Efficacy of Antimicrobial Agents in Lettuce Leaf Processing Water for Control of Escherichia coli O157:H7

Abstract

The objectives of this research were to study transfer and control of Escherichia coli O157:H7 during simultaneous washing of inoculated and uninoculated lettuce pieces and to determine the efficacy of antimicrobial agents (peroxyacetic acid, mixed peracid, and sodium hypochlorite) on reducing the transfer of E. coli O157:H7 through processing water with or without organic load. Lettuce leaf pieces (5 by 5 cm) were inoculated with a five-strain mixture of green fluorescent protein-labeled E. coli O157:H7 at 5.6 log CFU per piece. One inoculated lettuce piece was added to five uninoculated leaves during washing. Peroxyacetic acid and mixed peracid were tested at 10, 20, and 30 ppm, and chlorine was tested at 30 and 50 ppm. No organic load (liquefied lettuce leaves) and 10% organic load in processing water were compared. Without organic load, peroxyacetic acid at 30 ppm, mixed peracid at 10, 20, and 30 ppm, and chlorine at 30 and 50 ppm all significantly reduced E. coli O157: H7 in processing water by 1.83, 1.73, 1.50, 1.83, 1.34, and 1.83 log CFU/ml, respectively, compared with washing with water alone. These antimicrobials at all concentrations tested also significantly reduced transfer of the bacteria from an inoculated leaf to uninoculated leaves in the processing water by 0.96 to 2.57 log CFU per piece. A 10% organic load in the processing water reduced efficacy of antimicrobial agents. In this contaminated water, peroxyacetic acid at 10 and 20 ppm and chlorine at 30 ppm produced effects not significantly different from those of water alone. Therefore, it is important to understand the impact of organic load when validating the effectiveness of antimicrobial treatments.

Full text

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Journal of Food Protection, Vol. 72, No. 7, 2009, Pages 1392–1397
Copyright 䊚, International Association for Food Protection
Efficacy of Antimicrobial Agents in Lettuce Leaf Processing
Water for Control of Escherichia coli O157:H7
GUODONG ZHANG, LI MA, VANESSA H. PHELAN,
AND
MICHAEL P. DOYLE*
Center for Food Safety, University of Georgia, Griffin, Georgia 30223-1797, USA
MS 08-573: Received 18 November 2008/Accepted 7 February 2009
ABSTRACT
The objectives of this research were to study transfer and control of Escherichia coli O157:H7 during simultaneous
washing of inoculated and uninoculated lettuce pieces and to determine the efficacy of antimicrobial agents (peroxyacetic acid,
mixed peracid, and sodium hypochlorite) on reducing the transfer of E. coli O157:H7 through processing water with or without
organic load. Lettuce leaf pieces (5 by 5 cm) were inoculated with a five-strain mixture of green fluorescent protein–labeled
E. coli O157:H7 at 5.6 log CFU per piece. One inoculated lettuce piece was added to five uninoculated leaves during washing.
Peroxyacetic acid and mixed peracid were tested at 10, 20, and 30 ppm, and chlorine was tested at 30 and 50 ppm. No organic
load (liquefied lettuce leaves) and 10% organic load in processing water were compared. Without organic load, peroxyacetic
acid at 30 ppm, mixed peracid at 10, 20, and 30 ppm, and chlorine at 30 and 50 ppm all significantly reduced E. coli O157:
H7 in processing water by 1.83, 1.73, 1.50, 1.83, 1.34, and 1.83 log CFU/ml, respectively, compared with washing with water
alone. These antimicrobials at all concentrations tested also significantly reduced transfer of the bacteria from an inoculated
leaf to uninoculated leaves in the processing water by 0.96 to 2.57 log CFU per piece. A 10% organic load in the processing
water reduced efficacy of antimicrobial agents. In this contaminated water, peroxyacetic acid at 10 and 20 ppm and chlorine
at 30 ppm produced effects not significantly different from those of water alone. Therefore, it is important to understand the
impact of organic load when validating the effectiveness of antimicrobial treatments.
