Locally advanced rectal cancer (LARC) is treated with neoadjuvant chemo-radiotherapy (nCRT) followed by surgery. A minority of patients show complete response (CR) to nCRT and may avoid surgery and its functional consequences. Instead, most patients show non-complete response (non-CR) and may benefit from additional treatments to increase CR rates. Reliable predictive markers are lacking. Aim of this study was to identify novel signatures predicting nCRT responsiveness. We performed a combined analysis of tumor-associated microbiome and immune gene expression profiling of diagnostic biopsies from 70 patients undergoing nCRT followed by rectal resection, including 16 with CR and 54 with non-CR. Findings were validated by an independent cohort of 49 patients, including 7 with CR and 42 with non-CR. Intratumoral microbiota significantly differed between CR and non-CR groups at genus and species level. Colonization by bacterial species of Ruminococcus genera was consistently associated with CR, whereas abundance of Fusobacterium, Porhpyromonas, and Oscillibacter species predicted non-CR. Immune gene profiling revealed a panel of 59 differentially expressed genes and significant upregulation of IFN-gamma and -alpha response in patients with CR. Integrated microbiome and immune gene profiling analysis unraveled clustering of microbial taxa with each other and with immune cell-related genes and allowed the identification of a combined signature correctly identifying non-CRS in both cohorts. Thus, combined intratumoral microbiome-immune profiling improves the prediction of response to nCRT. Correct identification of unresponsive patients and of bacteria promoting responsiveness might lead to innovative therapeutic approaches based on gut microbiota pre-conditioning to increase nCRT effectiveness in LARC.

Diet is one of the main factors shaping the human microbiome, yet our understanding of how specific dietary components influence microbial consortia assembly and subsequent stability in response to press disturbances - such as increasing resource availability (feeding rate) - is still incomplete. This study explores the reproducible re-assembly, metabolic interplay, and compositional stability within microbial consortia derived from pooled stool samples of three healthy infants. Using a single-step packed-bed reactor (PBR) system, we assessed the reassembly and metabolic output of consortia exposed to lactose, glucose, galacto-oligosaccharides (GOS), and humanized GOS (hGOS). Our findings reveal that complex carbohydrates, especially those containing low inclusion (~1.25 gL-1) components present in human milk, such as N-acetyl-lactosamine (LacNAc), promote taxonomic, and metabolic stability under varying feeding rates, as shown by diversity metrics and network analysis. Targeted metabolomics highlighted distinct metabolic responses to different carbohydrates: GOS was linked to increased lactate, lactose to propionate, sucrose to butyrate, and CO2, and the introduction of bile salts with GOS or hGOS resulted in butyrate reduction and increased hydrogen production. This study validates the use of single-step PBRs for reliably studying microbial consortium stability and functionality in response to nutritional press disturbances, offering insights into the dietary modulation of microbial consortia and their ecological dynamics.

Analysis of the presence and the abundance of microorganisms related to diseases can be used to monitor marine environmental health. Our study evaluated the interference of taxonomic databases (SILVA, Greengenes v13.8, Greengenes2, and RDP) to monitor the distribution of bacterial genera potentially related to diseases in marine organisms (BGPRDs) from low- (Dois Rios Beach-DR), medium- (Abraão Beach-AB) and high (Guanabara Bay-GB) impacted marine environments. The frequency, richness, diversity, and composition of BGPRDs present in DR, AB and GB were significantly influenced by the different databases (p < 0.05). Consequently, the analyses revealed that the use of different databases resulted in controversial results regarding the distribution of BGPRDs in the DR, AB and GB. While Greengenes v13.8 and RDP showed that GB had the highest frequency of BGPRDs (p < 0.05), analysis based on Greengenes2 and SILVA revealed a greater frequency of BGPRDs in AB (p < 0.05). Additionally, there was no congruence of BGPRDs detected by each taxonomic database in DR, AB and GB. In highly-impacted GB, Arcobacter was the main BGPRD obtained with the Greengenes2 and RDP databases, whereas Synechococcus and Alteromonas represented the main BGPRD according to the Greengenes v13.8 and SILVA databases, respectively. Our results showed we cannot determine the exact composition and abundance of BGPRDs in low-, medium- and highly-impacted marine environments. These findings emphasize the critical influence of database choice on microbial community characterization and its implications for effective environmental monitoring and management strategies. Interestingly, alpha diversity indices of BGPRDs obtained from DR, AB and GB were consistent among the different databases and showed greater congruence than did the frequency, richness, distribution and abundance of BGPRDs. The use of diversity indices of BGPRDs can be an alternative to overcome the limitations caused by the bias of taxonomic annotations for biomonitoring marine environments.

Stroke is the second leading cause of death worldwide. Acute ischemic stroke (AIS) patients often exhibit hypercoagulable state and gut microbiota dysbiosis. However, the association between coagulation abnormalities and gut microbiota dysbiosis in AIS patients and their predictive value for poor functional outcomes in AIS has not been investigated. Our study enrolled 95 AIS patients and 81 healthy controls, using 16S rRNA sequencing to analyze gut microbiota composition. Baseline fibrinogen level was found to be an independent risk factor for poor functional outcomes at 90-day follow-up (odds ratio = 2.16, 95% confidence interval: 1.02-4.59, P = 0.044). AIS patients showed significant gut microbiota dysbiosis, with significantly increased Parabacteroides and Alistipes, and decreased Prevotella and Roseburia, associated with coagulation indices. Furthermore, compared with AIS patients with normal coagulation function, those in a hypercoagulable state exhibited a significant increase in Alistipes and a decrease in Prevotella. We identified gut microbial biomarkers consisting of 15 bacteria that predicted poor functional outcome in AIS patients at 90-day follow-up. Coagulation indices improved the predictive performance of these biomarkers. In training and validation cohorts, area under the curve (AUC) values were 0.930 and 0.890 for microbial biomarkers alone, 0.691 and 0.751 for coagulation indices alone, and 0.943 and 0.944 for coagulation indices combined with gut microbial biomarkers. Our study showed that AIS patients with hypercoagulable state had gut microbiota dysbiosis, with Alistipes and Prevotella significantly associated with coagulation indices. A classification model based on coagulation indices and gut microbial biomarkers accurately predicted poor functional outcome in AIS patients at 90-day follow-up.

