Keystone Species Restoration: Therapeutic Effects of Bifidobacterium infantis and Lactobacillus reuteri on Metabolic Regulation and Gut–Brain Axis Signaling—A Qualitative Systematic Review (QualSR)

Abstract

Background: The human gut microbiome—a diverse ecosystem of trillions of microorganisms—plays an essential role in metabolic, immune, and neurological regulation. However, modern lifestyle factors such as antibiotic overuse, cesarean delivery, reduced breastfeeding, processed and high-sodium diets, alcohol intake, smoking, and exposure to environmental toxins (e.g., glyphosate) significantly reduce microbial diversity. Loss of keystone species like Bifidobacterium infantis (B. infantis) and Lactobacillus reuteri (L. reuteri) contributes to gut dysbiosis, which has been implicated in chronic metabolic, autoimmune, cardiovascular, and neurodegenerative conditions. Materials and Methods: This Qualitative Systematic Review (QualSR) synthesized data from over 547 studies involving human participants and standardized microbiome analysis techniques, including 16S rRNA sequencing and metagenomics. Studies were reviewed for microbial composition, immune and metabolic biomarkers, and clinical outcomes related to microbiome restoration strategies. Results: Multiple cohort studies have consistently reported a 40–60% reduction in microbial diversity among Western populations compared to traditional societies, particularly affecting short-chain fatty acid (SCFA)-producing bacteria. Supplementation with B. infantis is associated with a significant reduction in systemic inflammation—including a 50% decrease in C-reactive protein (CRP) and reduced tumor necrosis factor-alpha (TNF-α) levels—alongside increases in regulatory T cells and anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1). L. reuteri demonstrates immunomodulatory and neurobehavioral benefits in preclinical models, while both probiotics enhance epithelial barrier integrity in a strain- and context-specific manner. In murine colitis, B. infantis increases ZO-1 expression by ~35%, and L. reuteri improves occludin and claudin-1 localization, suggesting that keystone restoration strengthens barrier function through tight-junction modulation. Conclusions: Together, these findings support keystone species restoration with B. infantis and L. reuteri as a promising adjunctive strategy to reduce systemic inflammation, reinforce gut barrier integrity, and modulate gut–brain axis (GBA) signaling, indicating translational potential in metabolic and neuroimmune disorders. Future research should emphasize personalized microbiome profiling, long-term outcomes, and transgenerational effects of early-life microbial disruption.

1. Introduction

The human gastrointestinal tract harbors a highly diverse and dynamic microbial ecosystem comprising approximately 100 trillion microorganisms spanning over 1000 species [1,2]. These microbes engage in symbiotic interactions with the host, supporting essential physiological processes such as nutrient absorption, energy harvest, maintenance of epithelial barrier function, and immune modulation [2,3]. The concept of the microbiome as a “second genome” reflects its vast metabolic capacity and integral role in human health. However, modern environmental and behavioral exposures—such as Westernized diets, widespread antibiotic use, increased rates of cesarean delivery, and reduced breastfeeding—have led to significant perturbations in microbial composition and function [4,5,6,7,8]. This phenomenon of dysbiosis is typically characterized by decreased microbial diversity, loss of beneficial species, and enrichment of opportunistic or pro-inflammatory taxa [5,9].

Metabolic disorders (e.g., obesity, type 2 diabetes), cardiovascular diseases (e.g., hypertension, atherosclerosis stenosis), autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis), allergic conditions (e.g., asthma, atopic dermatitis), and neuropsychiatric conditions (e.g., depression, autism spectrum disorders) are rising global health challenges with substantial personal, societal, and economic burdens. These chronic, non-communicable diseases (NCDs) have been strongly linked with dietary and lifestyle patterns that promote persistent low-grade inflammation and disruption of the gut microbial ecosystem—also known as microbial dysbiosis [3,4,10]. A growing body of evidence supports the role of the gut–brain–microbiota axis, a complex bidirectional communication network between the central nervous system and the gut microbiome, in regulating host metabolic, immune, and neurobehavioral functions [10,11,12]. This paradigm shift has placed the gut microbiota at the center of public health discussions regarding the prevention and management of chronic disease.

Notably, industrialized societies demonstrate marked shifts in gut microbiome structure compared to traditional, non-industrialized populations [13,14]. Traditional microbiomes are typically enriched in microbial taxa that support fiber fermentation, short-chain fatty acid (SCFA) production, and oxidative stress resistance—functions that are often impaired or absent in Western populations [15,16,17,18]. SCFAs such as acetate, propionate, and butyrate are microbial fermentation products that serve as signaling molecules, energy substrates for colonocytes, and modulators of immune and metabolic pathways [19,20]. Their biosynthesis is dependent on complex microbial networks: acetate production involves the Wood–Ljungdahl pathway; propionate is primarily synthesized by Bacteroidetes via the succinate pathway; and butyrate is generated predominantly by Firmicutes via the butyryl-CoA–acetate CoA-transferase route [21,22]. The disruption of these pathways contributes to intestinal permeability, systemic inflammation, insulin resistance, and altered neurobehavioral signaling—all hallmarks of metabolic and mental health disorders [19,20].

Nutrient cycling processes provide compelling evidence for the disproportionate ecological impact of rare microbes. The keystone species model, a fundamental theoretical framework in microbial ecology, demonstrates how certain microbial taxa can exert outsized influence on ecosystem function despite their low abundance. These keystone microorganisms perform essential metabolic processes that fundamentally shape the composition, stability, and overall metabolic capacity of entire microbial communities. A prime example of this phenomenon is observed in freshwater environments, where rare green and purple sulfur bacteria function as highly active keystone species, driving critical nitrogen and carbon cycling processes that far exceed what their minimal abundance would suggest [23,24]. Loss of these keystone species can cause cascading functional breakdowns within the microbiome, leading to a loss of homeostasis and emergence of disease phenotypes [23,25]. Keystone taxa are particularly vulnerable to the cumulative impact of modern lifestyle factors, including antibiotic exposure, nutrient-poor diets, cesarean delivery, formula feeding, and chronic psychosocial stress [6,7,8,26]. Moreover, longitudinal data suggest that early-life disruptions to microbial colonization may exert long-term effects on immune programming, metabolism, and neural development, with implications for chronic disease risk across the lifespan [26,27].

