reducing e. coli in lettuce - yara assi 07-11-13

Reducing E. coli in lettuce

by Yara Assi

University of BlamandSaint George University Medical

Center

outline

- Through the microscope

- Breakouts in History

- How lettuce is processed at SGHUMC

Yara Assi - Saint George University Medical Center

outline

Studies

-Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce.

-Modeling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging.

-Development of a risk-based methodology for estimating survival and growth of enteropathogenic Escherichia coli on iceberg lettuce exposed at short-term storage in foodservice centers.


Through the microscope

E. coli is the name of a germ, or bacterium, that lives in the digestive tracts of humans and animals.

There are many types of E. coli, and some can cause bloody diarrhea. One common type is called E. coli O157:H7. In some people, this type of E. coli may also cause severe anemia or kidney failure, which can lead to death.



What causes an E. coli infection?

You get an E. coli infection by coming into contact with the feces, or stool, of humans or animals.

Raw fruits and vegetables, such as lettuce, sprouts, or unpasteurized apple cider or other unpasteurized juices that have come in contact with infected animal feces.

E. coli from person-to-person contact

The bacteria can also spread from one person to another, usually when an infected person does not wash his or her hands well. E. coli can spread from an infected person's hands to other people or to objects.


Breakouts in History



CDC issued a travel advisory for travel to Germany due to the Escherichia coli (STEC) bacteria outbreak that began May 2, 2011.

This outbreak is being caused by an infection with a Shiga toxin-producing Escherichia coli (STEC) bacteria. Many people have been hospitalized, several requiring intensive care, and at least 18 people have died. New cases are still being reported although today the New York Times reported that the outbreak “appeared to be stabilizing.”



Epidemiology of Escherichia coli Outbreaks, United States, 1982–2002


In St George Hospital University Medical Center

Lettuce handling

Step 1:Lettuce is received every Tuesday and Friday

Step 2:Lettuce is stored at ~6 °C until needed



Lettuce handling

Step 3:

Lettuce is cleaned with tap water as a first step of preparation



Lettuce handling

Step 4:

Lettuce is sliced and soaked in soap



Lettuce handling

Step 5:

Lettuce is sanitized in chlorine based sanitizer and left for 30 min.

Reducing E. coli in lettuce

-Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce.

-Modeling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging.

-Development of a risk-based methodology for estimating survival and growth of enteropathogenic Escherichia coli on iceberg lettuce exposed at short-term storage in foodservice centers.


Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuceFrancisco López-Gálvez, Ana Allende, Maria V. Selma, Maria I. Gil *

* Research Group on Quality, Safety and Bioactivity of Plant Foods, CEBAS-CSIC, P.O. Box 164, Espinardo, Murcia, 30100, Spain

International Journal of Food Microbiology - 2009


Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce

1. Introduction

Fresh products are an important part of a healthy diet, but concerns about its safety have been raised as they represent the 2nd leading cause of foodborne diseases in the USA.

In 2006, the E. Choli outbreak functioned as a wake up call for product safety, and lettuce was the single most frequently implicated commodity.

It is very difficult to guarantee the microbiologic safety of the fresh-cut products as the processing omits any effective antimicrobial step.


1. Introduction

The best method to eliminate pathogens is to prevent contamination, disinfectants, can only reduce but not eliminate pathogens.

Water may be a useful tool for reducing potential contamination, it may also introduce or spread contaminants.

Plants’ cut surfaces represent points of entry as well as safe harbor for microbes, in which microorganisms are unaffected by water disinfectants.



1. Introduction

It’s important to test potential sanitizing agents taking into account their suitability to prevent cross-contamination.

Treatment of processing water with sanitizing agents should be managed with the goal of minimizing the effective dose to reduce safety hazards and detrimental effects on the product quality.



1. Introduction

The minimum effective doses of four commercial sanitizing agents were selected based on their capability to eliminate E. Coli in processing water containing high organic matter, simulating real conditions of the fresh-cut industry.