Acute ischemic stroke (AIS) patients often exhibit hypercoagulable state and gut microbiota dysbiosis. However, the relationship between hypercoagulable state and gut microbiota dysbiosis in AIS patients and their predictive value for poor functional outcomes has not been fully explored. Our study of 95 AIS patients showed that baseline fibrinogen level was an independent risk factor for poor functional outcome at 90-day follow-up in AIS patients. Hypercoagulable state in AIS patients correlates with gut microbiota dysbiosis. AIS patients with hypercoagulable state had increased Alistipes abundance and decreased Prevotella abundance. A classification model based on coagulation indices and gut microbial biomarkers accurately predicted poor functional outcome in AIS patients at 90-day follow-up.

Biofilters are widely utilized for treating odorous gases. Understanding the biosafety of biofilters in real-world applications necessitates a comprehensive investigation into their bioaerosol emission characteristics. In this study, bioaerosols captured from the inlet and outlet, along with packing material microbiome were investigated over a 12-month period from three full-scale biofilters. Their characteristics, size distribution, and sources were investigated and analyzed. Notably, over 96 % of the bioaerosols originated from the inlet air rather than from the packing materials. Full-scale biofilters not only reduced bacterial emissions by 5-17 % but also lowered the potential pathogenicity. Although biofilters effectively diminished bioaerosol emissions, there are still potential pathogenicity primarily originated from the inlet air. This study emphasizes the significance of considering upstream processes when evaluating the risks associated with bioaerosols in real-world applications.

Mounting evidence suggests that sustained microbial dysbiosis is associated with the development of multiple cancers, while the species-level bacterial taxa and metabolic dysfunction of oral microbiome in patients with head and neck squamous cell carcinoma (HNSCC) remains unclear. In this cross-sectional study, comprehensive metagenomic and 16S rRNA amplicon sequencing analyses of oral swab samples from 172 patients were performed. Unsupervised clustering algorithms of relative microbial abundance profiles revealed three distinctive microbiome clusters. Based on the metagenomic and 16S rRNA amplicon sequencing data, machine learning-based methods were used to construct the HNSCC diagnostic classifier, which exhibited high area under the curve values of 0.78-0.89. Our study provided the first exhaustive metagenomic and 16S rRNA amplicon sequencing analyses to date, revealing that microbial-metabolic dysbiosis is a potential risk factor for HNSCC progression and therefore providing a robust theoretical basis for potential diagnostic and therapeutic strategies for HNSCC patients.

Soil bacterial and fungal communities play fundamental roles in biogeochemical cycles and ecosystem stability. Urbanization alters soil properties and microbial habitats, driving shifts in community composition, yet the divergent responses of bacteria and fungi and their ecological consequences remain inadequately understood. To elucidate these differential responses, we investigated soil bacterial and fungal communities along an urbanization gradient, ranging from undisturbed reference forests to urban parks, across three distinct climatic regions. To capture different disturbance intensities, urban parks were classified by tree age into old parks (>60-year-old trees) and young parks (10-20-year-old trees). Climate had a strong influence on soil microbiota, yet urbanization still significantly altered both bacterial and fungal communities in all regions. Urban disturbances homogenized soil microbial communities: average similarity among bacterial communities increased from ∼79 % in forests to ∼85 % in young urban parks, indicating substantial homogenization, whereas fungal communities showed little homogenization. Urbanization also homogenized microbial functional traits, with a greater reduction in trait dissimilarity for bacteria than for fungi. Bacterial communities exhibited high adjustability to urban conditions, dominated by generalist taxa (∼90 %), whereas fungal communities consisted mostly of specialists (∼83 %). Despite these asynchronous responses-bacteria adjusting and homogenizing more than fungi-overlapping functional traits between bacteria and fungi help maintain functional resilience in urban ecosystems.

The assembly of the rhizosphere microbiome determines its functionality for plant fitness. Although the interactions between arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) play important roles in plant growth and disease resistance, research on the division of labor among the members of the symbionts formed among plants, AMF, and PGPR, as well as the flow of carbon sources, is still insufficient. To address the above questions, we used soybean (Glycine max), Funneliformis mosseae, and Pseudomonas putida KT2440 as research subjects to establish rhizobiont interactions and to elucidate the signal exchange and division of labor among these components. Funneliformis mosseae can attract P. putida KT2440 by secreting cysteine as a signaling molecule and can promote the colonization of P. putida KT2440 in the soybean rhizosphere. Colonized P. putida KT2440 can stimulate the l-tryptophan secretion of the host plant and can lead to the upregulation of genes involved in converting methyl-indole-3-acetic acid (Me-IAA) into IAA in response to l-tryptophan stimulation. Collectively, we decipher the tripartite mechanism of rhizosphere microbial community assembly via cross-kingdom interactions.

The effect of founders (the potential influence of initially colonizing species on the composition, functionality, and stability of communities) plays a crucial role in community assembly; many experimental studies on priority effects or artificially assembled species have suggested the existence of this effect, but direct experimental evidence at the community level remains limited. This study used sterilized and nonsterilized paocai soup (a traditional Chinese fermented vegetable soup) from the same source to simulate initial environments with and without founders. These were placed in beakers with varying opening sizes on an open rooftop for 15 days to explore the impact of founders on community assembly under different dispersal intensities. The 16S rRNA sequencing analysis revealed that communities with founders exhibited lower species richness (320) compared to communities without founders (645). Additionally, communities with founders showed reduced species turnover and richness variation (53.7%) compared to communities without founders (60.9%). Furthermore, the average variability degree (AVD) in communities with founders (0.446 ± 0.044) was significantly lower than in communities without founders (0.927 ± 0.466), indicating higher community stability. Finally, deterministic processes dominated communities with founders (with heterogeneous selection contributing 70%), whereas stochastic processes primarily governed communities without founders (homogeneous dispersal 10% and undominated processes 70%). These findings demonstrate that founders presence reduces dispersal impacts, decreases community diversity, enhances stability, and deterministic processes. The effect of founders fundamentally shapes the direction of community assembly. This study helps further understanding of how founders influence biodiversity maintenance and community assembly processes.

