ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 25 (2008) 597–606
www.elsevier.com/locate/fm

Identification of a non-pathogenic surrogate organism for chlorine

dioxide (ClO2) gas treatment$
Jeong-Mok Kima, Richard H. Lintonb,
a
Department of Food Science, Mokpo National University, Jeonnam 534-729, Republic of Korea
b
Department of Food Science, Purdue University, West Lafayette, IN 47907-2009, USA
Received 13 August 2007; received in revised form 7 February 2008; accepted 12 February 2008
Available online 10 March 2008

Abstract

The identification of non-pathogenic surrogate microorganisms is beneficial for determining and validating the efficacy of
antimicrobial treatments in food manufacturing environments. A surrogate organism was identified to aid in the decontamination
process of fresh produce when treated with chlorine dioxide (ClO2) gas. Thirty-two known strains of pathogenic and non-pathogenic
microorganisms and seven unknown microbial isolates from mushroom, tomatoes, and strawberries were evaluated. The primary goal
was to find alternative non-pathogenic organisms that had an equal or higher resistance compared to Escherichia coli O157:H7,
Salmonella spp., and Listeria monocytogenes. Among the strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927, E. coli
O157:H7 204P, STB2 (strawberry isolate), and vegetative cells of Bacillus cereus 232 in wet inoculum were found to be the most resistant
to gaseous ClO2 treatment at 0.3 mg/l for 1 min and D-values at 0.3 mg/l ClO2 were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. For
identification, the MR1 and STB2 strains were identified using a RibotyperTM with the EcoRI restriction enzyme of 16S rDNA sequence.
MR1 was identified as Hafnia alvei with a similarity value of 94% using the ribotype pattern and with a 93.6% similarity using an API
20E strip, and with a 99% similarity using 16S rDNA analysis. The Ped-2E9-based cytotoxicity assay was conducted for the MRI strain
extracellular toxin and whole cell toxicity and did not show cytotoxicity. Analysis, using multiplex PCR, was performed to verify absence
of the eaeA gene. H. alvei is a suitable non-pathogenic surrogate, with higher resistance to ClO2 gas compared to pathogens studied, that
may be useful to establish optimum conditions of ClO2 gas decontamination systems.
r 2008 Published by Elsevier Ltd.

Keywords: Chlorine dioxide gas; Surrogate; Hafnia alvei

1. Introduction have been the pathogenic bacteria of most concern on

Consumption of fresh fruits and vegetables has increased
in recent years due to convenience and health benefits.
Minimally processed produce may be washed, chopped,
peeled, sliced, or shredded prior to package and storage.
These products can be contaminated by food pathogens
during harvesting, processing and distribution. Pathogens
common to raw fruit and vegetables include bacteria,
viruses and protozoan cysts. In recent years, Escherichia
coli O157:H7, Salmonella spp. and Listeria monocytogenes,
$ et al., 1999; Han et al., 1999).
This paper is journal article 2007-18159 of the Purdue University
Agriculture Research Program.
Corresponding author. Tel.: +1 765 494 6481; fax: +1 765 494 7953.
E-mail address: linton@purdue.edu (R.H. Linton).
fresh produce.
Chlorine dioxide (ClO2) has been recognized as a
disinfectant since the early 1900s. It is more effective than
chlorine as a biocide over a wide pH range. In 1967, the
Environmental Protection Agency (EPA) first registered
aqueous ClO2 for use as a disinfectant and sanitizer
(EPA, 2008). This compound can be used as a sanitizer for
food surfaces and food contact surfaces and there have
been many food applications of ClO2 in the gaseous or
aqueous form. Aqueous ClO2 is becoming more widely
used in the food industry (Tsai et al., 1995, 2001; Kim
The Food and Drug Administration (FDA) approved
the use of the ClO2 for controlling microorganisms in chill
water for poultry processing, for fruit and vegetable

0740-0020/$ - see front matter r 2008 Published by Elsevier Ltd.