Lettuce is one of the most commonly consumed leafy
greens, with a farm value of over $1.5 billion in 2005 in
the United States (10). Lettuce is perceived by consumers
as healthful and nutritious. Contamination of vegetables by
human pathogens can occur at many locations in the farm-
to-fork continuum, including contamination of seeds and of
product during production, harvesting, postharvest han-
dling, transport distribution, storage, processing, and prep-
aration (13). Survival and growth of foodborne human
pathogens on fresh and fresh-cut produce has been widely
reported (3–5, 12, 14, 15, 21). The efficacy of different
antimicrobials used to kill foodborne pathogens on fresh
and fresh-cut produce has been studied extensively, and
most antimicrobials are minimally effective, reducing path-
ogen contamination by only 1 to 2 log CFU/g (3, 5, 9, 21).
Antimicrobial agents often are added to water in flumes
that convey or wash fresh fruits and vegetables. The addi-
tion of these agents reduces the number of microorganisms
in fruit and vegetable processing water. Reducing the num-
ber of microorganisms in recycled processing water helps
prevent the water from becoming a vehicle of cross-con-
tamination (7, 8, 11, 16, 18, 19). Antimicrobial chemicals
in processing water also can reduce microorganisms on the
surfaces of fruits and vegetables. However, processing wa-
ter antimicrobials are more effective for reducing micro-
organisms in water suspensions than on fruit and vegetable
surfaces (1, 2, 6, 11, 18–20).
* Author for correspondence. Tel: 770-228-7284; Fax: 770-229-3216;
E-mail: mdoyle@uga.edu.
This study was conducted (i) to investigate transfer of
Escherichia coli O157:H7 from an inoculated lettuce leaf
piece to uninoculated lettuce leaf pieces during washing,
(ii) to determine the efficacy of peroxyacetic acid, mixed
peracid, and chlorine for reducing the transfer of E. coli
O157:H7 under conditions of high organic load, and (iii)
to determine the efficacy of peroxyacetic acid, mixed per-
acid, and chlorine for reducing E. coli O157:H7 in lettuce
processing water.
MATERIALS AND METHODS
Bacterial strains and culture conditions. Five strains of E.
coli O157:H7 were used: ATCC 43888 (human feces), EO122
(cattle isolate), K3995 (spinach isolate), K4492 (lettuce, clinical
isolate), and F4546 (alfalfa sprout outbreak isolate). A plasmid
(pGFPuv) containing a gfp gene was introduced into each strain
using a CaCl
2
heat shock method (17). Expression of green fluo-
rescent protein (GFP) in labeled cells was evaluated by epifluo-
rescence microscopic examination of colonies. The five strains
were cross-streaked onto tryptic soy agar (Difco, Becton Dickin-
son, Sparks, MD) to confirm lack of cross-inhibitory activity. All
strains were grown at 37⬚C for 24 h on brain heart infusion agar
(BHIA; Difco, Becton Dickinson) or in brain heart infusion broth
(BHIB; Difco, Becton Dickinson) supplemented with ampicillin
(Roche Diagnostics, Indianapolis, IN) at a concentration of 100
␮g/ml (BHIA-amp and BHIB-amp, respectively). Colonies of
these GFP-labeled strains were viewed under a 396-nm wave-
length UV lamp for enumeration.
All E. coli O157:H7 strains were transferred to BHIB-amp
three times at 24-h intervals before they were used as inocula.
Cells from overnight culture (10 ml) were sedimented by centri-

J. Food Prot., Vol. 72, No. 7 E. COLI O157:H7 CONTROL IN LETTUCE PROCESSING WATER 1393
fugation at 5,000 ⫻gfor 10 min and resuspended in 10 ml of
0.1% sterile peptone water (Difco, Becton Dickinson). Approxi-
mately equal populations of each of the five strains were com-
bined. Dilutions were made in 0.1% sterile peptone water to create
a culture suspension for inoculation of approximately 10
6
CFU/
ml.
Antimicrobial agents. Peroxyacetic acid (Tsunami 100),
mixed peracid (Tsunami 200), and sodium hypochlorite (XY-12)
were provided by Ecolab, Inc. (St. Paul, MN).
Preparation of lettuce for inoculation. Iceberg lettuce (Lac-
tuca sativa L.) was purchased from a grocery store (Griffin, GA).
Two or three layers of outer leaves were removed from each head
of lettuce, and inner leaves were aseptically cut into pieces (ca. 5
by 5 cm), using as much of the leaf portion as possible and avoid-
ing stem areas.
