125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

An Update on EPA’s Initiatives & Activities of 

AOAC Expert Review Panel on 

Clostridium difficile: Is C. difficile becoming 

Less Difficile?

Jafrul Hasan, PhD, MPH

Team Leader, Office of Pesticide Programs 

Microbiology Laboratory Branch

Environmental Protection Agency

&

Chair, AOAC Expert Review Panel on 

C. difficile
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Outline

Background on Clostridium difficile

EPA Interim Guidance for C. difficile

Need for Standardized Methodology

C. difficile Initiatives at EPA

AOAC Expert Review Panel (ERP)
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Background: C. difficile

 Strict anaerobic spore-forming bacillus

 Major cause of nosocomial (hospital-acquired) 

gastrointestinal infections in developed world; 

C. difficile Infection (CDI) range from 

uncomplicated diarrhea to sepsis and even 

death 

 Fecal-oral transmission through contaminated 

environment and hands of healthcare personnel

 Exposure to antibiotics is major risk factor for 

disease; other risk factors: older age, 

hospitalization, antiulcer medications, etc. 

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125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Pathogenesis of C. difficile Infection (CDI)

 Ingestion (vegetative form or spores)

 Colonization in small intestine (spores germinate 

to vegetative form) 

 CDI can arise in large intestine if normal flora 

disrupted by antibiotic therapy

 C. difficile reproduces in intestinal crypts, 

releases toxins A (enterotoxin) and B 

(cytotoxin)-leading to colitis, pseudomembrane 

formation and watery diarrhea

Adapted from Sunenshine and Mcdonald, Cleveland Clin J Medicine, Vol 73, 2006

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125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

5

Yearly C. difficile–related Mortality by Listing on Death 

(per million) Certificates, United States, 1999–2004.

Source: Centers for Disease Control and Prevention

# of death

per million
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

C. Difficile Vegetative Cells

Courtesy: LS Wiggs, Public Health Information Library (#9999), CDC
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Visualization of Pure C. difficile Spores

 (a) Endospore stain of C. difficile

culture showing the pink 

vegetative cells (veg) and pink 

extracellular matrix (ex), with a few 

interspersed green spores (sp). (b) 

Purified C. difficile spores are 

stained green. (c) TEM of 

sectioned C. difficile spores, 

demonstrating the spore 

ultrastructure including the 

exosporium, coat, cortex, core, 

membrane, and ribosomes. Bar, 

100 nm. (d) Magnified section of 

outer surface of spore. Bar, 50 nm. 

 Source: Lawley et., al., J. 

Bacteriol., 2009, p. 5377, Vol. 191, 

No. 17

Copyright © 2009, American 

Society for Microbiology. 
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

EPA on C. difficile 

 EPA‟s Office of Pesticide Programs (OPP) has the responsibility under the 

Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) to regulate the 

registration of antimicrobial products, including products designed to treat 

surfaces contaminated with spores of C. difficile. Under FIFRA, the 

registrant of a product with a public health claim is required to submit 

efficacy, or effectiveness data, to EPA in support of the product's 

registration. 

 EPA‟s Antimicrobial Testing Program (ATP) has been testing hospital 

sterilants, disinfectants, and tuberculocides since 1991 to help ensure that 

products in the marketplace meet stringent efficacy standards. 

 EPA issued interim guidance in 2009 for the efficacy evaluation of 

antimicrobial products labeled for use to treat hard non-porous surfaces in 

healthcare settings contaminated with spores of C. difficile. 

 EPA is conducting post-registration evaluations of products registered for 

claims against C. difficile.
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

EPA Interim Guidance for C. difficile Claims

 Antimicrobial Products (dilutable liquids, powders, ready-to-use 

formulations, spray products, towelettes) to treat surfaces in health 

care settings

 Claim: Kills C. difficile spores on hard, non-porous surfaces in ≤10 

minutes

 Test Methods (Qualitative/Quantitative):

 AOAC Sporicidal Activity Test (AOAC SAT 966.04)

 Quantitative Three Step Method (TSM, ASTM E2414)

 Quantitative Carrier Test Method (QCT-2, ASTM 

E2197)

 Test Strains: ATCC 43598, ATCC 700792, ATCC 43599

 Must achieve ≥6 log reduction of viable spores with control 

carriers >106 spores/carrier

 No organic soil load; label must specify pre-clean step 
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

10

C. difficile Initiatives at EPA

(Since September, 2008)

 EPA‟s need for developing standardized 

methodology and regulatory guidance related to 

C. difficile was due to requests for registration

 Lacked standard method to generate spore 

suspensions of high titer (>8 log10/mL) & quality 

(≥90% spores/resistance to acid, etc) 

 Research at EPA addressed following attributes: 

● Strains ●Sporulation Media

● Recovery Medium ● Incubation Conditions

● Monitoring of sporulation ●Anaerobic Jar vs Chamber
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

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Findings-EPA Research 

 Strains: Non-toxigenic (700057) and Toxigenic (700792, 43598, 43599, 9689)

– 700792, 43598 and 43599 part of interim guidance.

