Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011

Abstract

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Figure 1. Transmission events and phylogeny of Clostridium difficile 078, the Netherlands 2002–11 (n=65)

A. Distribution of Dutch hospitals and pig farms included in this study. Only pig farms with a known location were plotted. Blue dots represent the hospitals (n = 16) where isolates from hospitalised patients were obtained, red dots represent pig farms (n = 12) where isolates from farmers and pigs were obtained. Brown dots represent the pig farms where pigs and farmers had identical C. difficile isolates. The green arrow indicates a potential (long-range) transmission event between two farms. B. Phylogenetic tree revealing likely transmission between pigs and humans. Shown is the reconstructed phylogenetic tree based on 774 core genome single-nucleotide polymorphisms (SNPs). Samples are colour-coded according to their source: pig (red), farmer (green) and clinical isolate (blue). Identical genotypes with an epidemiological link (i.e. same location/farm) are marked with brown boxes. Long-range transmission events (i.e. different locations) are marked with a green box. The tip labels are coded with the city name followed by two numbers that represent year of isolation (’08 ⩠ 2008). The CDM120 genome (purple) is used for the reference-based mapping, RT066 (purple) is used as an out-group to root the tree. The scale indicates the branch length that correspond to 10 SNP differences. The numbers for the internal nodes show the support from 100 non-parametric bootstraps of a maximum likelihood reconstruction (only bootstrap values > 50 are shown).

Figure 2. Phylogenetic cluster showing relatedness of Clostridium difficile clinical, pig and farmer isolates, the Netherlands, 2008–11 (n=4)

A. Geographical map showing the location of the isolates present in the phylogenetic cluster shown in panel B. Blue dot represents a hospital (Breda), red dots represent the two pig farms (R’donksv. and Oirschot). B. Zoom-in on a phylogenetic cluster containing highly related isolates from different sources (swine, farmer and clinical isolates). The numbers on the tree branches represent the number of single-nucleotide polymorphisms differences in the cluster, the tip labels are coded with city (Breda) followed by two numbers that represent year of isolation (’08 ⩠ 2008).

Figure 3. Phylogeny of Clostridium difficle 078 isolates showing the presence of antimicrobial resistance determinants, the Netherlands, 2002–11 (n=65^a)

NT = not phenotypically tested. Circular representation of the C. difficile 078 phylogeny with coloured dots representing the distribution of antimicrobial resistance (AMR) determinants. The legend shows the identified transposons together with the AMR determinants (between brackets) located on the transposon. The coloured dotted lines represent the source of the respective isolates (swine, farmer and clinical isolate). The presence of Tn6190 (tetM) is associated with tetracycline resistance; 078 isolates phenotypically tested as tetracycline-resistant are indicated with orange tree branches, streptomycin-resistant isolates are indicated with purple tree branches, isolates resistant to both tetracycline and streptomycin are indicated with blue tree branches. ^a Two isolates were sequenced in duplicate. Ne RT 066 sequence was included as root sequence. In total, 68 sequences are shown.