ORCA-EFCD consensus report on clinical recommendations for caries diagnosis. Paper III: caries diagnosis at the individual level.

The aim of the present consensus paper was to provide recommendations for clinical practice on the individual etiological and modifying factors to be assessed in the individual diagnosis of caries, and the methods for their assessment, supporting personalized treatment decisions. The executive councils of the European Organisation for Caries Research (ORCA) and the European Federation of Conservative Dentistry (EFCD) nominated ten experts each to join the expert panel. The steering committee formed three work groups which were asked to provide recommendations on 1) caries detection and diagnostic methods, 2) caries activity assessment and 3) forming individualised caries diagnoses. The experts responsible for "individualised caries diagnosis" searched and evaluated the relevant literature, drafted this manuscript and made provisional consensus recommendations. These recommendations were discussed and refined during the structured process in the whole work group. Finally, the agreement for each recommendation was determined using an anonymous eDelphi survey. The threshold for approval of recommendations was determined at 70% agreement. Ten recommendations were approved and agreed by the whole expert panel, covering medical history, caries experience, plaque, diet, fluoride and saliva. While the level of evidence was low, the level of agreement was typically very high, except for one recommendation on salivary flow measurement, where 70% agreed. It is recommended that all aspects of caries lesion progression and activity, recent caries experience, medical conditions and medications, plaque, diet, fluoride and saliva should be synthesized to arrive at an individual diagnosis. The expert panel merged evidence from existing guidelines and scientific literature with practical considerations and provided recommendations for their use in daily dental practice.

ORCA-EFCD consensus report on clinical recommendations for caries diagnosis. Paper II: caries lesion activity and progression assessment.

INTRODUCTION: This consensus paper provides recommendations for oral health professionals on why and how to assess caries activity and progression with special respect to the site of a lesion. METHODS: An expert panel was nominated by the executive councils of the European Organization for Caries Research (ORCA) and the European Federation of Conservative Dentistry (EFCD). The steering committee built three working groups that were asked to provide recommendations on 1) caries detection and diagnostic methods, 2) caries activity and progression assessment and 3) obtain individualized caries diagnoses. The experts of work group 2 phrased and agreed on provisional general and specific recommendations on caries lesion activity and progression, based on a review of the current literature. These recommendations were then discussed and refined in a consensus workshop followed by an anonymous Delphi survey to determine the agreement on each recommendation. RESULTS: The expert panel agreed on general (n=7) and specific recommendations (n=6). The specific recommendations cover coronal caries on pits and fissures, smooth surfaces, proximal surfaces, as well as root caries and secondary caries/ caries adjacent to restorations and sealants (CARS). 3/13 recommendations yielded perfect agreement. CONCLUSION: The most suitable method for lesion activity assessment is the visual-tactile method. No single clinical characteristic is indicative of lesion activity; instead, lesion activity assessment is based on assessing and weighing several clinical signs. The recall intervals for visual and radiographic examination need to be adjusted to the presence of active caries lesions and recent caries progression rates. Modifications should be based on individual patient characteristics.

ORCA-EFCD consensus report on clinical recommendation for caries diagnosis. Paper I: caries lesion detection and depth assessment.

OBJECTIVES: The aim of the present consensus paper was to provide recommendations for clinical practice considering the use of visual examination, dental radiography and adjunct methods for primary caries detection. MATERIALS AND METHODS: The executive councils of the European Organisation for Caries Research (ORCA) and the European Federation of Conservative Dentistry (EFCD) nominated ten experts each to join the expert panel. The steering committee formed three work groups that were asked to provide recommendations on (1) caries detection and diagnostic methods, (2) caries activity assessment and (3) forming individualised caries diagnoses. The experts responsible for "caries detection and diagnostic methods" searched and evaluated the relevant literature, drafted this manuscript and made provisional consensus recommendations. These recommendations were discussed and refined during the structured process in the whole work group. Finally, the agreement for each recommendation was determined using an anonymous Delphi survey. RESULTS: Recommendations (N = 8) were approved and agreed upon by the whole expert panel: visual examination (N = 3), dental radiography (N = 3) and additional diagnostic methods (N = 2). While the quality of evidence was found to be heterogeneous, all recommendations were agreed upon by the expert panel. CONCLUSION: Visual examination is recommended as the first-choice method for the detection and assessment of caries lesions on accessible surfaces. Intraoral radiography, preferably bitewing, is recommended as an additional method. Adjunct, non-ionising radiation methods might also be useful in certain clinical situations. CLINICAL RELEVANCE: The expert panel merged evidence from the scientific literature with practical considerations and provided recommendations for their use in daily dental practice.

