Principal Coordinate Analysis Plot Research Articles

Associative analysis of sludge microbiota and wastewater degradation efficacy within swine farm sludge systems

Industrial wastewater management is a significant global challenge. Sludge microbiota from swine farms may play a crucial role in enhancing wastewater treatment processes, thereby reducing water pollution from industrial activities. A deeper understanding of this complex community could lead to innovative approaches for improving wastewater treatment methods. Sludge samples were collected from the anaerobic, sedimentation, and thickening tanks of ten swine farms. The microbiota communities were analyzed using 16S rRNA full-length sequencing on the PacBio platform, with subsequent data analysis conducted on the QIIME2 platform utilizing the SILVA database. Compared to anaerobic and thickening tanks, the sedimentation tanks exhibited a unique profile of sludge microbiota, with higher abundances of the phyla Proteobacteria, Bacteroidota, and Caldatribacteriota. Additionally, sludges from farms already utilized in processing industrial water—specifically farms B, G, and J—contained higher concentrations of bacteria (>20 ng/μL), indicating the robustness of the bacterial load for practical industrial use. Furthermore, sludge from farms with higher alpha diversity, such as E, G, I, and J, exhibited enriched degradation profiles, including the degradation of aromatic compounds, polymers, industrial compounds, toluene, and vanillin. The farms were categorized based on wastewater ammonia nitrogen degradation levels, revealing a clustering effect of the microbiota from the sedimentation tanks in the Principal Coordinates Analysis (PCoA) plot. A higher relative abundance of the families Rhodocyclaceae, AKYH767, and Comamonadaceae, and a lower abundance of the families Anaerolineaceae and Christensenellaceae, were found in groups with high ammonia nitrogen reduction, suggesting potential targets for bioaugmentation strategies. In conclusion, this study underscores the critical role of microbial abundance, composition, and biodiversity in optimizing wastewater treatment and advocates for comprehensive microbiota analysis to identify suitable sludge for industrial applications.

Optimization of a DNA extraction protocol for improving bacterial and fungal classification based on Nanopore sequencing.

Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimized protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and full-length amplicon PCR-based approaches using Nanopore sequencing. Three lysis conditions were applied to a mock microbial community, including known bacterial and fungal species: ZymoBIOMICS lysis buffer (ML) alone, incorporating bead-beating (MLB) or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). In profiling of bacteria in comparison to reference data, MLB had more statistically different bacterial phyla and genera than the other two conditions. In fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect Malassezia and Cryptococcus. Also, a principal coordinates analysis plot by the Bray-Curtis metric showed a significant difference among groups for bacterial (P=0.033) and fungal (P=0.012) profiles, highlighting the importance of understanding the biases present in pretreatment. Overall, microbial profiling and diversity analysis revealed that ML and MLBE are more similar than MLB for both bacteria and fungi; therefore, using this specific pipeline, bead-beating is not recommended for whole gene amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, taxonomic classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human bacterial and fungal microbiome studies.

Sample collecting methods for bacterial community structure analysis of scalp hair: non-invasive swabbing versus intrusive hair shaft cutting

Human skin samples for microbiome analysis are traditionally collected using a non-invasive swabbing method. Here, we compared the differences in bacterial community structures on scalp hair and scalps with samples collected using non-invasive swabbing and cutting/removal of scalp hair in 12 individuals. Hair-related samples, such as hair shafts and hair swabs, had significantly higher alpha diversity than scalp swab samples, whereas there were no significant differences between hair shafts and hair swabs. The relative abundances of the three major phyla and five major operational taxonomic units were not significantly different between the hair shaft and hair swab samples. The principal coordinate analysis plots based on weighted UniFrac distances were grouped into two clusters: samples from hair-related areas and scalp swabs, and there were significant differences only between samples from hair-related areas and scalp swabs. In addition, a weighted UniFrac analysis revealed that the sampling site-based category was a statistical category but not a hair sampling method-based category. These results suggest that scalp hair bacteria collected using non-invasive swab sampling were comparable to those collected cutting/removal of scalp hair.

PSIX-10 Effects of Bacillus subtilis ATCC PTA-122264 on macronutrient digestibility and fecal characteristics, metabolites, and microbiota of healthy adult dogs subjected to an abrupt diet change

Abstract Probiotic use in dogs is increasing, with spore-forming Bacillus species gaining interest due to their viability during processing, storage, and in the gastrointestinal tract. B. subtilis is known for its adaptable metabolism that may be useful in pets subjected to abrupt dietary changes. The objective of this study was to determine dietary apparent total tract macronutrient digestibility (ATTD) and fecal characteristics, metabolites, and microbiota of healthy adult dogs supplemented with B. subtilis ATCC PTA-122264. All procedures were approved by the University of Illinois IACUC prior to experimentation. Dogs [n = 12; 6 ± 1 yr; 8.71 ± 0.91 kg body weight (BW)] were used in a replicated 3x3 Latin square design (n = 12/group) and fed to maintain BW. In each 42-d experimental period, dogs were allotted to one of three treatments: Control (kibble diet only), Low [kibble diet + 1×109 colony-forming units (CFU) of B. subtilis], and High (kibble diet + 5×109 CFU of B. subtilis). After a 22-d adaptation phase, a 5-d fecal collection phase was followed by a blood collection phase. On d 28, dogs were abruptly changed to a canned diet and fed for 14 d, with fecal samples collected on d 30 (2 d post change), 34 (6 d post change), 38 (10 d post change), and 42 (14 d post change). Fecal microbiota data were evaluated using QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P < 0.05 being considered significant. B. subtilis supplementation of the kibble diet did not influence food or energy intake, fecal output, ATTD, most serum metabolites, hematology, fecal characteristics, fecal bacterial alpha and beta diversity indices, and dysbiosis index, demonstrating the safety of the strain in dogs. However, fecal Streptococcus and Blautia abundances were higher in High dogs than Low or Control dogs, while E. coli abundance was less in Low dogs than High and Control dogs. The abrupt change to a canned diet led to significant alterations in fecal characteristics, dysbiosis index, and bacterial abundances, which were not influenced by B. subtilis supplementation. Bacterial alpha diversity measures were affected, with observed ASV (amplicon sequence variants) and the Fisher Index reducing, and the Shannon Index increasing by dietary change. The abrupt dietary change caused a distinct shift in microbial communities, as evidenced by principal coordinates analysis plots. The relative abundances of various fecal bacterial taxa, including Bifidobacterium, Faecalibaculum, Turicibacter, and Lactobacillus were decreased, while Collinsella, Ruminococcus gnavus group, and Escherichia-Shigella were increased with the dietary change. Our data support the notion that daily supplementation of 1 – 5×109 CFU of B. subtilis ATCC PTA-122264 in healthy dogs is safe and does not affect markers of general health and fecal characteristics, warranting further exploration.

GENETIC DIVERSITY OF Moringa oleifera Lam. IN SOUTHEASTERN MEXICO

Moringa oleifera Lam. is a plant with nutritional and nutraceutical properties. It was introduced from India to Mexico, and some of its ecotypes are cultivated for industrial use. These ecotypes show morphological variations, indicating genetic variation. Therefore, the purpose of this study was to evaluate eight simple sequence repeat (SSR) loci to assess the genetic diversity of 10 cultivated M. oleifera populations collected from the southeast of Mexico. All markers generated a total of 127 alleles, with 15.8 alleles per locus. The polymorphic information content of markers was 0.869, with an average heterozygosity of 0.194. At the population level, the Nei´s gene diversity was found to be 0.66, with 4.9 different alleles per locus and 5.2 genotypes on average per accession. The distribution of genetic variation was 20 % among populations, 58 % between individuals, and 22 % within individuals. The principal coordinate analysis (PcoA) plot and unweighted pair group method with arithmetic mean (UPGMA) linkage revealed three groups, corresponding to the three original genetic populations identified through structure analysis (Delta K = 3). Results showed significant differences in genetic diversity in M. oleifera populations stablished in Southeastern Mexico, which can be leveraged to increase the use of this germplasm.

Impact of rumen cannulation surgery on rumen microbiota composition in Hanwoo steers.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Comparison of mucosal microbiomes from biliary tract among patients with extrahepatic cholangiocarcinoma, patients with chronic cholecystitis, and healthy controls.

