[In the Lab: Listeria] From Contamination to Control

Foods and food components can become contaminated by Listeria at any stage in the food chain, but evidence from outbreaks of listeriosis highlights food-processing environments as key contamination sites. To get the full story on Listeria from contamination to control, QA contacted Dr. Don Liu, assistant research professor at Mississippi State University and author of the Handbook of Listeria monocytogenes, scheduled for publication in early 2008 by CRC Press.

WHAT IS LISTERIA? The term “Listeria” refers to a pathogenic Gram-positive, short, rod-shaped bacterium within the genus Listeria, i.e., Listeria monocytogenes, which is an adaptive, intracellular pathogen capable of causing a severe foodborne illness (listeriossis) in humans. There are five other species in this genus that do not usually cause disease in humans, but because they resemble L. monocytogenes, they can add complexity to the laboratory diagnosis of listeriosis.

A variety of food products (meat, milk, cheese, fish and vegetables) offer an ideal medium for Listeria growth and an effective agent for transmission of listeriosis, since Listeria bacteria are:

• relatively nutritionally undemanding and grow well on a number of non-selective microbiological media.
• highly adapted to grow at diverse temperatures (-0.15° C to 45° C), pH (4.3 to 9.4) and salt (10 percent NaCl and 200 ppm NaNO2) ranges.
• capable of enduring virtually all routine food processing procedures.

WHAT CAUSES LISTERIOSIS IN HUMANS? Listeriosis occurs in various animals, including humans, most often affecting the uterus at pregnancy, the central nervous system or the bloodstream. In humans, consumption of contaminated food is believed to be the principal route of infection, however this disease also can be transmitted by cross-infection during the neonatal period.

Infection most often is recognized in the immunocompromised, the elderly, the pregnant, and unborn or newly delivered babies. Although infection can be treated successfully with antibiotics, human infection has a mortality of 20-40 percent. This is attributable to the fact that listeriosis in humans often presents uncharacteristic influenza-like initial symptoms, and it is not always diagnosed until it is too late for antibiotic therapy.

A wide range of food types has been associated with transmission. These foods are generally characterized by their ability to support the multiplication of L. monocytogenes, having been processed with extended (usually refrigerated) shelf lives, consumed without further cooking and contaminated with high levels of L. monocytogenes.

The minimal infective dose for listeriosis is unclear, although it is likely to vary considerably between individuals.

WHERE DOES LISTERIA COME FROM AND HOW DOES IT GET INTO THE FOOD PLANT? Listeria bacteria are found throughout the natural environment, and are especially abundant in runoff water, sewage, soil and vegetation from which they invariably find their way into various food materials. Because of their ability to withstand extreme conditions, these bacteria remain largely unscathed after many food-manufacturing processes.

Contamination of foods may occur pre-harvest, especially in those that contact soil such as fruits and vegetables. In addition, an extremely wide range of mammals, birds, fish and invertebrates has been reported to carry Listeria spp. in the feces without apparent disease, so environments contaminated by animal feces frequently contain bacteria from this genus.

During post harvest, foods and food components can become contaminated at any stage from food processing to retailing or in consumers’ homes. However, evidence from listeriosis outbreaks highlights the risk of contamination from sites within food processing environments. Listeria can be introduced from the environment into a food processing plant especially where hygiene barriers are insufficient and it can persist for considerable periods of time.

HOW CAN LISTERIA BEST BE DETECTED AND IDENTIFIED IN THE FOOD PLANT? Listeria rods measure 0.4-0.5 by 1-2 ìm, and so cannot be seen with the naked eye. In addition, they are indistinguishable from other bacteria under microscope. For these reasons, specific identification of Listeria relies on the application of biochemical, serological or molecular methods. Further, because most clinical, food and environmental specimens contain low numbers of the bacteria, samples need to be processed and cultured (enriched and isolated) before Listeria can be reliably detected and identified.

There are several official, standardized protocols for L. monocytogenes enrichment and isolation, but the FDA protocol, which is designed for use with any food product, is the most common method in the U.S. Other primary protocols are USDA, designed for isolations from meat, poultry and egg products, and ISO, the most common in European countries.

