Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

AbstractClostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB) is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.

Download Full-text

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

PLoS Pathogens ◽

10.1371/journal.ppat.1009181 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009181

Author(s):

Michael J. Mansfield ◽

Benjamin J-M Tremblay ◽

Ji Zeng ◽

Xin Wei ◽

Harold Hodgins ◽

...

Keyword(s):

Selective Advantage ◽

Phylogenomic Analysis ◽

Toxin A ◽

Diagnostic Assays ◽

Surface Patches ◽

Clostridioides Difficile ◽

Distinct Cell ◽

Ncbi Short Read Archive ◽

Toxin Sequence ◽

Entire Sequence

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB) is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.

Download Full-text

Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

Scientific Reports ◽

10.1038/s41598-020-79123-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bryan Angelo P. Roxas ◽

Jennifer Lising Roxas ◽

Rachel Claus-Walker ◽

Anusha Harishankar ◽

Asad Mansoor ◽

...

Keyword(s):

Biofilm Formation ◽

Diarrheal Disease ◽

The United States ◽

Genomic Islands ◽

Rapid Expansion ◽

Phylogenomic Analysis ◽

Community Settings ◽

Hamster Model ◽

Clostridioides Difficile ◽

Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

Download Full-text

Impact of Two-Step Testing Algorithm on Reducing Hospital-Onset Clostridioides difficile Infections

Antimicrobial Stewardship & Healthcare Epidemiology ◽

10.1017/ash.2021.78 ◽

2021 ◽

Vol 1 (S1) ◽

pp. s42-s43

Author(s):

Bhagyashri Navalkele ◽

Wendy Winn ◽

Sheila Fletcher ◽

Regina Galloway ◽

Jason Parham ◽

...

Keyword(s):

Medical Center ◽

Molecular Testing ◽

Toxin A ◽

Clostridioides Difficile ◽

Contact Isolation ◽

Academic Center ◽

Mississippi Medical ◽

True Infection ◽

Testing Algorithm ◽

The University

Clostridioides difficile infection (CDI) is one of the leading causes of hospital–onset infections. Clinically distinguishing true CDI versus colonization with C. difficile is challenging and often requires reliable and rapid molecular testing methods. At our academic center, we implemented a 2-step testing algorithm to help identify true CDI cases. The University of Mississippi Medical Center is a 700+ bed academic facility located in Jackson, Mississippi. Hospital-onset (HO) CDI was defined based on NHSN Laboratory Identified (LabID) event as the last positive C. difficile test result performed on a specimen using a multistep testing algorithm collected >3 calendar days after admission to the facility. HO-CDI data were collected from all inpatient units except the NICU and newborn nursery. HO-CDI outcomes were assessed based on standardized infection ratio (SIR) data. In May 2020, we implemented a 2-step testing algorithm (Figure 1). All patients with diarrhea underwent C. difficile PCR testing. Those with positive C. difficile PCR test were reflexed to undergo enzyme immunoassay (EIA) glutamate dehydrogenase antigen (Ag) testing and toxin A and B testing. The final results were reported as colonization (C. difficile PCR+/EIA Ag+/Toxin A/B−) or true CDI case (C. difficile PCR+/EIA +/Toxin A/B +) or negative (C. difficile PCR−). All patients with colonization or true infection were placed under contact isolation precautions until diarrhea resolution for 48 hours. During the preintervention period (October 2019–April 2020), 25 HO-CDI cases were reported compared to 8 cases in the postintervention period (June 2020–December 2020). A reduction in CDI SIR occurred in the postintervention period (Q3 2020–Q4 2020, SIR 0.265) compared to preintervention period (Q4 2019–Q1 2020, SIR 0.338) (Figure 2). We successfully reduced our NHSN HO-CDI SIR below the national average after implementing a 2-step testing algorithm for CDI. The 2-step testing algorithm was useful for antimicrobial stewardship to guide appropriate CDI treatment for true cases and for infection prevention to continue isolation of infected and colonized cases to reduce the spread of C. difficile spores.Funding: NoDisclosures: None

Download Full-text

Prospective Evaluation of the mariPOC Test for Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxins A/B

Journal of Clinical Microbiology ◽

10.1128/jcm.01872-19 ◽

2020 ◽

Vol 58 (4) ◽

Author(s):