The efficacy of these sanitizing agents to avoid potential transmission of pathogens from inoculated fresh-cut lettuce to processing water and from contaminated water to uninoculated product, simulating cross-contamination, was also determined.

Prevention of Escherichia coli cross-contamination by different commercial sanitizers during wsshing of fresh-cut lettuce


2. Materials and methods

2.1. Bacterial strains and inoculum preparation

Cultures were hydrated in nutrient broth and then inoculated onto separate nutrient agar plates and incubated at 37 °C for 24 h.

2.2. Commercial sanitizers

The commercial sanitizers tested were:

•Chlorine, adjusted to pH 7

•Purac FCC 80,

(3) Citrox 14 W, and (4) Tsunami 100.




2.2. Minimum effective doses

The selection of the appropriate dose of each commercial sanitizer based on the manufacturer's recommendations was carried out simulating the standard conditions in a fresh-cut processing line.

The wash water used in this study had similar characteristics to those found in the washing tank in a fresh-cut processing line.




2.2. Minimum effective doses

Two concentrations of E. coli (3 and 5 log CFU/ml) were added to the processing water to study the efficacy of different sanitizer concentrations reducing the E. coli inoculum.

Processing water was treated with different concentrations of each sanitizer under constant agitation.




2.3. Escherichia coli cross contamination of fresh-cut lettuce

Core and outer leaves were removed and internal leaves were cut into pieces with a stainless steel knife.

In each experiment, fresh-cut lettuce were inoculated by adding E. coli and carefully mixed to ensure a homogeneous distribution of the inoculum.




2.3. Escherichia coli cross contamination of fresh-cut lettuce

For each sanitizer, inoculated fresh-cut lettuce were washed in sanitizing solution for 1 min under manual agitation at 8 °C.

Then, uninoculated fresh-cut lettuce were also washed in the same water for 1 min under the same conditions. Lettuce washed with tap potable water at 8 °C as control was also evaluated.




2.4. Microbial and physicochemical analyses

In each trial, the evaluation of microbial load, chemical oxygen demand (COD) and pH was carried out in processing water samples.

Additionally, microbial load of inoculated and uninoculated fresh-cut lettuces was determined in the cross-contamination trial.




3. Results

3.1. Selection of minimum effective doses

Processing water usually contains high organic load which reduces the efficacy of sanitizing agents and affects quality and safety of fresh-cut products.

COD is commonly used as an indirect measure of total organic load in the water.

When chlorine was added to the processing water, COD value was maintained almost constant, regardless of the applied concentration.




3. Results


However, COD increased when increasing the concentration of the other sanitizers and the increase in COD was extremely high when Citrox and Purac were added.

The pH value of chlorine was maintained within the recommended range (6.5–7.0) independently of the concentration.

pH values decreased when Tsunami, Citrox and Purac were added, particularly with increasing concentrations.



3. Results


Only chlorine at 40 and 60 mg/l and Tsunami at 375 and 500 mg/l reduced pathogen population to undetectable levels.



3. Results


None of the tested Purac and Citrox concentrations were able to eliminate E. coli load.

Therefore, the highest tested concentrations of Tsunami, Citrox and Purac were selected. Higher doses were not tested as they are not recommended by the manufacturers.

The selected concentrations of each sanitizer: chlorine 40 mg/l, Tsunami 500 mg/l, Purac 20,000 mg/l and Citrox 5,000 mg/l, were then compared in the same trial.



3. Results


As previously found chlorine at 40 mg/l and Tsunami at 500 mg/l were effective in inactivating the highest E. coli level inoculated in the processing water.

As observed for the preliminary experiments, COD increased significantly after the addition of Citrox and Purac.



3. Results

3.2. Efficiency of different commercial sanitizers to prevent Escherichia coli cross-contamination of fresh-cut lettuce

Washing inoculated fresh-cut lettuce confirmed that washing had little effect on microbial reduction on product surface.

