Linear Discriminant Analysis Effect Size Research Articles

Effects of lactic acid bacteria inoculants on the nutrient composition, fermentation quality, and microbial diversity of whole-plant soybean-corn mixed silage

The mixture of whole-plant soybean and whole-plant corn silage (WPSCS) is nutrient balanced and is also a promising roughage for ruminants. However, few studies have investigated the changes in bacterial community succession in WPSCS inoculated with homofermentative and heterofermentative lactic acid bacteria (LAB) and whether WPSCS inoculated with LAB can improve fermentation quality by reducing nutrient losses. This study investigated the effect of Lactobacillus plantarum (L. plantarum) or Lactobacillus buchneri (L. buchneri) on the fermentation quality, aerobic stability, and bacterial community of WPSCS. A 40:60 ratio of whole-plant soybean corn was inoculated without (CK) or with L. plantarum (LP), L. buchneri (LB), and a mixture of LP and LB (LPB), and fermented for 14, 28, and 56 days, followed by 7 days of aerobic exposure. The 56-day silage results indicated that the dry matter content of the LP and LB groups reached 37.36 and 36.67%, respectively, which was much greater than that of the CK group (36.05%). The pH values of the LP, LB, and LPB groups were significantly lower than those of the CK group (p < 0.05). The ammoniacal nitrogen content of LB was significantly lower than that of the other three groups (p < 0.05), and the ammoniacal nitrogen content of LP and LPB was significantly lower than that of CK (p < 0.05). The acetic acid content and aerobic stability of the LB group were significantly greater than those of the CK, LP, and LPB groups (p < 0.05). High-throughput sequencing revealed a dominant bacteria shift from Proteobacteria in fresh forage to Firmicutes in silage at the phylum level. Lactobacillus remained the dominant genus in all silage. Linear discriminant analysis effect size (LEFSe) analysis identified Lactobacillus as relatively abundant in LP-treated silage and Weissella in LB-treated groups. The results of KEGG pathway analysis of the 16S rRNA gene of the silage microbial flora showed that the abundance of genes related to amino acid metabolism in the LP, LB, and LPB groups was lower than that in the CK group (p < 0.05). In conclusion, LAB application can improve the fermentation quality and nutritional value of WPSCS by regulating the succession of microbial communities and metabolic pathways during ensiling. Concurrently, the LB inoculant showed the potential to improve the aerobic stability of WPSCS.

Non-traumatic osteonecrosis of the femoral head induced by steroid and alcohol exposure is associated with intestinal flora alterations and metabolomic profiles

ObjectiveOsteonecrosis of the femoral head (ONFH) is a severe disease that primarily affects the middle-aged population, imposing a significant economic and social burden. Recent research has linked the progression of non-traumatic osteonecrosis of the femoral head (NONFH) to the composition of the gut microbiota. Steroids and alcohol are considered major contributing factors. However, the relationship between NONFH caused by two etiologies and the microbiota remains unclear. In this study, we examined the gut microbiota and fecal metabolic phenotypes of two groups of patients, and analyzed potential differences in the pathogenic mechanisms from both the microbial and metabolic perspectives.MethodsUtilizing fecal samples from 68 NONFH patients (32 steroid-induced, 36 alcohol-induced), high-throughput 16 S rDNA sequencing and liquid chromatography with tandem mass spectrometry (LC-MS/MS) metabolomics analyses were conducted. Univariate and multivariate analyses were applied to the omics data, employing linear discriminant analysis effect size to identify potential biomarkers. Additionally, functional annotation of differential metabolites and associated pathways was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Subsequently, Spearman correlation analysis was employed to assess the potential correlations between differential gut microbiota and metabolites.ResultsHigh-throughput 16 S rDNA sequencing revealed significant gut microbial differences. At the genus level, the alcohol group had higher Lactobacillus and Roseburia, while the steroid group had more Megasphaera and Akkermansia. LC-MS/MS metabolomic analysis indicates significant differences in fecal metabolites between steroid- and alcohol-induced ONFH patients. Alcohol-induced ONFH (AONFH) showed elevated levels of L-Lysine and Oxoglutaric acid, while steroid-induced ONFH(SONFH) had increased Gluconic acid and Phosphoric acid. KEGG annotation revealed 10 pathways with metabolite differences between AONFH and SONFH patients. Correlation analysis revealed the association between differential gut flora and differential metabolites.ConclusionsOur results suggest that hormones and alcohol can induce changes in the gut microbiota, leading to alterations in fecal metabolites. These changes, driven by different pathways, contribute to the progression of the disease. The study opens new research directions for understanding the pathogenic mechanisms of hormone- or alcohol-induced NONFH, suggesting that differentiated preventive and therapeutic approaches may be needed for NONFH caused by different triggers.

