Listeria monocytogenes Challenge Testing of Ready-to-Eat Refrigerated Foods

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Version Number: 1
Issue Date: November 24, 2010

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Table of Contents

• 1. Purpose
  
  
• 2. Scope
  
  
• 3. Background
  
  
• 4. Safety Precautions
  
  
• 5. Suggested Experimental Design
  • 5.1 Listeria monocytogenes strains
  • 5.2 Preparation and Enumeration of Cells
    • Maintenance of cultures and inoculum preparation
    • Inoculum Level
  • 5.3 Sampling Design
    • Lethal treatments
  • 5.4 Preparation of Food Product
  • 5.5 Inoculation of Food Products
  • 5.6 Special Product Packaging Conditions
  • 5.7 Incubation of Inoculated Food Products
  • 5.8 Enumeration and Enrichment Methods
  • 5.9 Interpretation of Data and Documenting Results
    
    
• 6. Measures to Take if Food Product Supports Growth of Listeria monocytogenes
  
  
• 7. References

1. Purpose

The purpose of this document is to recommend an experimental design for challenge test studies to determine the growth potential of Listeria monocytogenes in ready-to-eat (RTE) foods, more specifically, to determine if a RTE food can or cannot support the growth of L. monocytogenes. In this document, those RTE foods in which the growth of L. monocytogenes can occur are defined as products where L. monocytogenes levels will increase more than 0.5 log cfu/g over the shelf-life of the product, under reasonable conditions of distribution, storage and use (Health Canada, 2010). Additionally, this document provides guidance on how to assess the efficacy of lethality treatments for L. monocytogenes in RTE refrigerated foods.

2. Scope

This document is intended for use by academic, private, industry and/or governmental laboratories involved in designing, implementing and interpreting the results of challenge test studies for L. monocytogenes.

Experiments conducted according to the recommendations of this document can be used to determine whether L. monocytogenes can survive and/or grow to a level of concern in refrigerated RTE foods. Examples of refrigerated RTE foods where L. monocytogenes challenge testing studies may be used include, but are not limited to processed meat or poultry products such as deli-meats that are cured or not cured, smoked fish products, complete meals, soft cheeses, soups, sauces, pasta and other prepared salads, and sandwiches. Wherever possible, the challenge study should be performed using "worst-case" scenario parameters, i.e., the conditions that would be most the permissive for growth of L. monocytogenes.

In addition to assessing the safety of a product in terms of the growth of L. monocytogenes, challenge studies can be used to validate a treatment or process that aims to reduce or eliminate the presence of the pathogen. Data collected from challenge studies can also be used to determine the shelf-life of a product.

This document can also guide food safety regulators and government inspection agencies in their evaluation of the design and interpretation of challenge studies involving L. monocytogenes.

An expert in food microbiology should be involved in all phases of the study, especially in the study design and interpretation of results.

3. Background

Outbreaks and sporadic cases of listeriosis caused by the ingestion of L. monocytogenes in RTE foods are numerous (Pagotto et al., 2006). High risk foods include ready-to-eat deli meats, hot dogs, pâté and soft cheeses (Health Canada, 2010). Recent examples of RTE foods that have caused illnesses are sliced deli meats, pasteurized milk, pre-packed sandwiches, cheese and meat frankfurters (PHAC, 2009; Anonymous, 2008; Dawson et al., 2006; Pagotto et al., 2006; Mead et al., 2006).

