Identification of novel, cryptic Clostridioides species isolates from environmental samples collected from diverse geographical locations

Abstract

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile . At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioide s species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB, were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile , and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates (n=5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates (n=4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides . These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.

Clostridioides difficilePCR ribotype 151 is polyphyletic and includes pathogenic isolates from cryptic clade C-II with mono-toxin pathogenicity loci that can escape routine diagnostics

“…We do note that positive identifications were made using the ECDC-endorsed multiplex PCR (relating to little or no mismatches in the primer sequence compared to the genome). A recent report investigating C. difficile isolates from environmental locations showed several atypical PaLoc locations in cryptic clade isolates and in silico PCR further suggested that routine diagnostic assays would return highly variable results if such isolates would be encountered in the clinic (3), which is in line with what we found in the current study. This underscores the potential of these isolates to be OneHealth pathogens.…”

“…Altogether, reports of cryptic clade isolates that cause human CDI are still very rare. With the increasing rate of community-onset CDI 39 and the discovery that cryptic clade isolates with atypical PaLoc configurations are widespread in the environment 3 , it is very likely that other CDI cases caused by isolates from this cryptic clade occur but are missed by standard diagnostics if only PCR is used. S1.…”

“…Phylogenetic analyses have revealed that most C. difficile isolates associated with human and animal disease fall within 5 clades 2 . In recent years, reports have appeared on (mainly environmental) isolates that are phylogenetically distinct from these 5 clades, but rather fall within at least 3 so-called cryptic clades (C-I, C-II and C-III) that can be considered a separate genomospecies [2][3][4][5] . Though cryptic clade isolates can contain toxin genes 2,4,6 , knowledge about the occurrence and clinical significance of these isolates is limited.…”

“…3 Numbers indicate positive (1) or negative (0) for 16S, gluD, tcdA , tcdB and cdt (in that order). For reference see main manuscript 4.…”

Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile

“…Water and soil are also an important reservoir for this pathogen. At the Flagstaff site (Arizona), researchers found potential novel strains belongings to clades C-I, C-II and C-III and a hypothetical additional clade (C-V) [ 124 ].…”