Exploring the bladder tissue microbiome in patients with muscle-invasive bladder cancer using 2bRAD-M sequencing

Abstract Objectives The 2bRAD sequencing for Microbiome (2bRAD-M) represents an innovative and streamlined approach for the reconstruction of microbial profiles at the species level. In our investigation, we conducted 2bRAD-M analysis to characterize the microbiome of bladder tissue in patients with muscle-invasive bladder cancer (MIBC). Methods 15 tumor tissues and 15 paired para-carcinoma tissues were obtained from the bladder excised during surgery. 2bRAD-M sequencing was used to assess the abundance of microorganisms in samples. Results The microbial community structure and biodiversity, as assessed at varying taxonomic ranks, exhibited a high degree of similarity between the tumor and paired non-tumor tissues. At the genus level, we observed a notably elevated abundance of Brachybacterium and Haloparvum, coupled with a diminished abundance of Anoxybacillus, Anoxybacillu_A, Deinococcus, NCEH01, and Pseudoxanthomonas_A in the tumor tissues. Meanwhile, at the species level, the non-tumor tissues exhibited an enrichment of Anoxybacillus_A rupiensis, Anoxybacillus flavithermus_G, Klebsiella quasipneumoniae, NCEH01 sp002304505, and Pseudoxanthomonas_A sp004284195. Linear discriminant analysis effect size (LEfSe) identified 29 discriminative features, characterized by significant variations (p<0.5, LDA≥2.0) in relative abundance between the two groups. Furthermore, an analysis of functional predictions utilizing Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) also uncovered disparities in the microbial functional composition. Conclusions This study identified several microorganisms that exhibit differences between MIBC tumor tissue and adjacent non-tumor tissue using 2bRAD-M sequencing, providing some insights into the potential association between the bladder microbiome and cancer.

Bacterial biota associated with the invasive insect pest Tuta absoluta (Meyrick)

Tuta absoluta (the tomato pinworm) is an invasive insect pest with a highly damaging effect on tomatoes causing between 80 and 100% yield losses if left uncontrolled. Resistance to chemical pesticides have been reported in some T. absoluta populations. Insect microbiome plays an important role in the behavior, physiology, and survivability of their host. In a bid to explore and develop an alternative control method, the associated microbiome of this insect was studied. In this study, we unraveled the bacterial biota of T. absoluta larvae and adults by sequencing and analyzing the 16S rRNA V3-V4 gene regions using Illumina NovaSeq PE250. Out of 2,092,015 amplicon sequence variants (ASVs) recovered from 30 samples (15 larvae and 15 adults), 1,268,810 and 823,205 ASVs were obtained from the larvae and adults, respectively. A total of 433 bacterial genera were shared between the adults and larval samples while 264 and 139 genera were unique to the larvae and adults, respectively. Amplicon metagenomic analyses of the sequences showed the dominance of the phylum Proteobacteria in the adult samples while Firmicutes and Proteobacteria dominated in the larval samples. Linear discriminant analysis effect size (LEfSe) comparison revealed the genera Pseudomonas, Delftia and Ralstonia to be differentially enriched in the adult samples while Enterococcus, Enterobacter, Lactococcus, Klebsiella and Wiessella were differentially abundant in the larvae. The diversity indices showed that the bacterial communities were not different between the insect samples collected from different geographical regions. However, the bacterial communities significantly differed based on the sample type between larvae and adults. A co-occurrence network of significantly correlated taxa revealed a strong interaction between the microbial communities. The functional analysis of the microbiome using FAPROTAX showed that denitrification, arsenite oxidation, methylotrophy and methanotrophy as the active functional groups of the adult and larvae microbiomes. Our results have revealed the core taxonomic, functional, and interacting microbiota of T. absoluta and these indicate that the larvae and adults harbor a similar but transitory set of bacteria. The results provide a novel insight and a basis for exploring microbiome-based biocontrol strategy for this invasive insect pest as well as the ecological significance of some of the identified microbiota is discussed.

The comparative plastisphere microbial community profile at Kung Wiman beach unveils potential plastic-specific degrading microorganisms.

Plastic waste is a global environmental issue that impacts the well-being of humans, animals, plants, and microorganisms. Microplastic contamination has been previously reported at Kung Wiman Beach, located in Chanthaburi province along with the Eastern Gulf of Thailand. Our research aimed to study the microbial population of the sand and plastisphere and isolate microorganisms with potential plastic degradation activity. Plastic and sand samples were collected from Kung Wiman Beach for microbial isolation on agar plates. The plastic samples were identified by Fourier-transform infrared spectroscopy. Plastic degradation properties were evaluated by observing the halo zone on mineral salts medium (MSM) supplemented with emulsified plastics, including polystyrene (PS), polylactic acid (PLA), polyvinyl chloride (PVC), and bis (2-hydroxyethyl) terephthalate (BHET). Bacteria and fungi were identified by analyzing nucleotide sequence analysis of the 16S rRNA and internal transcribed spacer (ITS) regions, respectively. 16S and ITS microbiomes analysis was conducted on the total DNA extracted from each sample to assess the microbial communities. Of 16 plastic samples, five were identified as polypropylene (PP), four as polystyrene (PS), four as polyethylene terephthalate (PET), two as high-density polyethylene (HDPE), and one sample remained unidentified. Only 27 bacterial and 38 fungal isolates were found to have the ability to degrade PLA or BHET on MSM agar. However, none showed degradation capabilities for PS or PVC on MSM agar. Notably, Planococcus sp. PP5 showed the highest hydrolysis capacity of 1.64 ± 0.12. The 16S rRNA analysis revealed 13 bacterial genera, with seven showing plastic degradation abilities: Salipiger, Planococcus, Psychrobacter, Shewanella, Jonesia, Bacillus, and Kocuria. This study reports, for the first time of the BHET-degrading properties of the genera Planococcus and Jonesia. Additionally, The ITS analysis identified nine fungal genera, five of which demonstrated plastic degradation abilities: Aspergillus, Penicillium, Peacilomyces, Absidia, and Cochliobolus. Microbial community composition analysis and linear discriminant analysis effect size revealed certain dominant microbial groups in the plastic and sand samples that were absent under culture-dependent conditions. Furthermore, 16S and ITS amplicon microbiome analysis revealed microbial groups were significantly different in the plastic and sand samples collected. We reported on the microbial communities found on the plastisphere at Kung Wiman Beach and isolated and identified microbes with the capacity to degrade PLA and BHET.

