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.

Organization for Caries Research-European Federation of Conservative Dentistry 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. 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.

European Organisation for Caries Research and the European Federation of Conservative Dentistry Consensus Report on Clinical Recommendations for Caries Diagnosis: Paper III - Caries Diagnosis at the Individual Level.

OBJECTIVES: 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. MATERIAL 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 which were asked to provide recommendations on (1) caries detection and diagnostic methods, (2) caries activity assessment, and (3) forming individualized 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. RESULTS: 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. CONCLUSION: 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. CLINICAL RELEVANCE: The expert panel merged evidence from existing guidelines and scientific literature with practical considerations and provided recommendations for their use in daily dental practice.

The effectiveness of partial pulpotomy compared with full pulpotomy in managing deep caries in vital permanent teeth with a diagnosis of non-traumatic pulpitis.

BACKGROUND: Pulpitis may be pain free or alternatively characterized by mild to severe pain and associated symptoms. Evidence has recently emerged that patients presenting with carious pulp exposure range of symptoms can be treated effectively with pulpotomy. OBJECTIVE: The current systematic review aimed to answer the following research question: "In patients with deep caries lesions in permanent teeth associated with no symptoms, reversible pulpitis or signs and symptoms indicative of irreversible pulpitis (P), is partial pulpotomy (I) as effective as full pulpotomy (C), in terms of a combination of patient and clinical reported outcomes (O), with "tooth survival" as the most critical outcome? METHODS: The systematic literature search was conducted in the following electronic databases: OVID, Scopus, PubMed (Including MEDLINE), and Cochrane Central Register of Controlled Trials (CENTRAL) supplemented with Grey literature and hand searching of relevant journals. The English language clinical trials comparing the patient and clinical reported outcomes between partial and full/complete were included. After a structured literature search, two authors independently performed study selection, extracted data and performed a risk of bias assessment; a third reviewer resolved disagreements. As there were only two studies with different exclusion criteria, no meta-analysis was performed and the quality of evidence was assessed by the GRADE approach. RESULTS: After study selection a total of two randomised clinical trials with a total of 156 teeth were included both for the management of teeth with irreversible pulpitis. There were no studies for asymptomatic teeth or teeth with reversible pulpitis. A "Low" risk of bias was noted for both studies with a high level of overall evidence. A meta-analysis was not carried out due to differences in inclusion criteria between the studies related principally to caries depth. Both studies reported a high rate of clinical success for pulpotomy with a pooled unadjusted success rate for full pulpotomy of 90% and 83% partial pulpotomy of at 1-year; however, no significant difference between the treatments was noted in either study. There was significantly reduced postoperative pain reported in the full pulpotomy group over 1-week compared with the partial pulpotomy in one but not in the other study. DISCUSSION: Pulpotomy as a definitive treatment modality is as effective in managing teeth exhibiting signs and symptoms indicative of irreversible pulpitis and challenges the established protocols to manage this condition. Although based on only two RCTs with a limited number of patients, no difference was shown in terms of clinical or radiographic outcome or postoperative pain between groups. Further well designed randomised clinical trials of longer duration are required in this area to improve the evidence available. CONCLUSION: There is no consistent difference in patient-reported pain between partial and full pulpotomy at day 7 postoperatively and the clinical success rate was similar after 1 year for both treatment modalities.

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.

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.

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.

STAndard Reporting of CAries Detection and Diagnostic Studies (STARCARDDS).

AIM: The aim of this paper is to present recommendations from an international workshop which evaluated the methodology and reporting of caries diagnostic studies. As a unique feature, this type of studies is focused on caries lesion detection and assessment, and many of them are carried out in vitro, because of the possibility of histological validation of the whole caries spectrum. This feature is not well covered in the existing reporting STARD guideline within the EQUATOR Network. PARTICIPANTS AND METHODS: An international working group of 13 cariology researchers was formed. The STARD checklist was reviewed and modified for caries detection and diagnosis purposes, in a three-step process of evaluation, consensual modification, and delivery during three 2-day workshops over 18 months. Special attention was paid to reporting requirements of caries studies that solely focus on reliability. RESULTS: The STARD checklist was modified in 14/30 items, with an emphasis on issues of sample selection (tooth selection in in vitro studies), blinding, and detailed reporting of results. CONCLUSION: Following STARCARDDS (STAndard Reporting of CAries Detection and Diagnostic Studies) is expected to result in complete reporting of study design and methodology in future caries diagnosis and detection experiments both in vivo and in vitro, thus allowing for better comparability of studies and higher quality of systematic reviews. CLINICAL RELEVANCE: Standardization of caries diagnostic studies leads to a better comparability among future studies, both in vivo and in vitro.

