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Abstract Recent advances in whole-genome sequencing (WGS) have increased the accessibility of this tool, offering substantial potential for pathogen surveillance, outbreak response, and diagnostics. However, the routine clinical adoption of WGS is hindered by factors such as high costs, technical complexity, and the requirement for bioinformatics expertise for data analysis. To address these challenges, we propose RapidONT, a workflow designed for cost-effective and accessible WGS-based pathogen analysis. RapidONT employs a mechanical shearing–based DNA extraction protocol, followed by library construction by using a multiplexing Oxford nanopore technologies (ONT) rapid barcoding kit. Flye software is used for de novo assembly without manual intervention, followed by basic assembly polishing using Medaka and Homopolish. The polished assemblies are then analyzed using the user-friendly web-based platform Pathogenwatch, which facilitates species identification, molecular typing, and antimicrobial resistance (AMR) prediction, all while requiring minimal bioinformatics expertise. The efficacy of RapidONT was evaluated using nine clinically relevant pathogens, encompassing a total of 90 gram-positive and gram-negative bacterial strains. The workflow demonstrated high accuracy in critical tasks such as multilocus sequence typing (MLST) and AMR identification, using only ONT R9.4.1 flowcell data. Notably, limitations were observed with Salmonella spp. and Neisseria gonorrhoeae. Furthermore, RapidONT enabled the generation of genomic information for 48 bacterial isolates by using a single flow cell, significantly reducing sequencing costs. This approach eliminates the need for extensive experimentation in obtaining crucial genomic information. This workflow facilitates broader WGS implementation in clinical pathogen analysis and diagnostics.

Abstract Background Blood cultures are essential for diagnosing bloodstream infections, but current phenotypic tests for antimicrobial resistance (AMR) provide limited information. Oxford Nanopore Technologies introduces nanopore sequencing with adaptive sampling, capable of real-time host genome depletion, yet its application directly from blood cultures remains unexplored. This study aimed to identify pathogens and predict AMR using nanopore sequencing. Methods In this cross-sectional genomic study, 458 positive blood cultures from bloodstream infection patients in central Taiwan were analyzed. Parallel experiments involved routine microbiologic tests and nanopore sequencing with a 15-h run. A bioinformatic pipeline was proposed to analyze the real-time sequencing reads. Subsequently, a comparative analysis was performed to evaluate the performance of species identification and AMR prediction. Results The pipeline identified 76 species, with 88 Escherichia coli, 74 Klebsiella pneumoniae, 43 Staphylococcus aureus, and 9 Candida samples. Novel species were also discovered. Notably, precise species identification was achieved not only for monomicrobial infections but also for polymicrobial infections, which was detected in 23 samples and further confirmed by full-length 16S rRNA amplicon sequencing. Using a modified ResFinder database, AMR predictions showed a categorical agreement rate exceeding 90% (3799/4195) for monomicrobial infections, with minimal very major errors observed for K. pneumoniae (2/186, 1.1%) and S. aureus (1/90, 1.1%). Conclusions Nanopore sequencing with adaptive sampling can directly analyze positive blood cultures, facilitating pathogen detection, AMR prediction, and outbreak investigation. Integrating nanopore sequencing into clinical practices signifies a revolutionary advancement in managing bloodstream infections, offering an effective antimicrobial stewardship strategy, and improving patient outcomes.

Fluconazole-resistant clade 4 Candida tropicalis causing candidemia in humans has been detected in tropical/subtropical areas, including those in China, Singapore, and Australia. We analyzed 704 individual yeasts isolated from fruits, soil, water, and farmers at 80 orchards in Taiwan. The most common pathogenic yeast species among 251 isolates recovered from farmers were Candida albicans (14.7%) and C. parapsilosis (11.6%). In contrast, C. tropicalis (13.0%), C. palmioleophila (6.6%), and Pichia kudriavzevii (6.0%) were prevalent among 453 environmental isolates. Approximately 18.6% (11/59) of C. tropicalis from the environment were resistant to fluconazole, and 81.8% (9/11) of those belonged to the clade 4 genotype. C. tropicalis susceptibility to fluconazole correlated with susceptibilities to the agricultural azole fungicides, difenoconazole, tebuconazole, and triadimenol. Tandem gene duplications of mutated ERG11 contributed to azole resistance. Agriculture environments are a reservoir for azole-resistant C. tropicalis; discontinuing agricultural use of azoles might reduce emergence of azole-resistant Candida spp. strains in humans.