Identification of KRAS mutation-associated gut microbiota in colorectal cancer and construction of predictive machine learning model.

Gut microbiota has demonstrated an increasingly important role in the onset and development of colorectal cancer (CRC). Nonetheless, the association between gut microbiota and KRAS mutation in CRC remains enigmatic. We conducted 16S rRNA sequencing on stool samples from 94 CRC patients and employed the linear discriminant analysis effect size algorithm to identify distinct gut microbiota between KRAS mutant and KRAS wild-type CRC patients. Transcriptome sequencing data from nine CRC patients were transformed into a matrix of immune infiltrating cells, which was then utilized to explore KRAS mutation-associated biological functions, including Gene Ontology items and Kyoto Encyclopedia of Genes and Genomes pathways. Subsequently, we analyzed the correlations among these KRAS mutation-associated gut microbiota, host immunity, and KRAS mutation-associated biological functions. At last, we developed a predictive random forest (RF) machine learning model to predict the KRAS mutation status in CRC patients, based on the gut microbiota associated with KRAS mutation. We identified a total of 26 differential gut microbiota between both groups. Intriguingly, a significant positive correlation was observed between Bifidobacterium spp. and mast cells, as well as between Bifidobacterium longum and chemokine receptor CX3CR1. Additionally, we also observed a notable negative correlation between Bifidobacterium and GOMF:proteasome binding. The RF model constructed using the KRAS mutation-associated gut microbiota demonstrated qualified efficacy in predicting the KRAS phenotype in CRC. Our study ascertained the presence of 26 KRAS mutation-associated gut microbiota in CRC and speculated that Bifidobacterium may exert an essential role in preventing CRC progression, which appeared to correlate with the upregulation of mast cells and CX3CR1 expression, as well as the downregulation of GOMF:proteasome binding. Furthermore, the RF model constructed on the basis of KRAS mutation-associated gut microbiota exhibited substantial potential in predicting KRAS mutation status in CRC patients.IMPORTANCEGut microbiota has emerged as an essential player in the onset and development of colorectal cancer (CRC). However, the relationship between gut microbiota and KRAS mutation in CRC remains elusive. Our study not only identified a total of 26 gut microbiota associated with KRAS mutation in CRC but also unveiled their significant correlations with tumor-infiltrating immune cells, immune-related genes, and biological pathways (Gene Ontology items and Kyoto Encyclopedia of Genes and Genomes pathways). We speculated that Bifidobacterium may play a crucial role in impeding CRC progression, potentially linked to the upregulation of mast cells and CX3CR1 expression, as well as the downregulation of GOMF:Proteasome binding. Furthermore, based on the KRAS mutation-associated gut microbiota, the RF model exhibited promising potential in the prediction of KRAS mutation status for CRC patients. Overall, the findings of our study offered fresh insights into microbiological research and clinical prediction of KRAS mutation status for CRC patients.

[Comparison of the microbiota diversity between autogenous and anautogenous Culex pipiens pallens].

To investigate the microbiota composition and diversity between autogenous and anautogenous Culex pipiens pallens, so as to provide insights into unraveling the pathogenesis of autogeny in Cx. pipiens pallens. Autogenous and anautogenous adult Cx. pipiens pallens samples were collected at 25 ℃, and the hypervariable regions of the microbial 16S ribosomal RNA (16S rRNA) gene was sequenced on the Illumina NovaSeq 6000 sequencing platform. The microbiota abundance and diversity were evaluated using the alpha diversity index, and the difference in the microbiota structure was examined using the beta diversity index. The microbiota with significant differences in the abundance between autogenous and anautogenous adult Cx. pipiens pallens samples was identified using the linear discriminant analysis effect size (LEfSe). The microbiota in autogenous and anautogenous Cx. pipiens pallens samples belonged to 18 phyla, 28 classes, 70 orders, 113 families, and 170 genera, and the dominant phyla included Proteobacteria, Bacteroidetes, and so on. At the genus level, Wolbachia was a common dominant genus, and the relative abundance was (77.6 ± 11.3)% in autogenous Cx. pipiens pallens samples and (47.5 ± 8.5)% in anautogenous mosquito samples, while Faecalibaculum (0.4% ± 0.1%), Dubosiella (0.5% ± 0.0%) and Massilia (0.5% ± 0.1%) were specific species in autogenous Cx. pipiens pallens samples. Alpha diversity analysis showed that higher Chao1 index and ACE index in autogenous Cx. pipiens pallens samples than in anautogenous samples (both P values > 0.05), and lower Shannon index (P > 0.05) and Simpson index (P < 0.05) in autogenous Cx. pipiens pallens samples than in anautogenous samples. LEfSe analysis showed a total of 48 significantly different taxa between autogenous and anautogenous Cx. pipiens pallens samples (all P values < 0.05). There is a significant difference in the microbiota diversity between autogenous and anautogenous Cx. pipiens pallens.

