Effect of storage temperature and atmosphere on the survival of Escherichia coli O157:H7 on cut and whole leaf lettuce and spinach

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Chapter 3: Effect of storage temperature and atmosphere on the survival of Escherichia coli O157:H7 on cut and whole leaf lettuce and spinach

ABSTRACT

Numerous food-borne outbreaks of Escherichia coli O157:H7 have been linked to leafy greens in recent years. An overwhelming amount of lettuce and spinach on the market is sold in modified atmosphere packaging as ready to eat salad mixes. The objectives of this study were to determine the effects of modified atmosphere, storage temperature, and inoculum size on survival of E. coli O157:H7 on cut and whole leaf lettuce and spinach. E. coli O157:H7 H1730 was inoculated onto cut and whole leaves of leaf lettuce and spinach. Samples were held under normal atmospheric conditions or in a modified atmosphere package at either 4ºC or 10ºC to simulate display and abuse temperatures. Leaves were sampled at Days 0, 1 and every other day until noticeable spoilage occurred (7 days for lettuce, 9 days for spinach). E. coli O157:H7 was able to survive at 4° and 10°C regardless of atmosphere and inoculum size for 7 days on cut and whole lettuce and 9 days on cut and whole spinach. Overall, Numbers of E. coli O157:H7 increased (1 log) throughout the storage period on spinach, and decreased on lettuce (1-1.5 log). Significantly higher (P < 0.05) numbers of E. coli O157:H7 were found on lettuce and spinach stored at 10ºC than when stored at 4ºC. There were no significant differences (P > 0.05) in Numbers of E. coli O157:H7 with respect to atmosphere, leaf type or inoculum size. If contamination of lettuce or spinach with E. coli O157:H7 occurred, the pathogen may survive well under typical packaging and storage conditions.

