A Focus on Listeria monocytogenes -- Updated Version

On this page... Classification Natural Reservoirs and Transmission Growth Characteristics Mechanism of Infection Listeriosis Fatality of L. monocytogenes Foodborne Disease Outbreaks Detection Methods Regulatory Status of L. monocytogenes Research at the USDA Agricultural Research Service References This technical fact sheet illustrates the following key points about Listeria monocytogenes: Leading cause of death among foodborne pathogens with a 20 to 25 percent fatality rate. Grows at refrigeration temperatures, high salt concentrations, and a low pH, unlike most pathogens. Spreads from cell-to-cell in infected tissue without directly contacting the extracellular environment. Causes two types of Listeriosis: invasive and non-invasive, which are most commonly caused by eating contaminated ready-to-eat foods. Research is focused on developing control methods in food processing plants and in the areas of genomics and proteomics. Listeria monocytogenes, one of the most virulent foodborne pathogens with 20 percent of clinical infections resulting in death4, is the causative agent of Listeriosis. Responsible for approximately 2,500 illnesses and 500 deaths in the United States (U.S.) annually,33,7 Listeriosis is the leading cause of death among foodborne bacterial pathogens with fatality rates exceeding even Salmonella and Clostridium botulinum.35 L. monocytogenes is prevalent worldwide and foodborne outbreaks have recently occurred in several countries. In 2007 the Centre for Food Safety, Hong Kong identified 20 outbreaks in Australia, New Zealand, United Kingdom (U.K.), and the U.S.5 Another 2007 outbreak was identified in the U.S. from the consumption of pasteurized milk which resulted in five cases including three deaths.8 In 2002, a multistate outbreak occurred in the U.S. from the consumption of contaminated turkey deli meat which resulted in 54 cases including 11 deaths total, 3 fetal deaths, and the recall of more than 30 million pounds of products.6 Due to the prevalence and high fatality rates of L. monocytogenes, current research efforts are focused on developing methods for its isolation, detection, control, and risk assessment in foods worldwide. Additional research in the area of genomics and proteomics has begun to develop a better understanding of how L. monocytogenes responds to its environment.24 As knowledge of its molecular and applied biology increases, progress can be made in the prevention and control of human infection. Ensuring that food is safe from L. monocytogenes requires the collaboration of government, food industry and consumers. This includes the government’s efforts to monitor and control food contamination, the food manufacturer’s role to maintain good manufacturing practices (GMPs), and the consumer’s responsibility to follow safe food handling practices and consumption guidelines. These efforts combined with a better understanding of the pathogenic process of L. monocytogenes will lead to improved control in foods. Classification L. monocytogenes is a gram positive, non-spore forming, motile, facultatively anaerobic, rod shaped bacterium. It is catalase positive, oxidase negative, and expresses a Beta hemolysin which causes destruction of red blood cells. This bacterium exhibits characteristic tumbling motility when viewed with light microscopy.15 Although L. monocytogenes is actively motile by means of peritrichous flagella at room temperature (20-25oC), the organism does not synthesize flagella at body temperatures (37oC).51 The genus Listeria belongs to the Clostridium sub-branch, together with Staphylococcus, Streptococcus, Lactobacillus and Brochothrix. The genus Listeria includes 6 different species (L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seegligeri, and L. grayi). Both L. ivanovii and L. monocytogenes are pathogenic in mice, but only L. monocytogenes is consistently associated with human illness.46 There are 13 serotypes of L. monocytogenes which can cause disease, but more than 90 percent of human isolates belong to only three serotypes: 1/2a, 1/2b, and 4b. L. monocytogenes serotype 4b strains are responsible for 33 to 50 percent of sporadic human cases worldwide and for all major foodborne outbreaks in Europe and North America since the 1980s.14 Back to top Natural Reservoirs and Transmission L. monocytogenes is ubiquitous in the environment and can survive under adverse conditions longer than most other non-spore forming food pathogens. Listeria has been isolated from many places such as: soil, wastewater, vegetation, stale water supplies, grazing areas, poorly prepared animal feed, and the intestines of healthy animals and humans.51 Soil is the most common and widespread medium for pathogen growth since it provides a cool and moist environment. In addition, the plant and fecal material in the soil supplies essential nutrients and facilitates the growth.14 L. monocytogenes is carried in the intestinal tract of 5 to 10 percent of healthy humans. In addition to humans, 17 avian species and more than 42 mammalian species (wild and domestic) harbor this bacterium. It has also been isolated from crustaceans, fish, oysters, ticks and flies.51 Multiple studies in healthy animals (exhibiting no symptoms of Listeriosis) such as cattle, sheep, goats, pigs, and poultry have shown that 50 percent of fecal samples collected from healthy animals contain L. monocytogenes.39 Therefore, healthy animals act as reservoirs of pathogenic strains of L. monocytogenes involved in the contamination of food products. Five Modes of Transmission Contaminated animal feed -- L. monocytogenes is easily spread to food animals from contaminated animal feed, most commonly by improperly fermented silage. Silage is the feed produced by controlled fermentation of high moisture herbage. Since Listeria is commonly found in soil, it often contaminates ensiled feeds. Under poor conditions, when oxygen is present and the pH is above 5.0, the bacteria replicate to very large numbers within the surface layers of the silage (i.e., top and sides of bunker silos).12 Most cases of animal Listeriosis have been associated with stacked and baled silage due to the introduction of oxygen during the wrapping step. Oxygen infiltration promotes pathogen growth by disturbing anaerobic fermentation.44 In contrast, L. monocytogenes is rarely a problem in well fermented and preserved silages because its growth is inhibited due to the production of sufficient lactic acid which lowers the pH below 5.0.42 Therefore, preventing Listeriosis on the farm is primarily based on proper silage management practices to ensure effective fermentation and to minimize soil contamination of harvested feeds. Contaminated manure -- On-farm transmission of L. monocytogenes occurs when contaminated