Is there a rise of prevalence for Molar Incisor Hypomineralization? A meta-analysis of published data.

Since Molar Incisor Hypomineralization was first described as a pathologic entity, public perception often suggests a considerable rise in prevalence of the respective disease. Since there are still considerable doubts regarding the etiology and-accordingly-prevention of MIH and respective therapeutic approaches are difficult this question is of considerable clinical and public interest. Accordingly, a systematic literature search in accordance with the PRISMA guidelines for systematic reviews on Medline, Cochrane Database, EMBASE, LILACS, Web of Science, Google scholar, Scopus was performed to retrieve original articles reporting the prevalence of MIH as defined by the European Academy of Pediatric Dentistry (EAPD). From initially 2360 retrieved titles, 344 full texts were assessed for possible inclusion and finally 167 articles of mainly moderate to high quality and based on data of 46'613 individuals were included in the meta-analysis. All studies published before 2001 had to be excluded since it was not possible to align the findings with the EAPD classification. Studies varied considerably regarding cohort size (25 to 23'320, mean 1'235)) and age (5.6-19 y, mean 9.8 y). Over all studies, the weighted mean for the prevalence for MIH was 12.8% (95% CI 11.5%-14.1%) and no significant changes with respect to either publication year or birthyear were found. A sub-analysis of eleven studies reporting on the prevalence in different age groups, however, revealed strong evidence for an increasing prevalence between the years 1992 (3%) and 2013 (13%).Therefore, based on data from cross-sectional studies a possible rise in prevalence of MIH remains unclear. Future prospective large-scale studies under standardized examination conditions with an emphasis on examiner calibration are needed to gain better understanding in the evolution of the prevalence of MIH.

Systematic Evaluation of Clinical, Nutritional, and Fecal Microbial Factors for Their Association With Colorectal Polyps.

INTRODUCTION: The identification of risk factors for precursor lesions of colorectal cancer (CRC) holds great promise in the context of prevention. With this study, we aimed to identify patient characteristics associated with colorectal polyps (CPs) and polyp features of potential malignant progression. Furthermore, a potential association with gut microbiota in this context was investigated. METHODS: In this single-center study, a total of 162 patients with CPs and 91 control patients were included. Multiple variables including information on lifestyle, diet, serum parameters, and gut microbiota, analyzed by 16S-rRNA gene amplicon sequencing and functional imputations (Picrust2), were related to different aspects of CPs. RESULTS: We observed that elevated serum alkaline phosphatase (AP) levels were significantly associated with the presence of high-grade dysplastic polyps. This association was further seen for patients with CRC. Thereby, AP correlated with other parameters of liver function. We did not observe significant changes in the gut microbiota between patients with CP and their respective controls. However, a trend toward a lower alpha-diversity was seen in patients with CRC. Interestingly, AP was identified as a possible clinical effect modifier of stool sample beta diversity. DISCUSSION: We show for the first time an increased AP in premalignant CP. Furthermore, AP showed a significant influence on the microbial composition of the intestine. Relatively elevated liver enzymes, especially AP, may contribute to the detection of precancerous dysplastic or neoplastic changes in colorectal lesions. The association between elevated AP, premalignant CP, and the microbiome merits further study.

2D and 3D Erosion Landscape Analysis of Endodontic-Treated Teeth Using EDTA and HEDP as Chelating Agents: A High-Resolution Micro-Computed Tomographic Study.

This study aimed to assess the amount of erosion during activated endodontic irrigation with either HEDP or EDTA via high-resolution micro-computed tomography. Two root canals of twenty premolars were prepared with ProTaper Next and irrigated with sodium hypochlorite. Palatal canals, which served as control groups, were sealed, while buccal canals were further irrigated with either EDTA (n = 10) or HEDP (n = 10), which served as test groups. Micro-CT was performed to measure erosion depth. For 2D and 3D measurements, non-parametric repeated ANOVA measurements and post hoc tests were performed. 2D analysis showed highly significant differences between the case groups at each position of the root (p ≤ 0.01). The cervical and apical positions showed significant differences in the EDTA group (p = 0.03). The 3D analysis also showed significant differences between both chelating agents (p < 0.01) and the case and control groups (p = 0.01). The mean erosion depths in the cervical, middle, and apical thirds of the EDTA group were 45.75, 41.79, and 32.25 µm, and for the HEDP group were 20.25, 16.40, and 15.96 µm, respectively. HEDP seems to have a significantly less erosive effect. Different irrigation protocols with harsher conditions, as might be the case during endodontic retreatment, could be assessed with micro-CT.