doi:10.1016/j.fm.2008.02.002
 ARTICLE IN PRESS
598 J.-M. Kim, R.H. Linton / Food Microbiology 25 (2008) 597–606
washing, and for meat and poultry disinfection (FDA,
1995; FDA–USDA–CDA, 1998). In 1988, the EPA
registered ClO2 gas as a sterilant for manufacturing
and laboratory equipments and environmental surfaces
(Kaczur and Cawlfield, 1993; EPA, 2008).
Decontamination systems using ClO2 gas for fresh
produce processing have been gaining interest for food
processors. Gaseous ClO2 is highly soluble water and it can
be considered an alternative-sanitizing agent for food
surfaces including produce. Because the gaseous form has
greater penetration ability compared to the liquid form,
ClO2 gas may be more effective for food surface sanitation.
In food processing applications, a treatment of 10 mg/l
ClO2 gas for 30 min completely inactivated spoilage
microorganisms on aseptic juice tank surfaces (Han et al.,
1999). ClO2 gas has also been shown to be effective for
controlling pathogens on lettuce (Lee et al., 2004), apples
(Du et al., 2003; Sapers et al., 2003), and green pepper
surfaces (Han et al., 2000). While studies on pathogen
inactivation on produce surfaces have been very encoura-
ging, identification of surrogate organisms would be
helpful to validate conditions used in actual food proces-
sing facilities.
Using surrogate microorganisms is desirable to deter-
mine and validate the efficacy of ClO2 for the decontami-
nation process of fresh produce because it is not prudent to
introduce pathogens into a processing facility. A surrogate
may be defined as ‘‘a non-pathogenic organism that
behaves similarly to the pathogenic organism when
exposed to the same conditions or treatment’’ (Liu and
Schaffner, 2007). There are many examples where surro-
gate organisms have been used in food processing. Bacillus
subtilis, Bacillus stearothermophillus and Clostridium
sporogenes have been used as surrogates for Clostridium
botulinum (Stewart et al., 2000; Ananta et al., 2001).
Generic E. coli (Duffy et al., 2000; Masschalck et al., 2000;
Leenanon and Drake, 2001) and Enterococcus faecium
(Franz et al., 2001) have been used as surrogates for
E. coli O157:H7. Listeria innocua (Goff and Slade, 1990;
Piyasena and McKellar, 1999; Sabanadesan et al., 2000;
Kozempel et al., 2002), Lactobacillus delbrueckii subsp.
(Siegumfeldt et al., 2000) and Leuconostoc mesenteroides
(Kalchayanand et al., 2002) have been used as surrogates
for L. monocytogenes. Enterobacteria aerogenes (Montville
et al., 2001), Lactobacillus bulgaricus, Lactococcus lactis,
Streptococcus thermophilus, L. innocua (Siegumfeldt et al.,
2000), E. faecium (Audisio et al., 1999; Andrade et al.,
1998), and E. coli (Masschalck et al., 2000) have all been
used as surrogates for Salmonella sp. No information is
available relative to the use of surrogate organisms during
ClO2 gas treatment.
The purpose of this study was to compare microbial
survival and resistance of selected pathogenic and non-
pathogenic strains to ClO2 gas treatment and to identify a
suitable surrogate organism for foodborne pathogens
common to fresh fruit and vegetables. Our overall goal
was to identify a non-pathogenic organism that has similar
or higher resistance to ClO2 gas treatment when compared
to the inactivation kinetics of selected pathogenic bacteria
(E. coli O157:H7, Salmonella sp., and L. monocytogenes)
important to the produce industry.
2. Materials and methods
2.1. Preparation of bacterial cultures
E. coli O157:H7 C7927 (human isolate from cider
outbreak), E. coli O157:H7 204P (heat resistant pork
isolate), E. coli O157:H7 ATCC 43895, E. coli O157:H7
G5303 (apple cider outbreak), and E. coli O157:H7 13B88
(Odwalla cider outbreak), E. coli ATCC 51739 and E. coli
K12 W3110, L. monocytogenes F4244 (human isolate from
Philadelphia outbreak), L. monocytogenes Scott A (human
feces), and L. monocytogenes LCDC-81-861 (Canadian
outbreak of coleslaw/cabbage), L. innocua ATCC 33090,
Salmonella Choleraesuis ATCC 13076, Salmonella Javiana,
Salmonella Typhimurium C133117, Salmonella Enterica
(PT30) BAA-1045, Salmonella Stanley, Salmonella Enter-
itidis E190-88 (human isolate), Salmonella Agona (alfalfa
sprouts), Salmonella Anatum Group E, Salmonella Gami-
narum F2712 (orange juice), Bacillus cereus 232, B. subtilis
ATCC 9372, Staphylococcus aureus ATCC 25923, Staphy-
lococcus faecalis ATCC 344, Pediococcus acidilactici AB1
and PH3, Lactobacillus acidophilus NRRL B1910, Lacto-
bacillus buchneri, Lactobacillus brevis, Leuconostoc citreum
TPB85, and Pseudomonas fluorescens (all from our
laboratory collection) were utilized in these experiments.
This collection of organisms was selected for a wide variety
of reasons. Some organisms were selected because they
were isolated from produce-related foodborne outbreaks
and others were selected based on their resistance to ClO2
gas treatments. During the past 8 years, we have been
studying the effects and resistance of ClO2 gas for a wide
variety of produce systems. We have collected strains that
have survived treatment to different gas concentrations and
experimental conditions.
Two consecutive transfers of each culture were com-
pleted prior to experimental use. All cultures were
maintained on tryptic soy agar (TSA) with 0.6% (w/v)
yeast extract (TSAYE; Difco Laboratories, Sparks, MD,
USA) at 4 1C. Each bacterial culture, except lactic acid
bacteria, was grown for 12–14 h at 37 1C with continuous
agitation (100 rpm) on a MaxQ 2000 platform shaker
(Barnstead Lab-line, Melrose, IL, USA). Lactic acid
bacteria (Leuconostoc sp., Pediococcus sp., Lactobacillus
sp.) were grown in MRS broth for 16 h at 37 1C in a 7%
