Fish Microbiology and Quality Assurance (2+2)

9. Isolation and identification of Listeria monocytogenes from seafoods

Listeria are Gram-positive, non-spore forming, catalase-positive, microaerophilic rods. They are motile at room temperature, but not at 37oC. Among the 6 species included under this genus, L. monocytogenes, L. ivanovii and L. seeligeri are considered as pathogens to man and animals. L. monocytogenes is a foodborne pathogen and occurs commonly in water, sediment, foods, etc. Pathogenic L. monocytogens is also found in seafoods and are known to cause meningitis, encephalitis, abscesses, abortion and death. It is differentiated from the nonpathogenic species by biochemical and virulence tests and serotyping. All virulent strains produce b-hemolysis on blood agar, produce a toxin called listeriolysin and the disease caused by L. monocytogenes is called as listeriosis. L. monocytogenes should be absent in fish and fishery products.

Materials required

1. Trypticase Soya Broth (TSB) + 0.6% Yeast extract
2. University of Vermont (UVM) Broth
3. Modified Fraser Broth (MFB)
4. Polymyxin-Acriflavine-Lithium chloride – Ceftazidime – Aseculin – Mannitol (PALCAM) agar
5. Columbia blood agar base
6. Nitrate broth
7. Carbohydrate fermentation broth base containing 0.5% solutions of glucose, esculin, mannitol, salicin, maltose, xylose and rhamnose
8. Triple Sugar Iron (TSI) agar
9. Urea agar
10. Glucose phosphate broth
11. Hydrogen peroxide
12. Polymyxin B
13. Aeriflavine hydrochloride
14. Ceftazidime
15. Nalidixic acid
16. Modified McBride’s agar (MMA)
17. Cycloheximide

Procedure
Isolation of L. monocytogenes involves pre and selective enrichment followed by subculturing enriched broth culture on selective media. Characteristic colonies developing on selective media are confirmed as L.monocytogenes based on the results of biochemical tests. About 25 g of fish sample is aseptically homogenised in 225 ml of TSB + 0.6% Yeast extract and incubated at 37^oC for 24 hours for pre-enrichment. 10 ml of pre-enriched sample is transferred to 90 ml of UVM broth and incubated at 37^oC for 24 hours for primary enrichment. Following this, 0.1 ml of enriched sample is transferred to 10ml of Modified Fraser Broth (MFB) and incubated at 37^oC for 24 hours for secondary enrichment. A loopful of secondary enriched sample is streaked onto the PALCAM agar or MMA plates and incubated at 37^oC for 24 to 48 hours. After incubation, the plates are observed for the following colony morphology:
On PALCAM agar plates: Green or black coloured colonies with a sunken centre and a black halo on a cherry red background.
On Modified McBride’s agar (MMA) plates: Appear as small (less than 2 mm) bluish grey or blue colonies.
Suspected typical colonies are purified onto TSA plates and further subjected to the following biochemical tests for confirmation:

Reactions of L. monocytogenes

Tests	Reactions	Results
Catalase Oxidase Gram reaction Motility at 37oC Motility at room temperature TSI reaction Urease MR VP Nitrate reduction Glucose Esculin Salicin Rhamnose Maltose Mannitol Xylose b-hemolysis CAMP reaction with Staphylococcus aureus	Effervescence No colour change Purple Non-motile Motile A/A No colour change Magenta red Portwine colour No colour change Acid; gas Acid Acid Acid Acid No acid No acid Haemolytic Haemolytic	Positive Negative Positive Negative Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive

Media composition
University of Vermont (UVM) Broth

Proteose peptone : 5.0 g
Tryptone : 5.0 g
Beef extract : 5.0 g
Yeast extract : 5.0 g
Sodium chloride : 20.0 g
Monopotassium dihydrogen phosphate: 1.35 g
Disodium hydrogen phosphate : 12.0 g
Esculin : 1.0 g
Distilled water :1000.0 ml
pH : 7.4 ± 0.2

Sterilize at 121^oC for 15 minutes, cool to 50⁰C, add filter sterilized Nalidixic acid (20 mg) and Acriflavine HCl (12mg) and mix well.

Modified Fraser Broth (MFB)

UVM : 52.0 g
Lithium chloride : 3.0 g
Ferric ammonium citrate : 0.5 g
Sodium chloride : 20.0 g
Acriflavine HCl : 0.025 g
Distilled water : 1000.0 ml
pH : 7.4 ± 0.2

Sterilize at 121^oC for 15 minutes, cool to 50⁰C, add filter sterilized Acriflavine HCl (25mg) and mix well.

Polymyxin Acriflavine Lithium chloride, Ceftazidime, Aesculin Mannitol (PALCAM) Agar

Peptone : 23.0 g
Starch : 1.0 g
Lithium chloride : 15.0 g
Aesculin : 0.8 g
Ammonium Ferric citrate : 0.5 g
Sodium chloride : 5.0 g
Mannitol : 10.0 g
Dextrose : 0.5 g
Phenol red : 0.08 g
Agar : 13.0 g
Distilled water : 1000.0 ml
pH : 7.0 ± 0.2

Sterilize at 121^oC for 15 minutes, cool to 50⁰C and add filter sterilized Polymyxin B (10mg), Ceftazidime (20mg) and Acriflavine HCl (5 mg). Mix well and pour into plates.

PALCAM medium allows diagnosis of Listeria spp. by their ability to hydrolyze aesculin. Inclusion of a ferric salt in the medium leads to the formation of black complex. In addition, PALCAM contains mannitol and phenol red. L. monocytogenes does not ferment mannitol and can easily be differentiated from those organisms, which hydrolyse aesculin and also ferment mannitol, thereby giving rise to a change of colour in the medium. Lithium chloride and ceftazidime suppresses Enterococcus spp., an occasional strain of Micrococcus and ceftazidime inhibits the other common commensals of fresh seafoods, chicken, pork and soft cheeses.

Modified McBride’s Agar (MMA)

Phenylethanol - 2.5 g
Glycine anhydride - 10.0 g
Lithium chloride - 0.5 g
Sodium chloride - 5.0 g
Tryptone - 10.0 g
Beef extract - 3.0 g
Agar - 15.0 g
Distilled water - 1000.0 ml
pH - 7.3 ± 0.2

Sterilize at 121^oC for 15 minutes, cool to 50⁰C and add 200 mg cycloheximide, and then pour into plates after mixing.