Draft genome sequence of Bifidobacterium breve DSM 32583, isolated from human milk.

Here, we describe the draft genome sequence of Bifidobacterium breve DSM 32583 isolated from human milk obtained from a healthy mother. Potentially, this B. breve strain could serve as a probiotic.

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors.

BACKGROUND: Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. METHODS: We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. FINDINGS: We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. INTERPRETATION: We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. FUNDING: Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

Non-canonical transcriptional start sites in E. coli O157:H7 EDL933 are regulated and appear in surprisingly high numbers.

Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.

Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes.

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Is Autologous Fecal Microbiota Transfer after Exclusive Enteral Nutrition in Pediatric Crohn's Disease Patients Rational and Feasible? Data from a Feasibility Test.

BACKGROUND: Exclusive enteral nutrition (EEN) is a highly effective therapy for remission induction in pediatric Crohn's disease (CD), but relapse rates after return to a regular diet are high. Autologous fecal microbiota transfer (FMT) using stool collected during EEN-induced clinical remission might represent a novel approach to maintaining the benefits of EEN. METHODS: Pediatric CD patients provided fecal material at home, which was shipped at 4 °C to an FMT laboratory for FMT capsule generation and extensive pathogen safety screening. The microbial community composition of samples taken before and after shipment and after encapsulation was characterized using 16S rRNA amplicon sequencing. RESULTS: Seven pediatric patients provided fecal material for nine test runs after at least three weeks of nutritional therapy. FMT capsules were successfully generated in 6/8 deliveries, but stool weight and consistency varied widely. Transport and processing of fecal material into FMT capsules did not fundamentally change microbial composition, but microbial richness was <30 genera in 3/9 samples. Stool safety screening was positive for potential pathogens or drug resistance genes in 8/9 test runs. CONCLUSIONS: A high pathogen burden, low-diversity microbiota, and practical deficiencies of EEN-conditioned fecal material might render autologous capsule-FMT an unsuitable approach as maintenance therapy for pediatric CD patients.

The Development of the Bacterial Community of Brown Trout (Salmo trutta) during Ontogeny.

Brown trout (Salmo trutta) is an important aquaculture species in Germany, but its production faces challenges due to global warming and a high embryo mortality. Climate factors might influence the fish's bacterial community (BC) and thus increase embryo mortality. Yet, knowledge of the physiological BC during ontogeny in general is scarce. In this project, the BC of brown trout has been investigated in a period from unfertilized egg to 95 days post fertilization (dpf) using 16S rRNA gene amplicon sequencing. Developmental changes differed between early and late ontogeny and major differences in BC occurred especially during early developmental stages. Thus, analysis was conducted separately for 0 to 67 dpf and from 67 to 95 dpf. All analyzed stages were sampled in toto to avoid bias due to different sampling methods in different developmental stages. The most abundant phylum in the BC of all developmental stages was Pseudomonadota, while only two families (Comamonadaceae and Moraxellaceae) occurred in all developmental stages. The early developmental stages until 67 dpf displayed greater shifts in their BC regarding bacterial richness, microbial diversity, and taxonomic composition. Thereafter, in the fry stages, the BC seemed to stabilize and changes were moderate. In future studies, a reduction in the sampling time frames during early development, an increase in sampling numbers, and an attempt for biological reproduction in order to characterize the causes of these variations is recommended.

Three Novel Antisense Overlapping Genes in E. coli O157:H7 EDL933.

The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping candidate genes are equipped with typical structural elements required for transcription and translation, i.e., promoters, transcription start sites, as well as terminators, all of which were experimentally verified. Translationally arrested mutants, unable to produce the overlapping encoded protein, were found to have a growth disadvantage when grown competitively against the wild type. Thus, the phenotypes found imply biological functionality of the genes at the level of proteins produced. The addition of 3 more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes emphasizes the underestimated coding capacity of bacterial genomes. IMPORTANCE The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated, since such genes are not allowed in genome annotations. However, ribosome profiling catches mRNA in the moment of being template for protein production. Using this technique and subsequent experiments, we verified 3 novel overlapping genes encoded in antisense of known genes. This adds more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes.

The Role of Gut and Oral Microbiota in the Formation and Rupture of Intracranial Aneurysms: A Literature Review.