550 Background: Cholangiocarcinoma (CCA) is a malignancy associated with a poor prognosis. Gut microbiome dysbiosis is considered to be related with multiple disease, including and cancer. However, relationship between mucosal microbiome dysbiosis and CCA remains unclear. In this study, we evaluated mucosal microbiome of patients with extrahepatic-CCA (EH-CCA) and compared it with patients with chronic cholecystitis (CC) and living liver donors who served as healthy control (HC), to understand the role of microbiome in development EH-CCA. Methods: A total of 18 patients with EH-CCA, 21 patients with CC, and 17 HC were included. During operation, swab sample were obtained from cancer tissue of patient with EH-CCA and from gallbladder in other participants, respectively. The bacterial 16S rRNA gene (V3/V4 region) was amplified using PCR and sequenced on the Illumina MiSeq platform. The QIIME 2 pipelines were used to analyze the raw data. Results: The median age were 71, 53, and 29 years for patients with EH-CCA, patients with CC, and HC, respectively. The mean number of operational taxonomic unit were significantly higher in the CCA (13,037 [SD 9,676]) compared to HC (5,045 [12,679]; p&lt;0.001). Among the sample from HC, the most abundant taxa at phylum level was Firmicutes (74.47%), followed by Bacteroidota (14.8%), and Proteobacteria accounted for only 2%. In the CC group, Firmicutes is most abundant (63.15%) phylum, followed by Proteobacteria (22.09%). In contrast, among the samples from CCA, Proteobacteria were the most common phylum (50.43%). Pairwise comparison of α-diversity measured by Chao1 index showed that richness was higher in samples from CC compared to other groups (vs HC, p=0.02; vs CCA, p&lt;0.001). In terms of α-diversity measured by Shannon index, samples from cancer have lower diversity compared to healthy controls ( p&lt;0.001), however, no significant differences were found between samples from cancer and CC ( p=0.1). The Principal Coordinate Analysis plot based on Bray-Curtis distance showed that all three groups formed significantly separate clusters (p=0.001). The linear discriminant analysis effect size showed that the most abundant taxa from samples from CCA were Enterobacter, Klebsiella, and Fusobacterium. In the samples from HC, Limosilactobacillus, Lachnospiraceae_NK4A136_group, and Faecalibacterium were most significant, while Lactobacillus and Pseudomonas were most abundant among the CC group. Conclusions: This study compared the mucosal microbiome from biliary tract among patients with CCA, patients with CC, and HC. All three groups showed significantly different microbiome profiles. In CCA sample, the proportion of Proteobacteria is high, and diversity were low, suggesting mucosal microbial dysbiosis. Further studies are needed to investigate the clinical implications of microbial dysbiosis in CCA.

98 Characterization of Factors Affecting the Fecal Microbiome in Young Canadian Pigs

Abstract Although established as an important contributor to pig health and performance, high diversity and variability of the microbiome has challenged identification of a beneficial community composition that could inform best management practice and gut modifier development. To address this challenge, rectal swabs were collected at intervals from near birth to one-week postweaning from 4 piglets in 10 litters from 13 conventional and 9 RWA barns across Canada. The 16S rRNA gene was sequenced in 1,997 fecal samples collected at ~4 days of age (4d), 1 day before weaning (W-1), and 7 days after weaning (W+7), piglets were also weighed at the time of sampling. Raw sequences were denoised and assigned to genera using DADA2. After quality control and filtering there was an average of 32,710 reads per sample, with 649 genera identified in the samples. Principal Coordinate Analysis (PCoA) plots were created using the vegan and phyloseq packages (cao model and MDS settings). Relationships between metadata factors and the microbiome were investigated using PERMANOVA analysis in the adonis2 package. In PCoA plots samples clustered by production stage, with an expected marked change of the bacterial community following weaning. Because production stage had a large effect on the microbiome the effects of metadata factors were investigated within sampling visit. Metadata factors investigated included farrowing pen (FP) location at the 4 d-old sampling, barn and rearing system [conventional versus raised without antibiotics (RWA)] and the lifetime health (LH) status including always healthy, received a health score 1 or greater at any one visit or did not complete the study. At ~4 d of age, PERMANOVA indicated that all these factors were significantly linked to the microbiome composition (P = 0.001), FP explained the most microbiome variation (R2 = 0.338), followed by barn (R2 = 0.217). Rearing system and LH explained much less of the variation, R2 = 0.009 and R2 = 0.005, respectively. At W-1, the microbiome was not significantly correlated to LH (P = 0.053); however, the other metadata factors were significant (P = 0.001) with similar R2 values to the previous timepoint (FP; R2 = 0.317, barn R2 = 0.210, and system R2 = 0.008). At W+7 all metadata factors were once again significant (P &amp;lt; 0.005). The correlation to FP had reduced to R2 = 0.245, cohort increased to R2 = 0.266, system effects doubled to R2 = 0.21, and LH remained very low at R2 = 0.006. However, interactions between LH and FP (R2 = 0.059) and system and LH (R2=0.003) were also observed. These results suggest that farrowing pen has a significant and lasting effect on the microbiome. The effect of being raised RWA or conventionally was very small in preweaning piglets, but that affect increased post weaning. The immediate environment early in life appears to have a large effect on piglet microbiomes and may present an opportunity for beneficial intervention.

