Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens

Abstract

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 10⁶ pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

FIG. 1.

Efficacy of health care disinfectants in inactivating pure C. difficile spores. (a) Viability of spores derived from distinct C. difficile genetic variants after exposure to various health care disinfectants. All C. difficile strains are virulent to humans. A summary of the disinfectants is listed in Table 1. Data are representative of 5 separate experiments. Error bars indicate standard deviations. The broken horizontal line indicates the detection limit of 2 CFU/ml. EtOH, ethyl alcohol. (b) TEM image of an untreated, viable spore. (c) TEM of a spore that was inactivated by 10% hydrogen peroxide exposure for 10 min.

FIG. 2.

C. difficile spores are inactivated by prolonged exposure to high-level hydrogen peroxide. (a) Viability of C. difficile M68 spores after exposure to 1% or 10% hydrogen peroxide for 1, 10, and 20 min. Data are representative of three separate experiments. Error bars indicate standard deviations. The broken horizontal line indicates the detection limit of 2 CFU/ml. (b) Spore strips containing either 10⁶ C. difficile M68 or G. stearothermophilus spores were exposed to 400 ppm hydrogen peroxide vapor for 1, 5, 20, or 60 min prior to culture of the bacteria to determine the efficacy of disinfection. Positive growth strip indicates that the spores were not inactivated by HPV. Six spore strips were tested per time point.

FIG. 3.

Environmental C. difficile spores are highly infective. Infective dose curves of pure and fecally derived C. difficile M68 spores in mice (4 per dose) after exposure to environmental spore contamination. Based on the Reed-Muench formulation (24), the environmental spore dose to infect 50% (ID₅₀) of the mice with a 1-hour exposure is 5 spores/cm² for fecal spores and 10 spores/cm² for pure spores, as indicated by the gray vertical dashed lines and the black arrow below the graph. Data are representative of two independent experiments. As a reference, the vertical arrow below the graph labeled “spore contamination” indicates the level of spores that was retrieved from the cage after fecal contamination by the infected mice corresponding to Fig. 4. The vertical arrow labeled “10⁶ spores/cage” indicates the level of spores that is equivalent to that on the indicator strips corresponding to Fig. 2.

FIG. 4.

Efficacies of health care disinfectants in reducing host transmission of C. difficile via environmental spore contamination. (a) Transmission efficiency of environmental C. difficile M68 spores to naïve mice after surface disinfection with the indicated agent (10 mice per agent) compared to that of the untreated group (18 mice untreated). (b) Effect of hydrogen peroxide vapor contact time on reducing transmission of C. difficile spores to naïve mice. Exposure time is defined as the time the HPV was at the peak level of 400 ppm. There were 10 mice corresponding to each time point (0, 1, 5, and 20 min). The estimated level of spore contamination was 23 ± 6 spores/cm².