Abstract Background Airflow obstruction is a hallmark of disease severity and prognosis in bronchiectasis. The relationship between lung microbiota, airway inflammation, and outcomes in bronchiectasis with fixed airflow obstruction (FAO) remains unclear. This study explores these interactions in bronchiectasis patients, with and without FAO, and compares them to those diagnosed with chronic obstructive pulmonary disease (COPD). Methods This prospective observational study in Taiwan enrolled patients with either bronchiectasis or COPD. To analyze the lung microbiome and assess inflammatory markers, bronchoalveolar lavage (BAL) samples were collected for 16S rRNA gene sequencing. The study cohort comprised 181 patients: 86 with COPD, 46 with bronchiectasis, and 49 with bronchiectasis and FAO, as confirmed by spirometry. Results Patients with bronchiectasis, with or without FAO, had similar microbiome profiles characterized by reduced alpha diversity and a predominance of Proteobacteria, distinctly different from COPD patients who exhibited more Firmicutes, greater diversity, and more commensal taxa. Furthermore, compared to COPD and bronchiectasis without FAO, bronchiectasis with FAO showed more severe disease and a higher risk of exacerbations. A significant correlation was found between the presence of Pseudomonas aeruginosa and increased airway neutrophilic inflammation such as Interleukin [IL]-1β, IL-8, and tumor necrosis factor-alpha [TNF]-α, as well as with higher bronchiectasis severity, which might contribute to an increased risk of exacerbations. Moreover, in bronchiectasis patients with FAO, the ROSE (Radiology, Obstruction, Symptoms, and Exposure) criteria were employed to classify individuals as either ROSE (+) or ROSE (−), based on smoking history. This classification highlighted differences in clinical features, inflammatory profiles, and slight microbiome variations between ROSE (−) and ROSE (+) patients, suggesting diverse endotypes within the bronchiectasis with FAO group. Conclusion Bronchiectasis patients with FAO may exhibit two distinct endotypes, as defined by ROSE criteria, characterized by greater disease severity and a lung microbiome more similar to bronchiectasis without FAO than to COPD. The significant correlation between Pseudomonas aeruginosa colonization and increased airway neutrophilic inflammation, as well as disease severity, underscores the clinical relevance of microbial patterns. This finding reinforces the potential role of these patterns in the progression and exacerbations of bronchiectasis with FAO.

Objectives: Predicting progression of nontuberculous mycobacterial lung disease (NTM-LD) remains challenging. This study evaluated whether sputum bacterial microbiome diversity can be the biomarker and provide novel insights into related phenotypes and treatment timing. Methods: We analyzed 126 sputum microbiomes of 126 patients with newly diagnosed NTM-LD due to Mycobacterium avium complex, M. abscessus complex, and M. kansasii between May 2020 and December 2021. Patients were followed for 2 years to determine their disease progression status. We identified consistently representative genera that differentiated the progressor and nonprogressor by using six methodologies. These genera were used to construct a prediction model using random forest with five-fold cross validation. Results: Disease progression occurred in 49 (38.6%) patients. Compared with nonprogressors, α-diversity was lower in the progressors. Significant compositional differences existed in the β-diversity between groups (P = 0.001). The prediction model for NTM-LD progression constructed using seven genera (Burkholderia, Pseudomonas, Sphingomonas, Candidatus Saccharibacteria, Phocaeicola, Pelomonas, and Phascolarctobacterium) with significantly differential abundance achieved an area under curve of 0.871. Conclusion: Identification of the composition of sputum bacterial microbiome facilitates prediction of the course of NTM-LD, and maybe used to develop precision treatment involving modulating the respiratory microbiome composition to ameliorate NTM-LD.

