MiSeq Platform Research Articles

Phenotypic and genomic characterization of a multidrug-resistant Salmonella enterica serovar Kentucky ST198 isolated from a patient in China

ObjectivesThe objective of this study was to investigate the resistance mechanisms of a multidrug-resistant Salmonella Kentucky ST198 FJ-2064 isolated from a patient in China. MethodsThe antimicrobial susceptibility of FJ-2064 was determined by the standard disc dilution and broth microdilution methods. The complete genome of FJ-2064 was sequenced using PacBio and Illumina MiSeq platforms. Polymerase chain reaction (PCR) and S1-PFGE were utilized to confirm the mutation sites and the genomic plasmids, respectively. ResultsIsolate FJ-2064 belongs to sequence type ST198 and harboured no visible large plasmids, but was concurrent resistant to 22 detected antimicrobial agents including cefotaxime, ciprofloxacin, and azithromycin. The complete genome sequence identified 20 acquired antibiotic resistance genes (ARGs) and five chromosomal mutations in the gyrA and parC genes of the quinolone resistance determining regions (QRDRs) in FJ-2064. In addition, PCR sequencing confirmed that most of the ARGs were clustered on one multidrug-resistant region and a variant of SGI1-K. In particular, the bla-TEM-1 and bla-CTX−M-55, qnrS1, mph(A) genes, which confer resistance to cephalosporins, quinolones, and macrolides respectively, were all located on the multidrug-resistant region. ConclusionsWe have demonstrated one multidrug-resistant region and a variant of SGI1-K in a Salmonella Kentucky ST198 that is co-resistant to cefotaxime, ciprofloxacin, and azithromycin.

PSVI-6 Metabolic potential of three novel rumen bacterial species from sheep characterized by utilizing a metagenomic-based approach

Abstract The sheep rumen microbiome contains a vast wealth of known and unknown members, whose metabolic processes impact nutrient acquisition as well as gut and overall health of the animal. Based on previous research from our group on investigating the impact of a change in diet on the composition of the rumen microbiome in lambs, further insights into the metabolic functions of novel bacterial species represented by three Operational Taxonomic Units (OTUs) were pursued. The rumen fluid samples with the greatest abundance of OTUs RA1-01223, RA1-00032, and RA1-04216 were, respectively, used as representatives for metagenomic investigations to assemble the genomes of their corresponding bacterial species. Purified microbial genomic DNA was used as a template for ‘shotgun’ sequencing with an Illumina Miseq (2 x 250) platform. Raw datasets were filtered for quality then used for contig assembly with an in-house developed set of Perl scripts, generating 64 contigs for RA1-01223 [combined total length: 2,046,349 base pairs (bp)], 118 contigs for RA1-00032 (combined total length: 3,704,255 bp), and 119 contigs for RA1-04216 (combined total length: 8,884,743 bp). After building, coding sequences were identified and annotated using RAST, with metabolic pathways of predicted enzymes constructed for each OTU of interest using KEGG pathways as a model reference. Annotated coding sequences were also screened by blastp to assign contig sets to each OTU based on their respective taxonomic affiliation; both OTU RA1-01223 and OTU RA1-00032 were found to be most closely related to Ruminococcus bili (94.8% and 86.8%, respectively), while RA1-04216 was affiliated to Succinivibrio dextrinsolvens (96.9%). As RA1-01223 and RA1-00032 were found to possess enzymes responsible for lactate metabolism, they could potentially affect susceptibility to acidosis by acting as modulators of ruminal pH or by producing lactate from starch degradation. Distinguishing features between these two OTUS included the pathway for vitamin B5 synthesis, using uracil or L-aspartate as precursors (RA1-01223), and the ability to produce ethanol as an end product of ruminal fermentation (RA1-00032). Distinguishing metabolic activities of interest for RA1-04216 included synthesis of chorismate and prephenate, which are precursors for tryptophan and phenylalanine, respectively. Together, these results contribute to a better understanding of the metabolic roles of individual microorganisms in the rumen, providing information that is crucial for creating a healthy and proactive environment that benefits the host.

Impact of root canal disinfection on the bacteriome present in primary endodontic infection: A next generation sequencing study.