Associations between the wastewater microbiome and community obesity rates: A study in Australia

Gut microbes, crucial for human health, are usually collected in urban wastewater systems. Seven wastewater treatment plants in Australia with distinct population obesity rates between 18% and 33%, were selected for wastewater sampling and analysis, human gut microbioms detected using metagenomic sequencing were investigated for their associations with the community obesity rate. To unravel this complex relationship, a range of algorithms, including linear discriminant analysis effect size (LEfSe), similarity percentage analysis (SIMPER), statistical analysis of metagenomic profiles (STAMP), linear models for microarray and RNA-Seq data analysis (Limma), Relief, ratio approach for identifying differential abundance (RAIDA), least absolute shrinkage and selection operator (LASSO), support vector machine (SVM), Boruta, DESeq2 and analysis of compositions of microbiomes with bias correction (ANCOM-BC), were used to identify potential bacterial biomarker for obesity in the wastewater microbiome. Among these algorithms, LEfSe, Limma, SIMPER and SVM are effective in identifying multiple microbial biomarkers. Specific human gut microbes, including Ruminococcus_E, Agathobacter, Fusicatenibacter, Anaerobutyricum, Blautia_A and Neisseria, were identified as potential consensus microbial biomarkers for obesity in the population. A high obesity rate is mainly characterized by a high abundance of pathogenic bacteria and microorganisms associated with xenobiotic biodegradation and metabolism, endocrine and metabolic diseases, and transcription pathways. This study and its findings underscore the innovative potential of leveraging human gut microbes in wastewater as biomarkers for monitoring obesity levels across communities, offering a novel, cost-effective, and indirect approach to public health surveillance.

Dietary apidaecin Api-PR19 addition improves growth performance, intestinal health and modulating gut microbiota in broilers.

This study investigated the effects of Apidaecin Api-PR19 as feed additive on growth performance, intestinal health, and small intestinal microbiota of broilers. A total of 360 1-d-old Arbor Acres broilers were randomly assigned to 3 groups with 6 replicates including control group with basal diet (CON), antibiotic growth promotor group with basal plus 10 mg/kg colistin sulfate and 50mg/kg roxarsone (AGP), and antibacterial peptide group with basal diet plus 330 mg/kg Apidaecin Api-PR19 (ABP). The trial lasted 35 d. Results showed that dietary Api-PR19 addition increased (p＜0.05) the average daily feed intake (ADFI), average daily gain (ADG) and decreased (p＜0.05) feed conversion ratio (FCR) during 1 to 21 d compared with the CON group. The digestibility of dry matter and crude protein were higher in AGP and ABP groups (p＜0.05) where greater trypsin activity was detected in duodenum (p＜0.05). The ratio of villus height to crypt depth (V/R) in duodenum and jejunum was increased at 35 d when broilers were given diets with ABP or AGP (p＜0.05). Besides, ABP treatments up-regulated (p＜0.05) the mRNA expression of EAAT3, GLUT2, ZO-1 and Claudin-1 in duodenum of broilers at 35 d of age. The results of immunohistochemistry showed that ABP treatment significantly increased (p＜0.05) duodenal sIgA content. In addition, 16S rRNA gene sequencing revealed that there were differences in the intestinal microbiota diversity and composition among three groups. Notably, the linear discriminant analysis effect size (LEfSe) showed that p_Firmicutes, g_Enterococcus, g_Carnobacterium, g_Kitasatospora and g_Acidaminococcus were dominant in ABP group. Redundancy analysis showed that these changes in gut microbiota in ABP group had correlation with growth performance, intestinal morphology, and content of sIgA. In general, these results indicated that dietary 330 mg/kg Apidaecin Api-PR19 supplementation promoted growth performance of broilers by improving intestinal development, nutrients absorption, immune function and modulating intestinal microbiota.

Changes and driving factors of microbial community composition and functional groups during the decomposition of Pinus massoniana deadwood.

Clarifying changes in the microbial community in deadwood at different stages of decomposition is crucial for comprehending the role of deadwood in the biogeochemical processes and the sustainability of forest development. However, there have been no reports on the dynamics of microbial community during the decomposition of Pinus massoniana. We used the "space-for-time" substitution to analyze the characteristics of microbial community changes and the key influencing factors in the P. massoniana deadwood during different decomposition stages by 16S and ITS rRNA gene sequencing. The results suggest that the microbial community structure of the early decomposition (decay class I) was significantly different from the other decay classes, while the diversity and richness of the microbial community were the highest in the late decomposition (decay class V). The Linear Discriminant Analysis Effect Size analysis revealed that most bacterial and fungal taxa were significantly enriched in decay classes I and V deadwood. During the initial stages of decomposition, the relative abundance of the bacterial functional group responsible for carbohydrate metabolism was greater than the later stages. As decomposition progressed, the relative abundance of saprophytic fungi gradually decreased, and there was a shift in the comparative abundance of mixed saprophytic-symbiotic fungi from low to high before eventually decreasing. Total organic carbon, total nitrogen, carbon-to-nitrogen ratio, total potassium, total phenol, condensed tannin, lignin, and cellulose were significantly correlated with microbial community structure, with the carbon-to-nitrogen ratio having the greatest effect. Our results indicate that the physicochemical properties of deadwood, microbial community structural composition and functional group changes were related to the decay class, among which the carbon-to-nitrogen ratio may be an important factor affecting the composition and diversity of microbial communities.

