1

1 Review

2 Gut Microbiota during the First 1000 Days of Life and

3 Implications in the Development of Autoimmune Diseases: A
4 Literature Review
5 Alexandru Cosmin Pantazi1,2, Adriana Luminita Balasa1,2,*, Cristina Maria Mihai1,2, Tatiana Chisnoiu1,2,*, Vasile
6 Valeriu Lupu3, Mustafa Ali Kassim Kassim4, Larisia Mihai1,2, Corina Elena Frecus1,2, Ancuta Lupu3,*, Antonio
7 Andrusca1,2, Constantin Ionescu4, Viviana Cuzic1,2, Simona Claudia Cambrea5

8 1
Pediatrics, Faculty of Medicine, “Ovidius” University, 900470 Constanta, Romania; [email protected] (A.C.P.);
9 [email protected] (A.L.B.); [email protected] (C.M.M.); [email protected] (T.C.); [email protected] (L.M.);
10 [email protected] (C.E.F.); [email protected] (A.A.); [email protected] (V.C.)
11 2
Pediatrics, County Clinical Emergency Hospital of Constanta, 900591 Constanta, Romania
12 3
Pediatrics, “Grigore T. Popa” University of Medicine and Pharmacy, 700115 Iasi, Romania;
13 [email protected] (V.V.L.); [email protected] (A.L.)
14 4
Faculty of Medicine, “Ovidius” University of Constanta, 900470 Constanta, Romania; [email protected]
15 (M.A.K.K.); [email protected] (C.I.)
16 5
Infectious Diseases, Faculty of Medicine, “Ovidius” University, 900470 Constanta, Romania;
17 [email protected]
18 * Correspondence: [email protected] (A.L.B.); [email protected] (T.C.);
19 [email protected] (A.L.);
20

21 Abstract: The first 1000 days of life represent a critical window for gut microbiome development,
22 which is essential for immune system maturation and overall health. The gut microbiome
23 undergoes major changes during this period due to shifts in diet and environment. Disruptions to
24 the microbiota early in life can have lasting health effects, including increased risks of
25 inflammatory disorders, autoimmune diseases, neurological disorders, and obesity. Maternal and
Citation: To be added by editorial
26 environmental factors during pregnancy and infancy shape the infant gut microbiota. In this
staff during production.
27 article we will review how maintaining a healthy gut microbiome in pregnancy and infancy is
28 Academic Editor: Firstname important for long-term infant health. Furthermore, we briefly include fungal colonization and its
29 Lastname effects on the host immune function which are discussed as part of gut microbiome ecosystem.
30 Received: date Additionally, we will describe how potential approaches such as hydrogels enriched with
31 Revised: date prebiotics and probiotics, gut microbiota transplantation (GMT) during pregnancy, age-specific
32 Accepted: date microbial ecosystem therapeutics, and CRISPR therapies targeting the gut microbiota hold
33 Published: date potential for advancing research and development. Nevertheless, thorough evaluation of their
34 safety, effectiveness, and lasting impacts is crucial prior to their application in clinical approach.

Copyright: © 2023 by the authors.

Submitted for possible open access
publication under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).

3 Nutrients 2023, 15, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/nutrients

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35 The article emphasizes the need for continued research to optimize gut microbiota and immune
36 system development through targeted early life interventions.

37 Keywords: microbiota; autoimmune; colonization; children

38 1. Introduction

39 The first 1000 days of a child represent a critical window for the maturation of the
40 immune system and the establishment of gut microbiota. This simultaneous
41 development has caught the attention of immunology researchers, making it an area of
42 study that is both intriguing and captivating. The human being lives in harmony with
43 microbiota, which is made not only from bacteria but also from viruses, fungi, and
44 bacteriophages [1].These microorganisms are present throughout the human body in
45 different sites such as skin, mouth, nasopharynx, and intestine [1,2]. Identifying the
46 bacterial composition of prenatal meconium has been challenging due to the potential
47 for microbiological contamination [3–5]. It is widely documented that the process of
48 microbial colonization starts quickly after birth, as evidenced by numerous studies [6,7].
49 During the initial 1000 days of an infant's life, breastfeeding influences the intestinal
50 microbiome which is dominated by Bifidobacterium. This group of bifidobacteria
51 includes Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum
52 spp [8,9]. The microbiome undergoes significant transformations during two crucial
53 developmental phases early in life: from birth until weaning and from weaning to
54 adulthood. These modifications are driven by the diversification of the diet, resulting in
55 considerable changes to the microbial composition [10]. The variance in microbiome
56 configurations depends on the infant's genetics plus the infant's environmental
57 properties, including humidity, pH, nutrients, and oxygenation [1]. This article aims to
58 provide insights into the changes that occur in the infant's gut microbiota during the first
59 1000 days of life.

