Emergence of a non-sporulating secondary phenotype in Clostridium (Clostridioides) difficile ribotype 078 isolated from humans and animals

Abstract

Clostridium (Clostridioides) difficile is a Gram positive, spore forming anaerobic bacterium that is a leading cause of antibiotic associated diarrhoea in the developed world. C. difficile is a genetically diverse species that can be divided into 8 phylogenetically distinct clades with clade 5 found to be genetically distant from all others. Isolates with the PCR ribotype 078 belong to clade 5, and are often associated with C. difficile infection in both humans and animals. Colonisation of animals and humans by ribotype 078 raises questions about possible zoonotic transmission, and also the diversity of reservoirs for ribotype 078 strains within the environment. One of the key factors which enables C. difficile to be a successful, highly transmissible pathogen is its ability to produce oxygen resistant spores capable of surviving harsh conditions. Here we describe the existence of a non-sporulating variant of C. difficile ribotype 078 harbouring mutations leading to premature stop codons within the master regulator, Spo0A. As sporulation is imperative to the successful transmission of C. difficile this study was undertaken to investigate phenotypic characteristics of this asporogenous phenotype with regards to growth rate, antibiotic susceptibility, toxin production and biofilm formation.

Figure 1

(A) Non sporulating variant of C. difficile 078 emerging from the wild type culture after 7 days incubation at 37 °C under anaerobic conditions on FAABL. (B) Both colony variants can be sub-cultured independently after initial separation (24 h incubation at 37 °C under anaerobic conditions on FAABL). Wild type cells give rise a small, rough, white colony type whist the non-sporulating variants produce much larger, smooth, grey colonies.

Figure 2

(A) Spore-stain image of H^s isolate at x 1000 magnification. (B) Spore-stain image of H^ns (non-sporulating) secondary phenotype at x 1000 magnification. Spores are identified by observation of green staining, while vegetative cells are red/pink.

Figure 3

Recovery of spores after alcohol shock with 70% (v/v) ethanol for 1 h at room temperature compared to the control, CFU obtained without alcohol shock, (C). (i) A^s isolate, (ii) A^ns isolate, (iii) H^s isolate, (iv) H^ns isolate.

Figure 4

Growth of A^s, A^ns, H^s and H^ns in BHI over 24 h at 37 °C under anaerobic conditions. A) A^s (o) and A^ns (●), B) H^s (□) and H^ns (■).

Figure 5

Biofilm formation of each isolate measured via Crystal Violet assay. Cultures were grown statically for 6 days in BHI under anaerobic conditions at 37 °C.

Figure 6

Maximum toxin production of each phenotype for animal and human isolates. Isolates were grown in BHI media under anaerobic conditions at 37 °C. Sampling began at 14.5 h after inoculation with samples taken every 3 h thereafter, for a total of 24 h: Maximum toxin production was observed at 14.5 h.

Figure 7

Motility of each isolate in BHI 0.175% agar for 24 h at 37 °C under anaerobic conditions. (A) Animal isolates: A^s and A^ns, (B) Human isolates: H^s and H^ns.

Figure 8

Susceptibility to metronidazole and vancomycin measured through zone of inhibition (mm). Isolates were grown anaerobically at 37 °C on FAABL for 24 h in the presence of antimicrobial discs containing 5 μg of (A) Metronidazole, (B) Vancomycin.

Figure 9

Alignment of sequences of spo0A for each isolate. M120 – reference 078, A^s- animal isolate phenotype 1, A^ns - animal isolate phenotype 2 (with stop codon at the 7^th codon- x), H^s - human isolate phenotype 1, H^ns - human isolate phenotype 2 with stop codon at the 162^nd codon - x).