manure is used to fertilize crops and soil. For example, a 1981 outbreak involving 42 cases of human Listeriosis in Nova Scotia was linked to consumption of coleslaw. This coleslaw was produced from cabbage harvested from fields fertilized with untreated manure from Listeria-infected sheep.39 Therefore, raw manure should not be used to fertilize produce intended for human consumption. Mastitic cows -- Although not particularly common, L. monocytogenes can cause mastitis and has been isolated from the udders and milk of mastitic cows. Prolonged excretion of L. monocytogenes in milk and the consumption of raw milk may be important factors in the transmission and epidemiology of Listeria infection.45 Contaminated plant and animal foods -- Since plant and animal foods are frequently contaminated with L. monocytogenes, the pathogen can become prevalent in food processing environments.20 For example, raw meat and poultry from infected, slaughtered animals allows continuous entry of the pathogen into the processing environment. It can cross-contaminate food contact surfaces, equipments, floors, drains, standing water and employees.22 In fact, each type of food processing establishment (e.g. dairy, meat, fresh plant produce) harbors its own unique population of L. monocytogenes which persists in the environment over time.20 In addition, contaminated waste from these food processing plants increases the spread of L. monocytogenes in the environment.14 Biofilms -- L. monocytogenes has the ability to attach to a wide variety of food contact surfaces and form biofilms.45 A biofilm is a community of bacteria that adheres to a surface and is frequently embedded in an extracellular matrix. The L. monocytogenes biofilm formation can occur on various kinds of food processing surfaces including stainless steel, glass, and rubber.22 The common areas and sites involved in biofilm development are: floors, freezers, processing rooms, cases and mats36, waste water pipes, bends in pipes, rubber seals, conveyor belts, and Buna-n and Teflon seals.30 Biofilms are difficult to eliminate by cleaning or disinfection, and are considered to be an important reservoir of microbial contamination by food scientists and the food industry. The ability of L. monocytogenes to grow and survive in almost every environment for long periods of time provides numerous opportunities for its transmission along the farm to table continuum. Back to top Growth Characteristics The four growth characteristics important to food processors are: temperature, acidity (pH), atmosphere and water activity, and salt. Temperature -- L. monocytogenes can grow at temperatures between 0oC to 45oC, with growth occurring more slowly at lower temperatures.14 The optimal growth temperatures are between 30oC and 37oC.45 Most bacteria grow poorly when temperatures fall below 4oC, but L. monocytogenes has the ability to survive and grow at refrigeration temperatures.10 As a result, it may be transmitted in ready-to-eat (RTE) foods that have been properly refrigerated. Therefore, it is of particular interest to food processors that use refrigeration methods to preserve semi- and post-processed foods. Freezing causes a limited reduction in the viable population of L. monocytogenes. During frozen storage, pathogen survival and injury are affected by the strain type, freezing medium, and freezing rate. Slow freezing at a temperature of -18oC is more lethal and injurious to the organism than rapid freezing at -198oC. Multiple freeze-thaw cycles are found to be more effective in killing than was a single cycle.45 L. monocytogenes is killed by pasteurization. The current minimum standards established by the Food and Drug Administration (FDA) for low-temperature, long-time pasteurization (61.7oC/30min) and high-temperature, short-time pasteurization (71.6oC/15sec) are adequate to render milk safe from the pathogen.45 The U.K. Department of Health advised that ready meals or similar products should receive a heat treatment of at least 2 minutes at 70oC, or equivalent, to ensure the destruction of L. monocytogenes. In the U.S., standards for the heat treatment of frozen dessert mixes require a process of 68.3oC for 30 minutes, or 79.4oC for 25 seconds to ensure the pathogen is destroyed.11 Acidity (pH) -- L. monocytogenes can survive and grow in foods having moderate to low acidity levels. It can initiate growth in the pH range of 4.4 - 9.6;14 however growth at low pH values is influenced by the incubation temperature and the type of acid. Many scientific studies have shown that the minimum pH at which growth occurs is well below 5.0 at the optimum incubation temperature(30-37oC). Pathogen growth in fermented and acidified foods such as soft cheeses and coleslaw respectively, has confirmed that L. monocytogenes can grow well in acidic foods.45 Atmosphere and Water Activity -- Listeria grows well under both aerobic and anaerobic conditions. The capacity for anaerobic growth at refrigeration temperatures makes it a potential threat to the safety of foods packaged under a vacuum or modified atmosphere where limited levels of oxygen are used.45 L. monocytogenes can grow optimally at a water activity (aw) of about 0.97. However, when compared with most food pathogens, it has a rather unique ability to multiply at values as low as 0.90. Survival of the pathogen under reduced moisture conditions depends on both the water activity and main solute in the medium. For instance, the bacterium survives for a shorter time in a sodium chloride-adjusted growth medium than in a sucrose or glycerol-adjusted medium. Also, an inverse relationship exists between thermal resistance of L. monocytogenes and water activity of the suspending medium14. For example, the pathogen was found to be about four times more heat resistant in a buffer solution having a water activity of 0.90 as compared with 0.98.45 This is a concern to food manufacturers who rely on low water activity and thermal treatment for preservation of their food products. Salt (NaCl) -- Salt, or sodium chloride (NaCl), is an important antimicrobial ingredient that affects the water activity of foods. L. monocytogenes, a halotolerant organism capable of surviving high salt concentrations (10 to 12 percent NaCl), can grow to high populations in moderate concentrations (6.5 percent NaCl).14 Due to its high osmotic tolerance, immersing products like cheese and salmon in brine solution is not a reliable preservation method. However, Listeria’s survival in high salt concentrations is temperature dependent and can be significantly increased by lowering the temperature. For instance, survival times of the pathogen in a media containing 25.5 percent NaCl was reported to be 3, 24, and >132 days when the storage temperatures were 37o C, 22oC and 4oC, respectively.45 Back to top