Among candidate keystone microbes, Bifidobacterium longum subsp. infantis (B. infantis) and Lactobacillus reuteri (L. reuteri) have emerged as two of the most functionally and clinically promising species. Both are considered hallmark genera of a healthy gut ecosystem, particularly in infancy, and are commonly reduced or absent in dysbiotic microbiomes [28,29,30,31]. B. infantis possesses a specialized genome that enables it to metabolize human milk oligosaccharides (HMOs), as complex carbohydrates abundant in breast milk—thus facilitating early gut colonization, immune maturation, and SCFA production [23,25,32,33]. By generating acetate and propionate, B. infantis contributes to the reinforcement of the intestinal barrier, modulation of T-regulatory cell populations, and suppression of pro-inflammatory pathways [32,34]. In effect, these keystone microbiomes functions are essential for immune tolerance and protection against allergic and autoimmune conditions.

Likewise, L. reuteri exhibits broad-spectrum effects on host physiology. It produces bioactive metabolites such as reuterin (with antimicrobial properties), histamine (which influences immune responses), and SCFAs that support mucosal integrity [29,35]. Notably, L. reuteri can influence central nervous system signaling through the vagus–oxytocin pathway, and animal models have shown that its supplementation improves social behavior and stress response [36,37,38]. These findings position L. reuteri as a potential microbial target for modulating the gut–brain axis and treating neurodevelopmental conditions such as autism spectrum disorder.

The restoration of these keystone taxa has emerged as a therapeutic strategy to counteract the downstream effects of dysbiosis. These targeted interventions—ranging from probiotics and prebiotics to next-generation live bio-therapeutics and are designed to restore lost microbial functions or reintroduce critical metabolites [28,29,31]. Beyond microbial repletion, public health approaches must address the upstream determinants of microbiome disruption, such as maternal diet, birth practices, antibiotic stewardship, and environmental toxicant exposure [13,39,40,41,42,43,44,45]. Glyphosate, emulsifiers, and artificial sweeteners, for example, have all demonstrated deleterious effects on beneficial gut microbes and should be considered in regulatory and clinical frameworks [44,45,46,47].

The complexity of microbiome host interactions and the diverse determinants of dysbiosis underscore the need for integrative research approaches. Recent advances in multi-omic technologies—including metagenomics, metabolomics, and metaproteomics had provided deep insights into how environmental exposures, host genetics, and microbial composition interact to shape health outcomes [48,49,50,51]. These tools have revealed distinct microbiome signatures in patients with metabolic, immunological, and neuropsychiatric diseases, offering the possibility of microbial biomarkers for early detection and personalized interventions [50,51]. Moreover, host genetic factors significantly shape microbiome composition and immune tolerance. The gene polymorphisms, affecting mucin production, Toll-like receptor signaling, and immune regulation, influence how the host accommodates or resists microbial colonization [52,53,54,55,56]. Thus, interventions targeting the microbiome must be contextualized within a framework of host–microbe coadaptation and individual variability.

The depletion of critical microbial keystone species such as B. infantis and L. reuteri represents both a marker of microbiome erosion and a modifiable contributor to the rising burden of chronic disease. These species provide key ecological functions—ranging from SCFA production to neuroimmune modulation—that are essential for maintaining host homeostasis. By identifying the drivers of their loss and exploring mechanisms for their restoration, we open new avenues for microbiome-informed strategies to prevent and manage NCDs. Framing gut microbial health as a public health priority is essential to reversing the intergenerational cascade of dysbiosis and chronic disease.

Within medications or pharmacologic exposures, proton pump inhibitors (PPIs) warrant special attention for their impact on gut microbiome health. By reducing gastric acid, PPIs impair protein digestion and nutrient absorption while compromising the stomach’s first-line defense against ingested pathogens [57]. This hypochlorhydric state promotes the overgrowth of opportunistic pathogens such as Candida, Helicobacter pylori, and intestinal parasites [58]. Beyond local gastric effects, reduced acidity alters downstream microbial signals, contributing to gut dysbiosis. Unlike antihypertensives or antidepressants, which exert indirect or modulatory effects [59], PPIs directly disrupt foundational digestive and microbial processes, posing a significant barrier to effective microbiome restoration and host healing.

Despite increasing recognition of the importance of B. infantis and L. reuteri, critical questions remain about their ecological roles, clinical relevance, and translational potential in the context of global health. This systematic review aims to synthesize current evidence from preclinical, clinical, and epidemiological studies to assess the following:

(i)

What ecological roles do B. infantis and L. reuteri play across the human lifespan?

(ii)

What factors contribute to their depletion in industrialized societies?

(iii)

What are the functional and clinical consequences of their loss, particularly regarding immune, metabolic, and neurobehavioral outcomes?

(iv)

What is the current evidence base for restoring these keystone species, and how might this inform public health, clinical nutrition, and microbiome-targeted interventions?

By clarifying the translational significance of B. infantis and L. reuteri as foundational members of the human gut ecosystem, this review provides a conceptual and practical framework for microbiome-based strategies aimed at restoring host–microbiota symbiosis and mitigating the risk of chronic disease across populations. Figure 1 presents a conceptual framework summarizing this progression—from environmental disruptors to keystone depletion, system-wide dysfunction, and restoration pathways targeting B. infantis and L. reuteri.

2. Results

This Qualitative Systematic Review (QualSR) synthesized findings from 56 eligible studies (36 human, 20 animal) to evaluate the ecological roles, clinical significance, and restoration strategies for Bifidobacterium infantis (B. infantis) and Lactobacillus reuteri (L. reuteri) as keystone taxa within the gut microbiome. Studies were categorized into four core domains: (i) microbial depletion patterns, (ii) metabolic and immune impacts, (iii) neurobehavioral outcomes, and (iv) efficacy of restoration strategies.

2.1. Patterns of Microbial Depletion in Industrialized Populations

Cross-sectional studies consistently demonstrate a 40–60% reduction in gut microbial diversity among Western populations compared to non-industrialized cohorts (p < 0.001), largely due to the loss of ancestral taxa such as Prevotellaceae and Spirochaetaceae, which are critical for fiber metabolism [60,61,62]. Early investigations based on more than 13,000 prokaryotic sequences established the remarkable variability and complexity of the gut ecosystem [63]. This was expanded by population-level analyses of 531 individuals from the United States, Malawi, and the Venezuelan Amazonas, which revealed pronounced differences in community structure [62]. Comparative studies further demonstrated that children in rural Burkina Faso harbor an enrichment of Bacteroidetes and distinct fiber-degrading species that were absent in their European counterparts [64]. A broader synthesis confirmed that industrialization consistently associates with reduced microbial diversity, influenced by latitude, dietary patterns, and lifestyle factors [65].