Comparison of the Gut Microbial Communities of Domestic and Wild Mallards (Anas platyrhynchos) Based on High-Throughput Sequencing Technology.

Mallards (Anas platyrhynchos) are currently one of the most popular species in rare bird breeding in several southern provinces of China, but there have been no studies comparing the gut microbial communities of domestic and wild mallards. In this study, 16S rRNA gene high-throughput sequencing technology was used to compare the composition and diversity of gut microbial communities in domestic and wild mallards. Alpha diversity analysis showed significant differences in gut microbial communities between the two groups of mallards, and the diversity and richness of gut microbial communities were significantly higher in wild mallards than in domestic mallards. Beta diversity analysis showed that the two groups of stool samples were mostly separated on the principal coordinate analysis (PCoA) plot. In domestic mallards, Firmicutes (68.0% ± 26.5%) was the most abundant bacterial phylum, followed by Proteobacteria (24.5% ± 22.9%), Bacteroidetes (3.1% ± 3.2%), Fusobacteria (2.2% ± 5.9%), and Actinobacteria (1.1% ± 1.8%). The dominant bacterial phyla in wild mallards were Firmicutes (79.0% ± 10.2%), Proteobacteria (12.9% ± 9.5%), Fusobacteria (3.4% ± 2.5%), and Bacteroidetes (2.8% ± 2.4%). At the genus level, a total of 10 dominant genera (Streptococcus, Enterococcus, Clostridium, Lactobacillus, Soilbacillus, Bacillus, Acinetobacter, Comamonas, Shigella, and Cetobacterium) with an average relative abundance greater than 1% were detected in the fecal samples of both groups. The average relative abundance of five potential pathogenic genera (Streptococcus, Enterococcus, Acinetobacter, Comamonas, and Shigella) was higher in domestic mallards than in wild mallards. The enrichment of pathogenic bacteria in the intestinal tract of domestic mallards should be of sufficient concern.

Distribution of bacterial community structures on human scalp hair shaft in relation to scalp sites.

Bacterial community structure on the human skin is specific to each individual and varies among different body sites. In this study, we investigated differences in bacterial community structure among 5 hair sampling sites and among 12 individuals. Significant differences were found between individuals in terms of alpha diversity and relative abundance of major bacterial phyla and genera, whereas no differences were found between hair sampling sites. The principal coordinate analysis plots of within-individual group tended to converge individually, whereas those of within-hair sampling site group did not cluster. In addition, weighted UniFrac analysis showed that the individual-based category was a statistically significant category but not the scalp hair sampling site-based category. These results suggest that the distribution of bacterial community structures on scalp hair shafts within individuals was relatively steady, even when the scalp hair sampling site was different.

Alterations of the salivary microbiota in gastroesophageal reflux disease

ObjectivesGastroesophageal reflux disease (GERD) is among the most prevalent gastrointestinal disorders. The oral microbiota plays an important role in human health and may be altered by the presence of GERD. Here, we aimed to investigate the alterations of salivary microbiota in GERD patients. MethodsWe collected clinical information and salivary samples from 60 individuals. All participants underwent combined pH/impedance monitoring measurement and submitted samples for salivary microbiota sequencing. According to acid exposure time and DeMeester score, participants were divided into two groups: GERD + (Group G) and GERD – (Group C). ResultsThere was no significant difference in alpha diversity between study groups. Regarding beta diversity, principal coordinate analysis plots indicated that the microbiota composition data of the participants were grouped within partial overlapping clusters. The statistical analysis of the distance matrices was performed using the Adonis test (p = 0.017). Based on linear discriminant analysis effect size, the relative abundances of the phylum Bacteroidetes, class Bacteroidia, order Bacteroidales, family Prevotellaceae, and genus unidentified_Prevotellaceae were enriched in Group G. Compared with Group C, the phylum Actinobacteria, classes unidentified_Actinobacteria and Bacilli, orders Micrococcales and Lactobacillales, families Micrococcaceae and Streptococcaceae, and genuses Rothia and Streptococcus were decreased in Group G. At the genus level, the abundances of Streptococcus and Rothia were negatively correlated with DeMeester score and acid exposure time. ConclusionsThis study revealed alterations of the salivary microbiota in GERD patients, suggesting that acid reflux changes the oral ecosystem.