Mechanism of Infection L. monocytogenes is ingested by eating contaminated food. Following ingestion, the host’s immune system targets the pathogen to prevent infection. Cell-mediated immunity (CMI) is the main host defense mechanism against L. monocytogenes. The majority of phagocytized bacteria are targeted by this defense before they can cause infection.10 However, if the immune system is compromised, bacteria cross the intestinal barrier and spread through intracellular mechanisms causing systemic infections with high mortality rates.51,32 Unlike other bacterial pathogens, L. monocytogenes is able to penetrate the blood-brain barrier and placental barrier leading to severe infection of the brain and fetus, respectively.13 L. monocytogenes invades the host by inducing macrophage phagocytic uptake by displaying D-galactose residues which adhere to the D-galactose receptors on the epithelial cells of the host’s gastrointestinal tract.51 Host cell invasion requires the action of internalins, a family of bacterial surface proteins that mediate the process of phagocytosis. Once phagocytosed, the pathogen is encapsulated by the host cell’s phagolysosome, a sub-cellular organelle with an acidic and toxic environment.48 Listeria survive the hostile environment of the phagolysosome because they produce two enzymes, catalase and superoxide dismutase, which neutralize the effects of the phagocytic oxidative burst (enzymes and reactive oxygen species that digest phagolysosomal contents.51 The pathogenic bacteria escape the phagolysosome within a few minutes by lysing the vacoule's entire membrane with Listeriolysin O (LLO), a hemolysin produced by Listeria and activated by the low pH environment. LLO allows L. monocytogenes to escape from the phagolysosome into the cytoplasm without damaging the plasma membrane of the infected cell. This enables the bacteria to live intracellularly, protected from extracellular immune system factors. All pathogenic strains of Listeria produce LLO, thus it is required for its pathogenesis.48 Once L. monocytogenes is within the growth-permissive cytoplasm, a bacterial protein, ActA, induces the polymerization of actin, a component of host cell cytoskeleton.43 Surrounded by a sheet of actin filaments, the bacteria reside and multiply with doubling times of about one hour. The actin sheet functions as a propulsive force which drives the bacteria to the host cell's outer membrane.51 Many bacteria migrate to the periphery of the cytoplasm where they push against the host cell's outer membrane to form elongated protrusions or filopods which are engulfed by adjacent cells. Once the bacterium enters the adjacent cell, the life cycle begins anew and the process is repeated, perpetuating the infection. Therefore, Listeria directly spreads from cell-to-cell (intracellularly) in an infected tissue without directly contacting the extracellular environment.48 Back to top Listeriosis Listeriosis is a foodborne bacterial disease caused by L. monocytogenes. The infective dose in unknown but is believed to be strain and host dependent. It is assumed that fewer than 1,000 total organisms may cause foodborne illness in susceptible populations.18 Two types of Listeriosis are associated with L. monocytogenes -- non-invasive and invasive. Non-invasive Listeriosis -- A milder form of the disease also referred to as febrile listerial gastroenteritis. Symptoms occur after a short incubation period from ingestion of high doses of L. monocytogenes by healthy individuals. Symptoms include: Diarrhea Fever Headache Myalgia (muscle pain) Invasive Listeriosis -- This type affects high-risk people including immune-compromised individuals, pregnant women, the elderly, and the very young. The incubation period is variable and ranges from 3 to 70 days. In infants, symptoms may include:50 Loss of appetite Lethargy Jaundice Vomiting Respiratory distress (usually pneumonia) Skin rash Shock Meningitis In adults, many different disease symptoms can occur depending on the organ system infected. Symptoms may include: Meningitis Pneumonia Septicemia Endocarditis Abscesses, skin lesions, conjunctivitis (milder forms) In pregnant women, Listeriosis is particularly harmful. Infected pregnant women may experience mild flu like symptoms, although they are at risk for:29 Premature delivery Miscarriage Spontaneous abortion Stillbirth Death of a newborn within a few hours of birth Treatment of Listeriosis Listeriosis infections are associated with high mortality rates, therefore effective antibiotic treatment is essential. Antibiotics for Listeriosis are:25 Penicillin G Ampicillin Gentamicin Chloramphenicol Ampicillin alone or in combination with gentamicin is the treatment of choice. For people allergic to the antibiotics listed above, or for those with certain disease states, trimethoprim, sulphamethoxazole, erythromycin, vanomycin, and the fluoroquinolone are used. Cephalosporins are not active against L. monocytogenes. No vaccinations for L. monocytogenes exist.49 L. monocytogenes Contamination in Foods Listeriosis is most commonly contracted by eating contaminated food. Foods that have been reported as vehicles of L. monocytogenes are: raw and pasteurized milk, cheeses (particularly soft-ripened varieties), ice cream, raw fruits and vegetables, fermented raw-meat sausages, raw and cooked poultry, raw meats (all types), and raw and smoked fish.18 RTE foods that support the growth of L. monocytogenes pose the highest risk to consumers and include:19 Milk, high fat and other dairy products (e.g. butter and cream) soft unripened cheeses (e.g. quesco fresco, cottage and ricotta cheese) Cooked crustaceans (e.g. shrimp and crab) Smoked seafood (smoked finfish and mollusks) Certain vegetables (e.g. cabbage) and non-acidic fruits (e.g. melons) Some deli type salad sandwiches (e.g. prepared from seafood, non-acidified) The presence of the bacterium in processed foods like cheese, ice cream and processed meats, is due to post-processing contamination.1 Certain foods commonly preserved by refrigeration offer a favorable environment for the multiplication of L. monocytogenes during manufacture, aging, transportation and storage. This poses a higher risk of contracting Listeriosis for susceptible populations.14 Prevention of Listeriosis The Centers for Disease Control and Prevention (CDC) has guidelines, Listeriosis: How can you Reduce your Risk, for both the general population and high risk individuals in order to prevent Listeriosis.9 The FDA Center for Food Safety and Applied Nutrition (CFSAN) has developed a Listeria: Frequently Asked Questions (FAQ) resource to help moms-to-be reduce their risk during pregnancy. Back to top Fatality of L. monocytogenes L. monocytogenes is potentially fatal to newborns, the elderly, and immune-compromised individuals such as those