The structural, magnetic, optical, and adhesion properties of Cobalt-Iron-Dysprosium (CoFeDy) films were investigated in this study. Glass substrates were coated with a Co60Fe20Dy20 alloy via sputtering, with a thickness ranging from 10 nm to 50 nm. Subsequently, the films underwent annealing at temperatures of 100 °C, 200 °C, and 300 °C for one hour. X-ray diffraction (XRD) analysis confirmed the amorphous nature of the deposited CoFeDy films under four distinct conditions. Notably, a thickness-dependent increase in low-frequency alternate-current magnetic susceptibility (χac) was observed. After annealing at 300 °C, CoFeDy films exhibited the highest χac compared to other temperatures. Surface roughness exhibited a decreasing trend with rising annealing temperature, as observed through atomic force microscopy (AFM) experiments. The maximum surface energy of CoFeDy films was achieved at a thickness of 50 nm following annealing at 300 °C. Higher surface energy was indicative of stronger adhesion efficiency. Furthermore, lower resistance and sheet resistance values were obtained through annealing at higher temperatures, suggesting that increasing thickness and reducing electron transport barriers enhanced electron conductivity. As film thickness increased, transmittance decreased due to the thickness effect, suppressing the photon signal. Consequently, rougher surfaces were associated with improved performance in magnetism, electrical adhesion, and optics, attributed to reduced domain pinning, enhanced carrier conductivity, and minimized light scattering.

Abstract Gut microbiota rearrangement induced by cold temperature is crucial for browning in murine white adipose tissue. This study provides evidence that DUSP6, a host factor, plays a critical role in regulating cold-induced gut microbiota rearrangement. When exposed to cold, the downregulation of intestinal DUSP6 increased the capacity of gut microbiota to produce ursodeoxycholic acid (UDCA). The DUSP6-UDCA axis is essential for driving Lachnospiraceae expansion in the cold microbiota. In mice experiencing cold-room temperature (CR) transitions, prolonged DUSP6 inhibition via the DUSP6 inhibitor (E/Z)-BCI maintained increased cecal UDCA levels and cold-like microbiota networks. By analyzing DUSP6-regulated microbiota dynamics in cold-exposed mice, we identified Marvinbryantia as a genus whose abundance increased in response to cold exposure. When inoculated with human-origin Marvinbryantia formatexigens, germ-free recipient mice exhibited significantly enhanced browning phenotypes in white adipose tissue. Moreover, M. formatexigens secreted the methylated amino acid Nε-methyl-L-lysine, an enriched cecal metabolite in Dusp6 knockout mice that reduces adiposity and ameliorates nonalcoholic steatohepatitis in mice. Our work revealed that host-microbiota coadaptation to cold environments is essential for regulating the browning-promoting gut microbiome.

ABSTRACT Candida tropicalis is the leading cause of non-albicans candidemia in tropical/sub-tropical areas. We detected a predominant genotype of azole-resistant C. tropicalis clinical strains in Taiwan from 2014 to 2018. To investigate the potential fomite transmission of drug-resistant C. tropicalis, we characterized yeasts recovered from 17 samples sourced from 6 different kinds of fruits from a supermarket in northern Taiwan in the present study. Fruits were gently washed with buffer, and the solution was then collected for centrifugation. The cell suspension was plated onto CHROMagar Candida medium. Representative yeast species was identified by rDNA sequencing. Drug susceptibilities of C. tropicalis were determined by the broth microdilution method. We found that different types of fruit surfaces had different distributions of yeast species. Washing fruit can significantly eliminate the number of yeasts from the surface. Of 123 identified isolates, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. Among 10 collected C. tropicalis, all 3 fluconazole-resistant ones were non-susceptible to voriconazole. Furthermore, the genotype of all azole-resistant C. tropicalis belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Hence, fruit can serve as a vehicle of azole-resistant C. tropicalis and other species, especially C. krusei, which is intrinsically resistant to fluconazole. IMPORTANCE Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