Passive treatment using microorganisms is a promising method for removing toxic metals from acid mine drainage (AMD). To provide a better understanding of the compositions of mine microbial communities and their relationship with pH and metal concentrations in mine water, we analyzed both physicochemical data and 16S rRNA gene amplicon sequences from 40 untreated drainages from 30 abandoned mines across all regions of Japan. In the 40 mine drainages, higher concentrations of metals (iron, lead, cadmium, copper, and manganese) were detected in mine drainages with acidic pH (< 4.5; 24 samples) than in those with circumneutral pH (> 6.0; 16 samples), except for arsenic. Comparison of principal coordinate analysis scatter plots between mine drainages of acidic and circumneutral pH revealed a relatively clear separation of microbial compositions except for one mine drainage sample. In the 24 AMDs, the dominant operational taxonomic units (OTUs) found at relative abundance levels >3 % were generally one of three related to Caballeronia arationis, Ferrovum myxofaciens, or Gallionella capsiferriformans, except for two mine drainage samples. Although the microbial communities of the 40 mine drainages were obtained from a single sampling time (triplicate analysis), they may reflect the typical microbial communities of each mine drainage, because analysis of drainage samples taken from the same sampling point over three different seasons (summer, autumn, and winter) revealed similar communities regardless of the season. Among mine drainages analyzed in this study, the OTU showing 96.8 % identity to C. arationis was the most prevalent bacterial OTU, which was found in 23 drainages (the top OTU in 15 mine drainages) with >3 % relative abundance.

Bacteria are the major component of poultry gastrointestinal tract (GIT) microbiota and play an important role in host health, nutrition, physiology regulation, intestinal development, and growth. Bacterial community profiling based on the 16S ribosomal RNA (rRNA) gene amplicon sequencing approach has become the most popular method to determine the taxonomic composition and diversity of the poultry microbiota. The 16S rRNA gene profiling involves numerous steps, including sample collection and storage, DNA isolation, 16S rRNA gene primer selection, Polymerase Chain Reaction (PCR), library preparation, sequencing, raw sequencing reads processing, taxonomic classification, α- and β-diversity calculations, and statistical analysis. However, there is currently no standardized protocol for 16S rRNA gene analysis profiling and data deposition for poultry microbiota studies. Variations in DNA storage and isolation, primer design, and library preparation are known to introduce biases, affecting community structure and microbial population analysis leading to over- or under-representation of individual bacteria within communities. Additionally, different sequencing platforms, bioinformatics pipeline, and taxonomic database selection can affect classification and determination of the microbial taxa. Moreover, detailed experimental design and DNA processing and sequencing methods are often inadequately reported in poultry 16S rRNA gene sequencing studies. Consequently, poultry microbiota results are often difficult to reproduce and compare across studies. This manuscript reviews current practices in profiling poultry microbiota using 16S rRNA gene amplicon sequencing and proposes the development of guidelines for protocol for 16S rRNA gene sequencing that spans from sample collection through data deposition to achieve more reliable data comparisons across studies and allow for comparisons and/or interpretations of poultry studies conducted worldwide.

Antibiotic resistance has become a global health concern, driving the need for sustainable alternatives in animal husbandry. This study explores the potential of natural feed additives as a viable solution to enhance poultry growth and health while reducing reliance on antibiotics. Chinese herbal medicines and probiotics have been widely studied as green, healthy, and safe antibiotic alternatives in livestock and poultry production. A total of 120 chickens were randomly divided into four groups: a control group and three treatment groups supplemented with 1% Pulsatilla chinensis powder, 3% fresh Acer truncatum, or 1% Clostridium butyricum. The results showed that Pulsatilla chinensis powder significantly increased gamma-glutamylcysteine (p < 0.05), UDP-N-acetylglucosamine (p < 0.05), tyramine (p < 0.01), and leucine (p < 0.05). Acer truncatum notably altered cecal metabolites, including L-tyrosine (p < 0.05), α-ketoisovaleric acid (p < 0.01), myristoleic acid (p < 0.01), glutathione (p < 0.05), and PGA1 (p < 0.05). Clostridium butyricum modified cecal metabolites such as L-glutamine (p < 0.05), riboflavin (p < 0.05), L-Carnitine (p < 0.05), ergocalciferol (p < 0.01), and α-tocotrienol (p < 0.05).

Antibiotic resistance poses a significant threat to human health, and wastewater treatment plants (WWTPs) are important reservoirs of antibiotic resistance genes (ARGs). Here, we analyze the antibiotic resistomes of 226 activated sludge samples from 142 WWTPs across six continents, using a consistent pipeline for sample collection, DNA sequencing and analysis. We find that ARGs are diverse and similarly abundant, with a core set of 20 ARGs present in all WWTPs. ARG composition differs across continents and is distinct from that of the human gut and the oceans. ARG composition strongly correlates with bacterial taxonomic composition, with Chloroflexi, Acidobacteria and Deltaproteobacteria being the major carriers. ARG abundance positively correlates with the presence of mobile genetic elements, and 57% of the 1112 recovered high-quality genomes possess putatively mobile ARGs. Resistome variations appear to be driven by a complex combination of stochastic processes and deterministic abiotic factors.

The persistence and specificity of fish host-microbial interaction during evolution is an important part of exploring the host-microbial symbiosis mechanism. However, it remains unclear how the environmental and host factors shape fish host-microbe symbiotic relationships in subtropical rivers with complex natural environments. Freshwater fish are important consumers in rivers and lakes and are considered keystone species in maintaining the stability of food webs there. In this study, patterns and mechanisms shaping gut microbiota community in 42 fish species from the Pearl River, in the subtropical zone of China, were investigated. The results showed that fish host specificity is a key driver of gut microbiota evolution and diversification. Different taxonomic levels of the host showed different degrees of contribution to gut microbiota variation. Geographical location and habitat type were the next most important factors in shaping gut microbiota across the 42 fishes, followed by diet and gut trait. Our results emphasized the contribution of stochastic processes (drift and homogenizing dispersal) in the gut microbial community assembly of freshwater fishes in the middle and lower reaches of the Pearl River. Phylosymbiosis is evident at both global and local levels, which are jointly shaped by complex factors including ecological or host physiological filtration and evolutionary processes. The core microbiota showed co-evolutionary relationships of varying degrees with different taxonomic groups. We speculate that host genetic isolation or habitat variation facilitates the heterogeneous selection (deterministic process), which occurs and results in different host-core bacterium specificity.

Freshwater fish are regarded as the dominant consumers in rivers and lakes. Due to their diverse feeding modes, fish significantly enhance the trophic link and nutrient recycling/retention in aquatic habitats. For this, they are often considered keystone species in maintaining the stability of food webs in rivers and lakes. A significant part of fish nutrition is essentially mediated by their gut microbiota, which can enhance fish tolerance to fluctuations in external resources and improve the efficiency of nutrients extracted from various food sources. As gut bacterial symbionts have a profound impact on the nutrition and development of their hosts, as well as their overall fitness, it is critical to answer the question of how hosts maintain these benefits by procuring or inheriting these vital symbionts, which is still largely unanswered, especially for freshwater fish. Our study provides new insights into the co-evolutionary relationship between wild fish and their symbiotic microbiome, the hidden diversity of gut microbiome, and the ecological adaptation potential of wild freshwater fish.

