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 Peroxyacids, glutaraldehyde,
Mycobacterium formaldehyde, chlorine dioxide,
Non-enveloped Viruses ethylene oxide

Mycobacterium Phenolics, Iodophors,

Non-enveloped Viruses Hypochlorites
Fungi

Vegetative Bacteria Quaternary Ammonium

Enveloped Viruses Compounds, Organic Acids

* There Are Exceptions!

 Antimicrobial Tests
(Required for EPA Registration)

Product Test Required Organisms

General disinfectant AOAC Use S. cholerasuis ATCC 10708

Dilution S. aureus ATCC 6538

Hospital disinfectant AOAC Use S. cholerasuis ATCC 10708

Dilution S. aureus ATCC 6538
P. aeruginosa ATCC 1542

Sporicidal AOAC B. subtilis ATOC 19659

Sporicidal Cl. sporogenes ATCC 3584
 Food Contact Surface Sanitizer
AOAC Germicidal Detergent Sanitizer Test

99 ml Sanitizer Add 1 ml of E.coli 30 second

Use-Solution or S.aureus Contact 0
25ºC (minimum of 7.5 x 107 Time
CFU/ml) 15

30

Enumerate
Survivors Neutralize 1 ml
Required
Efficacy:
99.999% Kill in
30 seconds at
25ºC
 Suggested Surrogates

Agent Surrogate

Variola major (Smallpox) Vaccinia virus

Yersinia pestis (Plague) 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
Solution strength
strength must
must be
be according
according to
to label
label
– Applications
Applications must
must be
be on
on the
the label
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 A few virons can

induce disease.

Anthrax Infectious dose

(inhaled) may be
10,000 spores

– Smallpox decontamination to 100

virons/m2 not acceptable
– Anthrax decontamination to 100
spores/m2 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 1066
 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 ClO22 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 2,500 ppm for 30 min.

Vegatative 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
70oF. 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 Quat Iodophor Chlorine
n Peracetic Acid Organic Acid Anionic

S. aureus E. coli
 All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
55oF. 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 Quat Iodophor Chlorine
n Peracetic Acid Organic Acid Anionic

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

S. aureus E. coli
 All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
40 oF. Well Water (250 ppm)
2 Minutes
L
o
g
6
5
RR
ee 4
dd
u
3
u
cc 2
tt
ii
1
oo 0
nn 0.20% 0.35% 0.13% 0.26% Acid Quat Iodophor Chlorine
Peracetic Acid Organic Acid Anionic

S. aureus E. coli
 Effect of Temperature Organic Load
and Concentration on Disinfectant
Activity
Quaternary Disinfectant:
(Log Reduction for P. aeruginosa)
20°C
20°C 10°C
10°C
x0.5
x0.5 x1.0
x1.0 x2.0
x2.0 x0.5
x0.5 x1.0
x1.0 x2.0
x2.0
.03%
.03% F
F PP PP FF FF PP
BSA
BSA
QUAT
1000 ppm .3%
.3% F
F P
P P
P F
F F
F F
F
5 minutes BSA
BSA

P
P (Pass)
(Pass) == >> 55 log
log reduction
reduction
F
F (Fail)
(Fail) == << 55 log
log reduction
reduction

Taylor et al 1999
 Effect of Temperature Organic Load
and Concentration on Disinfectant
Activity
Peracetic Acid Disinfectant:
(Log Reduction for P. aeruginosa)
20°C
20°C 10°C
10°C
x0.5
x0.5 x1.0
x1.0 x2.0
x2.0 x0.5%
x0.5% x1.0
x1.0 x2.0
x2.0
.03%
.03% P
P PP PP PP PP P
P
BSA
BSA
Peracetic
Acid .3%
.3% F
F P
P P
P F
F P
P P
P
300 ppm BSA
BSA
5 minutes
P
P (Pass)
(Pass) == >> 55 log
log reduction
reduction
F
F (Fail)
(Fail) == << 55 log
log reduction
reduction

Taylor et al 1999
 Treatment of a 48 Hour
Biofilm of Pseudomonas aeruginosa*

70
Average (CFU/cm2)

60 1 Minute Contact Time

50
40
30
20
10
0
80 ppm 160 ppm 320 ppm
Peracetic/Organic Acid Peracetic Acid Chlorine

*Fatemi
*Fatemi and
and Frank.
Frank. 1999.
1999. Journal
Journal of
of Food
Food Protection.
Protection. Vol.
Vol. 62
62 (761-765)
(761-765)
 Effect of Relative Humidity on
Sporicidal Activity (B. globigii) of
Formaldehyde

Log
Concentration RH Temp Reduction Time

400 mg/m33 30% 25°C 1 22’

400 mg/m33 98% 25°C 1 9’

Ref:
Ref: Emerging
Emerging Infectious
Infectious Disease
Disease Vol
Vol 99 No
No 66 p.
p. 625
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:
Ref: Emerging
Emerging Infectious
Infectious Disease
Disease Vol
Vol 99 No
No 66 p.
p. 626
626
 Inactivation Of Bacillus spp. By Boiling In
Tap Water
Initial log After 5’
CFU/ml 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
 General Method
Bacillus Food Soil
Spores • Egg Yolk Emulsion 8.8% protein
• Whole Milk 3.5% fat
• Flour Slurry 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