60
61 2. Development of Intestinal Microbiota During Pregnancy
62 During pregnancy, the mother's body experiences numerous physiological and meta-
63 bolic changes that intend to provide an optimal intrauterine environment for the devel-
64 oping fetus and to ensure proper growth, development, and accommodation [11]. These
65 gestational changes include endocrine, immunological, and metabolic modifications that
66 promote a pro-inflammatory state, leading to specific changes in the maternal microbiota
67 across different body sites, such as the vaginal area, intestine, and oral cavity [12]. The
68 development and growth of the fetus are significantly influenced by the in-utero environ-
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69 ment and fetal-maternal interactions [13]. According to a study, around 20% of stunting
70 cases are due to preterm birth, small-for-gestational age (SGA), or both, which have their
71 origins in the uterus [14]. Multiple studies indicate that the transmission of bacteria from
72 the mother influences the development and expansion of a healthy neonatal microbiome,
73 which can impact infant growth [15,16], immune system maturation [17–19], and neu-
74 rodevelopment [20,21]. Therefore, it is vital to maintain a healthy intestinal microbial
75 composition during pregnancy. It was found that the microbiota composition of pregnant
76 women is different in many aspects from non-pregnant women and changes throughout
77 pregnancy [22,23]. The gut microbiota in late pregnancy is reduced compared to the first
78 trimester, with a decrease in the number of Firmicutes and an increase in the presence of
79 Proteobacteria, Actinobacteria, and Streptococcus [22]. These shifts in the gut microbiota
80 can determine pregnant women more susceptible to developing gestational diabetes and
81 increase the incidence of macrosomia. Additionally, another study reported that the re-
82 duced alpha-diversity of the gut microbiota in late pregnancy is linked to adverse out-
83 comes such as low birth weight [24]. In the early days of life, the infant microbiota begins
84 to resemble that of an adult with a predominance of Firmicutes, Bacteroides [25], Bifi-
85 dobacterium, Parabacteroides, Escherichia, Shigella, Lactobacillus, and Prevotella [26].
86 During this period, the microbiota undergoes further diversification and becomes more
87 stable. This is an important phase, as it sets the stage for long-term health outcomes
88 [27,28].
89
90 3. First Gut Microbiota of Infant
91 Delivery plays a crucial role in establishing the initial colonization of the infant's gut
92 microbiota, the skin, mouth, and intestine of the newborns who were delivered vaginally
93 have been observed to contain Lactobacillus which is one of the most abundant micro-
94 organisms of the maternal vaginal flora [29]. After birth, the first inhabitants of the in-
95 fant's body come from the mother's microbiota found in the vaginal area, feces, breast
96 milk, mouth, and skin, as well as the surrounding environment [30–32]. During the initial
97 years of life, the microbial population that inhibits the gastrointestinal tract includes aer-
98 obic bacteria. However, in the years to come, these early colonizers are gradually dis-
99 placed by conventional anaerobic bacteria that eventually come to dominate the intesti-
100 nal microbiota [33]. Immediately after birth, the newborn’s intestinal microbiota is firstly
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101 overpopulated by the presence of Enterobacteriaceae and Staphylococcus [34], but it is

