The Delicate Skin of Preterm Infants: Barrier Function, Immune-Microbiome Interaction, and Clinical Implications

The skin of preterm infants is a delicate organ with critical structural and functional differences as compared to term born infants. Unique features contribute to an increased susceptibility to injury, infection, thermal instability, and water loss. During rapid, often accelerated adaption of the physical barrier function of preterm skin, a parallel and mutual development of host skin immunity and skin microbiome seem to be crucial for skin homeostasis. Recent advances in molecular biology have enabled researchers to gain a deeper understanding of the microbial community composition of preterm skin and the important relationship with microbiome composition of other body sites. Nevertheless, several questions remain to be answered, including niche factors and environmental influences on skin maturation. In line with that, evidence-based guidelines on skin care practice in preterm infants are missing. This review articles aims to provide an overview of the current knowledge of preterm infant skin development including immune and barrier function, host-microbial interactions, and potential clinical implications.


Introduction
The skin is the largest epithelial organ of the body and acts as physical barrier and immunological interface. It has a major role in thermoregulation, hydration, sensory perception, vitamin production, and control of substance absorption [1]. At birth, the skin undergoes a sudden transition from the warm, amniotic fluid environment in utero to the cold, dry, microbe-rich outside world. The skin of term neonates has a structural composition that is appropriate for the required adaptive fitness [2]. In contrast, the barrier function of preterm infants' skin is less functional which renders them susceptible to thermal, mechanical, and chemical injury as well as water loss and electrolyte imbalance and infection [2,3]. Environmental exposures, This article is licensed under the Creative Commons Attribution 4.0 International License (CC BY) (http://www.karger.com/Services/ OpenAccessLicense). Usage, derivative works and distribution are permitted provided that proper credit is given to the author and the original publisher. i.e., invasive measures, diagnostic procedures, devices, and treatments additionally increase the risk for the impairment of skin integrity in preterm infants [2]. The postnatal maturation of preterm skin is accelerated ex utero and driven by external cues, e.g., the dry, gaseous surroundings, and the maternal microbiome [1,[4][5][6]. During this timeframe, the immune system undergoes maturational processes which are mutually interacting with the dynamic microbiome establishment [7,8]. With the advent of culture-independent molecular-biological techniques, the ecological profiling of skin microbiome and their role in early life has become an emerging field of research. This article provides an overview on the current knowledge of the preterm infant skin with focus on: (1) barrier development and function, (2) skin microbiome interactions, and (3) potential clinical implications for preterm skin care practices and infection prevention measures.

Key Structural Differences of Skin between Preterm and Term Infants
The delicate skin of preterm infants differs remarkably in structure, composition, integrity, and immunological function from term infants' skin. Key hallmarks of developmental differences are outlined in Table 1. Environmental factors, such as amniotic fluid composition, endemic flora, hormonal exposure, and nutrient, supply significantly drive skin adaptation at the transition from aqueous immersion in utero to the gaseous outside world [9]. It is known that keratinization begins at 9 weeks gestation, and at 13 weeks gestation, stratification into different layers becomes apparent. However, maturation into a competent physical barrier is achieved at 34 weeks [1,2]. In preterm infants, the outermost layer of the epidermis, the stratum corneum (SC), is thinner than in term infants which increases permeability and the risk for heat and water loss as well as infection.

The Unique Role of Vernix Caseosa
Alongside with epidermis development, the formation of sebum glands starts at 16-18 weeks', followed by the production of a gelatinous biofilm called vernix caseosa (VC). Most extremely preterm infants have very little VC. As outlined in Table 2, VC consists of 80% water, lipid components from sebaceous and epidermal origin and proteins as well as fetal corneocytes which lack desmosomal attachments [2,10]. VC is essential for building a hydrophobic barrier to the amniotic fluid, preventing maceration and protecting from infection by a large variety of antimicrobial compounds, antioxidant production such as alpha-tocopherol and acidification [2,8,[11][12][13][14][15][16]. Finally, the presence of surfactant proteins A and D in the VC -probably in timely association with lung surfactant production -are important factors of innate immunity and suggest a protective role in the context of chorioamnionitis and balanced transition to the ex utero environment [17,18].

