Biological Agents and Factors Affecting Decontamination

Biological Agents
And
Factors Affecting Decontamination
Bruce R. Cords, Ph.D.
Ecolab Inc.

Biological Agents of Concern
B. anthracis Anthrax
Variola major
Smallpox
Yersinia pestis
Plague
Vibrio cholera
Cholera
Filovirdia
Ebola
Aphthovirus
Foot & Mouth Disease
Botulinum Toxin
Botulism

Resistance to Biocides
PRIONS
BACTERIAL SPORES
PROTOZOA CYST/OOCYSTS
(e.g. Crytosporidium)
MYCOBACTERIUM
NON-ENVELOPED VIRUSES
FUNGI
VEGETATIVE BACTERIA
LIPID-ENVELOPED VIRUSES

Relative Biocidal Activity
Bacterial Spores
Mycobacterium
Non-enveloped Viruses
Peroxyacids, glutaraldehyde,
formaldehyde, chlorine dioxide,
ethylene oxide
Mycobacterium
Non-enveloped Viruses
Fungi
Phenolics, Iodophors,
Hypochlorites
Vegetative Bacteria
Enveloped Viruses
Quaternary Ammonium
Compounds, Organic Acids
* There Are Exceptions!

Antimicrobial Tests
(Required for EPA Registration)
Product Test Required Organisms
General disinfectant
AOAC Use
Dilution
S. cholerasuis ATCC 10708
S. aureus ATCC 6538
Hospital disinfectant AOAC Use
Dilution
S. cholerasuis ATCC 10708
S. aureus ATCC 6538
P. aeruginosa ATCC 1542
Sporicidal
AOAC
Sporicidal
B. subtilis ATOC 19659
Cl. sporogenes ATCC 3584

Food Contact Surface Sanitizer
AOAC Germicidal Detergent Sanitizer Test
99 ml Sanitizer
Use-Solution
25ºC
Add 1 ml of E.coli
or S.aureus
(minimum of 7.5 x 10 7
CFU/ml)
30 second
Contact
Time
0
15
30
Required
Efficacy:
99.999% Kill in
30 seconds at
25ºC
Enumerate
Survivors
Neutralize
1 ml

Suggested Surrogates
Agent
Variola major (Smallpox)
Yersinia pestis (Plague)
Vaccinia virus
Surrogate
Yersinia pseudotuberculosis
Bacillus anthracis (Anthrax) B. subtilis B. cereus
B. globigii
Foot-and-Mouth Disease Virus ?
Norwalk Virus
Feline calicivirus

Regulatory Hurdle
− EPA does not allow real or implied claims
for any infectious agent that is not stated
on the product label.
− Very few commercial products have been
tested against the candidate biological
agents.

Legal Issues
1) It is a violation of Federal law to use an EPA registered
product in a manner inconsistent with its labeling.
– Solution strength must be according to label
– Applications must be on the label
2) It is a violation of Federal law for a manufacturer to
make real or implied claims for efficacy against
organisms which are not on the label.
– Creates problems when we encounter:
• FMDV
• Anthrax
• Norwalk
• SARS
• Avian Influenza H5N1

Anthrax/Bacillus anthracis
−
−
−
−
No products carry label claims (some have
crisis exemption)
Cutaneous, pulmonary, and
gastrointestinal transmitted by aerosols
and on environmental surfaces
Infectious dose - 5-10,000 spores (inhaled)
Effective agents (published literature)
– .25%-.50% (2500-5000 ppm) Peroxyacetic Acid
– 2% (20,000 ppm) Glutaraldehyde
– 4% (40,000 ppm) Formaldehyde
– 1% (10,000 ppm) Sodium Hypochlorite

Smallpox/Variola Major
Complex Coat
−
−
−
−
No products carry label claims
Transmitted through aerosols, contact
with infected person, and environmental
surfaces
Low infectious dose - a few virons
Effective agents (published literature)
– 1% (10,000 ppm) Sodium Hypochlorite
– 2% (20,000 ppm) Glutaraldehyde
– 2% (20,000 ppm) Formaldehyde

Levels of Decontamination Somewhat
Dependent on Infectious Dose
Smallpox
Anthrax
A few virons can
induce disease.
Infectious dose
(inhaled) may be
10,000 spores
– Smallpox decontamination to 100
virons/m 2 not acceptable
– Anthrax decontamination to 100
spores/m 2 probably a safe level

Plague/Yersinia pestis
− No products carry label claims
− Transmitted as an aerosol through
respiratory droplets or plague-infected
fleas. Organism does not survive more
than a few hours outside the host.
− Infectious dose - 50-1500
− Effective agents (published literature)
– 1% (10,000 ppm) Sodium Hypochlorite
– 70% Ethanol
– 2% (20,000 ppm) Glutaraldehyde
– Many general disinfectants

