Listeria Monocytogenes can be further characterized based on the presence of specific heat stable somatic (0) and heat-labile flagella (H) antigens. Based on the "O" and H" antigens, strains of Listeria Monocytogenes, isolated from pathological sources are subdivided into serotypes: 1/2a,1/2 b, 1/2c, 3a,3b,3c,4a,4ab,4b,4c,4d,4e,4f,5 and 6. Serotypes 1/2a, 1/2b and 4b are responsible for greater than 95% of all human infections (Frances Pouch Downee, Keith Ito, 2001).
Factors affecting growth and survival of Listeria Monocytogenes.
Listeria Monocytogenes is a psychotropic bacterium that is it has the ability to resist the cold temperature of refrigeration. However, Listeria Monocytogenes is also thermo tolerant when subjected to temperatures above the optimum.
The broad pH range for growth for Listeria Monocytogenes allows it to survive. pH 7.0 - 7.5 is the optimum pH for the growth of Listeria Monocytogenes (Dean, 1990).
The bacteria can resist a high concentration of salt that is an environment with a low water potential. It has been shown that the organism can tolerate environments of 25.6 % Nacl for at least 132 days at 22Â°C and 5 days at 37Â°C (Adams; 2001; Lovette, 1989).
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Moreover the presence of other microorganisms in the same medium (on the same contaminated food) can cause a decrease in the population of Listeria Monoctyogenes.
Also known as the Circling Disease or Silage sickness, Listeriosis is a sporadic bacterial infection caused by Listeria Monocytogenes. It is a worldwide disease and a serious food borne disease for humans. The term Listeriosis encompasses a wide variety of disease symptoms that are similar on animals and humans.
Persons of advanced age, pregnant women, new born and adults with infected immune systems are normally prone to attract this disease. A normal person without those criteria mentioned above can also be affected. He can be infected by consuming contaminated food. Babies may get infected at birth itself if their mother had consumed contaminated food during pregnancy.
According to the world health organization (WHO), outbreaks of Listeriosis have been reported from many countries, including Australia, S Switzerland, France and the United states. Two recent outbreaks of Listeria Monocytogenes in France in 2000 and in the USA in 199 caused by contaminated pork tongue and hot dogs.
Health risk of listeria:
Hayes (1992) considered Listeria Monocytogenes as a low grade pathogen since there is no clinical manifestation in healthy individuals upon ingestion of low numbers of viable cells. According to the center for food security and public health 1-10 % of the population is thought to carry Listeria Monocytogenes asymptomatically in the intestines (May, 2005).
Vomiting, Nausea, Cramps, Diarrhea, severe headache, constipation ad persistent fever are the symptoms that may occur suddenly. Meningitis encephalitis is the infection of the brain and its surrounding tissue. Septicemia is the poisoning of blood caused by listeria. The overall mortality rate in the group of susceptible people mention in sectionâ€¦. Are 20 -30%.
Listeria Monocytogenes can be identified in tissues using ELISA, PCR and other molecular techniques. It is treated with antibiotics depending on the form of the disease.
Mode of invasion and spread of Listeria in host cells:
Listeria Monocytogenes is acquires by ingestion. The bacterium must find and adhere to the intestinal mucosa or the intestinal crypt cells, which are the only undifferentiated mucosal cells. Once the bacterium is phagocytosed, it becomes enclosed in a phagolysosome, a sub cellular organelle. Normally the low pH and the contents of the phagosomes are toxic to microorganism, however, environment of low pH causes Listeria Monocytogenes to produce hemolysin, Listeriolysin O (LLO).LLO lyses the cell membrane of the phagolysosome and this causes release of the Listeria into the cytoplasm. According to F.S Southwick and D.L Purich all pathogenic strains of Listeria produce Listeriolysin-O which is important for their escape and pathogenesis.
Once in the cytoplasm, the bacteria multiply and proliferate and the bacteria become surrounded by an electron-dense material. The bacteria are then known to be polarized at one end. The electron-dense material give the bacterium an elongated protrusions form and filopods which are in turns ingested by adjacent cells and the cycle begins anew.
Spreading from cell to cell without directly being in contact with the extracellular environment is how Listeria Monocytogenes invade the cells of its hosts.
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Isolation of Listeria Monocytogenes:
Since there has been increasing interest in the presence or absence of Listeria Monocytogenes in foods, as a result of some substantial outbreaks of food borne listeriosis in North America and Europe, there has been a vital need to develop methods to isolate it in various type of food. Several methods such as ELISA, PCR and genetic hybridization technologies (Entis and Lerner, 1991, RMR Labs, 2000, certification, Report, 2000; Klein and Juneja, 2001) have been developed.
Department of agriculture (USDA) and the Food and Drug Administration (FDA), are the two US agencies that made use of different protocol for analysis of Listeria Monocytogenes.
The techniques for isolation of Listeria Monocytogenes involve two- stage enrichment, the pre-enrichment followed by enrichment and plating for isolation.