Abstract 5903: Comparison of the tissue and gut microbiome in extrahepatic cholangiocarcinoma patients and healthy liver donors

Abstract Background: Biliary tract cancer (BTC) is an aggressive malignancy associated with a poor prognosis. Most BTC patients are diagnosed at an unresectable stage; further, the efficacy of cytotoxic chemotherapy for BTC remains unsatisfactory. Recently, there is growing evidence that gut microbial dysbiosis is related to multiple diseases, including biliary disease and cancer. However, the association between tumor and gut microbiome environments and BTC, especially extrahepatic cholangiocarcinoma (EH-CCA), remains unclear. In the present study, we compared the microbiome from the tissue and stool samples of EH-CCA patients and healthy living liver donors (controls). Methods: A total of 24 people (13 EH-CCA patients and 11 healthy controls) were included. We collected fecal and swab samples, which were obtained from the EH-CCA tissues of the patients and normal gallbladder tissues of the controls during operation. The bacterial 16S rRNA gene (V3 and V4 region) was amplified using PCR and sequenced on the Illumina MiSeq platform. The QIIME 2 pipelines were used to analyze the raw data. Results: The median age of the patients and controls were 70 (range, 58-79) and 29 (range, 19-35) years, respectively. The swab samples from EH-CCA patients showed significantly lower alpha diversity than the samples from the controls (pShannon &amp;lt; 0.001). Proteobacteria was the most commonly found phylum (53%) in the EH-CCA patients; Firmicutes (79%) and Actinobacteria (13%) were the common phyla in the controls. The genera Streptococcus (25.1%), Escherichia_Shigella (16.9%), and Enterococcus (13.9%) were found in the patient samples, whereas Blautia (12%), Limosilactobacillus (12%), and Bifidobacterium (19%) were observed in the controls. The Principal Coordinate Analysis (PCoA) plot of the weighted UniFrac distances showed that the patient and control groups formed separate clusters. Among the fecal samples, Streptococcus (12.5%), bacterioides (8.2%) is most common in patients’ group, whereas Blautia (13.8%) and Faecalibacterium (11.5%) is most common in control group at the gene levels. The PCoA plot of the fecal samples showed significant differences in microbiota composition between the EH-CCA patients and healthy controls (P=0.001 by PERMANOVA). In the beta diversity analysis of all samples (swab plus fecal), the patient-swab samples formed distinct clusters, whereas the patient-fecal, control-swab, and control-fecal samples showed overlapping clusters.Conclusions: To the best of our knowledge, this study is the first that compares the microbiome of tissues and stools from EH-CCA patients and healthy controls; the EH-CCA patients and healthy controls showed different microbiota profiles. Further studies are needed to investigate the clinical implications of microbial dysbiosis in BTC and formulate novel microbiome-based treatments for BTC. Citation Format: Sora Kang, Jihee Kang, Gun-Seok Park, Seung-Hyun Ko, Jung-Hyun Park, Yunseon Jang, Ik-Chan Song, Hyewon Ryu, Seok-Hwan Kim. Comparison of the tissue and gut microbiome in extrahepatic cholangiocarcinoma patients and healthy liver donors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5903.

Omega-3-Rich Fish-Oil-Influenced Mouse Gut Microbiome Shaped by Intermittent Consumption of Beef.

Beef consumption can provide various amino acids, lipids, vitamins, and minerals; however, excessive intake causes metabolic disorders and increases the probability of obesity, atherosclerosis, and colorectal cancer. The intake of omega-3 fatty acids can ameliorate metabolic disorders by lowering blood glucose and triglyceride levels. In the present study, we investigated the effect of omega-3-rich fish oil on body performance and the gut microbiome in a beef-rich diet. Four-week-old C57BL/6 mice were distributed into four groups (chow diet [Chow], chow with beef diet [Beef], chow with omega-3 diet [Cw3], and chow with beef and omega-3 diet [Bw3]). We observed that body weight was unaltered between groups, and serum triglyceride levels were reduced in the omega-3 supplemented groups. The beta diversity indices, unweighted UniFrac distance (P = 0.001), and Jaccard distance (P = 0.001) showed statistically significant differences, and the principal coordinates analysis plot showed a clear separation between groups. In addition, the taxonomic comparison revealed that beef consumption increased numerous potentially pathogenic bacteria, including Escherichia-Shigella, Mucispirillum, Helicobacter, and Desulfovibrio, which were decreased following omega-3 supplementation. Metabolic comparison based on 16S rRNA revealed that energy and glucose metabolism were higher in omega-3 supplemented groups. Our findings suggest that the omega-3 supplementation under intermittent beef consumption contributes to changes in the gut microbiome and microbial metabolic pathways.

