The United States Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory initially developed and submitted the ASTM Standard E2839-11, Standard Test Method for Production of Clostridium difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents for approval. The EPA now intends to revise/update the Standard as follows: 1.Addition of a new Section (Section 14), Storage of Purified Spores. 2.Addition of seven laboratory collaborative data under the Precision and Bias Section (Section 15). 3.Addition of editorial changes as shown in tracked lines. During the ASTM Fall Meeting in Jacksonville, FL on October 24, 2013, Dr. Jafrul Hasan of EPA, the Technical Contact for this submission (Work Item 44172), presented an overview of the proposed changes to the ASTM Subcommittee E35.15 (Antimicrobial Agents). The recommended changes submitted here under the CONTENTS OF BALLOT ITEM (see below) for balloting were discussed and agreed upon with the attending members of the Subcommittee E35.15 at the ASTM Fall Meeting, 2013. Rationale for adding Section 14: Storage of Purified Spores: ASTM Standard E2839-11 instructs the user how to generate spore suspensions of Clostridium difficile (ATCC 43598) of high titer and purity. The Standard calls for resuspending the purified spores in phosphate buffered saline containing 0.1% (v/v) Tween-80 (ST-80); however, it does not address the long-term storage of a spore suspension. To address this issue, a study was conducted where a single preparation of a spore suspension was stored in ST-80 at -20C in 1.5 mL low retention (siliconized) microcentrifuge tubes for approximately one year. Over the one year study period, the titer of the spores was evaluated at least biweekly (for the first three months) and then monthly (after three months) using membrane filtration on pre-reduced BHIY-HT (Brain-Heart Infusion agar with Yeast extract, Horse blood and Sodium Taurocholate). As an additional measure of spore quality, the spores were exposed to sodium hypochlorite (1500150 ppm) in a suspension-based assay with contact time of 3 minutes. The log10 reduction (LR) in viable spores was determined. The untreated spore titer was very consistent over the one year storage period (average log10 density was 6.80 with a standard deviation of 0.37). Over the study period, the average log10 reduction in spores due to exposure of the sodium hypochlorite was also consistent with a mean LR value of 3.77with a standard deviation of 0.81. These results strongly support the storage of spores in 0.1% ST-80 at -20C for up to one year. Rationale for amending Section 15: Precision and Bias: In 2011, a seven laboratory collaborative was conducted (with oversight by an AOAC International Expert Review Panel on Clostridium difficile) to evaluate the spore titer, purity and acid resistance of spores produced in accordance with the ASTM E2839-11 (see reference 7). Each laboratory produced three independent spore suspensions in phosphate buffered saline containing 0.1% (v/v) Tween-80 (ST-80). The main test variables (i.e., acceptance criteria) were, 1) titer ( (8 log10 density (LD)/mL), 2) purity ( 95%), and 3) acid resistance (2 log10 reduction (LR) in viability upon exposure to 2.5 M HCl for 10 min). Across laboratories, use of ASTM E2839-11 yielded spore suspensions of a mean spore log density/mL of 9.5, 98.7 % spore purity and 0.99 LR for acid resistance. The average repeatability standard deviation (Sr, among replications variability within a lab) for mean LD was 0.1. The reproducibility standard deviation (SR, among-lab variability) for mean LD was 0.36 (see reference 7). The mean repeatability standard deviation (Sr) and the mean reproducibility standard deviation (SR) for spore titer employing ASTM E2839-11 across seven labs were acceptable (see reference 8).
Keywordsacid resistance; Clostridium difficile; density gradient medium; spore production; spore purity; sporicidal efficacy testing; vegetative cells;
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