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494
Journal of Food Protection, Vol. 68, No. 3, 2005, Pages 494–498
Copyright Q, International Association for Food Protection
Elimination of
Listeria monocytogenes
Biofilms by Ozone,
Chlorine, and Hydrogen Peroxide
JUSTIN B. ROBBINS, CHRISTOPHER W. FISHER, ANDREW G. MOLTZ,
AND
SCOTT E. MARTIN*
Department of Food Science and Human Nutrition, University of Illinois, 486 Animal Sciences Laboratory, 1207 West Gregory Drive,
Urbana, Illinois 61801, USA
MS 04-219: Received 28 May 2004/Accepted 17 October 2004
ABSTRACT
This study evaluated the efficacy of ozone, chlorine, and hydrogen peroxide to destroy Listeria monocytogenes planktonic
cells and biofilms of two test strains, Scott A and 10403S. L. monocytogenes was sensitive to ozone (O
3
), chlorine, and
hydrogen peroxide (H
2
O
2
). Planktonic cells of strain Scott A were completely destroyed by exposure to 0.25 ppm O
3
(8.29-
log reduction, CFU per milliliter). Ozone’s destruction of Scott A increased when the concentration was increased, with
complete elimination at 4.00 ppm O
3
(8.07-log reduction, CFU per chip). A 16-fold increase in sanitizer concentration was
required to destroy biofilm cells of L. monocytogenes versus planktonic cells of strain Scott A. Strain 10403S required an
ozone concentration of 1.00 ppm to eliminate planktonic cells (8.16-log reduction, CFU per milliliter). Attached cells of the
same strain were eliminated at a concentration of 4.00 ppm O
3
(7.47-log reduction, CFU per chip). At 100 ppm chlorine at
208C, the number of planktonic cells of L. monocytogenes 10403S was reduced by 5.77 log CFU/ml after 5 min of exposure
and by 6.49 log CFU/ml after 10 min of exposure. Biofilm cells were reduced by 5.79 log CFU per chip following exposure
to 100 ppm chlorine at 208C for 5 min, with complete elimination (6.27 log CFU per chip) after exposure to 150 ppm at 208C
for 1 min. A 3% H
2
O
2
solution reduced the initial concentration of L. monocytogenes Scott A planktonic cells by 6.0 log
CFU/ml after 10 min of exposure at 208C, and a 3.5% H
2
O
2
solution reduced the planktonic population by 5.4 and 8.7 log
CFU/ml (complete elimination) after 5 and 10 min of exposure at 208C, respectively. Exposure of cells grown as biofilms to
5% H
2
O
2
resulted in a 4.14-log CFU per chip reduction after 10 min of exposure at 208C and in a 5.58-log CFU per chip
reduction (complete elimination) after 15 min of exposure.
Listeria monocytogenes is a gram-positive, aerobic to
facultative anaerobic bacterium that is a major foodborne
pathogen. Owing to its ubiquitous nature, L. monocytogenes
is frequently isolated from food processing plants. A factor
promoting colonization within a processing environment is
adhesion to a wide range of surfaces including stainless
steel, rubber, glass, and polypropylene (13). L. monocyto-
genes has been found on cooler floors, freezers, processing
rooms, cases, mats (14), and lubricants used in the dairy
industry (20). It has been found in a variety of products
including poultry, milk, cheese, coleslaw, lettuce, and meat
products (14). Biofilms have been defined as ‘‘matrix en-
closed bacterial populations adherent to each other and/or
to surfaces or interfaces’’ (3). This includes cell aggregates
that are attached to, or imbedded into, solid surfaces or that
are suspended in, or on top of, liquids. Kumar and Anand
(9) defined a biofilm as a metabolically active matrix of
cells and extracellular compounds. Donlan and Costerton
(5) further refined the definition of a biofilm as ‘‘a micro-
bially derived sessile community characterized by cells that
are irreversibly attached to a substratum or interface, or to
each other, are embedded in a matrix of extracellular poly-
meric substance that they have produced, and exhibit an
altered phenotype with respect to growth rate and gene tran-
scription.’’
* Author for correspondence. Tel: 217-244-2877; Fax: 217-244-2517;
E-mail: semartn@uiuc.edu.
Chlorine as hypochlorous acid is an active form of
chlorine and is produced by the hydrolysis of chlorine gas
at acidic pH values (17). Studies have shown that chlorine
rapidly inactivates suspended L. monocytogenes cells (2, 6).
Lee and Frank (10) found that L. monocytogenes biofilms
grown on stainless steel surfaces for 8 days were nearly
twice as resistant to chlorine than cells grown for 4 days
and were significantly more heat resistant. Norwood and
Gilmour (16) examined a multispecies biofilm of L. mon-
ocytogenes, Pseudomonas fragi, and Staphylococcus xylo-
sus and found increased chlorine resistance in the multi-
species biofilm compared with monospecies biofilms.
Ozone (O
3
) is one of the most powerful oxidizing agents
known. It is a 52% stronger oxidant than chlorine and acts
more rapidly against a wide spectrum of microorganisms.
