Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Abstract

Background: The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results: Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions.

Conclusions: Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.

Figure 1

Global and group-by-group phylogenetic analysis of the genomes. (a) Phylogeny of all 486 genomes from included CDI cases. Clades corresponding to STs are shown as triangles, the height of which represents the diversity of each ST. Numbers in parentheses indicate the number of genomes belonging to each ST sequenced. The 15 STs or groups of closely related STs analyzed in detail are shaded, and indexed from 1 to 15. (b) Detailed phylogeny for samples in group 9. The ten genomes of ST54 are labeled from A to J and the five genomes of ST63 are labeled from K to O. The × co-ordinate of each genome indicates time of isolation. For each internal node of the tree (corresponding to a common ancestor), the mean inferred age is shown on the tree with the 95% credibility interval around this mean shown as a blue bar.

Figure 2

Conceptual representation of how the TMRCA relates to transmission. Two patients A and B are shown, and a transmission event happened from A to B as indicated by a red arrow. Within each patient, a colonizing population evolves between the time of infection and the time when the patient becomes symptomatic, at which point a single strain is isolated, represented by a red dot. The most recent common ancestor of the two isolates from A and B is shown as a blue dot.

Figure 3

Summary of results of the transmission analysis. Each horizontal panel corresponds to one of the 15 groups defined in Figure 1a. Each circle represents a CDI case, and is located on the x-axis according to the isolation date. Cases are linked by horizontal lines where the genomic analysis found that transmission was possible. The 15 genomes in group 9 are labeled from A to O as in Figure 1b. Within each group, cases that are connected with one another based on the epidemiological analysis are indicated by circles of the same unique color, and cases with no identified epidemiological connections are shown in gray. The same colors are used to label isolates in Additional file 3.

Figure 4

Distributions of the TMRCA for all pairs of genomes with the same ST and isolated within a month of each other. Top left: 48 pairs not from ST1 and with an epidemiological link based on shared time and space on a ward. Bottom left: 118 pairs not from ST1 and without an epidemiological link. Top right: 310 pairs from ST1 and with an epidemiological link. Bottom right: 149 pairs from ST1 and without an epidemiological link.