Good scientific practice is important in all areas of science. In recent years this has gained more and more attention, especially considering the 'scientific reproducibility crisis'. While most ...researchers are aware of the issues with good scientific practice, not all of these issues are necessarily clear, and the details can be very complicated. For many years it has been accepted to perform and publish sequencing based microbiome studies without including proper controls. Although in recent years more scientists realize the necessity of implementing controls, this poses a problem due to the complexity of the field. Another concern is the inability to properly interpret the information gained from controls in microbiome studies. Here, we will discuss these issues and provide a comprehensive overview of problematic points regarding controls in microbiome research, and of the current standards in this area.
Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been ...documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration MIC = 8 mg L
). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections.
Host glycans are paramount in regulating the symbiotic relationship between humans and their gut bacteria. The constant flux of host-secreted mucin at the mucosal layer creates a steady niche for ...bacterial colonization. Mucin degradation by keystone species subsequently shapes the microbial community. This study investigated the transcriptional response during mucin-driven trophic interaction between the specialised mucin-degrader
Akkermansia muciniphila
and a butyrogenic gut commensal
Anaerostipes caccae
.
A. muciniphila
monocultures and co-cultures with non-mucolytic
A. caccae
from the
Lachnospiraceae
family were grown anaerobically in minimal media supplemented with mucin. We analysed for growth, metabolites (HPLC analysis), microbial composition (quantitative reverse transcription PCR), and transcriptional response (RNA-seq). Mucin degradation by
A. muciniphila
supported the growth of
A. caccae
and concomitant butyrate production predominantly via the acetyl-CoA pathway. Differential expression analysis (DESeq 2) showed the presence of
A. caccae
induced changes in the
A. muciniphila
transcriptional response with increased expression of mucin degradation genes and reduced expression of ribosomal genes. Two putative operons that encode for uncharacterised proteins and an efflux system, and several two-component systems were also differentially regulated. This indicated
A. muciniphila
changed its transcriptional regulation in response to
A. caccae
. This study provides insight to understand the mucin-driven microbial ecology using metatranscriptomics. Our findings show that the expression of mucolytic enzymes by
A. muciniphila
increases upon the presence of a community member. This could indicate its role as a keystone species that supports the microbial community in the mucosal environment by increasing the availability of mucin sugars.
Fecal microbiota transplantation has proven to be an effective treatment for infections with the gram-positive enteropathogen
. Despite its effectiveness, the exact mechanisms that underlie its ...success are largely unclear. In this review, we highlight the pleiotropic effectors that are transferred during fecal microbiota transfer and relate this to the
lifecycle. In doing so, we show that it is likely that multiple factors contribute to the elimination of symptoms of
infections after fecal microbiota transplantation.
Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 ...mutations contribute to HCC development remains unclear. In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced beta-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored beta-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive beta-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. Our study provides insights into the effects of repairing AXIN1 mutations on beta-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting.
A multi-centre study was conducted at four hospitals located across the ...Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred.
CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization tCDC) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no ‘hypervirulent’ PCR ribotype 027 (RT027) was detected (95% CI, 0–0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0–0.073) or 1-year follow-up (0/38; 95% CI, 0–0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data.
In this endemic setting with a low prevalence of ‘hypervirulent’ strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting.
Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare ...single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.
A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential ...importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.
•The complete circular genome sequence is provided for a C. difficile isolate harboring three plasmids.•The plasmids pJMR5–1, pJMR5–4 and pJRM5-W are compatible in a single strain and are present in multiple copies per cell.•The pJMR5-plasmids belong to the previously characterized pCD-ECE1, pCD-ECE4 and pCD-WTI1 families.•Functional replicons were cloned from pJMR5–1 and pJMR5-W that are compatible with pCD630.
The Gram-positive enteropathogen
(
) is the major cause of healthcare-associated diarrhoea and is also an important cause of community-acquired infectious diarrhoea. Considering the burden of the ...disease, many studies have employed whole-genome sequencing of bacterial isolates to identify factors that contribute to virulence and pathogenesis. Though extrachromosomal elements (ECEs) such as plasmids are important for these processes in other bacteria, the few characterized plasmids of
have no relevant functions assigned and no systematic identification of plasmids has been carried out to date. Here, we perform an
analysis of publicly available sequence data to show that ~13 % of all
strains contain ECEs, with 1-6 elements per strain. Our approach identifies known plasmids (e.g. pCD6, pCD630 and cloning plasmids) and six novel putative plasmid families. Our study shows that plasmids are abundant and may encode functions that are relevant for
physiology. The newly identified plasmids may also form the basis for the construction of novel cloning plasmids for
that are compatible with existing tools.
The aim of this thesis was to elucidate how various microbial communities work, with a focus on next generation sequencing data. The introduction in chapter 1 focuses on the history of biology, how ...the field of systems biology arose, and how the rise of nucleic acid sequencing has shaped a completely new field (among others), the microbiome research. In chapter 2, an overview is given how the microbiota can be studied, in connection to metabolic syndrome and its sub-pathologies, including obesity, type II diabetes, elevated blood pressure, and dyslipidemia. We summarize which different methodologies (16S rRNA amplicon sequencing, metagenomics, metatranscriptomics) can be used to investigate the microbiome with different foci, and how as a next step the microbiome can be modelled, in vitro and in silico. Chapter 3 describes the genome and transcriptome of the rat gut commensal Romboutsia ilealis CRIBT. We characterized genomic properties, including those related to metabolism and sporulation abilities. The transcriptome allowed us to investigate the organism’s carbohydrate degradation abilities, including its potential regulation. Chapter 4 is an investigation of an in vitro fermentation system, inoculated with human faecal material and the potential prebiotic Isomalto/malto-polysaccharides. The metatranscriptome of this system gave an insight into which genes are involved in the carbohydrate degradation, and which different types of organisms are involved and potentially need to cooperate for a full utilization of this carbohydrate. In chapter 5, the cow rumen microbiota is investigated under different feeding regimes. The metatranscriptome of the cow rumen microbiota showed distinct patterns depending on the ratio of starch or cellulose enriched feed components, namely maize vs. grass silage. The increase in starch led to a decrease in methane emissions of the cow rumen microbiota, which was reflected in the metatranscriptomics data. Most notably, lower expression levels of genes encoding for proteins involved in methanogenic pathways of the rumen archaeon Methanobrevibacter smithii was observed. The last chapter, the general discussion, mainly discusses the research described in this thesis with a focus on the relevant issues with modelling microbial communities, as well as overall scientific integrity in relationship with microbiome research.
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