The enrichment procedures helps to keep the level of contaminating microorganisms to a reasonable numbers and allow multiplication of Listeria Monocytogenes to levels that are enough for detection of the organism. Half Fraser Broth and the University of Vermont broth (UVM) are examples of broths for the pre-enrichment procedures. They allow revival of injured Listeria cells. The Fraser broth is used with a selective Fraser broth supplement in enriching for the enrichment steps after the pre-enrichment and for detecting Listeria. Both the Fraser and Half Fraser Broth contain sodium phosphate and potassium phosphate which are buffering agents. The presence of ferric ions acts as an indicator since the bacteria produces 6, 7- dihydroxycoumarin that reacts with the ferric ions thus resulting in the blackening of the medium. Lithium chloride, nalidixic acid and acriflavine give the broth a higher concentration of salt and inhibit growth of enterococci.
However, it is only after 48 hours that there is blackening of the broth.
For plating, PALCAM, Oxford and Modified Oxford (MOX) are used as selective agars for isolation of Listeria Monocytogenes. Lithium chloride, polymyxin B sulphate and acriflavine HCl, present in the PALCAM medium Base and ceftazidine found in the PALCAM supplement ensure the selectivity of the medium. These elements suppress other bacteria present in food except Listeria.
For differentiation, the PALCAM medium provides esculin and mannitol. Hydrolysis of esculin by Listeria causes production of 6, 7, dihydroxycoumarin, which reacts with the ferric ions that are present in the PLACAM medium to form blacken halos.
The USDA method involve a two- stage enrichment procedure with a 24-48 hours primary enrichment with UVM medium followed by a second enrichment phase with Fraser broth. Black colonies on the MOX plates show the presence of Listeria Monocytogenes.
This method involves 48 hour enrichment at 30Â°C in buffered Listeria Enrichment Broth (BLEB). Pre enrichment procedure is optional which is done 4 hours at 30Â°C prior to the addition of the selective supplements. After 24 hours to 48 hours the culture is streaked onto Oxford, PALCAM, Lithium chloride-phenyl ethanol moxalactant (LPM). After 24-48 hours at 30Â°C, black-halo colonies prove the presence of Listeria.
The ISO method and the USDA method are alike with only the difference of using Half Fraser Broth for enrichment in the ISO method. The enrichment is done using Fraser Broth. On PALCAM or Oxford agar, the Listeria colonies are gray green with the black halo and black respectively.
As the phylogenic or the population genetics research is increasing, genomic DNA isolation from living material such as bacteria, fungi, plants, insects, animal cells and blood is important.
The conventional method of DNA extraction involves 3 steps namely:
Lysis of cell wall or cell membrane.
Removal of all unwanted content fro example proteins, polysaccharide and lipids.
Recovery of DNA.
Gram positive bacteria such as Listeria Monocytogenes has thick cell walls composed of peptidoglycan which is rigid, hence making it difficult to break down the cell wall. Therefore important treatment which involves the use of lysozyme, osmotic shock, detergent, boiling extraction is carried out. Commercial kit also available on the market is used for DNA extraction.
Molecular method for detection of Listeria Monocytogenes:
Frances.P.D and Keith.I stated "Methods for subtyping Listeria can be separated into two broad categories: (a) concentional methods, which include serotyping, phage typing, and bactteriocin typing and (b) molecular methods, which encompass multilocus enzyme electrophoresis(MEE), chromosomal DNA restriction endonucleases analysis (REA), ribotyping, DNA macrorestriction analysis by pulsed- field electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD) by PCR, and DNA sequence-based subtyping." (2001). RFLP ( restricted fragment length polymorphism) forms part of the molecular method for subtyping (Emily.T.yeh, 2004).
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Note: for this experiment RAPD PCR and RFLP was carried out to assess the genetic relatedness of the isolates of Listeria Monocytogenes.
In 1983, the introduction of PCR has been revolutionized the microbial ecology and has been of increasing use in food microbiology. The polymerase chain reaction makes use of enzymes to amplify millions of copies of target DNA in few hours. "PCR today plays a central role in genetic typing of organisms or individuals and molecular epidemiology (Viljoen, G.J et al., 2005).
However, false positive results may be obtained if the PCR reaction is contaminated by living cells since the PCR can amplify both sequences from both living and non living cells. (Malorny et al., 2003)
Restricted fragment length polymorphism (RFLP):
" one of the most commonly used tools for following the inheritance of genes or gene markers is that of the linked Restriction Fragment Length Polymorphism(RFLP)"stated by M.J.McPherson, et al.,1991.
The main technique involve in RFLP is making use of enzymes to digest the targeted DNA into different small fragments. Separation of the fragments is done by gel electrophoresis and comparison is done using a probe.
In many epidemiological investigations, RFLP has been successfully used. However, to compare and distinguish between isolates of closely related strains, a greater amount of DNA sample cannot be used to have a maximum variation. (G.Roger, 2005).