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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 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, $ 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: [email protected] (R.H. Linton).

0740-0020/$ - see front matter r 2008 Published by Elsevier Ltd. doi:10.1016/j.fm.2008.02.002

have been the pathogenic bacteria of most concern on 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 et al., 1999; Han et al., 1999). 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

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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 encouraging, identification of surrogate organisms would be helpful to validate conditions used in actual food processing facilities. Using surrogate microorganisms is desirable to determine and validate the efficacy of ClO2 for the decontamination 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 surrogate 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 nonpathogenic 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 Enteritidis E190-88 (human isolate), Salmonella Agona (alfalfa sprouts), Salmonella Anatum Group E, Salmonella Gaminarum F2712 (orange juice), Bacillus cereus 232, B. subtilis ATCC 9372, Staphylococcus aureus ATCC 25923, Staphylococcus faecalis ATCC 344, Pediococcus acidilactici AB1 and PH3, Lactobacillus acidophilus NRRL B1910, Lactobacillus 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 completed 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 Scientific 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)

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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 glovebox (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).

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Plates were exposed to 0.3 mg/l ClO2 gas for 30 s or 1 min in the chamber (65–70% relative humidity). After treatment, each well in the microtiter well plates was neutralized immediately with 100 ml of double strength (2  ) neutralizing buffer (NB), followed by 100 ml of 2  TSB. Neutralizing buffer has the ability to inactivate the bactericidal and bacteriostatic effect of chlorine compounds. 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 Spectrophotometer, 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 LuriaBertani (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 restriction 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 (bioMe´rieux 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 sequencing 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 supernatant 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) cellbased 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay (Beattie and Williams, 1999; Pedersen et al., 2002). Hybrid B lymphocytes Ped2E9 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

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

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

3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 1/3 0/3 0/3 0/3 0/3 0/3 3/3 3/3 0/3 3/3 3/3

3/3 3/3 2/3 2/3 0/3 3/3 2/3 0/3 0/3 0/3 0/3 0/3 1/3 3/3 1/3 0/3 2/3 3/3 3/3 3/3 0/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 3/3 3/3 0/3 3/3 3/3

0/3 3/3 0/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 2/3 0/3 0/3 0/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 2/3 1/3

0/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

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 included 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 recovered 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

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

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

3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3 3/3 0/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3 3/3 0/3 1/3 0/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 3/3

3/3 3/3 3/3 2/3 3/3 3/3 0/3 0/3 1/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3 2/3 3/3 2/3 1/3 0/3 0/3 0/3 0/3 0/3 3/3

1/3 2/3 2/3 1/3 1/3 2/3 0/3 0/3 1/3 1/3 0/3 2/3 3/3 3/3 3/3 1/3 1/3 1/3 1/3 1/3 3/3 0/3 0/3 0/3 0/3 1/3 1/3 0/3 0/3 0/3 0/3 0/3 1/3

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 melibiose. 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

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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

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

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

3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 1/3 2/3 3/3 3/3 3/3 3/3 3/3 0/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 1/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3 2/3 3/3 2/3 1/3 3/3 2/3 1/3 3/3 3/3 3/3 3/3 3/3 1/3 1/3 1/3 0/3 0/3 0/3 3/3 3/3 0/3 1/3 3/3 0/3 3/3 3/3 3/3

3/3 3/3 2/3 3/3 0/3 3/3 2/3 2/3 0/3 3/3 3/3 1/3 1/3 3/3 2/3 1/3 3/3 0/3 0/3 1/3 3/3 1/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 3/3 3/3 0/3 0/3 3/3 0/3 3/3 3/3 1/3

3/3 3/3 2/3 1/3 0/3 2/3 0/3 0/3 0/3 1/3 1/3 0/3 0/3 0/3 2/3 0/3 3/3 0/3 0/3 1/3 3/3 0/3 0/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 2/3 0/3 0/3 3/3 0/3 3/3 3/3 0/3

1/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 2/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

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 nonvirulent. H. alvei strains are generally considered nonpathogenic 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 possession 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 multiplexPCR assay (Table 5). Enzymatic properties associated with some pathogenic bacteria, including hemolysin, protease,

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elastase, and lecithinase activities, have not been detected in Hafniae (Janda et al., 2002). In this study, we conducted cytotoxicity assays to provide confidence that this stain of H. alvei is not pathogenic to humans. 3.4. Comparative resistance of the nalidixic acid resistant strain ClO2 gas One of the desirable and important criteria for surrogate organisms is that it should be easily distinguished from background microflora. Surrogates with stable antibiotic resistance markers can be recovered more easily when Table 4 Inactivation kinetics of E. coli O157:H7 204P, E. coli O157:H7 C7927, Bacillus cereus 232, mushroom isolate 1 (MR1), and strawberry isolate 2 (STB2) after exposure to 0.3 mg/l ClO2 gas Test organism