Cholera/Vibrio cholera
− No products carry label claims
− Transmitted by contact and in water and
food contaminated with excreta from
infection individuals
− Infectious dose 10 6
− Effective agents
– General disinfectants

Ebola/Filovirida
Enveloped RNA Virus
− No products carry label claims
− Transmitted by contact with infected
individuals or their blood/secretions.
Environmental?
− Low infectious dose
− Effective agents (published literature)
– 2% (20,000 ppm) Sodium Hypochlorite
– 2% (20,000 ppm) Glutaraldehyde
– .5% (5,000 ppm) Peracetic Acid
– 1% (10,000 ppm) Formaldehyde

Foot & Mouth
Disease/ Apthovirus
Non Enveloped RNA Virus
− Some products carry Label Claims
− Transmitted by aerosols, environmental
surfaces and direct contact between animals
− Low infectious dose
− Effective agents (published literature and
product approvals)
– .03% (300 ppm) Peracetic Acid
– .1% (10,000 ppm) Peroxymonosulphate
– .05% (500 ppm) Sodium Hypochlorite
– Other products (Ministry of Agriculture Fisheries
and Food; U.K.)

Is SARS a Potential Bioterrorism
Agent?
1. Available? Yes
2. Culturable? Yes
3. Infective? Highly
4. Deliverable? Unknown
5. Decontamination? Likely to be
susceptible to common disinfectants

Lessons Learned from the Anthrax Case
1. We were not prepared for
decontamination (e.g. Hart Building).
2) It took three treatments with ClO 2 to
achieve adequate results.
3) Many items were destroyed as opposed
to decontaminated.
4) Ambulance chasers with “cure-alls” were
abundant (e.g. spores vs. vegetative).

Bacillus anthracis
Spores vs. Vegetative Cells
Peracetic Acid 5-log
reduction
Spores
Vegatative
2,500 ppm for 30 min.
150 ppm for 30 sec.

Factors Contributing to Failure
of Decontamination
Disinfectant/Biocide:
− Selection of biocide not effective against
infectious agent
− Biocide too dilute
− Insufficient contact time
− Temperature too low*
− Relative humidity too low
− (gaseous disinfectants)

Factors Contributing To Failure
of Decontamination
Environmental Factors:
− Presence of organic matter*
− Inactivation of QAC’s by residual soaps
and detergents
− Incorrect application/coverage
− Inadequate treatment of water supply
− “Wettability” of surface

All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
70 o F. Well Water (250 ppm)
30 Seconds
L
o
g
6
5
R 4
e
d 3
u 2
c
t
1
i 0
o 0.20% 0.35% 0.13% 0.26% Acid
n Peracetic Acid Organic Acid Anionic
Quat
Iodophor Chlorine
S. aureus E. coli

All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
55 o F. Well Water (250 ppm)
30 Seconds
L
o
g
6
5
R 4
e
d 3
u 2
c
t
1
i 0
o 0.20% 0.35% 0.13% 0.26% Acid
n Peracetic Acid Organic Acid Anionic
Quat
Iodophor Chlorine
S. aureus E. coli

L
o
g
R
e
d
u
c
t
i
o
n
6
5
4
3
2
1
0
All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
40 o F. Well Water (250 ppm)
30 Seconds
0.20% 0.35% 0.13% 0.26% Acid
Peracetic Acid Organic Acid Anionic
S. aureus E. coli
Quat Iodophor Chlorine

All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
40 o F. Well Water (250 ppm)
2 Minutes
L
o
g
6
R
e
d
u
c
t
i
o
n
5
4
3
2
1
0
0.20% 0.35% 0.13% 0.26% Acid
Peracetic Acid Organic Acid Anionic
Quat Iodophor Chlorine
S. aureus E. coli

Effect of Temperature Organic Load
and Concentration on Disinfectant
Activity
Quaternary Disinfectant:
(Log Reduction for P. aeruginosa)
QUAT
1000 ppm
5 minutes
20°C 10°C
x0.5 x1.0 x2.0 x0.5 x1.0 x2.0
.03% F P P F F P
BSA
.3% F P P F F F
BSA
Taylor et al 1999
P (Pass) = > 5 log reduction
F (Fail) = < 5 log reduction

Effect of Temperature Organic Load
and Concentration on Disinfectant
Activity
Peracetic Acid Disinfectant:
(Log Reduction for P. aeruginosa)
Peracetic
Acid
300 ppm
5 minutes
20°C 10°C
x0.5 x1.0 x2.0 x0.5% x1.0 x2.0
.03% P P P P P P
BSA
.3% F P P F P P
BSA
Taylor et al 1999
P (Pass) = > 5 log reduction
F (Fail) = < 5 log reduction