INTRODUCTION

An increase in fresh produce consumption in recent years has coincided with an increase in the number of outbreaks caused by food-borne pathogens associated with produce (Sivapalasingam, S., Friedman, C.R., Cohen, L. and Tauxe, R.V., 2004). Since fresh-cut produce is often consumed raw and is only subjected to minimal processing, contamination with pathogens is of major concern in regards to incidences of food-borne illness (FDA, October 2004). Lettuce and spinach have been implicated as the vehicles responsible for many food-borne outbreaks and has been linked to E. coli O157:H7 (Beuchat, L. R., 1995, Sivapalasingam, S., Friedman, C.R., Cohen, L. and Tauxe, R.V., 2004). The infectious dose of E. coli O157:H7 is suspected to be very low (ranging from 1 to 100 colony-forming units) which increases the likelihood of food-borne illness if contamination with fresh produce occurs (Paton, J. C. and A. W. Paton, 1998).
Modified atmosphere packaging (MAP) is commonly used by the food industry to package lettuce and other fresh cut fruits and vegetables in order to extend their shelf life by delaying product respiration (FDA, 2001). MAP has been shown to be most effective with low storage temperature in order to prolong the shelf life of produce (Liesbeth Jacxsens, D., Frank, Van der Steen, Caroline and Debevere, Johan 29 June 2001). By increasing the shelf life of produce, pathogens which may be present, will have more time to grow as well as inhibit the growth of spoilage aerobes. Previous research has shown that E. coli O157:H7 can survive and/or grow in modified atmosphere packaging at low storage temperatures on produce (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993, Francis, G. A. a. O. B., D., 2001, Gunes, G. G. a. H., J.H., 2002, Hao, Y. a. B., RE, 1993). Temperature abuse during storage can further enhance the growth and survival of E. coli O157:H7 and this pathogen has been reported to grow at 8ºC and above (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993, Francis, G. A. a. O. B., D., 2001). Since E. coli O157:H7 has been shown to preferentially attach to cut edges of lettuce, it may additionally attach to cut spinach as well.
The majority of fresh produce items maintain ideal quality and freshness at temperatures near 0º C, but are usually kept in retail food displays and sold to consumers at around 4ºC (FDA, 2001). E. coli O157:H7 is able to survive on a variety of produce commodities at low temperatures ( < 5ºC) (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993, Beuchat, L. R., 1995, Hao, Y. a. B., RE, 1993, Li, Y., Brackett, R.E., Chen, J. and Beuchat, L.R., 2001). Li et al. studied the influence of temperature and modified atmosphere on growth of E. coli O157:H7 and observed that the pathogen was able to survive at 5ºC on shredded lettuce over 18 days (Li, Y., Brackett, R.E., Chen, J. and Beuchat, L.R., 2001). In their study, PD-961 EZ film bags (OTR 7,000 cc/m2/24 h and CO2 21,000 cc/m2/24 h) were used and bags were sealed under ambient conditions (Li, Y., Brackett, R.E., Chen, J. and Beuchat, L.R., 2001). Similar observations were found by Abdul-Raouf et al., which showed that numbers E. coli O157:H7 were detected on packaged shredded lettuce in polyolefin L-bags (OTR 3,000 cm3/m2/24 h and CO2 9,800 cm3/m2/24 h) stored at 5ºC survived throughout a 14 day storage period (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993).
E. coli O157:H7 also preferentially attaches to cut surfaces of lettuce leaves (Seo, K. H. and J. F. Frank, 1999, Takeuchi, K. and J. F. Frank, 2000, Takeuchi, K., Hassan, A.N. and Frank, J.F., 2001). Takeuchi and Frank observed an increased number of E. coli O157:H7 cells at the cut and damaged edges of lettuce compared to those at uncut surfaces (Takeuchi, K. and J. F. Frank, 2000). These observations were also concluded by Seo and Frank as well as Takeuchi et al. under modified atmospheres at 4 and 22ºC as a result of the respiration rate of the lettuce (Seo, K. H. and J. F. Frank, 1999, Takeuchi, K., Hassan, A.N. and Frank, J.F., 2001). Since most bagged lettuces are cut or chopped prior to packaging, this may create another advantage for E. coli O157:H7 to persist in bagged lettuces.
The objectives of this study were to determine the survival of low (3 log CFU/g) and high Numbers (6 log CFU/g) of E. coli O157:H7 on whole and fresh cut leaf lettuce and spinach surfaces when packaged under passively modified atmosphere, and stored at 4 and 10ºC until noticeable spoilage occurs.

MATERIALS AND METHODS

GFP Transformation

E. coli O157:H7 strain H1730, a clinical isolate associated with a lettuce outbreak (obtained from Dr. Larry Beuchat, University of Georgia) was stored at -80 ºC. The culture was activated through a series of three overnight transfers in tryptic soy broth (TSB; Difco, Detroit, MI) at 37ºC. Cells were streaked onto Sorbitol Mackonkey agar (SMAC; Difco, Detroit, MI) to identify E. coli O157:H7. Further serological confirmation of cells was completed using the RIM latex agglutination test (Remel, Lenexa, KS) to confirm the presence of the O:157 and H7 antigens.
The Clontech Plasmid (Clontech, Palo Alto, CA) PW250 (Invitrogen, Carlsbad, CA) coding for GFP expression and kanamycin resistance was inserted into electrocompetent E. coli O157:H7 strain H1730 using electroporation method per manufacturers instructions (Bio-Rad GenePulser Xcell, Bio-Rad Laboratories, Hercules, CA). In the presence of isopropyl-beta-D-thiogalactoside (IPTG), the lac promoter is “turned on”, inducing the expression of GFP, which fluoresces green under long wave UV light (Hereford, M., 2003).