Preterm Skin Is Sensitive to Handling
Differentiation of sensory nerve endings is documented at week 20 of gestation [19]. Preterm skin also contains elements of the hypothalamic-pituitary-adrenal axis for cortisol response to local stressors, e.g., discomforting procedures, irritants, temperature, and barrier disruption [20].

Barrier Maturation in Preterm Infants
Adaptive processes of the immature skin to the outside world include an accelerated cornification process of the SC. In term infants, the excess outermost layers of SC are shed in the first week of life (desquamation) and postnatal adaptation includes a rapid epidermal turnover, reduction of water loss, alterations in skin pH, such as an increase in acidity, as well as changes in sebaceous activity. In preterm infants, however, the desquamation process is enhanced due to the lack of protease inhibitors and enzyme regulators as compared to term infants [16]. The surface pH in preterm infants is > 6.0 and acidification is further delayed by the mandatory environmental humidity required to prevent water loss in the first weeks of life. The lacking "acid mantle" increases the risk for invasion of pathogens and may also play a role in wound healing as recovery rates after adhesive skin injury (via tape stripping) seem to be faster with an acidic skin pH of 5.5 as compared to pH 7.4 [21]. Filaggrin processing products -which are needed to maintain skin moisture and inhibition of pathogens [19] -are less expressed in preterm infants at birth compared to term infants but levels catch up at 3 months of chronological age. Moreover, cohesion between epidermis and dermis is reduced due to a smaller number of fibers and a wider interspace between fibers as compared to term infants. If the SC basal cell layer that generates the epidermis is damaged, healing results in wound contraction without formation of granulation tissue and significant scar formation [19]. In addition, immature skin has a higher expression of aquaporin 1 and 3 than mature skin in locations where they influence water transport and hydration. This might contribute to the Water 80% [2,9] Sebum lipid components and fetal corneocytes 10% Cholesterol (esters), ceramides, diacylglycerol, eicosanoids, and related oxylipin analogues, endocannabinoids, fatty alcohols, (free) fatty acids (including oleic, linoleic, and long-chain species), monoacylglycerol, phospholipids, sphingolipids, squalene, sterol esters, triacylglycerol, triglycerides, wax esters Fetal corneocytes are embedded in the hydrophobic lipid matrix and engulf most of the vernix water which results in high viscosity of VC [2,9,11,14] Proteins for structure and cellular function 10% Albumin, alpha 1-antitrypsin, annexin A1, arginase 1, apolipoprotein A1, A2 and C1, caspase 14 precursor, cystatin A, cytokeratin 1, 9, 10; epidermal secretory protein, fibronectin, glycosylasparaginase, haemoglobin α-,β-, and γ-chain; histone H3, H4; insulin-like growth factor binding protein 1; involucrin; keratin; lymphocyte antigen Ly6E; profilaggrin; proteasome subunit β type 2, psoriasin, squamous cell carcinoma antigen 1 and 2 (SCCA), pulmonary surfactant-protein A, B, D; RNase 4; serotransferrin, superoxide dismutase; thioredoxin; transthyretin; ubiquitin; uteroglobin related protein 1 and 2 (UGRP); Zn-alpha 2-glycoprotein [2,9,12,14,15] Cytokines interleukin-1 alpha and beta (IL-1α, IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor alpha (TNFα) [2, 9,  pathophysiology of increased transepidermal water loss (TEWL) [22]. Finally, preterm infants carry a higher risk for transdermal absorption of drugs and chemicals. Historical examples of accidental poisoning comprise the application of aniline dye with the consequence of methaemoglobinaemia, the use of iodine-containing antiseptics resulting in hypothyroidism [23] and high systemic levels of theophylline after transdermal application [24].

Perinatal Microbiome Establishment in Preterm Infants
An uneventful postnatal transition mainly relies on innate immune functions of the skin involving local immune cells orchestrating pro-and anti-inflammatory cytokine responses, specific antigen presenting cells, and early microbiome establishment [16]. It is well acknowledged that the priming of fetal development is dependent on metabolic signals derived from the maternal microbiome in utero [7]. Whether true colonization occurs before birth -beyond the context of premature rupture of membranes -continues to be a highly debated issue [25]. Recent advances in sequencing technologies have challenged the "sterile womb" paradigm since traces of nucleic acid or live bacteria were detected in the amniotic fluid and meconium. Given the highly sensitive detection methods, however, these findings might reflect contamination, and appropriate controls are missing [26]. In line with this, the successful generation of germ-free animals via caesarean section (CS) [27] supports the "sterile womb" hypothesis.