Vaginal microbiomes of breast cancer survivors treated with aromatase inhibitors with and without vulvovaginal symptoms

Genitourinary syndrome of menopause (GSM) is the leading cause of vaginal symptoms in breast cancer survivors treated with aromatase inhibitors. However, there are currently no effective treatment options available for women with a history of breast cancer. Recent research has established that changes in the vaginal microbiome may be linked to GSM. Most studies have assessed the microbiome without accounting for the estrogen status. It remains unknown whether the vaginal microbiome differ among patients with a low estrogenic state with and without vulvovaginal symptoms. To address such research questions, our study compares the vaginal microbiomes among breast cancer survivors treated with aromatase inhibitors with and without vulvovaginal symptoms. A total of 50 breast cancer survivors treated with aromatase inhibitors were recruited, among whom 25 had vulvovaginal symptoms and 25 had no vulvovaginal symptoms. Vaginal swabs were collected. DNA extraction, followed by sequencing of the V3–V4 regions of the 16S ribosomal RNA gene, were performed. Differential abundance analysis was conducted by linear discriminant analysis effect size. Taxonomy assignment, alpha diversity and beta diversity were examined. The relative abundance of genus Sneathia and genus Gardnerella was significantly increased in vulvovaginal symptoms group with no differences in bacterial diversity and richness.

Exploring the diversity of vaginal microbiota between healthy women and cervical cancer patients in India.

Introduction. Cervicovaginal diversity has been reported as a predictive biomarker for cervical cancer risk. We recently reported the bio-therapeutic potential of vaginal probiotics from healthy Indian women against vaginal pathogens, isolated from the invasive cervical cancer (ICC) patients.Gap Statement. The cervicovaginal microflora from cervical cancer patients has not yet been reported from Indian population.Aim. The present study aimed at comparing the cervicovaginal microbiome between healthy controls (HC) and ICC patients from the Indian population.Methodology. In total, 30 vaginal swabs (15 from HC and 15 from ICC) were subjected to 16S rRNA gene sequencing. Alpha diversity was evaluated by Shannon and Chao1 index; and beta diversity by principle coordinate analysis (PCoA) of weighted and unweighted UniFrac distances. The relative abundance of the microbial taxa was done according to linear discriminant analysis effect size (LEfSe).Results. Predominance of Staphylococcus spp. in ICC and Lactobacillus gasseri in HC groups was observed. Alpha-diversity was found to be higher in ICC as compared to HC but was statistically non-significant. LEfSe analysis revealed Bacteroides fragilis and Escherichia coli as the marker genera in ICC with a marked decrease in Lactobacillus sp. Contrarily, in HC, L. gasseri, L. iners and L. fermentum were found to be abundant.Conclusion. Differences in the vaginal microbiome between healthy and ICC women could help in the early prediction of cervical cancer risk and thus in designing prevention strategies.

Formation of In‐Cloud Aqueous‐Phase Secondary Organic Matter and Related Characteristic Molecules

AbstractThe formation process of in‐cloud aqueous‐phase secondary organic matter (aqSOM) and its characteristics are unclear. Herein, water‐soluble inorganic ions, oxalate, and water‐soluble organic carbon (WSOC) were determined in cloud water and aerosol (PM2.5) samples simultaneously collected at a remote mountain site in southern China during spring 2018 and winter 2020. The molecular compositions of water‐soluble organic matter (WSOM) in cloud water and aerosols were analyzed by a Fourier transform ion cyclotron resonance mass spectrometer in negative electrospray ionization (ESI‐) mode. The results showed that the mean concentration of WSOC was 6.27–8.54 mg C L−1 in cloud water and 0.60–1.37 μg C m−3 in aerosols. The strong correlation observed between WSOM and aqueous secondary matter (e.g., NO3− and oxalate), the positive matrix factorization results, and the elevated WSOM/K+ ratios observed in cloud water suggested enhanced aqSOM formation in cloud water. According to random forest analysis, the factors related to in‐cloud WSOM variation mainly included secondary ions, K+, cloud water pH, and atmospheric NOx. Additionally, 37 characteristic in‐cloud aqSOM molecules, classified as ‐Ox, ‐NOx, ‐N2Ox, and ‐N1‐2OxS, mainly consisting of dicarboxylic acids, nitrophenols, and dinitrophenols, were identified using linear discriminant analysis effect size (LefSe). The characteristic N‐ and S‐containing molecules in in‐cloud aqSOM with carbon numbers &gt;10 had low or extremely low volatility; therefore, they might contribute to secondary organic aerosol formation after droplet evaporation. The results revealed the modifying effects of in‐cloud processes on aerosol organic composition at the molecular level and could improve our understanding of aerosol–cloud interactions.

