Biology subjects

Kuijper, E. J.

Publications and source records attributed to Kuijper, E. J..

13 recordsLinked to original sources

bioRxiv2026

Characterization of the clade 4 non-toxigenic C. difficile isolate L-NTCD03 carrying the cfr(B) gene

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization - including the complete genome sequence - of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validated the relevance of the cfr(B) gene from this strain in antimicrobial resistance to clindamycin, linezolid, retapamulin and streptogramin A. We found the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

microbiology↗

bioRxiv2025

Manufacture of Clostridioides difficile spores for experimental infection of human volunteers

BackgroundInfections with C. difficile remain a significant global healthcare problem for which novel (preventive) treatment strategies are urgently needed. Controlled human infection models (CHIMs) can contribute to a better understanding of the disease and accelerate (novel) medicinal product development. MethodsA robust production process for C. difficile spores for experimental human administration, was implemented. Direct-release capsules containing viable C. difficile spores were formulated. The manufacturing process aligns with good manufacturing practices (GMP) guidelines, including quality controls and release by a qualified person (QP). ResultsSelected non-toxigenic and toxigenic C. difficile strains were used to create a master cell bank and working cell bank from which multiple batches of purified active substance were produced. Substance was then used to manufacture the final active product. The active product and its intermediate products passed all quality controls for identity, potency, purity and safety, and were individually released by the QP. Stability of active substance and product is confirmed up to 12 months. ConclusionAlthough there are no clear standardized international guidelines for challenge material production, we demonstrate that it is feasible to produce C. difficile human challenge material for small scale application in CHIM trials. This application of GMP principles to an unconventional production process of a bacterial spore-forming anaerobic challenge agent is an example for the production of challenge material as described in the auxiliary medicinal product guidelines of EMA and FDA.

microbiology↗

bioRxiv2024

Metagenomic global survey and in-depth genomic analyses of Ruminococcus gnavus reveal differences across host lifestyle and health status

Ruminococcus gnavus is a highly prevalent gut bacterium (present in >90% of healthy individuals), of which increased abundance is associated with chronic inflammatory diseases, most notably Crohns disease. Nevertheless, its global distribution has not been investigated and little is known about intraspecies genomic variation. Through a large-scale survey of 12,791 gut metagenomes, we recapitulated known associations with metabolic diseases and inflammatory bowel disease. We uncover a higher prevalence and abundance of R. gnavus in Westernized populations and observe relative abundances of up to 83% in newborns and infants. Next, we built a collection of existing and newly cultured R. gnavus isolates (N = 45) from both healthy individuals and Crohns disease patients and subjected these to PacBio circular consensus sequencing to greatly expand the number of complete R. gnavus genomes. Analysis of these genomes as well as publicly available high quality draft genomes (total > 300 genomes) revealed multiple clades which separated Crohns-derived isolates from healthy-derived isolates. Functional analyses of genes predicted to constitute R. gnavus virulence factors could not explain this separation. Bacterial GWAS revealed that Crohns-derived isolates were enriched in genes related to mobile elements and putative mucin foraging. Together, we present one of the largest complete genome collections of any commensal gut microbe and provide novel biological insights into the global distribution and genomic variation of R. gnavus.

microbiology↗

bioRxiv2023

Fungal-bacterial gut microbiota interactions in patients with Clostridioides difficile colonisation and infection

ObjectivesThe bacterial microbiota is well-recognised for its role in Clostridioides difficile colonisation and infection, while fungi and yeasts remain understudied. The aim of this study was to analyse the mycobiota and its interactions with the bacterial microbiota in light of C. difficile colonisation and infection. MethodsThe mycobiota was profiled by ITS2 sequencing of faecal DNA from infected patients (CDI; n = 29), asymptomatically colonised patients (CDC; n = 38) and hospitalised controls with C. difficile negative stool culture (Controls; n = 38). Previously published 16S rRNA gene sequencing data of the same cohort were used additionally for machine learning and fungal-bacterial network analysis. ResultsCDI patients were characterised by a significantly higher abundance of Candida spp. (MD 0.270 {+/-} 0.089, P = 0.002) and Candida albicans (MD 0.165 {+/-} 0.082, P = 0.023) compared to Controls. Additionally, they were deprived of Aspergillus spp. (MD -0.067 {+/-} 0.026, P = 0.000) and Penicillium spp. (MD -0.118 {+/-} 0.043, P = 0.000) compared to CDC patients. Network analysis revealed a positive association between several fungi and bacteria in CDI and CDC, although the analysis did not reveal a direct association between Clostridioides spp. and fungi. Furthermore, the microbiota machine learning model outperformed the models based on the mycobiota and the joint microbiota-mycobiota model. The microbiota classifier successfully distinguished CDI from CDC (AUROC = 0.884) and CDI from Controls (AUROC = 0.905). Blautia and Bifidobacterium were marker genera associated with CDC patients and Controls. ConclusionsThe gut mycobiota differs between CDI, CDC, and Controls, and may affect Clostridioides spp. through indirect interactions. The identification of bacterial marker genera associated with CDC and Controls warrants further investigation. Although the mycobiotas predictive value of C. difficile status was low, fungal-bacterial interactions might be considered when diagnosing and treating C. difficile infection.