Ozone is a protoplasm oxidant, and its bactericidal action
is extremely rapid. Restaino et al. (18) found more than 5-
log unit reductions in Salmonella Typhimurium and Esch-
erichia coli counts after exposure to 0.18 ppm O
3
, while L.
monocytogenes cells were found to be more resistant. Kim
and Yousef (8) reported that exposure of Pseudomonas fluo-
rescens, E. coli, and L. monocytogenes to ozone at 2.5 ppm
for 40 s caused 5- to 6-log decreases in counts. Hydrogen
peroxide (H
2
O
2
) is an effective disinfectant that is used in
the food industry. It is a strong oxidizing agent that has
been shown to damage bacterial proteins, DNA, and cel-
lular membranes (1). Lettuce contaminated with L. mono-
494
Journal of Food Protection, Vol. 68, No. 3, 2005, Pages 494–498
Copyright Q, International Association for Food Protection
Elimination of
Listeria monocytogenes
Biofilms by Ozone,
Chlorine, and Hydrogen Peroxide
JUSTIN B. ROBBINS, CHRISTOPHER W. FISHER, ANDREW G. MOLTZ,
AND
SCOTT E. MARTIN*
Department of Food Science and Human Nutrition, University of Illinois, 486 Animal Sciences Laboratory, 1207 West Gregory Drive,
Urbana, Illinois 61801, USA
MS 04-219: Received 28 May 2004/Accepted 17 October 2004
ABSTRACT
This study evaluated the efficacy of ozone, chlorine, and hydrogen peroxide to destroy Listeria monocytogenes planktonic
cells and biofilms of two test strains, Scott A and 10403S. L. monocytogenes was sensitive to ozone (O
3
), chlorine, and
hydrogen peroxide (H
2
O
2
). Planktonic cells of strain Scott A were completely destroyed by exposure to 0.25 ppm O
3
(8.29-
log reduction, CFU per milliliter). Ozone’s destruction of Scott A increased when the concentration was increased, with
complete elimination at 4.00 ppm O
3
(8.07-log reduction, CFU per chip). A 16-fold increase in sanitizer concentration was
required to destroy biofilm cells of L. monocytogenes versus planktonic cells of strain Scott A. Strain 10403S required an
ozone concentration of 1.00 ppm to eliminate planktonic cells (8.16-log reduction, CFU per milliliter). Attached cells of the
same strain were eliminated at a concentration of 4.00 ppm O
3
(7.47-log reduction, CFU per chip). At 100 ppm chlorine at
208C, the number of planktonic cells of L. monocytogenes 10403S was reduced by 5.77 log CFU/ml after 5 min of exposure
and by 6.49 log CFU/ml after 10 min of exposure. Biofilm cells were reduced by 5.79 log CFU per chip following exposure
to 100 ppm chlorine at 208C for 5 min, with complete elimination (6.27 log CFU per chip) after exposure to 150 ppm at 208C
for 1 min. A 3% H
2
O
2
solution reduced the initial concentration of L. monocytogenes Scott A planktonic cells by 6.0 log
CFU/ml after 10 min of exposure at 208C, and a 3.5% H
2
O
2
solution reduced the planktonic population by 5.4 and 8.7 log
CFU/ml (complete elimination) after 5 and 10 min of exposure at 208C, respectively. Exposure of cells grown as biofilms to
5% H
2
O
2
resulted in a 4.14-log CFU per chip reduction after 10 min of exposure at 208C and in a 5.58-log CFU per chip
reduction (complete elimination) after 15 min of exposure.
Listeria monocytogenes is a gram-positive, aerobic to
facultative anaerobic bacterium that is a major foodborne
pathogen. Owing to its ubiquitous nature, L. monocytogenes
is frequently isolated from food processing plants. A factor
promoting colonization within a processing environment is
adhesion to a wide range of surfaces including stainless
steel, rubber, glass, and polypropylene (13). L. monocyto-
genes has been found on cooler floors, freezers, processing
rooms, cases, mats (14), and lubricants used in the dairy
industry (20). It has been found in a variety of products
including poultry, milk, cheese, coleslaw, lettuce, and meat
products (14). Biofilms have been defined as ‘‘matrix en-
closed bacterial populations adherent to each other and/or
to surfaces or interfaces’’ (3). This includes cell aggregates
that are attached to, or imbedded into, solid surfaces or that
are suspended in, or on top of, liquids. Kumar and Anand
(9) defined a biofilm as a metabolically active matrix of
cells and extracellular compounds. Donlan and Costerton
(5) further refined the definition of a biofilm as ‘‘a micro-
bially derived sessile community characterized by cells that
are irreversibly attached to a substratum or interface, or to
each other, are embedded in a matrix of extracellular poly-
meric substance that they have produced, and exhibit an
altered phenotype with respect to growth rate and gene tran-
scription.’’
* Author for correspondence. Tel: 217-244-2877; Fax: 217-244-2517;
E-mail: semartn@uiuc.edu.
Chlorine as hypochlorous acid is an active form of
chlorine and is produced by the hydrolysis of chlorine gas
at acidic pH values (17). Studies have shown that chlorine
rapidly inactivates suspended L. monocytogenes cells (2, 6).
Lee and Frank (10) found that L. monocytogenes biofilms
grown on stainless steel surfaces for 8 days were nearly
twice as resistant to chlorine than cells grown for 4 days
and were significantly more heat resistant. Norwood and
Gilmour (16) examined a multispecies biofilm of L. mon-
ocytogenes, Pseudomonas fragi, and Staphylococcus xylo-
sus and found increased chlorine resistance in the multi-
species biofilm compared with monospecies biofilms.
Ozone (O
3
) is one of the most powerful oxidizing agents
known. It is a 52% stronger oxidant than chlorine and acts
more rapidly against a wide spectrum of microorganisms.
