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Anaerobe

Volume 69, June 2021, 102355
Anaerobe

Clostridioides difficile (including epidemiology)
Microbiological features, epidemiology, and clinical presentation of Clostridioides difficile strains from MLST Clade 2: A narrative review

https://doi.org/10.1016/j.anaerobe.2021.102355Get rights and content

Highlights

  • The C. difficile MLST Clade 2 has been regarded as the “hypervirulent” clade because it includes the NAP1/RT027/ST01 strain.

  • The 83 additional sequence types (STs) that compose the clade have received much less attention.

  • There is a profound lack of (meta)data for nearly two thirds of all known Clade 2 STs.

  • When available, phenotypic data reveals large differences across clade members and isolates from the same ST.

  • Robust evidence is lacking to conclude that hypervirulence is a widespread feature of this clade.

Abstract

Clostridioides difficile is an emerging One Health pathogen and a common etiologic agent of diarrhea, both in healthcare settings and the community. This bacterial species is highly diverse, and its global population has been classified in eight clades by multilocus sequence typing (MLST). The C. difficile MLST Clade 2 includes the NAP1/RT027/ST01 strain, which is highly recognized due to its epidemicity and association with severe disease presentation and mortality. By contrast, the remaining 83 sequence types (STs) that compose this clade have received much less attention. In response to this shortcoming, we reviewed articles published in English between 1999 and 2020 and collected information for 27 Clade 2 STs, with an emphasis on STs 01, 67, 41 and 188/231/365. Our analysis provides evidence of large phenotypic differences that preclude support of the rather widespread notion that ST01 and Clade 2 strains are “hypervirulent”. Moreover, it revealed a profound lack of (meta)data for nearly 70% of the Clade 2 STs that have been identified in surveillance efforts. Targeted studies aiming to relate wet-lab and bioinformatics results to patient and clinical parameters should be performed to gain a more in-depth insight into the biology of this intriguing group of C. difficile isolates.

Introduction

Clostridioides difficile is a Gram-positive, strictly anaerobic, spore-forming bacterium, and a leading cause of antibiotic-associated diarrhea worldwide [1,2]. C. difficile infection (CDI) is defined by the presence of diarrhea, which varies from mild to severe, and either by detection of C. difficile toxins in stool samples or pseudomembranous colitis in a colonoscopy or histopathologic studies [3]. Other potential complications of CDI include toxic megacolon or even death [4].

CDI typically develops among hospital patients with risk factors beyond receiving antibiotic therapy, such as older age, exposure to health care centers, and comorbidities such as inflammatory bowel disease and immunodeficiency, among others [5]. However, the epidemiology of CDI is changing, and a growing number of community-acquired (CA) cases have been seen among individuals considered to be at low-risk, including young adults and children [6]. Furthermore, many CDI cases now show more severe symptoms and require more prolonged hospitalizations; hence the burden of this disease on health systems continues to increase [5,7].

The major virulence factors of C. difficile are toxins TcdA and TcdB, which belong to the large clostridial glycosylating toxin family. Both toxins play a role in symptom development [8,9]. Nonetheless, as demonstrated by assays in animal models, only TcdB seems to be essential for virulence [10]. Other proteins, such as the surface layer protein (SlpA), cell wall protein 84 (Cwp84), flagellar components, and the binary toxin CDT, have also been shown to play roles in CDI pathogenesis [11,12].

Comparative analyses of global collections of C. difficile whole genome sequences have demonstrated that TcdB is much more diverse than TcdA (12 vs. 7 protein sequence subtypes), possibly as a result of a higher mutation rate or distinct recombination and lateral gene transfer events in the former protein [13,14]. Both genes appear to be under purifying selection, although tcdB shows more positively selected sites [13,14].

TcdB variants differ in their biological features, immunoactivities, and potential pathogenicity, and this heterogeneity correlates with the severity of clinical outcomes during CDI progression. Depending on the sequence of its glycosyltransferase domain (GTD), which influences toxins’ functionalities and target affinities, TcdB can induce two types of cytopathic effects (CPE): a classical, arborizing, CPE in which cells develop neurite-like protrusions and remain attached to cell culture plates [15], or a TcsL-like effect that is characterized by cell rounding and clumping and surface detachment [15,16].

Together with polymorphisms in toxin alleles, strain differences in virulence have been traced to antimicrobial resistance patterns [17], spore production and germination capabilities [18], and recently to phase-variable signal transduction systems [19], c-di-GMP levels [20], and the production of alarmones [21], among other factors.

The high diversity that distinguishes this species has encouraged the implementation of various typing techniques, some of which have proven useful in epidemiological investigations and the identification of strains with unique biological properties [22,23].

The first C. difficile typing techniques were based on phenotypic traits. However, they suffered from low discriminatory power and reproducibility and were replaced by genotypic approaches.

Genotypic typing methods can be classified according to the nature of their targets and as to whether they are band- or sequence-based (Table 1). Whereas pulsed-field gel electrophoresis (PFGE) is frequently used in North America, PCR ribotyping is the most frequently used C. difficile genotyping method in Europe. Multilocus sequence typing (MLST) is more suitable for evolutionary studies, outbreak detection, and transmission or population structure studies [24].

MLST allows isolate discrimination through sequencing of 405–500 bp DNA fragments of seven or eight housekeeping genes (for C. difficile, seven gene fragments, total length 3501 bp). The obtained sequences are compared with reference sequences uploaded to internet-accessible MLST databases (https://pubmlst.org/organisms/clostridioides-difficile/) and used to generate allelic profiles composed of allele numbers assigned to sequence variants of a given locus. Each unique allele profile is assigned a sequence type number that is stored in the database to facilitate interlaboratory comparison [23,24].

As indicated by MLST, the known population of C. difficile can be distributed in eight clades (Clades 1 to 5, plus Clades C-I, C-II, and C-III) [23,25]. Whereas isolates from Clades 1-5 are more often associated with humans [26,27], the so-called cryptic Clades C-I to C-III mainly include non-toxigenic isolates from the environment [25] and, to a lesser extent, toxigenic strains implicated in CDI [30]. New STs with different host affinities, ecological adaptations, and virulence potentials are continually being identified.

According to a globally-optimized eBURST cluster analysis [28] showed herein, the C. difficile MLST Clade 2 comprises 84 STs distributed in 15 clonal complexes (Fig. 1, Fig. 2). It includes the highly recognized NAP1/027/ST01 strain, and for this reason, it has been repeatedly referred to as the “hypervirulent clade” [29,30] although the virulence and pathogenicity potential of most non-NAP1/027/ST01 strains are poorly understood or unknown. To corroborate whether increased virulence is indeed a widespread trait among members of this clade, we compared in this review microbiological, epidemiological and, clinical features of various C. difficile Clade 2 sequence types (STs).

We collected scientific articles published between 1999 and 2020 in English from the Google Scholar, JSTOR, Scopus, and PubMed databases using keywords such as “C. difficile MLST”, “C. difficile Clade 2”, “C.difficile ST01”, “C.difficile ST67”, “C.difficile ST41”, “C. difficile typing techniques”, “C. difficile transmission”, “C.difficile antibiotic-resistance”, “C. difficile toxins”, “C.difficile emergent sequence types”. A total of 31 publications were selected to elaborate elementary aspects of C. difficile, including its main virulence factors, CDI pathogenesis, epidemiology, typing techniques and, phylogenetic classification. The number of publications obtained for each ST differed, with a marked predominance of studies on the ST01 strain (n = 40). This figure was followed by papers on ST67 and ST41 (ca. 20 publications for each one). Information for STs 188/231/365 was even more scarce (less than 10 publications). A few STs, such as ST62, ST95, ST97, ST114, ST123, ST154, ST192, ST223, ST229, ST264, and ST461-464, have only been analyzed with regards to their virulence factors and origins. No information was found for 55 Clade 2 STs.

Section snippets

Microbiological features

ST01 is by far the best-described Clade 2 member. ST01 strains have been reported to overproduce toxins TcdA and TcdB subtype 2 (TcdB2) in vitro [13,31,32], arguably as a result of an 18bp deletion and a single-base-pair deletion at position 117 in the gene for the transcriptional regulator TcdC [32,33]. Strains from this ST have been linked to 3- to 23-fold higher titers than those of the main hospital strains, according to results derived from Swedish [34] and Canadian studies [35]. However,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank Ricardo Gutiérrez, Enzo Guerrero, Jonathan Huffman, and William Huffman for their comments and suggestions to early versions of the manuscript. This work was supported by the Vicerectory of Research of the University of Costa Rica, grant B7606.

References (100)

  • M. Mac Aogáin et al.

    Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile

    J. Glob. Antimicrob. Resist.

    (2015)
  • F. Hidalgo-Villeda et al.

    Diversity of multidrug-resistant epidemic Clostridium difficile NAP1/RT027/ST01 strains in tertiary hospitals from Honduras

    Anaerobe

    (2018)
  • A. Cheknis et al.

    Molecular epidemiology of Clostridioides (Clostridium) difficile strains recovered from clinical trials in the US, Canada and Europe from 2006-2009 to 2012-2015

    Anaerobe

    (2018)
  • M.J.T. Crobach et al.

    European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection

    Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis.

    (2016)
  • R. Herbert et al.

    Two-year analysis of Clostridium difficile ribotypes associated with increased severity

    J. Hosp. Infect.

    (2019)
  • K.M. Tamez-Torres et al.

    Impact of Clostridium difficile infection caused by the NAP1/RT027 strain on severity and recurrence during an outbreak and transition to endemicity in a Mexican tertiary care center

    Int. J. Infect. Dis.

    (2017)
  • H. Cao et al.

    Genomic investigation of a sequence type 67 Clostridium difficile causing community-acquired fulminant colitis in Hong Kong

    Int. J. Med. Microbiol. IJMM.

    (2019)
  • G.Z. Saldanha et al.

    Genetic relatedness, virulence factors and antimicrobial resistance of C. difficile strains from hospitalized patients in a multicentric study in Brazil

    J. Glob. Antimicrob. Resist.

    (2020)
  • M. Krutova et al.

    The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027

    J. Microbiol. Immunol. Infect.

    (2018)
  • D.A. Collins et al.

    Community-associated Clostridium difficile infection in emergency department patients in Western Australia

    Anaerobe

    (2017)
  • A.C. Cheng et al.

    Laboratory-based surveillance of Clostridium difficile circulating in Australia, september - november 2010

    Pathology

    (2016)
  • V. Tkalec et al.

    High Clostridium difficile contamination rates of domestic and imported potatoes compared to some other vegetables in Slovenia

    Food Microbiol.

    (2019)
  • S. Hong et al.

    Phenotypic characterisation of Clostridium difficile PCR ribotype 251, an emerging multi-locus sequence type clade 2 strain in Australia

    Anaerobe

    (2019)
  • M.C. Wehrhahn et al.

    A series of three cases of severe Clostridium difficile infection in Australia associated with a binary toxin producing clade 2 ribotype 251 strain

    Anaerobe

    (2019)
  • D.A. Collins et al.

    Laboratory-based surveillance of Clostridium difficile strains circulating in the Australian healthcare setting

  • A.N. Diniz et al.

    Molecular epidemiology of Clostridioides (previously Clostridium) difficile isolates from a university hospital in Minas Gerais, Brazil

    Anaerobe

    (2019)
  • C. Quesada-Gómez et al.

    Proteogenomic analysis of the Clostridium difficile exoproteome reveals a correlation between phylogenetic distribution and virulence potential

    Anaerobe

    (2020)
  • J. Cloud et al.

    Clostridium difficile strain NAP-1 is not associated with severe disease in a nonepidemic setting

    Clin. Gastroenterol. Hepatol. Off. Clin. Pract. J. Am. Gastroenterol. Assoc.

    (2009)
  • K.A. Bauer et al.

    Impact of the NAP-1 strain on disease severity, mortality, and recurrence of healthcare-associated Clostridium difficile infection

    Anaerobe

    (2017)
  • S. Bletz et al.

    Defining and evaluating a core genome multilocus sequence typing scheme for genome-wide typing of Clostridium difficile

    J. Clin. Microbiol.

    (2018)
  • M.J.T. Crobach et al.

    Understanding Clostridium difficile colonization

    Clin. Microbiol. Rev.

    (2018)
  • S.H. Cohen et al.

    Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA)

    Infect. Control Hosp. Epidemiol.

    (2010)
  • S. Tratulyte et al.

    First genotypic characterization of toxigenic Clostridioides difficile in Lithuanian hospitals reveals the prevalence of the hypervirulent ribotype 027/ST1

    Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol.

    (2019)
  • S.M. Vindigni et al.

    Changing epidemiology and management paradigms

    Clin. Transl. Gastroenterol.

    (2015)
  • S. Khanna et al.

    The epidemiology of community-acquired Clostridium difficile infection: a population-based study

    Am. J. Gastroenterol.

    (2012)
  • E. Balsells et al.

    Global burden of Clostridium difficile infections: a systematic review and meta-analysis

    J. Glob. Health.

    (2019)
  • K. Aktories et al.

    Clostridium difficile toxin biology

    Annu. Rev. Microbiol.

    (2017)
  • D. Lyras et al.

    Toxin B is essential for virulence of Clostridium difficile

    Nature

    (2009)
  • A.M. Hoegh et al.

    A multiplex, internally controlled real-time PCR assay for detection of toxigenic Clostridium difficile and identification of hypervirulent strain 027/ST-1

    Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol.

    (2012)
  • M.M. Awad et al.

    Clostridium difficile virulence factors: insights into an anaerobic spore-forming pathogen

    Gut Microb.

    (2014)
  • E. Shen et al.

    Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B

    Commun. Biol.

    (2020)
  • M.J. Mansfield et al.

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

    BioRxiv

    (2020)
  • Z. Peng et al.

    Antibiotic resistance and toxin production of Clostridium difficile isolates from the hospitalized patients in a large hospital in Florida

    Front. Microbiol.

    (2017)
  • E.M. Garrett et al.

    Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence

    PLoS Biol.

    (2019)
  • E.B. Purcell et al.

    Cyclic diguanylate signaling in Gram-positive bacteria

    FEMS Microbiol. Rev.

    (2016)
  • C.W. Knetsch et al.

    Current application and future perspectives of molecular typing methods to study Clostridium difficile infections

    Euro Surveill. Bull. Eur. Sur Mal. Transm. Eur. Commun. Dis. Bull.

    (2013)
  • D. Griffiths et al.

    Multilocus sequence typing of Clostridium difficile

    J. Clin. Microbiol.

    (2010)
  • S. Janezic et al.

    Highly divergent Clostridium difficile strains isolated from the environment

    PloS One

    (2016)
  • R.A. Stabler et al.

    Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations

    PloS One

    (2012)
  • A.P. Francisco et al.

    Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach

    BMC Bioinf.

    (2009)
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