[Effects of periodontitis on bone and tryptophan metabolism of gut microbiota in estrogen-deficient mice].

Objective: To study the effects of periodontitis on bone and tryptophan metabolism of gut microbiota in the context of estrogen deficiency. Methods: Thirty-two female C57BL6/J mice were randomly divided into four groups based on table of random numbers (n=8 in each group): Sham group, in which mice were given sham surgery; Sham_Lig group, in which mice were given sham surgery and were induced to periodontitis by ligating the bilateral maxillary second molars with 5-0 silk threads at the fourth week; Ovx group, in which mice were given bilateral ovariectomy; Ovx_Lig group, in which mice were given bilateral ovariectomy and were induced to periodontitis at the fourth week. After 8 weeks of ligation, the mice of 4 groups were euthanized for collecting the samples of femur, tibia, mandible and skull. Those samples were scanned by micro-CT to measure the bone mineral density (BMD), bone volume versus total volume ratio (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp). The cecum contents of 4 groups of mice were collected for gut microbiota 16S rRNA gene sequencing. The tryptophan and its metabolites in intestinal tracts were detected by liquid chromatography-mass spectrometry. Pearson correlation analysis was performed to analyze the correlation between the abundance of gut microbiota and the content of tryptophan and its metabolites. Results: Femur BMD [(82.23±3.97) mg/cm3], BV/TV [(9.25±1.37) %] and Tb.Th [(70.95±5.70) μm] in Ovx_Lig group were significantly lower than Ovx group [(96.30±3.76) mg/cm3 (P=0.004); (14.45±1.55)% (P=0.022) and (87.58±8.02) μm (P<0.001), respectively]. The β-diversity analysis of gut microbiota based on Bray-Curtis distance showed that samples of Ovx_Lig group and Ovx group were obviously grouped. Linear discriminant analysis effect size (LEfSe) showed that Alistipes was the representative genus in Ovx_Lig group. The relative abundance of Alistipes in Ovx_Lig group [(0.42±0.14) %] were significantly higher than that in Ovx group [(0.17±0.05) %] (t=4.45, P<0.001). Tryptophan metabolism analysis showed that the content of kynurenic acid [(531.12±158.60) ng/g] in Ovx_Lig group were significantly higher than that in Ovx group [(400.42±57.96) ng/g] (t=2.19, P=0.046). And the content of Indole-3-carbaldehyde [(383.37±144.06) ng/g] in Ovx_Lig group were lower than Ovx group [(701.72±141.93) ng/g] (t=4.45, P<0.001). Correlation analysis showed that relative abundance of Alistipes was positively correlated with kynurenic acid (r=0.32, P=0.088), while negatively correlated with indole-3-carbaldehyde (r=-0.32, P=0.088). Conclusions: Periodontitis can induce bone destruction of femur in estrogen-deficient mice, the mechanism of which may be related to Alistipes in gut and the tryptophan metabolites kynurenic acid and indole-3-carbaldehyde.

Association between Gut Microbiota Composition and Long-Term Vaccine Immunogenicity following Three Doses of CoronaVac

Background: Neutralizing antibody level wanes with time after COVID-19 vaccination. We aimed to study the relationship between baseline gut microbiota and immunogenicity after three doses of CoronaVac. Methods: This was a prospective cohort study recruiting three-dose CoronaVac recipients from two centers in Hong Kong. Blood samples were collected at baseline and one year post-first dose for virus microneutralization (vMN) assays to determine neutralization titers. The primary outcome was high immune response (defined as with vMN titer ≥ 40). Shotgun DNA metagenomic sequencing of baseline fecal samples identified potential bacterial species and metabolic pathways using Linear Discriminant Analysis Effect Size (LEfSe) analysis. Univariate and multivariable logistic regression models were used to identify high response predictors. Results: In total, 36 subjects were recruited (median age: 52.7 years [IQR: 47.9–56.4]; male: 14 [38.9%]), and 18 had low immune response at one year post-first dose vaccination. Eubacterium rectale (log10LDA score = 4.15, p = 0.001; relative abundance of 1.4% vs. 0, p = 0.002), Collinsella aerofaciens (log10LDA score = 3.31, p = 0.037; 0.39% vs. 0.18%, p = 0.038), and Streptococcus salivarius (log10LDA score = 2.79, p = 0.021; 0.05% vs. 0.02%, p = 0.022) were enriched in low responders. The aOR of high immune response with E. rectale, C. aerofaciens, and S. salivarius was 0.03 (95% CI: 9.56 × 10−4–0.32), 0.03 (95% CI: 4.47 × 10−4–0.59), and 10.19 (95% CI: 0.81–323.88), respectively. S. salivarius had a positive correlation with pathways enriched in high responders like incomplete reductive TCA cycle (log10LDA score = 2.23). C. aerofaciens similarly correlated with amino acid biosynthesis-related pathways. These pathways all showed anti-inflammation functions. Conclusion: E. rectale,C. aerofaciens, and S. salivarius correlated with poorer long-term immunogenicity following three doses of CoronaVac.