Ozone is a protoplasm oxidant, and its bactericidal action
is extremely rapid. Restaino et al. (18) found more than 5-
log unit reductions in Salmonella Typhimurium and Esch-
erichia coli counts after exposure to 0.18 ppm O
3
, while L.
monocytogenes cells were found to be more resistant. Kim
and Yousef (8) reported that exposure of Pseudomonas fluo-
rescens, E. coli, and L. monocytogenes to ozone at 2.5 ppm
for 40 s caused 5- to 6-log decreases in counts. Hydrogen
peroxide (H
2
O
2
) is an effective disinfectant that is used in
the food industry. It is a strong oxidizing agent that has
been shown to damage bacterial proteins, DNA, and cel-
lular membranes (1). Lettuce contaminated with L. mono-
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/3/494/1673258/0362-028x-68_3_494.pdf by Brazil user on 12 January 2023
J. Food Prot., Vol. 68, No. 3 LISTERIA BIOFILM ELIMINATION 495
cytogenes, E. coli O157:H7, and Salmonella enterica and
treated with lactic acid, a hydrogen peroxide–based sanitiz-
er, and a mild heat treatment was found to significantly
reduce microbial contamination (12). Dominiguez et al. (4)
reported the bactericidal effect of hydrogen peroxide was
enhanced with mild heat. Attached cells of L. monocyto-
genes are more resistant to sanitizers than their planktonic
(unattached) counterparts (16). The most problematic phe-
nomenon associated with biofilms is enhanced resistance to
sanitizers (7, 15, 16). A biofilm that has withstood the
cleaning process can potentially shed bacteria. In the case
of foodborne pathogens, these shedding cells can continue
to contaminate product lines even after a sanitizer has been
used (20).
The purpose of this study was to examine the effec-
tiveness of ozone, chlorine, and hydrogen peroxide on the
destruction of planktonic and biofilm cells of L. monocy-
togenes.
MATERIALS AND METHODS
Bacterial strains. L. monocytogenes strain 10403S was ob-
tained from Dr. Daniel A. Portnoy, University of California,
Berkeley; L. monocytogenes strain Scott A was obtained from Dr.
Larry Beuchat, University of Georgia, Griffin.
Growth conditions. Frozen stocks of the cultures were pre-
pared by inoculating 10 ml of tryptic soy broth (TSB; Difco, Bec-
ton Dickinson, Sparks, Md.) with 0.1 ml of an overnight station-
ary-phase inoculum. These tubes were then vortexed, frozen, and
stored at 2208C. As needed, stocks were thawed and inoculated
into 250-ml Erlenmeyer flasks containing 90 ml of TSB (Difco,
Becton Dickinson) and grown at 378C in a gyratory shaking water
bath (New Brunswick Scientific, Edison, N.J.) to stationary phase,
which corresponded to an optical density at 600 nm (OD
600
)of
1.0 to 1.1 for approximately 12 h of growth.
Stainless steel chip preparation. The wash procedure was
modified from Lee and Frank (10). Stainless steel (4 grade) was
fabricated into chips (2.54 by 2.54 cm) giving a total surface area
of 6.45 cm
2
. Chips were vigorously washed in Fisherbrand Spark-
leen for manual washing (Fisher Scientific, Pittsburgh, Pa). After
washing, distilled rinses were carried out (33) in 400 ml of dis-
tilled water. Four chips were placed in Pyrex glass petri dishes
and autoclaved for 15 min at 1218C.
Attachment and biofilm development. The procedure was
a modification of that described by Leriche and Carpentier (11).
An overnight culture (early stationary phase; 0.1 ml) was placed
on each stainless steel chip and spread evenly over the surface of
the chip using a sterile inoculating loop. The glass petri dishes
were then placed in desiccators at 208C and at 100% relative hu-
midity to prevent evaporation of the medium from the chips. After
3 h the chips were removed and washed with 25 ml of sterile
potassium phosphate buffer (PPB; 50 mM adjusted to pH 7.0).
This wash step removed planktonic cells, leaving behind only at-
tached cells. After washing, 0.1 ml of sterile TSB was added to
each chip and the chips were placed back into the desiccators.
Every 24 h the wash step was repeated and additional medium
was added daily for 4 days (stationary phase).
Ozone treatment of planktonic cells. An infinity corona dis-
charge ozone generator (CD-7, Del Industries, San Luis Obispo,
Calif.) and an ozone sensor (1054B, Rosemount Analytical, Ir-
vine, Calif.) were used to generate and detect levels of ozone. The
sensor was calibrated in distilled water. When an OD
600
of 1.0
was obtained, cells were harvested by centrifugation (16,300 3g
for 10 min at 48C) and suspended into 100 ml of sterile PPB.
Ozone was diffused in a 1-liter spinner flask with 900 ml of sterile
PPB at the proper ozone concentration. The suspended culture was
then added to this flask and exposed to the ozone for 3 min. A
sample solution (10 ml) was removed from the flask and placed
into 90 ml of 0.1% peptone water and serially diluted and plated
on tryptic soy agar plates (TSA; Difco, Becton Dickinson). These
plates were incubated at 378C for 24 h. Results were reported as
CFU per milliliter.
Ozone treatment of biofilm cells. Biofilm inoculated chips
were submerged in the ozonated PPB for 3 min. After exposure,
the chips were immediately swabbed with a sterile cotton-tipped
applicator, which was first wetted in sterile 0.1% peptone water
(Difco, Becton Dickinson). Removed cells were suspended in 10
ml of 0.1% peptone water and serially diluted and plated on TSA.
These plates were incubated at 378C and were read after 24 h of
incubation. Results were reported as CFU per chip.
Chlorine treatment of planktonic and biofilm cells. Levels
of chlorine, as calcium hypochlorite (Fisher Scientific), were de-
termined by a colorometric commercial test kit for both planktonic
and biofilm cell experiments (LaMotte Co., Chestertown, Md.).
Chlorine solutions (ppm free available chlorine) at the desired
concentrations were prepared in distilled water (pH 10.8) or sterile
0.85% saline. The swabbing procedure was used for the removal
of cells following chlorine treatment of the chips. The same pro-
cedure for cultivating and harvesting cells as described above was
used. Exposure to chlorine was carried out in these sets of exper-
iments, but prior to swabbing residual active chlorine was neu-
tralized with 10 ml of a 10% sodium thiosulfate solution. Serial
dilutions and plating were performed as described above.
