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From: Food Quality & Safety magazine, April/May 2006

Controlling Listeria

Environmental Plate Technology Hunts Down All Species

by Cathering W. Donnelly, PhD

Recall of food products contaminated by Listeria monocytogenes are on the rise, due in part to federal regulatory agencies’ increased scrutiny of food processing plants. Each year in the United States, L. monocytogenes causes approximately 2,500 cases of illness, which result in 500 deaths (Mead, 1999). Susceptible persons include pregnant women and those with immune system compromise due to cancer, organ transplant therapy, kidney disease, diabetes, aging and AIDS.

Persons with compromised immune systems account for approximately 20 percent of the United States population, and this percentage continues to rise. The key reason for the heightened regulatory focus on L. monocytogenes is that it causes a higher rate of hospitalization than any other foodborne pathogen: 95 percent of the individuals who acquire Listeriosis are hospitalized, and this disease is the leading cause of death from a foodborne pathogen (Mead, 1999).

An excellent surveillance system is operational in the United States, and the Centers for Disease Control and Prevention (CDC) has achieved tremendous advances in disease surveillance through PulseNet and FoodNet. This enhanced surveillance, however, means an increased likelihood of tracing sources of infection to food product manufacturers. The good news for the food industry is that cases of Listeriosis are stable or on the decline due largely to food manufacturer intervention and prevention efforts.

When comparing data from 1996 to 1998, the incidence of Listeriosis in the United States declined by 40 percent in 2004 (CDC, 2005). Control of L. monocytogenes requires a focused commitment to maintaining the highest levels of plant sanitation and contamination prevention of food being processed. Risk assessment by FDA and USDA has identified those foods at highest risk for transmission of L. monocytogenes to susceptible persons (Health and Human Services [HHS]/USDA, 2003). Particularly for those manufacturers of ready-to-eat (RTE) foods that support high level growth of L. monocytogenes, there is a critical need for environmental control, which is best achieved by frequent environmental monitoring.

Species of Listeria, L. monocytogenes, Listeria innocua, and Listeria welshimeri, are very common and can be found almost anywhere in the environment. As indicator organisms, Listeria is useful in assessing the potential presence of L. monocytogenes in the processing plant environment. During food processing and manufacturing, there is the potential for L. monocytogenes to be continually introduced into that environment. The challenge for food manufacturers is to direct efforts to prevent the growth and establishment of L. monocytogenes within the plant environment through appropriate controls. These include good manufacturing practices (GMPs), sanitation and employee training. It is also critical to have in place a system that verifies these control procedures are functioning. This is most effectively accomplished through an environmental monitoring program. The contamination of food contact surfaces poses the greatest threat to product contamination. Contamination from other areas of the plant can serve as indirect sources of product contamination. Frequent testing is imperative to ensure that routine cleaning and sanitation procedures are working. Absence of Listeria is the desired outcome of this verification.

Failure to control L. monocytogenes may result in the establishment of niches, including biofilms, after which routine cleaning and sanitizing efforts become ineffective. Only through sampling and testing can identification of niche areas be accomplished. If these areas are left unchecked, they will serve as a source of product contamination (Tompkin, 2000).

Numerous studies have shown that colonization of L. monocytogenes in food processing plants and establishment of niches can lead to continuous contamination of food during processing. Investigation of a multistate outbreak of Listeriosis, occurring in 2000 and linked to delicatessen turkey meat, revealed contamination by a strain of L. monocytogenes, which may have persisted in the incriminated processing plant for at least 12 years, and caused intermittent contamination during that time period (Olsen, et. al. 2005). Focus sampling and testing efforts to niche areas where resident strains of L. monocytogenes may persist, such as drains, floors and mats (Tompkin, 2002).

The International Commission on Microbiological Specifications for Foods (2002) provides guidance on environmental sampling sites and zones, which are ranked according to risk for product contamination. In addition to environmental monitoring, establish a corrective action plan that functions to correct the situation when the prevalence of environmental contamination is beyond acceptable limits.

Standard methods for detection of Listeria from environmental sampling can take five days or more for a confirmed positive result (USDA/Food Safety Inspection Services [FSIS]; Microbiology Laboratory Guidebook [MLG] 8.04, 2005; Hitchins; 2002; ISO 11290).

Fortunately, an environmental Listeria (EL) plate provides a rapid and cost-effective tool for food manufacturers to improve the quantity and quality of data received to better detect, manage and prevent Listeria in their plant environment. Rapid results are provided in 29+/- 2 hours (including a one-hour repair step) from the time of sample collection.