with acquired immune deficiency syndrome (AIDS), cancer, organ transplants, and diabetes.38 Persons with AIDS are almost 300 times more susceptible to Listeriosis than people with normal immune systems.47 Listeriosis is a concern for pregnant women who are 20 times more likely to acquire the disease than other healthy adults. Listeriosis is rare in humans with an occurrence rate in the U.S. of approximately five cases per million people per year. Still, L. monocytogenes is the leading cause of death among foodborne pathogens with a 20 to 25 percent fatality rate.27 Epidemiological surveillance data show that the L. monocytogenes case-fatality rate varies by age, with higher case-fatality rates among newborns and the elderly.21 An 11 percent case-fatality rate is documented in persons of age 40 and under, a 63 percent case-fatality rate is recorded for persons over age 60.31 According to the CDC, the rate of Listeriosis has declined by 36 percent from 1996-2006, however outbreaks continue to occur and L. monocytogenes is still the major cause of foodborne fatalities in the U.S. Back to top Foodborne Disease Outbreaks Listeriosis has emerged as a major foodborne illness during the past 25 years.14 The first confirmed foodborne Listeriosis outbreak occurred in 1981 in Nova Scotia, Canada which was traced to contaminated coleslaw. Since then, many steps were taken to control L. monocytogenes in foods and it appeared that this pathogen was under control. However, recent outbreaks and recalls have indicated that this bacterium still poses a significant risk and is an emerging foodborne pathogen.13 Invasive Foodborne Outbreaks in North America, The six invasive outbreaks summarized below, demonstrate that most RTE foods can transmit Listeriosis. 1981 Nova Scotia, Canada Coleslaw Outbreak38 Contaminated Coleslaw Resulted in 41 cases, including 34 pregnant women, and 18 deaths Contamination source was Listeria-infected sheep manure used to fertilize cabbage 1985 Los Angeles Mexican-Style Soft Cheese Outbreak 26 Contaminated Mexican style soft cheese Resulted in 145 cases including 93 pregnant women, and 64 deaths Contamination was due to inadequate pasteurization and contamination of equipment 1998-1999 Multi states Hot Dogs and Deli Meats Outbreak26 Contaminated hot dogs and deli meats Resulted in over 100 cases including 15 deaths and 6 miscarriages Contamination source was construction dust at the processing plant which contaminated products in the packaging room 2002 Multi State Turkey Deli Meat Outbreak6 Contaminated RTE turkey deli meat Resulted in 54 cases including 11 total deaths and 3 fetal deaths Contamination occurred at the plant (post processing contamination) 2007 Massachusetts Pasteurized Milk Outbreak8 Contaminated pasteurized milk Resulted in 5 cases including 3 deaths Contamination occurred after pasteurization (post processing contamination) 2008 Toronto, Canada RTE Deli Meats Outbreak37 Contaminated RTE deli meats Resulted in 42 cases including 15 deaths (as of September, 2008) Contamination occurred at the processing plant For additional foodborne outbreak information and statistics in the U.S., visit FoodNet -- Foodborne Diseases Active Surveillance Network Invasive Outbreaks in Europe In Europe, 18 outbreaks of invasive Listeriosis were reported from 1991-2002, of which approximately half were linked to unpasteurized dairy products.20,34 These outbreaks were caused by: unpasteurized soft cheese in Switzerland (1983-87), unpasteurized milk in Austria (1986), and a brie-type cheese made from unpasteurized milk in France (1995). On the contrary, the 1999 Finland Pasteurized Butter Outbreak indicated that pasteurized dairy products can cause disease. In this case, it was found that pasteurized milk can be contaminated during subsequent stages of production.34 Non-invasive Outbreaks L. monocytogenes occasionally causes outbreaks with non-invasive symptoms in low risk (healthy) populations. In a 1994 Illinois outbreak, the consumption of chocolate milk containing high concentrations of L. monocytogenes lead to 45 cases. None of the victims contracted the fatal form of the disease and the one infected pregnant woman delivered a healthy baby 5 days later.13 Back to top Detection Methods A variety of conventional and rapid methods are currently available for the isolation and identification of L. monocytogenes in food samples. Conventional Methods Conventional methods are very sensitive and do not require sophisticated and expensive equipment. They are the standards against which other methods are compared and validated; however, these methods are labor intensive and time consuming. Several conventional methods for Listeria isolation from foods have gained acceptance from regulatory agencies. Worldwide, the most commonly used methods for Listeria isolation in foods are the ISO 11290 Standards.28 In the U.S., the three standard isolation methodologies are: the FDA method, the Association of Official Analytical Chemists (AOAC) official method, and the United States Department of Agriculture (USDA) method. The FDA methodology can be used for dairy products, seafoods, and vegetables16,28; the AOAC standards may be used for dairy products only3; and, the USDA method is recommended for red meat and poultry (raw or cooked RTE), eggs and egg products, and environmental samples.54 One standard method might be more suitable than the other depending on the nature of the sample. The conventional culture methods include an enrichment procedure which uses liquid culture media containing selective agents that vary with the method.28 Both FDA and ISO methods also include a pre-enrichment step that is intended for the recovery of sub-lethally injured L. monocytogenes cells, whereas in the USDA and AOAC methods, the samples are processed directly into enrichment broth.40 The ISO standard method 11290 (part 2), as well as optional protocols mentioned in the FDA and USDA methods, are used for the quantification of initial enrichment broth before starting incubation. Following selective enrichment, cultures are plated onto selective/differential agar plates for isolation of presumptive colonies of L. monocytogenes. The FDA method uses both Oxford agar and lithium chloride/phenylethanol/moxalactam (LPM or PALCAM) agar. The USDA method contains lithium chloride, colistin, and maxalactam in Modified Oxford (MOX) agar. Typical Listeria species grown on these plates can then be selected for further identification to the species level using a series of tests. These tests include: Gram staining Catalase Motilty Hemolysis Carbohydrate utilization Christie-Atkins-Munch-Peterson (CAMP) Rapid Methods Rapid methods include conventional and new commercially available tests. Most of these methods have relatively high detection limits and are very rapid, but