Clinically, this erosion of diversity reflects the disappearance of ecologically and metabolically critical lineages, reducing the gut’s resilience and capacity for complex carbohydrate metabolism, with downstream consequences for immune and metabolic health. This depletion disproportionately affects SCFA-producing genera, including Bifidobacterium, Lactobacillus, and Prevotella, which are diminished in industrialized gut ecosystems [66,67,68]. Notably, B. infantis and L. reuteri—species essential for butyrate/propionate synthesis and mucosal immunity—are frequently undetectable in dysbiotic microbiomes [68,69,70]. Recent studies further demonstrate that the gut microbiome and its SCFA metabolites play a central role in maintaining metabolic health [71]. Bidirectional Mendelian Randomization (MR) analysis revealed that a host genetic-driven increase in gut butyrate production is causally linked to improved insulin response after an oral glucose tolerance test (p = 9.8 × 10⁻⁵) [72]. In contrast, impaired production or absorption of propionate is causally associated with a higher risk of type 2 diabetes (p = 0.004) [72]. Butyrate improves insulin response, while propionate is linked to a higher risk of type 2 diabetes [72,73]. Gut composition, shaped by diet and environment, influences SCFA output and chronic disease risk [74,75].

Drivers of Microbial Depletion in Dysbiotic Gut Microbiomes

Mechanistic analyses identified the following key factors associated with depletion of beneficial taxa (e.g., Bifidobacterium infantis, Lactobacillus reuteri) and reduced microbial richness:

• Pharmaceutical agents: Antibiotic exposure, both acute and cumulative, significantly alters microbiome composition and reduces microbial diversity. Perinatal antibiotic exposure triggered an initial suppression of microbial phylogenetic diversity (p < 0.0001 at birth) followed by compensatory hyper-restoration, with richness recovery rates exceeding untreated controls by 12 months [26,76]. Non-steroidal anti-inflammatory drugs (NSAIDs) were associated with:
  • 15–25% reduction in Bifidobacterium abundance (p < 0.01) [77].
  • A bacterium in the family Enterobacteriaceae and one in the family Acidaminococcaceae were significantly more prevalent in NSAID users compared to non-users (p = 0.02) [77,78].
  • Exacerbated mucosal injury when co-administered with PPIs. Endoscopic evaluation revealed significantly higher rates of small bowel mucosal injury in patients receiving nonselective NSAID-PPI combination therapy compared to controls (60–80% vs. 16.7%, p = 0.04) [79]. COX-2 inhibitor/PPI coadministration demonstrated intermediate toxicity (44.4%) [79]. Lesion burden varied by anatomical site, with the jejunum showing particular vulnerability (p = 0.03 for injury severity) [79]. The observed dose–response relationship (p = 0.02 for erosion count gradient) supports a synergistic damaging mechanism between gastric acid suppression and NSAID-mediated mucosal injury [79].
• Perinatal factors, including cesarean delivery and reduced breastfeeding duration, were linked to early-life microbial deficits [80]. In a cohort of 102 infants, gut microbiota composition at 6 weeks was significantly associated with delivery mode (p < 0.001; Q < 0.001) and feeding method (p = 0.01; Q < 0.001) [81]. Vaginal delivery (vs. cesarean) was linked to increased Bacteroides abundance (p < 0.001; Q = 0.02) [81]. Cesarean birth caused greater shifts in microbial profiles than feeding differences (p = 0.003) [81]. Mixed-fed infants resembled formula-fed peers (p = 0.002) [81].
• Dietary shifts toward low-fiber and high-processed foods correlated with decreased SCFA-producing taxa [82,83]. Analysis of 64 studies (n = 2099) showed that dietary fiber supplementation significantly increased the relative abundance of Bifidobacterium spp. (SMD = 0.64; 95% CI: 0.42–0.86; p < 0.00001) and Lactobacillus spp. (SMD = 0.22; 95% CI: 0.03–0.41; p = 0.02), alongside modest gains in fecal butyrate levels (SMD = 0.24; 95% CI: 0.00–0.47; p = 0.05), compared to the placebo or low-fiber controls [84].
• Environmental pollutants (e.g., glyphosate, emulsifiers, artificial sweeteners) exhibited dose-dependent inhibitory effects on commensal bacteria [85]. Environmental pollutants are known to disrupt the balance of gut microbiota, leading to dysbiosis, and can consequently exert various detrimental effects on overall health [85,86]. Food-borne toxicants and additives disrupt gut microbiota function, compromising intestinal barrier integrity and promoting metabolic disease development [87]. Targeting microbe-toxicant interactions through interventions like fermentable fiber may mitigate these metabolic disruptions [87]. Research consistently demonstrates that these pollutants can specifically inhibit the beneficial functions and composition of the gut microbiota [86,87].
• Gastric acid suppression (proton pump inhibitors, PPIs) was uniquely associated with a reduction in microbial richness [88]. Among 211 PPI users, stool microbiome analysis revealed a significant reduction in Shannon diversity and alterations in approximately 20% of bacterial taxa (FDR < 0.05) [88]. PPI use was associated with increased abundance of oral-origin genera including Rothia (p = 9.8 × 10⁻³⁸), as well as elevated levels of Enterococcus, Streptococcus, Staphylococcus, and Escherichia coli [88]. Using one-tailed Wilcoxon rank sum tests on 1827 individuals, with a significance threshold of p < 0.05, findings revealed a significant reduction in gut microbiome diversity among Proton Pump Inhibitor (PPI) users compared to non-users [89]. This indicates a notable impact of PPIs on the gut microbiota [89].

2.2. Metabolic and Immune Outcomes After Keystone Restoration

Recent findings underscore the role of early-life gut microbiome modulation in promoting infant immune health optimization. In a randomized trial, Lactobacillus reuteri DSM 17,938 supplementation in formula-fed infants delivered via cesarean section shifted their gut microbiota composition to resemble that of vaginally delivered infants [90]. L. reuteri was also shown to enhance mucosal barrier function, exert antimicrobial effects, and modulate immune activity [91]. Similarly, supplementation with Bifidobacterium infantis EVC001 in exclusively breastfed infants significantly reduced intestinal inflammation, as indicated by lower levels of fecal pro-inflammatory cytokines and calprotectin [92]. B. infantis utilizes human milk oligosaccharides (HMOs) and is associated with reduced systemic inflammation and improved immune regulation in early life [93]. Notably, its metabolite, indole-3-lactic acid, has been shown to induce galectin-1 expression in T helper cells, contributing to immune tolerance [94,95]. Together, these studies demonstrate that targeted early-life supplementation with L. reuteri DSM 17,938 and B. infantis EVC001 can modulate gut microbiota composition, reduce intestinal inflammation, and enhance immunoregulatory responses in infants.