Maternal Microbiome Is a Crucial Influencer of Postnatal Skin Microbiome Development
Postnatal establishment of the microbiome has a strong bias toward maternal microbiome and is influenced by the mode of delivery, and most very low birth weight infants are born by CS [28]. The skin microbiome of CS delivered infants is dominated by maternal skin flora (Staphylococcus, Streptococcus, Corynebacterium, Proprionibacterium spp.) [29,30], while vaginally born infants acquire a Lactobacillus-dominated microbiome. These differences in skin microbiome may disappear after 1 month [29]. Initially, the composition of the microbiome is similar across all body sites, while site-specific maturation occurs after 4-8 weeks depending on gestational age and environmental exposures. Six weeks after delivery, the skin microbiome of mother and infant are more similar than their gut microbiome which diverges more rapidly [29,31]. Although microbiome in indoor hospital environments can match their occupants within hours [32,33], maternal strains are more likely to persist in infants compared to strains that are not maternally acquired -most likely due to share genetic, immunological, and environmental factors [34].

The Neonatal Skin Microbiome Is Dominated by Firmicutes
The preterm skin is a unique habitat with Firmicutes such as Coagulase-negative Staphylococcus (S.) spp.
(CoNS) being the pioneer champion colonizer. In the order of abundance, Actinobacteria (e.g., Corynebacterium spp.), Gammaproteobacteria (e.g., Escherichia coli, Enterobacter spp.), and Bacteroidetes (e.g., Prevotella spp.) also dominate the preterm skin colonization profile. In Table 3, the limited number of studies on preterm skin microbiome composition is summarized [29,31,[35][36][37]. Despite reduced richness and evenness of skin microbiome signatures in preterm infants, there is a marked overlap with patterns in term infants. In a bacterial source tracking model, it was demonstrated that the microbiome of each skin site was attributable to the infant's other body sites. The reciprocal exchange between body sites seems to have a higher impact on microbiome establishment than hospital environment [36]. Interestingly, identical colonizing strains in preterm infants may show different growth rates by site, e.g., the oral cavity as compared to skin [38]. Less is known on the site-specific role of skin pH, temperature, and composition of hair follicles -as resource-rich habitat for fungi and bacteria -for immune defense. First exploratory studies on the skin mycobiome in preterm infants found that fungal communities remain relatively static while bacterial colonization is highly dynamic [30].

The Neonatal Host Strongly Interacts with Skin Flora
In a physiological sense, host immunity and skin microbiome establishment develop in a synergistic fashion in order to establish a competent barrier function. Initial postnatal immunity is therefore primed towards "tolerizing" microbial antigens. In line with this, the transient influx of activated regulatory T cells is important for permissive colonization with skin commensals without eliciting aberrant inflammation [39] (Fig. 1). Reciprocally, the residential skin microbiome educates the cutaneous immune system. This was proven in germ-free mice models, where resident skin T cells exhibit an attenuated cytokine response when challenged by inflammatory stimuli [40]. Moreover, commensal microbes may occupy a niche and thereby hamper harmful microbes to gain DOI: 10.1159/000529026 access by competitive exclusion [41] or by alternative mechanisms, such as the ability of S. epidermidis, Corynebacteria, and the fungus Malassezia globosa to inhibit S. aureus virulence by secretion of proteases [42,43].

