Ethanolamine enhances adhesion, promotes microcompartment formation, and modulates gene expression in Levilactobacillus brevis ATCC 14869.

Ethanolamine is an abundant compound in the gastrointestinal tract and a valuable source of carbon and nitrogen for pathogenic bacteria harboring ethanolamine utilization (eut) genes. Eut-positive pathogens can consume free ethanolamine to outcompete commensal microbes, which often lack eut genes, and establish infection. Ethanolamine can also act as a host recognition signal for eut-positive pathogens to upregulate virulence genes during colonization. Therefore, reducing free ethanolamine titers may represent a novel approach to preventing infection by eut-positive pathogens. Interestingly, the commensal microorganism Levilactobacillus brevis ATCC 14869 was found to encode over 18 eut genes within its genome. This led us to hypothesize that L. brevis can compete with eut-positive pathogens by clearing free ethanolamine from the environment. Our results demonstrate that despite being unable to metabolize ethanolamine under most conditions, L. brevis ATCC 14869 responds to the compound by increasing the expression of genes encoding proteins involved in microcompartment formation and adhesion to the intestinal epithelial barrier. The improved intestinal adhesion of L. brevis in the presence of ethanolamine also enhanced the exclusion of eut-positive pathogens from adhering to intestinal epithelial cells. These findings support further studies to test whether L. brevis ATCC 14869 can counter enteric pathogens and prevent or reduce the severity of infections. Overall, the metabolic capabilities of L. brevis ATCC 14869 offer a unique opportunity to add to the armamentarium of antimicrobial therapies as well as our understanding of the mechanisms used by beneficial microbes to sense and adapt to host microenvironments.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models.

Microbial management is central to aquaculture's efficiency. Pediococcus acidilactici MA18/5M has shown promising results promoting growth, modulation of the immune response, and disease resistance in many fishes. However, the mechanisms through which this strain confers health benefits in fish are poorly understood, particularly in Pacific salmonid models. Briefly, the aims of this study were to i) assess the protective effects of P. acidilactici MA18/5M by examining gut barrier function and the expression of tight junction (TJ) and immune genes in vitro and in vivo, and ii) to determine the protective effects of this strain against a common saltwater pathogen, Vibrio anguillarum J382. An in vitro model of the salmonid gut was employed utilizing the cell line RTgutGC. Barrier formation and integrity assessed by TEER measurements in RTgutGC, showed a significant decrease in resistance in cells exposed only to V. anguillarum J382 for 24 h, but pre-treatment with P. acidilactici MA18/5M for 48 h mitigated these effects. While P. acidilactici MA18/5M did not significantly upregulate tight junction and immune molecules, pre-treatment with this strain protected against pathogen-induced insults to the gut barrier. In particular, the expression of ocldn was significantly induced by V. anguillarum J382, suggesting that this molecule might play a role in the host response against this pathogen. To corroborate these observations in live fish, the effects of P. acidilactici MA18/5M was evaluated in Chinook salmon reared in real aquaculture conditions. Supplementation with P. acidilactici MA18/5M had no effect on Chinook salmon growth parameters after 10 weeks. Interestingly, histopathological results did not show alterations associated with P. acidilactici MA18/5M supplementation, indicating that this strain is safe to be used in the industry. Finally, the expression pattern of transcripts encoding TJ and immune genes in all the treatments suggest that variation in expression is more likely to be due to developmental processes rather than P. acidilactici MA18/5M supplementation. Overall, our results showed that P. acidilactici MA18/5M is a safe strain for use in fish production, however, to assess the effects on growth and immune response previously observed in other salmonid species, an assessment in adult fish is needed.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

The Eµ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia.

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Revisiting the Definition of 'Healthy' Participants in Substantiation of Structure/Function Claims for Dietary Supplements.

Concepts and definitions of 'healthy' have been evolving within clinical treatment algorithms as well as reference standards such as Body Mass Index and Dietary Reference Intakes. Consumers' perception of the word 'healthy' is also changing to reflect longer life span, need to stay active and in a good state of mental well-being while managing multiple diseases. Guidelines from the US Food and Drug Administration indicate that substantiating evidence for support of Structure/Function (S/F) claims for dietary supplements is best derived from clinical research conducted in a 'healthy' population. S/F claims cannot be represented to diagnose, treat, cure or prevent any disease. However, in this context, the term 'healthy' is non-descriptive and largely interpreted as an absence of disease. Guidelines for treatment of disease have been broadened to include biomarkers of disease risk such that the pool of 'healthy' volunteers eligible to be enrolled in clinical trials for S/F claim substantiation is greatly diminished. This perspective presents the challenges faced by the food and dietary supplement industry and by researcher efforts designed to substantiate S/F claims and suggest the phrase 'physiologically stable' or 'apparently healthy' as descriptions better suited to replace the term 'healthy.'

Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease.

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD. In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

Perspective: microbial interventions in the urinary tract.

Despite multiple advances in medicine, the management of urinary tract infections (UTIs) in women has remained stalled for decades. To prevent the development of symptomatic recurrences, low-dose antibiotics are the mainstay, while alternative approaches have been attempted with limited success. The use of probiotics was first considered forty years ago, and while some promising studies have been published, additional evidence in larger patient groups is needed to recommend specific strains as a primary preventive regimen. Overall, the role of beneficial microbes in reducing the risk of UTI and other urological diseases, such as urolithiasis, remains a target for researchers. The aim of this perspective is to offer a viewpoint on the status of this approach and recommendations for how to develop novel probiotic therapies.

Recommendations to Improve Quality of Probiotic Systematic Reviews With Meta-Analyses.

Importance: Systematic reviews and meta-analyses often report conflicting results when assessing evidence for probiotic efficacy, partially because of the lack of understanding of the unique features of probiotic trials. As a consequence, clinical decisions on the use of probiotics have been confusing. Objective: To provide recommendations to improve the quality and consistency of systematic reviews with meta-analyses on probiotics, so evidence-based clinical decisions can be made with more clarity. Evidence Review: For this consensus statement, an updated literature review was conducted (January 1, 2020, to June 30, 2022) to supplement a previously published 2018 literature search to identify areas where probiotic systematic reviews with meta-analyses might be improved. An expert panel of 21 scientists and physicians with experience on writing and reviewing probiotic reviews and meta-analyses was convened and used a modified Delphi method to develop recommendations for future probiotic reviews. Findings: A total of 206 systematic reviews with meta-analysis components on probiotics were screened and representative examples discussed to determine areas for improvement. The expert panel initially identified 36 items that were inconsistently reported or were considered important to consider in probiotic meta-analyses. Of these, a consensus was reached for 9 recommendations to improve the quality of future probiotic meta-analyses. Conclusions and Relevance: In this study, the expert panel reached a consensus on 9 recommendations that should promote improved reporting of probiotic systematic reviews with meta-analyses and, thereby, assist in clinical decisions regarding the use of probiotics.

Multi-site microbiota alteration is a hallmark of kidney stone formation.

BACKGROUND: Inquiry of microbiota involvement in kidney stone disease (KSD) has largely focussed on potential oxalate handling abilities by gut bacteria and the increased association with antibiotic exposure. By systematically comparing the gut, urinary, and oral microbiota of 83 stone formers (SF) and 30 healthy controls (HC), we provide a unified assessment of the bacterial contribution to KSD. RESULTS: Amplicon and shotgun metagenomic sequencing approaches were consistent in identifying multi-site microbiota disturbances in SF relative to HC. Biomarker taxa, reduced taxonomic and functional diversity, functional replacement of core bioenergetic pathways with virulence-associated gene markers, and community network collapse defined SF, but differences between cohorts did not extend to oxalate metabolism. CONCLUSIONS: We conclude that multi-site microbiota alteration is a hallmark of SF, and KSD treatment should consider microbial functional restoration and the avoidance of aberrant modulators such as poor diet and antibiotics where applicable to prevent stone recurrence. Video Abstract.

Targetable lesions and proteomes predict therapy sensitivity through disease evolution in pediatric acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) genomes show that relapses often arise from subclonal outgrowths. However, the impact of clonal evolution on the actionable proteome and response to targeted therapy is not known. Here, we present a comprehensive retrospective analysis of paired ALL diagnosis and relapsed specimen. Targeted next generation sequencing and proteome analysis indicate persistence of actionable genome variants and stable proteomes through disease progression. Paired viably-frozen biopsies show high correlation of drug response to variant-targeted therapies but in vitro selectivity is low. Proteome analysis prioritizes PARP1 as a pan-ALL target candidate needed for survival following cellular stress; diagnostic and relapsed ALL samples demonstrate robust sensitivity to treatment with two PARP1/2 inhibitors. Together, these findings support initiating prospective precision oncology approaches at ALL diagnosis and emphasize the need to incorporate proteome analysis to prospectively determine tumor sensitivities, which are likely to be retained at disease relapse.

Age and ligand specificity influence the outcome of pathogen engagement on preleukemic and leukemic B-cell precursor populations.

Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid-based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.

Adaptation of the Th-MYCN Mouse Model of Neuroblastoma for Evaluation of Disseminated Disease.

High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease.

Delivery mechanism can enhance probiotic activity against honey bee pathogens.