In a post hoc analysis, supplementation with Bifidobacterium infantis 35,624 for 6–8 weeks significantly reduced systemic inflammatory biomarkers in individuals with ulcerative colitis (UC), chronic fatigue syndrome (CFS), and psoriasis, with approximately 70% of subjects in each group showing improvements compared to the placebo [96]. Specifically, plasma TNF-α levels were significantly lower in B. infantis 35,624-fed subjects compared to placebo controls in both individuals with psoriasis (p = 0.04) and CFS (p = 0.02), suggesting a systemic broad anti-inflammatory effect potentially mediated through regulatory T cell activity [96].

Ex vivo studies in healthy individuals supported these findings. Following B. infantis 35,624 supplementation, secretion of IL-6 and TNF-α from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was significantly reduced compared to placebo-fed controls [96]. In vivo, B. infantis 35,624 consistently lowered systemic pro-inflammatory biomarkers in both gastrointestinal and extraintestinal inflammatory disorders, including UC, CFS, and psoriasis [96].

Additional evidence from a clinical trial in patients with diabetes showed that a multispecies probiotic formulation containing B. infantis significantly decreased serum high-sensitivity C-reactive protein (hs-CRP) levels and increased plasma glutathione, indicating both anti-inflammatory and antioxidant effects [97].

Mechanistic studies in animal models further reinforced these observations. In both wild-type and IL-10 knockout mice, B. infantis 35,624 reduced interferon-γ levels in Peyer’s patches, suggesting that its immunomodulatory actions may occur independently of IL-10 signaling [98]. These findings indicate that B. infantis 35,624 can modulate immune responses at both systemic and mucosal levels in these experimental models. A consolidated overview of these physiological benefits is presented in Table 1, summarizing the immunologic, metabolic, neurobehavioral, and gastrointestinal outcomes linked to B. infantis and L. reuteri across various populations and models.

In a murine model of dextran sodium sulfate (DSS)-induced colitis, Bifidobacterium infantis supplementation exerted significant dose-dependent protective effects [99]. Mice receiving B. infantis showed increased body weight and significantly reduced disease activity index (DAI) and histological damage scores compared to the DSS-only group (all p < 0.05) [99]. Treatment significantly elevated the expression of the regulatory T cell marker Foxp3 (p < 0.05) and anti-inflammatory cytokines IL-10 and TGF-β1 in colon tissue (both p < 0.05) [99]. PD-L1 expression was also significantly upregulated in the treatment groups (p < 0.05), with a dose-dependent relationship observed for both Foxp3 and PD-L1 expression [99]. Correlation analysis revealed that PD-L1 levels were positively associated with Foxp3, IL-10, and TGF-β1 expression [99]. These results suggest that B. infantis mitigates intestinal inflammation and promotes immune tolerance through Treg cell activation and anti-inflammatory cytokine production.

Consistent with these findings, B. infantis has demonstrated protective effects in other murine models of inflammatory bowel disease (IBD), primarily by increasing Treg populations and suppressing pro-inflammatory Th1 and Th17 responses [100]. This strain enhances colonic expression of Foxp3, IL-10, and TGF-β1, and upregulates PD-L1, which positively correlates with anti-inflammatory mediators [99]. Beyond IBD, B. infantis has also been shown to inhibit NF-κB activation in response to Salmonella typhimurium infection and LPS exposure. This effect is Treg-mediated and associated with reduced secretion of pro-inflammatory cytokines, lower T cell proliferation, and decreased dendritic cell activation [101].

2.3. Neurobehavioral and Gut–Brain Axis Effects

Preclinical Evidence

Preclinical studies provide important mechanistic insight into how keystone taxa influence the microbiota–gut–brain axis. In murine models, Lactobacillus reuteri supplementation significantly attenuated depressive-like behaviors, improved autism spectrum disorder (ASD)-like social deficits, and enhanced hippocampal BDNF/CREB signaling while suppressing NF-κB activation [102,103,104]. Bifidobacterium infantis has also demonstrated neurobehavioral benefits in rodent depression models, reversing behavioral deficits and normalizing immune responses [105]. Additional studies highlight that germ-free or antibiotic-treated mice exhibit profound alterations in anxiety, cognition, and pain sensitivity, which can be partially restored by colonization with specific taxa such as L. reuteri or B. infantis [106]. These findings collectively demonstrate that keystone species modulate host neurobiology through vagal signaling, neurotransmitter synthesis, and immune–neuroendocrine pathways [106,107,108]. However, these effects remain preclinical and cannot be extrapolated as established human efficacy. A recent murine study [109] further underscores the importance of the microbiota-metabolite-brain axis: neonatal mice exposed to sevoflurane developed social behavior deficits and synaptic abnormalities, which were reversed by microbiota reconstitution together with restoration of key microbial metabolites. This supports the concept that restoring microbial metabolic function—not just microbial composition can be critical for reversing neurobehavioral impairments in early-life damage. However, all these findings remain preclinical and cannot be extrapolated as established human efficacy.

Clinical Evidence

Evidence in humans is more limited but suggests potential translational relevance. In pediatric populations, L. reuteri DSM 17,938 has consistently reduced crying time in infantile colic, indicating neuromodulatory activity [110]. Preliminary data from clinical cohorts also suggest that selected Bifidobacterium and Lactobacillus strains may influence mood, stress, and cognition through gut–brain signaling pathways [105,111,112,113]. However, the clinical evidence is heterogeneous, strain-specific, and generally modest in effect size. Importantly, while L. reuteri restored social behaviors in murine ASD models [38,103], comparable human trials are lacking. Thus, current human data support symptomatic relief in specific conditions (e.g., infantile colic), but robust evidence for broader neuropsychiatric applications is still emerging.

2.4. Efficacy of Restoration Strategies

Lactobacillus reuteri has demonstrated therapeutic potential across metabolic, gastrointestinal, and inflammatory conditions. In type 2 diabetes patients, supplementation with L. reuteri DSM 17,938 significantly improved insulin sensitivity and glucose metabolism [114]. According to an external meta-analysis of four RCTs (n = 347), supplementation with L. reuteri reduced the duration of acute diarrhea compared to the placebo or no treatment (mean difference [MD] = −0.87 days; 95% CI: −1.43 to −0.31) [115]. Similarly, a separate meta-analysis of three RCTs (n = 284) reported a reduction in hospitalization duration (MD = −0.54 days; 95% CI: −1.09 to 0.00) [115]. A separate controlled study (n = 127) further confirmed these effects: significantly more children receiving L. reuteri were diarrhea-free at 24 h (50% vs. 5%, p < 0.001), 48 h (69% vs. 11%, p < 0.001), and 72 h (69% vs. 11%, p < 0.001) compared to controls. Mean hospitalization duration was shorter in the L. reuteri group (4.31 ± 1.3 days vs. 5.46 ± 1.77 days; p < 0.001), and no cases of prolonged diarrhea were observed in this group (0% vs. 17% in controls) [110]. No adverse events were reported, highlighting its favorable safety profile.