Increased consumer demand for convenient and fresh foods with minimal preservatives and low thermal processing has led to increased sales of RTE foods worldwide. Many refrigerated RTE foods are treated with mild heat processes, with maximum temperatures typically reaching 70-95°C, packaged in a vacuum or with modified atmospheres (usually anaerobic), and then refrigerated (Peck, 2006). The combination of a heat treatment and refrigerated anaerobic storage is designed to prevent the growth of non-spore forming pathogens and spoilage organisms. However inadequate kill steps, post-process contamination, or characteristics of the product may allow for the survival and growth of pathogens. The pathogenic bacterium L. monocytogenes is of particular concern because of its ability to grow in the absence of oxygen, at refrigeration temperatures, and survive in the processing plant environment where it can contaminate foods during pre or post-processing (D'Amico and Donnelly, 2008). An extended shelf-life exacerbates the problem by providing additional time for L. monocytogenes to grow to numbers high enough to cause illness. In addition, an extended shelf-life provides more opportunity for temperature abuse of the product, enabling any L. monocytogenes present in the product to possibly grow to levels higher than 100 cfu/g, considered unacceptable in many jurisdictions (Health Canada, 2010; US FDA, 2008; Codex, 2009).

4. Safety Precautions

The Office of Laboratory Security, Public Health Agency of Canada, states that L. monocytogenes should be handled under biosafety level 2. Personnel must be fully informed about the hazards (refer to  Material Safety Data Sheets).

Containment equipment and facilities should be used for all activities involving clinical materials or cultures. Biosafety cabinets should be used for activities likely to generate aerosols. A laboratory coat, gloves and eye protection should be worn.

Potentially infectious materials should always be stored in sealed containers that are appropriately labelled. Containers should be stored and transported in unbreakable, leak-proof trays or boxes. If accidental spills occur, allow aerosols to settle, wear protective clothing, gently cover spill with paper towels and apply 1% sodium hypochlorite starting at the perimeter and working towards the centre (PHAC, 2001). Allow sufficient contact time (30 min) before clean up.

All materials should be autoclaved at 121°C for a minimum of 15 min (PHAC, 2001). Used glassware and other supplies in contact with infectious materials are to be placed in a sturdy, heat-resistant container when autoclaved. Disposable material such as gloves, cotton or tissue paper must be collected as biohazardous waste and autoclaved.

No pathogens or inoculated products should enter food production areas or be used on food production equipment.

5. Suggested Experimental Design

5.1 Listeria monocytogenes strains

To account for variation in growth and survival among strains of L. monocytogenes, challenge studies should generally be conducted with a pool (i.e., cocktail) of at least five different strains. If there is little knowledge of how the organism grows or responds to a particular food commodity, a cocktail of up to 10 different strains can be used (NACMCF, 2009). The inoculum should at least include strains of serotypes 1/2a, 1/2b and 4b. Strains isolated from the same food, or a food similar to the one being tested, should be included, where possible. Additionally, the use of strains isolated from outbreaks or sporadic cases should be included if they are available. It is important to carefully pre-screen and characterize the strains for growth, tolerance, possible treatment resistant characteristics (i.e. resistance to heat, salt, acidity, etc.), as well as possible competition between L. monocytogenes strains, prior to their inclusion in the cocktail (Gorski et al., 2006). Many of the organisms that are considered suitable for challenge testing have been carefully characterized and made available in international culture collections from where they should be obtained.  ATCC and  ILSI both house strain collections with a wide variety of isolates.

In certain circumstances, it will be necessary to use a surrogate organism (i.e., when conducting a challenge test in a food processing facility or a pilot plant to validate a process). Surrogates should be used only when there are no other options and they should not be used for controlled laboratory studies. The surrogate being used should demonstrate growth and resistance characteristics equal to or greater than that of L. monocytogenes. Listeria innocua can be used as a surrogate for L. monocytogenes (Scott et al., 2005).

5.2 Preparation and Enumeration of Cells

Maintenance of cultures and inoculum preparation

Organisms should be stored in the laboratory by a method that minimizes or eliminates transfers (i.e., in glycerol, stored at -80°C). This is important to avoid mutations or changes that may affect their growth or survival characteristics (Herruzo-Cabrera et al., 2004; Pagotto et al., 2005). AOAC International Guidelines for Laboratories (2006) recommends that no more than five passages of the reference strain should take place.