Hydrogen peroxide treatment of planktonic and biofilm
cells. Hydrogen peroxide (Fisherbrand, Fisher Scientific) at the
desired concentrations was prepared by appropriate dilution of the
30% stock solution in sterile 0.85% saline solution at 48C. The
same procedure for cultivating and harvesting cells as described
above was used. Exposure to hydrogen peroxide was carried out
in these sets of experiments, but prior to swabbing residual hy-
drogen peroxide was neutralized by adding the chips to 100 ml
of a 1% sodium pyruvate solution. Serial dilutions and plating
were performed as described above.
Statistical analyses. Statistical analyses were performed us-
ing a StatView 5121Version 1.2 (Brain Power Inc., Calabasas,
Calif.). A one-way analysis of variance was used to determine any
significant differences between the tested strains. The means and
standard errors of the means of triplicate and quadruplicate ex-
periments are shown in Table 1.
RESULTS AND DISCUSSION
Ozone inactivation of planktonic and biofilm cells.
As shown in Table 1, both strains of L. monocytogenes were
sensitive to ozone in both the planktonic and biofilm states.
In the unattached state, strain Scott A was completely de-
stroyed by exposure to 0.25 ppm O
3
(8.29-log reduction,
CFU per milliliter). Ozone’s destruction of Scott A biofilms
increased, when the concentration was increased, with com-
plete elimination at 4.00 ppm O
3
(8.07-log reduction, CFU
per chip). A 16-fold increase in sanitizer concentration was
required to destroy attached cells of L. monocytogenes ver-
J. Food Prot., Vol. 68, No. 3 LISTERIA BIOFILM ELIMINATION 495
cytogenes, E. coli O157:H7, and Salmonella enterica and
treated with lactic acid, a hydrogen peroxide–based sanitiz-
er, and a mild heat treatment was found to significantly
reduce microbial contamination (12). Dominiguez et al. (4)
reported the bactericidal effect of hydrogen peroxide was
enhanced with mild heat. Attached cells of L. monocyto-
genes are more resistant to sanitizers than their planktonic
(unattached) counterparts (16). The most problematic phe-
nomenon associated with biofilms is enhanced resistance to
sanitizers (7, 15, 16). A biofilm that has withstood the
cleaning process can potentially shed bacteria. In the case
of foodborne pathogens, these shedding cells can continue
to contaminate product lines even after a sanitizer has been
used (20).
The purpose of this study was to examine the effec-
tiveness of ozone, chlorine, and hydrogen peroxide on the
destruction of planktonic and biofilm cells of L. monocy-
togenes.
MATERIALS AND METHODS
Bacterial strains. L. monocytogenes strain 10403S was ob-
tained from Dr. Daniel A. Portnoy, University of California,
Berkeley; L. monocytogenes strain Scott A was obtained from Dr.
Larry Beuchat, University of Georgia, Griffin.
Growth conditions. Frozen stocks of the cultures were pre-
pared by inoculating 10 ml of tryptic soy broth (TSB; Difco, Bec-
ton Dickinson, Sparks, Md.) with 0.1 ml of an overnight station-
ary-phase inoculum. These tubes were then vortexed, frozen, and
stored at 2208C. As needed, stocks were thawed and inoculated
into 250-ml Erlenmeyer flasks containing 90 ml of TSB (Difco,
Becton Dickinson) and grown at 378C in a gyratory shaking water
bath (New Brunswick Scientific, Edison, N.J.) to stationary phase,
which corresponded to an optical density at 600 nm (OD
600
)of
1.0 to 1.1 for approximately 12 h of growth.
Stainless steel chip preparation. The wash procedure was
modified from Lee and Frank (10). Stainless steel (4 grade) was
fabricated into chips (2.54 by 2.54 cm) giving a total surface area
of 6.45 cm
2
. Chips were vigorously washed in Fisherbrand Spark-
leen for manual washing (Fisher Scientific, Pittsburgh, Pa). After
washing, distilled rinses were carried out (33) in 400 ml of dis-
tilled water. Four chips were placed in Pyrex glass petri dishes
and autoclaved for 15 min at 1218C.
Attachment and biofilm development. The procedure was
a modification of that described by Leriche and Carpentier (11).
An overnight culture (early stationary phase; 0.1 ml) was placed
on each stainless steel chip and spread evenly over the surface of
the chip using a sterile inoculating loop. The glass petri dishes
were then placed in desiccators at 208C and at 100% relative hu-
midity to prevent evaporation of the medium from the chips. After
3 h the chips were removed and washed with 25 ml of sterile
potassium phosphate buffer (PPB; 50 mM adjusted to pH 7.0).
This wash step removed planktonic cells, leaving behind only at-
tached cells. After washing, 0.1 ml of sterile TSB was added to
each chip and the chips were placed back into the desiccators.
Every 24 h the wash step was repeated and additional medium
was added daily for 4 days (stationary phase).
Ozone treatment of planktonic cells. An infinity corona dis-
charge ozone generator (CD-7, Del Industries, San Luis Obispo,
Calif.) and an ozone sensor (1054B, Rosemount Analytical, Ir-
vine, Calif.) were used to generate and detect levels of ozone. The
sensor was calibrated in distilled water. When an OD
600
of 1.0
was obtained, cells were harvested by centrifugation (16,300 3g
for 10 min at 48C) and suspended into 100 ml of sterile PPB.
Ozone was diffused in a 1-liter spinner flask with 900 ml of sterile
PPB at the proper ozone concentration. The suspended culture was
then added to this flask and exposed to the ozone for 3 min. A
sample solution (10 ml) was removed from the flask and placed
into 90 ml of 0.1% peptone water and serially diluted and plated
on tryptic soy agar plates (TSA; Difco, Becton Dickinson). These
plates were incubated at 378C for 24 h. Results were reported as
CFU per milliliter.