Cyanobacterial harmful algal blooms pose significant threats to global water supplies, ecosystems, and economies. Among the harmful cyanobacteria, Planktothrix, a resilient and toxin-producing filamentous cyanobacterium, has garnered increasing attention. However, an understanding of the entire microbiome, particularly the phycosphere surrounding Planktothrix blooms, remains largely unexplored. To the best of our knowledge, this is the first comprehensive study combining 16S rDNA and fungal internal transcribed spacer amplicon sequencing and shotgun metagenomics to elucidate Planktothrix bloom microbiomes and identify potential microbial or functional biomarkers for CyanoHABs. Our observations revealed that a summer bloom in Grand Lake St. Marys was initiated with Dolichospermum and then shifted to Planktothrix dominance. This transition was associated with nitrogen metabolism genes, suggesting that nitrogen plays a key role in bloom persistence through interactions among nitrogen-fixing bacteria, ammonia-oxidizing archaea, anammox bacteria, and denitrifiers. Additionally, metagenomic data revealed a strong positive correlation of toxin concentration with carbohydrate-nitrogen-sulfur-fatty acid associated metabolic pathways and a strong negative correlation with pollutant degradation pathways. Intriguingly, diazotrophic methane-related microbes were detected, which opens discussion on potential symbiosis that couples nitrogen and carbon metabolism. Toxin-degrading bacteria, such as Polynucleobacter and Acidovorax, were positively correlated with fungi like Vishniacozyma, proposing their cooperative roles during bloom events. Notably, Rhodobacter, a photosynthetic purple non-sulfur bacterium, showed strong negative correlations with both Planktothrix and the toxin-producing gene mcyE, positioning it as a promising biomarker for early bloom detection. Overall, this study advances the understanding of Planktothrix-dominated bloom ecology and highlights microbial signatures for proactive CyanoHAB management in freshwater systems.

Agarwood is a valuable resinous aromatic substance known to have healing properties. Only plants in the Thymelaeace family, specifically the genera Aquilaria and Gyrinops, produce it to defend themselves from insect, bacterial, and fungal attacks. Over-harvesting of natural agarwood has given rise to different artificial agarwood induction techniques. However, the contribution of endophytic microorganisms in this process remains largely unknown. Herein, we employed fire drills and fire drill + brine treatments and investigated their impact on metabolite constituents and endophytes over time. Gas chromatography-mass spectrometry was used to examine the metabolites, and 16 S and ITS amplicon sequencing were applied to check for endophytes. Metabolites from each treatment were related to 16 S and ITS amplicon sequencing results from different times to find out what effect the treatments had and what role endophytes played in making agarwood. Fire drills with 50-80 ml of 0.4 mmol brine treatment resulted in the highest production of essential oil from agarwood, whereas fire drills with 50-80 ml of 4.0 mmol brine treatment produced the highest number of metabolites. Agarwood fragrant compounds such as chromones, sesquiterpenes, and agarotetrol were prominent among the differentially expressed metabolites and were positively associated with the abundance of bacterial endophytes Acidobacteriota, Chlamydiae, Bacteroidota, and Actinobacteria; and the fungal endophytes Rozellomycota, Basidiomycota, Aphelidiomycota, and Mortierellomycota. Saprotrophs and pathogens were prevalent in the treatment groups; however, with time, their proportion declined as Symbiotroph's proportion inclined, indicating successive roles of these fungi in agarwood induction and propagation. Among them, Acidobacteriota, Chlamydiae, Bacteroidota, and Actinobacteria bacterial; and Rozellomycota, Basidiomycota, Aphelidiomycota, and Mortierellomycota fungal endophytes are correlated with enhanced levels of essential agarwood fragrances such as Chromones, Sesquiterpenes, and Agarospirol. Our results show how important bacterial and fungal endophytes are for making agarwood. They also show how these endophytes change when interacting with the host plant after a fire drill and a brine treatment. A moderate brine concentration (0.4 mmol) following a fire drill can thus be employed as a sustainable agarwood production practice.

This study investigates the influence of three regulators of human homeostasis-intestinal microbiota, immune profile, and circulating metabolites-on stroke severity. We conducted a study involving 33 patients with mild/moderate stroke (MS) and 32 with severe stroke (SS), all assessed during the acute phase (first 24 h). The analysis focused on microbiota composition (45 patients), serum metabolomics and inflammatory markers (20 patients). The patients with SS exhibited more pronounced insulin resistance associated with increased levels of branched-chain amino acids and their metabolites. SS patients showed an increase in inflammatory cytokines IL-6 and TNF-α, and surprisingly an increase in IL-10 and butyrate which are anti-inflammatory. SS patients also displayed a distinct microbiota profile, with statistically significant differences in β-diversity compared to the MS group, notably a higher prevalence of Pseudomonadota (formerly Proteobacteria). In summary, our data indicate that patients with SS, compared to those with MS, are characterized by a more inflammatory and insulin-resistant state, associated with three key regulators: microbiota, metabolites, and interleukins. These findings provide new insights into the regulatory components of the gut-brain axis, which could be developed into cost-effective and widely accessible therapies for SS.

