Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia

Abstract

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLS_B agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Keywords: AMR; Clostridioides difficile; WGS; epidemiology; microbial genomics.

Fig. 1.. Genetic diversity of STs as measured by pairwise cgSNP distances. Each graph shows the frequency distribution of pairwise genetic distances for all strains in a given ST. STs 2, 13, 14 and 49 together comprise RT014/020, while ST8 strains belong to RT002 and ST34 strains belong to RT056.

Fig. 2.. C. difficile RT014/020 cgSNP tree. Maximum-likelihood phylogeny of 169 C. difficile RT014/020 (STs 2, 13, 14, 49) genomes. Trees are based on evolution in 2716 (ST2), 1610 (ST13), 2157 (ST14) and 1275 (ST49) non-recombinant, non-repetitive cgSNPs in clonal frame. Trees are midpoint rooted, and the nodes are supported by 500 non-parametric bootstrap replicates. Tree scales are in single-nucleotide changes per quality- and recombination-filtered site. Clonal relationships (two or more strains sharing ≤2 cgSNPs) are indicated by red boxes with red letters referencing clonal strains detailed in Table 1.

Fig. 3.. C. difficile RT002 and RT056 cgSNP trees. Maximum-likelihood phylogeny of 77 C. difficile RT002 genomes and 36 C. difficile RT056 genomes. The tree is based on evolution in 1955 and 9684 (RT002 and RT056, respectively) non-recombinant, non-repetitive cgSNPs in clonal frame. The tree is midpoint rooted and the nodes are supported by 500 non-parametric bootstrap replicates. Tree scales are in single-nucleotide changes per quality- and recombination-filtered site. Clonal relationships (two or more strains sharing ≤2 cgSNPs) are indicated by red boxes with red letters referencing clonal strains detailed in Table 1.

Fig. 4.. Days between the isolation of clonal pairs compared for same-site collected pairs vs. pairs collected at different sites. Outlier A consisted of two strains isolated at the same site in SA, 714 days apart, while Outlier B consisted of two strains isolated at separate sites in NSW and SA, 1432 days apart.