. A dysbiosis occurs when the skin microbiome is altered from the status of a normal “healthy” microbiome. Dysbiosis is present in many changes of the skin physiology (overwashing, humidity, clothes, antimicrobials) leading to pathologies including for example erythema toxicum neonatorum, superficial folliculitis, body or feet odor, atopic dermatitis, and acne.

In general, we differentiate primary infections of the skin, skin diseases which become secondary superinfected (impetiginized) and skin diseases in which changes of the microbiota drive in addition the course of the individual disease.

(figure 3 and 4)

Erythema toxicum neonatorum

It is seen early in life in neonates with macular erythema, papules and pustules, mostly self-limited. It is almost always localized at hair follicle bearing skin (in newborns higher density than in adults) and most probably related to coccal microbes entering the opening of the follicle. A strong eosinophilic infiltrate is seen by histology but no evidence for allergic reactions are given. A reaction of the adaptive immune system in an early development stage is discussed.

Superficial and Deep Folliculitis

In both situations an overgrowth of staphyloccal species takes place in the follicular acro – and infrainfundibulum showing superficial pustules, infiltrated lesions with papules and finally abscesses leading to furuncle or carbuncle if not appropriately treated in time. All of these are primary infections of the skin. Primary skin infections may have acute (streptoccocal erysipelas) or delayed onset (borreliosis, tuberculosis) (see chapter 2.2.4 Folliculitis).

Impetigo contagiosa

Two types of impetigo are differentiated, the staphyloccocal and the streptococcal types. Impetigo staphylogenes is located at the intrafollicular epidermis and bacteria settle in and on the stratum corneum. Impetigo streptogenes is located at the interfollicular epidermis. Both are mostly seen in the first life decade, in particular in small children who often touch lesions and transfer the bacteria to other parts of the face and body. Impetigo is highly contagious, as its name implies. Topical disinfectants and antimicrobials are preferred as topical treatment in contrast to topical antibiotics. Evaluation of smears from the nares for resistant strains is recommended (see chapter 2.2.2 Impetigo contagiosa).

Pityriasis versicolor

Superficial infection with Malassezia furfur species is common and shows multiple asymptomatic scaly macules and patches, presenting in color from white to tanned to brown to pink skin. A follicular variant is M. furfur folliculitis on the trunk and Pityriasis folliculitis of the scalp. Seborrhoeic dermatitis is related to M. furfur superinfection, which is an important trigger. Fourteen different species are recognized of which M.furfur, M.globosa and M. sympodialis are the clinically important ones. The yeast M.furfur is almost found in about >80% of adults as a commensal without pathologic manifestation. However, conditions with warm and humid environments, clothes with low air circulation and increased sweating, immunosuppression, malnutrition, pregnancy, or Cushing disease are trigger factors. Scratching of lesions material on a glass slide and staining with methylene blue or Wood light examination help to diagnose the presence and amount of the yeast. Topical treatment includes selenium sulfide, zinc- pyrithione, ciclopirox olamine, azole and allylamine antifungals. In relapsing and widespread cases, oral treatment is preferred (see chapter 2.3.3 Pytiriasis versicolor).

Atopic Dermatitis

Barrier dysfunction (reduced very-long chain epidermal lipids, filaggrin dysfunction), inflammation and microbes (itch-scratch cycle with autoantigen development) contribute to the pathogenesis of AD (see chapter 1.1.2.1 Atopic dermatitis).

S. aureus clumping factor B binds to loricrin and cytokeratin 10 and promotes adhesion of S. aureus to the str. corneum also via fibronectin.

Antimicrobial peptides (AMPs) such as beta-defensins and cathelicidins are also reduced in AD lesions.

The interleukins IL-4 and IL-13 of the Th2 cell pathway play a major role and drive inflammation in AD. Th2 cytokines reduce expression of important skin barrier proteins: filaggrin, loricrin and involucrin. While healthy individuals are less colonized with S.aureus, 70% of patients with AD are colonized at lesional sites. Bacterial virulence factors, such as superantigens, proteases and cytolytic phenol-soluble modulins (PSMs) secreted by S. aureus, cause additional skin inflammation and may also contribute to bacterial persistence and /or epithelial penetration via scratching and, in the worst case, superinfection.

When sampling skin swabs during flares of AD the cutaneous microbial diversity has been shown to decrease remarkably, with predominance of S. epidermidis, S. aureus, and Malassezia species.