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.

Recommendations for bacterial ribosome profiling experiments based on bioinformatic evaluation of published data.

Ribosome profiling (RIBO-Seq) has improved our understanding of bacterial translation, including finding many unannotated genes. However, protocols for RIBO-Seq and corresponding data analysis are not yet standardized. Here, we analyzed 48 RIBO-Seq samples from nine studies of Escherichia coli K12 grown in lysogeny broth medium and particularly focused on the size-selection step. We show that for conventional expression analysis, a size range between 22 and 30 nucleotides is sufficient to obtain protein-coding fragments, which has the advantage of removing many unwanted rRNA and tRNA reads. More specific analyses may require longer reads and a corresponding improvement in rRNA/tRNA depletion. There is no consensus about the appropriate sequencing depth for RIBO-Seq experiments in prokaryotes, and studies vary significantly in total read number. Our analysis suggests that 20 million reads that are not mapping to rRNA/tRNA are required for global detection of translated annotated genes. We also highlight the influence of drug-induced ribosome stalling, which causes bias at translation start sites. The resulting accumulation of reads at the start site may be especially useful for detecting weakly expressed genes. As different methods suit different questions, it may not be possible to produce a "one-size-fits-all" ribosome profiling data set. Therefore, experiments should be carefully designed in light of the scientific questions of interest. We propose some basic characteristics that should be reported with any new RIBO-Seq data sets. Careful attention to the factors discussed should improve prokaryotic gene detection and the comparability of ribosome profiling data sets.

ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples.

BACKGROUND: One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. RESULTS: Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. CONCLUSION: Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

Systematic review and meta-analysis of diagnostic methods for occlusal surface caries.

AIM: This systematic review and meta-analysis aimed to assess the diagnostic performance of commonly used methods for occlusal caries diagnostics, such as visual examination (VE), bitewing radiography (BW) and laser fluorescence (LF), in relation to their ability to detect (dentin) caries under clinical and laboratory conditions. MATERIALS AND METHODS: A systematic search of the literature was performed to identify studies meeting the inclusion criteria using the PIRDS concept (N = 1090). A risk of bias (RoB) assessment tool was used for quality evaluation. Reports with low/moderate RoB, well-matching thresholds for index and reference tests and appropriate reporting were included in the meta-analysis (N = 37; 29 in vivo/8 in vitro). The pooled sensitivity (SE), specificity (SP), diagnostic odds ratio (DOR) and areas under ROC curves (AUCs) were computed. RESULTS: SP ranged from 0.50 (fibre-optic transillumination/caries detection level) to 0.97 (conventional BW/dentine detection level) in vitro. AUCs were typically higher for BW or LF than for VE. The highest AUC of 0.89 was observed for VE at the 1/3 dentin caries detection level; SE (0.70) was registered to be higher than SP (0.47) for VE at the caries detection level in vivo. CONCLUSION: The number of included studies was found to be low. This underlines the need for high-quality caries diagnostic studies that further provide data in relation to multiple caries thresholds. CLINICAL RELEVANCE: VE, BW and LF provide acceptable measures for their diagnostic performance on occlusal surfaces, but the results should be interpreted with caution due to the limited data in many categories.

Systematic review and meta-analysis of diagnostic studies of proximal surface caries.