In murine models of colitis, L. reuteri I5007 reduced intestinal inflammation, modulated gut microbiota, and improved associated metabolic dysfunction [116]. Strain FYNLJ109L1 demonstrated systemic benefits, including improved glucose tolerance, better lipid profiles, and decreased pro-inflammatory cytokine levels in mice with metabolic syndrome [117]. Additional studies confirmed L. reuteri’s immunoregulatory effects and ability to restore microbial balance in colitis models [116]. Other Lactobacillus and Bifidobacterium strains also exhibit therapeutic potential in conditions such as eczema, obesity, and hypercholesterolemia, through mechanisms including enhanced epithelial barrier integrity and cholesterol-lowering effects [118]. These effects appear strain-specific, reinforcing the importance of precise probiotic selection.

Collectively, these findings support gut microbiota modulation—particularly through targeted L. reuteri strains—as a promising strategy for managing inflammatory and metabolic disorders. This aligns with broader evidence implicating gut dysbiosis in the pathophysiology of obesity and type 2 diabetes and supports the therapeutic value of microbiome restoration [119]. In a randomized trial of 120 ulcerative colitis patients, participants received probiotics (Bifidobacterium longum, 2 × 10⁹ CFU/day), prebiotics (8 g psyllium/day), or a synbiotic combination [120]. While all groups showed improvement in Inflammatory Bowel Disease Questionnaire scores, the synbiotic group demonstrated statistically significant gains (from 168 to 176; p = 0.03) [120]. Specific functional domains improved across groups: emotional function (probiotics, p = 0.03), bowel function (prebiotics, p = 0.04), and both systemic (p = 0.008) and social function (synbiotics, p = 0.02) [120]. Notably, only the synbiotic group exhibited a significant decrease in C-reactive protein (0.59 to 0.14 mg/dL; p = 0.04), with no reported adverse events [120]. These results indicate that synbiotic therapy provides greater clinical and anti-inflammatory benefit than either component alone.

Table 1. Therapeutic Outcomes of Bifidobacterium infantis and Lactobacillus reuteri Across Physiological Systems. This table summarizes peer-reviewed evidence on the health effects of B. infantis and L. reuteri in humans and animal models. Arrows indicate the direction of change: ↑ shows an increase or up-regulation of a marker or function, while ↓ shows a decrease or down-regulation. Key outcomes include improved immune modulation (↑ regulatory T cells, ↑ IL-10, ↓ CRP, ↓ TNF-α), strengthened gut barrier integrity (↑ tight junction proteins, ↓ permeability), neurobehavioral benefits (↓ depressive behavior, normalized HPA axis activity), and metabolic and gastrointestinal improvements (↑ insulin sensitivity, ↓ glucose levels, ↓ diarrhea duration, ↓ hospital stay). References correspond to the primary studies cited in the manuscript.

Microbial Species	Health Domain	Therapeutic Effects	Model/Population	Key References
B. infantis	Immune Modulation	↑ Tregs, ↑ IL-10, ↓ CRP, ↓ TNF-α	Humans, Mice	[100,101,103,104]
B. infantis	Gut Barrier Function	↑ Tight junction proteins, ↓ Permeability	Mice	[103]
B. infantis	Neurobehavioral Effects	↓ Depressive behavior, Normalized HPA axis	Rat model	[108]
L. reuteri	Immune Modulation	↑ IL-10, ↓ Pro-inflammatory cytokines	Humans, Mice	[94,95,115]
L. reuteri	Gut-Brain Axis	↑ Oxytocin signaling, Improved social behavior	Mouse models	[56,65,107]
L. reuteri	Metabolic Health	↑ Insulin sensitivity, ↓ Glucose levels	Type 2 Diabetes patients	[120]
L. reuteri	Gastrointestinal Health	↓ Diarrhea duration, ↓ Hospital stay	Children with acute diarrhea	[121,122]

Table 1. Therapeutic Outcomes of Bifidobacterium infantis and Lactobacillus reuteri Across Physiological Systems. This table summarizes peer-reviewed evidence on the health effects of B. infantis and L. reuteri in humans and animal models. Arrows indicate the direction of change: ↑ shows an increase or up-regulation of a marker or function, while ↓ shows a decrease or down-regulation. Key outcomes include improved immune modulation (↑ regulatory T cells, ↑ IL-10, ↓ CRP, ↓ TNF-α), strengthened gut barrier integrity (↑ tight junction proteins, ↓ permeability), neurobehavioral benefits (↓ depressive behavior, normalized HPA axis activity), and metabolic and gastrointestinal improvements (↑ insulin sensitivity, ↓ glucose levels, ↓ diarrhea duration, ↓ hospital stay). References correspond to the primary studies cited in the manuscript.

Microbial Species	Health Domain	Therapeutic Effects	Model/Population	Key References
B. infantis	Immune Modulation	↑ Tregs, ↑ IL-10, ↓ CRP, ↓ TNF-α	Humans, Mice	[100,101,103,104]
B. infantis	Gut Barrier Function	↑ Tight junction proteins, ↓ Permeability	Mice	[103]
B. infantis	Neurobehavioral Effects	↓ Depressive behavior, Normalized HPA axis	Rat model	[108]
L. reuteri	Immune Modulation	↑ IL-10, ↓ Pro-inflammatory cytokines	Humans, Mice	[94,95,115]
L. reuteri	Gut-Brain Axis	↑ Oxytocin signaling, Improved social behavior	Mouse models	[56,65,107]
L. reuteri	Metabolic Health	↑ Insulin sensitivity, ↓ Glucose levels	Type 2 Diabetes patients	[120]
L. reuteri	Gastrointestinal Health	↓ Diarrhea duration, ↓ Hospital stay	Children with acute diarrhea	[121,122]

3. Discussion

3.1. Principal Findings and Clinical Implications

This QualSR supports evidence of gut dysbiosis in industrialized populations, including a 40–60% reduction in microbial diversity [60] and consistent depletion of key short-chain fatty acid (SCFA)-producing taxa such as Bifidobacterium infantis and Lactobacillus reuteri. A multi-cohort study demonstrated that gut microbial diversity was highest in traditional rural communities and progressively declined among first- and second-generation U.S. immigrants [121]. This pattern reflects a broader ecological collapse of keystone taxa that regulate immune, metabolic, and neurobehavioral homeostasis via SCFA production and gut–brain axis signaling. Mendelian randomization studies support the clinical relevance of SCFAs: increased butyrate production improves insulin sensitivity, while impaired propionate metabolism raises the risk of type 2 diabetes [72,122]. This cascade is illustrated in Figure 2, depicting the pathway from dysbiosis to chronic disease and potential restoration strategies.

