End-stage kidney disease (ESKD) is the final stage of chronic kidney disease (CKD), in which long-term damage has been caused to the kidneys to the extent that they are no longer able to filter the blood of waste and extra fluid. Peritoneal dialysis (PD) is one of the treatments that remove waste products from the blood through the peritoneum which can improve the quality of life for patients with ESKD. However, PD-associated peritonitis is an important complication that contributes to the mortality of patients, and the detection of bacterial pathogens is associated with a high culture-negative rate. The present study aimed to apply a metagenomic approach for the bacterial identification in the PD effluent (PDE) of patients with CKD based on 16S ribosomal DNA sequencing. As a result of this investigation, five major bacteria species, namely Escherichia coli, Phyllobacterium myrsinacearum, Streptococcus gallolyticus, Staphylococcus epidermidis and Shewanella algae, were observed in PDE samples. Taken together, the findings of the present study have suggested that this metagenomic approach could provide a greater potential for bacterial taxonomic identification compared with traditional culture methods, suggesting that this is a practical and culture-independent alternative approach that will offer a novel preventative infectious strategy in patients with CDK.

Brain abscess is medically challenging. In this study, we applied nanopore sequencing for 16S rRNA analysis and investigated its efficacy and diagnostic value for patients with brain abscesses. Genomic DNA was extracted from the pus samples (n = 27) of brain abscess, and 16S rRNA genes were amplified by PCR. Sequencing libraries were generated using a rapid barcoding kit, and the generated reads were analyzed using the EPI2ME16S workflow. A conventional culture study was performed. More sensitive identification of pathogens was made by 16S sequencing, faster than the culture study. The proportion of anaerobic bacteria identified by 16S sequencing was higher (75%) than that obtained by culturing (32%). Polymicrobial infections were identified in 10 cases (40%) by 16S sequencing, while the culture study identified multiple bacteria in only 2 cases (8%). 16S sequencing was useful for identifying the composition of polymicrobial infections, including rare pathogens, and for the initial diagnosis of space-occupying lesions.

Risk factors for infective endocarditis (IE) include congenital heart defects, poor dentition, immunosuppression, or recent instrumentation. The occupational hazard of a dog bite, combined with bicuspid aortic valve (BAV) led to IE. 16S ribosomal DNA was able to pinpoint the causative organism. A healthy 33-year-old postman presented in profound heart failure and sepsis due to aortic regurgitation and an aortic root abscess. He underwent emergency aortic valve replacement and was found to have a BAV and anomalous right coronary artery. Blood cultures remained negative. 16S ribosomal DNA polymerase chain reaction (PCR) revealed the causative organism was Capnocytophaga canimorsus. On review, he recalled receiving a dog bite followed by a febrile illness a few days later. Congenital BAVs may become infected by seemingly innocuous injuries. 16S rDNA PCR is a more sensitive and specific diagnostic test than culture. This case demonstrates its utility in providing appropriate antimicrobial management for IE.

We report here the case of a 49-year-old man admitted for a diagnostic work-up of bilateral lung nodules. Empyema rapidly developed after a chest computed-tomodensitometry guided lung puncture. Despite the lack of obvious aetiology after two biopsies, the diagnosis was reached by performing 16S ribosomal DNA (rDNA) sequence analysis, which identified Prevotella spp in the pleural liquid. The empyema and lung nodules resolved after appropriate antibiotic therapy.

UNASSIGNED: This study was performed to investigate the clinical significance of miR-4535 and miR-1915-5p in severe chorioamnionitis.
UNASSIGNED: Amniotic fluid samples from 37 patients with severe chorioamnionitis were subjected to miRNA array analysis and ddPCR™. Diagnostic values were assessed using the receiver operating characteristic curve. The patients were separated into three groups according to Blanc\'s criteria.
UNASSIGNED: The expression of miR-4535 and miR-1915-5p was significantly correlated with the copy number of 16S rDNA, had extremely high diagnostic accuracy for severe chorioamnionitis, and was linked to maternal and fetal inflammation.
UNASSIGNED: miR-4535 and miR-1915-5p serve as promising biomarkers for the diagnosis of severe chorioamnionitis.

In this chapter, we provide a methodological description of the process to perform gastrointestinal (GIT) microbiota profiling on human stool samples. The process includes: (i) collection of feces, (ii) isolation of DNA from fecal community bacteria, (iii) selection of both 16S rDNA sequencing target and next-generation sequencing platform, and (iv) analysis and interpretation of sequence data. The process culminates into a comprehensive report on the GIT microbiota composition and structure that may translate into clinically actionable results.

As next-generation sequencing technology has become more advanced, research on microbial 16S ribosomal DNA sequences has developed rapidly. Sequencing of 16S ribosomal DNA allows the composition of bacteria and archaea in a sample to be obtained and many analytical tools related to 16S ribosomal DNA sequences have been proposed; however, most do not include a user-friendly platform with a graphical user interface. Here, a comprehensive and easy-to-use online platform, Easy Microbiome Analysis Platform (EasyMAP), has been developed for analysis of 16S ribosomal DNA sequencing data. EasyMAP integrates the QIIME2, LefSe, and PICRUSt pipelines and includes temporal profiling analysis. Users can perform quality checks, taxonomy differential abundance analysis, microbial gene function prediction and longitudinal analysis with step-by-step guidance. EasyMAP is a user-friendly tool for comprehensive analysis of 16S ribosomal DNA sequencing data. The web server and documentation are freely available at http://easymap.cgm.ntu.edu.tw/.