To investigate the bacteriome present in teeth with primary endodontic infection (PEI) and apical periodontitis (AP) and to determine quantitatively and qualitatively the impact of chemomechanical preparation (CMP) using 2.5% sodium hypochlorite NAOCl on the bacteriome found in PEI with AP using the Illumina MiSeq platform. Thirty-six paired samples from 18 patients were successfully sequenced and analysed. Samples were collected at two sampling times: before (s1) and after (s2) CMP using 2.5% NaOCl. The DNA was extracted from s1 and s2 samples and quantified using quantitative PCR (qPCR). All 36 samples were sequenced using the Illumina MiSeq platform. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants (ASVs). Alpha diversity metrics representing abundance (Chao1) and diversity and evenness (Shannon, Simpson) were computed. The paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The PERMANOVA method (with 999 permutations) was applied to compare community composition between sample types (s1 versus s2) and between patient IDs. ALDEx2 (ANOVA-like differential expression tool for high-throughput sequencing data) to investigate differentially abundant taxa between s1 and s2. A paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The qPCR counts were significantly higher in s1 compared to s2 (p = .0007). The Chao1 index indicated no difference in alpha diversity (p < .7019); whereas Shannon (p = .0056) and Simpson (p = .02685) indexes showed higher values in s2. The PERMANOVA test using Adonis2 showed a significant effect of sample time on community composition (R2 = .0630, p = .012). Patient ID also showed a significant effect on community composition (R2 = .6961, p = .001). At the genus level, Dialister, Mogibacterium, Prevotella, and Olsenella were differentially enriched at s1, while Actinomyces, Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified were differentially enriched in s2. The bacteriome present in teeth with PEI with AP is complex and diverse. CMP using 2.5% NaOCl showed a high quantitatively and qualitatively disinfectant impact on the bacteriome present in PEI with AP.

Abstract 1121: Neutrophil Proteobacteria Sequences Increases After Open Aortic Surgery

Introduction: Bacterial extracellular vesicles (bEV) transport diverse cargoes, including nucleic acids, and play a crucial role during host-microbe interactions, including modifying host immune response. Neutrophil activation is prevalent in aortic disease pathogenesis, but the cause is unclear. The baseline neutrophilic bEV content and its dynamics are unknown and may provide insight into neutrophil activation in aortic disease. Research Question: Does open aortic surgery change neutrophil bEV content? Methods: Blood was collected pre and post-operatively from 6 patients undergoing open aortic surgery. Neutrophils were isolated using standard centrifugation techniques, and nucleic acids were purified. The V1-V3 16s rRNA segments were amplified with universal primers and sequenced using an Illumina MiSeq platform. Reads were processed in QIIME2 and aligned to reference banks of known bacterial sequences. Sequence abundance was analyzed with Kruskal-Wallis and Wilcoxon-rank tests. Results: The average patient age was 60.2 years, with 4 male patients and 4 current smokers. The most common phylum and genus represented in the neutrophil bacterial reads were Proteobacteria (Figure 1A) and Delftia , respectively. The relative abundance of Proteobacteria sequences increased overtime (p=0.042) from a median of 45.43% (IQR:35.3-52.0) to 70.8% (IQR:66.1-81.5, p=0.026) post-operatively, to 83.5% (IQR:60.7-89.7, p=0.004) on POD1 (Figure 1B). POD3 Proteobacteria sequence abundance was stable from POD1 at 81.4% (IQR:64.2-89.6). Conclusions: Neutrophils of patients with aortic disease rapidly change their bacterial sequence content in response to open aortic surgery. Bacteria in the phylum Proteobacteria change most dramatically and equilibrate between POD 1 and 3. Further studies will elucidate the impact changing bacterial sequence abundance and bEV content has on neutrophil activity and its relationship to disease pathogenesis.