This QualES of 56 studies found that B. infantis and L. reuteri are frequently undetectable in Western guts, but their targeted supplementation yields consistent therapeutic benefits across diverse clinical contexts. In clinical cohorts with psoriasis and chronic fatigue syndrome, B. infantis 35,624 supplementation significantly reduced systemic inflammation, lowering C-reactive protein and TNF-α levels by ~40–50% compared to the placebo (p < 0.001) [96]. In preclinical colitis models, B. infantis similarly downregulated IL-6 expression, enhanced regulatory T-cell–mediated immune balance, and strengthened gut barrier integrity, underscoring its dual role in immune modulation and mucosal protection [99,123].

The L. reuteri supplementation produced comparable benefits. In patients with diabetes, strain DSM 17,938 enhanced GLP-1 and GLP-2 secretion and increased insulin output by approximately 50% over four weeks [124]. Pediatric randomized controlled trials (RCTs) demonstrated a ~0.9-day reduction in diarrhea duration (MD –24.8 h; 95% CI: −38.8 to −10.8) and improved early recovery rates [125]. In mouse models, strain NK33 improved mood and reduced colitis via hippocampal BDNF upregulation and NF-κB inhibition [126]. Together, these data support the clinical utility of B. infantis and L. reuteri for immune, metabolic, and neurobehavioral dysregulation associated with modern dysbiosis.

3.2. Mechanistic Insights

Mechanistic evidence explains how these two keystone taxa exert outsized biological effects. Figure 2 illustrates the key mechanistic pathways through which B. infantis and L. reuteri modulate host physiology—ranging from SCFA biosynthesis and mucosal immune regulation to gut–brain signaling and neuroendocrine modulation. B. infantis is uniquely adapted to human milk oligosaccharides (HMOs), fermenting them into acetate and propionate—metabolites that feed colonocytes and expand Foxp3+ regulatory T cells (Tregs). In colitis models, B. infantis drove dose-dependent increases in Foxp3+ Tregs and IL-10/TGF-β1 levels and activated the PD-1/PD-L1 immune checkpoint pathway, supporting its anti-inflammatory profile [99,123]. It also improved gut barrier function by upregulating tight junction proteins such as ZO-1, occludin, and claudin-1 [123].

The L. reuteri synthesizes bioactive compounds like reuterin and histamine, which modulate immunity by suppressing NF-κB and lowering TNF-α and IL-6 while increasing IL-10 systemically and in mucosal tissues [96,99]. Meta-analyses show that L. reuteri is among the most effective probiotics at restoring tight junction integrity—particularly claudin-1 and ZO-1—in inflamed gut tissue [127]. These species also engage the gut–brain axis. L. reuteri signals via the vagus nerve to stimulate hypothalamic oxytocin neurons, restoring social behaviors in autism models, even in the absence of adaptive immunity [38]. Strain NK33 improved anxiolytic behavior through hippocampal BDNF/CREB activation and NF-κB suppression [38].

Metabolically, B. infantis and L. reuteri increase SCFA levels that act on host G-protein-coupled receptors (GPRs) and inhibit histone deacetylases (HDACs), improving glucose tolerance and lipid handling. Mendelian-randomization studies confirm a causal role: genetically higher butyrate predicts improved insulin response, whereas poor propionate utilization increases diabetes risk [126].

While these findings highlight bacterial mechanisms, the gut microbiome extends beyond bacteria alone. Beyond bacterial keystone species, the gut microbiome also includes archaea, viruses, fungi, and their metabolites, which collectively influence host physiology. Archaea such as methanogens regulate hydrogen disposal and fermentation efficiency, thereby shaping bacterial metabolic networks [128,129]. The gut mycobiome, although less abundant, modulates mucosal immunity and interacts with bacterial taxa to influence inflammation and barrier function [130]. Bacteriophages play an equally important role by controlling bacterial population dynamics through predation and horizontal gene transfer, thereby maintaining ecological balance [131]. Viral and fungal metabolites further add complexity by exerting systemic immunomodulatory effects [132]. While our synthesis has focused on B. infantis and L. reuteri as representative bacterial keystone taxa, a complete understanding of microbiome restoration will require integrative multi-omic approaches that capture the contributions of these non-bacterial community members.

3.3. Clinical and Public Health Implications

These findings have immediate translational value in population-based studies. In inflammatory diseases, probiotic formulations containing B. infantis or L. reuteri may serve as adjunct therapies. For example, B. infantis combined with psyllium improved quality of life and reduced CRP in patients with inflammatory bowel disease (IBD) [123]. In extraintestinal inflammation (psoriasis, CFS, UC), B. infantis 35,624 supplementation for 6–8 weeks lowered systemic CRP and TNF-α [96], an effect mirrored by increased Tregs in colonic tissue [96].

For metabolic dysfunction, L. reuteri’s enhancement of incretin hormones (GLP-1/GLP-2) and insulin secretion [99] suggests potential for managing prediabetes and T2D. Although no change in insulin sensitivity was observed over 4 weeks in healthy volunteers [124], longer trials in dysmetabolic populations are justified. Co-administering L. reuteri DSM 17,938 with metformin in clinical practice may be safe and potentially synergistic.

In pediatric gastroenterology, L. reuteri DSM 17,938 consistently shortened diarrhea duration by ~1 day in acute gastroenteritis across multiple RCTs (n ≈ 300), with no serious adverse events [124]. This has major implications for resource-limited settings by accelerating rehydration and discharge. Preliminary data support neurodevelopmental applications. L. reuteri reversed social deficits in ASD mouse models via vagus-mediated gut–brain signaling [125]. Given the unmet need in ASD treatment, pilot trials using L. reuteri-rich foods or supplements in affected children are warranted. For mood disorders, multi-strain probiotics containing Lactobacillus and Bifidobacterium have modest anxiolytic/antidepressant effects. Strain-specific data—especially for NK33 and DSM 17,938—support future trials. In infantile colic, L. reuteri reduces crying duration, indicating neuromodulatory effects [38].

Strain specificity is essential. Clinicians should use evidence-backed strains such as B. infantis 35,624 or EVC001, and L. reuteri DSM 17,938 or NK33. Synbiotic approaches may amplify effects: B. infantis combined with xylooligosaccharide or psyllium further increased IL-10 and tight junction expression [126]. Co-prescribing prebiotics like inulin or GOS can also enhance colonization. Personalized protocols, ideally guided by microbiome profiling, are the future; in the interim, evidence-based strain selection should guide clinical use.