This EL plate has a number of advantages. For one, it allows labs to test more often. Compared to real-time PCR ELISA-based and other rapid methods that require use of costly equipment, EL Plates are extremely cost-effective. Compared with traditional protocols, which require preparation of enrichment and plating media, the EL Plates are ready-made, allowing more testing and less media preparation time. This format is particularly suitable for small to very small food manufacturers who are limited by lab size, lack of incubator space, and lack of analytical equipment.

Another advantage is that results can be interpreted quantitatively, providing more information to identify hot spots, niches of resident strains and contamination sources for appropriate corrective action. Routine environmental monitoring and testing through use of Listeria as indicator organisms for the pathogen L. monocytogenes allows the establishment of a baseline, which can be used for comparative purposes, to observe trends or to detect a contamination problem. Once plant history and normal values have been established for a particular plant, threshold limits can be established and used to determine when corrective action is required. Online sampling during production, coupled with use of quantitative data, can indicate times when periodic cleaning and sanitation are necessary to reduce numbers and overall probability of product contamination.

The EL Plate protocol also provides a one-hour period of nonselective enrichment in buffered peptone water (BPW) to resuscitate potentially stressed Listeria, which may exist as a result of exposure to chemical sanitizers, heat, frozen sampling sites or dry/starvation environments. Failure to resuscitate injured Listeria may result in these organisms escaping detection if highly selective enrichment media are used for recovery (Donnelly, 2002). Injured Listeria can repair damage, go on to regain pathogenicity and grow to high levels in foods.

During routine microbiological analysis, it is essential to account for all Listeria, both healthy and injured. Failure to detect injured organisms may produce misleading negative results and provide a false sense of security with respect to risks that the presence of Listeria poses in the food processing environment. In addition, the EL Plate format requires no enrichment and is sealed by a protective film so it presents less cross-contamination risks. Many small companies routinely perform traditional indicator tests such as coliform counts to indicate the potential presence of E. coli, or standard plate counts to provide an indication of overall bacterial numbers. Companies elect these tests instead of specific pathogen testing because of concerns pathogens present in the laboratory environment. However, coliform media and standard plate count agar can support the growth of pathogens, and all growth media must be treated to prevent the spread of pathogenic organisms.

Regarding specific pathogen testing for L. monocytogenes, traditional regulatory protocols, such as those described by the USDA-FSIS, require use of primary and secondary liquid enrichment media prior to plating on selective media such as modified oxford agar (MOX). In liquid enrichment media, Listeria, if present in a sponge or other environmental sample, can grow to high numbers. During transfers, the potential exists for cross-contamination of the laboratory, which could lead to inadvertent plant contamination. Further, this format is ideal for shipment of suspect plates requiring follow-up to consulting laboratories, where sub-typing to identify potential for resident subtypes can be performed. EL plates can be easily coded for sample identification, and the sealed film format provides confidence that such transfer could be managed without concerns for cross-contamination or sample mix-up. The small size and lack of bulk of the EL plates means that many samples can be efficiently and cost-effectively shipped for follow-up confirmation and analysis.

There are also a couple of myths associated with L. monocytogenes testing that are important to dispel:

Myth #1: Listeria testing cannot be done in house

The food industry has many available technologies for the detection, isolation, identification and characterization of L. monocytogenes. Rapid and automated microbiological methods have provided tremendous advances for the food industry. Because Listeria contamination of foods results primarily from resident strains in food processing plants, it is ideal if these specific biotypes can be identified. This is achievable for large companies with a trained technical staff to perform pulsed-field gel electrophoresis (PFGE) or ribotype analysis.

Distinguishing transient biotypes, which may be associated with raw foods, from resident types implicated in human illness, allows manufacturers to focus efforts to eliminate niches. While this may be a practical approach for large manufacturers, it is much more cost-effective for smaller plants to target Listeria as indicator organisms and use this data to achieve environmental control. EL plates are ideal tools for routine environmental testing in verification of cleaning and sanitation efforts.

Depending on the level of expertise in the food company and how the facility is designed for production, specific testing for L. monocytogenes is not normally advised within the plant environment. This is because positive controls are required in testing and the potential exists for laboratory contamination or introduction of L. monocytogenes into the processing environment. However, with EL plates, the nonpathogenic species L. innocua can be used as a positive control, thus eliminating the need to use the pathogenic L. monocytogenes. If follow-up is desired, EL plates can be shipped to an outside lab for further analysis and confirmation of biotypes resulting from growth on the EL plates.

Myth #2: Non-enrichment methods are not as effective as enrichment methods

The advantage of the EL plate is that it is a no-enrichment, sample-ready culture medium with a chromogenic indicator, providing results within 29+2 hours. A comparative study of the EL Plate Method was conducted versus USDA and modified USDA procedures for recovery of Listeria from 192 environmental and food contact surfaces (Groves and Donnelly, 2005). The USDA procedure employs primary selective enrichment in the University of Vermont Medium (UVM) followed by secondary enrichment in Fraser broth and selective plating on MOX agar.

