Chapter 15: Listeria monocytogenes

• Potential Food Safety Hazard
• Control Measures
• FDA Guidelines
• Recommended Microbial Limits
• Caniadian Bacteriological Guidelines
• Growth
• Heat Resistance
• Analytical Procedures
• L. monocytogenes
• Other analytical procedures
• Commercial Test Products
• References

Contents

Potential Food Safety Hazard

The greatest threat of listeriosis is from ready-to-eat products that do not require further cooking at home. L. monocytogenes in raw food that will be cooked before consumption is less of a concern to the food industry since the bacteria are killed during cooking. L. monocytogenes has been isolated from raw fish, cooked crabs, raw and cooked shrimp, raw lobster, surimi and smoked fish (Ward et al., 1997).

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Control Measures

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FDA Guidelines

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Recommended Microbial Limits

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Growth

Contents

Heat Resistance

Table 15-3. Heat resistance of L. monocytogenes.

Temp.		D-Value	Medium	Reference
(ºC)	(ºF)	(min)
50	122	34.48	Blue crabmeat	Harrison and Huang, 1990
50	122	40.43	Blue crabmeat	Harrison and Huang, 1990
51.6	125	97.0	Lobster	Budu-Amoako et al., 1992
54.4	130	55.0	Lobster	Budu-Amoako et al., 1992
55	131	9.18	Blue crabmeat	Harrison and Huang, 1990
55	131	12.00	Blue crabmeat	Harrison and Huang, 1990
55	131	10.23	Crawfish  tail meat	Dorsa et al., 1993
56	132.8	48.09	Mussels, brine soaked	Bremer and Osborne, 1995
57.2	135	8.3	Lobster  meat	Budu-Amoako et al., 1992
58	136.4	16.25	Mussels, brine soaked	Bremer and Osborne, 1995
58	136.4	10.73	Salmon	Embarek, 1995
58	136.4	7.28	Cod	Embarek, 1995
59	138.2	9.45	Mussels, brine soaked	Bremer and Osborne, 1995
60	140	2.39	Lobster  meat	Budu-Amoako et al., 1992
60	140	1.31	Blue crabmeat	Harrison and Huang, 1990
60	140	2.61	Blue crabmeat	Harrison and Huang, 1990
60	140	1.98	Crawfish  tail meat	Dorsa et al., 1993
60	140	5.49	Mussels, brine soaked	Bremer and Osborne, 1995
60	140	4.48	Salmon	Embarek, 1995
60	140	1.98	Cod	Embarek, 1995
62	143.6	1.85	Mussels, brine soaked	Bremer and Osborne, 1995
62	143.6	2.07	Salmon	Embarek, 1995
62	143.6	0.87	Cod	Embarek, 1995
62.7	145	1.06	Lobster  meat	Budu-Amoako et al., 1992
65	149	0.19	Crawfish  tail meat	Dorsa et al., 1993
65	149	0.87	Salmon	Embarek, 1995
65	149	0.28	Cod	Embarek, 1995
68	154.4	0.15	Salmon	Embarek, 1995
68	154.4	0.15	Cod	Embarek, 1995
70	158	0.07	Salmon	Embarek, 1995
70	158	0.03	Cod	Embarek, 1995

Z-values: lobster meat 5.0ºc, blue crabmeat 8.40ºc in trypticase soy agar, crawfish tail meat 5.5ºc, mussels 4.25ºc, salmon 5.6ºc, cod 5.7ºc.

Contents

Analytical Procedures

Contents

L. monocytogenes (Hitchins, 1998)

Bergey's Manual of Determinative Bacteriology, 9th edition (Seeliger and Jones, 1986), lists eight species in the genus Listeria: L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii, L. grayi, L. murrayi, L. denitrificans. Of these, the species L. grayi and L. murrayi are considered here as subspecies of a single redefined species, L. grayi (Jones, 1992; Rocourt et al., 1992); L. denitrificans is now in the genus Jonesia (Jones and Seeglier, 1986). Thus, six species need to be differentiated (Table 15-4). Differentiation of the newly recognized subspecies of L. ivanovii is not needed in Listeria analysis (Boeolin et al., 1992). Brocothrix, which is closely related phylogenetically to Listeria, is distinguished by its inability to grow at 35ºC and its lack of motility. Distinguishing features of the Gram-positive asporogenic rods Erysipelothrix (Bille and Doyle, 1991) and Kurthia, which occur rarely in Listeria analysis, can be found in Bergey's manual (Seeliger and Jones, 1986).