Deciphering the Structural and Functional Diversity of Rhizobacteria from Stone Pine Inoculated with Plant Growth Promoting Rhizobacteria (PGPR) before and after Transplanted into Degraded Agricultural Soil

The use of plant growth-promoting rhizobacteria (PGPR) inoculated on plants has shown that it can increase the success of reforestation and accelerate soil recovery by improving soil microbial diversity. Three PGPR isolated from natural pine populations were selected for their metabolic capabilities and taxonomic affiliation (Z4.3; Bacillus sp., Z5.4; Arthobacter sp., and Z7.15; and Pseudomonas sp.) when inoculated alone or in combination (consortium) on stone pine seedlings before transplanting to the field. Before transplanting and after nine months, rhizospheric soil samples were collected for structural and functional metagenomic studies. First, the data were analyzed using EasyMAP. Neither alpha nor beta diversity showed significant differences between the samples, although unique taxa representative of each sample were detected. The predominant phylum in all cases was Proteobacteria, followed by Bacteroidetes and Acidobacteria. The linear discriminant analysis (LDA) effect size (LEfSe) found significantly over-represented taxa in some samples, highlighting different representatives of the order Sphingomonadales in several of them. Functional inference performed with PICRUSt also showed significantly over-represented functions in some samples. The study demonstrates that PGPR have a positive effect on plants and cause detectable changes in microbial communities in terms of both structure and function.

Bioindicator responses to extreme conditions: Insights into pH and bioavailable metals under acidic metal environments

Acid mine drainage (AMD) caused environmental risks from heavy metal pollution, requiring treatment methods such as chemical precipitation and biological treatment. Monitoring and adapting treatment processes was crucial for success, but cost-effective pollution monitoring methods were lacking. Using bioindicators measured through 16S rRNA was a promising method to assess environmental pollution. This study evaluated the effects of AMD on ecological health using the ecological risk index (RI) and the Risk Assessment Code (RAC) indices. Additionally, we also examined how acidic metal stress affected the diversity of bacteria and fungi, as well as their networks. Bioindicators were identified using linear discriminant analysis effect size (LEfSe), Partial least squares regression (PLS-R), and Spearman analyses. The study found that Cd, Cu, Pb, and As pose potential ecological risks in that order. Fungal diversity decreased by 44.88% in AMD-affected areas, more than the 33.61% decrease in bacterial diversity. Microbial diversity was positively correlated with pH (r = 0.88, p = 0.04) and negatively correlated with bioavailable metal concentrations (r = −0.59, p = 0.05). Similarly, microbial diversity was negatively correlated with bioavailable metal concentrations (bio_Cu, bio_Pb, bio_Cd) (r = 0.79, p = 0.03). Acidiferrobacter and Thermoplasmataceae were prevalent in acidic metal environments, while Puia and Chitinophagaceae were identified as biomarker species in the control area (LDA>4). Acidiferrobacter and Thermoplasmataceae were found to be pH-tolerant bioindicators with high reliability (r = 1, P < 0.05, BW > 0.1) through PLS-R and Spearman analysis. Conversely, Puia and Chitinophagaceae were pH-sensitive bioindicators, while Teratosphaeriaceae was a potential bioindicator for Cu–Zn–Cd metal pollution. This study identified bioindicator species for acid and metal pollution in AMD habitats. This study outlined the focus of biological monitoring in AMD acidic stress environments, including extreme pH, heavy metal pollutants, and indicator species. It also provided essential information for heavy metal bioremediation, such as the role of omics and the effects of organic matter on metal bioavailability.

Abstract 758: Comparative Microbiome Analysis of Paired Mucosal and Fecal Samples in Korean Colorectal Cancer Patients

Abstract Background Beyond the known risk factors such as age over 50, unhealthy diet, ethnicity, and smoking, emerging evidence suggests that gut microbial dysbiosis plays a crucial role in the early stages of colorectal cancer (CRC) by contributing to chronic inflammation and various intestinal dysfunctions. Even though various bacterial species have been studied, the gut microbiome profile as a representative biomarker in CRC patients remains largely unreported. This study presents a comparative analysis of the microbiome profiles of fecal and mucosal samples from CRC patients, followed by metagenomic profiling investigation to validate dominant bacteria in each sample type of CRC patients. Methods The study involved 12 patients receiving CRC resection at Myongji Hospital in South Korea, recruited from May 2020 to June 2021. Fecal samples were collected pre-surgery, and mucosal samples during surgery. Paired-end sequencing was performed using Illumina MiSeq (Illumina, San Diego, USA) platform. 16s rRNA gene-based microbiome profiling was analyzed using QIIME2 (V.2020.11), a plugin-based platform for microbiome analysis. Sequencing reads were filtered, denoised, and clustered into amplicon sequence variants (ASVs) using DADA2 included in QIIME2. Taxonomic classification was assigned in the QIIME2 pipeline using a pre-trained classifier based on the Silva 138.1 reference sequence. Linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed with LDA score ≥3.0 and p-value of 0.05. Results The study subjects included 12 Korean patients, with an average age of 71 ± 10 years. The gender distribution included 3 men and 9 women whose body mass index was 24.6 ± 2.4 (mean ± SD). There was significant difference observed in UniFrac beta-diversity between the two groups (p=0.001). The relative taxonomic patterns of the microbiota between CRC stool and tissue showed that Bacteroides and Fusobacterium were more dominant in tissue than stool at both phylum and genus levels. Further taxonomic differences between the samples revealed that Firmicutes, Bacilli, and Lactobacillacease were over-represented in CRC patient stool, while Pseudomonadales (p&amp;lt;0.001) and Pseudomonas (p&amp;lt;0.001) were identified in the mucosal tissue. Conclusion Our findings revealed that notable distinction was observed in the beta-diversity, indicating a unique microbial composition in tissue. Specifically, higher prevalence of Bacteroides and Fusobacterium at the genus level were identified in tissue. Additionally, our analysis highlighted Pseudomonadales and Pseudomonas emerged in CRC mucosal tissues. Furthermore, by comparing with a healthy control group, we aim to further elucidate potential markers unique to CRC patients. Citation Format: Sanghun Lee. Comparative Microbiome Analysis of Paired Mucosal and Fecal Samples in Korean Colorectal Cancer Patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 758.