102 later replaced by Bifidobacterium and some lactic acid bacteria [35]. This Bifidobac-
103 terium-dominated microbiota, also known as "Bifidus flora," remains until the introduc-
104 tion of complementary food [36,37]. As the neonate approaches weaning, the relative
105 abundance of Bacteroides gradually increases, leading to the competitive exclusion of Bi-
106 fidobacterium within the intestinal microbiota. After weaning, the Bifidus flora in an in-
107 fant's gut is replaced by adult-type microorganisms, which mainly include bacteria such
108 as Bacteroides, Prevotella, Ruminococcus, Clostridium, and Veillonella [36]. By three
109 years of age, the gut microbiota becomes similar to that of an adult [38]. The gut micro-
110 biota's functions also change significantly before and after the introduction of solid foods.
111 In the first year of life, the early microbiota is enriched with bacteria that can utilize lac-
112 tate. Solid food promotes the growth of bacteria that can use a wider variety of carbohy-
113 drates, synthesize vitamins, and degrade xenobiotics [36,38,39]. By this period, the in-
114 fant's gut has been exposed to a variety of environmental factors such as breastfeeding,
115 type of delivery and exposure to antibiotics which can have a significant impact on the
116 further development of the infant's intestinal flora [40–42].
117
118 4. Factors Influencing the Intestinal Microbiota during the First 1000 Days of Life
119 4.1. Maternal factors
120 Infants with normal gestational age present a microbiota which develops more
121 quickly towards that of an adult when compared to preterm infants [43]. Premature in-
122 fants are characterized by lower diversity of the intestinal microbiota compared to full-
123 term infants [44]. Type of birth, especially vaginal delivery determines in newborns mi-
124 crobial species from the vaginal area of the mother and perianal regions, including Lacto-
125 bacillus, Prevotella, or Sneathia spp. [45], while infants who are delivered through a C-
126 section have limited exposure to these bacteria [46]. Maternal vaginal microbiota is an-
127 other important contributing factor, when the woman is pregnant, microbial variety
128 within the vaginal microbiota decreases in abundance, on the other hand, the abundance
129 of Lactobacillus spp. increases, potentially strengthening their defensive function [47,48].
130 Uterine bacteria may confer potential benefits to both the developing fetus and the new-
131 born. Specifically, these microbes may foster tolerance towards microorganisms that pro-
132 mote postnatal well-being, including those affiliated with the Lactobacillus genus [49].
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133 However, some studies suggest that dysbiosis that occurs at the time of pregnancy
134 leads to an increased risk of premature birth [50]. A relation between microbiota and
135 preterm births was observed [51]. The study found that women who delivered preterm
136 exhibited significantly different vaginal microbiota compared to women who gave birth
137 to full-term infants. Explicitly, women who gave birth to preterm had decreased levels of
138 Lactobacillus. These findings suggest that the composition of the vaginal microbiota may
139 be a useful indicator of the risk of preterm birth. Another possible factor is maternal
140 health status, this includes obesity, gestational diabetes, and inflammation, which can
141 participate in the instability of the constituents and mixture of the infant's intestinal mi-
142 crobiota which have been seen during the analysis of the newborn meconium, addition-
143 ally, maternal inflammation during pregnancy has been associated with an increased risk
144 of gut dysbiosis in the infants [52–54]. Maternal diet has been observed to influence the
145 microbiota, as a high-fat diet in mothers affected the initial colonization of bacteria [55].
146 When the mother consumed a high-fat diet, the meconium microbiome of the neonate
147 was significantly depleted in Bacteroides, and this depletion continued until the infant
148 was 6 weeks old [55].
149 The gut microbiota is significantly influenced by a maternal high-fat diet during
150 early life, not just maternal obesity [56]. Microbiota in maternal milk may come from the
151 mother's gut microbiota [57,58]. Therefore, if the mother's gut microbiota is imbalanced
152 due to an unhealthy diet, this could potentially be transferred to the milk and affect the
153 infant's gut microbiome during breastfeeding. However, there is currently limited re-
154 search on the long-term effects of maternal lifestyle and health during pregnancy and
155 breastfeeding on the infant's gut microbiota.
156 Exposure to antibiotics during pregnancy has a significant impact on the micro-
157 biome, leading to a reduction in microbial load and changes in composition, as well as
158 long-term effects on the development of the infant gut microbiome [59]. Another study
159 has shown that antibiotics can have an impact on the microbiome of breast milk, with
160 high levels of Bifidobacterium found in breast milk from mothers who did not receive an-
161 tibiotics [60]. Furthermore, antibiotic use during lactation can result in a decrease in the
162 microbial community of breast milk, including lactobacilli and bifidobacteria, and is asso-
163 ciated with lower bacterial diversity in breast milk [61,62].
164
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165 4.2. Environmental Factors