In the methodology for isolating L. monocytogenes, suspected foods are sampled and the samples are composited, if required, according to compliance instructions. Analytical portions (25 g) are enriched for Listeria species in selective enrichment broth at 30ºC for 48 h. The enrichment culture is streaked at 24 and 48 h on two different, but complementary, differential selective agars to isolate Listeria species. Isolates are purified on nonselective agar and speciated directly by a battery of conventional tests or by such tests in kit form, or are preliminarily identified as members of the genus Listeria by genus specific rapid test ELISA or DNA probe kits. Alternatively, isolate cultures are rapidly speciated, within 1 h, as L. monocytogenes (or not L. monocytogenes) by a chemiluminescent labeled DNA probe kit or by equivalent L. monocytogenes specific DNA probes or probe kits. Serotyping, virulence testing, and enumeration of Listeria by direct plating on selective agar or by MPN enrichment and selection are optional methods.

In this version of the Listeria methodology several important modifications have been made:

The AOAC Official First Action enrichment formula for milk and dairy products (AOAC, 1995a) has been modified by increasing its buffering strength. Preliminary collaborative and other studies showed that the degree of buffering is not crucial in a 2 d (as opposed to a 7 d) enrichment when conventional isolation and identification techniques are used. Nevertheless, rapid DNA probe methods require more stringent control of pH to counteract effects of competitors. With this addition, the methodology is better positioned for future incorporation of rapid DNA probe and other methods that may be less sensitive than conventional cultural methodology. The nondairy food enrichment has also been modified to make it identical to the milk and dairy product enrichment by reducing the acriflavin concentration from 15 to 10 mg/ml Furthermore, the addition of sodium pyruvate and the delayed addition of selective agents until the fourth hour of enrichment will speed the recovery of damaged cells.

The differential selective agar PALCAM (Van Netten et al., 1989) has been introduced as a companion selective agar to Oxford agar (OXA), optionally replacing LPM plus esculin and Fe³⁺ or plain LPM as the required second selective agar. This substitution brings the methodology closer to that used outside the U.S. and decreases reliance on the delicate Henry illumination technique now used only optionally to confirm the purity of Listeria isolates. Other selective agar media are currently under consideration as alternative substitutes for LPM. The normal mouse pathogenicity test has been replaced by the more sensitive and slightly faster immunocompromised mouse pathogenicity test of Stelma et al. (1987).

Several rapid DNA probe and ELISA kits have been sanctioned for use in the generic or specific identification of Listeria isolates on selective isolation or purification agars. In line with the recent taxonomic evidence and the redefinition of L. grayi, use of the nitrate reduction test is now optional.

Table 15-4. Differentiation of Listeria species

Species	Hemolytic (b )a	Nitrate Reduced	Acid produced from			Virulent (mouse)
			Mannitol	Rhamnose	Xylose
L. monocytogenes	+	-	-	+	-	+
L. ivanovii	+	-	-	-	+	+
L. innocua	-	-	-	Vb	+	-
L. welshimeri	-	-	-	Vb	+	-
L. seeligeri	+	-	-	-	+	-
L. grayic	-	V	+	V	-	-

^aSheep blood stab
^bV, variable
^cL. grayi now includes the former nitrate-reducing, rhamnose variable species L. murrayi.

1. Equipment and materials
1. Balance for weighing sample
2. Cover slip, glass
3. Erlenmeyer flask, 500 ml
4. Fermentation tubes (Durham)
5. Grease pencil or magic marker
6. Incubators, 30 and 35ºC
7. Immersion oil
8. Inoculating loops
9. Inoculating needle
10. Microscope slides
11. Needle, 26 gauge, 3/8 inch (1 cm)
12. Phase contrast microscope with oil immersion phase objective (100X)
13. Petri plates
14. Pipets, 25, 10, and 1 ml
15. Tubes, 16 x 125 mm or other appropriate sizes, screw cap
16. Blender and jars or Stomacher and Stomacher bags
17. Tuberculin syringe, sterile, disposable
18. Dissecting or low power microscope with illuminator (Figure 15-1)
2. Media and reagents

NOTE: Alternative sources may be used when the products are equivalent.

