SummaryCell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10−4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile .

The kelp forest of the Pacific temperate rocky marine coastline of Monterey Bay in California is a dominant habitat for large brown macro-algae in the order of Laminariales. It is probably one of the most species-rich, structurally complex and productive ecosystems in temperate waters and well-studied in terms of trophic ecology. However, still little is known about the microorganisms thriving in this habitat. A growing body of evidence suggests that bacteria associated with macro-algae represent a huge and largely untapped resource of natural products with chemical structures that have been optimized by evolution for biological and ecological purposes. Those microorganisms are most likely attracted by algae through secretion of specific carbohydrates and proteins that trigger them to attach to the algal surface and to form biofilms. The algae might then employ those bacteria as biofouling control, using their antimicrobial secondary metabolites to defeat other bacteria or eukaryotes. We here analyzed biofilm samples from the brown macro-algae Macrocystis pyrifera sampled in November 2014 in the kelp forest of Monterey Bay by a metagenomic shotgun and amplicon sequencing approach, focusing on Planctomycetes and Verrucomicrobia from the PVC superphylum. Although not very abundant, we were able to find novel Planctomycetal and Verrucomicrobial species by an innovative binning approach. All identified species harbor secondary metabolite related gene clusters, contributing to our hypothesis that through inter-species interaction, microorganisms might have a substantial effect on kelp forest wellbeing and/or disease-development.

To trace the routes and frequencies of transmission of Clostridioides difficile in a tertiary-care hospital in Madrid (Spain), we sequenced the genomes from all C . difficile isolates collected over 36 months (2014–2016) that were indistinguishable from any other isolate by PCR ribotyping. From a total of 589 C . difficile infection cases, we cultivated and PCR-ribotyped 367 C . difficile isolates (62%), of which 265 were genome-sequenced. Based on close relatedness of successively collected isolates (≤2 SNPs difference in their genomes), whole-genome sequencing revealed a total of 17 independent, putative transmission clusters, caused by various C . difficile strains and each containing 2 to 18 cases, none of which had been detected previously by standard epidemiological surveillance. Proportions of linked isolates varied widely among PCR ribotypes, from 3% (1/36) for ribotype 014/020 to 60% (12/20) for ribotype 027, suggesting differential aptitudes for nosocomial spread. Remarkably, only a minority (17%) of transmission recipients had direct ward contact to their presumed donors and specific C . difficile genome types frequently went undetectable for several months before re-emerging later, suggesting reservoirs for the pathogen outside of symptomatic patients. Taken together, our analysis based on genome sequencing suggested considerable within-hospital epidemic spread of C . difficile , even though epidemiological data initially had been inconspicuous.

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Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea.Local transmissions and international outbreaks of this pathogen have been 50 previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce EnteroBase, a publicly accessible database (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome 55 multilocus sequence typing (cgMLST). We demonstrate that the identification of highly related genomes is 89% consistent between cgMLST and single-nucleotide polymorphisms. EnteroBase currently contains 13,515 quality-controlled genomes which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. Hierarchical clustering can be used to identify populations of C. difficile at 60 all epidemiological levels, from recent transmission chains through to pandemic and endemic strains, and is largely compatible with prior ribotyping. Hierarchical clustering thus enables comparisons to earlier surveillance data and will facilitate communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile. 65 toxin variant Clostridium difficile PCR ribotype 017 reveals the evolution of two

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission.