Biology subjects

Smits, W. K.

Publications and source records attributed to Smits, W. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of novel cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called post-glycosylation modifications (PGMs) often resulting in exclusive molecular structures. In Clostridioides difficile a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-{beta}-D-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase, that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar assembly.

microbiology↗

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

Antimicrobial susceptibility testing of Clostridioides difficile: a dual-site study of three different media and three therapeutic antimicrobials

ObjectivesIncreasing resistance to antimicrobials used for the treatment of Clostridioides difficile infections necessitates reproducible antimicrobial susceptibility testing. Current guidelines take a one-size-fits-all approach and/or offer limited guidance. We investigated how the choice of medium affects measured MIC values across two sites. MethodsWe determined MIC values for the antimicrobials fidaxomicin, metronidazole and vancomycin for a representative collection of European C. difficile strains (n=235) using agar dilution on three different media: Brucella Blood Agar (BBA), Fastidious Anaerobe Agar supplemented with horseblood (FAA-HB) and Wilkins-Chalgren (WC) agar. The study was conducted at two sites to compare reproducibility. Useability (ease of preparation of the media as well as read-out of the assay) was assessed through a survey. ResultsWe found that all media result in highly consistent aggregated MIC data for all antibiotics, with MIC50 and MIC90 within 2-fold of each other across sites. For fidaxomin, MIC values on WC were lower than on the other media. Metronidazole showed the lowest MIC on BBA, and the highest on WC. For vancomycin, there was little difference between media. Though absolute values for individual isolates differed between sites, identified resistant isolates were similar. Results obtained on FAA-HB were most consistent between sites and results obtained on WC showed the most divergence. FAA-HB was positively evaluated in the usability survey. ConclusionsThis study shows medium-dependent differences in C. difficile MICs for at least two antimicrobials across two sites. We suggest the use of FAA-HB to align with general EUCAST recommendations for susceptibility testing of anaerobic bacteria and deposited reference strains for standard susceptibility testing of C. difficile to increase interlaboratory reproducibility.

microbiology↗

bioRxiv2024

Elucidation of the glycan structure of the b-type flagellin of Pseudomonas aeruginosa PAO1

Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin, often undergoes post-translational modifications, with glycosylation being a common occurrence. In Pseudomonas aeruginosa PAO1, the b-type flagellin is O-glycosylated with a structure that includes a rhamnose, a phospho-group and a previous unknown moiety. This structure resembles the well-characterized glycan (Type A) in Clostridioides difficile strain 630, which features an N-acetylglucosamine linked to an N-methylthreonine via a phosphodiester bond. This study aimed to characterize the b-type glycan structure in Pseudomonas aeruginosa PAO1 using a set of mass spectrometry experiments. For this purpose, we used wildtype P. aeruginosa PAO1 and several gene mutants from the b-type glycan biosynthetic cluster. Moreover, we compared the mass spectrometry characteristics of the b-type glycan with those of in vitro modified Type A-peptides from C. difficile strain 630{Delta}erm. Our results demonstrate that the thus far unknown moiety of the b-type glycan in P. aeruginosa consists of an N,N-dimethylthreonine. These data allowed us to refine our model of the flagellin glycan biosynthetic pathway in both P. aeruginosa PAO1 and C. difficile strain 630.

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↗

bioRxiv2024

Proteolytic activity of surface exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile

To survive in the host, pathogenic bacteria need to be able to react to the unfavourable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

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

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

Phosphorylation is a post-translational modification that can affect both house-keeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile the extent and nature of phosphorylation events is poorly characterized, though a protein-kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of stationary growth phase. Among the overall more than 1500 phosphosites, our approach identifies expected targets and phosphorylation sites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV) and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. ImportanceIn this manuscript, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in regulation in this organism has been published; the current study is expected to form the basis for research on this post-translational modification in C. difficile.

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

Practical observations on the use of fluorescent reporter systems in C. difficile

ObjectivesFluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. MethodsWe performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. ResultsWe show that the intrinsic fluorescence of C. difficile cells increases during growth, independent from sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. ConclusionsThe present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/448366v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@a19b90org.highwire.dtl.DTLVardef@1ec1db9org.highwire.dtl.DTLVardef@939510org.highwire.dtl.DTLVardef@11f623f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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↗

bioRxiv2020

Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

In many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B ({sigma}B). In C. difficile {sigma}B has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-{sigma}B antibody to demonstrate time-limited overproduction of {sigma}B in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced {sigma}B gene expression. Inducible {sigma}B overproduction uncouples {sigma}B expression from its native regulatory network and allowed for the refinement of the previously proposed {sigma}B regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile {sigma}B was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to {sigma}B-dependent gene activation. ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of {sigma}B by evaluating the transcriptional effects of {sigma}B overproduction, provide biochemical evidence of direct transcriptional activation by {sigma}B, and show that {sigma}B-dependent genes can be activated by antimicrobials. Together our data suggest that {sigma}B is a key player in dealing with toxic radicals.

microbiology↗

bioRxiv2019

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold. In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors. ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor. Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain. Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

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↗