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PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.

Key Points As the number of environmental small subunit (SSU) ribosomal RNA gene sequences has greatly surpassed the number of cultured microorganisms, reconciliation of the established taxonomy and classification of the uncultured microorganisms are crucial. Rational taxonomic boundaries have been proposed for the high taxa (that is, genus and above) of the Bacteria and the Archaea on the basis of 16S rRNA gene sequence identities. These are : 94.5% for genus, 86.5% for family, 82.0% for order, 78.5% for class and 75.0% for phylum. The application of these thresholds to the clustering of the SILVA database confirms that the current number of formally described taxa at any rank (for example, ∼30 phyla) is negligible compared with the total number of detected taxa (for example, ∼1,300 phyla). In addition, the study of the annual rate of taxa discovery enables a new extrapolation of the total number of species (4 × 10 5 ) and high taxa on Earth (for example, 1 × 10 5 genera), which indicates that most common terrestrial and aquatic habitats will be exhaustively described within the next 5 years. Taxon recovery tests that were carried out using partial 16S rRNA gene sequences show that short reads are not suitable for accurate richness estimations and accurate classifications of high taxa. On the basis of the general taxonomic thresholds and phylogenetic considerations, we suggest a new biodiversity unit known as the candidate taxonomic unit (CTU), which is compatible with the hierarchy that was established in the Bacteriological Code. The ability to specify a taxonomic rank for particular clades is a major advance in understanding tree topologies and goes beyond the classic phylogenetic delineation. The usefulness of CTUs has been intensively tested in the reclassification of the phylum Spirochaetes and the classification of 15 candidate divisions and environmental clades that are presented in this Analysis article, which also provide new insights into the coherence of classes, phyla and superphyla. By providing explicit and well-documented guidelines, it is hoped that this work will facilitate the implementation of the many changes in the current taxonomy that are necessary to develop a common taxonomic classification of high taxa of bacteria and archaea on the basis of SSU rRNA gene sequences. The vast increase in the number of 16S ribosomal RNA gene sequences that are now available has led to an urgent need to implement taxonomic boundaries and classification principles that can apply to both cultured and uncultured microorganisms. In this Analysis article, the authors use 16S rRNA gene sequence identities to propose rational taxonomic boundaries for high taxa of bacteria and archaea and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.

Background Recent advances have increased the importance of the human microbiome, including the skin microbiome. Despite the hand microbiome research, the factors affecting the composition of the hand microbiome and their personal characteristics are incompletely known. Objectives Despite changing environmental factors and personal variation, we aimed to indicate the interpersonal distinction between skin microbiota using simple and rapid molecular methods. Methods Over a non‐consecutive 10‐day period, samples were taken from 10 adult individuals, and ribotyping analysis of the 16S and 23S genes of S. epidermidis was performed on each skin sample. Additionally, EcoRI and HindIII enzyme reactions and variable number tandem repeat (VNTR) reactions of S. epidermidis obtained from DNA samples were performed. The skin microbiomes of individuals were evaluated along with the microbiome profiles left on the surfaces they touched. Results In the environmental samples taken, it has been observed that people preserve their core skin microbiota characters and carry them to their environment. It was determined that the highest similarity rate was 77.14%, and the lowest similarity rate was 31.74%. Conclusion Our study showed that the core skin microbiota retains its characteristics and leaves traces in environments. The fact that the personal microbiome remains unchanged despite environmental differences and has characteristic features has shown that it can be used in forensic sciences to distinguish individuals from each other. These results with simple and rapid methods further increased the importance and significance of the study. The findings indicate that personal skin microbiota can provide a significant contribution to criminal investigations by increasing accuracy and reliability, especially in forensic analyses.

Background Clostridioides difficile is a major cause of hospital-acquired diarrhea and a driver of nosocomial outbreaks, yet rapid, accurate ribotype identification remains challenging. We sought to develop a MALDI-TOF MS–based workflow coupled with machine learning to distinguish epidemic toxigenic ribotypes (RT027 and RT181) from other strains in real time. Results We analyzed MALDI-TOF spectra from 379 clinical isolates collected across ten Spanish hospitals and identified seven discriminant biomarker peaks. Two peaks (2463 and 4993 m/z) were uniquely associated with RT027, while combinations of five additional peaks reliably identified RT181. Our classifiers–implemented both in the commercial Clover MSDAS platform and the open-access AutoCdiff web tool–achieved up to 100% balanced accuracy in ribotype assignment and proved robust in real-time outbreak simulations. Conclusions This study demonstrates that MALDI-TOF MS combined with tailored machine learning can deliver rapid, high-precision ribotype identification for C. difficile . The freely available AutoCdiff models ( https://bacteria.id ) offer an immediately deployable solution for clinical laboratories, with the potential to enhance outbreak surveillance and control.

The human skin microbiome has important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that although the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne, and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may have roles in determining virulence properties of P. acnes strains, and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain-level analysis of the human microbiome to define the role of commensals in health and disease.