AIM: This systematic review and meta-analysis aimed to assess the diagnostic accuracy and reliability of commonly used caries detection methods for proximal caries diagnostics. Visual examination (VE), bitewing radiography (BWR), laser fluorescence (LF), and fibre-optic transillumination (FOTI) were considered in detail. MATERIAL AND METHODS: PRISMA guidelines for the reporting of systematic reviews and meta-analyses were applied. The mnemonic PIRDS (problem, index test, reference test, diagnostic and study type) concept was used to guide the literature search. Next, studies that met the inclusion criteria were stepwise selected and evaluated for their quality with a risk of bias (RoB) assessment tool. Studies with low/moderate bias and sufficient reporting were considered for meta-analysis. The pooled sensitivity (SE), specificity (SP), diagnostic odds ratio (DOR), and area under the ROC curve (AUC) were calculated. RESULTS: From 129 studies meeting the selection criteria, 31 in vitro studies and five clinical studies were finally included in the meta-analysis. The AUC values for in vitro VE amounted to 0.84 (caries detection) and 0.85 (dentin caries detection). BWR ranged in vitro from 0.55 to 0.82 (caries detection) and 0.81-0.92 (dentin caries detection). LF showed higher AUC values for overall caries detection (0.91) and dentin caries detection (0.83) than did other methods. Clinical data are limited. CONCLUSION: The number of diagnostic studies with low/moderate RoB was found to be low and indicates a need for high-quality, well-designed caries diagnostic studies. CLINICAL RELEVANCE: BWR and LF showed good diagnostic performance on proximal surfaces. However, because of the low number of includable clinical studies, these data should be interpreted with caution.

Salmonella persistence in soil depends on reciprocal interactions with indigenous microorganisms.

Fresh fruits and vegetables have numerous benefits to human health. Unfortunately, their consumption is increasingly associated with food-borne diseases, Salmonella enterica being their most frequent cause in Europe. Agricultural soils were postulated as reservoir of human pathogens, contributing to the contamination of crops during the growing period. Since the competition with the indigenous soil microbiota for colonization sites plays a major role in the success of invading species, we hypothesized that reduced diversity will enhance the chance of Salmonella to successfully establish in agricultural environments. We demonstrated that the abundance of Salmonella drastically decreased in soil with highly diverse indigenous prokaryotic community, while in soil with reduced prokaryotic diversity, Salmonella persisted for a long period. Furthermore, in communities with low diversity, Salmonella had an impact on the abundance of other taxa. The high physiological plasticity allows Salmonella to use agricultural soils as alternative habitat which might provide a route of animal/human infections. In addition, adjusted transcriptional profile with amino acid biosynthesis and the glyoxylate cycle most prominently regulated, suggests an adaptation to the soil environment. Our results underline the importance of the maintenance of diverse soil microbiome as a part of strategy aiming at reduced risk of food-borne salmonellosis outbreaks.

Sonic irrigant activation for root canal disinfection: power modes matter!

BACKGROUND: Sonic irrigant activation has gained widespread popularity among general dentists and endodontists alike in recent years. This in vitro study aimed to evaluate the impact of three power modes of a sonic activation device (EDDY) on its antimicrobial effectiveness in infected root canals. METHODS: The root canals of straight, human roots (n = 120) were prepared to size 40/.06. In a short-term infection experiment, the root canals were inoculated with different microbial species for three days. The following irrigation protocols, using 4 ml of normal saline as irrigant, were performed: negative control, manual rinsing, sonic irrigant activation at power modes "low", "medium" and "high". In a second, long-term experiment, testing the same irrigation protocols, inoculation lasted 21 days and sodium hypochlorite was used as irrigant. Sequential infection control samples were assessed using culture assays. The statistical analysis included one-way analysis of variance of log10-scaled counts of colony-forming units (CFU) with post-hoc comparisons using Bonferroni corrections and Chi2 tests (α = 0.05). RESULTS: In the short-term experiment, the sonic irrigation protocols decreased the number of CFUs by 1.88 log10 units compared with the negative control (p < 0.001). The power modes "medium" and "high" achieved the most effective reduction of the microbial load. In the long-term experiment, microbial regrowth occurred after 7 days unless the device was used at its highest power setting. CONCLUSIONS: The power modes of the sonic irrigation device have a significant impact on the effectiveness for endodontic disinfection. The sonic irrigation device should always be used at the highest power setting in order to maximize its antimicrobial effectiveness.

A novel short L-arginine responsive protein-coding gene (laoB) antiparallel overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 Sakai originated by overprinting.