Ozone treatment of biofilm cells. Biofilm inoculated chips
were submerged in the ozonated PPB for 3 min. After exposure,
the chips were immediately swabbed with a sterile cotton-tipped
applicator, which was first wetted in sterile 0.1% peptone water
(Difco, Becton Dickinson). Removed cells were suspended in 10
ml of 0.1% peptone water and serially diluted and plated on TSA.
These plates were incubated at 378C and were read after 24 h of
incubation. Results were reported as CFU per chip.
Chlorine treatment of planktonic and biofilm cells. Levels
of chlorine, as calcium hypochlorite (Fisher Scientific), were de-
termined by a colorometric commercial test kit for both planktonic
and biofilm cell experiments (LaMotte Co., Chestertown, Md.).
Chlorine solutions (ppm free available chlorine) at the desired
concentrations were prepared in distilled water (pH 10.8) or sterile
0.85% saline. The swabbing procedure was used for the removal
of cells following chlorine treatment of the chips. The same pro-
cedure for cultivating and harvesting cells as described above was
used. Exposure to chlorine was carried out in these sets of exper-
iments, but prior to swabbing residual active chlorine was neu-
tralized with 10 ml of a 10% sodium thiosulfate solution. Serial
dilutions and plating were performed as described above.
Hydrogen peroxide treatment of planktonic and biofilm
cells. Hydrogen peroxide (Fisherbrand, Fisher Scientific) at the
desired concentrations was prepared by appropriate dilution of the
30% stock solution in sterile 0.85% saline solution at 48C. The
same procedure for cultivating and harvesting cells as described
above was used. Exposure to hydrogen peroxide was carried out
in these sets of experiments, but prior to swabbing residual hy-
drogen peroxide was neutralized by adding the chips to 100 ml
of a 1% sodium pyruvate solution. Serial dilutions and plating
were performed as described above.
Statistical analyses. Statistical analyses were performed us-
ing a StatView 5121Version 1.2 (Brain Power Inc., Calabasas,
Calif.). A one-way analysis of variance was used to determine any
significant differences between the tested strains. The means and
standard errors of the means of triplicate and quadruplicate ex-
periments are shown in Table 1.
RESULTS AND DISCUSSION
Ozone inactivation of planktonic and biofilm cells.
As shown in Table 1, both strains of L. monocytogenes were
sensitive to ozone in both the planktonic and biofilm states.
In the unattached state, strain Scott A was completely de-
stroyed by exposure to 0.25 ppm O
3
(8.29-log reduction,
CFU per milliliter). Ozone’s destruction of Scott A biofilms
increased, when the concentration was increased, with com-
plete elimination at 4.00 ppm O
3
(8.07-log reduction, CFU
per chip). A 16-fold increase in sanitizer concentration was
required to destroy attached cells of L. monocytogenes ver-
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/3/494/1673258/0362-028x-68_3_494.pdf by Brazil user on 12 January 2023
J. Food Prot., Vol. 68, No. 3496 ROBBINS ET AL.
TABLE 1. Log reduction of Listeria monocytogenes cells after 3 min of exposure to varying levels of ozone in PPB at 24
8
C
Mean log reduction (CFU/chip or CFU/ml) 6SEM
Ozone
concentra-
tion (ppm)
Scott A
Biofilm Unattached
10403S
Biofilm Unattached
0.25
0.50
1.00
2.00
4.00
1.48 60.09
4.03 60.24
4.34 60.08
4.51 60.03
8.07 60.04
a
8.29 60.05
a
ND
a,b
ND
a
ND
a
ND
a
2.65 60.11
2.75 60.57
4.02 60.54
3.97 60.21
7.47 60.07
a
1.93 60.21
3.47 60.06
8.16 60.13
a
ND
a
ND
a
a
Complete elimination was observed.
b
ND, not detected.
FIGURE 1. (Top) Effects of three concen-
trations of calcium hypochlorite on plank-
tonic cells of Listeria monocytogenes
10403S treated at 20
8
C. (Bottom) Effects
of three concentrations of calcium hypo-
chlorite on biofilm cells of Listeria mono-
cytogenes 10403S treated at 20
8
C.
sus unattached cells of strain Scott A. Strain 10403S re-
quired an ozone concentration of 1.00 ppm to eliminate
planktonic cells (8.16-log reduction, CFU per milliliter).
Attached cells of the same strain were eliminated at a con-
centration of 4.00 ppm O
3
(7.47-log reduction, CFU per
chip). A fourfold increase in sanitizer concentration was
required to destroy biofilm cells of L. monocytogenes ver-
sus unattached cells of strain 10403S.
Chlorine inactivation of planktonic and biofilm
cells. The results for the chlorine inactivation of planktonic
and biofilm cells of L. monocytogenes 10403S are shown
J. Food Prot., Vol. 68, No. 3496 ROBBINS ET AL.
TABLE 1. Log reduction of Listeria monocytogenes cells after 3 min of exposure to varying levels of ozone in PPB at 24
8
C
Mean log reduction (CFU/chip or CFU/ml) 6SEM
Ozone
concentra-
tion (ppm)
Scott A
Biofilm Unattached
10403S
Biofilm Unattached
0.25
0.50
1.00
2.00
4.00
1.48 60.09
4.03 60.24
4.34 60.08
4.51 60.03
8.07 60.04
a
8.29 60.05
a
ND
a,b
ND
a
ND
a
ND
a
2.65 60.11
2.75 60.57
4.02 60.54
3.97 60.21
7.47 60.07
a
1.93 60.21
3.47 60.06
8.16 60.13
a
ND
a
ND
a
a
Complete elimination was observed.
b
ND, not detected.