CoNS -Beneficial or Harmful?
Early colonization with commensal CoNS is an important aspect for the priming of physiological immune function. On the other hand, preterm birth is associated with several antenatal and postnatal events that may disturb microbiome establishment such as invasive measures, procedures, and antibiotics. In this context, skin colonizing CoNS may breach barriers and are the most frequent cause of nosocomial infections [44][45][46]. Likewise, CoNS abundance in the developing gut microbiome has been suggested a feature of dysbiosis preceding nosocomial infection [47,48]. We therefore hypothesize that the host-microbiome interaction of the skin and the virulence factors of the pathogen determine whether CoNS bacteria act as immunological educators in a milieu of "tolerance" or may become harmful pathogens. In the latter, CoNS causing sepsis in preterm infants are often methicillin-resistant (mecA gene) and frequently transmitted from the hospital environment [49]. In addition, most harmful CoNS are able to form a biofilm which does not allow the antibiotics to diffuse in host cells and provides immunological escape mechanisms [46]. On the other hand, the neonatal period is a distinct window to develop immunological tolerance to commensal CoNS. An early life colonization model demonstrated that the selective inhibition of neonatal regulatory T cell influx completely abrogated the tolerance to S. epidermidis [50]. In contrast, a strong pro-inflammatory response and impaired wound healing were demonstrated in adult mice which were exposed to S. epidermidis for the first time in adulthood [50]. The host may also provide selective pressure on certain microorganisms by presenting microbial-derived antigens through Langerhans cells or dermal dendritic cells and releasing antimicrobial compounds by keratinocytes, sebocytes, and fibroblasts [51]. For example, sebaceous glands can generate sapienic and linoleic acid in order to disrupt membranes in bacterial cells of pathogenic S. aureus. Commensal CoNS, however, are able to incorporate linoleic acid into their membranes in order to promote their own survival [52]. In line with that, S. epidermidis can produce lipoteichoic acid or TNF-α induced protein 3 which inhibit Toll-like receptor-3 mediated inflammatory injury as well as growth of S. aureus [53][54][55]. Niche shifts in the microbial ecosystem, such as aerobic or anaerobic conditions, may influence whether a microbe activates the immune system or evades recognition. Under anaerobic conditions, e.g., Corynebacterium acnes produces short-chain fatty acids which limit pro-inflammatory pathways in keratinocytes and prevent colonization with pathogenic S. epidermidis [56]. Different S. epidermidis strains (ST2, ST59) found in skin swabs of preterm infants may mutually interact in an antagonistic, outcompeting fashion by dominating sites at different timepoints. These strains differ in their ability to stick to plastic surfaces such as indwelling lines which is an important virulence factor of S. epidermidis [57].
The recent adoption of culture-independent techniques and next-generation sequencing enables researchers to gain a deeper insight into community composition and interactions of the involved skin microbes [58]. These new techniques have overcome former limitations of sequencing skin swabs of preterm infants which often contain a low microbial biomass. They may help gain a strain-level understanding of skin colonization dynamics and to design new modes of prevention [57].

Clinical Implications and Translation into Practice
Despite growing awareness of a key role of skin barrier function on short-term and potentially long-term outcome of preterm infants, evidence-based guidelines on skin care in this cohort are missing, and care practices vary significantly among centers [59].

Reduction of TEWL Is Achieved by Humidified Incubators
In preterm infants, TEWL inversely correlates with gestational age and is strongly influenced by external conditions, such as relative humidity, air temperature, or conventional phototherapy [1]. The optimum humidity of incubators for preterm infants -based on their level of maturity -remains to be defined. An increase in relative humidity decreases TEWL and heat loss but may delay skin barrier maturation at the same time [1,60]. Unitbased protocols for adjustments of incubator humidity therefore need to be provided. A pragmatic approach may be given by 80% humidity in infants <28 weeks' gestation in the first week of life and a decrease by 1-5% per day until a humidity of 40% is reached. This strategy of care inside the incubator should, however, not interfere with promoting maternal kangaroo-mother-care even in the most vulnerable babies [61,62]. Thermal stress needs to be avoided by servo control settings of the incubator temperature. Monitoring of the infant's peripheral and core temperature during incubator periods as well as skin-toskin care is important. Semipermeable wraps, adhesive or nonadhesive, may decrease TEWL and maintain skin integrity in preterm infants undergoing incubator care [59]. However, removal may strip outermost skin layers and even disrupt skin barrier function with a transient postremoval increase in TEWL at the affected sites [63]. With regard to bathing of preterm infants at the earliest of seven postnatal days when vital signs are stable and umbilical cord is already fell off, a frequency of bathing every 4 days has comparable effects on bacterial skin colonization and Neonatology 2023;120:295-307 DOI: 10.1159/000529026 risk of skin infection as bathing every 2 days but less risk for temperature instability [59]. With sponge bathing exposing more wet skin to the ambient air, tube bathing is superior in terms of temperature instability and hypothermia in preterm infants [64].