CO2 water-jacketed incubator (Model 3120, Forma Scien-
tific Inc., Marjetta, OH, USA).
2.2. Preparation of isolated cultures
Mushrooms (MR), tomatoes (TM), and strawberry
(STB) isolates found to be resistant to high levels of ClO2
gas, were obtained after treatment of ClO2 gas (0.5–2 mg/l)
 ARTICLE IN PRESS
J.-M. Kim, R.H. Linton / Food Microbiology 25 (2008) 597–606
for 5 min. After ClO2 treatment of these products, each
sample was mixed with neutralizing buffer in a sterile
stomaching bag and shaken for 15 min at 250 rpm using
platform shaker (Innova 2100, New Brunswick Scientific,
Edison, NJ, USA). Dilutions prepared in 0.1% peptone
water (Difco Laboratories, Sparks, MD, USA) were plated
on TSA plates. Surviving colonies from ClO2 treatment
were isolated by size, color and shape using microscopy.
Isolates from fresh produces were cultivated in tryptic soy
broth (TBS) at 25 1C with shaking for 24–48 h.
2.3. Production of ClO2 gas
ClO2 gas was prepared using a CDG generator (CDG
Technology Inc., New York, NY, USA) based on the
reaction of 4% chlorine gas in nitrogen reacting with
sodium chlorite. This treatment system has been previously
described by Han et al. (2004). Nitrogen flushed the entire
system before the production of ClO2. ClO2 gas in nitrogen
from the generator flowed through 8–10% sodium chlorite
solution (Sigma-Aldrich, St. Louis, MO, USA) in a flask to
scrub chlorine gas residue. Then, the gas was mixed with
filtered air for dilution and introduced into the arcyl glove-
box (L  W  H, 89 cm  61 cm  56 cm, Terra Universal
Inc., Fullerton, CA, USA) that had a separate sealable
chamber (0.027 m3) for removing treated samples over
time. During treatment, the ClO2 gas in the glove-box was
circulated using a fan. Gas concentration inside the
treatment chamber was continuously monitored using an
Optek-Control 4000 ClO2 gas analyzer (Optek-Danulat
Inc., Germantown, WI, USA).
2.4. Screening of ClO2 resistant strains from the
selected microorganisms
Ten milliliters of each bacterial culture was centrifuged
at 7000  g (Rotor JA-14, Beckman, Palo Alto, CA, USA)
for 10 min. The supernatant was discarded and the
cell pellet was washed and resuspended in 10 ml sterile
0.1% peptone solution (Difco, Sparks, MD, USA). The
centrifugation and washing procedure was carried out
twice to remove cell constituents and residual growth
media in an effort to reduce organic demand as much as
possible. Initial concentration of each bacterial culture
was approximately 109 CFU/ml. Ten-fold serial dilutions
of the initial culture were conducted using 0.1% peptone
solution. From the dilution series, 20 ml of bacteria was
inoculated into non-pyrogenic polystyrene 96-well Costars
flat bottom cell culture plates (Corning Incorporated,
Corning, NY, USA), which provides optimum optical
clarity, to give final cell concentrations ranging from
approximately 100–107 CFU/ml. Ninety-six-well microtiter
plates were treated immediately after inoculation (called
the ‘‘wet inoculum’’) or were dried for 5–6 h in the
biosafety cabinet (Labconco Purifier, Kansas City, MO,
USA) prior to treatment (called the ‘‘dried inoculum’’)
(Pao et al., 2007).
599
Plates were exposed to 0.3 mg/l ClO2 gas for 30 s or 1 min
in the chamber (65–70% relative humidity). After treat-
ment, each well in the microtiter well plates was neutralized
immediately with 100 ml of double strength (2  ) neutrali-
zing buffer (NB), followed by 100 ml of 2  TSB.
Neutralizing buffer has the ability to inactivate the
bactericidal and bacteriostatic effect of chlorine com-
pounds. It contains 0.16 g/l of sodium thiosulfate which
inactivates the effect of chlorine compounds. Neutralizing
buffer is not toxic to microorganisms, even when used in
procedures that call for concentrations up to 10 times the
single strength buffer (Downes and Ito, 2001).
Plates were then incubated in a 37 1C incubator.
Microbial growth was measured after 12–24 h using optical
density measurements (BenchmarkTM Microplate Spectro-
photometer, Bio-Rad, Hercules, CA, USA) at 490 nm.
2.5. D-value determination
Initial concentration of the selected five cultures
(E. coli O157:H7 204P and C7927, B. cereus 232, MR1,
STB2) from the screening test described above was
approximately 109 CFU/ml. An aliquot of 0.2 ml of
each culture was inoculated into sterile non-pyrogenic
polystyrene 24-well Costars flat bottom cell culture
plates (Corning Incorporated, Corning, NY, USA).
Plates were then exposed to gaseous 0.3 mg/l ClO2 for 0,
0.5, 1, 3, 5, and 10 min (65–70% relative humidity) in
the chamber. After treatment, each well in the plates
was neutralized immediately with 0.9 ml of 2  NB,
followed by 0.9 ml of 0.1% peptone solution. Each well
was serially diluted 10-fold and plated on TSA plates.
Surviving populations were counted after incubating at
37 or 25 1C for 1–2 days.
D-values (time required for a 90% reduction in the
number of survivors) were calculated by taking the
negative inverse of the slope from the linear regression
between the treatment time and the logarithm of the
microbial population (Microsoft Excel, 2002). The slopes
were determined by linear regression. At least three
replicates were performed for each strain on ClO2 gas
treatment. Analysis of variance (ANOVA) followed by
Duncan’s multiple range test with a significance level of
po0.05 was performed using SAS 9.1.3 (SAS Institute Inc.,
Cary, NC, USA).
2.6. Ribotyping of mushroom and strawberry isolates
The MR1 and STB2 isolates were grown in Luria-
Bertani (LB) broth for 18 h at 37 1C and streaked onto
LB agar for isolation of single colonies. Isolated colonies
were characterized using an automated Riboprinter