After enrichment and isolation, Listeria can be identified through biochemical, serological and molecular procedures:

• Commercially available biochemical test systems are routinely applied in clinical and food-testing laboratories. But biochemical tests always have the potential for strain vagaries or inconsistencies.
• Many immuno-based kits also have been marketed, most of which detect Listeria spp., not L. monocytogenes specifically. The AOAC Research Institute database (www.aoac.org) has a list of commercially available test kits for Listeria.
• Results of biochemical and serological testing, however, may be ambiguous at times, because they assess the phenotypic properties of Listeria, which often vary with changing external conditions, growth phase and spontaneous genetic alterations. In addition, phenotypic tests are very time-consuming, delaying result availability.
• To address the issues of phenotype procedures, a new generation of genotype-based methods, targeting the nucleic acids (DNA or RNA) of Listeria, has been developed. As these nucleic acids are intrinsically more stable than proteins and less prone to influences by outside factors, these procedures are much more precise and less variable than the phenotype-based methods — and they are much faster and more sensitive.
• A plethora of genotype-based tests that direct at a variety of genetic elements have been designed and described, with testing evolved from unsophisticated non-amplified DNA hybridization procedures to the all-encompassing nucleic acid amplification technologies — e.g., polymerase chain reaction (PCR) — coupled with a real-time detection capacity.

WHERE IS LISTERIA IN A FOOD PLANT? Listeria is capable of attaching to any food-processing surface including wood, steel and concrete. There is evidence that some pathogenic Listeria genotypes show enhanced binding to these surfaces and readily form biofilms that enhance their resistance to disinfectants and sterilizing agents commonly used in the industry.

Although Listeria can survive and grow in virtually all types of foods, ready-to-eat cheese and meats are especially important sources of transmission for listeriosis, since Listeria often replicates in these food products under storage conditions before being consumed.

WHY IS IT SO DIFFICULT TO ELIMINATE? Contamination by a single strain of L. monocytogenes can affect a single environment for several years. Sanitation efforts are usually insufficient for elimination because listeriae survive in niches within plants where sanitation is not sufficiently effective and this bacterium can persist and remain undetectable for years.

• L. monocytogenes can survive pH levels as low as 3.0, thus the use of low pH during processing of cured dried sausages and other fermented specialties is unlikely to completely eliminate this bacterium.
• Pasteurization (usually at 68° C) and cold storage (at 4° C) are typical temperature-related measures applied to foods, but some L. monocytogenes strains are known to survive this processes.
• Although heating food at very high temperatures will result in elimination of all bacteria, it generally isn’t desirable as it can adversely affect food taste and appearance.
• L. monocytogenes grows readily at 4° C, thus prolonged storage of food in refrigerators favors the replication of this bacterium, which can cause a serious problem if the stored food is consumed without further cooking. In addition, there is evidence that L. monocytogenes thermotolerance may actually increase after prior exposure to sublethal temperatures.
• Salt is an essential ingredient used for preparation of dried sausage and corned beef. As L. monocytogenes can survive at 40 percent NaCl for a long period and grow at up to 10 percent NaCl, it is unlikely to be terminally affected by conventional food-processing salt treatment.
• Listeria’s capacity to endure harsh pH and salt conditions is advantageous to its survival in the environment and food-processing facilities, since hypochloric acid (bleach), chlorine, potassium hydroxide, sodium hydroxide and quaternary salts are frequently applied as detergents and sanitizers to remove organic residuals from food processing surfaces, equipment and plants.

SO WHAT CAN BE DONE? As Listeria species grow poorly in nutrient-deficient, unpolluted seawater and springwater and thrive on nutrient-rich, contaminated waters, sewage and sludge, an obvious strategy to lower the occurrence of Listeria in the environment is to remove as much residual nutrient in the runoff waters from farms and processing plants as possible, thus leading to slower growth of Listeria bacteria therein.

Implementation of routine screening procedures to monitor the presence of Listeria in various environmental specimens will also enable one to take prompt, corrective action when Listeria bacteria reach unacceptable levels, and prevent the spread of listeriosis in animals and humans.