166 It is widely recognized that the mode of delivery plays a crucial role in determining
167 the initial bacterial colonization of infants [45]. Children born via C-section have an in-
168 creased risk for the development of specific diseases such as obesity, allergy, asthma, and
169 atopy possibly due to altered immune development [63,64]. Infants born vaginally are
170 colonized by bacteria that are normally located in the maternal vagina [45]. On the other
171 hand, infants born via C-section are colonized by bacteria that are similar to those
172 present on the maternal skin and in the oral cavity [46,65]. Furthermore, extensive studies
173 that have monitored the composition of microbiota in infants from birth until 2 years of
174 age suggested a correlation between Caesarean section (C-section) delivery and delayed
175 colonization of the Bacteroidetes phylum, as well as lower overall microbial diversity for
176 up to 2 years of age [66]. Moreover, at 7 years of age, differences were observed between
177 the microbiotas of infants born via C-section and those born vaginally [67]. The bacterial
178 microbiota is influenced by gestational age [68]. Premature infants are exposed to high
179 levels of antibiotics and frequently encounter longer hospitalization, mechanical ventila-
180 tion, and parenteral nutrition. These circumstances can cause permanent alterations to
181 the normal colonization and development processes of the gut microbiota [1,69]. Notably,
182 adopting a Mediterranean-style diet, supplementing with probiotics and prebiotics, and
183 nursing are all important for maintaining a varied gut microbiota and supplying new-
184 borns with needed nutrients can all assist to reduce the chance of developing allergies
185 [70].
186 Breastfeeding can be considered among the most crucial factors in the normal de-
187 velopment of infant microbiota. Type of nutrition, whether through breastfeeding or for-
188 mula, significantly affects the composition of gut microbiota in the first days of life. Hu-
189 man milk is rich in nutrients such as fats, proteins, and carbohydrates, together with im-
190 munoglobulins and endocannabinoids. Breast milk's constituents are responsible for se-
191 lecting the types of bacteria that colonize the gastrointestinal tract. For instance, immune
192 regulatory cytokines like TGF-β and IL-10 in breast milk facilitate the host immune sys-
193 tem's tolerance to intestinal bacteria and enhance the production of IL-10 in infants
194 [71,72]. The oligosaccharides in human milk (HMO) particularly galactooligosaccharide
195 (GOS) are incompletely broken down in the small intestine and instead primarily ferment
196 in the colon by Bifidobacterium, generating short-chain fatty acids [73,74]. Research by
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197 Matsuki et al. [34] has shown that the growth of Bifidobacterium in infants' gut leads to a
198 decrease in the number of HMOs present in feces, with a concurrent increase in acetic
199 acid and lactic acid. These results imply that HMOs have a beneficial impact on the de-
200 velopment of a Bifidobacterium-dominated gut microbiota [34]. It is recommended to en-
201 courage mothers to breastfeed their infants, in a cross-sectional study involving 1,008
202 mothers with children aged 9 to 14 months, factors influencing the duration of breast-
203 feeding were investigated [75]. The study findings revealed that perinatal education and
204 mother-baby friendly hospitals were associated with extended breastfeeding duration.
205 Notably, the conventional teaching of pregnant mothers and proactive engagement in
206 perinatal education emerged as crucial factors in promoting breastfeeding longevity. The
207 formula-fed infants show an abundance of Bacteroides, Clostridium coccoides and Lacto-
208 bacillus [76]. Other studies found that the fecal analysis from formula-fed infants is most
209 likely to contain staphylococci, Escherichia coli, and clostridia [77, 78]. However, in re-
210 cent times, milk formula has undergone enhancements, including the addition of certain
211 oligosaccharides, to enable the development of a microbiota rich in Bifidobacterium in in-
212 fants [79].
213 Administering antibiotics during the early stages of life can have a significant im-
214 pact on the development of gut microbiota. It causes a shift in the composition of gut mi-
215 crobiota towards a higher proportion of Proteobacteria and a lower proportion of Acti-
216 nobacteria populations [80,81]. This also results in reduced overall diversity of the in-
217 fant's microbiota and a selection of drug-resistant bacteria [82,83]. In a study by Tanaka
218 et al. [80] the impact of early postnatal exposure to antibiotics on the development of in-
219 testinal bacteria was observed in 26 infants. Among these infants, five were given antibi-
220 otics orally for the first four days of life, while three received antibiotics intravenously be-
221 fore delivery. The study discovered that the infants who were exposed to antibiotics had
222 fewer types of bacteria, with reduced colonization of Bifidobacterium and abnormal colo-
223 nization of Enterococcus during the first week. Additionally, an overgrowth of entero-
224 cocci and a lack of growth of Bifidobacterium were observed in the antibiotic-exposed
225 group. One month later, the antibiotic-exposed group was observed to have a higher
226 population of Enterobacteriaceae compared to the control group. These findings suggest
227 that exposure to antibiotics at an early stage of life can significantly impact the develop-
228 ment of neonatal intestinal microbiota [80]. Solid foods represent another influencing fac-
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229 tor for the composition of gut microbiota in infants, when solid foods containing indi-
230 gestible carbohydrates are introduced into an infant's diet, their gut quickly acquires a
231 functional gene pool that is largely dominated by carbohydrate metabolism genes, simi-
232 lar to that of an adult [38]. However, during the first year of life, the functional capabili-
233 ties of the infant gut microbiotas undergo significant changes. Initially, the microbiotas
234 are abundant in bacteria with genes that facilitate lactate utilization, while solid foods
235 lead to the growth of microbiotas with bacteria having genes that enable the utilization of
236 various carbohydrates, biosynthesis of vitamins, and degradation of xenobiotics [39,46].
237 Factors affecting the infant’s gut microbiota during the first 1000 days of life are de-
238 scribed in Table 1.
239
240 Table 1. Summary of the factors affecting the infant’s intestinal microbiota during the first 1000
241 days of life.
Description Factor Category
Full-term infants develop microbiota more quickly than Gestational Maternal
preterm infants, who have lower intestinal microbiota diver- Age
sity [43,44].
Vaginal delivery exposes newborns to maternal vaginal and Mode of De- Maternal
perianal microbes, while C-section limits this exposure [45,46]. livery
Microbial variety in the vaginal microbiota decreases during Maternal Maternal
pregnancy, potentially impacting the infant's microbiome Vaginal Mi-
[47,65]. Dysbiosis may increase the risk of premature birth crobiota
[51].
Obesity, gestational diabetes, and inflammation can impact the Maternal Maternal
infant's intestinal microbiota and increase the risk of gut dys- Health Status
biosis [52–54].
A high-fat diet can impact the initial colonization of bacteria in Maternal Diet Maternal
offspring, regardless of the mother's obesity status [55,56].
Antibiotics during pregnancy can reduce microbial load and Antibiotic Ex- Maternal
alter the composition of the infant's gut microbiome. They can posure Dur-
also impact breast milk microbiome [60–62]. ing Preg-
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nancy
Premature infants exposed to high levels of antibiotics and Gestational Environmen-
long hospital stays have altered gut microbiota development Age tal
[1,68,69].
Breastfeeding significantly impacts the formation of the gut Feeding Environmen-
microbiota, promoting the growth of Bifidobacterium [71,72]. Method tal
Formula feeding results in a different gut microbiota composi-
tion, but recent improvements aim to encourage Bifidobac-
terium growth [76–78].
Early exposure to antibiotics leads to altered gut microbiota Antibiotic Ex- Environmen-
composition, reduced overall diversity, and a selection for posure tal
drug-resistant bacteria [79–83].
The introduction of solid foods containing indigestible carbo- Introduction Environmen-
hydrates affects the infant's gut microbiota, leading to a func- of Solid tal
tional gene pool similar to an adult [38,39,46]. Foods
242
243 5. Intestinal Microbiota in Health and Disease
244 In recent years, the role of intestinal microecosystems has been widely studied in
245 health and disease, with numerous studies highlighting the significant impact that gut
246 microbiota can have on overall health. Recent studies have shown that intestinal micro-
247 biota plays a crucial role in immune system development [6,17,84–86]. By interacting
248 with the immune cells of the gut, the microbiota helps to establish immune tolerance and
249 prevent the development of inflammatory and autoimmune disorders [87]. The bacterial
250 species Bacteroides fragilis has been shown to promote the development of regulatory T
251 cells (Treg), which are essential for maintaining immune tolerance [88]. Short chain fatty
252 acids (SCFAs) produced by gut bacteria play a crucial role in linking the gut microbiota
253 and immune system maturation. A recent study examined fecal samples from 100 moth-
254 ers and their children to investigate changes in gut microbiota and SCFA composition as
255 children age [89]. The results revealed positive correlations between certain bacteria and
256 specific SCFAs, suggesting that these changes may have implications for preventing
257 early-life immunological disorders. A novel area of interest in this field is the impact of
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258 maternal microbiota on the offspring's immune system development. Recent studies
259 have suggested that vertical transmission of maternal microbiota, particularly during
260 vaginal birth, could be essential for the proper development of the neonatal immune sys-
261 tem [90]. However, further research is needed to elucidate the specific mechanisms by
262 which maternal microbiota influences the immune system and identify potential thera-
263 peutic targets for immune-related disorders. It was reported a possible relationship be-
264 tween heart failure and microbiota [91]. The correlation between heart failure and no-