Preparation of Inoculum

Transformed cells were frozen and maintained as stock cultures at -80 ºC in a 30% glycerol/ TSB. Prior to each experiment, the culture was activated through three overnight transfers in TSB supplemented with 100 µg/ml of kanamycin (Fisher Scientific, Fair Lawn, NJ) and 10 µg/ml of IPTG (Acros organics, NJ) at 37ºC. Cells were then streaked onto Tryptic Soy Agar (TSA; Difco, Detroit, MI) containing 100 µg/ml of kanamycin and 10 µg/ml of IPTG (TSA-K). Typical colonies were confirmed by fluorescence under long wave UV light.
The activated culture was centrifuged (4000xg, 15 min) and, the cells were washed twice and resuspended in sterile distilled water to achieve desired concentration (6 log or 3 log CFU/ml).
Two Inoculation levels of E. coli O157:H7 inoculum on lettuce and spinach were studied. A high inoculum (6 log CFU/ml) of E. coli O157:H7 was prepared as previously described; a lower inoculum (3 log CFU/ml) was prepared by serially diluting the high inoculum in 0.1% sterile peptone water.

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Lettuce, Leaf inoculation procedures

Green leaf lettuce was purchased at a local supermarket in Blacksburg, VA on the day of inoculation. Lettuce was stored at 4ºC for no longer than 6 hours prior to initiation of experiments. Prior to inoculation, outer leaves of lettuce were removed, and lettuce was washed with a 100 ppm sodium hypochlorite solution. Solution used was prepared by adding 0.17 ml commercial solution of sodium hypochlorite to 1 liter of distilled water. Lettuce was then submerged in chlorine for one minute and dried in a salad spinner for approximately 2.5 minutes (Chua, D., Goh, K., Saftner, R.A. and Bhagwat, A.A. , 2008).
E. coli O157:H7 H1730 was inoculated onto cut and whole leaves of green leaf lettuce via spot inoculation methods described by Lang et. al. (Lang, M. M., Harris, L.J. and Beuchat, L.R., 2004). Single whole leaves (10g per sample) were placed in a single layer on a sterile cutting board in a laminar flow bio-safety cabinet to facilitate drying.
Inoculum (50 µl) was deposited at three points onto the surface of each intact lettuce leaf (150 µl total), to yield initial E. coli O157:H7 concentrations of approximately 3 log CFU/g (low inoculum) or 6 log CFU/g (high inoculum). Cut and intact tissues were evaluated. To prepare cut samples, the cuticle in the middle of the leaf was disrupted by making a shallow incision with a sterile scalpel careful not to cut through the entire leaf. For cut samples, inoculum (150 µl) was deposited at the incision. After lettuce pieces were inoculated, they were held in the bio-safety cabinet to facilitate drying of the inoculum for one hour at 22 + 2ºC.
Spinach Inoculation Procedures (Cut and Whole)
Fresh Express bagged spinach was purchased at a local supermarket in Blacksburg, VA. Spinach was stored at 4ºC for no longer than 6 hours prior to initiation of experiments.
E. coli O157:H7 H1730 was inoculated onto cut and whole leaves of spinach leaves via spot inoculation methods described by Lang et. al. (Lang, M. M., Harris, L.J. and Beuchat, L.R., 2004). Single whole spinach leaves were placed in a single layer on a sterile cutting board in a laminar flow bio-safety cabinet to facilitate drying. Inoculum (50 µl) was deposited at three points onto the surface of each leaf (150 µl total), to yield initial E. coli O157:H7 concentrations of 3 log CFU/g (low inoculum) or 6 log CFU/g (high inoculum). Cut and intact tissues were evaluated. In order to prepare cut samples, the cuticle in the middle of the leaf was disrupted by making a shallow incision with a sterile scalpel. For cut samples, inocula was deposited at the incision. Following inoculation, samples were held in the laminar flow hood for one hour at 22 + 2ºC to facilitate drying Packaging of Lettuce Un-inoculated (control) and inoculated cut and whole leaf lettuce samples (10 grams per sample) were transferred aseptically into polyethylene PD 900 bags (O2 transmission rate = 200 cc/100 sq. in. and a CO2 transmission = 632 cc/100 sq. in.) (Cryovac, Duncan, SC). In order to simulate retail packaging conditions, the bags were flushed completely (100%) with Nitrogen before sealing the bags using a Koch vacuum packaging machine (Koch, San Mateo, CA, model # UV250).
Packages of lettuce were then held at 4ºC and 10ºC until noticeable spoilage occurred. Spoilage of lettuce was defined as described by Abdul-Raouf, et. al as a loss of typical color, loss of turgidity, development of sliminess and weeping of tissue fluid (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993). Each sample bag was destroyed upon analysis and a fresh, unopened bag was used for each sampling time. Bags were never resealed to continue the test. An equal number of bags were sealed under normal atmospheric gas conditions, this was done by putting 6 holes in each bag using a standard hole puncher.