lack sufficient sensitivity for direct testing. Moreover, food samples need to be culture-enriched before analysis.28. Some of these method types are: Immunoassay Methods -- These methods are based on antibodies specific to L. monocytogenes and were developed to identify this bacterium in foods. They are easy to apply, generate rapid test results and allow the use of difficult sample matrices. Some of the commercially available and validated tests are: a. Colorimetric Monoclonal Enzyme-linked Immunosorbent Assay (Listeria Tek) b. Enzyme Linked Immunofluorescent Assay (VIDAS LIS) Nucleic Acid-based Methods -- Very specific and can perform as fast as immunological assays. Some of the validated methods are: a. DNA hybridization -- Used for the differentiation of L. monocytogenes from other Listeria species by means of probes directed against specific genes. GeneTrakRand GeneQuenceR test kits are DNA hybridization-based methods. b. Polymerase Chain Reaction (PCR) -- A rapid and specific nucleic acid amplification method useful for detection of L. monocytogenes. It is more sensitive than nucleic acid and immunoassay based assays but sample enrichment is still required. The USDA has adopted the PCR-based BAXR system as their screening test for L. monocytogenes.53 Other detection kits in use are: LightCyclerR, GeneVisionR and the TaqManR. Subtyping methods -- Subtyping is useful in outbreak investigations, environmental tracking, and public health surveillance. Some of the subtyping methods are: a. Serotyping b. Phage typing c. Multilocus enzyme electrophoresis d. Chromosomal DNA restriction endonuclease analysis e. Nucleic acid sequence-based typing As a result of processing treatments such as heating, freezing, and acidification, L. monocytogenes may exist in foods in an injured state. Before these injured bacteria can be detected, they need special culture conditions that facilitate the repair of damaged cells. Although many detection methods have been developed for L. monocytogenes, no single method can be used for all foods.40 Back to top Regulatory Status of L. monocytogenes U.S. Regulations The FDA currently maintains a policy of "zero tolerance" for L. monocytogenes in all RTE foods except RTE meat and poultry products which are under the jurisdiction of the USDA. Zero tolerance means that any detectable level of L. monocytogenes in cooked, RTE foods is unacceptable from a public health perspective. The FDA uses an analytical method that can detect 1.0 colony forming unit (cfu) of L. monocytogenes per 25 grams of food sample to determine whether the pathogen is present in the food. Based on this method, the agency considers a food to be adulterated if any detectable level of L. monocytogenes is found.17 FDA has recently announced a new draft compliance policy guide for the control of L. monocytogenes in RTE foods that will create different policies for different categories of products based on the associated risk. This policy is intended to reflect current knowledge that L. monocytogenes can be reduced, but cannot always be eliminated from the finished product or the plant environment even though industry works towards eradication. The FDA will revise its tolerance level from zero to 100 cfu/g for RTE foods that do not support the growth of the pathogen.19 This will apply to: Foods with a pH less than or equal to 4.4 (acidified deli salads, pickled products) Foods with a water activity less than or equal to 0.92 (e.g. cereals, crackers, hard cheeses) Frozen foods (e.g. ice cream) Zero tolerance will remain the same for RTE foods that support pathogen growth such as milk, soft cheeses, smoked seafood, and non-acidic fruits. This guidance will be similar to the international standards adopted by Europe, Canada and other nations. The zero tolerance policy of the USDA Food Safety and Inspection Service (FSIS) applies to the detection of L. monocytogenes in RTE meat and poultry products. Products are deemed to be adulterated if they contain any detectable levels of L. monocytogenes under the provisions of the Federal Meat inspection Act and the Poultry Inspection Act.17 International Regulations Other countries, including some major trading partners of the U.S., have "non-zero tolerance" policies and have established regulatory limits for L. monocytogenes in RTE foods.17 For example, the Canadian regulations have divided food into three risk categories. Products most frequently linked to human Listeriosis are placed in Category 1 and are regulated more stringently than in Categories 2 and 3. RTE foods that have not been associated with an outbreak, and do not allow growth of L. monocytogenes during a 10-day period of refrigerated storage, may contain up to 100 cfu/g of the organism.41 Many European countries have adopted the EU regulations which set the limit of <100 cfu/g in all kinds of RTE foods (other than those intended for infants and special medical purposes) throughout their shelf life. Zero tolerance applies to foods intended for infants.52 The Food Standards Australia New Zealand (FSANZ) has divided packaged RTE foods into two risk categories. Zero tolerance applies to category 1 and includes foods that: are capable of supporting the growth of L. monocytogenes; have been implicated in human Listeriosis; and/or, are consumed by high risk groups, especially infants. A tolerance level of 100 cfu/g is given for Category 2 foods which do not support pathogen growth.23 Due to the ubiquitous nature of L. monocytogenes in food processing environments, it is difficult to maintain its absolute absence in all kinds of RTE foods.5 According to the FDA, there is no epidemiological evidence that demonstrates whether a zero or non-zero tolerance policy leads to better control of L. monocytogenes in foods.17 However, based on current available scientific evidence, the American Society for Microbiology (ASM) has found that a change from "zero tolerance" to a tolerance level of 100 cfu/g should result in a 50 percent to 99.5 percent decrease in Listeriosis, depending on changes in prevalence. ASM suggests that a change in tolerance levels will result in a net public health benefit, even under the most conservative assumptions.2 Back to top Research at the USDA Agricultural Research Service The USDA Agricultural Research Service (ARS) is actively involved in food safety research related to L. monocytogenes under the National Food Safety Program 108. This research program provide the means to ensure that the food supply is safe and secure for consumers and that food and feed meet foreign and domestic regulatory requirements. The following ARS research units conduct research on L. monocytogenes: Microbial Food Safety Research Unit Poultry Microbiological Safety Research Unit Bacteriological, Epidemiology and Antimicrobial Resistance Research