265 table alterations in the gut microbiome was observed, with decreased bacterial diversity,
266 a heightened proliferation of potentially detrimental bacteria, and reduced production of
267 short-chain fatty acids by gut bacteria. Additionally, it highlighted the potential rise in in-
268 testinal permeability among individuals with heart failure, enabling the entry of bacterial
269 products into the bloodstream.
270 Necrotizing enterocolitis is a condition marked by inflammation and necrosis of the
271 intestine, which can advance to systemic infection, multiorgan failure, and eventual mor-
272 tality [92]. A risk factor in the pathogenesis of this disease is the intestinal microbiota
273 [93]. Various microorganisms have been labelled as probable contributors to the patho-
274 genesis of necrotizing enterocolitis. These include Gram-negative enteric bacteria, as well
275 as Gram-positive bacteria like. Certain viruses such as Rotavirus, Norovirus, Cy-
276 tomegalovirus, and Echovirus have also been associated with the condition. Addition-
277 ally, fungi such as Candida spp. may also play a role in the pathogenesis of necrotizing
278 enterocolitis [94]. The intestinal microbial composition of infants who develop necrotiz-
279 ing enterocolitis (NEC) differs significantly from those who do not which has been seen
280 repeatedly in the literature [95,96]. Another study has shown that alterations in fecal bac-
281 teria can be detected as early as 72 hours before the onset of NEC [97]. Allergic diseases
282 have been associated with dysbiosis in children [98–102]. Recent studies have empha-
283 sized the significant role of the gut microbiota in influencing the pathogenesis of respira-
284 tory pathology, by the "gut-lung axis" phenomenon [103–105], while other studies have
285 explained the connection between intestinal microbiota and allergies [106]. A study
286 showed that fungi play a role in gut microbiome ecology and host immune function
287 [107]. Fungal colonization causes significant changes in the bacterial community and has
288 a separate effect on immune development. Although fungi alone did not cause colitis, co-
289 colonization with bacteria and fungi increased inflammation in the colon. The findings
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290 suggest that therapies aimed at modulating early-life microbiomes should consider the
291 role of fungi. Emerging evidence suggests that gut microbiota may also have a significant
292 impact on the development of neurological disorders during early life [108]. The gut-
293 brain axis is a unique communication pathway that connects the central nervous system
294 with the gastrointestinal tract, and the gut microbiota is thought to play a key role in
295 modulating this communication [109].
296 Altered gut microbiota composition has been observed in children with autism
297 spectrum disorder (ASD), and some researchers have proposed that dysbiosis could con-
298 tribute to the behavioral symptoms of ASD [110]. A novel area of interest is the potential
299 use of microbiota-targeted interventions, such as fecal microbiota transplantation, in the
300 treatment of ASD and other neurological disorders [111]. Nonetheless, further research is
301 needed to establish the safety and efficacy of such interventions. The intestinal micro-
302 biota has also been implicated in the development of obesity during early life [11,41,112].
303 An imbalance of the intestinal microecosystem characterized by a diminished abundance
304 of Bacteroides and an increased abundance of Firmicutes, is correlated with increased
305 adiposity and weight gain [113]. A recent study has highlighted the link between heart
306 failure and gut microbiome changes [91]. The report detailed the correlation between
307 heart failure and substantial changes in gut microbiome, characterized by a reduction in
308 the fundamental intestinal microbes, a decline in bacterial variety, an upsurge in the pro-
309 liferation of possibly harmful bacteria, and a decreased production of short-chain fatty
310 acids by intestinal bacteria. Moreover, children suffering from heart failure could face in-
311 creased permeability in their intestines, which paves the way for bacterial byproducts to
312 penetrate into the bloodstream and further exacerbate the progression of the disease.
313 Considering the pivotal function of microbiota in immunological responses, it can be
314 postulated that the phenomena of dysbiosis might engender conditions more propitious
315 for the proliferation and dispersion of deleterious microorganisms, such as Shigella spp.
316 This hypothesis garners support from a retrospective investigation spanning a ten-year
317 period that encompassed 376 Shigella-afflicted patients [114]. The research discerned a
318 heightened susceptibility to Shigella spp. in children below the age of five years. Further,
319 it underscored the substantial role played by environmental factors such as ambient tem-
320 perature, humidity, and precipitation in modulating the prevalence of Shigella spp. infec-
321 tions. These environmental factors may trigger dysbiosis, leading to increased vulnerabil-
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322 ity of the child's intestine to Shigella species. A recent review showed that while the exact
323 mechanisms are still not entirely clear on how post-infectious irritable bowel syndrome
324 (PI-IBS) starts, alterations in gut microbiota, immune responses, and gut-brain interac-
325 tions likely contribute to the development of PI-IBS [115], suggesting the connection be-
326 tween microbiota and PI-IBS.
327
328 6. Conventional Treatment for Mitigating Dysbiosis in Infants
329 Consuming live microorganisms, referred to as probiotics, in appropriate quantities
330 has been shown to offer health benefits to the host [116]. These products have demon-
331 strated efficacy in preventing and addressing a variety of gut microbiota-related disor-
332 ders, such as antibiotic-associated diarrhea [117], inflammatory bowel disease [118], and
333 allergies [119,120]. A meta-analysis which reviewed ten studies on allergic rhinitis [120],
334 found that five of them reported a substantial reduction in symptom scores and an en-
335 hancement in the quality of life for patients with allergic rhinitis, suggesting a positive
336 impact of probiotics. Bifidobacterium and Lactobacillus, essential microorganisms during
337 early childhood, appeared effective in treating allergic rhinitis in the study. Further re-
338 search is needed to substantiate these findings. Prebiotics, non-digestible components
339 found in food like fibers and carbohydrates, selectively encourage the growth and activ-
340 ity of the human gut's micro-ecosystem [121]. These components have been investigated
341 as a possible intervention for gut microbiota-related pathologies. Prebiotics have also
342 demonstrated a positive effect in reducing the risk of allergies; for example, a study on
343 infants showed that prebiotic supplementation lowered the risk of atopic dermatitis by
344 50% compared to placebo treatment [122].
345 Fecal microbiota transplantation is a technique that can normalize the gut micro-
346 biota of affected individuals by replacing it with a healthy individual's microecosystem
347 [123]. This method has proven effective in treating various conditions, such as recurrent
348 Clostridium difficile infection [124], inflammatory bowel disease [125], and even obesity
349 [126]. Breastfeeding has been identified as a factor that significantly impacts the develop-
350 ment of microbiota during an infant's early life, as it provides essential nutrients
351 [127,128]. Multiple studies have reported that Bifidobacterium [97] and Enterobacteri-
352 aceae [129] are the most prevalent types of intestinal flora in breastfed infants. To pro-
353 mote the diversity of this micro-ecosystem, it is recommended that expecting mothers
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354 breastfeed their children and receive education on this practice. A cross-sectional study
355 examining factors influencing breastfeeding duration in a sample of 1,008 mothers with
356 children aged 9 to 14 months found that perinatal education and mother-baby friendly
357 hospitals increased breastfeeding duration [74]. Conventional teaching of expecting
358 mothers and active pursuit of perinatal education were critical factors. Sustained educa-
359 tion before and after birth is necessary to create a favorable environment for exclusive
360 breastfeeding. Conversely, formula-fed infants exhibited an increased abundance of Bac-
361 teroides, Clostridium coccoides, and Lactobacillus [75]. Other studies have found that fe-
362 cal samples from formula-fed infants are more likely to contain Staphylococcus, Es-
363 cherichia coli, and Clostridia [76,77]. Finally, the correlation between microbiota and di-
364 gestive disorders such as esophageal cancer, gastritis, celiac disease, Helicobacter Pylori
365 infection and also systemic lupus erythematosus has been found associated [129–133].
366
367 7. Exploring Promising Interventions for Mitigating dysbiosis in the first 1000 days
368 of life
369
370 7.1. Prebiotic and probiotic-enriched hydrogels
371 Hydrogels are three-dimensional networks of hydrophilic polymers capable of re-
372 taining large amounts of water [134]. They have been explored as drug-delivery systems
373 for various applications [135]. One of these applications is the use of these hydrogels as a
374 carrier for probiotics which has been found in the literature on many occasions [136,137].
375 A promising intervention would involve the development of hydrogels containing prebi-
376 otics and probiotics to promote the growth of beneficial gut bacteria and counteract nega-
377 tive influences on the microbiota. The viability of these beneficial bacteria can be compro-
378 mised by various factors, including the acidic environment of the gastric juice which has
379 a severe impact on bacteria [138,139]. By encapsulating probiotic cells within a hydrogel,
380 a physical barrier can be created to protect them from harmful environmental conditions,
381 thereby enhancing their survival rate. Notably, hydrogels have been found to improve
382 the viability of probiotic bacteria not only under gastrointestinal conditions but also dur-
383 ing storage at various temperatures or exposure to heat treatment [140]. Thus, the utiliza-
384 tion of hydrogels appears to be a viable and optimistic strategy to amplify the effective-
385 ness of probiotics, thereby securing their advantageous impacts on the health of infants.
28 Nutrients 2023, 15, x FOR PEER REVIEW 14 of 27
29