1. Acetic acid, 5 N
2. Acriflavin HCl
3. Agar (Difco)
4. N-(1-naphthyl) ethylene diamine (R48)
5. a -naphthol reagent (R48)
6. Blood agar base No. 2 (Unipath)
7. Cycloheximide
8. Sheep blood, defibrinated; sheep blood agar (M135)
9. Ethanol, absolute
10. Fluorescent antibody (FA) buffer (Difco)
11. Glycine anhydride
12. Gram stain kit
13. Hydrogen peroxide solution, 3% for catalase test (R12)
14. KOH, 40% solution (R65)
15. Listeria typing sera set (Difco)
16. Lithium chloride-phenylethanol-moxalactam (LPM) agar (M81); LPM + esculin/iron (M82)
17. Nalidixic acid (sodium salt)
18. Nitrate broth (M108) and nitrite detection reagents (R48)
19. Nutrient broth (M114)
20. Physiological saline solution, 0.85% (R63)
21. Purple carbohydrate fermentation broth base (M130), containing 0.5% (w/v) solutions of dextrose, esculin, maltose, rhamnose, mannitol, and xylose
22. SIM medium (Becton-Dickinson Microbiology Systems:BDMS) (M137) or motility test medium (MTM, Difco) (M103)
23. Sulfanilic acid reagent (R48)
24. Trypticase soy agar with 0.6% yeast extract (TSAYE) (M153)
25. Trypticase soy broth with 0.6% yeast extract (TSBYE) (M157)
26. Tryptose broth and agar (Difco) (M167)
27. Oxford medium (OXA) (M118)
28. Enrichment broth (M52)
29. PALCAM agar (M118a)
30. Carrageenan (Sigma type II)
3. Enrichment procedure

See current compliance instructions for information about sampling plans and compositing in Listeria analysis. Be sure the sample represents the outer surface as well as the interior of the food. Refrigeration at 4ºC is recommended for handling, storing, and shipping materials to be analyzed for L. monocytogenes, which will grow, although slowly, at this temperature. However, if the laboratory sample is already frozen, it should be kept frozen until analysis.

Add 25 ml liquid or 25 g cream or solid test material to 225 ml enrichment broth (EB) without selective agents in blender or stomacher. Blend or stomach as required for thorough mixing. Place enrichment culture in blender jar or stomacher bag, or transfer to 500 ml Erlenmeyer flask; incubate 4 h at 30ºC, add selective agents acriflavin, nalidixic acid, and cycloheximide and continue incubating another 44 h, for a total of 2 d, at 30ºC.

4. Isolation procedure

At 24 and 48 h, streak EB culture onto both OXA (Curtis et al., 1989) and LPM (Lee and McClain, 1986) or LPM plus esculin/Fe³⁺ agars. PALCAM agar (Van Netten et al., 1989) may be substituted for LPM agars. Incubate OXA and PALCAM plates (use of a CO₂-air atmosphere is optional) at 35ºC for 24-48 h, and LPM plates at 30ºC for 24-48 h. Examine LPM plates for suspect colonies by using beamed white light powerful enough to illuminate plate well, striking plate bottom at 45º angle (Figure 15-1). When examined in this oblique transmitted light (Henry illumination) from an eye position directly above the plate (i.e., at 90º to the plate) either directly or via low power microscope or dissecting microscope (with mirror detached), Listeria spp. colonies on LPM agar appear sparkling blue (bluish crushed glass) or white. The use of positive and negative control colonies (not the test plate) to attune the observer's eyes is strongly recommended. Although components of the optical systems used may vary, the important points are the 45º angle of incident light and the 90º angle of emergent light.

Esculin and ferric iron salt may be added to LPM (M82) to eliminate the need for Henry illumination. In this case, proceed as with OXA isolates. With OXA, the Henry illumination method is inapplicable because on OXA, Listeria colonies have a black halo. Certain other bacteria can form black haloes, but color development takes longer than 2 d. Listeria behaves similarly on OXA and PALCAM.

Transfer 5 or more typical colonies from OXA and PALCAM or LPM to TSAYE, streaking for purity and typical isolated colonies. Purification on TSAYE is mandatory in the conventional analysis because isolated colonies on OXA, PALCAM, and LPM may still be in contact with an invisible weak background of partially inhibited competitors. At least 5 isolates are necessary because more than one species of Listeria may be isolated from the same sample. Incubate TSAYE plates at 30ºC for 24-48 h. The plates may be incubated at 35ºC if colonies will not be used for a wet mount motility observation (see 5-b, below).

5. Identification procedure

Identify purified isolates by the following classical tests (5, a-j). Rapid kits are available to facilitate biochemical testing to genus or species level (see 5-k and 5-l).

