Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective

Abstract

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3′)-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven “hypervirulent” RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

Design and Development of Molecular Beacon-Based Real-Time PCR Assays to Identify Clostridioides difficile Types of Main Evolutionary Clades

“…In the last decade, CDI cases have increased worldwide, particularly in the community [4,5,[13][14][15]. Whole genome sequencing (WGS) has shown that C. difficile strains from humans, animals, food, and the environment may be genetically closely related or indistinguishable, and that certain types of C. difficile are potentially transmitted between animals and humans [3,4,9,17,[23][24][25]28,29,31,33,57,60,63]. For these reasons, genetic analysis has been increasingly associated with a One Health approach to CDI for a better control and prevention of this infection [4,17,33,60,61].…”

“…More recently, RT 023 has emerged and spread in Europe and United Kingdom (UK) [12]. This RT is rarely detected in hospitals and is more frequently isolated from CA-CDI cases, animals, and environments [9,15,23].…”

Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile

“…All contig sequences were deposited to NCBI GenBank under BioProject No. PRJNA1011814 [ 22 ]. The accession numbers for the sequences of contigs are listed in Table S1 .…”

“…Different strains of C. difficile were recovered from diverse environmental sources, as previously described [49]. After identification and phenotypic characterisation, 166 C. difficile isolates were subjected to WGS, and further phylogenetic and epidemiological characterisation revealed a clear relationship between those C. difficile strains [22]. MLST Sequence Types (STs) were extracted in accordance with the C. difficile MLST database of the PubMLST website (https://pubmlst.org/organisms/clostridioides-difficile, accessed on 15 November 2022).…”

“…In this study, the whole genomes of 166 environmental C. difficile isolates from diverse environmental samples were sequenced and annotated as previously described [ 22 ] and used as a source for exploiting the prevalence and genetic diversity of integrated prophages in their genomes. Additionally, the predicted prophages could be correlated with the well-described sequence types (STs) and RTs of those environmental C. difficile strains.…”