Unit Microbial Food Safety Research Unit This unit of the ARS in collaboration with Rutgers University and Decisionalysis Risk Consultants, Inc. of Canada has developed an internet resource called the Predictive Microbiology Information Portal (PMIP). PMIP provides information on research, regulations, and other resources related to L. monocytogenes in RTE foods. ARS researchers at the Microbial Food Safety Research Unit have sequenced a whole genome of four strains of Listeria related to foodborne Listeriosis outbreaks. This is an important step to understand the bacterium’s virulence and its persistence in the food supply. Some of the L. monocytogenes research projects being conducted at this unit are: Validation of the Effect of Interventions and Processes on Persistence of Pathogens on Foods Project Objectives59 Elucidate the ecology (persistence, predominance, behavior, and community analysis) of pathogens in various food matrices; specifically focus on foods considered high risk by the stakeholder regulatory agencies (FSIS and FDA), for example RTE foods, or foods with a short shelf life. Develop and validate intervention strategies used either alone or in combination with other processes for pathogen control. Elucidate/define (including at the molecular level) the pathogens physiological responses to various intervention strategies and processes. Examine the influence of the inherent food macro and micro-environments. Accomplishments59 Designed a commercial microarray containing genes representative of the whole genome of L. monocytogenes. Analyzed the protein expression of L. monocytogenes in a model food such as the purge from vacuum-packaged frankfurters stored at refrigeration temperature. Heating/Drying of marinated turkey jerky at 165oF or 180oF resulted in a substantial reduction of L. monocytogenes. Application of lauric arginate using the Sprayed Lethality in Container method reduced L. monocytogenes levels on the surfaces of vacuum-packaged roast beef, turkey breast, and frankfurters within 24 h at 4oC Listeria monocytogenes Contamination of Deli Meat Slicers: Risk and Communication Project Objectives57 Quantify the degree of L. monocytogenes transfer during deli slicing of cured ham based on slicer blade composition, blade surface roughness, product temperature and product composition. Characterize the prevalence and levels of L. monocytogenes in deli-sliced meats and the likelihood and mechanism of cross-contamination occurring during deli slicing due to specific food service handling behaviors. Develop a compartmental mechanistic mathematical risk model of L. monocytogenes associated with the deli slicing process, including cross-contamination. Use the model to identify candidate risk mitigation strategies. Develop educational and outreach strategies, including the development of specific risk communication messages, targeting deli slicing machine operators to reduce the risk of contamination and growth of L. monocytogenes Accomplishments57 Acquisition of four model 2612 Hobart slicers, (including three types of blades) as well as one Berkel slicer (donated by Berkel) that was recently re-designed for enhanced cleanability. Set up three Hobart slicers and the Berkel slicer at a local delicatessen for one year of use and monthly sampling. Identified product contact areas on the slicers for sampling. Optimized blade inoculation method using surface-inoculated ham. Collected initial data on the potential and extent of sequential transfer of L. monocytogenes from the slicer blade to ham and other areas of the slicer during slicing. Microbial Modeling and Bioinformatics for Food Safety and Security Project Objectives62 Evaluate, validate, and develop new innovative, robust and valid predictive models for the responses of microbial pathogens, including foodborne threat agents, in select food matrices, as a function of: temperature, food formulation, competitive microflora, physiological history, and surface transfer. Develop novel approaches to assess model performance and robustness, leading to more efficient strategies for producing and extrapolating models to different classes of food. Determine the probability distribution of lag phase duration (LPD) for foodborne pathogens, as a function of the previous bacterial physiological history, to allow risk managers to estimate worst-and best-case scenarios for pathogen behavior, depending on likely sources of contamination. To identify molecular markers that discriminate bacterial lag, growth and stationary phases, thus leading to more mechanistic models and greater certainty for LPD prediction. Accomplishments62 Developed predictive models describing the behavior of L. monocytogenes in ham containing sodium lactate (1.0-4.2 percent) and sodium diacetate (0.05-0.2 percent) at storage temperatures of 0°-45°C. Developed models to describe the rates of inactivation of L. monocytogenes during sausage manufacturing. Poultry Microbiological Safety Research Unit This ARS research unit works with the poultry industry, regulatory and public health agencies to determine the epidemiology of human pathogens in poultry and to develop intervention technologies to prevent or diminish these pathogens. Some of the L. monocytogenes research projects being conducted at this unit are: Improved Methods for Control of Listeria monocytogenes within Biofilms in Meat and Poultry Processing Environments Project Objectives56 Develop and optimize a product for use against L. monocytogenes biofilm in meat and poultry processing equipment. Develop standard methodologies for biofilm removal for registration of Environmental Protection Agency (EPA) biofilm claims for products used in food processing. Accomplishments56 Implemented new methods to screen for detection efficacy against L. monocytogenes biofilms, with concomitant testing of biofilm sustainability and bacterial cell culturability from the biofilms. Suggested that protocol development and validation is critical to the establishment of standards for registration of EPA biofilm claims for used in food processing. The results of this project will yield an optimized formulation of a disinfectant for meat and poultry RTE food processing and production equipment that is more effective in reducing L. monocytogenes biofilm growth than currently used disinfectants, thus minimizing the risks to food safety from pathogenic contamination. Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Foodborne Bacterial Pathogens in the Chicken Project Objectives (selected)63 Developed approaches for processing biofilms to provide for quantitative measurement of bacterial populations especially of L. monocytogenes in situ. Accomplishments (selected)63 Developed methods to quantitatively assess L. monocytogenes under conditions that mimic poultry environments. Bacteriological, Epidemiology and Antimicrobial Resistance Research Unit The goal of the Bacterial Epidemiology and Antimicrobial Resistance Unit is to study antimicrobial resistance in zoonotic food borne pathogens and commensal bacteria. Some of the Listeria research projects being conducted at this unit are: Controlling Listeria monocytogenes in Further Processed Meat Project Objectives61 Determine the effect of commonly used marinade ingredients applied in raw product or as a post cook dip on the growth of L. monocytogenes on turkey deli loaves, ham deli loaves and beef frankfurters. Measure the effect such treatments have on consumer acceptability of the fully cooked products. Accomplishments61 Planned, conducted and completed research to test the effect of sodium tripolyphosphate, sodium lactate, sodium diacetate and sodium citrate individually and in different combinations to control the growth of L. monocytogenes in RTE meat products. Combination of sodium diacetate and sodium lactate was found to be effective in control of L. monocytogenes in turkey deli loaves and beef frankfurters. The best treatment combination (sodium diacetate and sodium lactate) for pathogen control did not result in an acceptable level of sensory quality. Microbial Ecology of Human Pathogens Relative to Poultry Processing Project Objectives58 Determine the role that outside environmental sources of L. monocytogenes play in the presence of this pathogen in poultry further processing facilities. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments. Evaluate the influence of animal agriculture on Campylobacter in the environment. Accomplishments58 Results will provide information on developing strategies of using antimicrobials in a manner that does not encourage development of resistance. Evaluation of FSIS Hazard Analysis Critical Control Point (HACCP) inspection model program: Planned, conducted and completed a collaborative study with FSIS and Stan Bailey to examine the microbiological quality of broiler carcasses processed in plants under the HACCP inspection model program (HIMP) of FSIS inspection. Epidemiology, Ecology, and Molecular Genetics of Antimicrobial Resistance in Pathogenic and Commensal Bacteria from Food Animals Project Objectives55 Use antibiotic resistance data obtained from the Collaboration on Animal Health and Food Safety Epidemiology (CAHFSE) and the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) programs and poultry studies to identify sources, reservoirs and amplifiers of resistant food borne and commensal bacteria, as well as the path of dissemination of these resistant bacteria in food producing animals and poultry. Map the spread of antimicrobial resistance throughout the U.S.USDA VetNet. Analyze and differentiate antimicrobial resistance mechanisms, both phenotypically and genotypically, and rapidly identify resistant strains. Accomplishments55 Developed a technique to identify and track the different antimicrobial resistance and virulence genes in all bacteria. A DNA microarray to detect 100 antimicrobial resistance genes was tested and expanded to detect 775 resistance and virulence genes simultaneously. L. monocytogenes isolates were found to be resistant to ciprofloxacin, ceftriaxone, oxacillin, tetracycline, and clindamycin.60 Demonstrated the variability in levels of resistance between Listeria species and the presence of transferable resistance genes which indicated that transfer between bacterial populations is possible.60 FSRIO Research Projects Database For additional USDA Listeria monocytogenes Research Projects, please visit the FSRIO Research Projects Database. For additional Listeria monocytogenes research projects conducted by other U.S. government and International agencies, please search the FSRIO Research Projects Database. Back to top References 1. American Meat Institute Fact Sheet. 2008. Listeria monocytogenes. Retrieved December 8, 2008, from http://www.meatami.com/ht/a/GetDocumentAction/i/11478.pdf. 2. American Society For Microbiology. 2004. Establishing a Regulatory Limit on Listeria monocytogenes. Retrieved December 13, 2008, from http://www.asm.org/Policy/index.asp?bid=29757. 3. Association of Official Analytical Chemists (AOAC) Official Methods of Analysis. 2005. Listeria monocytogenes in Milk and Dairy Products. Retrieved December 5, 2008 from http://www.eoma.aoac.org/methods/info.asp?ID=48309. 4. Bennion, R.J., F. Sorvillo, M.E. Wise, S. Krishna, and L. Mascola. 2008. Decreasing Listeriosis Mortality in the United States, 1990-2005. Clin Infect Diseases. 47:867-864. Retrieved November 5, 2008, from http://www.journals.uchicago.edu/doi/abs/10.1086/591131. 5. Center for Food Safety. 2008. Listeria monocytogenes and its Control. Retrieved November 1, 2008, from http://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_12_01.html. 6. Centers for Disease Control and Prevention. 2005. Multistate Outbreak of Listeriosis Linked to Turkey Deli Meat. 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FreeMD. 2008. Listeriosis Treatment. Retrieved December 13, 2008, from http://www.freemd.com/listeriosis/treatment.htm . 26. Harris, L. 2007. Listeria monocytogenes. Retrieved December 16, 2008, from www.cdfa.ca.gov/ahfss/Animal_Health/PHR250/2007/25007Lis-mjr.pdf. 27. Hitchins, A. D. and R.C. Whiting. 2001. Food-borne Listeria monocytogenes Risk Assessment. Food Additives and Contaminants. 18:1108-1117. Abstract Retrieved December 3, 2008, from http://cat.inist.fr/?aModele=afficheN&cpsidt=14158376. 28. Janzten, M.M., J. Navas, A. Corujo, R. Moreno, V. Lopez, and J.V. Martinez-Suarez. 2006. Review. Specific Detection of Listeria monocytogenes in Foods Using Commercial Methods: From Chromogenic Media to Real-time PCR. Spanish J. Agri. Research. 4:235-247. Retrieved December 9, 2008 from http://www.inia.es/gcontrec/pub/235-247-35-R_Specific_1157976609093.pdf. 29. KidsHealth. 2008. Listeria Infections. Retrieved December 12, 2008, from http://kidshealth.org/parent/infections/bacterial_viral/listeria.html. 30. Kumar, C.G. and S.K. Anand. 1998. Significance of Microbial Biofilms in a Food Industry: A Review. Intl. J. Food Microbiol. 42:9-27. Retrieved November 25, 2008, from http://tinyurl.com/cvhwhb. 31. Labbe, R.G. and S. Gracia, eds. 2001. Guide To Foodborne Pathogens. Johnwiley & Sons, Inc., New York. Preview Retrieved December 7, 2008 from http://tinyurl.com/crz7h7. 32. Linden S.K., H. Bierne, C. Sabet, C.W. Png , T.H. Florin, M.A. McGuckin and P. Cossart. 2008. Listeria monocytogenes Internalins Bind to the Human Intestinal Mucin MUC2. Arch Microbiol. 190:101-104. Retrieved November 26, 2008, from http://www.ncbi.nlm.nih.gov/pubmed/18327567. 33. Low, J.C. and W.Donachie. 1997. A Review on Listeria monocytogenes and Listeriosis. Science Direct. Vet J. 153:9-29. Retrieved November 3, 2008, from http://www.ncbi.nlm.nih.gov/pubmed/9125353. 