Specific vaginal and gut microbiome and the anti-tumor effect of butyrate in cervical cancer women

ObjectiveTo investigate the vaginal and gut microbes changes during the carcinogenesis of cervical and the auxiliary diagnostic value. To investigate the effect of microbiome-specific metabolites butyric on cervical cancer cells. MethodsWe studied 416 vaginal 16S rRNA sequencing data and 116 gut sequencing data. Reads were processed using VSEARCH. We used Shannon index, Chao1 index, Simpson diversity index, β diversity index, Linear discriminant analysis Effect Size (LEfSe), co-abundance network and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to explore microbiome differences between groups. We constructed random forest models based on genus and verified its discriminant effect. Finally, we used the cell counting kit-8 (CCK-8) method to detect cell proliferation capacity and flow cytometry to detect apoptosis and induction of cell cycle progression. ResultsCompared to the non-cancerous population, patients with cervical cancer had unique microbial community characteristics in both vaginal and gut ecological niches. Our predictive model based on genus in two ecological regions achieved high accuracy in the diagnosis of cervical cancer (vaginal model AUC=91.58 %; gut model AUC=99.95 %). Butyric inhibited cervical cancer cell proliferation in a concentration-dependent manner and promoted apoptosis of cancer cells. ConclusionSignificant differences were found in vaginal and gut microbes in patients with cervical cancer compared to the non-cancerous population. The prediction models constructed at the genus level in both ecological sites have good diagnostic value. Microorganisms may be involved in cervical cancer progression in a metabolite-dependent way, and targeting butyric may provide therapeutic options for cervical cancer.

Combining fecal microbiome and metabolomics reveals diagnostic biomarkers for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most predominant subtypes of esophageal cancer. The characteristics of the gut microbiome and its metabolites from patients with ESCC have not been adequately studied and discussed. In this study, 40 fecal samples (20 from ESCC patients and 20 from healthy controls) were analyzed by 16S rRNA gene sequencing and untargeted metabolomics. The data sets were analyzed individually and synthesized using various bioinformatics methods. Alpha and beta diversity indicated significant differences in microbial diversity and abundance between ESCC and healthy control feces. At the genus level, the abundance of Phascolarctobacterium, Sutterella, and Streptococcus was significantly increased in ESCC. At the genus level, linear discriminant analysis effect size identified two biomarkers: Bacteroides_stercoris and Prevotella_copri. Untargeted metabolomics analysis revealed 307 differential metabolites between ESCC and healthy control feces, with indoles and derivatives, tropane alkaloids, lipids, and lipid-like molecules in higher relative abundance in ESCC feces than in healthy control feces. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that unsaturated fatty acids (FAs), ascorbate and aldarate metabolism, and hypoxia-inducible factor 1 signaling pathway were significantly associated with differential metabolite. Phenylethanolamine and despropionyl p-fluoro fentanyl could be used as reliable biomarkers to differentiate ESCC from healthy control. The correlation analysis showed that Prevotella may be involved in the synthesis of fatty acyl, carboxylic acids and derivatives, benzenes and substituted derivatives, organic oxygenates, and indoles and derivatives as metabolites. Fusicatenibacter and Lachnospira may be involved in the degradation of indoles and derivatives. Alistipes, Agathobacter, and Parabacteroides may be involved in the synthesis of indoles and derivatives with strong contributions. There is an intricate relationship between the gut microbiome and the levels of several metabolites (e.g., fatty acyls, carboxylic acids and derivatives, indoles, and derivatives). Microbial-associated metabolites can be used as diagnostic biomarkers in therapeutic exploration. Further analysis revealed that Prevotella, Alistipes, Agathobacter, and Parabacteroides might promote ESCC by regulating the synthesis of indoles and their derivatives. The results of this study provide favorable evidence for the early diagnosis of ESCC and subsequent individualized treatment and targeted interventions.IMPORTANCEWe describe for the first time the differences in fecal microbiome composition and metabolites between patients with esophageal squamous cell carcinoma (ESCC) and healthy controls by 16S rRNA gene sequencing and untargeted metabolomics. The results of this study provide a favorable basis for the early diagnosis of ESCC and subsequent targeted interventional therapy.