Treatment of a 48 Hour
Biofilm of Pseudomonas aeruginosa*
70
60
1 Minute Contact Time
50
40
30
20
10
0
80 ppm 160 ppm 320 ppm
Peracetic/Organic Acid Peracetic Acid Chlorine
*Fatemi and Frank. 1999. Journal of Food Protection. Vol. 62 (761-765)

Effect of Relative Humidity on
Sporicidal Activity (B. globigii) of
Formaldehyde
Log
Concentration RH Temp Reduction Time
400 mg/m 3 30% 25°C 1 22’
400 mg/m 3 98% 25°C 1 9’
Ref: Emerging Infectious Disease Vol 9 No 6 p. 625

Effect of Relative Humidity on
Sporicidal Activity (B. subtilis v
niger) of Peracetic Acid Vapor
Log
Concentration RH Temp Reduction Time
1mg/L 80% RT >5 log 10’
1mg/L 60% RT >4 log 10’
1mg/L 40% RT >3 log 10’
Ref: Emerging Infectious Disease Vol 9 No 6 p. 626

Inactivation Of Bacillus spp. By Boiling In
Tap Water
Initial log
CFU/ml
After 5’
Boiling
C UNC C UNC
B. anthracis 4.95 4.92 0 2.01
(sterne)
B. cereus 4.62 4.59 0 1.46
(commercial)
B. cereus 4.54 4.76 0 0.48
ATCC 9592
B. thuringensis 4.63 4.46 0 1.47
ATCC 35646
C = Covered
UNC = Uncovered
Temp above surface: C = 98.9°C UNC = 77.3°C
Ref: Emerging Infectious Disease Vol 10 No 10 2004

National Center for Food Protection
and Defense
Study No: 910F2376745 – Ecolab
Development of Time/Temperature Concentration
Matrix for Inactivation of Infectious Bioterrorism
Agents by Chemical Biocides
Student:
Hilgren – Ecolab
Co-Advisors: Swanson – Ecolab
Diez – University of Minnesota

Bacillus
Spores
General Method
Food Soil
• Egg Yolk Emulsion
• Whole Milk
• Flour Slurry
8.8% protein
3.5% fat
7.6% starch
Spore/soil mixture dried on
stainless steel carrier
Carrier immersed in candidate
biocide for 10 minutes

Impact of Food Soil on Inactivation
of B. cereus spores by Peroxyacetic
Acid (Preliminary Results)
Conditions = 10 minute exposure
20°C
B. cereus ATCC 10987
Log Reduction
5,000 ppm 10,000 ppm
No Soil >7.0 >7.0
Whole Milk 1.5 4.0
Egg Yolk 2.0 4.0
Flour 2.0 7.0
Ref: Ecolab, NCFPD Project

Impact of Food Soil and Temperature on
Inactivation of B. cereus Spores by
Peroxyacetic Acid (Preliminary Results)
Conditions =
10 minute exposure
10,000 ppm Peroxyacetic acid
B. cereus ATCC #10987
Log Reduction
10°C 20°C 30°C
No Soil 6.0 >7.0 >7.0
Whole Milk 1.5 4.0 >7.0
Egg Yolk 2.0 4.0 5.5
Flour 1.5 7.0 7.0
Ref: Ecolab, NCFPD Project

Impact of Food Soil on Inactivation
of B. cereus spores by Sodium
Hypochlorite (Preliminary Results)
Conditions = 10 minute exposure
20°C
B. cereus ATCC 10987
Log Reduction @ 50,000 ppm
No Soil >7.0
Whole Milk 1.2
Egg Yolk 1.9
Flour >7.0
Note: Flour @ 1,000 ppm - >7.0 log reduction
Ref: Ecolab, NCFPD Project

Other Results to Date:
− Quarternary Ammonium Chloride
− Not effective at 75,000 ppm (7.5%), 20°C
− Normal disinfection level = 200-800 ppm
− Iodophor
− Not effective at 19,000 ppm (1.9%), 20°C
− Normal disinfection level = 25 ppm
− Acidified Sodium Chlorite
− Somewhat effective
− Egg yolk and milk results not as good as flour
− Hydrogen Peroxide
− Effective at 25-30%; 20°C
− Not effective at 10°C
− Mixed Peroxyacid System
− Results similar to peroxyacetic acid

Future Testing to Include:
− B. anthracis (nonvirulent strain)
− Yersinia Pestis