FIGURE 1. (Top) Effects of three concen-
trations of calcium hypochlorite on plank-
tonic cells of Listeria monocytogenes
10403S treated at 20
8
C. (Bottom) Effects
of three concentrations of calcium hypo-
chlorite on biofilm cells of Listeria mono-
cytogenes 10403S treated at 20
8
C.
sus unattached cells of strain Scott A. Strain 10403S re-
quired an ozone concentration of 1.00 ppm to eliminate
planktonic cells (8.16-log reduction, CFU per milliliter).
Attached cells of the same strain were eliminated at a con-
centration of 4.00 ppm O
3
(7.47-log reduction, CFU per
chip). A fourfold increase in sanitizer concentration was
required to destroy biofilm cells of L. monocytogenes ver-
sus unattached cells of strain 10403S.
Chlorine inactivation of planktonic and biofilm
cells. The results for the chlorine inactivation of planktonic
and biofilm cells of L. monocytogenes 10403S are shown
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J. Food Prot., Vol. 68, No. 3 LISTERIA BIOFILM ELIMINATION 497
FIGURE 2. (Top) Effects of two concen-
trations of hydrogen peroxide on plankton-
ic cells of Listeria monocytogenes 10403S
treated at 20
8
C. (Bottom) Effects of three
concentrations of hydrogen peroxide on
biofilm cells of Listeria monocytogenes
10403S treated at 20
8
C.
in Figure 1. At 100 ppm chlorine at 208C, the number of
planktonic cells was reduced by 5.77 log CFU/ml after 5
min of exposure and by 6.49 log CFU/ml after 10 min of
exposure. Biofilm cells of L. monocytogenes 10403S were
reduced by 5.79 log CFU per chip following exposure to
100 ppm chlorine at 208C for 5 min, with complete elimi-
nation (6.27 log CFU per chip) after exposure to 150 ppm
at 208C for 1 min. Norwood and Gilmour (16) found that
10 ppm chlorine was sufficient to inactivate 10
7
CFU/ml L.
monocytogenes Scott A planktonic cells, but that 500 ppm
chlorine was required to cause a statistically significant fall
in number of biofilm-associated cells. The discrepancy in
the chlorine concentration necessary to inactivate plankton-
ic cells in this study may be explained by the pH of the
chlorine solution. In the present study, the pH of the chlo-
rine solution in distilled water was 10.8, while in the Nor-
wood and Gilmour (16) study, the planktonic inactivation
was performed in phosphate-buffered saline, at an unde-
fined pH. The discrepancy in the chlorine concentration
necessary to inactivate biofilm cells may be explained by
use of a different strain (Scott A versus 10403S) and the
use of a multispecies biofilm. The results of this study con-
cur with studies by Mustapha and Liewen (15) and Stop-
forth et al. (19), who found 100 and 200 ppm chlorine (as
sodium hypochlorite) to be effective in the inactivation of
L. monocytogenes biofilms attached to stainless steel.
Hydrogen peroxide inactivation of planktonic and
biofilm cells. The results for the hydrogen peroxide inac-
tivation of planktonic and biofilm cells of L. monocytogenes
Scott A are shown in Figure 2. A 3% H
2
O
2
solution re-
duced the initial concentration by 6.0 log CFU/ml after 10
min of exposure at 208C, and a 3.5% H
2
O
2
solution reduced
the planktonic population by 5.4 and 8.7 log CFU/ml (com-
plete elimination) after 5 and 10 min of exposure at 208C,
respectively. Exposure of L. monocytogenes Scott A cells
grown as biofilms on stainless steel chips to 5% H
2
O
2
re-
sulted in a 4.14-log CFU per chip reduction after 10 min
of exposure at 208C and in a 5.58-log CFU per chip re-
duction (complete elimination) after 15 min of exposure.
Six percent H
2
O
2
caused 5.06 and 5.58 log CFU per chip
(complete elimination) reductions following exposure for
J. Food Prot., Vol. 68, No. 3 LISTERIA BIOFILM ELIMINATION 497
FIGURE 2. (Top) Effects of two concen-
trations of hydrogen peroxide on plankton-
ic cells of Listeria monocytogenes 10403S
treated at 20
8
C. (Bottom) Effects of three
concentrations of hydrogen peroxide on
biofilm cells of Listeria monocytogenes
10403S treated at 20
8
C.
in Figure 1. At 100 ppm chlorine at 208C, the number of
planktonic cells was reduced by 5.77 log CFU/ml after 5
min of exposure and by 6.49 log CFU/ml after 10 min of
exposure. Biofilm cells of L. monocytogenes 10403S were
reduced by 5.79 log CFU per chip following exposure to
100 ppm chlorine at 208C for 5 min, with complete elimi-
nation (6.27 log CFU per chip) after exposure to 150 ppm
at 208C for 1 min. Norwood and Gilmour (16) found that
10 ppm chlorine was sufficient to inactivate 10
7
CFU/ml L.