Pilot Study on the Geographical Mapping of Genetic Diversity among European Chestnut (Castanea sativa Mill.) Cultivars in Southern Italy.

Knowledge of the spatial distribution of European chestnut (Castanea sativa Mill.) cultivar diversity is essential for managing and conserving the genetic resources of this fruit tree species in Southern Italy. To this goal, the present work investigated the feasibility of mapping, through spatial representation, the distribution of genetic diversity of traditional chestnut varieties in the area of the Roccamonfina Regional Park in the Campania Region. After Principal Coordinates Analysis (PCoA) of molecular-genetic data, chestnuts formed varietal groups in a leopard spot on PCoA plots with a relatively high degree of genetic diversity. Successively, a Geographic Information System (GIS) tool utilized these molecular-genetic data to create a genetic divergence surface by geospatial interpolation on the geographic map of the Regional Park corresponding to each chestnut variety. The regions containing more biodiversity richness resulted in differentially colored from those containing cultivars less genetically distant from each other; thus, the area in study was consistently colored according to the allelic richness as evaluated by molecular-genetic markers. The combined use of tools for molecular and spatial analysis allowed for drafting genetic landscapes with the aim of extracting useful information for the safeguarding of the chestnut biodiversity at risk.

DOP47 Gut microbiome contributes to the development of immune checkpoint inhibitor-related colitis

Abstract Background The gut microbiota is implicated in shaping immune checkpoint inhibitors (ICIs) efficacy, but relatively little is known about how the microbiota influences immune-related adverse events (irAEs). Our study aimed to explore the association and causal relationship between gut microbiome and immune checkpoint inhibitor-related colitis. Methods We collected faecal samples from 32 patients with advanced malignant tumor patients developing irAE after anti-PD-1 therapy from May 2020 to Aug 2021. Faecal samples were analyzed by 16s rDNA amplicon sequencing method on Illumina MiSeq (PE300) sequencing platform. We also performed faecal microbiota transplantation (FMT) for antibiotic-treated mice with samples from patients with colitic irAE and without irAE. Results We compared the microbiota between 8 colitic irAE and 24 non-colitic irAE patients. Principal coordinates analysis (PCoA) plot showed significant difference of microbiota composition by Adonis analysis (P=0.003) (Fig. 1A). LEfSe analysis detected a lower abundance of Bacteroidota and a higher abundance of Proteobacteria at the phylum level and a lower abundance of Bacteroides and Bifidobacterium and higher abundance of Enterococcus at the genus level in colitic irAE (Fig. 1B). The total abundance of major butyrate-producing bacteria was lower in colitic irAE compared with non-colitic irAE patients (P=0.018) (Fig. 1C). Functional prediction analyses revealed different abundances of environmental information processing, genetic information processing, and metabolic pathways between colitic and non-colitic irAE patients (Fig. 1D). By FMT experiments in mice, we sought to explore the causal relationship between microbiota immune-related colitis. After injection of anti-PD-1 and anti-CTLA-4, three of nine colitic-irAE-FMT mice developed fatal severe colitis, while all nine non-irAE-FMT mice survived without colitis (P=0.052). The three colitic mice showed more rapid weight loss and higher disease activity index (DAI) scores compared with other mice (Fig. 2A and B). The histological colitis score in the irAE-FMT-Sac. mouse (14 points) was higher than those in the non-irAE-FMT mice (3.89 ± 1.17 points) and colitic-irAE-FMT-survival mice (3.67 ± 1.03 points) (Fig. 2C). The mean IHC scores of irAE-FMT-Sac. mouse from high to low were CD8 3.8 ± 1.3 points, CD4 2.0 ± 1.9 points, and CD20 1.1 ± 1.3 points (Fig. 2D), which also supported the occurrence of immune-related colitis. Figure 1. The gut microbiotas of patients with colitic and non-colitic type irAEs. Figure 2. Comparison of colitis in colitic-irAE-FMT and non-irAE-FMT mice. Conclusion Gut microbiota might play important role in the development of immune-related colitis, possibly by modulating metabolic pathways.

Analysis of serum antioxidant capacity and gut microbiota in calves at different growth stages in Tibet.