microbiology↗

bioRxiv2022

Clostridioides difficile PCR ribotype 151 is polyphyletic and includes pathogenic isolates from cryptic clade C-II with mono-toxin pathogenicity loci that can escape routine diagnostics

We report a patient case with pseudomembranous colitis associated with a mono-toxin producing Clostridioides difficile belonging to the very rarely diagnosed PCR ribotype (RT) 151. The infection was difficult to diagnose, since the isolate and the feces sample tested negative for toxin-encoding genes using a routine commercial test. This prompted us to sequence n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. By including whole genome sequence data from other sources, we could further place these isolates into the phylogenetic tree of C. difficile and assign them to their respective clades. These analyses revealed that 1) RT151s are polyphyletic with isolates falling into clades 1, and cryptic clades C-I and C-II 2) RT151 contains both non-toxigenic and toxigenic isolates and 3) RT151 C-II isolates contained mono-toxin pathogenicity loci (PaLoc). Additional analysis with PacBio circular consensus sequencing revealed that the isolate from our patient case report contains a novel PaLoc insertion site, lacked tcdA and a had significantly divergent tcdB sequence that might explain the failure of the diagnostic test. The study is noteworthy as 1) RT151 encompasses both typical and cryptic clades and 2) conclusive evidence for CDI due to clade C-II isolates was hitherto lacking.

microbiology↗

bioRxiv2022

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile

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 describe a human clinical isolate that carries three 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. Together, our data advance our understanding of C. difficile plasmid biology. HighlightsThe complete circular genome sequence is provided for a C. difficile isolate harboring three plasmids These three plasmids (pJMR5-1, pJMR5-4 and pJRM5-W) are therefore compatible in a single strain Sequence analysis suggest a modular nature of plasmid families to which the pJMR-plasmids belong A functional replicon was cloned from pJMR5-1 (pCD-ECE1 family) and pJMR5-W (pCD-WTSI1 family) and plasmids carrying this replicon are compatible with plasmid pCD630

microbiology↗

bioRxiv2021

Performance of Core Genome Multilocus Sequence Typing Compared to Capillary-Electrophoresis PCR Ribotyping and SNP Analysis of Clostridioides difficile

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary-electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better, but backward compatibility with CE-PCR ribotyping must be assessed. Using a well-characterized collection of diverse strains (N=630; 100 unique ribotypes [RTs]), we aimed to investigate PCR ribotyping prediction from core genome multilocus sequence typing (cgMLST). Additionally, we compared the discriminatory power of cgMLST (SeqSphere & EnteroBase) and whole genome MLST (wgMLST) (EnteroBase) with single nucleotide polymorphism (SNP) analysis). A unique cgMLST profile (>6 allele differences) was observed in 82/100 ribotypes, indicating sufficient backward compatibility. Intra-RT allele difference varied per ribotype and MLST clade. Application of cg/wgMLST and SNP analysis in two outbreak settings with ribotypes RT078 and RT181 (known with a low intra-ribotype allele difference) showed no distinction between outbreak- and non-outbreak strains, in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize and offers higher discrimination. However, in some ribotype complexes adjusted cut-off thresholds and epidemiological data are necessary to recognize outbreaks. We propose to decrease the current threshold of 6 to 3 alleles to better identify outbreaks.

molecular biology↗

bioRxiv2021

Asymptomatic gut colonization by extended-spectrum beta-lactamase-producing Escherichia coli is not associated with an altered gut microbiome or metabolome in Dutch adults

BackgroundGut colonization by antibiotic resistant E. coli strains, including extended-spectrum beta-lactamase (ESBL)-producing E. coli is a risk factor for developing overt infection. The gut microbiome can provide colonization resistance against enteropathogens, but it remains unclear whether it confers resistance against potentially pathogenic ESBL-producing E. coli. MaterialsFrom a Dutch cross-sectional population study (PIENTER-3), feces from 2751 individuals were used to culture ESBL-producing bacteria. Of these, we selected 49 samples which were positive for an ESBL-producing Escherichia coli (ESBL+), and negative for a variety of variables known to affect microbiome composition. These were matched in a 1:1 ratio to ESBL- samples based on age, sex, having been abroad in the past six months and ethnicity. Shotgun metagenomic sequencing was performed and taxonomic species composition and functional annotations (microbial metabolism and carbohydrate-active enzymes) were determined. Targeted quantitative metabolic profiling (1H NMR-spectroscopy) was performed to investigate metabolomic profiles. ResultsNo differences in alpha or beta diversity were observed, nor in relative abundance, between ESBL+ and ESBL- individuals based on bacterial species level composition. Machine learning approaches based on microbiota composition did not accurately predict ESBL status (area under the receiver operating characteristic curve (AUROC)=0.53), neither when based on functional profiles. The metabolome did also not convincingly differ between ESBL groups as assessed by a variety of approaches, including machine learning through random forest (AUROC=0.61). ConclusionUsing a combination of multi-omics and machine learning approaches, we conclude that asymptomatic gut carriage of ESBL-producing E. coli is not associated with an altered microbiome composition or function. This may suggest that microbiome-mediated colonization resistance against ESBL-producing E. coli is not as relevant as it is against other enteropathogens.

microbiology↗

bioRxiv2020

Heme is crucial for medium-dependent metronidazole resistance in clinical isolates of C. difficile

Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile. We find that nearly all isolates demonstrate a heme-dependent increase in the minimal inhibitory concentration for metronidazole, which in some cases leads to isolates being qualified as resistant (MIC > 2 mg/L). Moreover, whole genome sequence analysis reveals a single nucleotide polymorphism in the heme responsive gene hsmA, which defines a metronidazole resistant lineage of PCR ribotype 010 / multilocus sequence type 15 isolates that also includes pCD-METRO containing strains. Together our data demonstrate that heme is crucial for medium-dependent metronidazole resistance in C. difficile.

microbiology↗

bioRxiv2019

Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

When studying the microbiome using next generation sequencing, DNA extraction method, sequencing procedures and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing, and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with maximum three-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for estimating richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs and variation induced by DNA extraction methods was lower than inter-subject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we demonstrate the importance of including negative controls when analyzing low bacterial biomass samples.\n\nIMPORTANCEMethod choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls, and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiome studies. Our methods were not only applied to commonly studied samples for microbiota analysis, e.g. feces, but also for more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g. urine and tumor biopsies) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiome studies, especially when low bacterial biomass is expected.

