Biology subjects

Harmanus, C.

Publications and source records attributed to Harmanus, C..

9 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

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↗

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↗

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

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↗

bioRxiv2017

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

microbiology↗