monocytogenes Scott A planktonic cells, but that 500 ppm
chlorine was required to cause a statistically significant fall
in number of biofilm-associated cells. The discrepancy in
the chlorine concentration necessary to inactivate plankton-
ic cells in this study may be explained by the pH of the
chlorine solution. In the present study, the pH of the chlo-
rine solution in distilled water was 10.8, while in the Nor-
wood and Gilmour (16) study, the planktonic inactivation
was performed in phosphate-buffered saline, at an unde-
fined pH. The discrepancy in the chlorine concentration
necessary to inactivate biofilm cells may be explained by
use of a different strain (Scott A versus 10403S) and the
use of a multispecies biofilm. The results of this study con-
cur with studies by Mustapha and Liewen (15) and Stop-
forth et al. (19), who found 100 and 200 ppm chlorine (as
sodium hypochlorite) to be effective in the inactivation of
L. monocytogenes biofilms attached to stainless steel.
Hydrogen peroxide inactivation of planktonic and
biofilm cells. The results for the hydrogen peroxide inac-
tivation of planktonic and biofilm cells of L. monocytogenes
Scott A are shown in Figure 2. A 3% H
2
O
2
solution re-
duced the initial concentration by 6.0 log CFU/ml after 10
min of exposure at 208C, and a 3.5% H
2
O
2
solution reduced
the planktonic population by 5.4 and 8.7 log CFU/ml (com-
plete elimination) after 5 and 10 min of exposure at 208C,
respectively. Exposure of L. monocytogenes Scott A cells
grown as biofilms on stainless steel chips to 5% H
2
O
2
re-
sulted in a 4.14-log CFU per chip reduction after 10 min
of exposure at 208C and in a 5.58-log CFU per chip re-
duction (complete elimination) after 15 min of exposure.
Six percent H
2
O
2
caused 5.06 and 5.58 log CFU per chip
(complete elimination) reductions following exposure for
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/3/494/1673258/0362-028x-68_3_494.pdf by Brazil user on 12 January 2023
J. Food Prot., Vol. 68, No. 3498 ROBBINS ET AL.
10 and 15 min, respectively, at 208C. Unlike chlorine, the
efficacy of hydrogen peroxide treatment is relatively unaf-
fected by high organic loads. Lin et al. (12) showed that
treatment of iceberg lettuce with 2% H
2
O
2
at 508C resulted
in a 3-log reduction of L. monocytogenes.
Results from this study show that ozone, chlorine, and
hydrogen peroxide were able to kill both planktonic and
biofilm cells of L. monocytogenes. In general, biofilm cells
were more resistant to the three disinfectants than were
planktonic cells.
ACKNOWLEDGMENT
This work was supported by U.S. Department of Agriculture award
98-35201-6217.
REFERENCES
1. Bell, K. Y., C. N. Cutter, and S. S. Sumner. 1997. Reduction of
foodborne microorganisms on beef carcass tissue using acetic acid,
sodium bicarbonate and hydrogen peroxide spray washes. Food Mi-
crobiol. 14:439–448.
2. Brackett, R. E. 1987. Antimicrobial effect of chlorine on Listeria
monocytogenes. J. Food Prot. 50:999–1003.
3. Costerton, J. W., Z. Lewandowski, D. Caldwell, D. Korber, and H.
Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711–745.
4. Dominguez, L., J. F. F. Garayazabal, E. R. Ferri, J. A. Vazquez, E.
Gomez-Lucia, C. Ambrosio, and G. Suarez. 1987. Viability of Lis-
teria monocytogenes on milk treated with hydrogen peroxide. J.
Food Prot. 50:636–639.
5. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mech-
anisms of clinically relevant microorganisms. Clin. Microbiol. Rev.
15:167–193.
6. El-Kest, S. E., and E. H. Marth. 1988. Inactivation of Listeria mon-
ocytogenes by chlorine. J. Food Prot. 51:520–524.
7. Frank, J. F., and R. A. Koffi. 1990. Surface-adherent growth of Lis-
teria monocytogenes is associated with increased resistance to sur-
factant sanitizers and heat. J. Food Prot. 53:550–554.
8. Kim, J. G., and A. E. Yousef. 2000. Inactivation kinetics of food
spoilage and pathogenic bacteria by ozone. J. Food Sci. 65:521–528.
9. Kumar, C. G., and S. K. Anand. 1998. Significance of microbial
biofilms in the food industry: a review. Int. J. Food Microbiol. 42:
9–27.
10. Lee, S. H., and J. F. Frank. 1991. Inactivation of surface-adherent
Listeria monocytogenes by hypochlorite and heat. J. Food Prot. 54:
4–6.
11. Leriche, V., and B. Carpentier. 2000. Limitation of adhesion and
growth of Listeria monocytogenes on stainless steel surfaces by
Staphylococcus sciuri biofilms. J. Appl. Microbiol. 88:594–605.
12. Lin, C. M., S. S. Moon, M. P. Doyle, and K. H. McWatters. 2002.
Inactivation of Escherichia coli O157:H7, Salmonella enterica se-
rotype Enteritidis and Listeria monocytogenes on lettuce by hydro-
gen peroxide and lactic acid and by hydrogen peroxide with mild
heat. J. Food Prot. 65: 1215–1220.
13. Lunden, J. M., M. K. Miettinen, T. J. Autio, and H. J. Korkeala.
2000. Persistent Listeria monocytogenes strains show enhanced ad-
herence to food contact surface after short contact times. J. Food
Prot. 63:1204–1207.
14. Martin, S. E., and C. W. Fisher. 1999. Listeria monocytogenes, p.
1228–1238. In R. Robinson, C. Batt, and P. Patel (ed.), The ency-
clopedia of food microbiology. Academic Press, New York.
15. Mustapha, A., and M. B. Liewen. 1989. Destruction of Listeria mon-
ocytogenes by sodium hypochlorite and quaternary ammonium san-
itizers. J. Food Prot. 52:306–311.
16. Norwood, D. E., and A. Gilmour. 2000. The growth and resistance
to sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm. J. Appl. Microbiol. 88:512–520.
17. Odlaug, T. E. 1981. Antibacterial activity of halogens. J. Food Prot.
44:608–613.
18. Restaino, L., E. W. Frampton, J. B. Hemphill, and P. Palnikar. 1995.
Efficacy of ozonated water against various food-related microorgan-
isms. Appl. Environ. Microbiol. 61:3471–3475.