E. coli population present on inoculated fresh cut lettuce was reduced when washing with chlorine and Tsunami.

Citrox and Purac were almost as efficient as chlorine and Tsunami, barely reducing E. coli counts.

The lowest reduction was observed when inoculated fresh-cut lettuce was washed in tap water.



3. Results


E. coli population in the wash water increased after washing inoculated fresh-cut lettuce in tap water.

When chlorine and Tsunami were added, E. coli was not detected in the water after washing.



3. Results


The COD and pH levels of each washing solution changed after washing fresh-cut lettuce and significant differences were observed among the sanitizing agents.

Washing fresh-cut lettuce increased COD levels.

However, Tsunami and mainly Citrox and Purac significantly increased COD and decreased pH values.



4. Discussion

Processing wash water, if not properly sanitized, can become a source of microbiological contamination.

There is strong evidence that the use of sanitizing agents helps to minimize the potential cross-contamination and thus, maintaining the quality of processing water.

Beside pH and temperature, organic load can have a significant impact on the efficacy of those disinfectants which can be affected by organic material.



4. Discussion

Another important issue to take into account for the selection of the most suitable sanitizers should be the COD contribution. Sanitizers that increase COD determines water quality.

The organic load of the processing water increased when Citrox and Purac were added, while Tsunami did to a lower extent.

Chlorine and Tsunami showed the same efficacy of inactivating E. coli from the processing water with high organic loads.



4. Discussion

The minimum effective dose of chlorine needed to inactivate the E. coli in processing water was much lower than that commonly used in fresh cut processing operations (150–200 mg/l).

Citrox and Purac at the tested doses were not effective for water disinfection. In spite of their limited direct microbial benefit on the produce, sanitizing agents should be used to maintain the quality of the water and to prevent cross-contamination of the product.



4. Discussion

Citrox and Purac, at the highest recommended doses, were not effective in preventing cross contamination between inoculated and uninoculated fresh-cut lettuce.

Some disadvantages are associated with the use of Tsunami when compared to the use of chlorine, such as the cost, pH of the aqueous solution and the increase of the organic load of the wash water.

Thus, the use of chlorine, minimizing the effective dose, still represents the most suitable option to guarantee microbial disinfection of processing water.



Modeling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging*

Guiomar D. Posada-Izquierdo a,*, Fernando Pérez-Rodríguez a, Francisco López-Gálvez b, Ana Allende b,

María V. Selma b, María I. Gil b, Gonzalo Zurera a

a Department of Food Science and Technology, University of Cordoba, International Campus of Excellence in the AgriFood Sector ceiA3, Campus Rabanales, Edificio Darwin e C1,

14014 Córdoba, Spain

b Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, 30100 Murcia, Spain


Modeling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging

1. Introduction

Production and consumption of fresh-cut or minimally-processed (MP) fruit and vegetables has increased which poses serious risk since they do not undergo a process lethal to pathogenic bacteria.

E. Coli is a serious concern for the fresh-cut produce industry since vegetables may become contaminated in the field by contact with contaminated animal waste, dust, soil, irrigation water, and inadequately treated manure.


1. Introduction

Epidemiological data suggest that the pathogen could transmit through the food chain, contaminating the final product (i.e. fresh-cut vegetables) and causing illness.

In the last three decades, many outbreaks of foodborne illness have been associated with the consumption of fresh and fresh-cut produce contaminated with E. coli.



1. Introduction

The cross-contamination during washing, could have a crucial role.

Studies show that chlorinated water, the most used disinfectant agent for the fresh-cut industry, as well as other commercial sanitizers, does not completely eliminate pathogens in produce.



1. Introduction

Studies have shown that E. coli is able to survive during processing, and then recover and grow during refrigerated storage.

The exposure of bacteria to stress conditions like chlorine washing can affect their subsequent growth, depending on the intensity and duration of the stress. However, most studies investigating bacterial growth on MP vegetables have employed pathogenic bacteria cultured under non-stressful conditions



1. Introduction

Modified atmosphere packaging (MAP) can extend the shelf-life of cut lettuce primarily by providing a sufficiently low O2 partial pressure (pO2) to retard browning.