Emollient Use to Improve Barrier Integrity
In term infants, it is recommended not to remove the VC within the first 6 h of life. In addition, natural oils such as sunflower seed oil, coconut oil, and almond oil have long been used in infants for traditional oil massages and concomitant skin care in low and middle-income countries [65]. Meanwhile, these natural oils are increasingly used to reinforce epithelial integrity and have been ascribed several positive effects such as reduction of skin irritations, TEWL and heat loss as well as promotion of weight gain, shortening of the length of NICU stay, and prevention of infections in preterm infants [65][66][67][68][69][70]. The largest trial using sunflower seed oil as intervention in >13,000 newborn infants from India showed improvements in weight gain and also reductions in hospital admissions as well as neonatal morbidities in the per-protocol analysis [71]. In contrast, mustard seed oil may have harmful effects and cause skin barrier damage [68]. Randomized-controlled trials in different cohorts of clinically stable preterm infants reported improved SC hydration compared to untreated infants [66,68,69,72]. While some of these studies also found improved skin integrity as measured by Neonatal Skin Condition Scores [67,72] as well as accelerated skin maturation [72] or antibacterial properties provided by coconut oil [73], others documented delayed skin barrier maturation in infants with daily oil application [66]. It should be noted, however, that positive effects of emollients are mainly seen in studies performed in low-income countries. The general evidence of emollients is low due to large heterogeneity in study designs, sample size, and geographical location [74]. Research in highincome countries has focused on application of creams and ointments which were mostly petrolatum and panthenol-glycerin based [75,76]. These studies reported an increased risk of nosocomial infections in those infants randomized to prophylactic ointment therapy, including a large cohort of 1,191 extremely low birth weight infants of the Vermont Oxford Network Neonatal Skin Care Study Group [76]. Topical petrolatum ointment increased the risk of candidemia and CoNS infection in these preterm study populations [75,76]. So far, data on skin emollients therapy in which included periviable preterm neonates (22-24 weeks) are scarce [67,76].

Measures of Preventing Infection from Skin Flora
Key preventive measures to reduce the risk of infection from skin colonizing bacteria are often introduced as "hygienic bundles" including (1) avoidance of invasive strategies, (2) hand hygiene, (3) strict adherence to aseptic protocols prior to line insertion and line care, (4) prevention of understaffing and overcrowding, (5) early start of enteral feeds and the use of breast milk [43,77].
Antiseptics may effectively reduce skin colonization with pathogens, and chlorhexidine gluconate and octenidine dihydrochloride are among the most common antiseptics used in preterm and term neonates [78,79]. Given the risk of absorption and subsequent systemic effects as well as potential development of skin necrosis, alcoholbased antiseptics must be avoided. As there is no international consensus guideline on which antiseptic to use in preterm infants practices vary significantly. While many units use antiseptics locally [80], others have implemented bathing of neonates and have analyzed the effect of full-body cleansing with these antiseptics in preterm infants [81,82]. One study reported a significant decrease in the burden of gram-positive bacteria in moderate preterm infants in the first 72 h following chlorhexidine bathing [82], whereas a meta-analysis did not find conclusive evidence for any beneficial effect [81]. Of note, antiseptic bathing is associated with an increased risk of skin irritation as well as systemic absorption and subsequent toxic effects [44]. Other concerns involve potential disturbance of the skin pH and impairment of antimicrobial and immunological properties as well as elimination of commensal bacteria and subsequent disturbance of the skin microbiome [83]. Even when applied locally, side effects of antiseptics need to be observed cautiously. Besides skin irritation, which especially occurs in the most immature preterm infants, individual antiseptic regimens have the potential to promote the development of bacterial resistance to antiseptics and disinfectants. In an observational study of two NICUs in the UK and Germany, using chlorhexidine gluconate and octenidine, respectively, we recently demonstrated that long-term use of chlorhexidine for skin disinfection may select for chlorhexidine and octenidine tolerance in CoNS [84]. These data highlight further issues to be taken into consideration when developing future infection control policies [84].