Roosa Savolainen ◽

Juha M. Koskinen ◽

Silja Mentula ◽

Janne O. Koskinen ◽

Suvi-Sirkku Kaukoranta

Keyword(s):

Glutamate Dehydrogenase ◽

Random Access ◽

Pcr Assay ◽

Toxin A ◽

Predictive Values ◽

Content Type ◽

Clostridioides Difficile ◽

Membrane Enzyme ◽

Access Test ◽

B Test

ABSTRACT The objective of this study was to evaluate a novel automated random-access test, mariPOC CDI (ArcDia Ltd., Finland), for the detection of Clostridioides difficile glutamate dehydrogenase (GDH) and toxins A and B directly from fecal specimens. The mariPOC test was compared with both the GenomEra C. difficile PCR assay (Abacus Diagnostica Oy, Finland) and the TechLab C. diff Quik Chek Complete (Alere Inc.; now Abbot) membrane enzyme immunoassay (MEIA). Culture and the Xpert C. difficile assay (Cepheid Inc., USA) were used to resolve discrepant results. In total, 337 specimens were tested with the mariPOC CDI test and GenomEra PCR. Of these specimens, 157 were also tested with the TechLab MEIA. The sensitivity of the mariPOC test for GDH was slightly lower (95.2%) than that obtained with the TechLab assay (100.0%), but no toxin-positive cases were missed. The sensitivity of the mariPOC test for the detection of toxigenic C. difficile by analyzing toxin expression was better (81.6%) than that of the TechLab assay (71.1%). The analytical specificities for the mariPOC and the TechLab tests were 98.3% and 100.0% for GDH and 100.0% and 99.2% for toxin A/B, respectively. The analytical specificity of the GenomEra method was 100.0%. The mariPOC and TechLab GDH tests and GenomEra PCR had high negative predictive values of 99.3%, 98.3%, and 99.7%, respectively, in excluding infection with toxigenic C. difficile. The mariPOC toxin A/B test and GenomEra PCR had an identical analytical positive predictive value of 100%, providing highly reliable information about toxin expression and the presence of toxin genes, respectively.

Download Full-text

Clostridioides difficile Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation

Frontiers in Microbiology ◽

10.3389/fmicb.2020.01998 ◽

2020 ◽

Vol 11 ◽

Author(s):

Conceição S. Martins ◽

Deiziane V. S. Costa ◽

Bruno B. Lima ◽

Renata F. C. Leitäo ◽

Gildênio E. Freire ◽

...

Keyword(s):

Toxin A ◽

Clostridioides Difficile ◽

Pathway Inhibition

Download Full-text

2359. Prospective Feasibility Study for Novel Ultrasensitive Multiplexed Immunoassay for Clostridiodes difficile Toxins A and B

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2037 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S812-S813

Author(s):

Lauren Watson ◽

Michele L Zimbric ◽

Catherine Shaughnessy ◽

Shraddha Kale ◽

Jeff Debad ◽

...

Keyword(s):

Diagnostic Tests ◽

Standard Of Care ◽

Positive Sample ◽

Nucleic Acid Amplification ◽

Serum Samples ◽

Toxin A ◽

Toxin B ◽

Diagnostic Assays ◽

Stool Samples ◽

Multiplexed Immunoassay

Abstract Background The diagnosis of Clostridiodes difficile infection is challenging. A wide array of diagnostic tests are used in practice; however, each available test has important limitations. We examined the feasibility and analytical performance of a novel ultrasensitive multiplexed immunoassay designed by Meso Scale Diagnostics (MSD) compared with five current diagnostic assays for detection of C. difficile toxin A and B. Methods Stool, serum and urine samples from 44 admitted inpatients were collected within 72 hours of a standard of care nucleic acid amplification test (NAAT) result (23 positive, 21 negative). These specimens underwent five standard diagnostic assays: enzyme immunoassay for toxins A and B (EIA), cytotoxin cell assay, bacterial culture isolation, and two different NAATs to determine presence of viable C. difficile cells, toxins, and toxin-encoding genes (Table 1). The concentration (fg/mL) of toxin A and toxin B in all stool samples was then quantified using MSD’s multiplexed immunoassay (Table 1). Results At least one of the five standard diagnostic tests for C. difficile was positive in 16 of the 23 clinically positive patients. The MSD multiplex immunoassay detected toxin A and/or toxin B in 15 of these 16 samples and quantified low levels of toxin A in one clinically positive sample that was negative for all other tests. In contrast, only 2 of the 16 positive samples were positive by EIA, demonstrating the benefits of the ultrasensitive assay over standard immunoassay methods. All clinically negative specimens were negative in all tests. Toxin detection in urine and serum samples was negligible. In stool samples, the MSD test had an estimated sensitivity of 93% (95% CI: 70–99%) and specificity of 93% (95% CI: 78–98%) compared with the clinically used NAAT. Conclusion The MSD multiplex toxin assay is a feasible test to move forward for further evaluation. Ultimately, future studies should examine the performance of this test compared with standard of care in a prospective randomized trial assessing clinical outcomes. Disclosures All authors: No reported disclosures.