(RiboPrinterTM Microbial Characterization System,
Dupont, Wilmington, DE, USA) with the EcoRI restric-
tion enzyme to cut the DNA of an isolated strain
into fragments. Ribopatterns were compared with the
RiboPrinterTM database for culture identification.
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2.7. API test
The API 20E biochemical strip (bioMérieux Inc.,
Durham, NC, USA) was performed according to the
instructions of the manufacturer for MR1 isolated strain
and was further characterized by Gram-staining, catalase
and oxidase reactions. Identification was performed using
the database (V4.0) with the analytical profile index.
2.8. 16S rDNA sequencing of PCR product
Extracted DNA from MR1 isolate was amplified with
primer 16SUF (50 -AGAGTTTGATCCTGGCTCAG) and
16SUR (50 -TACGGCTACCTTGTTACGACTT). The
PCR reaction was performed by using PuReTaqTM
Ready-To-GoTM PCR Beads (GE Healthcare, Amersham,
UK) in strips. The PCR product was purified using the
QIAquicks PCR purification kit (Qiagen, MD, USA)
according to the manufacturer’s protocol. DNA sequen-
cing reactions were performed using a DYEnamic ET
Terminator Cycle Sequencing kit (Amersham Biosciences,
Piscataway, NJ, USA) and analyzed on an Applied
Biosystems model 3700 sequencer (Applied Biosystems,
Foster City, CA, USA). The sequence data were analyzed
using National Center for Biotechnology Information
(NCBI; Bethesda, MD, USA) BLAST system.
2.9. Cytotoxicity assay of the isolate
The MR1 isolated colony onto LB agar was evaluated
using a cytotoxicity assay. The culture was grown in LB
broth at 37 1C for 16–18 h under shaking conditions
(100 rpm). The culture was centrifuged and the supernatant
containing the extracellular supernatant was aliquoted
and the pelletted cells were resuspended in Ped-2E9
cells-phosphate buffered saline (PBS). Both the super-
natant and the cell were tested for cell cytotoxicity by
Ped-2E9-based assay (Bhunia et al., 1994; Bhunia and
Westbrook, 1998) and Chinese hamster ovary (CHO) cell-
based 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) cytotoxicity assay (Beattie and Williams,
1999; Pedersen et al., 2002). Hybrid B lymphocytes Ped-
2E9 cells are highly susceptible to pathogenic bacteria.
Alkaline phosphatase (AP) is present in the cytoplasm of
many mammalian cells and tissues. Therefore, colorimetric
cytotoxicity assay was developed where AP release from
hybrid B lymphocytes (Ped-2E9) line was measured as a
sensitive indicator for bacteria cell cytotoxicity (Bhunia
and Westbrook, 1998). Briefly, the Ped-2E9 murine
hybridoma cells and CHO cells were cultured at 37 1C,
7% CO2 and were treated with 100 ml of either the
supernatant or the whole cell. The cell viability was
microscopically determined by trypan-blue staining. For
Ped-2E9 cytotoxicity assay, all tubes were incubated at
37 1C for 1 h, and it was determined by the AP release assay
(Bhunia and Westbrook, 1998). MTT-assay was done on
CHO cells, using the MTT-assay kit (Roche, Indianapolis,
IN, USA) based on the manufacturer’s recommendations
and following the procedure suggested by Gray et al.
(2005).
2.10. Multiplex PCR for eaeA gene
Isolated Hafnia alvei was screened for the presence of the
virulence gene eaeA. The PCR assay for the virulent gene
was performed as described previously (Maldonado et al.,
2005). E. coli O157:H7 strain EDL 933 was used as
the positive control, since this strain carries the targeted
eaeA gene.
2.11. Preparation of nalidixic acid resistant strain
The MR1 isolated colony was transferred into 10 ml of
TSB and incubated at 37 1C for 24 h. Following, these cells
were adapted to nalidixic acid by growing them in TSB
containing 50 mg/ml of nalidixic acid (TSBN) (Sigma
Chemical Co., St. Louis, MO, USA). Three successive
24 h transfers were made in TSBN prior to use as inocula
for experiments.
3. Results and discussion
3.1. Surrogate selection
In the initial screening experiments for surrogates, both
dried and wet inocula were tested to determine if inoculum
type had an effect on microbial efficacy due to gaseous
ClO2 (Tables 1 and 2). These tables provide post-treatment
survival and growth data for different organisms/strains
that were tested for the initial screening. When the optical
density value was greater than 0.1 after 24 h incubation, it
was considered positive growth, indicating that cells
survived the treatment.
Survival of cells treated with 0.3 mg/l ClO2 gas for
0.5 min was determined for the dry inoculum (Table 1)
and for the wet inoculum (Table 2). For the dry inoculum,
E. coli O157:H7 204P, Salmonella Agona, S. Anatum
GroupE, and S. Gaminarum F2712 were the only strains
that could be recovered with an initial inoculum of
103–104/well, and S. Gaminarum F2712 could be recovered
with an initial inoculum of 102–103/well. These strains
were identified as the most resistant organisms/strains
after treatment with ClO2 gas. With identical treatment
conditions, cells from the wet inoculum were more resistant
compared to cells from the dry inoculum. As an example,
E. coli O157:H7 204P from a dry inoculum showed
no growth with an initial inoculum of 102–103 CFU/well
or lower, whereas, cells from the wet inoculum with
an inoculum level of 100–101 CFU/well, showed growth in
the well.
Most test organisms from a wet inoculum were
recovered after 24 h incubation, even at the lowest initial
inoculum level (100–10 CFU/well) after treatment
with 0.3 mg/l ClO2 gas for 0.5 min (Table 2). L. buchneri,
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Table 1
Survival of selected microorganisms from the dry inoculum treated with 0.3 mg/l ClO2 gas for 0.5 min at 65–70% RH