Managed honey bee (Apis mellifera) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems-standard pollen patty infusion and a novel spray-based formulation-affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus, Bifidobacterium, Lactobacillus, and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.

The Impact of Nutrition, Physical Activity, Beneficial Microbes, and Fecal Microbiota Transplant for Improving Health.

The recognition that microbes are integral to human life has led to studies on how to manipulate them in favor of health outcomes. To date, there has been no conjoint recommendation for the intake of dietary compounds that can complement the ingested organisms in terms of promoting an improved health outcome. The aim of this review is to discuss how beneficial microbes in the form of probiotics, fermented foods, and donor feces are being used to manage health. In addition, we explore the rationale for selecting beneficial microbial strains and aligning diets to accommodate their propagation in the gut. A pilot clinical trial design is presented to examine the effects of probiotics and exercise in patients with phenylketonuria (PKU); it is the most common inborn error of amino acid metabolism, and it is a complication that requires lifelong dietary intervention. The example design is provided to illustrate the importance of using omics technology to see if the intervention elevates neuroactive biogenic amines in the plasma; increases the abundance of Eubacterium rectale, Coprococcus eutactus, Akkermansia muciniphila, or Butyricicoccus; and increases Escherichia/Shigella in the gut, all as markers of improved health. By emphasizing the combined importance of diet, microbial supplements, and the gut microbiome, we hope that future studies will better align these components, not only to improve outcomes, but also to enhance our understanding of the mechanisms.

Capacity of a Microbial Synbiotic To Rescue the In Vitro Metabolic Activity of the Gut Microbiome following Perturbation with Alcohol or Antibiotics.

The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.

How Do Lactobacilli Search and Find the Vagina?

With the advent of omics technology and the improved culturing of anaerobic microbes, there is a good understanding of the microorganisms present in a healthy and diseased vagina. This has led to the identification of a select few Lactobacillus species associated with health. However, the origin of these species and how they reach the vagina remains unclear, as does their timing of colonization. In an effort to create badly needed therapies for women, these gaps in knowledge need to be addressed.

Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection.

Paenibacillus larvae is a spore-forming bacterial entomopathogen and causal agent of the important honey bee larval disease, American foulbrood (AFB). Active infections by vegetative P. larvae are often deadly, highly transmissible, and incurable for colonies but, when dormant, the spore form of this pathogen can persist asymptomatically for years. Despite intensive investigation over the past century, this process has remained enigmatic. Here, we provide an up-to-date synthesis on the often overlooked microbiota factors involved in the spore-to-vegetative growth transition (corresponding with the onset of AFB disease symptoms) and offer a novel outlook on AFB pathogenesis by focusing on the 'collaborative' and 'competitive' interactions between P. larvae and other honey bee-adapted microorganisms. Furthermore, we discuss the health trade-offs associated with chronic antibiotic exposure and propose new avenues for the sustainable control of AFB via probiotic and microbiota management strategies.

Functional response to a microbial synbiotic in the gastrointestinal system of children: a randomized clinical trial.

BACKGROUND: Oral microbial therapy has been studied as an intervention for a range of gastrointestinal disorders. Though research suggests that microbial exposure may affect the gastrointestinal system, motility, and host immunity in a pediatric population, data have been inconsistent, with most prior studies being in neither a randomized nor placebo-controlled setting. The aim of this randomized, placebo-controlled study was to evaluate the efficacy of a synbiotic on increasing weekly bowel movements (WBMs) in constipated children. METHODS: Sixty-four children (3-17 years of age) were randomized to receive a synbiotic (n = 33) comprising mixed-chain length oligosaccharides and nine microbial strains, or placebo (n = 31) for 84 days. Stool microbiota was analyzed on samples collected at baseline and completion. The primary outcome was a change from baseline of WBMs in the treatment group compared to placebo. RESULTS: Treatment increased (p < 0.05) the number of WBMs in children with low baseline WBMs, despite broadly distinctive baseline microbiome signatures. Sequencing revealed that low baseline microbial richness in the treatment group significantly anticipated improvements in constipation (p = 0.00074). CONCLUSIONS: These findings suggest the potential for (i) multi-species-synbiotic interventions to improve digestive health in a pediatric population and (ii) bioinformatics-based methods to predict response to microbial interventions in children. IMPACT: Synbiotic microbial treatment improved the number of spontaneous weekly bowel movements in children compared to placebo. Intervention induced an increased abundance of bifidobacteria in children, compared to placebo. All administered probiotic species were enriched in the gut microbiome of the intervention group compared to placebo. Baseline microbial richness demonstrated potential as a predictive biomarker for response to intervention.