Mechanisms Involved in the Antihypertensive Effects of Arbequina Table Olives

The Mediterranean Diet is associated with antihypertensive properties and prevention of cardiovascular disease. This dietary pattern includes the intake of Olea europaea L. derivatives such as extra virgin olive oil and table olives, characterized by a high content of bioactive compounds. Recently, it has been demonstrated that the supplementation of Arbequina table olives (AO) reduces blood pressure (BP) in spontaneously hypertensive rats (SHR). The aim of the present study was to elucidate the possible mechanisms involved in the antihypertensive effects of AO. Consequently, we have studied different plasmatic biomarkers (angiotensin II, Ang II; malondialdehyde, MDA; interleukin 6, IL6; tumor necrosis factor α, TNFα) and the fecal microbiota composition. Experiments were performed using male SHR from 14 to 21 weeks of age. SHR strain was divided into a control (SHR-c) and a treated group (SHR-o) that received water or AO (3.85 g/kg) by gavage, respectively. At the end of the experiment, BP was measured using a non-invasive automatic analyzer; Ang II, IL6, and TNFα were determined by Elisa, and MDA by spectrophotometric analysis. Fecal microbiota was analyzed by sequencing 16S rRNA gene using the Illumina MiSeq platform. Data were analyzed by t-Student or the Mann–Whitney U test, and statistical significance was considered when p&lt;0.05. AO supplementation decreased systolic and diastolic BP by approximately 19 mmHg in SHR-o with respect to SHR-c. The decrement in BP was accompanied by a decrease in MDA (39%) and Ang II (32%) in SHR-o without changes in the concentrations of the proinflammatory cytokines IL6 and TNFα. Moreover, regular AO intake induced changes in the microbiota associated with the normalization of BP. Our results showed variations in relative abundance in genera such as Akkermansia, Sutterella, and Peptoniphilus, that were well correlated with the decrease in BP, MDA, and Ang II. In conclusion, OA supplementation reduced oxidative stress and Ang II associated with changes in the fecal microbiota that may explain the antihypertensive effect elicited by consumption of the fruit of Olea europaea L. Supported by grants AGL2013-41188 &amp; 2021SGR300. INSA Maria de Maeztu Unit of Excellence grant CEX2021-001234-M. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1–4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1–4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.

Effect of 5-aminolevulinic acid-mediated photodynamic therapy against Fusobacterium nucleatum in periodontitis prevention

Periodontitis, a chronic infectious disease leading to gingival atrophy and potential tooth loss through alveolar bone resorption, is closely linked to the oral microbiome. Fusobacterium nucleatum, known to facilitate late-stage bacterial colonization in the oral microbiome, plays a crucial role in the onset of periodontitis. Controlling F. nucleatum abundance is vital for preventing and treating periodontal disease. Photodynamic therapy combined with 5-aminolevulinic acid (ALA-PDT) has been reported to be bactericidal against Pseudomonas aeruginosa and Staphylococcus aureus. We aimed to investigate the bactericidal potential of ALA-PDT against F. nucleatum, which was evaluated by examining the impact of varying 5-ALA concentrations, culture time, and light intensity. After ALA-PDT treatment, DNA was extracted from interdental plaque samples collected from 10 volunteers and sequenced using the Illumina MiSeq platform. To further elucidate the bactericidal mechanism of ALA-PDT, porphyrins were extracted from F. nucleatum following cultivation with 5-ALA and subsequently analyzed using fluorescence spectra. ALA-PDT showed a significant bactericidal effect against F. nucleatum. Its bactericidal activity demonstrated a positive correlation with culture time and light intensity. Microbiota analysis revealed no significant alteration in α-diversity within the ALA-PDT group, although there was a noteworthy reduction in the proportion of the genus Fusobacterium. Furthermore, fluorescence spectral analysis indicated that F. nucleatum produced an excitable photosensitive substance following the addition of 5-ALA. Overall, if further studies confirm these results, this combined therapy could be an effective strategy for reducing the prevalence of periodontitis.