Inoculation of lettuce leaves. Lettuce leaf pieces were
placed on a sterile surface in a laminar flow biosafety cabinet, and
100 ␮l of the five-strain mixture of culture suspension was spot
inoculated with a micropipettor onto the adaxial side of each leaf
piece to achieve an initial E. coli O157:H7 population of 5.6 log
CFU per inoculated lettuce piece. The inoculated leaf pieces were
placed in a sterile plastic container with a lid and held at 4⬚C for
approximately 2 h to allow bacterial attachment before treatment.
A minor cut (ca. 2 mm) on one side was made on all inoculated
leaf pieces to differentiate these pieces from uninoculated leaf
pieces during treatment.
Organic load preparation. Two outer layers of iceberg let-
tuce leaves were discarded. Green leaves (100 g) were placed in
a sterile blender jar with 100 g of sterile water tempered to 4⬚C.
Leaves were blended on high speed until they were liquefied and
particulates were small enough to be suctioned through a pipette.
This organic load preparation was blended immediately before
use.
Antimicrobial use solution: chemistry without organic
load. The appropriate amount of test antimicrobial was pipetted
into 250 ml of sterile deionized water in a 500-ml volumetric
flask, and additional sterile deionized water was added to the 500-
ml mark.
Antimicrobial use solution: chemistry with 10% organic
load. The appropriate amount of test antimicrobial was pipetted
into 250 ml of sterile deionized water as above, 50 ml of the
organic load preparation was added, and additional sterile deion-
ized water was added to the 500-ml mark.
Antimicrobial use solution: concentration of antimicro-
bial agent. Concentrations of free chlorine and total peracid in
use solutions were determined by an iodine–sodium thiosulfate
redox titration (Oxidizer Kit 322, Ecolab). The following anti-
microbial agents were evaluated: water; peroxyacetic acid at 10,
20, and 30 ppm; mixed peracid at 10, 20, and 30 ppm; and sodium
hypochlorite at 30 and 50 ppm at pH 6.8. All antimicrobials were
evaluated without organic load and with a 10% organic load prep-
aration.
Treatment of lettuce leaves with antimicrobial agents. All
testing was conducted in a refrigerated room (4 to 5⬚C). The use
solutions (with or without organic load) were poured into the mix-
ing vessel (modified version of the CDC Biofilm Reactor, Bio-
Surface Technologies Corp., Bozeman, MT) and stirred at 125
rpm with a magnetic stir bar on a stir plate. Five uninoculated
lettuce pieces and one inoculated lettuce piece were placed in the
mixing vessel and agitated for 1.5 min treatment. Lettuce pieces
were then removed aseptically and separately placed into Whirl-
Pak bags (Nasco, Fort Atkinson, WI) containing 10 ml of 0.5%
sodium thiosulfate neutralizing agent (Fisher Scientific, Fair
Lawn, NJ). Lettuce pieces were then individually macerated at
230 rpm for 30 s, and serial dilutions were plated in duplicate on
BHIA-amp and incubated at 35 ⫾2⬚C for 48 h.
One milliliter of each treated use solution from the mixing
vessel was pipetted into 9 ml of 0.5% sodium thiosulfate. Serial
dilutions were plated in duplicate and incubated under the con-
ditions described above.
Control: test substance neutralization. Triplicate neutrali-
zation checks were performed on each type of chemistry. If more
than one use solution concentration was used, the most concen-
trated solution was tested. For control A, an uninoculated lettuce
piece was dipped into the test substance use solution for 1.5 min
and then removed and placed into a small Whirl-Pak bag con-
taining 10 ml of the neutralizing agent (0.5% sodium thiosulfate).
Subsequently, 0.1 ml of E. coli O157:H7 test system suspension
(10
5
CFU/ml) was added and mixed. For control B, an uninocu-
lated lettuce piece was dipped into the test substance diluent (ster-
ile deionized water) for 1.5 min and then removed and placed into
a small Whirl-Pak bag containing 10 ml of the neutralizing agent.
Subsequently, 0.1 ml of E. coli O157:H7 test system suspension
(10
5
CFU/ml) was added and mixed. For control C, 0.1 ml of E.
coli O157:H7 test system suspension (10
5
CFU/ml) was added to
10 ml of sterile peptone water and mixed. Leaf pieces from con-
trols A, B, and C were held at room temperature for 30 min before
the microbiological assay. Portions (0.25 ml in quadruplicate and
0.1 ml in duplicate) of each control were plated on BHIA-amp
and incubated at 35 ⫾2⬚C for 48 h.
