**1. Introduction**

Although the cause of the growing prevalence of allergic diseases remains unclear, the Old Friends Hypothesis (also called the Biodiversity Hypothesis) has recently been proposed [1]. The hypothesis states that the development of many diseases, including allergy, might be attributed to a lack of exposure to the "right" bacterial strains. Assuming this to be the case, it may be possible that identifying such bacteria could halt the allergic march and foster the development of novel prevention and treatment methods for allergic diseases.

The composition of the human microbiome is not only characteristic of each individual, but also evolves over time [2–7]. In various parts of the child's body, the microbiome begins to form and differentiate within the first six weeks post natum [2,3]. The most pronounced changes in the gut microbiome are usually observed during first two or three years of

**Citation:** Ło´s-Rycharska, E.; Goł˛ebiewski, M.; Sikora, M.; Grzybowski, T.; Gorzkiewicz, M.; Popielarz, M.; Gawryjołek, J.; Krogulska, A. A Combined Analysis of Gut and Skin Microbiota in Infants with Food Allergy and Atopic Dermatitis: A Pilot Study. *Nutrients* **2021**, *13*, 1682. https://doi.org/10.3390/nu13051682

Academic Editor: Carla Mastrorilli

Received: 24 March 2021 Accepted: 12 May 2021 Published: 15 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

human life [4,5,8]. Following this, the composition of the gut microbiome remains relatively stable, unless significant environmental changes occur [5]. The early gut microbiome is believed to be shaped by a range of factors, including maternal race-ethnicity, age, diet, antibiotic treatment, marital status, the mode of delivery, health complications after birth, infant feeding method, solid introduction, probiotics intake, exposure to tobacco smoke and pets [2,4,6,7].

Similarly, the skin microbiota evolves over the years according to changes in skin structure and function [9], becoming similar to that of adults by the age of 12–18 months [10]. A possible critical window during which microbial-based intervention would be possible is early infancy (i.e., the first six months of life) [10–12]. The composition of skin microbiota is site-specific and, again, depends on a range of factors, including local conditions such as invaginations, pockets, niches and surfaces, as well as moisture, sebum secretion, temperature and exposure to external factors [9,10]. In the neonatal period, the mode of delivery appears to play an important role in shaping the skin microbiome [2]. The microbiome, considered a complex biocenosis where various organisms interact, influences the immune system in a multifaceted way, modulating both innate and adaptive immunity, and shaping allergy development through interactions with the host genome [13–15].

The gut and the skin are crucial barriers through which environmental factors interact with the human body; the two compartments are also massively colonized with distinct microbial communities. Furthermore, they constitute complex immune organs that are fully integrated into the overall immune system. Proper skin and gut functioning is essential for homeostasis, and decreases the risk of allergy. Hence, exposure to the many environmental factors that shape the microbiome of the gut and skin epithelium can alter the epithelial surfaces and thus drive the type 2 immune response, which is known to underlay most, if not all, atopic diseases. Many studies indicate the existence of a gut–skin axis, which is modulated by microbiota and its metabolites [16–18].

Generally, the first symptoms of allergy involve the skin and/or the gastrointestinal tract [19], manifesting as atopic dermatitis (AD) and/or food allergy (FA). These disorders are difficult to diagnose in early infancy, as there are no specific markers, and diagnosis is based mainly on their symptoms. Although previous studies have yielded varying results, it has been found that gut microbiota differed between otherwise healthy subjects and those who were food sensitized (FS) or food allergic (FA) [20,21]. It has been shown that the composition of the microbiota in the neonatal period may be associated with the development of allergies up to the age of one year. In later life, alpha-diversity, betadiversity, and the richness of individual bacteria are shaped differently in non-allergic and allergic children [3]. It has also been shown that the microbiome structure at three months is associated with the development of sensitization up to one year of age [11]. It also appears that the microbiome of the gut in infancy is a prognostic factor of allergy resolution by eight years of age [12].

The bacterial community structure has also been found to differ between IgE-mediated and non-IgE-mediated FA children [22], and differences can be seen between the microbiota associated with specific food allergies [23–25]. Fieten et al. report no differences in microbial diversity between FA and non-FA children; however, a fecal microbial signature that discriminated between the presence and absence of FA was found in children with AD [26].

Similarly to the gut microbiome, the skin microbiota has also been found to differ between patients with AD and those without [9], and other studies have reported decreased skin microbiome diversity in AD patients [16,27]. The significance of the skin microbiome in AD pathogenesis was confirmed by Laborel-Preneron et al., who showed that *Staphylococcus aureus* colonizing atopic skin promoted inflammation in children aged one to three years, while commensal *S. epidermidis* strains might mitigate this effect [28]. Similarly, Kong et al. found that the abundance of *Staphylococcus* (particularly *S. aureus*) was greater in children with AD, and that microbial diversity increased over the course of treatment [29]. Interestingly, while the development of AD appears to be related to colonization by *S. aureus*, it has also been proven that exacerbations of skin lesions are connected to a reduction

in skin microbiome diversity, while improvement is not directly related to a reduction in *S. aureus*, but rather to an increase in bacterial diversity [16]. In addition, prospective studies have identified changes in gut and/or skin microbiota in children, even prior to the development of food sensitivity (FS), FA or AD, with changes being apparent as early as in the third month of life [11,16,30–33]. It has also been proposed that decreased *Staphylococcus* richness in the skin microbiome during early infancy might be a prognostic factor of AD development before 12 months of age, and that colonization by commensal *Staphylococci* might protect against eczema [32].

Current evidence suggests that while the gut microbiota may demonstrate a reduction in microbial diversity before AD onset, no such relationship has been noted for the skin microbiota [16,23]. Furthermore, although gut dysbiosis has been described in AD [23,27,34], it is not clear whether the modulation of gut microbiota can impact skin microbiota and vice versa. The role of the skin microbiome in FA development is also unknown.

To gain a more holistic understanding of how the microbiome of the human body interacts with the immune system, and how it can influence the development of FA and/or AD, it is necessary to study the skin and gut together as part of one combined study. The present study is the first such study to compare the microbiota of two compartments, i.e., gut and skin, in young infants with early onset of FA and/or AD. Indeed, of all the studies regarding the relationship between microbiota and allergy published to date, only one [35] has analyzed multiple compartments.

The present study characterizes the alpha and beta diversity of the fecal, i.e., gut, and skin bacterial communities from infants with FA and/or AD and compares them with those of healthy control subjects.

We hypothesize that:

**Hypothesis 1.** *Gut and skin alpha diversity differ between children with an allergy (FA and/or AD) and those without;*

**Hypothesis 2.** *The communities within a single compartment differ according to the clinical status of the child;*

**Hypothesis 3.** *OTUs characteristic for each compartment-clinical status communities exist;*

**Hypothesis 4.** *Relationships exist between the gut and skin microbiota, and these differ with regard to clinical status.*

To confirm these hypotheses, V3-V4 16S rRNA gene libraries were generated and sequenced, and the resulting sequences were analyzed bioinformatically.