1. Examine TSAYE plates for typical colonies. With the oblique Henry illumination system, already described, colonies appear blue-gray to blue. The use of known controls on TSAYE is recommended.
2. Pick typical colony from culture plate incubated at 30ºC or lower and examine in a wet mount, using 0.85% saline for suspending medium, with the oil immersion objective of a phase contrast microscope. Choose a colony with enough growth to make a fairly heavy suspension; emulsify thoroughly. If too little growth is used, the few cells present will stick to the glass slide and appear nonmotile. Listeria spp. are slim, short rods with slight rotating or tumbling motility. Always compare with known culture. Cocci, large rods, or rods with rapid, swimming motility are not Listeria spp. Alternatively, use the 7 d motility test medium (see 5-i).
3. Test typical colony for catalase. Listeria species are catalase positive.
4. Gram stain 16-24 h cultures. All Listeria spp. are short, Gram positive rods; however, with older cultures the Gram stain reaction can be variable and cells may appear coccoidal. The cells have a tendency to palisade in thick stained smears, leading to false rejection as a diphtheroid.
5. Pick typical colony to a tube of TSBYE for inoculating carbohydrate fermentation and other test media. Incubate at 35ºC for 24 h. This culture may be kept at 4ºC several d and used repeatedly as inoculum.
6. Inoculate heavily (from TSAYE colony) 5% sheep blood agar (or horse blood agar) by stabbing plates that have been poured thick and dried well (check for moisture before using). Draw grid of 20-25 spaces on plate bottom. Stab one culture per grid space. Always stab positive controls (L. ivanovii and L. monocytogenes) and negative control (L. innocua). Incubate for 48 h at 35ºC. Stabs should pass right through the agar layers. Try to avoid hitting bottom of plate too hard and fracturing agar.
7. Examine blood agar plates containing culture stabs with bright light. L. monocytogenes and L. seeligeri produce slightly cleared zone around stab. L. innocua shows no zone of hemolysis, whereas L. ivanovii produces well defined clear zone around stab. Do not try to differentiate species at this point, but note nature of hemolytic reaction. Resolve doubtful reactions by the CAMP test.
8. Nitrate reduction test (optional). For this test, use TSBYE culture to inoculate nitrate broth (M108). Incubate at 35ºC for 5 d. Add 0.2 ml reagent A, followed by 0.2 ml reagent B (R48). A red violet color indicates presence of nitrite, i.e., nitrate has been reduced. If no color develops, add powdered zinc and let stand 1 h. A developing red violet color indicates that nitrate is still present and has not been reduced. Only L. grayi ssp. murrayi reduces nitrates, and the test is only necessary to distinguish it from L. grayi ssp. grayi.

As an alternative procedure (R48), add 0.2 ml reagent A followed by 0.2 ml reagent C. An orange color indicates reduction of nitrate. If no color develops, add powdered zinc as above. Development of an orange color indicates unreduced nitrate.

9. Inoculate SIM or MTM from TSBYE. Incubate for 7 d at room temperature. Observe daily. Listeria spp. are motile, giving a typical umbrella-like growth pattern. MTM gives better defined umbrellas. Alternatively, observe 30ºC TSBYE cultures by phase contrast microscopy (1000X) for tumbling motility.
10. From TSBYE culture, inoculate the following carbohydrates as 0.5% (w/v) solutions in purple carbohydrate broth (the use of Durham tubes is optional): dextrose, esculin, maltose, rhamnose, mannitol, and xylose. Incubate 7 d at 35ºC. Positive reacting Listeria spp. produce acid with no gas. Consult Table 15-4 for xylose rhamnose reactions of Listeria spp. All species should be positive for dextrose, esculin, and maltose. All Listeria spp. except L. grayi should be mannitol negative. If pigmentation of the isolate on OXA, PALCAM or LPM plus esculin/Fe³⁺ is unequivocal, the esculin test may be omitted.
11. Purified isolates can be rapidly identified by conventional tests (additional tests may be needed to speciate completely) using commercial kits: 20 S^TM or API-ZYM (Analytical Products, Plainview, NY); Vitek Automicrobic Gram Positive and Gram Negative Identification cards (BioMerieux, Hazelwood, MO); or API Listeria (BioMerieux sa Marcy-l'Etoile, France), which does not require an additional CAMP test. The MICRO-ID^TM kit (Organon Teknika Corp., Durham, NC) permits speciation of Listeria isolates if their CAMP reactions are known. AOAC INTERNATIONAL, Gaithersburg, MD, has adopted the Micro-ID and the Vitek Automicrobic System as Official First Action Methods.
12. Several nonconventional rapid methods are available for identifying Listeria isolates to genus or species (L. monocytogenes) level. These methods, particularly the L. monocytogenes specific methods, accelerate identification. Isolates in pure broth culture may be identified to genus level by using commercial ELISA kits (Organon; Bioenterprises Pty Ltd, Roseville, NSW, Australia) or nonradiolabeled DNA probe kits (GeneTrak, Framingham, MA) (Klinger et al., 1988; Mattingly et al., 1988). If such kits are used to screen enrichment cultures for Listeria spp., cultures should still be streaked on selective agars regardless of screening results.