In recent years, there has been a growing interest in the role of the microbiome in cardiovascular and cerebrovascular diseases. Emerging research highlights the potential role of the microbiome in intracranial aneurysm (IA) formation and rupture, particularly in relation to inflammation. In this review, we aim to explore the existing literature regarding the influence of the gut and oral microbiome on IA formation and rupture. In the first section, we provide background information, elucidating the connection between inflammation and aneurysm formation and presenting potential mechanisms of gut-brain interaction. Additionally, we explain the methods for microbiome analysis. The second section reviews existing studies that investigate the relationship between the gut and oral microbiome and IAs. We conclude with a prospective overview, highlighting the extent to which the microbiome is already therapeutically utilized in other fields. Furthermore, we address the challenges associated with the context of IAs that still need to be overcome.

Gut Microbial Disruption in Critically Ill Patients with COVID-19-Associated Pulmonary Aspergillosis.

OBJECTIVES: COVID-19 disease can be exacerbated by Aspergillus superinfection (CAPA). However, the causes of CAPA are not yet fully understood. Recently, alterations in the gut microbiome have been associated with a more complicated and severe disease course in COVID-19 patients, most likely due to immunological mechanisms. The aim of this study was to investigate a potential association between severe CAPA and alterations in the gut and bronchial microbial composition. METHODS: We performed 16S rRNA gene amplicon sequencing of stool and bronchial samples from a total of 16 COVID-19 patients with CAPA and 26 patients without CAPA. All patients were admitted to the intensive care unit. Results were carefully tested for potentially confounding influences on the microbiome during hospitalization. RESULTS: We found that late in COVID-19 disease, CAPA patients exhibited a trend towards reduced gut microbial diversity. Furthermore, late-stage patients with CAPA superinfection exhibited an increased abundance of Staphylococcus epidermidis in the gut which was not found in late non-CAPA cases or early in the disease. The analysis of bronchial samples did not yield significant results. CONCLUSIONS: This is the first study showing that alterations in the gut microbiome accompany severe CAPA and possibly influence the host's immunological response. In particular, an increase in Staphylococcus epidermidis in the intestine could be of importance.

RoBDEMAT: A risk of bias tool and guideline to support reporting of pre-clinical dental materials research and assessment of systematic reviews.

OBJECTIVES: To develop a risk of bias tool for pre-clinical dental materials research studies that aims to support reporting of future investigations and improve assessment in systematic reviews. METHODS: A four-stage process following EQUATOR network recommendations was followed, which included project launch, literature review, Delphi process and the tool finalization. With the support of the European Federation of Conservative Dentistry (EFCD) and the Dental Materials Group of the International Association for Dental Research (DMG-IADR), a total of 26 expert stakeholders were included in the development and Delphi vote of the initial proposal. The proposal was built using data gathered from the literature review stage. During this stage, recent systematic reviews featuring dental materials research, and risk of bias tools found in the literature were comprehensively scanned for bias sources. The experts thus reached a consensus for the items, domains and judgement related to the tool, allowing a detailed guide for each item and corresponding signalling questions. RESULTS: The tool features nine items in total, spread between 4 domains, pertaining to the following types of bias: bias related to planning and allocation (D1), specimen preparation (D2), outcome assessment (D3) and data treatment and outcome reporting (D4). RoBDEMAT, as presented, features signalling questions and a guide that can be used for RoB judgement. Its use as a checklist is preferred over a final summary score. CONCLUSION: RoBDEMAT is the first risk of bias tool for pre-clinical dental materials research, supported and developed by a broad group of expert stakeholders in the field, validating its future use. CLINICAL SIGNIFICANCE: This new tool will contribute the study field by improving the scientific quality and rigour of dental materials research studies and their systematic reviews. Such studies are the foundation and support of future clinical research and evidence-based decisions.

Taxonomy Informed Clustering, an Optimized Method for Purer and More Informative Clusters in Diversity Analysis and Microbiome Profiling.

Bacterial diversity is often analyzed using 16S rRNA gene amplicon sequencing. Commonly, sequences are clustered based on similarity cutoffs to obtain groups reflecting molecular species, genera, or families. Due to the amount of the generated sequencing data, greedy algorithms are preferred for their time efficiency. Such algorithms rely only on pairwise sequence similarities. Thus, sometimes sequences with diverse phylogenetic background are clustered together. In contrast, taxonomic classifiers use position specific taxonomic information in assigning a probable taxonomy to a given sequence. Here we introduce Taxonomy Informed Clustering (TIC), a novel approach that utilizes classifier-assigned taxonomy to restrict clustering to only those sequences that share the same taxonomic path. Based on this concept, we offer a complete and automated pipeline for processing of 16S rRNA amplicon datasets in diversity analyses. First, raw reads are processed to form denoised amplicons. Next, the denoised amplicons are taxonomically classified. Finally, the TIC algorithm progressively assigning clusters at molecular species, genus and family levels. TIC outperforms greedy clustering algorithms like USEARCH and VSEARCH in terms of clusters' purity and entropy, when using data from the Living Tree Project as test samples. Furthermore, we applied TIC on a dataset containing all Bifidobacteriaceae-classified sequences from the IMNGS database. Here, TIC identified evidence for 1000s of novel molecular genera and species. These results highlight the straightforward application of the TIC pipeline and superior results compared to former methods in diversity studies. The pipeline is freely available at: https://github.com/Lagkouvardos/TIC.