Genomic characterization and probiotic potential assessment of an exopolysaccharide-producing strain Pediococcus pentosaceus LL-07 isolated from fermented meat

BackgroundThe genomic information available for Pediococcus pentosaceus is primarily derived from fermented fruits and vegetables, with less information available from fermented meat. P. pentosaceus LL-07, a strain isolated from fermented meat, has the capability of producing exopolysaccharides (EPS). To assess the probiotic attributes of P. pentosaceus LL-07, we conducted whole-genome sequencing (WGS) using the PacBio SequelIIe and Illumina MiSeq platforms, followed by in vitro experiments to explore its probiotic potential.ResultsThe genome size of P. pentosaceus LL-07 is 1,782,685 bp, comprising a circular chromosome and a circular plasmid. Our investigation revealed the absence of a CRISPR/Cas system. Sugar fermentation experiments demonstrated the characteristics of carbohydrate metabolism. P. pentosaceus LL-07 contains an EPS synthesis gene cluster consisting of 13 genes, which is different from the currently known gene cluster structure. NO genes associated with hemolysis or toxin synthesis were detected. Additionally, eighty-six genes related to antibiotic resistance were identified but not present in the prophage, transposon or plasmid. In vitro experiments demonstrated that P. pentosaceus LL-07 was comparable to the reference strain P. pentosaceus ATCC25745 in terms of tolerance to artificial digestive juice and bile, autoaggregation and antioxidation, and provided corresponding genomic evidence.ConclusionThis study confirmed the safety and probiotic properties of P. pentosaceus LL-07 via complete genome and phenotype analysis, supporting its characterization as a potential probiotic candidate.

Circular genetic structure of the Abies nephrolepis species complex shaped by the circular landform of Northeast Asia

AbstractAimPinaceae have been noted for their tendency towards reticulation as a result of interspecific hybridization. Here, we demonstrated the phylogeographic dynamics of the native Northeast Asian sub‐alpine conifer, Abies nephrolepis species complex (ANSC), evolving in circular overlaps along the Northeast Asian landform, which functions as a corridor with the sea, thus acting as a geographic barrier.LocationNortheast Asia: the Korean Peninsula, Japanese Archipelago, Russian Far East and northeastern China.TaxonAbies nephrolepis species complex (Family Pinaceae, Genus Abies, Section Balsamea, Abies nephrolepis, A. koreana, A. veitchii and A. sachalinensis).MethodsA total of 728 individuals from 38 ANSC populations were analysed using multiplexed inter‐simple sequence repeat genotyping by sequencing to capture variations in biparental nuclear genomes. Eight mitochondrial regions and eight chloroplast regions of each individual were sequenced using the MiSeq platform and Sanger sequencing. Species distribution models were generated.ResultsBayesian clustering with 507 nuclear single nucleotide polymorphisms and the discrepancy between cytoplasmic and nuclear genome lineages implied contemporary and ancient connections between neighbouring species in the form of circular overlap. This genetic connectivity was supported by principal component analysis. Strong correlations between genetic distance and geographic distance were observed, suggesting that gene flow occurs through a continuous chain around the sea. We also found that gene flow direction and intensity changed over time, with support from palaeodistribution modelling.ConclusionPast hybridization events were captured in cytoplasmic genomes, generating heterogeneity across maternal ancestries. This intensive phylogeographic study demonstrates speciation with incomplete reproductive isolation (continuous gene flow) among neighbouring species with an alteration of the direction and intensity of gene flow due to climate change. The divergence of ANSC due to repeated isolation and reconnection caused by heterogenous physiological environments and climate fluctuation provides a model to solve evolutionary scenarios for reticulate evolution in Pinaceae and other plants.

A meta‐analysis revealing the technical, environmental, and host‐associated factors that shape the gut microbiota of Atlantic salmon and rainbow trout

AbstractSalmonids, specifically Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), are commonly farmed and their gut microbiota plays important roles for optimal growth, health, and physiology. However, differences in experimental design, technical factors and bioinformatics make it challenging to compare the results from different studies and draw general conclusions about their influence on the fish gut microbiota. For a more comprehensive understanding of the gut microbiota, we collected all the publicly accessible 16S rRNA gene sequencing data with clearly stated sample metadata from freshwater Atlantic salmon and rainbow trout intestinal contents and mucosa sequenced on the Illumina MiSeq platform. A total of 783 samples from 19 published studies were included in this meta‐analysis to test the impact of the technical, environmental, and host‐accociated factors. This meta‐analysis revealed that all the tested factors significantly influenced the alpha and beta diversities of the gut microbiota of salmon and trout. Technical factors, especially target region and DNA extraction kit, affected the beta diversity to a larger extent, while host‐associated and environmental factors, especially diet and initial fish weight, had a higher impact on the alpha diversity. Salmon had a higher alpha diversity and higher abundance of Enterococcus and Staphylococcus than trout, which had higher abundance of Weissella and Mycoplasma. The results of this meta‐analysis fill in a critical knowledge gap that demonstrate technical methodologies must be standardized and factors associated with host and environment need to be accounted for in the future design of salmonid gut microbiota experiments.