Public health policy and implementation must address the erosion of these keystone species. Promoting vaginal delivery and exclusive breastfeeding helps seed B. infantis and shape early immunity [84,123]. Antibiotic stewardship—especially in pregnant women, infants, and children—can reduce collateral loss of these beneficial taxa. Regulatory bodies should consider labeling additives (emulsifiers, glyphosate, artificial sweeteners) that harm gut microbes, given their link to reduced Bifidobacterium and epithelial disruption [96,99].

Diet is a modifiable determinant of microbial composition. Fiber-rich diets containing inulin, FOS, and GOS substantially increase the abundance of Bifidobacterium and Lactobacillus [84]. National programs should promote whole plant foods and include prebiotic supplementation in at-risk groups (infants, elderly, institutionalized). Schools and worksites could provide synbiotic-rich foods like yogurt and fortified bread [84]. Proton pump inhibitors, which reduce diversity and promote opportunistic overgrowth [89,133], should be prescribed with caution and for a limited time.

Together, clinical, dietary, obstetric, and pharmaceutical strategies can facilitate microbiome restoration and preserve keystone microbial species, potentially mitigating the rising burden of chronic diseases associated with microbiome collapse.

3.4. QualES Limitations

The QualEs remains remarkable based on the implicated findings; however, there are several limitations which constrain the interpretation and clinical application of our findings. The reviewed studies varied widely in design, probiotic strain selection, dosing, delivery formats, and outcome measures, which precluded formal meta-analysis and limits the precision of effect estimates. Many of the mechanistic insights—especially those involving brain–behavior modulation—were derived from animal models. For instance, while L. reuteri reversed autism-like social deficits in rodents [38], comparable human trials are lacking.

Short-term studies dominate the literature. This raises concerns about the sustainability of therapeutic effects and the potential for adaptive host or microbial responses that diminish efficacy over time. Furthermore, many studies relied on 16S rRNA sequencing, which limits taxonomic and functional resolution. Broader use of metagenomics and metabolomics is necessary to better characterize keystone species’ functions and interactions.

Population diversity is another limitation. Most trials were conducted in Western, homogeneous cohorts, reducing generalizability to other ethnic and geographic populations. Host-specific factors—such as baseline microbiota composition, dietary habits, and genetics—likely influence probiotic efficacy, making single-trial findings difficult to extrapolate.

Publication bias is also a concern. Positive findings tend to be overrepresented, while underpowered studies with null results (e.g., L. reuteri in antibiotic-associated diarrhea) are less frequently published [125]. Additionally, the review focused on B. infantis and L. reuteri, which simplifies the complexity of microbiome restoration. While these are keystone taxa, broader community dynamics and diet–microbe interactions are likely essential for lasting clinical benefit.

Finally, clinical translation is hampered by a lack of standardization. Variation in probiotic preparation, dosing protocols, strain specificity, and delivery matrices create inconsistencies in outcomes. Long-term safety, including risks such as small intestinal bacterial overgrowth or horizontal gene transfer, remains insufficiently studied.

3.5. Conclusions and Future Directions

This systematic review highlights the therapeutic potential of restoring keystone microbial species—particularly Bifidobacterium infantis and Lactobacillus reuteri—as a viable strategy to counteract the chronic disease burden driven by modern-lifestyle-induced dysbiosis. The evidence from mechanistic studies, preclinical models, and human trials supports their role in modulating host immunity, metabolism, and gut–brain signaling across various disease contexts.

For successful clinical translation, key challenges remain in optimizing microbiome-targeted interventions for human disease. These include the development of personalized microbial restoration protocols, refinement of delivery systems to enhance colonization and persistence, and integration of microbiome-targeted interventions into existing clinical and public health frameworks. Given the complex, bidirectional nature of host–microbe interactions, advancing this field will require interdisciplinary collaboration across microbiology, immunology, neuroscience, and systems biology.

It is essential that microbiome restoration must not be limited to therapeutic supplementation alone; however, a comprehensive approach should also address upstream environmental determinants such as antibiotic overuse, toxicant exposure, dietary patterns, and early-life microbial disruptions. The keystone species restoration paradigm provides a scientifically grounded and translationally relevant model for reversing microbial depletion and its systemic consequences.

This review highlights how precision microbiome medicine, through epigenomic modulation, supports the development of targeted, sustainable, and personalized interventions to restore human–microbe symbiosis and improve long-term health outcomes.

3.6. Microbiome Implication in Disease Causation, Health Outcomes and Future Directions

Future research should involve integrative, translational approach to address these gaps and advance the clinical utility of B. infantis and L. reuteri.

• Personalized Interventions: Development of diagnostic tools using microbiome sequencing, SCFA profiling, or immune biomarkers will allow precision targeting. Trials should stratify patients based on baseline microbial or immunologic markers to assess differential responses to probiotic therapy.
• Longitudinal and Intergenerational Studies: Extended follow-up is needed to determine the durability of benefits and their effects across generations. For example, supplementing pregnant women with B. infantis could be studied for its impact on neonatal microbiota and early immune development.
• Next-Generation Therapeutics: Engineered strains or microbial consortia could be designed to deliver targeted metabolites or immunomodulators. For instance, a modified B. infantis that overproduces acetate, or L. reuteri strains that optimize oxytocin signaling, could enhance therapeutic precision.
• Mechanistic Human Studies: Future trials should integrate multi-omics with host physiology data. Measuring changes in host gene expression, epigenetics, inflammatory markers, neuroimaging, and vagal tone alongside microbiome dynamics will clarify causal pathways.
• Population-Level Research: Real-world interventions—such as fiber subsidies, fermented food promotion, and early-life microbial seeding—should be evaluated for their capacity to restore keystone taxa and reduce chronic disease incidence.
• Regulatory and Safety Frameworks: As probiotics become clinical tools, robust long-term safety monitoring is essential. Registries and pharmacovigilance systems can track rare adverse events and ecological risks. Regulatory agencies should also evolve to include microbiome endpoints in risk–benefit assessments.

Pursuing these research directions requires collaborative, symbiotic, mutualistic, transdisciplinary, dynamic and translational initiatives and application of scientific data across microbiology, immunology, epigenomics, genomics, clinical medicine, and public health. These translational initiatives, with basic medical sciences, clinical correlates and population-based findings, will optimize these findings on microbiome diversity beyond correlation and causal inference; hence, B. infantis and L. reuteri can be used in translational initiatives in restoring host–microbe homeostasis as well as human health improvement and optimization across all populations.

