Table 1.
Sequence type, ribotyping, year of isolation, and presence of the main toxin genes in the three Brazilian clade 2 strains isolated between 2011 and 2015 in Belo Horizonte, Minas Gerais.
Table 2.
Animal species samples (n = 544) included for Clostridioides (Clostridium) difficile detection between February 2018 and January 2020 at the laboratory of anaerobic bacteria from the veterinary university of Minas Gerais.
Fig 1.
(A) A/B toxin production was measured at two different time-points using the Ridascreen Clostridioides difficile Toxin A/B® (R-Biopharm, Germany). (B) Clostridioides difficile spore counting on the log10 scale at the experimental time-points: 72 h and 120 h. (C) Diameter of the bacterial lawn of RT883, RT884, RT885, and RT027 at 24, 48, and 72 h. Data are reported as the means ± SD from three independent experiments.
Fig 2.
Kaplan‑Meier survival curve of hamsters infected with 104 spores of RT883, RT884, RT885, reference control strain (CD196/RT027), and negative control.
Syrian golden hamsters previously treated with clindamycin were orally inoculated with spores from the indicated strains. Hamsters were monitored at 8-h intervals for signs of Clostridioides difficile infection for 15 days, and the number of deceased animals was recorded.
Fig 3.
cgMLSA maximum likelihood phylogeny.
The cgMLSA phylogeny tree based in 2.643 genes, containing 2,531,373 positions rooted by midpoint is represented. The colour strip represents the C. difficile Clades, where Clade 1, 2, 3 and 5 are represented by respectively the colours cyan, red, purple and green. The values in the branches correspond to the percentage of support (0 to 100%) based on 1000 ultrafast bootstrap replicates. The figure represents that RT883, 884, and 885 are very similar to each other and are related to other clade 2 reference strains (R20291, CD196, BI1, 2007855). Reference strains from clade 1 (CD630), clade 3 (M68, CF5), and clade 5 (M120) were also added for comparison purpose.
Fig 4.
Clostridioides difficile PaLoc tcdC gene structure.
(A) A Partial multiple alignment of the tcdC gene. The C→A mutation of the RT883 and 884 isolates at position 191 that generates a “TAA” stop codon is highlighted by a red arrow, while the point deletion that resulted in a frameshift in 885 is highlighted by an orange arrow. (C) Amino acid sequences encoded by the tcdC genes in RT883, 884, and 885 are shown; the stop codons are represented by red asterisks. Positions after the first stop codons are represented in gray. C) Multiple alignment of the tcdC gene in RT883, RT884 and RT885. Asterisks at the bottom denotes conserved sequences in all isolates. The orange arrow represents the frameshift position in RT885.
Fig 5.
Phylogenetic analysis by Bayesian estimation of the amino acid sequences of tcdB alleles from the three Brazilian clade 2 strains (RT883, 884, and 885).
The distribution of alleles according to different clades is shown on color strip in the right. Posterior probability branch support is denoted by a color code, from red (65% support) to green (100% support). (*) A novel allele (tcdB 23) was identified in RT883, while tcdB alleles 8 and 10 were identified in RT884 and RT885, respectively.
Fig 6.
Distribution of Clostridioides difficile ribotypes from toxigenic strains isolated from humans (n = 86) and animals (n = 35) in Brazil between 2018 and 2020.
Fig 7.
Neighbour-joining tree generated from concatenated sequences of the seven housekeeping genes of all the 16 STs identified in the present study.
The novel STs are marked with asterisks (*). ST1, ST2, ST11, ST32, ST47, ST123, ST41, and ST67 (●) were added for comparison purposes. Software: The Molecular Evolution and Genetic Analysis 7.0.26 (MEGA7). B) eBURST analysis of Clostridioides difficile using STs available in the MLST database in December 2021. Each ST is represented by a circle, and lines connect single-locus variants. Khaki circles represent predicted founders. Detail showing the relation of four out of five STs from clade 2 detected in the present study (black arrows), including three Brazilian STs (462, 463, and 464). The remaining new sequence type (ST869) was a singleton.