Previous laboratory results found that the sensitivity of the USDA method could be improved through dual primary enrichment in both UVM and Listeria repair broth (LRB) media (Pritchard and Donnelly, 1999). LRB is a highly nutritious repair/ enrichment medium which supports both repair and high level growth of Listeria. In their studies on enrichment of dairy environmental samples in the UVM and Listeria repair broth, combining these two primary enrichment media into a single tube of Fraser broth for dual secondary enrichment yielded a significantly higher percentage (p < 0.05) of Listeria-positive samples than did use of either LRB or UVM alone. Silk et al. (2002) performed a comparison of growth kinetics for healthy and heat-injured L. monocytogenes in eight enrichment media, and LRB was shown to support higher maximum growth and faster mean repair times when compared with buffered Listeria, enrichment broth (BLEB), (M52) and UVM media (used in the FDA and USDA-FSIS procedures, respectively). In working with EL plates, they were found to have specificity and accuracy equal to that of the standard methods, and higher sensitivity, even when compared against dual primary enrichment using LRB to increase sensitivity and promote recovery of injured Listeria. The EL plate required less time to achieve the results. Further, because this system did not use enrichment, there was less risk of introducing contamination into the lab, thus making this method a viable alternative for in-house testing.

The plate format created less biohazardous waste and required less incubator space, making it an ideal test format for effective environmental monitoring for Listeria. Reducing the presence of Listeria, particularly persistent strains that have established residence in processing plants, will lead to improvements in product quality, regulatory compliance, and ultimately, further achievements in improved food safety and public health. n

References:

  • CDC 2005. “Preliminary FoodNet Data on the Incidence of Infection with Pathogens Transmitted Commonly Through Food-10 Sites.” United States, 2004. MMWR 54 (14): 352-356.
  • Donnelly, C. W. 2002. “Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury.” JAOAC Int. 85:495-500.
  • Groves, E. and C.W. Donnelly. 2005. “Comparison of 3M Petrifilm Environmental Listeria Plate vs. Standard Methods in detecting Listeria from Environmental Surfaces.” IAFP Annual Meeting, Abstract
  • HHS/USDA. 2003. “Quantitative assessment of the relative risk to public health from food borne Listeria monocytogenes among selected categories of ready-to-eat foods.” http://www.foodsafety.gov/~dms/lmr2-toc.html.
  • Hitchins, A.D. 2003. “Detection and Enumeration of Listeria monocytogenes in Foods.” Chapter 10. Revised 2003. FDA Bacteriological Analytical Manual (BAM).
  • ICMSF (International Commission on Microbiological Specifications for Foods). 2002. Microorganisms in Foods 7. “Microbiological testing in food safety management.” New York, NY:Kluwer Academic/Plenum Publishers.
  • Inside Microbiology 2003: “Getting a handle on Listeria.” Food Safety Magazine. http://www.foodsafetymagazine.com/issues/0212/colmicro0212.htm.
  • Mead, P.S., L. Slutsker, V. Dietz, et al. 1999. “Food-related illness and death in the United States.” Emerg Infect Dis. 5:607-625.
  • NF EN ISO 11290-1. 1996. “Horizontal method for detection and enumeration of
  • Listeria monocytogenes.” Part 1: Detection method.
  • NF EN ISO 11290-2. 1998. “Horizontal method for detection and enumeration of Listeria monocytogenes.” Part 2: enumeration method.
  • Olsen, S.J. et al. 2005. “Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat” Clin. Infect. Dis. 40:962-967.
  • Pritchard, T. J. and C.W. Donnelly. 1999. “Combined secondary enrichment of primary enrichment broths increases Listeria detection.” J. Food Prot. 62:532-535.
  • Silk, T/M., T.M.T. Roth and C.W. Donnelly. 2002. “Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths.” J Food Protect. 65:1333-1337.
  • Tompkin, R.B. 2002. “Control of Listeria monocytogenes in the Food Processing Environment.” J. Food Protect. 65:709-725.
  • Tompkin, R.B., V.N.Scott, D.T. Bernard, W.H. Sveum and K.S. Gombas. 1999. “Guidelines to prevent post-processing contamination from Listeria monocytogenes.” Food Protection Trends. 19:551-562.
  • USDA/FSIS, 2005. “Isolation and identification of Listeria monocytogenes from red meat, poultry, egg and environmental samples” (revision 4), chapter 8. In USDA/FSIS Microbiology Laboratory Guidebook (MLG), 3rd ed. Food Safety and Inspection Service, Washington, D.C.

Catherine W. Donnelly, Ph.D., is Professor of Nutrition and Food Science at the University of Vermont (UVM). Reach her at cdonnell@zoo.uvm.edu.

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