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence. Two hypervirulent ribotypes of the enteric pathogen Clostridium difficile , RT027 and RT078, have independently acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose, suggesting a correlation between the emergence of these ribotypes and the widespread adoption of trehalose in the human diet. The rise of an intestinal epidemic Clostridium difficile is an intestinal pathogen and a major cause of antibiotic-associated diarrhoea. In epidemics in recent years, hypervirulent ribotypes that cause severe disease have emerged, but the factors that contribute to their emergence are unclear. In this study, Robert Britton and colleagues show that two phylogenetically distinct hypervirulent ribotypes, RT027 and RT078, have independently acquired mechanisms to metabolize low concentrations of the disaccharide trehalose. The team also show that this ability to metabolize trehalose correlates with disease severity in a humanized mouse model. These data suggest a correlation between the emergence of these ribotypes and the widespread adoption and use of trehalose as a sugar additive in the human diet.

Dysbiosis is a hallmark of inflammatory bowel disease (IBD), but it is unclear which specific intestinal bacteria predispose to and which protect from IBD and how they are regulated. Peptidoglycan recognition proteins (Pglyrps) are antibacterial, participate in maintaining intestinal microflora, and modulate inflammatory responses. Mice deficient in any one of the four Pglyrp genes are more sensitive to dextran sulfate sodium (DSS)-induced colitis, and stools from Pglyrp-deficient mice transferred to wild type (WT) germ-free mice predispose them to much more severe colitis than stools from WT mice. However, the identities of these Pglyrp-regulated bacteria that predispose Pglyrp-deficient mice to colitis or protect WT mice from colitis are not known. Here we identified significant changes in β-diversity of stool bacteria in Pglyrp-deficient mice compared with WT mice. The most consistent changes in microbiome in all Pglyrp-deficient mice were in Bacteroidales, from which we selected four species, two with increased abundance (Prevotella falsenii and Parabacteroides distasonis) and two with decreased abundance (Bacteroides eggerthii and Alistipes finegoldii). We then gavaged WT mice with stock type strains of these species to test the hypothesis that they predispose to or protect from DSS-induced colitis. P. falsenii, P. distasonis, and B. eggerthii all enhanced DSS-induced colitis in both WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora. By contrast, A. finegoldii (which is the most abundant species in WT mice) attenuated DSS-induced colitis both in WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora, similar to the colitis protective effect of the entire normal microflora. These results identify P. falsenii, P. distasonis, and B. eggerthii as colitis-promoting species and A. finegoldii as colitis-protective species.

BackgroundMost gut microbiome studies have been performed using stool samples. However, the small intestine is of central importance to digestion, nutrient absorption, and immune function, and characterizing its microbial populations is essential for elucidating their roles in human health and disease.AimsTo characterize the microbial populations of different small intestinal segments and contrast these to the stool microbiome.MethodsMale and female subjects undergoing esophagogastroduodenoscopy without colon preparation were prospectively recruited. Luminal aspirates were obtained from the duodenum, jejunum, and farthest distance reached. A subset also provided stool samples. 16S rRNA sequencing was performed and analyses were carried out using CLC Genomics Workbench.Results16S rRNA sequencing identified differences in more than 2000 operational taxonomic units between the small intestinal and stool microbiomes. Firmicutes and Proteobacteria were the most abundant phyla in the small intestine, and Bacteroidetes were less abundant. In the small intestine, phylum Firmicutes was primarily represented by lactic acid bacteria, including families Streptococcaceae, Lactobacillaceae, and Carnobacteriaceae, and Proteobacteria was represented by families Neisseriaceae, Pasteurellaceae, and Enterobacteriaceae. The duodenal and FD microbial signatures were markedly different from each other, but there were overlaps between duodenal and jejunal and between jejunal and FD microbial signatures. In stool, Firmicutes were represented by families Ruminococcaceae, Lachnospiraceae, Christensenellaceae, and Proteobacteria by class Deltaproteobacteria.ConclusionsThe small bowel microbiome is markedly different from that in stool and also varies between segments. These findings may be important in determining how compositional changes in small intestinal microbiota contribute to human disease states.

Sequencing of the Arabidopsis thaliana root microbiome shows that its composition is strongly influenced by location, inside or outside the root, and by soil type. Root dwellers: bacterial communities in the plant root microbiome The association between a land plant and the soil microbes of the root microbiome is important for the plant's well-being. A deeper understanding of these microbial communities will offer opportunities to control plant growth and susceptibility to pathogens, particularly in sustainable agricultural regimes. Two groups, working separately but developing best-practice protocols in parallel, have characterized the root microbiota of the model plant Arabidopis thaliana . Working on two continents and with five different soil types, they reach similar general conclusions. The bacterial communities in each root compartment — the rhizosphere immediately surrounding the root and the endophytic compartment within the root — are most strongly influenced by soil type, and to a lesser degree by host genotype. In natural soils, Arabidopsis plants are preferentially colonized by Actinobacteria, Proteobacteria, Bacteroidetes and Chloroflexi species. And — an important point for future work — Arabidopsis root selectivity for soil bacteria under controlled environmental conditions mimics that of plants grown in a natural environment. Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation 1 , 2 , 3 . Colonization of the root occurs despite a sophisticated plant immune system 4 , 5 , suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant–microbe interactions derived from complex soil communities.