Packaging of Spinach

Un-inoculated (control) and inoculated cut and spinach leaf samples were transferred aseptically into polyethylene PD 961 bags (O2 transmission rate = 450 cc/100 sq. in. and a CO2 transmission = 1355 cc/100 sq. in.) to achieve 10 grams in each bag (Cryovac, Duncan, SC). In order to simulate retail packaging conditions, spinach leaves were sealed in packages air using a Koch vacuum packaging machine under a 30% vacuum. During storage, the respiration of the spinach modified the atmosphere within the packaged. Packages of spinach were then held at either 4ºC or 10ºC and sampled until noticeable spoilage occurred. Spoilage of lettuce was defined as described by Abdul-Raouf, et. al as a loss of typical color, loss of turgidity, development of sliminess and weeping of tissue fluid (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993).
Each bag was destroyed at sampling time, bags were not resealed to continue the experiment. An equal number of bags were sealed under atmospheric gas conditions by altering bags with 6 perforations using a standard hole puncher.

Analysis of the gaseous atmospheres inside the packages

On each sampling day (0, 1, 3, 5, 7), headspace gas within each package was withdrawn and analyzed to determine the percentage of O2 and CO2 in the packages over time using an oxygen and carbon dioxide gas analyzer (PBI-Dansensor, PBI Development, Denmark, Model 58042610 ).

Enumeration of E. coli O157:H7

Samples (10g each treatment and un-inoculated control) were aseptically transferred from packaging material into a 400 ml stomacher bag with 90 ml sterile peptone water and pummeled for 3 minutes. Serial dilutions of each homogenized sample were made in 0.1% peptone water and were surface plated (0.1 ml per plate) in duplicate onto TSA–K agar and incubated at 37ºC for 24 hours. Typical GFP-expressing E. coli O157:H7 colonies were identified by fluorescence under long wave UV light. Random colonies were selected for further confirmatory testing by streaking cells onto Sorbitol Mackonkey Agar (SMAC) and using the RIM latex agglutination test for confirmation of O:157 and H7 antigens.
Statistical Analysis:
All experiments were repeated three times. Data were analyzed using the Statistical Analysis System (SAS Institute, Cary, NC) for analysis of variance. Significant differences between means were determined by using Duncan’s multiple range tests. A significance level of 0.05 was used for all analyses.

