Germ Warfare
Sponsored by DuPont Qualicon

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular opportunistic pathogen, which is ubiquitous to the environment. Listeria is often transmitted through contaminated food and causes a rare but serious disease called listeriosis. One fourth of the food poisoning-related deaths are caused by Listeria.

Several studies have found that Listeria enters the body through the intestines and can spread to the liver while eluding the immune system by entering hepatocytes and proliferating intracellularlly. Infected resident phagocytes often spread through the blood to the brain and uterus. Outbreaks of Listeria are often related to contaminated ready-to-eat (RTE) foods. The most recent outbreak dates from September 2008 in Canada. Fifty-two cases have been linked to Maple Leaf Foods, where 20 people have died from severe listeriosis. The company recalled 191 products, which has lead to millions dollars of damage. The food industry is constantly evolving to minimize the outbreaks. Recently, several companies have received governmental approval for the use of bacteriophage technology to ensure food safety.

The threat

Studies suggest that 1 to 10 percent of the healthy human population may have their gastrointestal tracts colonized by Listeria.

The groups at risk for contracting listeriosis are pregnant women and their neonates, immunocompromised adults (e.g. acquired immune deficiency syndrome and organ transplant patients) and the elderly (60 years and older). There is controversy in the classification of Listeria. Host susceptibility plays a major role in the presentation of clinical disease upon exposure to Listeria. Most cases of listeriosis involve patients that have a physiological or pathological defect that affects cell-mediated immune response. This reasoning justifies the classification of Listeria as an opportunistic pathogen. Nevertheless, European and North American governmental safety regulations still regard Listeria as a pathogenic organism and a threat to overall public health. Severe infections during pregnancy can lead to pre-mature birth or miscarriage, and meningitis in newborn children. In immunocompromised adults and the elderly, infection can cause septicemia and meningitis. Approximately 2,500 cases of serious listeriosis and 500 deaths are reported each year in the United States alone. This number is assumptively lower than the actual number of infections due to the frequent misdiagnosing by physicians. Listeria is often overlooked as a possible cause of illness due to several important factors. Listeria is difficult to detect due to its unique growth capabilities. It grows best at refrigeration temperatures (1 to 10°C). Even when detected, phenotypic characteristics resemble that of Streptococci and other diphteroids, which are then often discarded as harmless contamination by microbiology lab technicians. Nearly one-fourth of all estimated foodborne disease-related deaths are contributed Listeria. Nevertheless, Listeria infection is relatively uncommon in comparison with other food borne pathogens (Salmonella typhimurium and Escherichia coli). Unique to Listeria is the usually high fatality rate, which can be as high as 40 percent among at-risk people.

Food industry

Certain features make Listeria especially problematic for the food industry. Listeria can proliferate (respectively) at temperatures from 1 to 45°C and survive at extremely low to extremely high temperatures for a certain time (minus 20 to 80°C). Listeria is also halotolerant, meaning that it can survive and grow in salt solutions where other bacteria cannot. Even relatively low pHs, frequently encountered in food processing, seem to have little effect against Listeria. Affected products include; fish, shellfish and fish products; raw meat, poultry and their products, including hot dogs and pâté; raw and processed vegetables; ripened soft cheeses; ice cream; retail cook-chill meats; salads including coleslaw; raw and partially pasteurized milk as well as raw and liquid egg. RTE products are especially vulnerable to contamination by Listeria since the products are not always re-heated before consumption.

Post-pasteurization processed products (e.g. sliced and repackaged deli meats) have been deemed high risk by the United States Department of Agriculture (USDA), for the susceptible population. Outbreaks of Listeria in the United States have lead to several nationwide recalls of RTE products.

Fighting Listeria

Listeria cannot simply be treated with antibiotics. There is no known vaccine and due to the low occurrence, it is very unlikely that pharmaceutical companies are interested in developing one. The sporadic occurrence of Listeria has limited the opportunity for it to develop extremely resistant strains due to the lack of feedback from patient to source. However, the globally increasing risk of resistant bacteria has the medical world worried about the limited resources of combating listeriosis. The food industry, on the contrary, has plentiful means to prevent Listeria contamination. Countless foodgrade listeriocidal agents and antilisteral processes are available. The majority of food-producing companies cannot make use of these techniques due to product formulation and lack of financial means. The food industry, like the medical society, is searching for a silver bullet that will improve the odds when fighting Listeria. Recent publications have re-introduced bacteriophages as possible anti-listeral agents. Bacteriophages are inanimate obligate parasites that have a broad or specific range of prokaryotic target organisms. Natural bacteriophages are the most abundant microorganisms in our environment and are present in high numbers in water and foods of various origins. Bacteriophages contribute to bacterial homeostasis in nature, where they keep bacteria under control.

Food contamination

Listeria infections can be sporadic due to contact with animals and agricultural materials.