The use of MAP is also intended to inhibit or retard the growth of spoilage and some pathogenic microorganisms, particularly due to the low O2 concentration.




2.1. Bacterial strains and inoculum preparation

A five-strain cocktail of E. coli strains isolated from human and foods associated with hemorrhagic colitis and Heamolytic Uremic Syndrome (HSU) was used in the study.

2.2. Preparation of vegetables and inoculation procedure

Iceberg lettuce was purchased from a local wholesale market at the day of harvest and transported to the laboratory within 1 h under refrigerated conditions.

The outer leaf layers, were manually removed and discarded while internal leaves were cut into pieces. Inoculation was performed by immersion of the lettuce pieces in water containing the inoculums cocktail for 1 min.




2.3. Sanitizing treatment and rinsing

The inoculated lettuce pieces were submitted to a standard chlorination treatment in cold tap water containing chlorine for 30 s.

Fresh-cut iceberg lettuce was washed in the sanitizing solution then rinsed in tap water.

Finally, lettuce was manually centrifuged to remove excess water for 1 min.




2.4. Packaging and storage conditions

After processing, centrifuged lettuce was distributed in plastic bags.

Bags were divided in three different batches and each batch was stored under a specific temperature (8, 13, 16 ◦C).

2.5. Sampling and microbiological analysis

Plates were incubated at 37 C for 24 h before counting in an automated plate counter.

2.6. Headspace analysis

Headspace gas composition (O2 and CO2) in individual packages were monitored using an O2 analyzer



3. Results

3.1. Behaviour of E. Coli in fresh-cut lettuce subjected to chlorine washing and MAP and stored at different temperatures.

The chlorination treatment followed by a rinsing step in water reduced the initial level of E. coli.

The obtained data indicated that surviving E. coli were able to grow at 8, 13, and 16 C while at 4 C, E. coli populations were reduced (data not shown).



3. Results

3.1. Behaviour of E. Coli in fresh-cut lettuce subjected to chlorine washing and MAP and stored at different temperatures.

The initial O2 content decreased in the headspace of the washed fresh-cut lettuce during the storage.

The generation of anaerobic atmosphere is associated with the biochemical activity of the vegetable tissue and native microflora growth but not with E. coli since anaerobic conditions occurred without apparent E. coli growth in the analyzed lettuce as observed at 8 C for 6 days.



3. Results

Pathogen populations increased in samples stored at 8 C.

However, this increment only took place between days 15 and 27, approximately.

Prior to this, there was a prolonged lag phase during which populations remained near inoculation levels.

For samples stored at 13 C, a significant increase of E. coli was observed after three days of storage. After that, E. coli reached an average maximum population density. This density was reached after 8 days, after which time populations began to decrease.

The fastest growth of E. coli was observed at 16 C. Population densities increased, and after reaching stationary phase, counts slightly decreased.



3. Results

3.2. Primary model and kinetic parameters

Overall, the prediction bands contained >82% out of the observed data which means that the approach taken to computing such bands was acceptable despite the high variability found between repetitions and analysis points.



4. Discussion

4.1. E. coli growth in leafy green vegetablesPrevious studies have shown that cells of E. coli that had penetrated into the lettuce leaf tissue at the cut edges were more likely to show cell viability after treatment with chlorine.

A study showed that starved E. coli treated with chlorine grew more rapidly than non-treated E. coli presenting shorter lag phase and higher growth rate. Another study found that treatment with chlorinated water could effectively reduce the number of total bacteria on fresh-cut lettuce more than 2 logs and resulted in a lower maximum specific growth rate, but shortened the lag time of the bacteria growth on fresh-cut lettuce.



4.1. E. coli growth in leafy green vegetables

Storage at 4 °C produced a slight decrease of the E. coli population after 15 days of storage. Other studies also observed a slight decrease of E. coli after storage for non-sanitized and sanitized produce, reporting log-decreases of 1 log cfu/g.