Download Full-text

Proteomic profiling of precipitated Clostridioides difficile toxin A and B antibodies

Vaccine ◽

10.1016/j.vaccine.2019.10.096 ◽

2020 ◽

Vol 38 (8) ◽

pp. 2077-2087

Author(s):

Penelope J. Adamson ◽

Jing J. Wang ◽

Natalie G. Anosova ◽

Alex D. Colella ◽

Timothy K. Chataway ◽

...

Keyword(s):

Proteomic Profiling ◽

Toxin A ◽

Clostridioides Difficile

Download Full-text

Characterization of a clinical Clostridioides difficile isolate with markedly reduced fidaxomicin susceptibility and a V1143D mutation in rpoB

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dky375 ◽

2018 ◽

Vol 74 (1) ◽

pp. 6-10 ◽

Cited By ~ 9

Author(s):

Julian Schwanbeck ◽

Thomas Riedel ◽

Friederike Laukien ◽

Isabel Schober ◽

Ines Oehmig ◽

...

Keyword(s):

Single Molecule ◽

Sequence Data ◽

Normal Growth ◽

Amplicon Sequencing ◽

Spore Formation ◽

Compensatory Mechanisms ◽

Toxin A ◽

Clostridioides Difficile

Abstract Objectives The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility. Methods Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630Δerm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility. Results Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64 mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630Δerm, this mutation decreased fidaxomicin susceptibility (MIC >64 mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate. Conclusions To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

Download Full-text

The Clostridioides difficile species problem: global phylogenomic analysis uncovers three ancient, toxigenic, genomospecies

10.1101/2020.09.21.307223 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel R. Knight ◽

Korakrit Imwattana ◽

Brian Kullin ◽

Enzo Guerrero-Araya ◽

Daniel Paredes-Sabja ◽

...

Keyword(s):

Species Boundaries ◽

Toxin Gene ◽

Phylogenomic Analysis ◽

Species Problem ◽

Bayesian Analyses ◽

Clostridioides Difficile ◽

Ecological Versatility ◽

Gene Architecture ◽

Close Relatives ◽

Global Population

AbstractClostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work impacts the diagnosis of CDI worldwide.

Download Full-text

Clostridium difficile

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0128 ◽

2020 ◽

pp. 1115-1120

Author(s):

David W. Eyre ◽

Mark H. Wilcox

Keyword(s):

Clostridium Difficile ◽

Gut Microbiome ◽

Severe Disease ◽

Toxin A ◽

Vegetative Cells ◽

Clostridioides Difficile ◽

Colonic Flora ◽

Healthcare Environments ◽

Poor Outcomes ◽

Colonization Rates

Clostridium difficile (recently renamed as Clostridioides difficile) is a Gram-positive spore-forming anaerobic bacillus that is ubiquitous in nature, and particularly common in healthcare environments. Its spores are part of the colonic flora in about 2–3% of healthy adults, with colonization rates increasing, typically up to 10–20%, during hospitalization. Disease occurs when the organism shifts from quiescent spores to replicating vegetative cells with toxin (A and B) production; this happens when there is inhibition of the resident colonic flora (gut microbiome) by prescribed antibiotics, although cases can occur when no such precipitant is identified. C. difficile infection is now recognized as the most important bacterial enteric pathogen in wealthier countries, epidemics, and outbreaks of which are common, most notoriously now due to the ribotype 027 (NAP-1) strain that is associated with more severe disease and poor outcomes.

Download Full-text