386 However, before the use of prebiotic- and probiotic-enriched hydrogels can become a vi-
387 able intervention, several important factors must be considered such as the biocompati-
388 bility and safety of these hydrogels must be thoroughly evaluated to ensure that they do
389 not pose any risks to the developing infant.
390
391 7.2. Gut microbiota transplantation (GMT) during pregnancy
392 Another promising novel intervention for optimizing the maternal gut microbiota
393 during pregnancy is gut microbiota transplantation (GMT). While fecal microbiota trans-
394 plantation (FMT) is a well-established treatment for recurrent Clostridioides difficile in-
395 fection (CDI) in adults [141–144], GMT during pregnancy could represent a promising
396 approach to enhance pregnancy outcomes and minimize the risk of adverse effects on the
397 infant's microbiota. A study conducted by Saeedi et al. [145] have elaborated on the
398 promising effect of FMT. However, according to guidelines published by the Interna-
399 tional Consensus Conference on fecal microbiota transplantation, it stated that FMT
400 should be avoided unless it was strictly needed for pregnant women with severe CDI in-
401 fection in whom standard therapy has been found ineffective [123]. The lack of data re-
402 garding the safety and efficacy of GMT during pregnancy raises concerns about the risks
403 associated with this intervention. The safety of GMT during pregnancy must be thor-
404 oughly evaluated to ensure that it does not pose any risks to the health of the mother or
405 developing fetus. Additionally, the potential long-term effects of GMT on the infant's mi-
406 crobiota and overall health must be carefully studied.
407
408 7.3. Microbial ecosystem therapeutics (MET) for infants
409 Microbial ecosystem transplantation (MET) is an advanced and more precise approach to
410 fecal microbiota transplantation (FMT). Unlike FMT, which involves the transfer of an
411 entire fecal sample. MET involves the purification and cultivation of select beneficial bac-
412 teria from the sample which results in a well-defined and stable microbial ecosystem that
413 can be transplanted into the recipient [142,143]. An innovative application of MET could
414 be the development of age-specific formulations for infants during the first 1000 days of
415 life. These formulations could be tailored to meet the specific needs of the infant's gut mi-
416 crobiota, promoting healthy development, and preventing or treating dysbiosis. By sup-
417 porting immune and metabolic development, these age-specific MET formulations could
30 Nutrients 2023, 15, x FOR PEER REVIEW 15 of 27
31

