Diverse sources of C. difficile infection identified on whole-genome sequencing

Abstract

Background: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions.

Methods: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location.

Results: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile.

Conclusions: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).

Figure 1. Genetic Variation and Epidemiologic Relationships among 957 Isolates Obtained from Patients with Clostridium difficile Infection

Panel A shows the number of single-nucleotide variants (SNVs) between each sample obtained during the period from April 1, 2008, through March 31, 2011, and the most closely related previous sample obtained after September 1, 2007. Panel B shows the percentages of isolates that were classified as genetically related, according to the different SNV thresholds, along with the epidemiologic links between related isolates.

Figure 2. Number of SNVs between Cases on the Basis of Multilocus Sequence Typing and Epidemiologic Data

Shown is the breakdown of 957 isolates obtained from patients with C. difficile infection from April 1, 2008, through March 31, 2011. Patients sharing a sequence type and epidemiologic contact with a case since September 1, 2007, are shown in Panels A through E: direct ward contact (Panel A), hospital-wide contact with a shared medical specialty (Panel B), hospital-wide contact with no shared medical specialty (Panel C), only ward-contamination contact (Panel D), and both ward-contamination and hospital-wide contact (Panel E). Patients with no previous case of or hospital contact with a case of the same sequence type are shown in Panel F. For multiple contacts of the same type that occurred with different potential sources of infection, the lowest number of SNVs associated with that type of contact is shown.

Figure 3. Timing and Size of C. difficile Genetic Clusters

Isolates with more than 10 SNVs from any previous sample were defined as distinct genetic subtypes and are plotted on separate horizontal lines, according to the date of the first isolation in Oxfordshire. Isolates that were obtained from September 2007 through March 2008 (run-in period) were included only as potential sources for later C. difficile infections, since patients could have acquired C. difficile from a case before the study started. (Models estimating the total population diversity are provided in Fig. S5 in the Supplementary Appendix.)

Figure 4. Trends in Genetically Related and Distinct Cases, According to Date

Shown are cases of C. difficile infection that were genetically distinct (>10 SNVs) from all previous cases (on the basis of samples obtained after September 1, 2007) (Panel A), cases that were genetically related (≤2 SNVs) (Panel B), and cases that were genetically related and either had a hospital link (Panel C) or had no hospital link (Panel D). The study population was 600,000 persons, so the rate of 30 cases per month corresponds to 5 cases per 100,000 population per month. The blue lines indicate the model-based estimates of the per-year rate ratio. (Details regarding the regression models that were used in the analysis and trends in genetically related cases grouped according to the presence or absence of ribotype 027 are provided in the Supplementary Appendix.)