The neutralizing agent was considered to have effectively
neutralized the test substance when the average plate count from
control C equaled that of control A ⫾10%. The neutralizing agent
was not detrimental to the culture suspension when the average
plate count from control C equaled that of control B ⫾10%.
Control: test substance diluent (sterile deionized water)
sterility. Portions (0.25 ml in quadruplicate and 0.1 ml in dupli-
cate) of sterile deionized water were plated on BHIA-amp and
incubated at 35 ⫾2⬚C for 48 h.
Control: E. coli O157:H7–free lettuce pieces. An uninoc-
ulated lettuce piece was aseptically placed into a Whirl-Pak bag,
10 ml of neutralizing agent was added, and the bag contents were
homogenized in a laboratory blender (Stomacher 400, Seward,
Worthington, UK) at 230 rpm for 30 s. Portions (0.25 ml in qua-
druplicate and 0.1 ml in duplicate) of homogenate were plated on
BHIA-amp and incubated at 35 ⫾2⬚C for 48 h.
Control: E. coli O157:H7–free organic load. Portions (0.25
ml in quadruplicate and 0.1 ml in duplicate) of organic load were
plated on BHIA-amp and incubated at 35 ⫾2⬚C for 48 h.
All chemical solutions were stored at 4⬚C 1 day before the
experiment. The entire experiment was conducted in a room with
temperature set at 4⬚C.
Statistical analysis. Data were analyzed using the general
linear models procedure of SAS (SAS 9.1.3; SAS Institute, Inc.,
Cary, NC at ␣⫽0.05. Duncan’s multiple range tests were used
to determine significant differences (␣⫽0.05) between mean val-
ues. The entire study was repeated three times.

J. Food Prot., Vol. 72, No. 71394 ZHANG ET AL.
TABLE 1. E. coli O157:H7 on lettuce leaves and in processing water with and without antimicrobials and without organic load
a
Antimicrobial agent Concn (ppm)
Mean (⫾SD) E. coli O157:H7 population
b
Inoculated leaves after
treatment (log CFU/piece)
Posttreatment processing water
(log CFU/ml)
Uninoculated leaves after
treatment (log CFU/piece)
Peroxyacetic acid 10 3.31 ⫾0.11
AB
0.88 ⫾0.84
AB
0.20 ⫾0.34
BC
20 3.21 ⫾0.36
ABC
0.76 ⫾1.32
AB
0.44 ⫾0.27
B
30 2.38 ⫾0.18
BC
ND
B
ND
C
Mixed peracid 10 2.27 ⫾0.55
BC
0.10 ⫾0.17
B
0.07 ⫾0.12
BC
20 2.10 ⫾1.84
C
0.33 ⫾0.58
B
0.18 ⫾0.18
BC
30 ND
D
ND
B
ND
C
Chlorine 30 3.42 ⫾0.35
AB
0.49 ⫾0.84
B
0.19 ⫾0.32
BC
50 2.60 ⫾0.44
ABC
ND
B
0.07 ⫾0.11
BC
Water 3.68 ⫾0.23
A
1.83 ⫾0.24
A
2.54 ⫾0.19
A
a
E. coli O157:H7 population on inoculated untreated leaves was at 5.6 log CFU per piece.
b
Within a column, means with the same letter are not significantly different at ␣⫽0.05. ND, not detected. Detection limits were 1
CFU/ml of processing solution and 10 CFU per leaf piece.
RESULTS
E. coli O157:H7 populations for control A were 2.97,
2.92, and 2.98 log CFU/ml for 30 ppm of peracetic acid,
30 ppm of mixed peracid, and 50 ppm of chlorine, respec-
tively, and 2.98 and 2.96 log CFU/ml for controls B and
C, respectively. These values were approximately the same,
indicating that the neutralizing agent effectively neutralized
the test substance and was not detrimental to E. coli O157:
H7. The sterile deionized water used for all solutions, the
lettuce leaves, and the prepared organic load were all neg-
ative for E. coli O157:H7.