418 have long-term benefits for infant health. Further investigation is needed to assess the
419 safety, efficacy, and long-term consequences of MET in infants. However, the potential
420 benefits of this approach make it a promising avenue for future research in the field of in-
421 fant gut microbiota therapeutics.
422
423 7.4. Gut microbiota targeted CRISPR therapies
424 Recent advances in gene-editing technologies have paved the way for novel interventions
425 that specifically target harmful bacteria in the gut microbiota without affecting beneficial
426 ones. The CRISPR/Cas9 system is a promising candidate for developing gut microbiota-
427 targeted therapies [144]. The idea behind this approach is to engineer CRISPR systems to
428 target and eliminate specific bacteria, thus promoting healthy gut microbiota in the first
429 1000 days of life. This approach could potentially reduce the risk of various health issues
430 associated with dysbiosis, such as metabolic disorders and immune dysfunction. Further-
431 more, research is ongoing on editing the microbiota using CRISPR [145,146]. However,
432 several challenges need to be overcome before CRISPR-based therapies can be translated
433 into clinical practice, including the efficient delivery of the CRISPR systems to the gut,
434 specificity of targeting, and off-target effects. Further research is needed to determine the
435 feasibility and safety of this approach in humans. Some interventions for mitigating dys-
436 biosis in the first 1000 days of life are described in Table 2.
437
438
439 Table 2. Summary of the promising interventions for mitigating dysbiosis in the first 1000 days of
440 life.
Challenges/Considera- Potential Benefits Description Intervention
tions
Thorough evaluation of Enhanced survival rate of Development of hydrogels Prebiotic and
biocompatibility and probiotics, improved ef- containing prebiotics and probiotic-en-
safety of hydrogels, rig- fectiveness of probiotics, probiotics to promote the riched hydro-
orous testing in clinical promotion of healthy in- growth of beneficial gut bac- gels
trials testinal microbiota teria and counteract negative
influences on the microbiota
32 Nutrients 2023, 15, x FOR PEER REVIEW 16 of 27
33