The gut microbiome is recognized as a critical factor in advancing precision nutrition and medicine for health and in developing dietary recommendations and targeted therapies for gastrointestinal (GI) health and diseases. However, conventional sampling methods, such as fecal analysis and colonoscopy, often fail to capture microbial information from specific regions of the GI tract or require invasive procedures, thereby limiting accuracy and clinical utility. As a non-invasive alternative, passive sampling capsules have been developed for site-specific microbiome analysis by employing pH-sensitive enteric coatings that delay sampling until the capsule reaches the targeted intestinal region. Although this approach has been successful in the small intestine, colonic sampling remains challenging due to the high interpersonal variability in intestinal pH, which makes it difficult to rely solely on a pH-triggering mechanism. To overcome this challenge, a dual bacterially and pH triggered polymeric enteric coating was created by blending lactulose and N,N-dimethylaminoethyl methacrylate, enabling complete dissolution within the colonic region. Through systematic characterization of multiple polymer blend compositions using Fourier Transform Infrared Spectroscopy, Thermogravimetric Analysis, and Differential Scanning Calorimetry, an optimized design was identified that provides both suitable physical integrity and rapid (∼2 h) degradation in the presence of colonic bacteria, across a pH range of 5 to 8. The optimized blend was subsequently applied as a double-layer enteric coating on a sampling capsule, enabling the dissolution of the outer layer in the small intestine and complete dissolution of the inner layer in the colon. In-vitro and in-vivo pig model studies were conducted to validate the capsule's sampling performance and to ensure the preservation of the microbial environment. Furthermore, 16S rRNA sequencing revealed a taxonomic similarity between samples collected by the capsule and the colonic microbiome (residing between the ileum and fecal matter). Overall, this technology provides an effective approach to targeted microbial sampling and may pave the way for more comprehensive colonic microbiome analyses and improved diagnostic capabilities for GI diseases. STATEMENT OF SIGNIFICANCE: Precise monitoring of the gut microbiome is vital for understanding health and disease, yet current sampling techniques often lack precision or require invasive procedures. Our work introduces a novel, non-invasive capsule that targets the colon using a dual-trigger polymer system activated by both pH and colonic bacteria. This design enables localized sampling of gut microbiota, overcoming the limitations of fecal analysis, endoscopy, and earlier pH-triggered capsule designs. By capturing microbial communities directly from the colon, our technology provides deeper insights into colonic health and conditions such as inflammatory bowel disease and colorectal cancer. This breakthrough represents a significant advancement in precision nutrition and medicine for human health, and advanced diagnostics and targeted therapies to support dietary guidance, clinical practice and biomedical research.

Antibiotics in early life can promote adiposity via interactions with the gut microbiota, but they represent only one possible route of antimicrobial exposure. Dietary preservatives exhibit antimicrobial activity, contain chemical structures accessible to microbial enzymes, and may therefore similarly disrupt microbial contributions to metabolic development.

Here, we test the hypothesis that preservatives alter the gut microbiota with consequences for host metabolism.

We screened common dietary preservatives for in vitro and ex vivo activity against a panel of gut bacteria and whole fecal microbial communities, profiling outcomes via optical density measurements and 16S rDNA sequencing. We then exposed adult mice to diet-relevant doses of 4 preservatives [acetic acid, butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA), and sodium sulfite] or ampicillin (positive control) for 7 d. Finally, we examined the effects of early-life EDTA and low-dose ampicillin exposure starting in gestation in a mouse model, tracking differences in growth and metabolism.

Preservatives altered microbial growth and community structure in vitro, ex vivo, and in vivo, but with compound-specific changes in gut microbiota composition distinct from those of ampicillin. Long-term EDTA exposure from gestation reduced calorie absorption and cecal acetate, resulting in 32% lower gains in body fat in females for a given food intake (±12% standard error, linear mixed effects model). Females exposed to ampicillin exhibited a similar 42% (±11%) reduction in food-adjusted gains in adiposity, along with larger brains and smaller livers. By contrast, among males, EDTA had no detectable metabolic impacts whereas ampicillin exposure increased food-adjusted gain in body fat by 108% (±12%).

Our results highlight the potential for everyday doses of common preservatives to affect the gut microbiota and impact metabolism differently in males and females. Thus, despite their generally regarded as safe designation, preservatives could have unintended consequences for consumer health via their impact on the gut microbiota.

Studies on human gut microbiota have recently highlighted a significant decline in bacterial diversity associated with urbanization, driven by shifts toward processed diets, increased antibiotic usage, and improved sanitation practices. This phenomenon has been largely overlooked in the Colombian Amazon, despite rapid urbanization in the region. In this study, we investigate the composition of gut bacterial microbiota and intestinal protozoa and soil-transmitted helminths (STHs) in both urban and rural areas of Leticia, which is located in the southern Colombian Amazon. Despite their geographic proximity, the urban population is predominantly non-indigenous, while indigenous communities mostly inhabit the rural area, resulting in notable lifestyle differences between the two settings. Our analyses reveal a reduction in bacterial families linked to non-processed diets, such as Lachnospiraceae, Spirochaetaceae, and Succinivibrionaceae, in the urban environment compared to their rural counterparts. Interestingly, Prevotellaceae, typically associated with non-processed food consumption, shows a significantly higher abundance in urban Leticia. STH infections were primarily detected in rural Leticia, while intestinal protozoa were ubiquitous in both rural and urban areas. Both types of parasites were associated with higher gut bacterial richness and diversity. Additionally, microbial metabolic prediction analysis indicated differences in pathways related to unsaturated fatty acid production and aerobic respiration between rural and urban bacterial microbiomes. This finding suggests a tendency towards dysbiosis in the urban microbiota, possibly increasing susceptibility to non-communicable chronic diseases. These findings provide new insights into the impact of urbanization on gut microbiota dynamics in the Amazonian context and underscore the need for further research to elucidate any associated health outcomes.

The Zoige uranium mine is situated in the harsh, cold northern region of Sichuan, characterized by its high altitude and fragile ecosystem. Uncovering the organisms that thrive in such extreme climates, particularly microorganisms, is of paramount importance for advancing bioremediation efforts. Herein, the potential functional microbiota for uranium sequestration in Zoige uranium mine soil was explored using high-throughput sequencing combined with bioinformatics analysis. Analysis of the physicochemical properties of soils showed that the concentration of uranium ranged from 35.20 to 40.62 µg·g-1 around the uranium mine. Bacterial communities differed significantly in soils around the Zoige uranium mine, with the most abundant phyla being Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, Gemmatimonadota, Verrucomicrobia, and Firmicutes. Notably, Actinobacteria was considered a biomarker for distinguishing soils with high uranium by linear discriminant analysis effect size. Meanwhile, the correlation analysis demonstrated that Firmicutes and Cyanobacteria were significantly and positively associated with uranium in soil samples, with the correlation coefficients being 0.8601 and 0.7832, respectively. Furthermore, the phylogenetic investigation of communities by reconstruction of unobserved states analysis revealed that the bacterial microbiota was mainly enriched in biosynthesis function in these soils. Interestingly, the abundance of functional genes involved in amino acid biosynthesis increased whereas that related to fatty acid biosynthesis decreased with an increase in uranium content. Taken together, Actinobacteria, Firmicutes, and Cyanobacteria were the potential functional microbiota for uranium sequestration via amino acid and fatty acid biosynthesis pathways in Zoige uranium mine soil. These findings are conducive to obtaining functional strains for developing microbial remediation technologies for uranium contamination.IMPORTANCEBased on the significance of the Zoige uranium mine and its unique ecological environment, this study emphasizes the necessity of in situ bioremediation. Herein, the potential functional microbiota for uranium sequestration in Zoige uranium mine soil was explored using high-throughput sequencing and bioinformatics analysis. Actinobacteria, Firmicutes, and Cyanobacteria were the potential functional microbiota in Zoige uranium mine soils. These microbes interacted and tolerated uranium via amino acid and fatty acid biosynthesis pathways. These findings provide insights into the functional microbiota of uranium sequestration, which are conducive to developing microbial resources and bioremediation technology for treating uranium contamination.