ABSTRACT Enteroviruses (EVs) are a group of viruses that cause various human illnesses. While the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) method can generate VP1 gene fragments for enterovirus genotyping, it is unable to detect recombinant strains. Recent advances in viral genome sequencing using next-generation sequencing technologies have enabled comprehensive analyses. However, the high cost poses a challenge for widespread adoption. To address this issue, this study proposes a cost-effective approach for generating complete enterovirus genome sequences using the Oxford Nanopore MinION sequencer. This protocol not only facilitates the generation of accurate genome sequences for various enterovirus strains but also allows for the differentiation of co-infections from viral isolates. In addition, the method can generate polyprotein sequences as well as peptide sequences of the upstream ORF (uORF) whose expression can impact virus infection. Through the analysis of complete enterovirus genomes, this study successfully identified seven enterovirus A71 isolates obtained during the 2018 enterovirus outbreak in Malaysia and Taiwan as recombinants between enterovirus A71 and coxsackievirus A2. Furthermore, our study has made a significant discovery by establishing a strong correlation between uORF trees and the epidemics of EVA71. This finding highlights the potential of uORF sequences as valuable indicators for monitoring and understanding the spread of EVA71 infections. We also identified notable amino acid changes in the transmembrane domain of the uORF protein within a newly identified lineage. These findings provide crucial insights into the molecular characteristics and evolutionary dynamics of EVA71, offering valuable information for future research and intervention strategies. IMPORTANCE By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study’s findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Purpose: 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a byproduct of DNA oxidation resulting from free radical attacks. Paradoxically, treatment with 8-OHdG accelerates tissue healing. The aim of this study is to quantify the 8-OHdG response after a single session of exercise in both trained and untrained adults.Methods: A systematic review and meta-analysis of exercise intervention studies measuring changes in blood 8-OHdG following resistance exercise and aerobic exercise were conducted. The literature search included Web of Science, PubMed, BASE, and Scopus, with publications up to February 2023 included. Subgroup analysis of training status was also conducted.Results: Sixteen studies involving 431 participants met the eligibility criteria. Resistance exercise showed a medium effect on increasing circulating 8-OHdG levels (SMD = 0.66, p < 0.001), which was similar for both trained and untrained participants. However, studies on aerobic exercise presented mixed results. For trained participants, a small effect of aerobic exercise on increasing circulating 8-OHdG levels was observed (SMD = 0.42; p < 0.001). In contrast, for untrained participants, a large effect of decreasing circulating 8-OHdG levels was observed, mostly after long-duration aerobic exercise (SMD = −1.16; p < 0.05). Similar to resistance exercise, high-intensity aerobic exercise (5–45 min, ≥75% VO2max) significantly increased circulating 8-OHdG levels, primarily in trained participants.Conclusion: Pooled results from the studies confirm an increase in circulating 8-OHdG levels after resistance exercise. However, further studies are needed to fully confirm the circulating 8-OHdG response to aerobic exercise. Increases in 8-OHdG after high-intensity aerobic exercise are observed only in trained individuals, implicating its role in training adaptation.Systematic Review Registration: [https://Systematicreview.gov/], identifier [CRD42022324180]

This data article describes a dataset of images of common Chinese deities. The dataset is divided into five categories according to the types of deities, and a total of 1314 original images were captured by smart phones from Chinese temples and through Google search engine. Each category were split into training, validation and test subsets in a ratio of 70:20:10. We rotated the pictures by 30°, 60°, 90°, 120°, 150°, and 180°; and zoomed in and out to augment the images for training and validation sets. After data enhancement, the total number of images reaches 10,786. Two models, EfficientNet-B0 and MobileNetV2, are used to identify five kinds of god images. After data augmentation, the accuracy, precision, recall, specificity and F1-score of EfficientNet-B0 were 96.15%, 96.44%, 96.18%, 96.16% and 97.60%, respectively; the accuracy, precision recall, specificity and F1-score of MobileNetV2 were 92.31%, 92.89%, 92.37%, 92.33% and 95.19%, respectively. This dataset can be used as a reference for traditional Chinese god statue images, and can also be used for object detection and image classification through machine learning and deep learning methods.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