A single lettuce leaf piece inoculated with E. coli
O157:H7 at 5.6 log CFU transferred contamination in 500
ml of water at approximately 2 log CFU/ml with or without
the presence of organic material. Although the contamina-
tion levels were not significantly different, peroxyacetic
acid at 10 and 20 ppm held the level of contamination in
the solution to 1 log CFU/ml less than that of water when
no additional organic material was present. All other anti-
microbial solutions had significantly less E. coli O157:H7
than did water when no additional organic material was
present. In posttreatment solutions without organic load
containing mixed peracid at 10 and 20 ppm, E. coli O157:
H7 levels were 1.5 log CFU/ml less than those in water. E.
coli O157:H7 was not detected (detection limit of 1 CFU/
ml) in posttreatment solutions when mixed peracid and per-
oxyacetic acid were at 30 ppm or chlorine was at 50 ppm.
The average E. coli O157:H7 population detected was 0.5
log CFU/ml after chlorine treatment at 30 ppm, which was
more than 1 log CFU/ml less than that for water alone
(Table 1).
The presence of 10% organic material reduced the ef-
fectiveness of several antimicrobial treatments for control
of E. coli O157:H7 transfer to the washing solutions. There
were no significant differences between E. coli O157:H7
levels in water and in chlorine at 30 ppm, mixed peracid
at 10 ppm, and peroxyacetic acid at 10 and 20 ppm. In
posttreatment solutions with 10% organic load, E. coli
O157:H7 was not detected in mixed peracid at 20 and 30
ppm. Peroxyacetic acid at 30 ppm had E. coli O157:H7
levels that were significantly less than those in water (␣⫽
0.05) by 1.7 log CFU/ml. Chlorine at 30 ppm and 50 ppm
had E. coli O157:H7 levels that were 0.8 and 1.3 log CFU/
ml, respectively, less than those in water only (Table 2).
In contrast to the results for the posttreatment solutions,
the E. coli O157:H7 populations transferred to uninoculated
leaves were significantly smaller for all antimicrobial treat-
ments than for water only with or without added organic
material. When one leaf piece inoculated with E. coli O157:
H7 at 5.6 log CFU was mixed with five uninoculated leaf
pieces in 500 ml of untreated water, the mean population
on the uninoculated leaves after treatment was greater than
2.5 log CFU per leaf piece. When no added organic ma-
terial was present, the mean population on uninoculated
leaves in antimicrobial solutions was at least 2 log units
less than that for water only, and no E. coli O157:H7 was
detected on uninoculated leaves treated with peroxyacetic
acid or mixed peracid at 30 ppm. There was no significant
difference between the results for those treatments and the
leaf results for mixed peracid at 10 and 20 ppm and chlo-
rine at 30 or 50 ppm (Table 1).
The presence of 10% organic material added to the
antimicrobial solutions reduced the effectiveness of limiting
transfer of E. coli O157:H7 to uninoculated leaves; how-
ever, all antimicrobial treatments resulted in significantly
lower cell numbers on uninoculated leaves compared with
the numbers on leaves in untreated water. Chlorine at 30
and 50 ppm and peroxyacetic acid at 10 ppm had mean cell
numbers 1 log or more lower than those for untreated water.
Peroxyacetic acid at 20 and 30 ppm and mixed peracid at
10, 20, and 30 ppm had mean cell numbers ⬎2 log less
than those in untreated water (Table 2).
For E. coli O157:H7 on inoculated lettuce leaves after
treatment without organic load, a significant reduction (␣
⫽0.05) of 1.9 log CFU per leaf piece was achieved by
washing with water alone. A reduction of 3.2, 3.5, and
⬎4.6 log CFU per leaf piece was achieved by peroxyacetic
acid at 30 ppm, mixed peracid at 20 ppm, and mixed per-
acid at 30 ppm, respectively, and this reduction was sig-
nificantly different from that achieved with water alone.