Nonradioactive DNA probe tests specific for identification of L. monocytogenes at the isolation and purification culture steps are available (Datta et al., 1987; Datta et al., 1991; Peterkin, 1991; Merker, 1998, Chapter 24). These methods are highly recommended, but with the indicated provisos. Identification of colonies from isolation or purification agar or in pure broth cultures with the Accuprobe^TM (Gen-Probe, Inc., San Diego, CA) Listeria culture confirmation test (Alden et al., 1990) or the L. monocytogenes assay (GeneTrak) is recommended. Culture-positive enrichments will sometimes test positive with these kits, especially at 48 h. These rapid nonradioactive probe tests are specific for L. monocytogenes. Purified isolates identified as L. monocytogenes should be retained for regulatory reference.

6. Serology (optional)

Table 15-5 shows serological relationships of Listeria spp. Most L. monocytogenes isolates obtained from patients and the environment are type 1 or 4, and more than 90% can be serotyped with commercially available sera. All nonpathogenic species, except L. welshimeri, share one or more somatic antigens with L. monocytogenes. Serotyping alone without thorough characterization, therefore, is not adequate for identification of L. monocytogenes.

Table 15-5. Serology of Listeria species

Species	Serotypes
L. monocytogenes	1/2A, 1/2B, 1/2 C, 3A, 3B, 3C, 4A, 4AB, 4B, 4C, 4D, 4E, "7"
L. ivanovii	5.00
L. innocua	4AB, 6A, 6B, Una
L. welshimeri	6A, 6B
L. seeligeri	1/2B, 4C, 4D, 6B, Un

^aUn, undefined

Serology is useful when epidemiological considerations are crucial. Use TSBYE culture to inoculate tryptose broth. Make 2 successive transfers of cultures incubated in tryptose broth for 24 h at 35ºC. Make a final transfer to 2 tryptose agar slants and incubate 24 h at 35ºC. Wash both slants in a total of 3 ml Difco fluorescent antibody (FA) buffer and transfer to sterile 16 x 125 mm screw cap tube. Heat in water bath at 80ºC for 1 h. Centrifuge at 1600 x g for 30 min. Remove 2.2-2.3 ml of supernatant fluid and resuspend pellet in remainder of buffer. Follow manufacturer's recommendations for sera dilution and agglutination procedure. If flagellar (H) and sub-factor (O) serotyping is required, see Merker (1998), Chapter 11 on serological methods. Phage typing, DNA restriction fragment analysis, and enzyme allele analysis are also very helpful in epidemiological studies of listeriosis outbreaks.

7. Immunocompromised mouse pathogenicity (optional)

The classical tests for Listeria pathogenicity are the Anton conjunctivitis test (rabbits) and inoculation of mice and of embryonated eggs (Bille and Doyle, 1991). An immunocompromised mouse test, using intraperitoneal (i.p.) injection is recommended because of its greatly improved sensitivity (Stelma et al., 1987). Confirmation of L. monocytogenes animal pathogenicity is not routine for clinical isolates and is optional for regulatory isolates. An isolate should be identified as L. monocytogenes if it meets all the other criteria outlined in this chapter.

Carrageenan (Sigma type II) dissolved in distilled water (40 mg/ml) is injected i.p. into 18-20 g mice (200 mg/kg) 24 h before the Listeria challenge. Grow isolate for 24 h at 35ºC in TSBYE. Transfer to 2 tubes of TSBYE for another 24 h at 35ºC. Place a total of 10 ml culture broth from both tubes into 16 x 125 mm tube and centrifuge at 1600 x g for 30 min. Discard supernatant and resuspend pellet in 1 ml phosphate buffered saline (PBS). This suspension will contain approximately 10¹⁰ bacteria/ml; dilute to 10⁵ bacteria/ml and determine actual concentration by a pour or spread plate count. Inject (i.p.) 16-18 g immunocompromised Swiss white mice (5 mice/culture) with 0.1 ml of the concentrated suspension, i.e., about 10⁴ bacteria per mouse. Observe for death over 5 d period. Nonpathogenic strains will not kill, but 10⁴ pathogenic cells will kill, usually within 3 d. Use known pathogenic and nonpathogenic strains and Carrageenan treated uninoculated mice as controls. Use 5 mice per control group. Carrageenan controls should be challenged with 0.1 ml PBS.