Tree blossoms have been widely used on the prevention and treatment of a variety of diseases in traditional Chinese medicine for thousand years [1,2]. The growth of flowers is not only for their ornamental value, but also for nutritional, medicinal, cooking, cosmetic and aromatic properties. They are a rich source of many compounds, which play an important role in various metabolic processes of the human body [3]. Edible flowers can promote the global demand for more attractive and delicious food, and can improve the nutritional content of gourmet food [4]. Flowers are beneficial for anti-anxiety, anti-cancer, anti-inflammatory, antioxidant, diuretic and immune-modulator, etc. It is very important to identify edible flowers correctly, because only a few are edible [5].The shapes or colors of different flowers may be very similar. Visual evaluation is one of the classification methods, but it is error-prone and time-consuming [6]. Flowers are divided into flowers from herbaceous plants (flower) and flower trees (blossom). Now there is a public herbaceous flower dataset [7], but lack of dataset for Chinese medicinal blossoms. This article presents and establishes the dataset for twelve most commonly and economically valuable blossoms used in traditional Chinese medicine. The dataset provide a collection of blossom images on traditional Chinese herbs help Chinese pharmacist to classify the categories of Chinese herbs. In addition, the dataset can serve as a resource for researchers who use different algorithms of machine learning or deep learning for image segmentation and image classification.

Abstract Background Human enteroviruses contain over 100 serotypes. We have routinely conducted enterovirus surveillance in northern Taiwan; but about 10% of isolates could not be serotyped using traditional assays. Next-generation sequencing (NGS) is a powerful tool for genome sequencing. Methods In this study, we established an NGS platform to conduct genome sequencing for the serologically untypable enterovirus isolates. Results Among 130 serologically untypable isolates, 121 (93%) of them were classified into 29 serotypes using CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)-based RT-PCR to amplify VP1 genes (VP1-CODEHOP). We further selected 52 samples for NGS and identified 59 genome sequences from 51 samples, including 8 samples containing two virus genomes. We also detected 23 genome variants (nucleotide identity

Third-generation cephalosporin-resistant Escherichia coli (CREC), particularly strains producing extended-spectrum β-lactamases (ESBLs), are a global concern. Our study aims to longitudinally assemble the genomic characteristics of CREC isolates from fecal samples from an index patient with recurrent CREC-related urinary tract infections and his family and swabs from his home environment 12 times between 2019 and 2021 to investigate the distribution of antibiotic resistance genes. CREC identified using the VITEK 2 were subjected to nanopore whole-genome sequencing (WGS). The WGS of 27 CREC isolates discovered in 137 specimens (1 urine, 123 feces, and 13 environmental) revealed the predominance of ST101 and ST131. Among these sequence types, blaCTX-M (44.4%, n = 12) was the predominant ESBL gene family, with blaCTX-M-14 (n = 6) being the most common. The remaining 15 (55.6%) isolates harbored blaCMY-2 genes and were clonally diverse. All E. coli isolated from the index patient’s initial urine and fecal samples belonged to O25b:H4-B2-ST131 and carried blaCTX-M-14. The results of sequence analysis indicate plasmid-mediated household transmission of blaCMY-2 or blaCTX-M-55. A strong genomic similarity was discovered between fecal ESBL-producing E. coli and uropathogenic strains. Furthermore, blaCMY-2 genes were widely distributed among the CREC isolated from family members and their home environment.

Abstract Background Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. Results ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. Conclusion The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.

The Oxford Nanopore MinION is an affordable and portable DNA sequencer that can produce very long reads (tens of kilobase pairs), which enable de novo bacterial genome assembly. Although many algorithms and tools have been developed for base calling, read mapping, de novo assembly, and polishing, an automated pipeline is not available for one-stop analysis for circular bacterial genome reconstruction. In this paper, we present the pipeline CCBGpipe for completing circular bacterial genomes. Raw current signals are demultiplexed and base called to generate sequencing data. Sequencing reads are de novo assembled several times by using a sampling strategy to produce circular contigs that have a sequence in common between their start and end. The circular contigs are polished by using raw signals and sequencing reads; then, duplicated sequences are removed to form a linear representation of circular sequences. The circularized contigs are finally rearranged to start at the start position of dnaA/repA or a replication origin based on the GC skew. CCBGpipe implemented in Python is available at https://github.com/jade-nhri/CCBGpipe. Using sequencing data produced from a single MinION run, we obtained 48 circular sequences, comprising 12 chromosomes and 36 plasmids of 12 bacteria, including Acinetobacter nosocomialis, Acinetobacter pittii, and Staphylococcus aureus. With adequate quantities of sequencing reads (80×), CCBGpipe can provide a complete and automated assembly of circular bacterial genomes.