Pre-Processing. Is important to adapt appropriate farming and husbandry practices that reduce and eliminate the occurrence of L. monocytogenes in vegetables and farm animals. For instance, use of organic fertilizers free of L. monocytogenes contamination in vegetable growing will lead to cleaner raw materials for downstream processing. Use of clean containers and water for pre-processing handling also will ensure a lowered level of contamination.

In order to reduce the incidence of listeriosis in animals, there are several issues that need to be addressed.

As animals invariably become infected with Listeria bacteria via contaminated feed and water, it is vital to ensure that only dry silage and water free of Listeria bacteria are fed to the animals. In addition, frequent cleaning of animal housing and exercise areas may help decrease the incidence of listeriosis.

It is necessary to implement a monitoring system to test any animals showing clinical manifestations of listeriosis, then separate and treat infected animals. Prompt diagnosis and treatment will help limit the spread of the bacteria as well as subsequent infections from cases of abortive listeriosis.

Future development and application of vaccines against L. monocytogenes and L. ivanovii will help prevent outbreaks of listeriosis in animals.

During Processing. Direct contact with contaminated processing equipment is a major cause of L. monocytogenes occurrence in food products. Adoption of Hazard Analysis and Critical Control Point (HACCP) measures during processing (e.g., thorough cleansing of in-process products and food-contact surfaces, clear separation of staff functions and scrupulous personal hygiene) has led to significant reduction of contamination in finished food products.

Use of heat treatment (e.g., hot steam, hot air or hot water at 80° C) is effective in cleaning and sanitizing skinning, slicing and brining equipment, and in eradicating L. monocytogenes from food plants.

Visitors and staff job rotation are potential risk factors for increasing L. monocytogenes contamination, thus control of personnel and designated hygiene and uniform program is critical for control.

Optimization and application of more effective, innovative combinations of detergents and sanitizers may help eliminate L. monocytogenes in food-processing environments.

Post Processing. Implementation of monitoring and quality control procedures represents the most effective post-processing measures against foodborne pathogens including L. monocytogenes.

Ensure that the finished food products meet recommended tolerable levels of L. monocytogenes in the specified product categories before releasing to retail markets. Implement a one-week post-processing storage plan for ready-to-eat (RTE) meat products, as this has been found to inhibit listerial growth, and decrease L. monocytogenes in many RTE products.

Continue publicizing best practices in home food handling, where storage of foods at incorrect refrigeration temperatures often contributes to heightened risk of listeriosis.

The author is staff editor of QA magazine and can be contacted at llupo@giemedia.com.

In August 2006, FDA-approved spraying meat with phages as a safe way (GRAS status) to eliminate Listeria in food. Phages (or bacteriophages) are viruses that invade only bacterial cells and cause bacteria to lyse, or dissolve, and die. Thus, they provide an alternative to antibiotics for treating bacteria without other unwanted side effects such as buildup of resistance. Being species-specific, phages used to destroy Listeria bacteria do not recognize other cell types and are harmless to human hosts.

Listeria Facts

• L. monocytogenes infections usually occur in urban populations and in the absence of specific contact with animals.
• Human listeriosis has a marked seasonality, with a peak in cases occurring during the late summer and autumn.
• The incidence of infection increases with age so that the mean age of adult infections is over 55 years.
• Men are more commonly infected than women older than 40, and since women are infected in the child-bearing years, the overall sex distribution is biased towards males in the elderly immunocompromised.
• In 1986, the U.S. reported an incidence of 7.1 cases/million persons/year. That number has been reduced to 3.1. Estimates on the incidence of listeriosis in European countries range from between less than 1 to more than 7 cases/million/year.
• The incubation period in humans between consumption of contaminated foods and clinical recognition of the disease varies widely between individuals from 1-90 days, with an average for intra-uterine infection of around 30 days.
• The contamination level in foods associated with infection has revealed an average contamination level of 102-106 CFU/mL/g in the majority of the cases.
• Contamination by a single strain of L. monocytogenes can affect a single environment for several years.
• The most frequent reason for a L. monocytogenes contamination of raw foods is poor hygiene during milking or slaughter. But contamination rates of raw meat and meat products can be similar or even higher than that found in dairies.
• Contamination rates of vegetables vary. If a plant is contaminated, more than 50 percent of the vegetables can contain L. monocytogenes, even after heat treatments at 90° C for 4 seconds.