34. Lunden, J., R. Tolvanen, and H. Korkeala. 2004. Human Listeriosis Outbreaks Linked to Dairy Products in Europe. J. Dairy Sci. 87: E6-E11. Retrieved December 13, 2008, from http://jds.fass.org/cgi/reprint/87/13_suppl/E6. 35. Mead, P.S., L. Slutsker, V.Dietz, L.F. McCaig, J.S. Bresee, and C. Shapiro. 1999. Food-Related Illness and Death in the United States. Emerg. Infect. Dis. 5:607-625. Retrieved November 2, 2008, from http://www.cdc.gov/ncidod/eid/Vol5no5/mead.htm. 36. Moltz, A.G. and S.E. Martin. 2005. Formation of Biofilms by Listeria monocytogenes under various growth conditions. J. Food Prot. 68:92-97. Abstract Retrieved November 15, 2008, from http://tinyurl.com/cndfxp. 37. Munroe, S. 2008. Deadly Listeria Outbreak in Canada Likely to Grow. Retrieved December 14, 2008, from http://canadaonline.about.com/b/2008/08/25/deadly-listeria-outbreak-in-canada-likely-to-grow.htm. 38. Muriana, P. and K. Kushwaha. Food Pathogens of Concern: Listeria monocytogenes. Retrieved December 8, 2008, from http://fapc.okstate.edu/files/factsheets/fapc136.pdf. 39. Nightingale, K.K., Y.H. Schukken, C.R. Nightingale, E.D. Fortes, A.J. Ho, Z.her, Y.T. Grohn, P.L. McDonough, and M. Wiedmann. 2004. Ecology and Transmission of Listeria monocytogenes Infecting Ruminants and in the Farm Environment. Appl. Environ. Microbiol. 70: 4458-4467. Retrieved November 14, 2008, from http://aem.asm.org/cgi/reprint/70/8/4458. 40. OIE Terrestrial Manual. 2008. Listeria monocytogenes. Retrieved December 16, 2008, from http://www.oie.int/eng/Normes/mmanual/2008/pdf/2.09.07_LISTERIA_MONO.pdf. 41. Peters, T. 2001. Canadian Regulations and Guidelines for Listeria. Retrieved December 20, 2008, from http://tinyurl.com/bn5q7g. 42. Pioneer Technical Insight. 2008. Listeria in Silage. Retrieved November 13, 2008, from http://genetic.co.nz/pdf%20files/maize%20pdfs/337.pdf. 43. Portnoy, D.A., V. Auerbuch and I.J. Glomski. 2002. The Cell Biology of Listeria monocytogenes Infection: The Intersection of Bacterial Pathogenesis and Cell-Mediated Immunity. J. Cell Biol. 158:409-414. Retrieved November 22, 2008, from http://jcb.rupress.org/cgi/reprint/158/3/409. 44. Riemann, P.H. and D.O. Cliver, eds. 2006. Foodborne Infections and Intoxications. Academic Press, New York. Preview Retrieved November 11, 2008, from http://tinyurl.com/dalz4m. 45. Ryser, E.T. and E.H. Marth, eds. 1999. Listeria, Listeriosis and Food Safety. Marcel Dekker, Inc. New York. Preview Retrieved November 9, 2008, from http://tinyurl.com/c7osnq. 46. Seafood Network Information Center. 2007. Compendium of Fish and Fishery Product Processes, Hazards, and Controls Chapter 15: Listeria monocytogenes. Retrieved November 7, 2008, from http://seafood.ucdavis.edu/haccp/compendium/chapt15.htm. 47. Seekwellness. 2008. Listeriosis. Retrieved December 13, 2008, from http://www.seekwellness.com/nutrition/listeria.htm. 48. Southwick, F.S. and D.L. Purich. 1996. More About Listeria. Retrieved November 27, 2008, from http://www.med.ufl.edu/biochem/Dlpurich/morelist.html. 49. Temple, M.E. and M.C. Nahata. 2000. Treatment of Listeriosis. The Annals of Pharmacotherapy. 34: 656-661. Abstract Retrieved December 15, 2008, from http://www.theannals.com/cgi/content/abstract/34/5/656. 50. The New York Times Health Guide. 2009. Listeriosis. Retrieved Januray 3, 2009, from http://health.nytimes.com/health/guides/disease/listeriosis/overview.html. 51. Todar, K. 2008. Listeria monocytogenes. Todar's Online Textbook of Bacteriology. Retrieved November 5, 2008, from http://textbookofbacteriology.net/Listeria.html. 52. Todd, E. Harmonizing International Regulations for Listeria monocytogenes in Ready-to-Eat Foods. Retrieved December 21, 2008, from http://www.fsis.usda.gov/PDF/Slides_092806_ETodd3.pdf. 53. United States Department of Agriculture (USDA). 2008. FSIS Procedure for the Use of a Listeria monocytogenes Polymerase Chain Reaction (PCR) Screening Test. Retrieved December 19, 2008, from http://www.fsis.usda.gov/PDF/MLG_8A_03.pdf. 54. USDA. 2008. Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Egg and Environmental Samples. Retrieved December 17, 2008, from http://www.fsis.usda.gov/PDF/MLG_8_06.pdf. 55. USDA Agricultural Research Service. 2008. Annual Report 2007: Epidemiology, Ecology, and Molecular Genetics of Antimicrobial Resistance in Pathogenic and Commensal Bacteria from Food Animals. Retrieved December 22, 2008, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=410525&showpars=true&fy=2007. 56. USDA Agricultural Research Service. 2008. Annual Report 2007: Improved Methods for Control of Listeria monocytogenes within Biofilms in Meat and Poultry Processing Environments. Retrieved December 27, 2008, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=409192&showpars=true&fy=2007. 57. USDA Agricultural Research Service. 2008. Annual Report 2007: Listeria monocytogenes Contamination of Deli Meat Slicers: Risk and Communication. Retrieved December 23, 2008, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=410149&showpars=true&fy=2007. 58. USDA Agricultural Research Service. 2008. Annual Report 2007: Microbial Ecology of Human Pathogens Relative to Poultry Processing. Retrieved December 28, 2008, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=410492&showpars=true&fy=2007. 59. USDA Agricultural Research Service. 2008. Annual Report 2006: Validation of the Effect of Interventions and Processes on Persistence of Pathogens on Foods. Retrieved December 23, 2008, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=409249&showpars=true&fy=2006. 60. USDA Agricultural Research Service. 2008. Comparative antimicrobial susceptibilty of Listeria monocytogenes, L. innocua and L. welshimeri. Retrieved December 27, 2008, from http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=227924. 61. USDA Agricultural Research Service. 2009. Annual Report 2008: Controlling Listeria monocytogenes in Further Processed Meat. Retrieved January 4, 2009, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=410065&showpars=true&fy=2008. 62. USDA Agricultural Research Service. 2009. Annual Report 2008: Microbial Modeling and Bioinformatics for Food Safety and Security. Retrieved January 4, 2009, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=410312&showpars=true&fy=2008. 63. USDA Agricultural Research Service. 2009. Annual Report 2008: Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Foodborne Bacterial Pathogens in the Chicken. Retrieved January 4, 2009, from http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=411839&showpars=true&fy=2008. Click Here to Provide Feedback on FSRIO's Fact Sheets Back to top

This document was created by Vaishali Dharmarha with contributions from Tara Smith. Users are encouraged to provide feedback and comments. This document was created in Dec 2008.