Results and Discussion

The ability of high and low levels of E. coli O157:H7 to survive on cut and whole green leaf lettuce and spinach stored at 4 and 10ºC under passively modified atmospheric conditions for 7 days (lettuce) and 9 days (spinach) of storage was investigated. E. coli O157:H7 was not detected in un-inoculated control samples of lettuce and spinach. Researchers took care to re-create conditions similar to those used in industry practices over the course of this study. E. coli O157:H7 was not detected in un-inoculated control samples of packaged lettuce and spinach.
The concentrations of oxygen (O2) and carbon dioxide (CO2) achieved within the packages are shown in Tables 3.1 and 3.2. Lettuce was sealed under modified atmosphere with initial concentrations of O2 and CO2, between 0.5% – 0.7% (Table 3.1). On each sampling day (0, 1, 3, 5, 7), gases within each packaged were analyzed. The packaged lettuce inoculated with the high inoculum showed that the levels of oxygen increased to 12% and the carbon dioxide levels increased to 0.9% during the 7 day storage period. The concentrations of gases in packaged lettuce when inoculated at low levels achieved an atmosphere of 14 % O2 and 0.5% CO2 during the storage period of 7 days. The O2 and CO2 concentration was not significantly affected by storage temperature.
Spinach leaves were sealed in bags to achieve initial concentrations of O2 between 14% – 16% and CO2 of 0.6% – 1.3% (Table 3.2). On each sampling day (0, 1, 3, 5, 7, 9), gases within each package were analyzed. The concentrations of oxygen (O2) and carbon dioxide (CO2) achieved within the packages are shown in Table 3.2. The packaged spinach inoculated with the high inoculum showed that the levels of oxygen increased to 15.9% and the carbon dioxide levels increased to 1.4% during the 9 day storage period. The concentrations of gases in packaged spinach when inoculated at low levels achieved an atmosphere of 16.1 % O2 and 1.2% CO2 during the storage period of 9 days. The difference in gas concentration between the two temperatures was not significant.
Effect of modified atmospheric packaging on survival of E. coli O157:H7
The modified atmosphere packaging did not significantly affect the survival of E. coli O157:H7. Lettuce stored under modified atmospheric conditions remained acceptable for consumption as long as lettuce packaged under atmospheric conditions. Regardless of temperature, leaf type and inoculum size, Numbers of E. coli O157:H7 consistently declined from throughout the 7 day storage period on lettuce packaged under modified and normal atmospheric conditions.
Within each inoculum size, temperature and leaf type there was no significant difference in bacterial counts of E. coli O157:H7 on bagged lettuce stored under modified atmospheric conditions than those stored under normal atmospheric conditions (P > 0.05) (Figures 3.1 and 3.2). This observation suggests that the gas composition inside the bags did not directly effect the survival or reduction of E. coli O157:H7 on the lettuce.
E. coli O157:H7 decreased significantly by day 1 with a total reduction of 2 log CFU/g by day 7 on lettuce stored under modified atmospheric conditions at 4ºC when inoculated at both low and high levels (Fig. 3.1). Under normal atmospheric conditions an overall reduction in numbers of E. coli O157:H7 was significant by day 1 with a total reduction of 1.5 logs observed by day 7.
Numbers of E. coli O157:H7 slightly increased throughout the 9 day storage period on spinach packaged in both modified and atmospheric conditions. Within each inoculum, temperature and leaf type there was no significant difference in bacterial counts of E. coli O157:H7 on bagged spinach stored under modified atmospheric conditions than those stored under normal atmospheric conditions (P > 0.05). This observation suggests that the gas composition inside the bags did not directly effect the survival of E. coli O157:H7. Spinach stored under modified atmospheric conditions remained acceptable for consumption as long as spinach packaged under normal atmospheric conditions.
E. coli O157:H7 increased 0.5 log CFU/g within 9 days on cut spinach leaves stored under modified atmospheric conditions at 4ºC when inoculated at both low and high levels (Fig. 3.3). There was no change, however, at 4ºC on cut spinach inoculated at low and high levels under normal atmospheric conditions. A significant increase (P <
.05) of 0.5 logs was observed on cut spinach inoculated at low levels at 10ºC under both modified and typical atmospheric conditions throughout the 9 day storage period (Figure 2). At 10ºC when inoculated at high levels a significant increase (P < .05) of approximately 1 log CFU/g was observed on cut spinach at both modified and atmospheric atmospheres. Whole spinach packaged under both modified and normal atmospheric conditions did not have any effect on the number of E. coli O157:H7 cells when inoculated at both high and low inoculum levels at 4 and 10ºC over 9 days.
Other studies have shown significant changes in microbial Numbers on fresh produce under modified atmospheres (Gunes, G. G. a. H., J.H., 2002, Hao, Y. a. B., RE, 1993). Gunes et. al studied survival of E. coli O157:H7 on fresh cut apples in modified atmosphere packaging and found that at a storage of 21% O2 and 30% CO2 at 20ºC significantly inhibited growth of E. coli O157:H7 (Gunes, G. G. a. H., J.H., 2002). However, inhibition was most likely due to the high concentration of CO2 present. CO2 at levels as low as 10% can be bactericidal (M.A Del Nobile, B., A., Benedetto, E. and Massignan, L. , 2005).
In this study, gas atmospheres within packages that were modified had no significant effect to the growth or survival of E. coli O157:H7. Similar observations have been reported that indicate that modified atmospheres have little to no effect on the survival of E. coli O157:H7 (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993). Abdul-Raouf et al. concluded that there was no significant effect on numbers of E. coli O157:H7 under modified atmospheres containing 3% O2 and 97%N2 stored at 5 and 12ºC (Abdul-Raouf, U. M., Beuchat, L.R. and Ammar, M.S., 1993). The concentration of CO2 in their study was not determined.