4.2 Importance of confidence interval and prediction limit in application of growth models

Results in our study showed that growth variability was larger at lower temperature, even though the high temperatures also presented a considerable variability.



5.Conclusion

E. coli growth was strongly influenced by temperature as evidenced by the estimated maximum growth rates, which increased as the temperature augmented.

Experimental variability might be captured by using a high number of replicates, revealing that E. coli can present variable response for similar experimental conditions, especially at low temperatures (8 C) in which stressing conditions are more severe.

Although in some cases, no growth occurred at 8 C, risk is still high due to the highly infectious properties of E. coli.



Development of a risk-based methodology for estimating survival and growth of enteropathogenic Escherichia coli on iceberg lettuce exposed at short-term storage in foodservice centersM.Y. Rodríguez-Caturla, A. Valero , R.M. García-Gimeno, G. Zurera⁎

Department of Food Science and Technology, International Campus of Excellence in the AgriFood Sector ceiA3, University of Cordoba, Campus Rabanales s/n Edif. Charles Darwin,

14014 Cordoba, Spain


Development of a risk-based methodology for estimating survival and growth of enteropathogenic Escherichia coli on iceberg lettuce exposed at short-term storage in foodservice centers1.Introduction

E. coli is a potentially hazardous bacterium which may cause severe gastrointestinal illnesses in humans associated with contaminated foods.

Leafy green vegetables are prone to contamination because the current industrial sanitizing treatments do not guarantee the total elimination of the pathogen.

In addition, they are characterized by high water activity levels, which make them more vulnerable to cross contamination.


1.Introduction

Throughout this work, an evaluation of growth of the strain of E. coli in iceberg-lettuce exposed at short-term storage was performed through the estimation of the log difference (logdiff) at different contamination levels (4.5, 3.5 and 2.5 log cfu/g).

Development of a risk-based methodology for estimating survival and growth of enteropathogenic Escherichia coli on iceberg lettuce exposed at short-term storage in foodservice centers


2. Material and methods

2.1 Experimental design at short-term storage conditions

Ready-to-eat iceberg-lettuce was purchased from a local supermarket and refrigerated at 4 °C.

Three initial contamination levels of E. coli were used (4.5, 3.5 and 2.5 log cfu/g) and the growth of the strain was evaluated during 6 h and was monitored at six periods (30, 60, 90, 120, 180 and 360 min) of incubation.

The samples of lettuce were analyzed at each time interval and incubated at 44 °C for 18–24 h.

Five static temperatures (8, 12, 16, 20 and 24 °C) were chosen for the evaluation of the potential outgrowth of E. coli in iceberg-lettuce.




2.2 Inoculation of ready-to-eat iceberg-lettuce with E. coli

The samples were inoculated with inoculum, which was randomly distributed over the lettuce surface.

The Petri dishes were aerobically stored and microbial counts were determined.

The internal temperature of the bags of lettuce together with the external temperature of the refrigerator was measured

The internal and external temperature records were collected.




2.3. Microbial analyses

Both control and inoculated samples were analyzed.

2.4. Data collection in foodservice centers

t/T data were collected from foodservice centers destined to high-risk population groups.

They were divided into school canteens (8 centers), long-term care facilities -LTCF (6 centers) and hospitals (5 centers).




2.4. Data collection in foodservice centers

Technical staff was responsible to take temperature measurements for lettuce-based salads.

Three steps were monitored: refrigeration of raw material in cameras; processing in chill rooms and distribution of ready-to-eat salads.

For each step, an individual temperature measurement was collected daily



3. Results and discussion

3.1. Growth of E. coli on ready-to-eat iceberg-lettuce at static temperatures

Throughout the experiment, variations in temperature inside the samples were between 0.2 and 0.5 °C.