J. Food Prot., Vol. 72, No. 7 E. COLI O157:H7 CONTROL IN LETTUCE PROCESSING WATER 1395
TABLE 2. E. coli O157:H7 on lettuce leaves and in processing water with and without antimicrobials and in the presence of 10%
organic load
a
Antimicrobial agent Concn (ppm)
Mean (⫾SD) E. coli O157:H7 population
b
Inoculated leaves after
treatment (log CFU/piece)
Posttreatment processing water
(log CFU/ml)
Uninoculated leaves after
treatment (log CFU/piece)
Peroxyacetic acid 10 3.99 ⫾0.45
A
1.61 ⫾0.09
AB
1.26 ⫾0.70
BC
20 3.25 ⫾0.69
A
1.27 ⫾0.63
AB
0.68 ⫾0.79
CD
30 1.66 ⫾1.47
BC
0.10 ⫾0.17
CD
0.07 ⫾0.12
D
Mixed peracid 10 3.42 ⫾0.43
A
1.24 ⫾0.81
AB
0.57 ⫾0.39
CD
20 2.57 ⫾0.46
AB
ND
D
0.27 ⫾0.31
D
30 0.90 ⫾0.85
C
ND
D
0.13 ⫾0.12
D
Chlorine 30 2.86 ⫾0.41
AB
1.15 ⫾1.00
ABC
1.68 ⫾1.00
B
50 2.88 ⫾0.29
AB
0.59 ⫾1.02
BCD
0.80 ⫾1.39
BCD
Water 3.93 ⫾0.56
A
1.96 ⫾0.26
A
2.64 ⫾0.15
A
a
E. coli O157:H7 population on inoculated untreated leaves was at 5.6 log CFU per piece.
b
Within a column, means with the same letter are not significantly different at ␣⫽0.05. ND, not detected. Detection limit was 1 CFU/
ml of processing water.
FIGURE 1. Comparison of antimicrobial agents at 30 ppm in
processing water without organic load for their effect on E. coli
O157:H7 in processing water and on inoculated and uninoculated
lettuce leaves. ND, not detected. The experiment was repeated
three times. One sample was evaluated for inoculated lettuce be-
fore treatment, processing water after treatment, and inoculated
lettuce after treatment in each replicate. Five samples were eval-
uated for uninoculated lettuce after treatment in each replicate.
Error bars represent the standard deviation.
The 2.18-log reduction achieved by washing with chlorine
at 30 ppm and the 3.0-log reduction with 50 ppm of chlo-
rine was not significantly different than that achieved with
water alone. The reduction of E. coli O157:H7 on inocu-
lated leaves was significantly greater for the mixed peracid
solution at 30 ppm than for any other treatment. When no
added organic material was present, E. coli O157:H7 was
not detected, representing a ⬎5-log reduction from the ini-
tial level of 5.56 log CFU per leaf piece. A similar trend
was observed for treatments with 10% organic load, with
slightly lower efficacy of all antimicrobial agents (Table 2).
DISCUSSION
Compared with water without antimicrobial agents,
peroxyacetic acid and mixed peracid at 30 ppm were more
effective for reducing the numbers of E. coli O157:H7 cells
in processing water, with or without 10% organic load, and
on inoculated lettuce leaves. However, peracid agents at 10
and 20 ppm (which are below the specified label use con-
centration) were much less effective than 30 ppm for re-
ducing E. coli O157:H7 in processing water and on inoc-
ulated lettuce leaves (Tables 1 and 2). According to the
Federal Insecticide, Fungicide and Rodenticide Act
(http://www.epa.gov/oecaagct/lfra.html), it is a violation of
Federal Law to use an Environmental Protection Agency–
registered product in a manner that is inconsistent with its
labeling. The results of this study demonstrate that improp-
er use of antimicrobial agents (e.g., reduced concentration)
under produce processing conditions will not achieve the
intended purpose of controlling pathogenic microorganisms
in processing water.
E. coli O157:H7 on inoculated leaves contaminated
processing water and was transferred to uninoculated leaves
in the processing water for all treatments except 30 ppm of
mixed peracid and 30 ppm of peroxyacetic acid. E. coli
O157:H7 contamination reached 2.5 and 2.6 log CFU per
leaf piece on uninoculated leaf pieces when they were
washed with leaf pieces inoculated at 5.6 log CFU per leaf
piece in water without and with 10% organic load, respec-
tively (Tables 1 and 2). In comparison with washing with
water only, peroxyacetic acid, mixed peracid, and chlorine
treatments at all concentrations resulted in significantly
lower numbers of E. coli O157:H7 cells on uninoculated
leaves (Tables 1 and 2). Proper levels of antimicrobials in
processing water are necessary to prevent transfer of path-
ogens from contaminated leaves to uncontaminated leaves
during washing.