The hypoxic environment at high altitudes poses a major physiological challenge to animals, especially young animals, as it disturbs the redox state and induces intestinal dysbiosis. Information about its effects on Holstein calves is limited. Here, serum biochemical indices and next-generation sequencing were used to explore serum antioxidant capacity, fecal fermentation performance, and fecal microbiota in Holstein calves aged 1, 2, 3, 4, 5, and 6 months in Tibet. Serum antioxidant capacity changed with age, with the catalase and malondialdehyde levels significantly decreasing (p < 0.05), and superoxide dismutase levels significantly increasing (p < 0.05) with age. No significant differences (p > 0.05) in total volatile fatty acid levels were noted between the groups. In all groups, Firmicutes, Bacteroidetes, and Actinobacteria were the three most dominant phyla in the gut. Gut microbial alpha diversity significantly increased (p < 0.05) with age. Principal coordinate analysis plot based on Bray-Curtis dissimilarity revealed significant differences (p = 0.001) among the groups. Furthermore, the relative abundance of various genera changed dynamically with age, and the serum antioxidant capacity was associated with certain gut bacteria. The study provides novel insights for feeding Holstein calves in high-altitude regions.

Gut metabolomic profiles in paediatric ulcerative colitis patients prior to and after receiving faecal microbiota transplants.

Ulcerative colitis (UC) is an immune-mediated inflammation of the colonic mucosa. Gut microbiota dysbiosis may play a significant role in disease pathogenesis by causing shifts in metabolomic profiles within the gut. To identify differences and trends in the metabolomic profile of paediatric UC patients pre- and post-faecal microbiota transplants (FMT). Forty-six paediatric patients with mild-to-moderate UC and 30 healthy paediatric patients were enrolled in this study. Baseline stool samples were collected prior to FMT initiation and at months 1, 3, 6, and 12 post-FMT. Pediatric Ulcerative Colitis Activity Index (PUCAI) scores were calculated at baseline and months 1, 3, 6, and 12 after FMT. The average Bray-Curtis dissimilarities to healthy subjects decreased after FMT. In principal coordinate analysis plots, UC patients' centroids drew nearer to healthy individuals. The variance explained by phenotype (Healthy versus UC) reduced and remained significant. From 1 to 3 months after FMT, PUCAI trends were statistically significant and decreasing. PUCAI scores remain flat starting 6 months after FMT. This study concludes that paediatric UC patients have a significantly different baseline metabolite profile than healthy controls. Although being time limited, FMT significantly altered these metabolite profiles and shifted them towards that of healthy controls.

Effects of a Saccharomyces cerevisiae fermentation product on fecal characteristics, metabolite concentrations, and microbiota populations of dogs subjected to exercise challenge.

The objective of this study was to determine the fecal characteristics, microbiota, and metabolites of dogs fed a Saccharomyces cerevisiae fermentation product (SCFP) and subjected to exercise challenge in untrained and trained states. Thirty-six adult dogs (18 male, 18 female; mean age: 7.1 yr; mean body weight: 29.0 kg) were randomly assigned to control or SCFP-supplemented (250 mg/dog/d) diets and fed for 10 wk. After 3 wk, dogs were given an exercise challenge (6.5 km run), with fresh fecal samples collected pre- and post-challenge. Dogs were then trained by a series of distance-defined running exercise regimens over 7 wk (two 6.4 km runs/wk for 2 wk; two 9.7 km runs/wk for 2 wk; two 12.9 km runs/wk for 2 wk; two 3.2 km runs/wk). Dogs were then given exercise challenge (16 km run) in the trained state, with fresh fecal samples collected pre- and post-challenge. Fecal microbiota data were evaluated using QIIME2, while all other data were analyzed using the Mixed Models procedure of SAS. Effects of diet, exercise, and diet*exercise were tested with P < 0.05 considered significant. Exercise challenge reduced fecal pH and ammonia in both treatments, and in untrained and trained dogs. After the exercise challenge in untrained dogs, fecal indole, isobutyrate, and isovalerate were reduced, while acetate and propionate were increased. Following the exercise challenge in trained dogs, fecal scores and butyrate decreased, while isobutyrate and isovalerate increased. SCFP did not affect fecal scores, pH, dry matter, or metabolites, but fecal Clostridium was higher in controls than in SCFP-fed dogs over time. SCFP and exercise challenge had no effect on alpha or beta diversity in untrained dogs. However, the weighted principal coordinate analysis plot revealed clustering of dogs before and after exercise in trained dogs. After exercise challenge, fecal Collinsella, Slackia, Blautia, Ruminococcus, and Catenibacterium were higher and Bacteroides, Parabacteroides, Prevotella, Phascolarctobacterium, Fusobacterium, and Sutterella were lower in both untrained and trained dogs. Using qPCR, SCFP increased fecal Turicibacter, and tended to increase fecal Lactobacillus vs. controls. Exercise challenge increased fecal Turicibacter and Blautia in both untrained and trained dogs. Our findings show that exercise and SCFP may affect the fecal microbiota of dogs. Exercise was the primary cause of the shifts, however, with trained dogs having more profound changes than untrained dogs.