Microbial counts of E. coli strain on ready-to-eat iceberg-lettuce at 8, 12, 16, 20 and 24 °C at 4.5, 3.5 and 2.5 log cfu/g are represented in Fig. 1.

There were not significant differences between the inoculum levels (P>0.05) although a lower increase at 24 °C was observed when the initial contamination was 2.5 log cfu/g (Fig. 1C).



Fig. 1. Observed growth of E. coli O158:H23 in RTE iceberg-lettuce at 4.5 (A), 3.5 (B) and 2.5 log cfu/g (C) at static temperatures and short-term storage (6 h).




3.1. Growth of E. coli on ready-to-eat iceberg-lettuce at static temperatures

The results indicate that maintenance of the lettuce at 8 °C slightly reduced E. coli population from ~3 h until the end of the study while growth was observed from 12 to 24 °C.

However, if chill chain is not maintained, E. coli can grow up to 1 log cfu/g at temperatures above 16 °C, even from the lowest contamination levels.

The previous studies conclude that the ability of E. coli strains to survive and grow during long periods at low temperatures may be related to product type, packaged atmosphere, storage temperature and strain type.




3.2. Simulation model: outgrowth of E. coli at different t/T scenarios in

foodservice centers

Mean storage temperatures ranged from 4.5 to 5.6 °C, although maximum values above 10 °C were observed in refrigeration cameras of LTCF and hospitals.

Minimal and maximal values of processing and distribution conditions were between12.6 and 29 °C

Hospitals presented shorter values regarding the time, in comparison to LTCF and school canteens.





foodservice centers

A more strict control of temperatures in hospitals evaluated was carried out, since salads were refrigerated in chill rooms prior to distribution (11.1 °C)





foodservice centers

Distributions of the whole model outputs for logdiff in school canteens, LTCF and hospitals are represented in Fig. 4.

At low contamination levels (2.5 log cfu/g) distributions were more left-skewed since values closer to 0 were obtained.

In LTCF and hospitals, there were significant differences among the three inocula tested (P<0.05) while in school canteens results were not significantly different (P>0.05).





foodservice centers

It is well known that the temperature of fresh produce should be maintained approximately below 5 °C to reduce the proliferation of spoilage organisms and human pathogens

Regulation about safety of ready-to-eat foods states that a short-term storage without temperature control can be allowed during handling, elaboration, transport and distribution of ready-to-eat foods.



4. Conclusion

E. coli can grow on iceberg lettuce at temperatures above 8 °C, even at low contamination levels of 2.5 log cfu/g.

A slight decrease of E. coli population at 8 °C was observed, which confirms that continuous refrigeration has been an effective preservation method for E. coli inhibition.

Also, the results indicated the ability of this EPEC strain to grow at temperatures above 16 °C.



4. Conclusion

The t/T scenarios analyzed in hospitals resulted in a lower increase of E. coli in ready-to-eat salads.

This study was based on an original approach to describe E. coli survival and growth on iceberg-lettuce, not using conventional kinetic parameters in estimating the procedures.



References

CDC . Retrieved from http://www.cdc.gov/ecoli/

López-Gálvez, F. (2009). Prevention of escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce. Murcia, Spain.: Research Group on Quality, Safety and Bioactivity of Plant Foods, CEBAS-CSIC.

Posada-Izquierdo, G. D. (2012). Modeling growth of escherichia coli o157:h7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging. Córdoba, Spain: Department of Food Science and Technology, University of Cordoba.

Rodríguez-Caturla , M. Y. (2012). Development of a risk-based methodology for estimating survival and growth of enteropathogenic escherichia coli on iceberg-lettuce exposed at short-term storage in foodservice centers.. Cordoba, Spain.: Department of Food Science and Technology, University of Cordoba.


Thank you


reducing e. coli in lettuce - yara assi 07-11-13

Health & Medicine

lettuce prevention

iceberg lettuce

coli infection

washing of fresh

lettuce francisco lpezglvez

introduction fresh products

commercial process conditions

microscope breakouts