Do magnification loupes affect the precision of cavity preparations made by undergraduates? A randomized crossover study.

BACKGROUND: Evidence on the effect of magnification devices on procedure quality in restorative dentistry is scant. This study therefore aimed to assess, under simulated clinical conditions, if magnification loupes affect the quality of preparations carried out by undergraduate dental students. METHODS: 59 undergraduate dental students underwent two visual acuity tests, based on which they were divided into a "low visual acuity group" (visus < 1) and a "good visual acuity group" (visus ≥ 1). In a randomized crossover experiment, participants performed a two-dimensional S and a three-dimensional O figure preparation with a dental handpiece on standardized acrylic blocs designed for preclinical restorative training. Each participant carried out the preparation tasks twice, once with magnification loupes (2.5×) and once without. Two blinded investigators independently evaluated parameters of preparation precision. Data were analyzed using Spearman rank correlation coefficients, intra-class correlation coefficients, and Wilcoxon rank-sum tests (α = 0.05). RESULTS: Participants from the "low visual acuity group" did not show a statistically significant improvement in accuracy when they used magnification loupes for the S figure preparation (p ≥ 0.0625). Participants from the "high visual acuity group" obtained a higher level of accuracy (p ≤ 0.0012) when they used magnification loupes for the S figure preparation. The use of magnification loupes had no statistically significant effect on the accuracy parameters of the O figure cavity preparations (p ≥ 0.1865). Participants with high visual acuity achieved only a marginally better accuracy than participants with a visus < 1. CONCLUSIONS: This study suggests that loupes with 2.5× magnification increase the accuracy of two-dimensional preparations while they have no significant effect, favorable or otherwise, on the accuracy of complex, three-dimensional cavity preparations of untrained dental students.

Complete Genome Sequence of the Type Strain Citrobacter rodentium DSM 16636.

The type strain Citrobacter rodentium DSM 16636 was characterized in 1995. This species is widely used in rodents to study the virulence of locus-of-enterocyte-effacement-type pathogens, such as enterohemorrhagic Escherichia coli. The type strain had not been sequenced yet. Here, we report the closed genome (5.3 Gbp) and its plasmid (39.3 kbp).

Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing.

Current methods of high-throughput RNA sequencing of prokaryotes, including transcriptome analysis or ribosomal profiling, need deep sequencing to achieve sufficient numbers of effective reads (e.g., mapping to mRNA) in order to also find weakly expressed genetic elements. The fraction of high-quality reads mapping to coding RNAs (i.e., mRNA) is mainly influenced by the large content of rRNA and, to a lesser extent, tRNA in total RNA. Thus, depletion of rRNA increases coverage and thus sequencing costs. RiboZero, a depletion kit based on probe hybridisation and rRNA-removal was found to be most efficient in the past, but it was discontinued in 2018. To facilitate comparability with previous experiments and to help choose adequate replacements, we compare three commercially available rRNA depletion kits also based on hybridization and magnetic beads, i.e., riboPOOLs, RiboMinus and MICROBExpress, with the former RiboZero. Additionally, we constructed biotinylated probes for magnetic bead capture and rRNA depletion in this study. Based on E. coli, we found similar efficiencies in rRNA depletion for riboPOOLs and the self-made depletion method; both comparable to the former RiboZero, followed by RiboMinus, succeeded by MICROBExpress. Further, our in-house protocol allows customized species-specific rRNA or even tRNA depletion or depletion of other RNA targets. Both, the self-made biotinylated probes and riboPOOLs, were most successful in reducing the rRNA content and thereby increasing sequencing depth concerning mRNA reads. Additionally, the number of reads matching to weakly expressed genes are increased. In conclusion, the self-made specific biotinylated probes and riboPOOLs are an adequate replacement for the former RiboZero. Both are very efficient in depleting rRNAs, increasing mRNA reads and thus sequencing efficiency.