D-value (min)

Linear regression coefficient (R2)

E. coli O157:H7 204P E. coli O157:H7 C7927 Bacillus cereus 232 Mushroom isolate 1 (MR1) Strawberry isolate 2 (STB2)

1.7270.07a 1.9570.13a 1.5770.12a 3.5370.95b 1.6870.03a

0.945 0.956 0.909 0.914 0.944

introduced into fresh produce containing inherent microflora. A nalidixic acid (NA) resistant strain of H. alvei was produced and compared to the original surrogate strain (not resistant to NA) to ensure that resistance to ClO2 was not changed. The D-values for H. alvei and NA-resistant H. alvei after exposure to 0.5 mg/l ClO2 gas were 1.37 and 1.38 min, respectively (Fig. 2). The NA-resistant strain was very stable and did not provide any difference in resistance to ClO2 compared to the parent strain. 4. Conclusion The effect of ClO2 gas treatment and resistance of non-pathogenic bacteria (E. coli, E. coli K12, L. innocua, B. subtilis, lactic acid bacteria) and isolated strains (from fresh produce) were compared to pathogen E. coli O157:H7, L. monocytogenes, Salmonella sp., B. cereus, and S. aureus. Of the 39 strains tested, wet inoculum of 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 dioxide treatment for 1 min. The MR1 strain was identified as H. alvei. 10.00

Data represent three separate experiments; regression R2 values are given. D-values with different lowercase letters in the column are significantly different (po0.05).

Hafnia alvei NA-resistant Hafnia alvei

9.00 8.00 Log10CFU ml-1

7.00

Marker

6.00 5.00 2

R = 0.9206 D value = 1.37

4.00 3.00 2.00

STB2

2

R = 0.9058 D value = 1.38

1.00 0.00

MR1

Fig. 1. Ribotype pattern obtained with EcoRI restriction enzyme of 16S rDNA sequence of strawberry isolate 2 (STB2) and mushroom isolate 1 (MR1) strains.

0

2

6 8 4 Treatment time (min)

10

12

Fig. 2. Microbial survival curves for inactivation of Hafnia alvei and nalidixic acid (NA) resistant Hafnia alvei after exposure to 0.5 mg/l ClO2 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 isolates

Identification by ribotyping

Similarity (%)

DIDa

Ped2E9-ALP cytotoxicityextracellular supernatant (%)

CHO-MTT cytotoxicityextracellular supernatant (%)

Ped2E9-ALP cytotoxicitywhole cell (%)

Average cell viability (%)

Presence of eaeA gene

MR1 STB2

Hafnia alvei Enterococcus faecalis

94 67

18,066 15,203

0.1770.1 NDb

0.0070.0 ND

0.0070.0 ND

88.89 ND

Negative ND

a

DuPont identification number. ND: not determined.

b

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In food manufacturing environments, the application of surrogates has been useful to evaluate adhesion, environmental 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 populations, 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 USDACSREES 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 identification and cytotoxicity. References Albelda-Puig, M.C., Ucar-Carorran, A., Perez-Arquillus, C., YanjuelaMartinez, J., Rivas-Pala, T., Herrera-Marteache, A., 1986. Isolation and identification of species of Enterobacteriaceae in hamburgers. Alimentaria 169 (23–24), 33–36. Albert, M.J., Faruque, S., Ansaruzzaman, M., Islam, M., Haider, K., Alam, K., Kabir, I., Robins-Browne, R., 1992. Sharing of virulenceassociated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei. J. Med. Microbiol. 37, 310–314. Ananta, E., Heinz, V., Schluter, O., Knorr, D., 2001. Kinetic studies on high-pressure inactivation of Bacillus stearothermophillus spores suspended in food matrices. Innovative Food Sci. Emerg. Technol. 2, 261–272. Andrade, N.J., Ajao, D.B., Zottola, E.A., 1998. Growth and adherence on stainless steel by Enterococcus faecium cells. J. Food Prot. 61, 1454–1458. Audisio, C.M., Oliver, G., Apella, C.M., 1999. Antagonistic effect of Enterococcus faecium J96 against human and poultry pathogenic Salmonella spp. J. Food Prot. 62, 751–755. Beattie, S.H., Williams, A.G., 1999. Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay. Lett. Appl. Microbiol. 28, 221–225. Bhunia, A.K., Westbrook, D.G., 1998. Alkaline phosphatase release assay to determine cytotoxicity for Listeria species. Lett. Appl. Microbiol. 26, 305–310.

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