BACKGROUND: The composition and relative abundance of bacterial species change throughout the development of dental caries; however, how these changes relate to clinical symptoms remains elusive. In this study, we explored the relationship between clinical symptoms and specific microorganisms in advanced dentinal caries.
METHODS: A total of 111 permanent premolars and molars were used to simulate the progression from caries to pulpitis indirectly. Clinical symptoms were evaluated, and teeth were diagnosed according to the diagnostic criteria of the American Association of Endodontics. Samples were collected for 16S ribosomal DNA sequencing. Associations between the microbiota and clinical symptoms/diagnosis and the relationship between alpha diversity and clinical symptoms/diagnosis were evaluated independently by the linear discriminant analysis effect size and Spearman rank correlation analyses.
RESULTS: The 16S ribosomal DNA sequences were assigned to 13,852 operational taxonomic units. The linear discriminant analysis effect size and Spearman correlations unveiled negative associations between the relative abundance of Bacteroidia and Gammaproteobacteria and referred pain, Gammaproteobacteria and the electric pulp test response, and Actinomyces and Propionibacterium and diagnosis (r < 0.0, P < .05). Conversely, the relative abundance of Lactobacillus was positively correlated to referred pain, the cold test, the percussion response, and diagnosis (r > 0, P < .05). Lactobillus reuteri, a probiotic bacterium, was more abundant in teeth with referred pain and teeth diagnosed with symptomatic irreversible pulpitis. The Spearman correlation between alpha diversity and clinical symptoms/diagnosis was not significant (P > .05).
CONCLUSIONS: Clinical symptoms and diagnosis were significantly associated with specific microorganisms in the most advanced layers of dentinal caries.

With increasing age, the rumen microbiota of new-born ruminants become central in the translation of fibrous feed substances into essential nutrients. However, the colonization process of the microbial community (especially fungal community) remains poorly understood in ruminants at pre-weaning stages. In this study, the rumen bacterial and fungal colonization processes were investigated in goats at eight stages using amplicon sequencing. For bacteria, we found 36 common core genera at D0, D3, D14, D28, and D56, including mainly Bacillus, Alloprevotella, Bacteroides, Prevotella_1, Lactococcus, and Ruminococcaceae_NK4A214. Firmicutes was the dominant phylum among the total microbiota in newborn goat kids (prior to nursing), while Bacillus, Lactococcus, and Pseudomonas were predominant genera. Interestingly, the proportion of Bacillus was as high as 55% in newborn animals. After milk nursing, the predominant phylum changed to Bacteroidetes, while the proportion of Bacillus and Lactobacillus was very low. CowPi was used to predict the functional gene pathways and we found increases in the abundance of genes associated with amino acid related enzymes, DNA repair and recombination proteins, aminoacyl tRNA biosynthesis, and peptidases after D3. With regard to fungi, we found that there were 51 common genera at day 0 (D0), D3, D14, D28, and D56, including mainly Cryptococcus, Aspergillus, and Caecomyces. Aspergillus occupied approximately 47% at day 0, but then it decreased from day 3 to day 14. This study indicates that the core microbes of rumen emerged shortly after birth, but the abundance was very different from the core genus of the adult rumen. In addition, we also report a detailed scheme of the bacterial and fungal colonization process in rumens and propose three distinct stages during the rumen colonization process in pre-weaning goats, which will offer a reference for the development of milk substitutes for small ruminants.

OBJECTIVE: To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis.
METHODS: We studied 272 eyes of 272 patients with keratitis. Eyes with keratitis were classified as \"definite\" (N = 118), \"likely\" (N = 71), or \"non-bacterial\" (N = 83) to have bacterial keratitis. The diagnostic efficacy of real-time PCR and conventional testing was determined by receiver operating characteristic analysis. The copy numbers of bacterial DNA and clinical characteristics were retrospectively analyzed for association with concordant culture results in the \"definite\" cases.
RESULTS: The level of bacterial DNA was significantly associated with the diagnostic probability of the three diagnostic categories. The level of bacterial DNA had comparable diagnostic efficacy with the area under the curve (AUC) at 0.67, by culture at 0.65, and by smear testing at 0.73. The efficacy was significantly improved by combining the DNA level with the conventional culture testing with an AUC of 0.81. Analysis of the \"definite\" cases showed culture positivity in 51.8% (58 eyes), and of these, 41 eyes (70.7%) were higher than the cutoff PCR values and 40 eyes were identified by 16S r-DNA sequencing. In the culture-negative eyes, the level of bacterial DNA was significantly lower (P = 0.0008). Eyes with higher bacterial DNA levels had significantly concordant outcomes with sequencing and culture results (P = 0.006). Previous antibiotic treatments decreased the bacterial DNA amount by 0.09-fold, and it was a significant factor for discordance (P = 0.006).
CONCLUSIONS: Quantification of the bacterial DNA level and conventional testing improves the diagnostic efficacy of infectious bacterial keratitis.