From a frozen stock of an isolate, streak for colony isolation onto a non-selective agar plate (i.e., Trypticase soy agar) and incubate for 24-48h at 37°C. Inoculate a non-selective nutrient broth (i.e., Trypticase Soy Broth with 0.6% yeast extract or Brain Heart Infusion) with cells from a single colony grown on the non-selective agar media and incubate the inoculated broth for 24-26h at 37°C to obtain stationary cells at approximately 1 x 10⁹ cells/ml. This should be done separately for each strain. From this broth, enough aliquots of frozen stocks generated from the single colony should be stored in order to complete all challenge studies without multiple passages of an isolate. The strain viability and retention of significant phenotypic characteristics should be verified before starting any challenge studies.

Since the products being tested are refrigerated, strains should be sub-cultured and stored in Trypticase Soy Broth at refrigeration temperatures (4°C) for approximately 48h, or until the cells enter early stationary phase (Scott et al., 2005). Each strain should be washed by centrifugation and resuspended in a carrier such as Phosphate Buffered Saline (PBS), 0.1% Peptone Water (PW) or a homogenized portion of the food. Equal numbers of each of the strains to be used in the cocktail should be thoroughly mixed together and dilutions made in either PBS or PW to achieve the desired concentration. In some situations, the strains may need to be centrifuged to increase the concentration. After the mixed working inoculum is prepared, the viable and injured populations should be determined by direct plating of a dilution series on both selective and non-selective agars.

The challenge strains should be in the same physiological state that contaminating cells are likely to be in, usually the stationary phase. In some situations, it may be necessary to adapt the challenge strains, for example, to a lower pH using broth with glucose or acidulants, or to a lower a_w using humectants found in the product formulation, or to colder temperatures by storing the cultures at refrigeration temperatures, or to increase the heat resistance by growing at higher than optimal temperatures (NACMCF, 2009). An expert food microbiologist should be consulted as strain adaptation responses may not be straightforward (Koutsoumanis and Sofos, 2004; Doyle et al., 2001).

When the manufacturing process or conditions of the product are likely to cause injury to the organism if it is present, then injured cells should be used in the challenge study (Microbiological Methods Committee, 2010). Sub-lethal treatments of drying, heating, freezing etc., can be used to stress the organism. Adaptation and stressing should be performed prior to making the mixed inoculum, to ensure that each strain maintains equal representation. Further information on adapting strains can be found in the Compendium of Analytical Methods, Part 4: Procedure for the Development and Management of Food Microbiological Methods-Procedure for Stressing Microorganisms in Artificially Contaminated Samples (Microbiological Methods Committee, 2010).

Inoculum Level

The inoculum level used in the L. monocytogenes challenge study depends on whether the objective of the study is to determine the product stability and shelf-life, or to validate a lethality step designed to reduce microbial numbers. It may be necessary to conduct challenge studies using multiple inoculum levels to determine the margin of safety in the process (Scott et al., 2005).

Typically, to determine product stability and shelf-life, the inoculum should be diluted so that a final concentration of approximately 10²-10³ cfu/g of product is attained (Table 1). A challenge test where the inoculum contains too many organisms may overload the preservative system associated with the product, whereas too few organisms may give a false-negative result. In addition, the detection limits of the enumeration method must be taken into account. If it is deemed necessary, lower levels of inoculation may be used (i.e., <100 cfu/g), if this level of contamination is more in keeping with levels of natural contamination. However, consistent inoculation and enumeration may be difficult at these low levels. Enumeration can be made more accurate by i) increasing the sample size, ii) using a Most Probable Number (MPN) method or iii) by increasing the number of replicate samples to be analyzed (NACMCF, 2009; Corry et al., 2010).

A challenge test to validate a lethal treatment will require a higher initial inoculum level, in the range of 10⁶-10⁷ cfu/g of product (Table 1) (FDA, 2001; NACMCF 2009; Scott et al, 2005). However, some lethality studies may be designed to inactivate low levels of microorganisms that have contaminated the product in the post-processing stage, after an initial lethality treatment. In this case, an inoculation level of 10⁴-10⁵ cfu/g might be more appropriate (Scott et al., 2005), followed by inoculation at 10³ cfu/g and an enrichment method to detect the presence or absence of L. monocytogenes (Table 1). These levels of inoculation would indicate if the lethality treatment can achieve the required 3-log reduction at low levels of contamination (Health Canada, 2010).