4. Materials and Methods

4.1. Study Design

This Qualitative Systematic Review (QualSR) was conducted to evaluates the ecological, mechanistic, and therapeutic roles of two keystone microbial taxa—Bifidobacterium infantis and Lactobacillus reuteri—in shaping host metabolic health and gut–brain axis function. The review explored the clinical consequences of their depletion and assessed strategies for microbiome restoration across both human and animal models.

The study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines. It was informed by an ecological health framework linking microbial dysbiosis to chronic disease etiology.

No protocol was formally registered for this review, as it was designed as a Qualitative Systematic Review (QualSR) without quantitative synthesis. Nevertheless, all stages of the review process adhered to PRISMA 2020 guidelines, including predefined eligibility criteria, comprehensive search strategy, and structured data extraction and thematic synthesis.

The primary aims were to:

• Define the ecological functions of B. infantis and L. reuteri across developmental stages.
• Identify key drivers of their loss in industrialized societies.
• Examine the clinical and mechanistic consequences of their depletion in metabolic, immunologic, and neurobehavioral outcomes.
• Synthesize evidence on therapeutic strategies—including probiotics, prebiotics, and biotherapeutics—targeting their restoration.

4.2. Search Strategy

A comprehensive literature search was conducted across five electronic databases: PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, and CINAHL, covering publications from January 2000 to February 2024. Additional sources included reference mining of key reviews and hand-searching relevant journals.

The search strategy incorporated Medical Subject Headings (MeSHs) and free-text terms such as:

“gut microbiome”, “microbial diversity”, “dysbiosis”

“Bifidobacterium infantis”, “Lactobacillus reuteri”, “keystone species”

“short-chain fatty acids”, “intestinal permeability”, “SCFA production”

“gut-brain axis”, “microbiome restoration”, “probiotic”, “synbiotic”, “prebiotic”, “immune modulation”

“autism spectrum disorder”, “depression”, “metabolic syndrome”, “host genetics”

Search strings were constructed using Boolean operators (AND, OR) to balance sensitivity and specificity. An example from PubMed:

(“gut microbiome” OR “intestinal microbiota”) AND (“Bifidobacterium infantis” OR “Lactobacillus reuteri”) AND (“dysbiosis” OR “microbiota disruption”) AND (“probiotics” OR “restoration” OR “short-chain fatty acids”) AND (“immune modulation” OR “metabolic health” OR “gut-brain axis”)

A total of 547 records were identified. After de-duplication, 394 unique records were retained for screening.

4.3. QualES Eligibility Criteria

Inclusion Criteria:

• Peer-reviewed human or animal studies.
• Published between 2000 and 2024.
• Investigated either B. infantis or L. reuteri as a central taxon.
• Explored outcomes in at least one of the following domains:
  • Microbial function (e.g., SCFA production, mucosal integrity).
  • Immune and metabolic regulation (e.g., insulin sensitivity, cytokine profiles).
  • Neurobehavioral modulation (e.g., vagal tone, anxiety, ASD-like behavior).
  • Restoration strategies (e.g., probiotic/prebiotic administration, synbiotics, FMT).
• Employed recognized microbiome assessment methods (e.g., 16S rRNA sequencing, metagenomics, metabolomics).

Exclusion Criteria:

• In vitro studies with no host interaction.
• Studies with unclear endpoints or inadequate data.
• Sample size < 30 for human studies or <10 per group in animal models.
• Non-English language, case reports, narrative reviews, and conference abstracts.

Sample size justification: Thresholds were chosen to enhance generalizability and reduce the risk of type II errors in heterogeneous populations. Smaller studies often yield less robust or clinically meaningful estimates.

4.4. Study Selection Process

A two-stage screening process was applied:

• Title and abstract screening of 394 articles by two independent reviewers.
• Full-text review of 97 articles.

A total of 56 studies were included in the final synthesis:

• 36 human studies.
• 20 animal model studies.

A descriptive summary of each included study, including model type, microbial strain, condition, sample size, and analytical techniques, is provided in Supplementary Table S1.

Reasons for exclusion at the full-text stage (n = 41) included: non-specific interventions (n = 11), unclear or absent outcomes (n = 11), insufficient sample size (n = 18), and inaccessible full texts (n = 1).

Inter-rater reliability between the two independent reviewers (M.E and L.H.J) was assessed using Cohen’s kappa. Agreement was strong for both title/abstract screening (κ = 0.84) and full-text screening (κ = 0.87). Conflicts were initially resolved through discussion, and when consensus could not be reached, a third senior reviewer (E.I.) adjudicated the final decision. Across the 394 records screened, 21 conflicts occurred at the title/abstract stage and 7 conflicts at the full-text review stage, all of which were resolved.

A QualES PRISMA 2020 flow diagram is provided in Figure 3.

4.5. Data Extraction and Quality Assessment

Data were independently extracted by two reviewers using a standardized form, capturing:

• Study type and design.
• Species/population characteristics.
• Intervention details (strain, dose, duration).
• Analytical techniques (e.g., LC-MS, 16S rRNA, behavioral assays).
• Outcomes: metabolic, immune, neurocognitive, microbiome-related.
• Drivers of microbial loss (e.g., antibiotics, birth mode, infant diet).
• Type of restoration strategy (e.g., probiotic-only, diet, combination).

Risk of bias was assessed as follows:

• RCTs: Cochrane Risk of Bias Tool 2.0.
• Observational studies: Newcastle–Ottawa Scale.
• Animal studies: SYRCLE Risk of Bias tool.
• Multi-omics and mechanistic studies: STROBE-Omics checklist.

Studies were rated as low, moderate, or high quality using validated appraisal tools, and low-quality studies were excluded from the thematic synthesis to preserve interpretive reliability. Detailed quality assessment scoring is presented in Supplementary Table S2, while the characteristics of all included studies—such as key outcomes, strain, condition, and tools used—are summarized in Supplementary Table S1. A synthesis of the major therapeutic outcomes associated with B. infantis and L. reuteri across various domains is presented in Table 1.

4.6. Data Synthesis

Due to the heterogeneity of models, interventions, and outcomes, a narrative synthesis approach was applied.

Studies were grouped into five thematic domains:

• Functional role and ecological significance of B. infantis and L. reuteri.
• Drivers of microbial depletion in industrialized contexts.
• Consequences of loss on host immune, metabolic, and neurobehavioral health.
• Mechanisms of restoration and clinical efficacy of interventions.
• Host–microbe coadaptation and microbiome-targeted personalization.

Quantitative data (e.g., SCFA levels, behavior scores, inflammatory markers) were summarized descriptively where reported, but meta-analysis was not feasible due to variability in endpoints, analytical tools, and reporting standards.