Lack of data on safety Potential enhancement of Transfer of select beneficial Gut micro-
and efficacy, careful pregnancy outcomes, po- bacteria to optimize maternal biota trans-
study of potential long- tential minimization of gut microbiota during preg- plantation
term effects on infant's the risk of adverse effects nancy and minimize the risk (GMT) during
microbiota and overall on infant's microbiota of adverse effects on the in- pregnancy
health fant's microbiota
A thorough investiga- Potential support of im- Tailored formulations for in- Microbial
tion of safety, efficacy, mune and metabolic de- fants during the first 1000 ecosystem
and long-term conse- velopment, potential days of life to meet specific therapeutics
quences long-term benefits for in- needs of infant gut micro- (MET) for in-
fant health biota, promoting healthy de- fants
velopment and preventing or
treating dysbiosis
Efficient delivery of Reduced risk of health is- CRISPR/Cas9 system engi- Gut micro-
CRISPR systems to gut, sues associated with dys- neered to target and eliminate biota targeted
specificity of targeting, biosis, such as metabolic specific harmful bacteria, pro- CRISPR thera-
and off-target effects disorders and immune moting healthy gut micro- pies
need to be overcome dysfunction biota
441
442 8. Conclusion
443 In conclusion, the first 1000 days of life are crucial for the development of the in-
444 fant's gut microbiota and immune system. The microbiome undergoes significant trans-
445 formations during this period, driven by changes in diet and environmental factors. Dis-
446 turbances to the microbiota during this window can have long-term effects on the infant's
447 health, including the development of inflammatory and autoimmune disorders, neuro-
448 logical disorders, and obesity. Maternal factors, including gestational age, mode of deliv-
449 ery, maternal vaginal microbiota, maternal health status, maternal diet, and exposure to
450 antibiotics during pregnancy and lactation, influence the infant's gut microbiota. Envi-
451 ronmental factors, such as mode of delivery, gestational age, and breastfeeding, also play
452 a role. Research continues to explore early-life interventions to optimize gut microbiota
453 and immune system development. Maintaining a healthy intestinal microbial community
 34 Nutrients 2023, 15, x FOR PEER REVIEW 17 of 27
35

454 during pregnancy and infancy is vital for the long-term health outcomes of the infant. Po-
455 tential novel approaches, such as prebiotic- and probiotic-enriched hydrogels, GMT dur-
456 ing pregnancy, age-specific MET formulations, and gut microbiota-targeted CRISPR ther-
457 apies, offer promising avenues for future research and development. However, rigorous
458 investigation of their safety, efficacy, and long-term consequences is necessary before
459 their implementation in clinical practice. Further research is required to fully compre-
460 hend the complicated relationship between the intestinal microecosystem and the devel-
461 oping infant and to develop effective interventions to promote optimal gut health during
462 this critical window.
463
464 Author Contributions: All authors contributed equally to this review. All authors have read and
465 agreed to the published version of the manuscript. C.M.M, T.C., V.V.L, M.A.K.K, L.M., C.E.F.,
466 A.L., A.A., C.I., V.C., S.C.C. and A.L.B. contributed equally with A.C.P. to this article
467 Funding: This research received no external funding.
468 Institutional Review Board Statement: Not applicable
469 Informed Consent Statement: Not applicable.
470 Data Availability Statement: Data sharing not applicable.
471 Conflicts of Interest: The authors declare no conflict of interest.

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