The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies.

Gut microbial metabolism of L-carnitine, which leads to the production of detrimental trimethylamine N-oxide (TMAO), offers a plausible link between red meat consumption and cardiovascular risks. Several microbial genes, including cntA/B, the cai operon, and the recently identified gbu gene cluster, have been implicated in the conversion of dietary L-carnitine into TMA(O). However, the key microbial genes and associated gut microbes involved in this pathway have not been fully explored. Utilizing the oral carnitine challenge test (OCCT), which specifically measures TMAO production from L-carnitine intake and identifies TMAO producer phenotypes, we compared the abundance of microbial genes between low- and high-TMAO producers across three independent cohorts. Our findings consistently revealed that the gbu gene cluster, rather than cntA/B or the cai operon, was significantly enriched in high-TMAO producers. We further analyzed 292 paired multi-omic datasets from OCCT and shotgun metagenomic sequencing, which demonstrated a significant positive correlation between the abundance of fecal gbu genes and L-carnitine-induced TMAO production, with gbuB showing the strongest correlation. Interestingly, these fecal gbu genes were found to increase with L-carnitine supplementation and decrease with a plant-based diet. Notably, we verified a previously uncultured gbu-containing bacterium, JAGTTR01 sp018223385, as the major contributor to TMA formation in the human gut. We isolated these gbu-containing gut microbes and confirmed their role in TMA/TMAO production using anaerobic incubation and a gnotobiotic mouse model. Using an in-house collection of gbu-containing isolates, we developed a qPCR-based method to quantify fecal gbuB and validated its correlation with L-carnitine-mediated TMAO production as measured by OCCT. Overall, these findings suggest that gbu-containing gut microbes are crucial for TMAO increases following L-carnitine intake and may serve as biomarkers or targets for personalized nutrition.

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.

Drug-resistant Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB), has become a serious threat to global public health. Yet the development of novel drugs against MTB has been lagging. One potentially powerful approach to drug development is computation-aided repositioning of current drugs. However, the effectiveness of this approach has rarely been examined. Here we select the "TB drugome" approach--a protein structure-based method for drug repositioning for tuberculosis treatment--to (1) experimentally validate the efficacy of the identified drug candidates for inhibiting MTB growth, and (2) computationally examine how consistently drug candidates are prioritized, considering changes in input data. Twenty three drugs in the TB drugome were tested. Of them, only two drugs (tamoxifen and 4-hydroxytamoxifen) effectively suppressed MTB growth at relatively high concentrations. Both drugs significantly enhanced the inhibitory effects of three first-line anti-TB drugs (rifampin, isoniazid, and ethambutol). However, tamoxifen is not a top-listed drug in the TB drugome, and 4-hydroxytamoxifen is not approved for use in humans. Computational re-examination of the TB drugome indicated that the rankings were subject to technical and data-related biases. Thus, although our results support the effectiveness of the TB drugome approach for identifying drugs that can potentially be repositioned for stand-alone applications or for combination treatments for TB, the approach requires further refinements via incorporation of additional biological information. Our findings can also be extended to other structure-based drug repositioning methods.

A plethora of algorithmic assemblers have been proposed for the de novo assembly of genomes, however, no individual assembler guarantees the optimal assembly for diverse species. Optimizing various parameters in an assembler is often performed in order to generate the most optimal assembly. However, few efforts have been pursued to take advantage of multiple assemblies to yield an assembly of high accuracy. In this study, we employ various state-of-the-art assemblers to generate different sets of contigs for bacterial genomes. A tool, named CISA, has been developed to integrate the assemblies into a hybrid set of contigs, resulting in assemblies of superior contiguity and accuracy, compared with the assemblies generated by the state-of-the-art assemblers and the hybrid assemblies merged by existing tools. This tool is implemented in Python and requires MUMmer and BLAST+ to be installed on the local machine. The source code of CISA and examples of its use are available at http://sb.nhri.org.tw/CISA/.

Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES.