Novel Clade C-I Clostridium difficile strains escape diagnostic tests, differ in pathogenicity potential and carry toxins on extrachromosomal elements

Gabriel Ramírez-Vargas,César Rodríguez,Josué Orozco-Aguilar,Adriana Badilla,Diana López-Ureña,Jörg Overmann,Thomas Riedel,Esteban Chaves-Olarte,Tatiana Murillo,Carlos Quesada-Gómez,Priscilla Rojas,Gabriel González

doi:10.1038/s41598-018-32390-6

Abstract

The population structure of Clostridium difficile currently comprises eight major genomic clades. For the highly divergent C-I clade, only two toxigenic strains have been reported, which lack the tcdA and tcdC genes and carry a complete locus for the binary toxin (CDT) next to an atypical TcdB monotoxin pathogenicity locus (PaLoc). As part of a routine surveillance of C. difficile in stool samples from diarrheic human patients, we discovered three isolates that consistently gave negative results in a PCR-based screening for tcdC. Through phenotypic assays, whole-genome sequencing, experiments in cell cultures, and infection biomodels we show that these three isolates (i) escape common laboratory diagnostic procedures, (ii) represent new ribotypes, PFGE-types, and sequence types within the Clade C-I, (iii) carry chromosomal or plasmidal TcdBs that induce classical or variant cytopathic effects (CPE), and (iv) cause different levels of cytotoxicity and hamster mortality rates. These results show that new strains of C. difficile can be detected by more refined techniques and raise questions on the origin, evolution, and distribution of the toxin loci of C. difficile and the mechanisms by which this emerging pathogen causes disease.

Highlights

Clostridium difficile infections (CDI) are characterized by high morbidity and mortality rates and have been traditionally regarded as nosocomial diseases[1]
By PCR we determined that the three isolates carry tcdB and that two of them, HMX-152 and hospitals San Juan de Dios (HSJD)-312, have cdtB (Fig. 1, lower panel)
Mapping Illumina reads to the genome of the reference strain 630 confirmed that HMX-149, HMX-152, and HSJD-312 lack tcdA and tcdC (Fig. 2A) and revealed that the pathogenicity locus (PaLoc) subtypes in HMX-152 and HSJD-312 are closely related

Summary

Introduction

Clostridium difficile infections (CDI) are characterized by high morbidity and mortality rates and have been traditionally regarded as nosocomial diseases[1]. The large clostridial toxins TcdA and TcdB have been traditionally regarded as the main virulence factors of C. difficile These glycosyltransferases are encoded in a so-called pathogenicity locus (PaLoc) along with genes that likely influence their synthesis and secretion[10]. As part of a routine surveillance of C. difficile in TcdB+ stool samples from diarrheic patients, we identified three isolates, termed HMX-149, HMX-152, and HSJD-312, that consistently gave negative results in a PCR-based screening for tcdC This unusual finding motivated us to thoroughly characterize them using phenotypic tests, whole-genome sequencing, protein analyses, and experiments in cell cultures and infection biomodels to confirm whether they belong to the Clade C-I and to gain insight into their biology and pathogenicity

Methods

Results

Conclusion