Microorganisms Post-treatment growth for the following initial microbial levels (CFU/well, 20 ml)

107–108 106–107 105–106 104–105 103–104 102–103 101–102 100–101

E. coli O157:H7 C7927 3/3a 3/3 3/3 0/3 0/3 0/3 0/3 0/3
E. coli O157:H7 204P 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3
E. coli O157:H7 ATCC 43895 3/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3
E. coli O157:H7 G5303 3/3 3/3 2/3 1/3 0/3 0/3 0/3 0/3
E. coli O157:H7 13B88 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
E. coli ATCC 51739 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
E. coli K12 W3110 3/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3
Listeria monocytogenes F4248 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Listeria monocytogenes ScottA 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Listeria monocytogenes LCDC-81-861 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Listeria innocua ATCC 33090 3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3
Salmonella Choleraesins ATCC 13076 3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3
Salmonella Javiana 3/3 3/3 1/3 0/3 0/3 0/3 0/3 0/3
Salmonella Typhimurium C133117 3/3 3/3 3/3 2/3 0/3 0/3 0/3 0/3
Salmonella Enterica (PT30) BAA-1045 3/3 3/3 1/3 0/3 0/3 0/3 0/3 0/3
Salmonella Stanley 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Salmonella Enteritidis E190-88 3/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3
Salmonella Agona 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3
Salmonella Anatum GroupE 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Salmonella Gaminarum F2712-OJ 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3
Bacillus cereus 232 3/3a 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Bacillus subtilis ATCC 9372 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
Staphylococcus aureus ATCC 25923 3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3
Staphylococcus faecalis ATCC 344 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Pediococcus acidilactici AB1 3/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3
Pediococcus acidilactici PH3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Lactobacillus acidophilus NRRL B1910 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Lactobacillus buchneri 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Lactobacillus brevis 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Leuconostoc citreum TPB85 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 1 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 2 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 4 3/3 3/3 3/3 2/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 5 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
a
Number of positive wells where growth was observed out of thee replicate samples.

L. citreum TPB85, and TM3 were easily destroyed.
The next sensitive group of microorganisms inclu-
ded E. coli K12 W3110, L. monocytogenes F4244,
S. aureus ATCC 25923, L. brevis, and TM1 (Table 2).
Organisms containing an initial inoculum level of
102–103 CFU/well before treatment could not be recove-
red after 24 h incubation following ClO2 treatment.
At this concentration and time, we still could not
differentiate among strains tested for ClO2 resistance
because most of the other strains were recovered at this
treatment condition.
Table 3 provides data for the for the more resistant cell
inoculum type (wet inoculum) that was treated for a longer
treatment time (1 min) at 0.3 mg/l ClO2 gas. This was done
so that we could better differentiate resistance of tested
organisms better than at the 30 s treatment time. Resistance
of ClO2 gas treatment varied among the organisms/strains
tested. Treatment at 0.3 mg/l ClO2 gas for 1 min led to
a complete inactivation of the wet inoculum (100–101 CFU/
well of initial inocula level) for E. coli O157:H7 C7927 and
204P, B. cereus 232, STB2, and MR1 (Table 3). At an
initial level above 101–102 CFU/well, these organisms
showed growth in one or two of the three replicate
samples. In the same treatment conditions, lactic acid
bacteria (Pediococcus sp., Lactobacillus sp., L. citreum)
were more sensitive and could not be recovered. The MR1
and STB2 isolates showed a similar resistance to ClO2
gas compared to E. coli O157:H7 C7927 and 204P, and
B. cereus 232.
3.2. Inactivation of E. coli O157:H7, Bacillus cereus, and
produce isolates
From the initial screening experiment, the five strains
(E. coli O157:H7 204P and C7927, B. cereus 232, MR1,
STB2) that showed the highest resistance after ClO2 gas
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Table 2
Survival of selected microorganisms from the wet inoculum treated with 0.3 mg/l ClO2 gas for 0.5 min at 65–70% RH