BACKGROUND: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail. RESULTS: A small open reading frame of Escherichia coli O157:H7 strain Sakai (EHEC), designated laoB (L-arginine responsive overlapping gene), is embedded in reading frame -2 in the antisense strand of ECs5115, encoding a CadC-like transcriptional regulator. This overlapping gene shows evidence of transcription and translation in Luria-Bertani (LB) and brain-heart infusion (BHI) medium based on RNA sequencing (RNAseq) and ribosomal-footprint sequencing (RIBOseq). The transcriptional start site is 289 base pairs (bp) upstream of the start codon and transcription termination is 155 bp downstream of the stop codon. Overexpression of LaoB fused to an enhanced green fluorescent protein (EGFP) reporter was possible. The sequence upstream of the transcriptional start site displayed strong promoter activity under different conditions, whereas promoter activity was significantly decreased in the presence of L-arginine. A strand-specific translationally arrested mutant of laoB provided a significant growth advantage in competitive growth experiments in the presence of L-arginine compared to the wild type, which returned to wild type level after complementation of laoB in trans. A phylostratigraphic analysis indicated that the novel gene is restricted to the Escherichia/Shigella clade and might have originated recently by overprinting leading to the expression of part of the antisense strand of ECs5115. CONCLUSIONS: Here, we present evidence of a novel small protein-coding gene laoB encoded in the antisense frame -2 of the annotated gene ECs5115. Clearly, laoB is evolutionarily young and it originated in the Escherichia/Shigella clade by overprinting, a process which may cause the de novo evolution of bacterial genes like laoB.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933 (EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and a peptide, RyhP.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

Probiotic Enterococcus faecalis Symbioflor® down regulates virulence genes of EHEC in vitro and decrease pathogenicity in a Caenorhabditis elegans model.

Enterohemorrhagic E. coli O157:H7 (EHEC) shorten the lifespan of Caenorhabditis elegans compared to avirulent bacteria. Co-feeding EHEC with Enterococcus faecalis Symbioflor® significantly increased the worms' lifespan. The transcriptome of EHEC grown in vitro with or without Symbioflor® was analyzed using RNA-seq. The analysis revealed downregulation of several virulence-associated genes in the presence of Symbioflor®, including virulence key genes (e.g., LEE, flagellum, quorum-sensing). The downregulation of the LEE genes was corroborated by lux-transposon mutants. Upregulated genes included acid response genes, due to a decrease in pH exerted by Symbioflor®. Further genes indicate cellular stress in EHEC (e.g. prophage/mobile elements involved in excision, cell lysis, and cell division inhibition). Thus, the observed protection of C. elegans during an EHEC infection by the probiotic Symbioflor® is suggested to be caused by triggering concomitant transcriptomic changes. To verify the biological relevance of this modulation, exemplary genes found to be influenced by Symbioflor® were knocked out (fliD, espB, Z3136, Z3917, and L7052). The lifespan of nematodes changed when using knock-outs as food source and the effect could be complemented in trans. In summary, Symbioflor® appears to be a protective probiotic in the nematode model.

Bacterial invasion into radicular dentine-an in vitro study.

OBJECTIVES: We wanted to investigate differences in invasiveness into radicular dentinal tubules by monocultured and co-cultured bacteria frequently found in infected root canals. METHODS: Fifty-one human roots were incubated for 8 weeks with monocultured Streptococcus gordonii ATCC 10558, Streptococcus sanguinis ATCC 10556, and with five capnophiles/anaerobes as well as with capnophiles/anaerobes co-cultured with a streptococcal species. Thereafter, bacterial samples were cultured from the inner, middle, and outer third of the root dentine of longitudinally broken teeth (n = 5). In addition, scanning electron microscopy (SEM) images were obtained. RESULTS: Single gram-positive species were able to penetrate into the middle and outer third of the root dentine. Fusobacterium nucleatum ATCC 25586 was not found in any of the dentine specimens. Prevotella intermedia ATCC 25611 and Porphyromonas gingivalis ATCC 33277 were found in the inner and middle third. The bacterial load of streptococci was higher in all thirds in co-cultures compared to single infections. In co-cultures with streptococci, Actinomyces oris ATCC 43146 was found in the outer third in 9/10 samples, whereas P. intermedia ATCC 25611 was not detectable inside dentine. Co-culture with S. sanguinis ATCC 10556 enabled F. nucleatum ATCC 25586 to invade dentine; SEM images showed that F. nucleatum ATCC 25586 had a swollen shape. CONCLUSIONS: Invasiveness of bacteria into dentinal tubules is species-specific and may change depending on culturing as a single species or co-culturing with other bacteria. CLINICAL RELEVANCE: Oral streptococci may promote or inhibit invasion of capnophiles/anaerobes into radicular dentine.