Infants already showing features of AD at 1 year of age have significantly less commensal Staphylococci in the antecubital fossa compared with the age of 2 months and when compared with unaffected infants. Early exposure to Staphylococcus may reduce the development of AD (adaptive immune response). In addition, AD patients are more prone to suffer from herpes (eczema herpeticatum) or malassezia infections. However, topical treatment with antibiotics is not the solution, but the interruption of the inflammatory cascade by the itch-scratch cycle and downregulating of Th2 cell mediators as well as the stabilization of the skin barrier. In general, one has to differentiate between a proinflammatory associated status and progressive change of microbiota in AD and a real superinfection.

Acne

Acne vulgaris originates in the pilosebaceous unit of the skin starting in (pre)puberty. Increased and modified sebum production, disturbance of follicular keratinocyte differentiation and release of inflammatory mediators are the primary driving forces of acne.

The second step in the pathogenic cascade is colonization of this unit by Cutibacterium acnes, a commensal bacterium followed by additional triggering of inflammation. It is not a primary infection per se, as for example a folliculitis with overgrowth of Staph.aureus.

The environment and biochemistry of the pilosebaceous unit is unique and the intrafollicular microbial colonization does not correlate with the interfollicular epidermal surface composition. C. acnes has the metabolic potential to substantially alter its local environment. It produces numerous enzymes and proteinases including lipases that alltogether alter the sebaceous lipid compositon and contribute to the production and release of antimicrobial and immunomodulatory molecules (i.e upregulation of Toll-like receptor 2 on follicular keratinocytes or macrophages, acquisition of Th4 and Th17 cells).

However, there are multiple strains of C.acnes in the follicular canal and on the skin responsible for different courses and severity of the disease. Via multi locus sequence typing (MLST), a standard method today of analyzing and subtyping bacteria, one differentiates the clade of subtypes IA1, IA2, IB, and IC responsible for the disease course.

In particular, topical antibiotic treatment with erythromycin, clindamycin, nadifloxacin or tetracyclines lead to development of resistance. Therefore, microbial killing substances such as benzoylperoxide or azelaic acid and stabilizers of the intrafollicular keratinization such as retinoids are preferred.

New therapeutic advances are the transplantation of enzymatic differently armoured C.acnes strains which compete with the pathologic ones which may lead to improvement of the disease.

(Figure 5)

Psoriasis

Psoriasis is characterized by hyperproliferation of keratinocytes with increased desquamation and increased inflammation. One should expect that the high epidermal turnover does not allow a settlement of pathologic microbiota, but the disturbed barrier function of psoriais gives rise to an altered microbiome. Moreover, throat and nasal Streptococcal infection have been shown to trigger initiation and exacerbation of psoriasis suggesting a microbial contribution to the disease.

At first, psoriasis-associated skin microbiota displays a higher diversity and more heterogeneity compared to healthy skin bacterial communities. So called specific microbial signatures are associated with lesional and non-lesional psoriatic skin compared to healthy skin.

A relative enrichment of Staph. aureus and Staph. pettenkofferi was strongly associated with both lesional and non-lesional psoriatic skin. In contrast, S. epidermidis and C. acnes were underrepresented in psoriatic lesions in particular on the arm, gluteal fold, and trunk.

However, both were normal in non-lesional skin. The Staph.aureus overgrowth in certain lesion types and anatomic sites drives Th 17 cell response and release of cytokines of IL-17 but also IL-23R and IL-22 types. This more or less drives additional inflammation but it is not an infection per se.

Skin aging

There is a now more evidence about skin aging and the skin microbiome. Recently, it was found out a negative relationship between microbiome diversity and transepidermal water loss, and a positive association between microbiome diversity and age.

Standard procedures in cutaneous microbiology are swabs from visible altered skin sites or nares or fluids. In addition, extraction of follicular material by pressure or by a needle or cyanoacrylate stripes is possible.

Material can also be taken by nail cutting (mycosis, pseudomonas) or hair plugging (bacteria or intratrichal or extratrichal mycosis) and, finally, deeper scratching to extract parasites from skin lesions (scabies, larva migrans). For mycoses with yeasts the Wood light can be used. For discrimination of deeper dermal infections such as MOTT or classical tuberculosis, Borrelia, deep mycoses (coccidiomycosis) or parasites (worms) a punch or spindle biopsy is necessary for immunohistochemistry or gene sequencing.

Usually, a standard identification of the microbe is performed. Resistance patterns for antibiotic systemic treatment are necessary when a relevant deeper infection or pathological virulent microbe is apparent. Subtyping of phylae, subspecies and clades are reserved for research purposes at the moment. Those current implemented methods are next generation 16S sequencing and shotgun metagenomics sequencing.

Currently, there is emerging research on the skin microbiome and its connection with the gut and intestine, referred to as the gut-skin axis and its effects on dermatologic conditions including the response to immune targeted therapy in melanoma.