In performing phenotypic characterization tests, the organisms used for the inoculation of test media should be from fresh transfers and in good physiological condition. The methods described in this chapter are based primarily on methods developed for the characterization and identification of organisms which have usually been isolated on nutrient-rich media. While zoologists may be content to classify all insects or all vertebrates, prokaryote systematics generally deals with all prokaryotes and in an evolutionary context attempts to extend this study back to the origin of life itself. To test for acetamide hydrolysis, streak the surface of an acetamide agar slant with a sample from a dilute suspension. Incubate for up to 7 days. Care should be taken to ensure that the basal medium to which the carbohydrates are added does not contain fermentable or oxidizable sugars, and control media without any added carbohydrates should always be included during testing of cultures. Methanogenic members of the Archaea also fluoresce under UV light. Some facultative anaerobes, such as Escherichia coli and Staphylococcus aureus, grow aerobically by respiration with oxygen and grow anaerobically by fermentation.

Phenotypic Characterization and the Principles of Comparative Systematics

Following the initial isolation and description in 1926 Listeria monocytogenes has been shown to be of world-wide prevalence and is associated with serious disease in a wide variety of animals, including man. Our knowledge of this bacterial pathogen and the various forms of listeriosis that it causes has until recently been extremely limited, but recent advances in taxonomy, isolation methods, bacterial typing, molecular biology and cell biology have extended our knowledge. It is an exquisitely adaptable environmental bacterium capable of existing both as an animal pathogen and plant saprophyte with a powerful array of regulated virulence factors. Most cases of listeriosis arise from the ingestion of contaminated food and in the UK the disease is particularly common in ruminants fed on silage. Although a number of forms of listeriosis are easily recognized, such as encephalitis, abortion and septicaemia, the epidemiological aspects and pathogenesis of infection in ruminants remain poorly understood. The invasion of peripheral nerve cells and rapid entry into the brain is postulated as a unique characteristic of its virulence, but relevant and practical disease models are still required to investigate this phenomenon. This review offers an up to date introduction to the organism with a description of virulence determinants, typing systems and a detailed account of listeriosis in animals. Experimental and field papers are reviewed and further sections deal with the diagnosis, treatment and control of listeriosis in animals. A final part gives an overview of listeriosis in man.

http://dzumenvis.nic.in/taxonomy/pdf/a review of listeria monocytogenes.pdf

A Review of Listeria monocytogenes and Listeriosis

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.

Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review

Epidemiology of human listeriosis and seafoods.

Two hundred seventy-nine cases of human listeriosis (92 pregnancy-related cases and 187 non-pregnancy-related cases) caused by a serovar 4b and phagovar 2389:2425:3274:2671:47:108:340 strain were identified in France between March and December 1992. Epidemiological investigations included a case-control study (not described here) and microbiological analyses of foods. Results of the case-control study and characterization of food isolates identified pork tongue in jelly, a ready-to-eat meat product, as the major vehicle of this outbreak, and to a lesser extent, delicatessen products contaminated secondarily during handling in food stores. As far as serotyping, phage typing, DNA macrorestriction pattern analysis (obtained by pulsed-field gel electrophoresis [PFGE]), and ribotyping are concerned, this epidemic strain is phenotypically and genomically closely related to strains responsible for major outbreaks of listeriosis previously observed in Europe and North America. The epidemic strain sensu stricto as defined by PFGE (2/1/3) displayed the same serovar, phagovar, ribovar, and ApaI and NotI PFGE patterns as the epidemic strains from outbreaks in Switzerland, California, and Denmark, but it consistently showed differences in the SmaI PFGE profile. This information greatly contributed to the identification of the major food vehicle (pork tongue in jelly) and further allowed exclusion of other foods (cheese) as possible sources of this major listeriosis epidemic.

Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992.

Typing Listeria monocytogenes : a comparison of random amplification of polymorphic DNA with 5 other methods

The rDNA gene restriction patterns of 134 isolates of Listeria species were determined with pKK3535--a pBR322 derived plasmid containing an Escherichia coli rRNA operon--used as a probe following digestion of chromosomal DNA by EcoRI endonuclease. Nineteen reference and type strains representing all species and serotypes of Listeria showed 17 distinct ribotypes. One hundred and fifteen wild strains of Listeria monocytogenes were ribotyped and the results were compared to those of serotyping, phage typing, multilocus enzyme electrophoresis (MEE) and restriction endonuclease analysis (REA). Ninety-six Listeria monocytogenes serotype 4b wild strains displayed six distinct ribotypes (I-VI), 72% (69/96) of them clustering in two very close rDNA patterns (I and II) of eight and nine bands, respectively. The same 96 strains displayed six REA patterns and eight MEE electrotypes. Among the 96 Listeria monocytogenes 4b isolates, the 34 epidemic strains defined by phage typing and by epidemiological data all belonged to one ribotype (ribotype I) representing 56% of the strains belonging to this ribotype. These same 34 epidemic strains were also grouped by REA and MEE typing in a unique profile (REA-A) and MEE electrotype (ET 1). Twenty-two Listeria monocytogenes strains of serogroup 1/2 analyzed by rDNA typing showed nine distinct ribotypes. For the 96 Listeria monocytogenes 4b strains studied, the discriminatory index was highest for phage typing and for any combination including phage typing. Ribotyping appears to be a well reproducible molecular typing method and could be a useful complement to other typing methods for the epidemiological study of listeriosis.

F Ischer

Papers

Typing Listeria monocytogenes : a comparison of random amplification of polymorphic DNA with 5 other methods

Characterization of Listeria strains from a foodborne listeriosis outbreak by rDNA gene restriction patterns compared to four other typing methods.