19. Stopforth, J. D., J. Samelis, J. N. Sofos, P. A. Kendall, and G. C.
Smith. 2002. Biofilm formation by acid-adapted and nonadapted Lis-
teria monocytogenes in fresh beef decontamination washings and its
subsequent inactivation with sanitizers. J. Food Prot. 65:1717–1727.
20. Wirtanen, G., M. Saarela, and T. Mattila-Sandholm. 2000. Biof-
ilms—impact in hygiene in food industries, p. 327–372. In J. D.
Bryers (ed.), Biofilms II process analysis and applications. Wiley-
Less, New York.
J. Food Prot., Vol. 68, No. 3498 ROBBINS ET AL.
10 and 15 min, respectively, at 208C. Unlike chlorine, the
efficacy of hydrogen peroxide treatment is relatively unaf-
fected by high organic loads. Lin et al. (12) showed that
treatment of iceberg lettuce with 2% H
2
O
2
at 508C resulted
in a 3-log reduction of L. monocytogenes.
Results from this study show that ozone, chlorine, and
hydrogen peroxide were able to kill both planktonic and
biofilm cells of L. monocytogenes. In general, biofilm cells
were more resistant to the three disinfectants than were
planktonic cells.
ACKNOWLEDGMENT
This work was supported by U.S. Department of Agriculture award
98-35201-6217.
REFERENCES
1. Bell, K. Y., C. N. Cutter, and S. S. Sumner. 1997. Reduction of
foodborne microorganisms on beef carcass tissue using acetic acid,
sodium bicarbonate and hydrogen peroxide spray washes. Food Mi-
crobiol. 14:439–448.
2. Brackett, R. E. 1987. Antimicrobial effect of chlorine on Listeria
monocytogenes. J. Food Prot. 50:999–1003.
3. Costerton, J. W., Z. Lewandowski, D. Caldwell, D. Korber, and H.
Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711–745.
4. Dominguez, L., J. F. F. Garayazabal, E. R. Ferri, J. A. Vazquez, E.
Gomez-Lucia, C. Ambrosio, and G. Suarez. 1987. Viability of Lis-
teria monocytogenes on milk treated with hydrogen peroxide. J.
Food Prot. 50:636–639.
5. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mech-
anisms of clinically relevant microorganisms. Clin. Microbiol. Rev.
15:167–193.
6. El-Kest, S. E., and E. H. Marth. 1988. Inactivation of Listeria mon-
ocytogenes by chlorine. J. Food Prot. 51:520–524.
7. Frank, J. F., and R. A. Koffi. 1990. Surface-adherent growth of Lis-
teria monocytogenes is associated with increased resistance to sur-
factant sanitizers and heat. J. Food Prot. 53:550–554.
8. Kim, J. G., and A. E. Yousef. 2000. Inactivation kinetics of food
spoilage and pathogenic bacteria by ozone. J. Food Sci. 65:521–528.
9. Kumar, C. G., and S. K. Anand. 1998. Significance of microbial
biofilms in the food industry: a review. Int. J. Food Microbiol. 42:
9–27.
10. Lee, S. H., and J. F. Frank. 1991. Inactivation of surface-adherent
Listeria monocytogenes by hypochlorite and heat. J. Food Prot. 54:
4–6.
11. Leriche, V., and B. Carpentier. 2000. Limitation of adhesion and
growth of Listeria monocytogenes on stainless steel surfaces by
Staphylococcus sciuri biofilms. J. Appl. Microbiol. 88:594–605.
12. Lin, C. M., S. S. Moon, M. P. Doyle, and K. H. McWatters. 2002.
Inactivation of Escherichia coli O157:H7, Salmonella enterica se-
rotype Enteritidis and Listeria monocytogenes on lettuce by hydro-
gen peroxide and lactic acid and by hydrogen peroxide with mild
heat. J. Food Prot. 65: 1215–1220.
13. Lunden, J. M., M. K. Miettinen, T. J. Autio, and H. J. Korkeala.
2000. Persistent Listeria monocytogenes strains show enhanced ad-
herence to food contact surface after short contact times. J. Food
Prot. 63:1204–1207.
14. Martin, S. E., and C. W. Fisher. 1999. Listeria monocytogenes, p.
1228–1238. In R. Robinson, C. Batt, and P. Patel (ed.), The ency-
clopedia of food microbiology. Academic Press, New York.
15. Mustapha, A., and M. B. Liewen. 1989. Destruction of Listeria mon-
ocytogenes by sodium hypochlorite and quaternary ammonium san-
itizers. J. Food Prot. 52:306–311.
16. Norwood, D. E., and A. Gilmour. 2000. The growth and resistance
to sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm. J. Appl. Microbiol. 88:512–520.
17. Odlaug, T. E. 1981. Antibacterial activity of halogens. J. Food Prot.
44:608–613.
18. Restaino, L., E. W. Frampton, J. B. Hemphill, and P. Palnikar. 1995.
Efficacy of ozonated water against various food-related microorgan-
isms. Appl. Environ. Microbiol. 61:3471–3475.
19. Stopforth, J. D., J. Samelis, J. N. Sofos, P. A. Kendall, and G. C.
Smith. 2002. Biofilm formation by acid-adapted and nonadapted Lis-
teria monocytogenes in fresh beef decontamination washings and its
subsequent inactivation with sanitizers. J. Food Prot. 65:1717–1727.
20. Wirtanen, G., M. Saarela, and T. Mattila-Sandholm. 2000. Biof-
ilms—impact in hygiene in food industries, p. 327–372. In J. D.
Bryers (ed.), Biofilms II process analysis and applications. Wiley-
Less, New York.
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/3/494/1673258/0362-028x-68_3_494.pdf by Brazil user on 12 January 2023