Microorganisms Post-treatment growth for the following initial microbial levels (CFU/well, 20 ml)

107–108 106–107 105–106 104–105 103–104 102–103 101–102 100–101

E. coli O157:H7 C7927 3/3a 3/3 3/3 3/3 3/3 3/3 3/3 1/3
E. coli O157:H7 204P 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3
E. coli O157:H7 ATCC 43895 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3
E. coli O157:H7 G5303 3/3 3/3 3/3 3/3 3/3 3/3 2/3 1/3
E. coli O157:H7 13B88 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
E. coli ATCC 51739 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3
E. coli K12 W3110 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Listeria monocytogenes F4248 3/3 3/3 3/3 3/3 2/3 0/3 0/3 0/3
Listeria monocytogenes ScottA 3/3 3/3 3/3 3/3 3/3 3/3 1/3 1/3
Listeria monocytogenes LCDC-81-861 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Listeria innocua ATCC 33090 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3
Salmonella Choleraesins ATCC 13076 3/3 3/3 2/3 3/3 3/3 3/3 3/3 2/3
Salmonella Javiana 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
Salmonella Typhimurium C133117 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
Salmonella Enterica (PT30) BAA-1045 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3
Salmonella Stanley 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Salmonella Enteritidis E190-88 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Salmonella Agona 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Salmonella Anatum GroupE 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Salmonella Gaminarum F2712-OJ 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
Bacillus cereus 232 3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3
Bacillus subtilis ATCC 9372 3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3
Staphylococcus aureus ATCC 25923 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Staphylococcus faecalis ATCC 344 3/3 3/3 3/3 3/3 3/3 3/3 2/3 0/3
Pediococcus acidilactici AB1 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3
Pediococcus acidilactici PH3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 1/3
Lactobacillus acidophilus NRRL B1910 3/3 3/3 3/3 3/3 3/3 3/3 1/3 1/3
Lactobacillus buchneri 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3
Lactobacillus brevis 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Leuconostoc citreum TPB85 3/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 1 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3
Tomato isolate (TM) 3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 5 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3
a
Number of positive wells where growth was observed out of three replicate samples.

treatment were selected and further tested to determine
their inactivation kinetics (D-values). Linear regression
of the log number of surviving cells versus ClO2 gas
treatment time had R2 values 40.90, indicating a good
linear fit to the data. The D-values for MR1, E. coli
O157:H7 C7927 and 204P, STB2, and B. cereus 232 were
3.53, 1.95, 1.72, 168, and 1.57 min, respectively (Table 4).
The D-value for MR1 was approximately twice the
D-value for the E. coli O157:H7 204P and B. cereus 232,
and, the D-value for STB2 was similar to that found
for E. coli O157:H7 204P. The higher resistance of MR1
to ClO2 treatment compared to the other pathogens
tested, makes this a suitable and conservative candidate
as a surrogate.
3.3. Ribotyping, API 20E, 16S rDNA analysis,
and cytotoxicity
MR1 and STB2 were evaluated using a RobotyperTM
with the EcoRI restriction enzyme of 16S rDNA sequence
based on the profile similarity to the RoboPrinterTM
Dupont Identification (DID) database. Ribotype patterns
were obtained with EcoRI for MR1 and STB2 (Fig. 1).
MR1 was identified as H. alvei with a similarity to DID
18,066 with a percent similarity value of 94% by ribotype
pattern (Table 5). The API 20E system also gave strong
identification of H. alvei (93.6% similarity). In biochemical
profiles by API 20E, the MR1 was characterized as a
Gram-negative rod, catalase positive and oxidase negative.
The isolate fermented glucose, mannitol, rhamnose and
arabinose, but not inositol, sorbitol, sucrose, and meli-
biose. The MR1 colony was characterized by amplifying
and sequencing a 16S rDNA fragment using unspecific
screening primers.
The sequence was compared to the bacterial sequences
in the NCBI database in order to identify the species. The
16S rDNA sequence of MR1 isolate belonged to H. alvei
with a similarity value of 99%. Generally, a match with
99% 16S rDNA sequence homology renders species-level
identification (Han, 2006). STB2 was identified poorly as
 ARTICLE IN PRESS
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Table 3
Survival of selected microorganisms from the wet inoculum treated with 0.3 mg/l ClO2 gas for 1 min at 65–70% RH

Microorganisms Post-treatment growth for the following initial microbial levels (CFU/well, 20 ml)