Sorghum bicolor, an important global crop, adapted to thrive in hotter and drier conditions than maize or rice, has deep roots that interact with a stratified soil microbiome that plays a crucial role in plant health, growth, and carbon storage. Microbiome studies on agricultural soils, particularly fields growing S. bicolor, have been mostly limited to surface soils (<30 cm). Here we investigated the abiotic factors of soil properties, field location, depth, and the biotic factors of sorghum type across 38 genotypes of the soil microbiome. Utilizing 16S rRNA gene amplicon sequencing, our analysis reveals significant changes in microbial composition and decreasing diversity at increasing soil depths within S. bicolor fields, regardless of genotype or field, with microbial richness and diversity declining to a minimum at the 60-90 cm layer and increasing beyond the 90 cm depth. Notably, specific microbial families, such as Thermogemmatisporaceae and an unclassified family within the ABS-6 order, were enriched in deeper soil layers beyond 30 cm. These findings highlight the importance of soil depth in agricultural soil microbiome studies.IMPORTANCESorghum bicolor is a valuable model for studying the microbiome in deep soils, which is crucial for enhancing carbon sequestration in agricultural systems. As we look to crops with deeper roots for improved carbon storage, it is essential to move beyond the traditional focus on surface soils in agricultural settings. This study shifts that focus by investigating microbial dynamics at greater soil depths, revealing significant changes in microbial composition and diversity with increasing depth, revealing the critical role of deep-soil microbiomes in nutrient cycling and carbon sequestration in agricultural fields with the deep-rooted crop S. bicolor. By exploring these processes beyond surface soils, this research supports the development of sustainable agricultural practices that can better harness the potential of deep-rooted crops for long-term carbon storage.

Aleurone, derived from the bran layer of grains like wheat and barley, has demonstrated positive effects on energy metabolism in pigs, mice, and untrained horses, influencing glucose-insulin dynamics and gut microbiome composition. Training itself enhances insulin sensitivity in horses, similar to the improvements in performance capacity observed in human athletes. This study aimed to investigate whether aleurone supplementation provides additional benefits to training by modulating insulin metabolism and gut microbiota in Standardbred mares.

Sixteen Standardbred mares (aged 3-5 years) participated in a cross-over study with two 8-week training periods separated by 8 weeks of detraining. Each horse received either 200 g/day aleurone supplementation or a control diet. Insulin metabolism was evaluated using oral (OGTT) and intravenous (FSIGTT) glucose tolerance tests, measuring parameters such as Maximumglucose, AUCglucose, Maximuminsulin, AUCinsulin, Time to peakinsulin (OGTT), Acute Insulin Response to Glucose (AIRg), glucose effectiveness (Sg), and disposition index (DI) (FSIGTT). Fecal samples underwent metagenomic analysis to assess alpha and beta diversity and microbial composition.

Training alone: Training significantly improved OGTT parameters by decreasing Maximuminsulin (P = 0.005) and AUCinsulin (P = 0.001), while increasing Time to peakinsulin (P = 0.03), indicating enhanced insulin sensitivity. FSIGTT results also showed a decrease in logAIRg (P = 0.044). Training with Aleurone: Aleurone supplementation further reduced FSIGTT AIRg (P = 0.030), logAIRg (P = 0.021) while increasing glucose effectiveness (Sg; P = 0.031). These findings suggest aleurone improves insulin sensitivity, glucose disposal, and fasting glucose regulation beyond training. Microbiome analysis revealed training decreased Pseudomonas, associated with dysbiosis, while aleurone reduced inflammation-associated Desulfovibrio. Beta diversity metrics showed no significant changes.

Aleurone supplementation enhances training-induced improvements in glucose metabolism and fecal microbiota composition, which could offer potential benefits for equine athletes by optimizing metabolic flexibility. It also supports improvements in glucose and insulin dynamics, particularly by further enhancing insulin sensitivity and glucose-mediated disposal. Future studies should investigate the mechanisms of aleurone at the muscle and gut level and explore its potential applications for metabolic disorders such as Equine Metabolic Syndrome.

Variation in gastric cancer (GC) incidence across different geographic areas persists, even when there are similar prevalence rates of Helicobacter pylori (H. pylori) infection. An extensive examination of the gastric microbiota in populations from both high- and low-risk regions of GC could help explain the geographical disparities in GC incidence.

This study enrolled a total of 130 patients with superficial gastritis (SG) and precancerous lesions of gastric cancer (PLGC) from a high-risk area for GC and 205 patients from a low-risk area. Gastric microbial profiling was performed using 16 S rRNA gene sequencing to analyze differences in microbial composition between regions and lesion types.

The study revealed significant differences in gastric microbial profiles between patients from high- and low-risk regions, particularly in PLGC patients. PLGC patients from the low-risk region exhibited higher microbial richness than those from the high-risk area, with marked distinctions in microbial community structure between the two regions. Specific differences in microbial composition were observed at the phylum and genus levels between different regions. Six bacterial genera, including Selenomonas and Peptostreptococcus, were identified as enriched in PLGC patients from the high-risk area. Additionally, there was a noticeable imbalance in the microbiota of the gastric mucosal lining during the progression of gastric lesions.

This comparative analysis highlights the potential impact of the gastric microbiome in the development of GC and suggests that regional differences in microbial profiles may provide clues to the varying incidence rates of GC.

Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of female death. Altered interactions between the host and the gut microbiota appear to play an influential role in carcinogenesis. Several studies have shown different signatures of the gut microbiota in patients with breast cancer compared to healthy women. Currently, there is disagreement regarding the different DNA isolation and sequencing methodologies for studies on the human microbiota, given that they can influence the interpretation of the results obtained. The goal of this work was to compare (1) three different DNA extraction strategies to minimize the impact of human DNA, and (2) two sequencing strategies (16S rRNA and shotgun) to identify discrepancies in microbiome results. We made use of breast tissue and fecal samples from both healthy women and breast cancer patients who participated in the MICROMA study (reference NCT03885648). DNA was isolated by means of mechanical lysis, trypsin, or saponin. The amount of eukaryotic DNA isolated using the trypsin and saponin methods was lower compared to the mechanical lysis method (mechanical lysis, 89.11 ± 2.32%; trypsin method, 82.63 ± 1.23%; saponin method, 80.53 ± 4.09%). In samples with a predominance of prokaryotic cells, such as feces, 16S rRNA sequencing was the most advantageous approach. For other tissues, which are expected to have a more complex microbial composition, the need for an in-depth evaluation of the multifactorial interaction between the various components of the microbiota makes shotgun sequencing the most appropriate method. As for the three extraction methods evaluated, when sequencing samples other than stool, the trypsin method is the most convenient. For fecal samples, where contamination by host DNA is low, no prior treatment is necessary.

Most infants with febrile urinary tract infection (UTI) do not have an underlying anatomical risk factor. Thus, other non-anatomical risk factors should be considered. Since the most common pathogens arise from the fecal microbiota, our aim was to investigate whether the gut microbiota composition differs between febrile infants younger than 2 months with or without UTI.

In this prospective, case-control, pilot study, we performed 16S ribosomal ribonucleic acid amplicon sequencing to characterize gut microbiota of febrile neonates with and without UTI admitted to the pediatric ward at Shamir Medical Center between February 2019 and May 2021.

The study cohort included 42 febrile neonates: 17 with and 25 without febrile UTI. We found a significant difference in beta diversity (i.e. between-sample/study group similarity indices) between the UTI and non-UTI group (p = 0.016). There were also distinct differences in the relative abundance of the 20 most prevalent genera. Furthermore, several genera were significantly enriched in the UTI group, with others dominating the non-UTI group. Streptococci were underrepresented in the UTI group. There was no difference in alpha diversity (i.e. within-sample diversity/richness) between groups.

Febrile neonates with UTI have a different fecal microbiota composition (beta-diversity), but not alpha diversity, in comparison to febrile neonates without UTI. A larger study is warranted to confirm these findings and their potential applications.

Metagenomics holds promise as a comprehensive, nontargeted tool for environmental monitoring. However, one key limitation is that the quantitative capacity of metagenomics is not well-defined. Here, we demonstrated a quantitative metagenomic technique and benchmarked the approach for wastewater-based surveillance of antibiotic resistance genes. To assess the variability of low-abundance oligonucleotide detection across sample matrices, we spiked DNA reference standards (meta sequins) into replicate wastewater DNA extracts at logarithmically decreasing mass-to-mass percentages (m/m%). Meta sequin ladders exhibited strong linearity at input concentrations as low as 2 × 10-3 m/m% (R2 > 0.95), with little to no reference length or GC bias. At a mean sequencing depth of 94 Gb, the limits of quantification (LoQ) and detection were calculated to be 1.3 × 103 and 1 gene copy per μL DNA extract, respectively. In wastewater influent, activated sludge, and secondary effluent samples, 27.3, 47.7, and 44.3% of detected genes were ≤LoQ, respectively. Volumetric gene concentrations and log removal values were statistically equivalent between quantitative metagenomics and ddPCR for 16S rRNA, intI1, sul1, CTX-M-1, and vanA. The quantitative metagenomics benchmark here is a key step toward establishing metagenomics for high-throughput, nontargeted, and quantitative environmental monitoring.

The widespread commercialization of genetically modified (GM) crops makes it important to assess the potential impact of Bacillus thuringiensis (Bt) on non-target organisms. Pardosa astrigera is an important predator in agroforestry ecosystems, and female and male spiders may react differently to Bt toxins due to their different activity habits and nutritional requirements. In this study, we found that exposure to Cry2Aa protein did not affect the survival and body weight of P. astrigera during growth and development. However, according to 16S rRNA sequencing results of the P. astrigera adults, Cry2Aa protein not only changed the diversity of symbiont bacteria, but also changed its symbiont composition. During feeding on prey without Bt artificial feed, the dominant communities in female and male adults were Actinobacteria and Corynebacterium-1, respectively. Feeding on prey containing Cry2Aa protein, Firmicutes were the dominant phyla. At the genus level, Cry2Aa protein significantly increased the relative abundance of Enterococcus and became the dominant genus in females only. In addition, Bacillus, Weissella and other symbiotic bacteria had significant changes in females. In terms of species composition, sex differences resulted in the absence of different types of symbiotic bacteria. Functional analysis of enrichment pathways showed significant changes in various metabolic pathways such as "Carbohydrate metabolism" and "Nucleotide metabolism", and there are differences between the sexes. These findings provide new data information and support for revealing the different strategies of spiders to cope with Cry2Aa protein based on sex differences, and also provide new data information and support for environmental safety assessment of GM crops.

Background/Objectives: Dietary fibre promotes health, partly by mediating gut microbiota and short-chain fatty acid (SCFA) production. Pregnancy alters the relationship between dietary composition and the gut microbiota, and it is unclear if fibre intake during late pregnancy alters the abundance of SCFA bacteria and circulating SFCA concentrations. The aim of this study was to determine the impact of dietary fibre on faecal microbiome composition and circulating concentrations of SCFA acetate, butyrate, and propionate in late pregnancy. We also aimed to assess the impact of propionate treatment on placental function using cultured placental explants. Methods: 16S rRNA gene amplicon sequencing was performed on faecal DNA collected at 28 weeks of gestation from participants enrolled in the SPRING cohort study consuming a low or adequate fibre diet. Circualting SCFA were assessed. Placental explants were treated with sodium propionate. Results: Fibre intake did not impact microbial diversity or richness but did impact the abundance of specific bacterial genera. Pregnant participants with low-fibre diets had a greater abundance of Bacteroides and Sutterella, and dietary fibre intake (mg/day) negatively correlated with genera, including Sutterella, Bilophila, and Bacteroides. SCFA concentrations did not differ between groups but circulating concentrations of acetate, propionate, and butyrate did correlate with the abundance of key bacterial genera. Propionate treatment of placental explants did not alter mRNA expression of fatty acid receptors, antioxidants, or markers of apoptosis, nor did it impact pAMPK levels. Conclusions: This study demonstrates that the impact of dietary fibre on SCFA concentrations in pregnant women is modest, although this relationship may be difficult to discern given that other dietary factors differed between groups. Furthermore, this study demonstrates that propionate does not impact key pathways in placental tissue, suggesting that previous associations between this SCFA and placental dysfunction may be due to other maternal factors.