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Survival on cut vs. whole leaf

There were no significant differences found (P > 0.05) between numbers of E. coli O157:H7 on cut and whole (intact) leaves irrespective of storage temperature, inoculum size, or atmosphere within packages of leaf lettuce. These results suggest that under these conditions, leaf type (cut or whole) may have little effect on the survival of E.
coli O157:H7. There was, however, a significant difference in E. coli O157:H7 numbers between cut and whole leaf lettuce on day 7 when inoculated at low levels at a storage temperature of 4ºC irrespective of atmospheric conditions. This may have occurred for several reasons, one possibility being competition with indigenous micro flora in the cut lettuce resulting in fewer numbers of E. coli O157:H7.
There were no significant differences found (P ≥ 0.05) between cut and whole (intact) leaves on numbers of E. coli O157:H7 irrespective of storage temperature, inoculum size, or atmosphere within packages of spinach leaves. These results suggest that leaf type (cut or whole) has no effect on the survival of E. coli O157:H7.
There are no studies that have explored the survival of E. coli O157:H7 on cut versus whole spinach leaves. Studies have been conducted on attachment to various lettuce surfaces. Greater numbers of E. coli O157:H7 attach to cut edges of lettuce than to undamaged whole leaves (Seo, K. H. and J. F. Frank, 1999, Takeuchi, K. and J. F. Frank, 2000, Takeuchi, K., Hassan, A.N. and Frank, J.F., 2001, Takeuchi, K., Matute, C.M., Hassan, A.N. and Frank, J.F., 2000). Takeuchi and Frank discovered that penetration of E. coli O157:H7 cells was greater into the cut edges than uncut surfaces of lettuce compared to higher temperatures (Takeuchi, K. and J. F. Frank, 2000). In another study, Takeuchi et. al discovered an increased penetration of cells at cut edges at 21% O2 at low temperatures (4ºC) (Takeuchi, K., Hassan, A.N. and Frank, J.F., 2001). They also found that attachment and penetration were reduced at 2.7% O2 as opposed to 21% O2 (Takeuchi, K., Hassan, A.N. and Frank, J.F., 2001). They speculated that modifying the O2 content may affect the degree of penetration of E. coli O157:H7 into lettuce leaves. This may explain why results from the current study do not follow previous literature on the effect of cut and damaged lettuce and spinach leaves on numbers of E. coli O157:H7. In the current study, O2 concentrations did not exceed 17% in any given bag of inoculated spinach and 14% in any give bag of inoculated lettuce. These results suggest that survival of E. coli O157:H7 on cut and whole leaf lettuce and spinach may be affected by O2 concentrations within modified atmospheric packaging.

TABLE OF CONTENTS 
Abstract
Acknowledgements
Dedication
Attribution
Table of Contents
Chapter 1: Introduction and Justification
Chapter 2: Literature Review
Escherichia coli O157:H7
E. coli O157:H7 Outbreaks Associated with Meat Products
Food-borne Disease Outbreaks Associated with Produce
E. coli O157:H7 Outbreaks Linked to Produce Consumption
Spinach
Lettuce
Modified Atmosphere Packaging
Attachment on Cut vs. Whole Surfaces
Green Fluorescent Protein
References
Chapter 3: Effect of storage temperature and atmosphere on the survival of Escherichia coli O157:H7 on cut and whole leaf lettuce and spinach
Abstract
Introduction
Materials and Methods
Results and Discussion
References
Chapter 4: Conclusion 
General Conclusions
Limitations and Pitfalls
Future Research
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