Kangaroo Care and Skin Microbiome Development
Neonatal units worldwide strongly encourage kangaroo care by parents, allowing for regular skin-to-skin contact with parents or other family members [85]. However, the role of skin-to-skin contact on preterm infants' microbiome DOI: 10.1159/000529026 composition has not been studied in detail. An ongoing randomized controlled trial in preterm infants 28 0/7 to 32 6/7 weeks' gestation (immediate parent-infant skin-toskin study) is currently investigating the effects of kangaroo care implemented immediately after birth on cardiorespiratory stability as well as several other outcomes including microbiome profile and infection control [86].

New Targets for Skin Protection
Understanding developmental physiology of the skin with state-of-the art tools is of utmost importance for future targets of clinical trials. Complex natural compounds such as amniotic fluid, VC, and human milk should be regarded as part of the delicate continuum between the intrauterine world and the sudden exposure to the outside world. This continuum is prematurely disturbed in the context of preterm birth. A thorough investigation of the complex composition of these natural compounds in line with insights into strain colonization dynamics of preterm skin and the impact of microbial richness will guide future targets of personalized prevention strategies. In line with that, the impact of external influences such as the use of antiseptics, humidification, phototherapy but also systemic antibiotic treatment and the effect of antibiotic stewardship programs in the NICU needs to be elucidated. The introduction of artificial intelligence may enable to sharpen the risk profiles for skin-associated infections in preterm infants. Recent advances showing that the application of natural bacteria to the skin modulates skin microbiome composition, underscore the importance for the manipulation of target microbes by preserving the rest of the community. So far, mechanistic studies are largely limited to in vitro models using murine cells that differ immunologically and structurally from the human skin. Future efforts will focus on the development of promising organoid models and systems biology approaches to predict the effects of temporary modulation of the skin barrier at the microbiome, immunological, and metabolic level without the risk of adverse events.

Skin Health and Long-Term Outcome
A disturbed microbiome development has been associated with adverse short-term outcomes but correlation with long-term outcomes remains unknown. The EPIFLO-RE observational cohort study in France recently reported data on gut microbiome signatures of preterm infants <32 weeks' during the neonatal period characterized by a clustering-based method. The investigators linked cluster patterns with 2-year outcome (death or neurodevelopmental delay using a Global Ages and Stages questionnaire score). These first explorative data linked Enterococcus dominated microbiome patterns with impaired 2-year outcome [87]; however, large-scale studies in different cohorts are needed. Kangaroo care of vulnerable babies needs to be promoted as bidirectional microbial transmission between the mother and infant appears to be of paramount importance in the process of developmental skin immunity [88]. The role of microbiome inheritance from the father remains poorly documented. Psychological stress of parents and infants decreases epidermal cell proliferation, size, and density of corneo-desmosomes in infants' skin and affects differentiation which negatively impacts barrier function. Furthermore, psychosocial stress reduces AMP expression in the epidermis leading to more severe skin infections [89,90]. Hence, more research is needed to study the impact of kangaroo care on skin health and long-term outcome of preterm infants [62].

Multiple Organ-Axes of the Skin?
In adults, the establishment of a balanced skin microbiome is intimately connected to changes within the gut microbiome through the gut-skin axis [91]. This is an emerging concept proposing the interaction of the gut and skin through microbiome, metabolites, immune mediators, and dietary components. Disturbances of the gutskin axis can influence the manifestation of various diseases including acne and food allergies. An example of the gut-skin axis influencing disease in adults was described in a case series of two alopecia patients who had successful hair regrowth after receiving a fecal microbiome transplant for the treatment of recurrent Clostridium difficile infections [92]. Finally, the close embryological connection between the epidermis and the brain -both are ectodermal derivatives -may provoke more interaction between skin science and neurobehavioral research.
In conclusion, future investigations need to elucidate both the host-microbiome interaction and the impact of surrounding factors on skin health of preterm infants. New targets of protection might include recent knowledge on the complex composition of physiological substrates, e.g., the antimicrobial properties of amniotic fluid and VC. Systems biology and artificial intelligence can help personalize treatment approaches that aim for long-term health.