Treatments with 30 ppm of peroxyacetic acid and
mixed peracid reduced the population of E. coli O157:H7
on inoculated leaves by ⱖ1 log CFU per leaf piece more
than did treatment with chlorine at 30 ppm with or without
10% organic load; however, only the 30-ppm mixed peracid
treatment result was significantly different from that of
chlorine (Figs. 1 and 2). In the postwash water containing
10% organic load, only peroxyacetic acid at 30 ppm, mixed
peracid at 20 and 30 ppm, and chlorine at 50 ppm were

J. Food Prot., Vol. 72, No. 71396 ZHANG ET AL.
FIGURE 2. Comparison of antimicrobial agents at 30 ppm in
processing water in the presence of 10% organic load for their
effect on E. coli O157:H7 in processing water and on inoculated
and uninoculated lettuce leaves. ND, not detected. The experiment
was repeated three times. One sample was evaluated for inocu-
lated lettuce before treatment, processing water after treatment,
and inoculated lettuce after treatment in each replicate. Five sam-
ples were evaluated uninoculated lettuce after treatment in each
replicate. Error bars represent the standard deviation.
significantly more effective than water for reducing E. coli
O157:H7. In the presence of 10% organic load in process-
ing water, peroxyacetic acid and mixed peracid at 30 ppm
significantly reduced the contamination of uninoculated
leaves by E. coli O157:H7 (ca. 0.1 log CFU per leaf piece),
whereas chlorine at 30 ppm left 1.68 log CFU per leaf piece
on uninoculated leaves (Tables 1 and 2). Results of this
study revealed that mixed peracid at 30 ppm in the presence
of organic load was more effective for inactivating E. coli
O157:H7 in processing water and preventing contamination
of uninoculated leaves than was chlorine at 30 ppm.
The organic load had a greater effect on the efficacy
of chlorine than on that of peroxyacetic acid and mixed
peracid. The 10% organic load in the processing water re-
duced the efficacy of chlorine at 30 ppm but had only minor
effects on the mixed peracid and peroxyacetic acid treat-
ments at 30 ppm. For example, E. coli O157:H7 counts in
posttreatment water with 30 ppm of chlorine, peroxyacetic
acid, or mixed peracid but without organic load were 0.49
log CFU/ml, not detected, and not detected, respectively,
but with 10% organic load were 1.15 and 0.1 log CFU/ml
and not detected, respectively (Tables 1 and 2). The organic
load also negatively impacted the effectiveness of chlorine
at 30 ppm but not the effectiveness of mixed peracid or
peroxyacetic acid for preventing the transfer of E. coli
O157:H7 to the uninoculated leaves. E. coli O157:H7 was
not detected on uninoculated leaves after treatment with 30
ppm of peroxyacetic acid or mixed peracid without organic
load, but the pathogen counts increased by approximately
0.1 log CFU per leaf piece in the presence of 10% organic
load. In contrast, treatment with 30 ppm of chlorine resulted
in an increase of E. coli O157:H7 on uninoculated leaves
from 0.19 log CFU per leaf piece without organic load to
1.68 log CFU per leaf piece with 10% organic load (Tables
1 and 2). Thus, the reuse of processing water and subse-
quent buildup of organic matter both influence the effec-
tiveness of antimicrobial treatments.
The results of this work revealed the potential impact
of organic load on the effectiveness of antimicrobial treat-
ment used to reduce the transfer of E. coli O157:H7 from
contaminated leaves to the processing water and to uncon-
taminated leaves. Although this study did not replicate con-
ditions that exist during processing, it illustrates the need
to evaluate more than just the antimicrobial concentration
when validating the effectiveness of produce processing
controls. Factors such as organic load, fluid/produce ratio,
antimicrobial type and concentration, and other variables
during processing can have a profound effect on the poten-
tial for spreading contamination throughout a production
lot. Additional research on the critical factors beyond an-
timicrobial type and concentration is needed to enhance
pathogen control during produce processing.
ACKNOWLEDGMENTS
This study was funded in part by Ecolab, Inc. Joy Herdt, Sally Foong
Cunningham, and Katherine Swanson (Ecolab) contributed to the design
of the experiment and reviewed the manuscript. We also thank Kellie
McKoon for her technical assistance.
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