Characterization of the Tumor-Associated Microbiome Landscapes of HPV-Related Anogenital Cancers

<h3>Purpose/Objective(s)</h3> There is an increasing interest in the composition of tumor-associated microbiomes and their effect on treatment response and patient outcomes. This study aims to explore the differences between the tumor-associated microbiome landscapes of patients diagnosed with HPV-related anogenital cancers. We have previously shown that <i>Lactobacillus iners</i> is associated with poor treatment response among cervical cancer patients, so we studied the presence of <i>L. iners</i> among other anogenital cancers. <h3>Materials/Methods</h3> Tumor swabs were collected from patients diagnosed with cervical cancer (n=85), anal cancer (n=20), vaginal cancer (n=7), and vulvar cancer (n=3) receiving chemoradiation therapy (CRT) from 2 institutions. Samples were collected and analyzed at baseline, week 1, 3, and 5 during CRT and at 3 months post-CRT using 16Sv4 rRNA gene sequencing. Multivariate community-level differences between the tumor microbiomes was conducted using principal coordinates analysis (PCoA) plots and pairwise PERMANOVA. Linear Discriminant Effect Size (LEfSe) analysis was used to identify specific taxa abundant among the different tumor types at baseline and end of treatment. <h3>Results</h3> PERMANOVA revealed significant compositional differences between the cancer types (p= 0.001). There were significant compositional differences between all cancer types (q<0.01 for all), except between vaginal and vulvar cancers (q= 0.12). LEfSe revealed that the microbiome of each cancer was dominated by different bacteria. <i>L. iners</i> was enriched in cervical samples, Clostridia and Bacteroidaceae were enriched in anal samples, Proteobacteria was enriched in vaginal samples, and Peptostreptococcales-Tissierellales and Staphylococcales were enriched in vulvar samples at baseline. Moreover, by the end of treatment, Bifidobacteriales were enriched in cervical samples, while Proteobacteria, Corynebacteriales, and Clostridia were enriched in anal samples, and Bacteroidota was enriched in vulvar samples. Of interest, L. iners was present in two out of three non-responders with anal cancer, and one out of two non-responders with vaginal cancer. However, the proportions of Liners in these patients were less than those in patients with cervical cancer. <h3>Conclusion</h3> Our results show that the tumor microbiomes of HPV-related anogenital cancers differ significantly from each other and have distinct reactions to CRT. These findings could lay the groundwork for interventions tailored to the tumor-specific microbiome to enhance the effectiveness of CRT.

Genetic diversity analysis using SSR marker in Naga King chilli (Capsicum chinense Jacq.) genotypes

The present investigation has been undertaken with the objective to study the genetic variability and diversity among the Naga king chilli genotypes using SSR marker at Medziphema during kharif season of 2018. All total 89 bands were produced by 30 primers, 37 bands being polymorphic and producing average percentage polymorphic bands of 41.66 %. The Primer CAMS-826 showed highest percentage of polymorphic bands (75%), followed by primer CAMS 032, CAMS-117, CAMS- 864 and CAMS-647 which revealed (66.66 %) and primer CAeMS 009, CAMS 163, CAMS 871, HmpsE 047 AND HpmsE 014 exhibited 50% polymorphism which indicates that the primer can be used for genetic diversity study of C. chinense genotypes. Genetic parameters analyzed using Popgene® software indicated that Peren population has higher diversity among the four region populations. Jaccard’s similarity coefficient between the genotypes ranged from 0.64 to 0.96, indicating moderate variation between the genotypes. Clustering analysis depicted three clusters where germplasm collected from Peren and Medziphema showed close relatedness and Principal Coordinate Analysis (PCo A) plot obtained also supports cluster analysis data. Germplasm evaluation is of immense important for genetic crop improvement.