microbiology↗

bioRxiv2019

Plasmid-mediated metronidazole resistance in Clostridioides difficile

BackgroundMetronidazole is used to treat mild- to moderate Clostridioides difficile infections (CDI). No clear mechanism for metronidazole resistance has been described for C. difficile. A patient treated in the Leiden University Medical Center suffered from recurrent CDI caused by a PCR ribotype (RT) 020 strain which developed resistance to metronidazole (MIC = 8 mg/L). Resistance is also seen in animal isolates, predominantly of RT010.\n\nMethodsSix metronidazole susceptible and 12 metronidazole resistant isolates from human and animal origin, including the patient isolates, were analyzed by whole genome sequence (WGS) analysis. 585 susceptible and resistant isolates collected in various international studies were tested for the presence of plasmid by PCR. Plasmid copy number was determined by quantitative PCR.\n\nFindingsStable metronidazole resistance correlated with the presence of a 7kb plasmid, pCD-METRO. pCD-METRO was not detected in 562 susceptible isolates, but was found in toxigenic and non-toxigenic metronidazole resistant strains from multiple countries (n=22). The introduction of a pCD-METRO-derived vector into a susceptible strain led to a [~]25 fold increase in the metronidazole MIC. The pCD-METRO replicon sustained a plasmid copy number of [~]30, which is higher than currently known replicons for C. difficile.\n\nInterpretationWe describe the first plasmid-mediated resistance to a clinically relevant antibiotic in C. difficile. pCD-METRO is an internationally disseminated plasmid capable of conferring metronidazole resistance in C. difficile, including epidemic ribotypes. Our finding that pCD-METRO may be mobilizable can impact diagnostics and treatment of CDI.\n\nFundingNetherlands Organisation for Scientific Research; Netherlands Center for One Health; European Center for Disease Prevention and Control\n\nResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSOn October 19, 2017, a PubMed search was performed with the terms metronidazole resistance and clostridium OR clostridioides, without language restrictions. A single relevant paper was found describing a strain displaying stable metronidazole resistance not obtained by serial passaging, but no mechanism was identified in this study. On the same day, a PubMed search using terms plasmid and resistance and clostridium difficile OR clostridioides difficile did not yield relevant literature on plasmid-mediated resistance in C. difficile.\n\nAdded value of this studyThis study is the first report of plasmid-mediated resistance in C. difficile, and more generally, the first to ascribe a clinically relevant function to a C. difficile plasmid. Specifically, we report the sequence and annotation of the plasmid pCD-METRO and show that it confers stable resistance to metronidazole, is detected in both toxigenic and non-toxigenic strains of human and animal origin (including epidemic types), is internationally disseminated, is maintained at a higher copy number than characterized C. difficile plasmids and can be acquired horizontally.\n\nImplications of all the available evidenceMetronidazole is widely used as a treatment for mild-to-moderate CDI, though treatment failure occurs in up to [~]30 % of patients. Our data show that carriage of pCD-METRO results in stable metronidazole resistance in C. difficile and suggest that pCD-METRO is mobilizable from an as-of-yet unknown bacterium. Our findings warrant a further investigation into the role of this plasmid in metronidazole treatment failure and the influence of metronidazole use on the international dissemination of pCD-METRO. It also offers an opportunity to improve treatment success and reduce the dissemination of antimicrobial resistance by screening C. difficile isolates or donor fecal material prior to fecal microbiota transplant.

microbiology↗

bioRxiv2019

An in silico survey of Clostridioides difficile extrachromosomal elements

The gram-positive enteropathogen Clostridioides difficile 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 to identify factors that contribute to virulence and pathogenesis. Though extrachromosomal elements such as plasmids are important for these processes in other bacteria, the few characterized plasmids of C. difficile have no relevant functions assigned and no systematic identification of plasmids has been carried out to date. Here, we perform an in silico analysis of publicly available sequence data, to show that [~]13% of all C. difficile strains contain extrachromosomal elements, with 1-6 elements per strain. Our approach identifies known plasmids (e.g. pCD6, pCD630 and cloning plasmids) and 6 novel putative plasmid families. Our study shows that plasmids are abundant and may encode functions that are relevant for C. difficile physiology. The newly identified plasmids may also form the basis for the construction of novel cloning plasmids for C. difficile that are compatible with existing tools.\n\nRepositoriesThe assembled circular type plasmids have been deposited at the European Nucleotide Archive (ENA) under accession numbers ERZ940801 and ERZ940803-ERZ940808.

microbiology↗

bioRxiv2018

Genome location dictates the transcriptional response to sub-inhibitory concentrations of PolC-inhibitors in Clostridium difficile

Clostridium difficile is a potentially lethal gut pathogen that causes nosocomial and community acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA-polymerase of gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E (N2-(3<,4-Dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine; MorE-DCBG) is a DNA polymerase inhibitor in pre-clinical development as a novel therapeutic against C. difficile infection. This synthetic purine shows preferential activity against C. difficile PolC over those of other organisms in vitro and is effective in an animal model of C. difficile infection. In this study we have determined its efficacy against a large collection of clinical isolates. At concentrations below the minimal inhibitory concentration, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response of C. difficile to replication inhibition and observed an overrepresentation of up-regulated genes near the origin of replication in the presence of PolC-inhibitors, but not when cells were subjected to sub-inhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal versus terminus-proximal gene copy number upon exposure to PolC-inhibitors. Moreover, we show that certain genes differentially regulated under PolC-inhibition are controlled by the origin-proximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition in C. difficile.

microbiology↗