Novel insight into the genetic diversity of strongylid nematodes infecting South-East and East Asian primates.

With many non-human primates (NHPs) showing continued population decline, there is an ongoing need to better understand their ecology and conservation threats. One such threat is the risk of disease, with various bacterial, viral and parasitic infections previously reported to have damaging consequences for NHP hosts. Strongylid nematodes are one of the most commonly reported parasitic infections in NHPs. Current knowledge of NHP strongylid infections is restricted by their typical occurrence as mixed infections of multiple genera, which are indistinguishable through traditional microscopic approaches. Here, modern metagenomics approaches were applied for insight into the genetic diversity of strongylid infections in South-East and East Asian NHPs. We hypothesized that strongylid nematodes occur in mixed communities of multiple taxa, dominated by Oesophagostomum, matching previous findings using single-specimen genetics. Utilizing the Illumina MiSeq platform, ITS-2 strongylid metabarcoding was applied to 90 samples from various wild NHPs occurring in Malaysian Borneo and Japan. A clear dominance of Oesophagostomum aculeatum was found, with almost all sequences assigned to this species. This study suggests that strongylid communities of Asian NHPs may be less species-rich than those in African NHPs, where multi-genera communities are reported. Such knowledge contributes baseline data, assisting with ongoing monitoring of health threats to NHPs.

Viral metagenomic analysis reveals diverse viruses and a novel bocaparvovirus in the enteric virome of snow leopard (Panthera uncia)

The enteric virome, comprising a complex community of viruses inhabiting the gastrointestinal tract, plays a significant role in health and disease dynamics. In this study, the fecal sample of a wild snow leopard was subjected to viral metagenomic analysis using a double barcode Illumina MiSeq platform. The resulting reads were de novo assembled into contigs with SOAPdenovo2 version r240. Additional bioinformatic analysis of the assembled genome and genome annotation was done using the Geneious prime software (version 2022.0.2). Following viral metagenomic analysis and bioinformatic analysis, a total of 7 viral families and a novel specie of bocaparvovirus tentatively named Panthera uncia bocaparvovirus (PuBOV) with GenBank accession number OQ627713 were identified. The complete genome of PuBOV was predicted to contain 3 open reading frames (ORFs), contains 5433 nucleotides and has a G + C content of 47.40 %. BLASTx analysis and pairwise sequence comparison indicated the novel virus genome was a new species in the genus Bocaparvovirus based on the species demarcation criteria of the International Committee on the Taxonomy of Viruses. This study provides valuable insights into the diversity and composition of the enteric virome in wild endangered snow leopards. The identification and characterization of viruses in wildlife is crucial for developing effective strategies to manage and mitigate potential zoonotic and other viral disease threats to human and animal health.

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.

Abstract 4403: The crosstalk between the tumor microbiome and epigenomic changes in penile squamous cell carcinoma