The honeybee (Apis mellifera) is a key pollinator critical to global agriculture, facing threats from various stressors, including the ectoparasitic Varroa mite (Varroa destructor). Previous studies have identified shared bacteria between Varroa mites and honeybees, yet it remains unclear if these bacteria assemble similarly in both species. This study builds on existing knowledge by investigating co-occurrence patterns in the microbiomes of both Varroa mites and honeybees, shedding light on potential interactions. Leveraging 16S rRNA datasets, we conducted co-occurrence network analyses, explored Core Association Networks (CAN) and assess network robustness. Comparative network analyses revealed structural differences between honeybee and mite microbiomes, along with shared core features and microbial motifs. The mite network exhibited lower robustness, suggesting less resistance to taxa extension compared to honeybees. Furthermore, analyses of predicted functional profiling and taxa contribution revealed that common central pathways in the metabolic networks have different taxa contributing to Varroa mites and honeybee microbiomes. The results show that while both microbial systems exhibit functional redundancy, in which different taxa contribute to the functional stability and resilience of the ecosystem, there is evidence for niche specialization resulting in unique contributions to specific pathways in each part of this host-parasite system. The specificity of taxa contribution to key pathways offers targeted approaches to Varroa microbiome management and preserving honeybee microbiome. Our findings provide valuable insights into microbial interactions, aiding farmers and beekeepers in maintaining healthy and resilient bee colonies amid increasing Varroa mite infestations.

Schistosoma mansoni (S. mansoni) infection is endemic in Ugandan fishing communities. We investigated its potential impact on Hepatitis B (Hep B) vaccine responses and its role in mediating the association between the gut microbiome and long-term effectiveness of the vaccine.

Participants were tested for S. mansoni infections at baseline and received the Hep B vaccine at baseline, month 1, and month 6. Those with infections were treated. Stool samples were collected at baseline and analyzed using 16S rRNA sequencing. The Wilcoxon rank-sum test was used to compare alpha diversity between groups. A linear regression model was applied to estimate the association between one-year Hep B vaccine responses and the baseline gut microbiome by infection status, adjusting for age and sex.

A total of 107 participants were included (44 from the fishing community and 63 from the Kampala community). There was no significant difference in microbiome composition by location or infection status at baseline or discharge. In the linear regression analysis, S. mansoni infection (β = 1.24, p = 0.025) and a higher alpha diversity (β = 0.001, p = 0.07) were associated with higher Hep B vaccine responses, while older age was associated with a lower Hep B vaccine response (β = -0.06, p = 0.0013).

S. mansoni infection status before vaccination may modify the association between the gut microbiome and Hep B vaccine response. Potential interventions could focus on infection control as well as improving microbiome richness before implementing vaccine programs in fishing communities.

Microbial metagenomic identification is generally attributed to the specificity and type of the bioinformatic tools, including classifiers and visualizers. In this study, the performance of two major classifiers, Centrifuge and Kraken2, and two visualizers (Recentrifuge and Krona) has been thoroughly investigated for their efficiency in the identification of the microorganisms using the Whole-Genome Sequence (WGS) database and four targeted databases including NCBI, Silva, Greengenes, and Ribosomal Database Project (RDP). Two standard DNA metagenomic library replicates, Zymo and Zymo-1, were used as quality control. Results showed that Centrifuge gave a higher percentage of Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica identification than Kraken2. Compared to Recentrifuge, Kraken2 was more accurate in identifying Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, and Cryptococcus neoformans. The results of the rest of the detected microorganisms were generally consistent with the two classifiers. Regarding visualizers, both Recentrifuge and Krona provided similar results regarding the abundance of each microbial species regardless of the classifier used. The differences in results between the two mentioned classifiers may be attributed to the specific algorithms each method uses and the sequencing depth. Centrifuge uses a read mapping approach, while Kraken2 uses a k-mer-based system to classify the sequencing reads into taxonomic groups. In conclusion, both Centrifuge and Kraken2 are effective tools for microbial classification. However, the choice of classifier can influence the accuracy of microbial classification and, therefore, should be made carefully, depending on the desired application, even when the same reference database is used.

Microbiome studies aimed at combating the citrus greening devastation caused by Liberibacter asiaticus abound. However, the role of farming practices, such as the massive use of herbicides, pesticides, and inorganic fertilizers on specific taxa and plant population immunity remains an important inquiry. To test our hypothesis that agricultural practices in managed Citrus groves induce root microbiome dysbiosis, potentially rendering citrus readily susceptible to citrus greening disease (CGD), we compared the CGD and root microbiome status of citrus plants in a rare > 130-year-old grove (no anthropogenic influence) to those of managed Valencia groves (symptomatic and asymptomatic). Citrus greening disease was detected by qPCR using the HLBa/HLBs/HLBp primer/probe combination, while root microbiome community structure was determined using 16S rDNA amplicon sequencing. The prevalence of CGD among citrus growing in the undisturbed, healthy soils was zero (Ct values > 36), while symptomatic and asymptomatic Valencia from managed groves was 100% positive (Ct < 34). Known beneficial plant symbionts (Actinomycetales, Bradyrhizobium, Verrucomicrobia, etc.) from Phylum Actinobacteria and Proteobacteria were depleted in the rhizosphere of the managed sites. This dysbiotic shift was characterized by enrichment with Acidobacterium, Nitrospira, and Sphingomonas spp. In highly infected Valencia oranges, beneficial taxa of the Alphaproteobacteria declined significantly (from 20-25% to 10-15%), while Bacillus sp. (a Firmicutes) was enriched 13-fold. Simpson and Shannon diversity indices were similar for all plant microbiomes except the heavily infected Valencia, which exhibited low diversity (p < 0.05), indicating that diversity indices alone are not reliable measures of soil health or rhizobiome homeostasis. Large reservoirs of known and novel putative beneficial rhizosphere microbes in undisturbed sites supported zero CGD, despite proximity to the managed sites where diverse non-beneficial taxa coincided with high disease rates. Supplementing the use of agrochemicals with carefully designed microbial products for plant disease control and sustainable soil health deserves acute attention.