– 43598 (Toxin B + only) was advanced based on use by 

industry, ease of production of spores, and safety 

 Sporulation Media: 

• Liquid: Reinforced Clostridial Medium, Columbia Broth, Brain Heart 

Infusion broth, and Cooked meat medium

• Solid: TSA with 5% blood , and CDC Anaerobic Blood Agar (CABA) plates

– CABA found to be most effective sporulation medium

 Recovery Media: TSA with 5% blood, CDC agar, Egg Yolk Agar, Columbia 

Agar, Cycloserine-Cefoxitin Fructose Agar-w/ horse blood and taurocholate 

(CCFA-HT)

– CCFA – HT selected as most efficient recovery medium

 Explored the most appropriate length of incubation for spore generation:

– 7-10 days optimum incubation / monitored sporulation rate using phase 

contrast microscopy

 Optimized incubation conditions in Anaerobic Jar and Anaerobic Chamber 
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

EPA Method

Journal of AOAC I, Jan-Feb, 2011, vol 94 (1), p 259-272

Spread 100 μL of actively growing C. difficile (ATCC 43598) in 

RCM onto 10 pre-reduced CABA plates

Incubate plates inverted anaerobically for 7-10 days at 36±l°C

Harvest spores from plates using ST-80/heat inactivate at 65±2°C for 10 min

Purify spores using HistoDenz, a density gradient column. Determine 

spore titer by plating on CCFA-HT, and perform quantitative acid 

resistance test 
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Monitoring percent sporulation of C. difficile (ATCC 43598) during 

anaerobic incubation at 36 ± 1oC under phase contrast microscopy 

(magnification 1000 ×) 

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Day 2

Day 8

Day 10
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

A: Non-purified C. difficile spores (ATCC 43598) 

B. purified (≥99% ) spores by a density gradient medium,

free from vegetative cells and cell fragments 

A B
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

C. Separated vegetative cells, and, 

D. Separated cell fragments upon purification by 

HistoDenzTM, a density gradient medium

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C

D
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Product of EPA Research: New ASTM 

Standard Method 

 ASTM Standard Method E2839 E-2839-11 -11: Debuts 

First Standard Method for sporulation for C. 

difficile (ATCC 43598)

 Spores are repeatedly being generated using 

EPA Method (ASTM Method E2839-11) 

 EPA uses spore production procedure to launch 

efficacy testing of products registered to treat 

surfaces contaminated with spores of C. difficile 

(post-registration evaluation adapted for 

registrant study).
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

AOAC ERP- C. difficile

 Under EPA‟s Office of Pesticide Programs contract with 

AOACI, an AOAC Expert Review Panel (ERP) on C. 

difficile was formed in January, 2010. Upon a “Call for 

Experts” was issued by AOAC, members selected from 

regulatory, academia and industry. 

 ERP was charged to develop and address the issues 

identified in the “Fitness for Purpose” Statement

 Panel Members are:

–Judith Noble-Wang, CDC

–Dave Rottjakob, ATS Laboratories

–Syed Sattar, Univ of Ottawa

–Lynne Sehulster, CDC

–Jafrul Hasan (Chair, EPA) 

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125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

ERP: Fitness for Purpose (cont.)

Methodology should include: 

1) Culture and maintenance of C. difficile

2) Generation of high titer spore suspensions 

3) Inoculation of carriers and enumeration of spore load

4) Product neutralization and confirmation 

5) Addition of soil load to the inoculum. Assays such as resistance 

to acid (e.g., as per AOAC Method 2008.05) or sodium 

hypochlorite may be utilized to qualify spore preparations for use. 

6) Efficacy test methods should demonstrate adequate repeatability 

(within lab variability) and reproducibility (between labs variability) 

based on performance of comparable antimicrobial test methods. 

Validated methodology is highly desirable.

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125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Summary of ERP Activities (2010-2011) 

 Held ~12 teleconferences and one „Face-to-Face‟ meeting (Aug, 

2010) 

 Developed “Fitness for Purpose” 

 Conducted literature search and reviewed 42 methods 

 Down selected 17 relevant methods for in-depth review and 

deliberation (publications/submitted manuscript/protocol)

 Assembled 90 pages of reviews from panel members

 1

st „Face-to-Face‟ meeting (Aug, 2010): Presented/discussed 

reviews in depth. Concurred on Phase I and II approach.

