Esculin hydrolysis negative and TcdA-only producing strains of Clostridium (Clostridioides) difficile from the environment in Western Australia

Abstract

Background and aims: Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β-glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies.

Methods and results: A total of 17 environmental isolates of C. difficile from garden soil and compost, and gardening shoe soles in Perth, WA, failed to produce black colonies on ChromID agar. MALDI-TOF MS analysis confirmed these strains as C. difficile. Four strains contained only a tcdA gene (A⁺ B^- CDT^- ) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low-level resistance to clindamycin (MIC = 8 mg/L). The four tcdA-positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β-glucosidase gene by PCR. Whole-genome sequencing showed the 17 strains belonged to novel multi-locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C-III. Four isolates carried a full-length tcdA but not tcdB nor binary toxin genes.

Conclusions: ChromID C. difficile agar is used for the specific detection of C. difficile in the samples. To date, all strains except RT 023 strains from clinical samples hydrolyse esculin. This is the first report to provide insights into the identification of esculin hydrolysis negative and TcdA-only producing (A⁺ B^- CDT^- ) strains of C. difficile from environmental samples.

Significance and impact of the study: White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false-negative results, however, whether these strains are truly pathogenic remains to be proven.

Keywords: Clostridium (Clostridioides) difficile; clade-III; environment; esculin hydrolysis negative; putative β glucosidase gene; toxin A variant; whole-genome sequencing.

FIGURE 1

C. difficile colonies on C. difficile ChromID agar. Standard black colonies produced by esculin hydrolysis‐positive strains of C. difficile clade 1 (RT012), clade 2 (RT027), clade 4 (RT017), clade 5 (RT078) and garden strain^‡ RT010, on ChromID. Colourless colonies produced by esculin hydrolysis‐negative strains of C. difficile clade 3 (RT023) and garden strain QX597

FIGURE 2

Growth kinetics of esculin hydrolysis‐negative and reference strains. The growth of strain clos di 21 (RT023), VPI10463 (RT087), CD630 (RT012), VPI11186 (RT038) and esculin hydrolysis strains was measured by OD₆₀₀ up to 36 h

FIGURE 3

Global phylogenetic context of seven novel sequence types. MLST phylogeny based on concatenated allele sequences for seven novel sequence types (cryptic clade C‐III) and well‐characterized representatives of MLST clade 1 (ST54, RT012), clade 2 (ST1, RT027), clade 3 (ST22, RT023), clade 4 (ST37, RT017), clade 5 (ST11, RT078), as well as other cryptic clades C‐I (ST360) and C‐II (ST200). The scale shows the number of substitutions per site

FIGURE 4

PaLoc structure of tcdA‐positive isolates of C. difficile from environmental samples. Mono‐toxin PaLoc of environmental isolates (e.g. HGP14) shared synteny and a high nucleotide sequence similarity to the PaLoc of clinical isolate RA09‐70 (clade 5)