It is recommended that an inoculum volume representing no more than 1% of the product weight or volume be added to the product.

Table 1. Examples of Suggested Inoculation Levels

Recommended level	Purpose	Reference
1-10 cfu/g inoculation level	Growth challenge studies	Uyttendaele et al. (2004)
Target the inoculation level at 50 cfu/g, should not exceed 100 cfu/g	Growth challenge studies	Beaufort et al. (2008)
102 - 103 cfu/g of product	Growth studies / product stability	FDA (2001)
102 cfu/g	Assessment of Listeria monocytogenes growth in foods	Augustin et al. (2010)
102 - 103 cfu/g	Growth studies Lower levels could be used if detection methods are sufficiently sensitive	NACMCF (2009)
102 - 105 cfu/g	Evaluating antimicrobial agent or post-processing lethality tests (low level inoculation)	Scott et al. (2005)
106 - 107 cfu/g	Validating a process lethality step; based on target level of reduction (i.e., 5 log or 3 log)	FDA (2001); NACMCF (2009); Scott et al. (2005)

5.3 Sampling Design

Sampling plans should be designed with practical considerations in mind, as well as statistical validity. To optimize the experimental design, it is recommended to consult a statistician with experience in experimental designs for food microbiology. The following are general recommendations for sampling design.

A minimum of three replicates, preferably five, should be analyzed at each sampling time, including time zero (immediately after inoculation). Sampling times should be set so that sufficient (minimum 3-4) data sets can be collected before the product becomes overtly spoiled or reaches the end of its shelf-life (Scott et al., 2005). Testing could take place more frequently at the beginning of the study, depending on the expected behaviour of the organism.

Additional product analyses should be performed on duplicate samples to allow evaluation of how changes in intrinsic characteristics would be expected to affect the survival and growth of L. monocytogenes over the life of the product. Analyses at time zero and the end of shelf-life should include a_w, pH, salt content, preservative level, aerobic plate count and gas analysis for modified-atmosphere-packaged products. Depending on the type of product, other analyses could include protein content, fat content, titratable acidity, moisture content, lactic acid bacteria count, psychrotrophic count, spore count, anaerobe count, etc.

In addition, a number of "blank" controls for analysis of background microflora, physical-chemical properties, modified atmosphere, etc., will be needed to monitor changes throughout the testing period. "Blank" controls should be inoculated with sterile water. Depending on the study design, inoculated controls without the antimicrobial treatment or other factors may be needed.

Two to three batches of product should be tested to account for product variation. The number of batches and samples tested should be increased for products with greater variation in composition, water activity, pH, etc., between batches.

The sample size for each data point should be as large as possible to reduce variation around the data points. A minimum sample size of 25 g or mL should be used for qualitative detection. For enumeration, the sample should be in a 1:5 dilution with the liquid diluent. In food matrices that require a higher dilution to allow for ease of spreading the food/diluent slurry on the agar plate, a 1:10 dilution can be used. More information on detection and enumeration can be found in section 5.8.

The sampling method should be appropriate for the food and the way in which it was inoculated. This may involve rinsing/washing the surface of the sample and analyzing the rinsate. Ideally, the entire sample should be weighed and blended with diluent (Notermans, 1993). Liquids can be mixed by blending, stomaching or pulsifying and an aliquot analyzed.

Lethal treatments

When validating lethal treatments, the product formulation and the treatment parameters within the typical range that are most likely to result in survival should be used. This will provide information on the 'worst case scenario' and minimum and maximum control limits for normal production can be set accordingly. Doyle et al (2001), provide a comprehensive review on the factors that influence the heat resistance of L. monocytogenes. This information should be used when designing the strain adaptation and product preparation aspects of the study. For example, if validating a heat treatment, perform the test using product with moisture values at the low end of the typical range encountered during production of the product, as pathogens have greater heat resistance at lower moisture values. Likewise, lower water activity can protect L. monocytogenes against high hydrostatic pressure processing (Hayman et al., 2008).

The inactivation kinetics should be determined by analysing products at several points throughout the treatment (Scott et al., 2005). To account for low levels of cells that may have survived the treatment and are subsequently able to multiply during the shelf-life, the product should be analysed at time zero after treatment, the mid-shelf-life and the end of the shelf-life, for the presence and levels of the microorganism, using enumeration (direct plating and MPN), as well as enrichment methods for presence/absence.

For all challenge studies, a minimum of two separate (preferably three) replicates of the experiments should be performed.

5.4 Preparation of Food Product

The critical parameters and process variability of the product should be known (i.e., mean values and standard deviation for pH, a_w, salt content, preservative concentration, etc.). Data may need to be collected to ensure the challenge test conditions encompass this variability (Scott et al., 2005). It is usually recommended to use the "worst case" conditions within the typical range for each critical parameter, i.e., test the formulation that is the most permissive for growth. For example, when studying the growth of L. monocytogenes, if the typical pH range of a product is 5.5 - 5.9, product with a pH of 5.9 should be used. This may involve changing the pH, a_w or other characteristics of the product being used for the study.

The point in the process when the food is inoculated with the challenge strains should be as similar as possible to the point at which contamination is likely to occur. Consideration of the impact of competing background microflora on the growth of L. monocytogenes should be taken into acount (NACMCF, 2009) and levels of spoilage microorganisms should be monitored throughout the shelf-life for possible interactions.

5.5 Inoculation of Food Products

When inoculating food with the challenge strains, the method should reflect the way contamination is likely to occur and the conditions of the product at that point. The distribution of the inoculum does not have to be homogeneous throughout the product if this is unlikely to occur naturally (Beaufort et al., 2008).

It is very important that the critical parameters of the product are not altered by the addition of the inoculum. This may mean decreasing a_w or pH values of the liquid being added, increasing the concentration of the inoculum, or using liquid already being added to the formulation to suspend the challenge strains. A post-inoculation drying and attachment period may be needed.

Surface inoculation of solid foods to simulate post-heating contamination can be performed by dipping the food into the inoculation suspension for a standardized period. The inoculum can also be surface-smeared over the food by using a sterile bent glass rod or a sterile pipette if a consistent level of inoculum can be delivered. Alternatively, with the aid of a sterile needle, inoculum can be evenly delivered to packaged products through a septum placed on top of the packaging material. A spray pistol inoculation is an additional alternative method that can be used to distribute the inoculum onto the product. The inoculum should be spread evenly over the surface of the product or the packaged product and gently massaged to evenly distribute it. If applicable, inoculum can be added directly during mixing, grinding or moulding.

Liquid products are most easily inoculated by adding the smallest volume of inoculum that is practical, followed by thorough mixing of the product.

To confirm the level of inoculation on the food product, a sample should be taken and enumerated immediately after the inoculation is performed, prior to storage or performing a lethal treatment. Enumeration methods are described in section 5.8.

5.6 Special Product Packaging Conditions

The inoculated product should be packaged as intended for retail sale. In products with modified-atmosphere packaging, care should be taken during inoculation to avoid disruption of the head space atmosphere and any change in composition of the gaseous environment. This may be difficult if the contents of the pack are under pressure. In this case, the product could be inoculated before packaging or re-packaged after inoculation, provided that this does not result in a safety hazard. A cover or septum which closes immediately after inoculation could also be used. The atmosphere should be defined and analysed throughout the test period to confirm that it does not change. If it is feasible, the product could be inoculated prior to the packaging step. If pre-packaging inoculation is the method of choice, the inoculated product should not come in contact with the packaging equipment used on products for distribution.

Following inoculation, product samples should carry labels warning of a biological hazard, and should remain under the control of the investigator. Inoculated product should not enter food production areas.

5.7 Incubation of Inoculated Food Products

For inoculated pack studies, it is recommended that the total incubation time should be at least equivalent to the anticipated shelf-life of the product (or until the product is clearly unfit for human consumption). If it is feasible, the product should be incubated up to one and a half times the anticipated shelf-life (Table 2). As a minimum, enumeration to determine the growth or survival of L. monocytogenes should be incorporated at time zero (immediately after inoculation of the product), the mid and end-point of the shelf-life, and if possible, at one and a half times the shelf-life. If the product is composed of different components, testing should cease on the day after overt spoilage of any of the components.

In testing the effect of storage temperature, an appropriate range of temperatures should be used (e.g., 4, 10 and 25°C). The temperatures chosen for the challenge study should accurately reflect the anticipated storage conditions and possible consumer temperature abuse (Table 2).

5.8 Enumeration and Enrichment Methods

Quantitative determination for L. monocytogenes is done according to the Health Products and Food Branch, Health Canada, official method MFLP-74, Enumeration of Listeria monocytogenes in foods (Pagotto et al., 2002). A supplement to the method is available at. If low levels of L. monocytogenes are expected, it is recommended to use an MPN enumeration method (ideally with 10 replicates) in addition to the direct plating method described above.

Lethality studies that require an enrichment step should use official method MFHPB-30, Isolation of Listeria monocytogenes from all food and environmental samples (Pagotto et al., 2001). Enrichment steps should be used when the expected levels of surviving cells are below the detection limit of direct plating. Methods of enrichment and enumeration from other organizations (i.e., AOAC International, ISO, CEN/AFNOR etc.) that have been validated as per the criteria in Health Canada's Compendium of Analytical Methods can also be used.

Table 2. Examples of Suggested Incubation Temperatures and Storage Times^*

5.9 Interpretation of Data and Documenting Results

To determine the log increase of L. monocytogenes over the shelf-life of the product, calculate the difference between the median concentration at the end of the shelf-life and the median concentration at day zero (in units of log₁₀ cfu/g). Calculate the difference separately for each batch. Example calculations are given in the Technical Guidance Document on Shelf-life Studies for Listeria monocytogenes in Ready-to-Eat Foods (Beaufort et al., 2008).

Graphical plotting (semi-log) of data over time will provide information on whether the L. monocytogenes population has increased, decreased or remained stable. Combining population data with data on spoilage organisms, product parameters, and other factors, provides information that can be used to determine the safety of the product, determine the shelf-life and understand the critical factors underlying the control of L. monocytogenes. These data should enable food processors to determine whether the limit for L. monocytogenes of 100 cfu/g will be reached during the shelf-life of the product.

Calculating the difference between the concentration (log cfu/g) of L. monocytogenes after a lethal treatment and the concentration (log cfu/g) before the treatment will give the log reduction. The Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2010) recommends a minimum 5-log reduction treatment during processing (usually achieved by heating), and a minimum 3-log reduction treatment post-processing.

All aspects of the challenge study should be documented in a report. This will include information on the selected strains and their preparation, the properties and intended shelf-life of the food product(s) tested, inoculation method, justification of the storage conditions and length, sampling design and method, enumeration and isolation methods, raw data and calculations as well as the conclusions and interpretation. The reasoning and statistical data behind each decision should also be documented.

6. Measures to Take if Food Product Supports Growth of Listeria monocytogenes

For guidance on recommended procedures and practices to reduce the risk of L. monocytogenes in RTE food products, refer to the Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2010). In general, food safety is managed by adherence to good hygienic practices and HACCP-based procedures.

It may be possible to reformulate the food product to prevent the growth of L. monocytogenes by altering the composition of the product, i.e., decreasing pH, decreasing water activity with humectants, adding approved preservatives, etc. A reassessment of the lethality treatments and/or the shelf-life may be necessary. An evaluation of the microbiological quality of individual ingredients can also provide useful information.

7. References

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