Abstract
Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked.
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Introduction
The genus Listeria is placed in the Clostridium sub-branch of Gram-positive bacteria based upon the low G + C content of its genome. There are six species currently recognized: Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri and Listeria grayi. Only two species of the genus are generally considered to be pathogenic, L. monocytogenes in humans and L. ivanovii in other mammals. However, there have been some reports of L. seeligeri and L. ivanovii causing illness in humans. Pathogenic infection by L. monocytogenes results in listeriosis and usually affects individuals pre-disposed through an underlying disease affecting the immune system, such as cancer or AIDS, and also other susceptible individuals such as the elderly, pregnant women, newborn babies or fetuses. Symptoms of the disease are flu-like, yet may result in severe complications, such as meningitis, septicaemia, spontaneous abortion or listeriosis of the newborn. The number of cases of listeriosis average 40–44 per year in Australia and around 100 per year from 1993 to 1997 in the USA . Although the incidence of listeriosis seems small compared to other food-borne diseases, the associated mortality is high at around 30%.
Although Murray first suspected an oral route for the bacterial infection observed in animals in 1924, it was not until 1981 that for the first time an outbreak of listeriosis in Canada was linked to a contaminated food source . Since the recognition of L. monocytogenes as a food-borne pathogen, there have been rapid advances in the development of suitable methods for isolation and identification.
Initial attempts to isolate Listeria from food based on clinical procedures such as direct plating onto blood agar were unsuccessful. Significant developments have occurred not only in selective culture enrichment procedures but also in the availability of many new and rapid detection methods based on antibody and molecular technologies. A major challenge for food testing has always been the interference of the tests by inhibitory food components and hence novel methods, such as immuno-capture, were developed to purify analytes from inhibitory food components as well as to increase sensitivity.
There has been a constant search for more rapid and sensitive methods, particularly in the food industry, where pressure from regulators to provide contaminant free food and the need to release perishable product onto the market prior to their expiry date are ever present. Culture and immunoassay methods are widely used because they are simple, inexpensive and allow a high sample throughput. Antibody-based tests target L. monocytogenes-specific proteins and utilization of culture tests using colorimetric or fluorescent substrates in media to detect virulence factor activity have been introduced to differentiate between pathogenic and non-pathogenic species.
Many tests endorsed and regulated by government agencies such as the Food and Drug Administration (FDA) or US Department of Agriculture (USDA) in the United States, or the Australian and New Zealand Food Administration (ANZFA) in Australia, do not differentiate between Listeria species. From the perspective of food hygiene the presence of a non-pathogenic species such as L. innocua may indicate potential contamination with L. monocytogenes. However, epidemiological studies have revealed that only L. monocytogenes and only strains belonging to serotypes 1/2a, 1/2b and 4b were implicated in 90% of outbreaks of listeriosis. It is unclear why only three of the 13 serotypes are implicated in food-borne outbreaks whilst other serotypes are also found as food contaminants. All serotypes possess the same virulence factors and hence have the same potential to cause disease. In light of these epidemiological data many food diagnostic tests have been developed to differentiate L. monocytogenes from the other species in food.
Although the majority of tests used for food testing are based on culture methods or antibodies, the trend in the food industry is towards the use of molecular methods. There are some disadvantages compared to culture methods such as equipment and reagent costs and the requirement of highly trained personnel. There is no regulatory approval for the majority of these tests, which prohibits their use in many food-testing laboratories. Recently, however, some molecular techniques such as PCR and DNA hybridization have become a feasible alternative to culture and serological techniques. The major advantage that molecular techniques offer over conventional methods is that these are based on differences within the genome and do not rely on the expression of certain antigenic factors or enzymes to facilitate identification. They are extremely accurate, reliable and some can be performed in the same time frame as immunoassay methods (). There is a wide range of molecular methods available for the identification and characterization of Listeria. Based on the multitude of publications that have appeared over the last two decades on molecular testing describing the adaptation of conventional PCR methods to the food testing laboratory, there is little doubt that many of these techniques will be applied routinely in the near future. The available technology in this area is both diverse and rapidly changing and here we review the most important developments for isolating and identifying Listeria spp. and L. monocytogenes.