107–108 106–107 105 –106 104–105 103–104 102 –103 101–102 100–101

E. coli O157:H7 C7927 3/3a 3/3 3/3 3/3 3/3 3/3 1/3 0/3
E. coli O157:H7 204P 3/3 3/3 3/3 3/3 3/3 3/3 2/3 0/3
E. coli O157:H7 ATCC 43895 3/3 3/3 3/3 3/3 2/3 2/3 0/3 0/3
E. coli O157:H7 G5303 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3
E. coli O157:H7 13B88 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
E. coli ATCC 51739 3/3 3/3 3/3 3/3 3/3 2/3 0/3 0/3
E. coli K12 W3110 3/3 3/3 3/3 3/3 2/3 0/3 0/3 0/3
Listeria monocytogenes F4248 3/3 3/3 3/3 3/3 2/3 0/3 0/3 0/3
Listeria monocytogenes ScottA 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3
Listeria monocytogenes LCDC-81-861 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3
Listeria innocua ATCC 33090 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3
Salmonella Choleraesins ATCC 13076 3/3 3/3 2/3 1/3 1/3 0/3 0/3 0/3
Salmonella Javiana 3/3 3/3 3/3 2/3 1/3 0/3 0/3 0/3
Salmonella Typhimurium C133117 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Salmonella Enterica (PT30) BAA-1045 3/3 3/3 3/3 2/3 2/3 2/3 0/3 0/3
Salmonella Stanley 3/3 3/3 3/3 1/3 1/3 0/3 0/3 0/3
Salmonella Enteritidis E190-88 3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3
Salmonella Agona 3/3 3/3 3/3 2/3 0/3 0/3 0/3 0/3
Salmonella Anatum GroupE 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
Salmonella Gaminarum F2712-OJ 3/3 3/3 3/3 3/3 1/3 1/3 0/3 0/3
Bacillus cereus 232 3/3a 3/3 3/3 3/3 3/3 3/3 2/3 0/3
Bacillus subtilis ATCC 9372 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3
Staphylococcus aureus ATCC 25923 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Staphylococcus faecalis ATCC 344 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3
Pediococcus acidilactici AB1 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
Pediococcus acidilactici PH3 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
Lactobacillus acidophilus NRRL B1910 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
Lactobacillus buchneri 3/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3
Lactobacillus brevis 3/3 3/3 1/3 0/3 0/3 0/3 0/3 0/3
Leuconostoc citreum TPB85 3/3 2/3 2/3 0/3 0/3 0/3 0/3 0/3
Leuconostoc mesenteroides 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3
Pseudomonas fluorescens 3/3 3/3 3/3 3/3 3/3 2/3 0/3 0/3
Tomato isolate (TM) 1 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 3 3/3 3/3 3/3 1/3 0/3 0/3 0/3 0/3
Tomato isolate (TM) 5 3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3
Strawberry isolate (STB) 1 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3
Strawberry isolate (STB) 2 3/3 3/3 3/3 3/3 3/3 3/3 1/3 0/3
Mushroom isolate (MR) 1 3/3 3/3 3/3 3/3 3/3 3/3 2/3 0/3
Mushroom isolate (MR) 2 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3
a
Number of positive wells where growth was observed out of three replicate samples.

Enterococcus faecalis by ribotyping to DID 15,023 with a
percent similarity value of 67%. No further identification
was done for STB2.
H. alvei, a member of the Enterobacteriaceae, has been
isolated from animals and natural environments such as
soil, water, sewage, and foods (Farmer et al., 1985;
Gamage et al., 1997; Farmer, 2003). The gastrointestinal
tract of animals is a very common ecologic habitat for
hafniae (Rhodes et al., 1998). Gordon and FitzGibbon
(1999) found that H. alvei was the third most common
enteric species identified following E. coli and E. cloacae.
Hafniae is commonly found in food products. Almost 50%
of all enteric isolates recovered from refrigerated meats
were to be H. alvei (Albelda-Puig et al., 1986; Ridell and
Korkeala, 1997). There are also reported isolates of
H. alvei from cheese and honey (Morales et al, 2003), fish
(Kim et al., 2001), and animal manure samples (Derlet and
Carlson, 2002).
It is important to show that a surrogate organism is non-
virulent. H. alvei strains are generally considered non-
pathogenic to humans. However, H. alvei has been
shown to possess the virulence-associated gene eaeA, and,
eaeA-positive H. alvei strains may be diarrheagenic (Albert
et al., 1992; Schauer and Falkow, 1993). Ismaili et al.
(1996) screened numerous clinical isolates for the posses-
sion of the eaeA gene, and, these isolates did not possess
the eaeA gene. Our H. alvei isolate did not carry the
virulence-associated eaeA gene as shown by the multiplex-
PCR assay (Table 5). Enzymatic properties associated with
some pathogenic bacteria, including hemolysin, protease,
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604 J.-M. Kim, R.H. Linton / Food Microbiology 25 (2008) 597–606

