A Simplified Method for Environmental Culturing for Clostridium difficile

Background:  Current methods for environmental culturing of C. difficile (CD) are labor-intensive, involve multiple steps that increase chances for laboratory contamination, and are limited to reference laboratories equipped with anaerobic chambers.

Objective:  To develop a simplified method for environmental culturing of CD that can be readily performed in any setting.

Methods:  Environmental CD contamination was simulated by taping off 2  41.5 x 41.5 cm­­ adjacent areas on a laboratory bench. Two different patient strains of toxigenic CD spore stocks, one epidemic (BI) and one non-epidemic were applied to each area at a target density of 10^-1, 10⁰, 10¹, 10², and 10³ cfu/cm² followed by 10-minute disinfection with 10% (5250 ppm) bleach . Viable spore counts were determined by plating serial 10X dilutions of spore stock solution on tryptone yeast extract agar supplemented with 50mM L-arginine and 0.1% sodium taurocholate (TcTYR) followed by anaerobic  incubation x 72 hours at 37 °C and colony counting.  Spore inocula were spread evenly across each area and allowed to dry. The entire area was sampled  using a 3.4 x3.4 cm sterile 70% isopropyl alcohol wipe (Kendall) using sterile gloves and aseptic technique.  These prepared areas were sampled at baseline, after each successive spore inoculation, and after bleach disinfection .  Each alcohol wipe was aseptically transferred to a Hungate culture tube containing 5 mL pre-reduced cefoxitin cycloserine mannitol broth with taurocholate and lysozyme (CCMB-TAL, Anaerobe Systems) and  incubated at 37^o C x 72 hours in a standard incubator.  Negative controls consisted of alcohol wipes handled in open air for 60 seconds using sterile gloves. Fermentation was monitored at 15-minute intervals using time-lapse photography. 10 µl of fermenting cultures were passed to pre-reduced 5% sheep blood agar and incubated anaerobically in jars to confirm the growth of CD.

Results:   For both strains, all spore inoculated areas tested (minimum 10^-1 spores/cm2 or total inoculum ≈100 spores) were positive for CD.  Negative control, pre-contamination, and post bleach decontamination samples were negative.  All fermenting cultures grew CD.  The time-to-fermentation was directly proportional to the log10 of the input inoculum (Figure 1) for both strains.

Conclusions:  This single-step method for environmental CD sampling is simple and can be utilized in any laboratory setting capable of anaerobic culture. Time-to-fermentation for samples confirmed to contain toxigenic CD provides a semi-quantitative measure of CD density, particularly if the sampled area is standardized.