 √Conducted Pre-Phase 1 evaluation for optimal recovery medium

 Phase I: Completed 7 labs collaborative for comparing two 

sporulation protocols

 2

nd „Face-to-Face‟ meeting held Nov 8-9, 2011

 Two Publications in the horizon: A white paper on ERP 

activities/Collaborative study comparing two sporulation protocols

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125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Pre-Phase 1 Study: Five C. difficile agar media were evaluated by ATS Lab, CDC and EPA. 

Colonies of C. difficile (ATCC 43598) after 48 hr incubation at 36°C

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√CCFA-HT √ BHIY-LT CDBA-T

√ BHIY-HT CCFA-L
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Update on Phase I Study 

●Evaluated EPA and University of Ottawa methods; three 

replications per method. 

●Major attributes compared for repeatability and reproducibility: 

Spore titer, spore purity and HCL resistance. Feedback 

evaluated. 

-Participating labs: 

 ATS Lab, Study Director: Dave Rottjakob

 Bioscience Lab, Study Director: John Mitchell

 CDC, Study Director: Judith Noble-Wang

 EcoLab, Study Director: Lisa Hellickson

 EPA, Study Director: Jafrul Hasan

 Lonza, Study Director: Milady Brutofsky

 STERIS, Study Director: Dan Klein

 University of Ottawa, Study Director: Syed Sattar

● Data collection completed: October 31, 2011

● ERP Face-to-face meeting completed: Nov 8-9, 2011
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Phase I collaborative: Comparison of Two 

Sporulation Methods 

EPA

Ref.: J. of AOAC I, Jan-Feb, 2011, 

vol. 94 (1), p 259-272.

 Stock Culture: Frozen vegetative 

stock ≤-70°C

 √Sporulation/Incubation (using solid 

medium). Freshly grown culture onto 

Commercially available CDC 

Anaerobic Blood Agar (CABA) 

plates/Incubate 7-10 days/Ensure 

≥90% spores

 Harvest: Scraping followed by 

centrifugation (4,500 × g) in salinetween, pre-purification heating at 

65±5°C for 10 min 

 √Purification: Physical separation by

commercially available Density 

Gradient medium.

 Qualify: by HCl resistance

 Enumeration: BHIY-HT, >8 log10; 

≥90% spores

Univ. of Ottawa

 Stock Culture: Liquid spore stock at 

4°C 

 √Sporulation/Incubation (using liquid 

medium) Transfer culture successively 

from Columbia Broth to 500 mL 

Clospore liquid medium/Incubate 5 

days/Ensure ≥60% spores 

 Harvest: Centrifugation (4,000 × g), 

wash in distilled water

 √Purification: Enzymatic digestion by 

trypsin/ lysozyme. Post-purification 

heating at 70°C for 10 min. 

 Qualify by HCl resistance.

 Enumeration: BHIY-HT, 8.9-9.7 log10; 

≥90% spores 

Ref: J. of AOAC I, Mar-Apr, 2011, 

vol. 94 (2), p 1-9.
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

ERP Face-to-face Meeting on Nov 8-9, 2011 at EPA

• Discussed Phase I multi-lab collaborative data for the two sporulation 

methods for titer (LD/mL), % purity and resistance to HCL. 

• Discussed statistical analyses including the repeatability and 

reproducibility. 

• Analyzed responses to questionnaire.

• Discussed/Demonstrated One Step Method (OSM), modified from 

Three Step Method (AOAC Method 2008.05), for future consideration 

by the ERP for efficacy testing (?). 

• The two methods were found to result in spore suspensions of similar 

titer, purity, and resistance to HCl. 

• More data to be developed on relative resistance of spore suspensions 

from both methods to AI (e.g., NaOCl and H2O2/Peracetic acid).

• More resistant spore suspensions from one method (preferable) may 

be advanced for efficacy testing (?)

• Call for Laboratories; Proposed Dates: Feb-May, 2012 
125th AOAC Annual Meeting & Exposition

September 18-21, 2011

New Orleans, Louisiana

Acknowledgements

 EPA

• Dr. Stephen Tomasino

• Susan Lawrence

• Knoxley Japal

• Erick Christensen

 AOAC Expert Review Panel

– Dr. Judith Noble-Wang, CDC

– Dave Rottjakob, ATS Laboratories

– Dr. Syed Sattar, Univ of Ottawa

– Dr. Lynne Sehulster, CDC

 AOAC INTERNATIONAL

• Jennifer Diatz

• Maria Nelson

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