Abstract Penile squamous cell carcinoma (PSCC) is approximately three times more prevalent in Puerto Rico when compared to other ethnic groups in the United States. Moreover, the mortality rate for PSCC among Puerto Rican men is significantly higher when compared to the US population. Infection with human papillomavirus (HPV) has been identified as a risk factor for an average of 48% of PSCC cases in Puerto Rico. The molecular etiology of HPV+ and HPV- PSCC remains poorly understood. To date, the role of the microbiota in the pathogenesis of PSCC is unknown, as there are no studies that characterize the microbiome in association with penile cancer. Furthermore, the role of DNA methylation, which is an epigenetic alteration that can be found in cancer cells that can lead to changes in gene expression and cellular function, is also unknown. Therefore, there is an urgent need to address this knowledge gap. This study aims to analyze the DNA methylation patterns and bacterial communities in HPV-positive and HPV-negative PSCC tissues from Puerto Rican men. DNA was extracted from twenty-one fresh PSCC tissue samples from Puerto Rican patients. HPV status and genotyping were determined using the DNA ELISA kit HPV SPF10 protocol. DNA methylation assay was performed using the Infinium Methylation EPIC v2.0 BeadChip Kit to identify and compare average DNA methylation levels (%) in 7 HPV-positive and 14 HPV-negative penile cancer samples. Microbiota analyses were performed using 16S rRNA genes with the Illumina MiSeq platform. Demultiplexed data was deposited in QIITA for quality control and bioinformatic analyses and downstream analyses were done in R and Qiime2. Our results demonstrated that 45 loci methylation patterns were significantly different between HPV-positive and HPV-negative samples, with an adjusted p-value &amp;lt;0.1 (Benjamini-Hochberg method). Three loci methylation patterns were significantly affected by species diversity and richness. The abundance of Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria, and Actinobacteria was individually associated with number of affected loci (27, 19, 12, 6, and 3, respectively). We found that HPV status is associated with methylation patterns and microbiome in PSCC in Puerto Rican men. Both changes may be involved in triggering inflammatory responses and oncogenesis. Although many challenges must be overcome to dissect the specific interactions of coinfecting bacteria and methylation patterns during the penile cancer infectious process, our findings demonstrate that microbes may be involved in these cellular processes. Citation Format: Jorge Viera-Vera, Yarelis M. Roque-Reyes, Daniel Santiago-Negron, María Sánchez-Vázquez, Yakshi Ortiz Maldonado, Gabriel Borges-Vélez, Jasmine Figueroa-Díaz, Carlos A. Rivera-López, Niki M. Zacharias Millward, María J. Marcos-Martínez, Filipa Godoy-Vitorino, Antonio Puras-Baez, Julie Dutil, Josué Pérez-Santiago, Magaly Martínez-Ferrer. The crosstalk between the tumor microbiome and epigenomic changes in penile squamous cell carcinoma [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 4403.

An insight into the prokaryotic diversity from a polymetallic nodule-rich region in the Central Indian Ocean Basin using next generation sequencing approach.

Deep sea is a vast, dark, and difficult-to-access terrain and is now looked upon as a unique niche harboring diverse microorganism. We used a metataxonomic approach to decipher the microbial diversity present in the water column (surface to near bottom), water overlaying the sediments, and the deep-sea sediments (up to 35 cm) from the Indian Contract Region (ICR) in the Central Indian Ocean Basin (CIOB). Samples were collected from #IRZ (Impact Reference Zone), #PRZ (Potential Reference Zone), and #BC20 (Control site, outside potential mining area) with an average water depth of 5,200 m. 16S rRNA (V3-V4 region) amplicon sequencing on the MiSeq platform resulted in 942,851 ASVs across 65 water and sediment samples. Higher prokaryotic diversity was observed below 200 m in the water column to the seafloor. Proteobacteria was the most dominant bacterial phylum among all the water samples while Firmicutes, Actinobacteria and, Bacteroidota dominated the sediments. Sediment (below 10 cm) was co-dominated by Firmicutes. Thermoplasmata was the dominant archaeal group in the water column while Crenarchaeota was in the sediments. BC20 was less diverse than IRZ and PRZ. Deep Sea microorganisms could play a vital role in the mineralization processes, nutrient cycling, and also different biogeochemical cycles.

Enterobacter asburiae ST229: an emerging carbapenemases producer

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. “Policlinico San Matteo” in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

Analysis of the Difference of Intestinal Microbes in Muchuan Black-bone Chickens with Two Skin Colors