elastase, and lecithinase activities, have not been detected introduced into fresh produce containing inherent micro-
in Hafniae (Janda et al., 2002). flora. A nalidixic acid (NA) resistant strain of H. alvei was
In this study, we conducted cytotoxicity assays to produced and compared to the original surrogate strain
provide confidence that this stain of H. alvei is not (not resistant to NA) to ensure that resistance to ClO2 was
pathogenic to humans. not changed.
The D-values for H. alvei and NA-resistant H. alvei after
3.4. Comparative resistance of the nalidixic acid resistant exposure to 0.5 mg/l ClO2 gas were 1.37 and 1.38 min,
strain ClO2 gas respectively (Fig. 2). The NA-resistant strain was very
stable and did not provide any difference in resistance to
One of the desirable and important criteria for surrogate ClO2 compared to the parent strain.
organisms is that it should be easily distinguished from
background microflora. Surrogates with stable antibiotic 4. Conclusion
resistance markers can be recovered more easily when
The effect of ClO2 gas treatment and resistance of
Table 4 non-pathogenic bacteria (E. coli, E. coli K12, L. innocua,
Inactivation kinetics of E. coli O157:H7 204P, E. coli O157:H7 C7927, B. subtilis, lactic acid bacteria) and isolated strains (from
Bacillus cereus 232, mushroom isolate 1 (MR1), and strawberry isolate 2 fresh produce) were compared to pathogen E. coli
(STB2) after exposure to 0.3 mg/l ClO2 gas
O157:H7, L. monocytogenes, Salmonella sp., B. cereus,
Test organism D-value (min) Linear regression and S. aureus. Of the 39 strains tested, wet inoculum of
coefficient (R2) E. coli O157:H7 C7927 and 204P, B. cereus 232, MR1 and
STB2 were found to be most resistant to 0.3 mg/l chlorine
E. coli O157:H7 204P 1.7270.07a 0.945
E. coli O157:H7 C7927 1.9570.13a 0.956 dioxide treatment for 1 min. The MR1 strain was identified
Bacillus cereus 232 1.5770.12a 0.909 as H. alvei.
Mushroom isolate 1 (MR1) 3.5370.95b 0.914
Strawberry isolate 2 (STB2) 1.6870.03a 0.944
10.00
Data represent three separate experiments; regression R2 values are given. Hafnia alvei
9.00
D-values with different lowercase letters in the column are significantly NA-resistant Hafnia alvei
different (po0.05). 8.00

7.00
Log10CFU ml-1

6.00

Marker 5.00
2
R = 0.9206
4.00
D value = 1.37
3.00

STB2 2.00 2
R = 0.9058
D value = 1.38
1.00

0.00
MR1 0 2 4 6 8 10 12
Treatment time (min)

Fig. 1. Ribotype pattern obtained with EcoRI restriction enzyme of 16S Fig. 2. Microbial survival curves for inactivation of Hafnia alvei and
rDNA sequence of strawberry isolate 2 (STB2) and mushroom isolate 1 nalidixic acid (NA) resistant Hafnia alvei after exposure to 0.5 mg/l ClO2
(MR1) strains. gas. Error bars represent standard deviation of the mean.

Table 5
Ribotyping, cytotoxicity assays, and presence of eaeA virulence gene for unknown isolates resistant to ClO2 gas treatment

Bacterial Identification by Similarity DIDa Ped2E9-ALP CHO-MTT Ped2E9-ALP Average cell Presence of
isolates ribotyping (%) cytotoxicity- cytotoxicity- cytotoxicity- viability (%) eaeA gene
extracellular extracellular whole cell (%)
supernatant (%) supernatant (%)

MR1 Hafnia alvei 94 18,066 0.1770.1 0.0070.0 0.0070.0 88.89 Negative

STB2 Enterococcus 67 15,203 NDb ND ND ND ND
faecalis
a
DuPont identification number.
b
ND: not determined.
 ARTICLE IN PRESS
J.-M. Kim, R.H. Linton / Food Microbiology 25 (2008) 597–606
In food manufacturing environments, the application of
surrogates has been useful to evaluate adhesion, environ-
mental transmission, and efficacy of sanitation treatments.
An effective microbial surrogate should be non-pathogenic
and it should have similar inactivation characteristics and
kinetics to target pathogens. A surrogate should also be
genetically stable, capable of achieving high-density popu-
lations, easily distinguished from background microflora,
easily enumerated using sensitive and inexpensive detection
systems, and susceptibility to injury similar to that of target
pathogens (Busta et al., 2003).
H. alvei could serve as a potential surrogate to assist in
determining the effectiveness of ClO2 gas treatment for
pathogen inactivation within a fresh produce processing
setting. The conditions of processing to inactivate H. alvei
in specific food products may vary depending on the food
matrix and processing parameters. Additional data is
needed to determine the resistance of H. alvei for specific
food products, as compared to the resistance of other
pathogenic and non-pathogenic organisms. More studies
should be done to confirm the use of this surrogate
organism for determining optimum processing conditions
in ClO2 processing systems.
Acknowledgments
This research was jointly supported by a USDA-
CSREES Integrated Food Safety Grant and by Mokpo
National University, Korea. The authors thank Drs. Arun
Bhunia, Padmapriya Banada, and Bruce Applegate from
Purdue University, for assistance with organism identifica-
tion and cytotoxicity.
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