However, the majority of listeriosis cases are related to the consumption of infected foods. Raw meat products are likely to be contaminated with foodborne pathogens but are practically always heated to temperatures that kill the bacteria before consumption. The at-risk products are those that fall into the RTE category. Vegetables that have been fertilized by natural manure and that can be eaten raw are highly susceptible to transmit the pathogen. The focus, however, lies on meat and dairy products because of the amount of processing the products generally undergo. Each processing step increases the risk of contamination.

Take, for example, a beef deli meat. The process from cow to sandwich often involves five to six different parties before consumption. Once the meat-processing plant has received the raw meat, it is processed and undergoes a common listeriocidal process: cooking. When the beef is cooked (pasteurized), it is totally free from Listeria. Once the roast beef exits the oven, it is prone to re-contamination from environmental sources (e.g. contaminated workers, aerosols and condense drip). When the meat is packaged, some companies apply a post-pasteurization anti-listeral process such as heat treatment of the product surface before packaging. After packaging, the beef is stored at refrigeration temperatures until shipping. The beef will stay at refrigeration temperatures until it reaches the primary or secondary consumer. As mentioned before; Listeria can and will proliferate at refrigeration temperatures. This poses an additional risk without even considering the possible temperature abuse/fluctuation the packaged beef might undergo during transportation. Deli meats are especially vulnerable to recontamination because they are often sliced and repackaged by the retailer. If contamination occurs during the slicing process, the Listeria cells will systemically spread to all slices of the consecutive batches until the slicing machine is cleaned. In summary, there are many possible sources of recontamination. Processed and unprocessed RTE foods are vulnerable and some might have more sources than others.

Listeria Prevention

Not all hope is lost. There are many possible ways of preventing recontamination of Listeria. Depending on the type of product, formulation limitation, a food-processing company can opt for several different approaches. There are three general concepts of prevention; mechanical pasteurization, physical containment and (bio-)chemical treatment. Mechanical pasteurization mainly involves high-temperature processing, but can also make use of ultraviolet radiation and high pressure. Physical containment is usually a bacteriostatic process where the materials and machinery used for packaging prevent proliferation of possible pathogens. Modified-atmosphere packaging (MAP) is a widely used technique which removes all the oxygen and adds nitrogen to inhibit the growth of aerobic bacteria. The last category is the use of bacteriocial or bacteriostatic (bio-)chemicals — many of which are techniques from antiquity, like the use of acids and high salt concentrations. New age chemicals like highly purified herbs and smoke extracts are often very effective but can affect the quality of the product. There is need for an universal product which evolves along with the bacterium in question.

Bacteriophage technology

Bacteriophage technology is an old, yet revived emerging technique to deal with Listeria.

Bacteriophages are viruses that infect bacteria. They are inanimate obligate intracellular parasites and lack their own metabolism. Phages are extremely host-specific and are the natural enemies of all sorts of bacteria. Listeriophages are, like Listeria, ubiquitous to the environment and can be isolated from the same niches where its host resides. A rather difficult concept to understand is the unusual mechanism of lytic phages. Once infected by a phage, the cell can still function normally depending on which cycle the phage enters (lytic or lysogenic). A phage-infected Listeria cell will not lyse immediately due to the phage life cycle. Thus, a lytic phage-infected cell is technically still alive, depending on the aggressiveness of the phage and the activity of the host’s metabolism. A higher host metabolism (correlated with optimal growth temperatures) will lead to quicker lysis of the cell and vice versa. Only lytic phages are useful to the food industry due to these obvious reasons.

Companies (EBI food safety, Wageningen, the Netherlands, and Intralytix, Baltimore, Md.) have isolated and improved several lytic phages specific for different pathogens. Selected bacteriophages can act as a listeriocidal and listeriostatic agent, by merely spraying a high titer solution on virtually any given product. There are several considerations for applying bacteriophage technology, which have been carefully listed by G.G. Greer in a review (see table 1).


Table 1: Considerations for Developing Bacteriophage Approach to the Control of Foodborne Bacteria

1. Self-perpetuating
Limited host ranges
2. Selective modification of bacterial flora
Possible phage-resistant bacterial mutants
3. Stable in foods and able to survive processing
Requires relatively large numbers of target bacteria
4. Natural
Barriers in food environments
5. Ubiquitous
Tansduction of undesirable
6. Ease of preparation and application
Lysogenic conversion to temperate phages
7. Non-toxic to eurkaryotic cells
8. No effect on food quality
Consumer perception
Source: Greer GG. “Bacteriophage”

Author's bio

Henk Robert Hoogenkamp is a biochemistry graduate student in The Netherlands at HAN University Nijmegen in Life Science. He also works as a research analyst at Nijmegen Centre for Molecular Life Science where his current project is on soft tissue engineering in children with congenital defects.