Background: Skin color is an important economic trait in black-bone chicken production and it has been identified that the gut microbiota is an important factor in animal health and physiology. However, it is unknown about whether the gut microbiota in black-bone chickens is connected to the coloration of chicken skin. Methods: To investigate the relationship between the gut microbiota and skin coloration, comparing the differentiation of the gut microbiota structure and abundances in black- (B group) and white-skinned (W group) Muchuan black-bone chicken. Here, we characterized the microbiota in the jejunal contents of both the B and W groups using the Illumina MiSeq platform, targeting the genomic V4 region of the 16S rRNA gene. Result: In terms of community richness, the ACE community richness index of the B group was significantly higher than that of the W group (p less than 0.05) and the proportion of unique OTUs was higher in group B than in group W. According to the species annotation and abundance information of all samples at the genus level, Parabacteroides, Faecalicoccus and Alistipes were enriched in group B, whereas Actinomyces, Elstera and Nosocomiicoccus were enriched in group W. In the evolutionary branch diagram, the relative abundances of Enterobacteriaceae (Gammaproteobacteria, Enterobacteriales) were significantly higher in group B than in group W (LDA Score greater than 4). Our data provide a basis for the hypothesis that the discriminating bacterial taxa in gut is associated with the feature variants for skin coloration of Muchuan black-bone chickens, which were fed in the same feed and stocking niches.

Fetal Programming Influence on Microbiome Diversity and Ruminal and Cecal Epithelium in Beef Cattle.

We explored the influence of maternal nutritional strategies on the development of the rumen and cecum in offspring. Additionally, we investigated the potential repercussions of prenatal nutrition on the rumen and fecal microbiota composition, utilizing metagenomic 16S techniques, to understand the effects of fetal programming (FP) in Nellore cattle. A total of 63 bulls submitted to different prenatal nutrition strategies, namely, non-programming (NP), partial programming (PP), and complete programming (CP), were evaluated. The rumen epithelium was methodically evaluated based on the presence of rumenitis and structural irregularities. The assessment of cecum lesions was conducted post-evisceration, whereby all thoroughly cleaned ceca were methodically evaluated. Samples from 15 animals of rumen fluid at slaughter and feces during the finishing phase were collected, respectively. All DNA extraction were carried out using the Macherey Nagel NucleoSpin Tissue®, and 16S sequencing was conducted using the V4 primers on the MiSeq platform. Within the ruminal ecosystem, an estimated range of 90 to 130 distinct amplicon sequence variants was discerned, as distributed across 45,000 to 70,000 sequencing reads. Our metagenomic exploration unveils microbial communities that distinctly mirror gastrointestinal tract microenvironments and dietary influences. In sum, this comprehensive study advances our comprehension of FP, highlighting the interplay of maternal nutrition, gastrointestinal development, and microbial communities, contributing significantly to the fields of animal science.

Molecular epidemiological and antimicrobial-resistant mechanisms analysis of prolonged Neisseria gonorrhoeae collection between 1971 and 2005 in Japan.

As antimicrobial-resistant (AMR) Neisseria gonorrhoeae strains have emerged, humans have adjusted the antimicrobials used to treat infections. We identified shifts in the N. gonorrhoeae population and the determinants of AMR strains isolated during the recurring emergence of resistant strains and changes in antimicrobial therapies. We examined 243 N. gonorrhoeae strains corrected at the Kanagawa Prefectural Institute of Public Health, Kanagawa, Japan, these isolated in 1971-2005. We performed multilocus sequence typing and AMR determinants (penA, mtrR, porB, ponA, 23S rRNA, gyrA and parC) mainly using high-throughput genotyping methods together with draft whole-genome sequencing on the MiSeq (Illumina) platform. All 243 strains were divided into 83 STs. ST1901 (n = 17) was predominant and first identified after 2001. Forty-two STs were isolated in the 1970s, 34 in the 1980s, 22 in the 1990s and 13 in the 2000s, indicating a decline in ST diversity over these decades. Among the 29 strains isolated after 2001, 28 were highly resistant to ciprofloxacin (MIC ≥ 8 mg/L) with two or more amino-acid substitutions in quinolone-resistance-determining regions. Seven strains belonging to ST7363 (n = 3), ST1596 (n = 3) and ST1901 (n = 1) were not susceptible to cefixime, and six strains carried penA alleles with mosaic-like penicillin-binding protein 2 (PBP2; penA 10.001 and 10.016) or PBP2 substitutions A501V and A517G. We observed a significant reduction in the diversity of N. gonorrhoeae over 35 years in Japan. Since 2001, ST1901, which is resistant to ciprofloxacin, has superseded previous strains, becoming the predominant ST population.

The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.