8. CAMP test

The Christie-Atkins-Munch-Peterson (CAMP) test (Table 15-6 and Figure 15-2) is useful in confirming species, particularly when blood agar stab test results are equivocal. To perform the test, streak a b -hemolytic Staphylococcus aureus and a Rhodococcus equi culture in parallel and diametrically opposite each other on a sheep blood agar plate. Streak several test cultures parallel to one another, but at right angles to and between the S. aureus and R. equi streaks. After incubation at 35ºC for 24-48 h, examine the plates for hemolysis. Hemolysis is more easily read when the blood agar is thinner than usual. The L. monocytogenes reaction is often optimal at 24 h rather than 48 h. To obtain enough R. equi to give a good streak of growth, incubate the inoculum slant culture longer than 24 h. Use of known control Listeria spp. on a separate sheep blood agar plate is recommended. Sheep blood agar plates should be as fresh as possible.

Streak weakly b -hemolytic S. aureus FDA strain ATCC 49444 (CIP 5710; NCTC 7428) or strains ATCC 25923 and R. equi (ATCC 6939; NCTC 1621) vertically on sheep blood agar. Separate vertical streaks so that test strains may be streaked horizontally between them without quite touching them. After 24- and 48-h incubation at 35ºC, examine plates for hemolysis in the zone of influence of the vertical streaks. CAMP test cultures are available from several national culture collections, including the American Type Culture Collection (ATCC), Rockville, MD.

Figure 15-2 shows arrangement of streak cultures on a CAMP plate. Hemolysis of L. monocytogenes and L. seeligeri is enhanced near S. aureus streak; L. ivanovii hemolysis is enhanced near R. equi streak. Other species are nonhemolytic and do not react in this test. The CAMP test differentiates L. ivanovii from L. seeligeri and can differentiate a weakly hemolytic L. seeligeri (that may have been read as nonhemolytic) from L. welshimeri. Isolates giving reactions typical for L. monocytogenes except for hemolysin production should be CAMP tested before they are identified as nonhemolytic L. innocua. A factor easily prepared from S. aureus cultures may be used to enhance hemolysis by L. monocytogenes and L. seeligeri in sheep blood agar plates. b -lysin disks (REMEL, Lenexa, KS) may be used for the same purpose.

Table 15-6. CAMP test reactions of Listeria species.

Species	Hemolytic interaction
	S. aureus (S)	R. equi (R)
L. monocytogenes	+	-*
L. ivanovii	-	+
L. innocua	-	-
L. welshimeri	-	-
L. seeligeri	+	-

*Rare strains are S+ and R+, but the R+ reaction is much less pronounced than that of L. ivanovii.

9. Interpretation of analyses data for speciation

The importance of completely characterizing each isolate cannot be overemphasized. Partial characterization, even if accurate, may be misleading. Since all Listeria spp. are negative for indole, oxidase, urease, and H₂S production, and are positive for methyl red and Voges-Proskauer, these tests are discretionary.

All Listeria spp. are small, catalase positive, Gram positive rods that are motile in wet mounts and in SIM. They utilize dextrose, esculin, and maltose; some species utilize mannitol, rhamnose, and xylose with production of acid. L. grayi utilizes mannitol with acid production. L. monocytogenes, L. ivanovii, and L. seeligeri produce hemolysis in sheep blood stabs and consequently are CAMP test positive. Of the three, only L. monocytogenes fails to utilize xylose and is positive for rhamnose utilization. The difficulty in differentiating L. ivanovii from L. seeligeri can be resolved by the CAMP test. L. seeligeri shows enhanced hemolysis at the S. aureus streak. L. ivanovii shows enhanced hemolysis at the R. equi streak. Of the nonhemolytic species, L. innocua may give the same rhamnose xylose reactions as L. monocytogenes but is negative in the CAMP test. L. innocua is sometimes negative for utilization of both rhamnose and xylose. L. welshimeri, which is rhamnose negative, may be confused with a weakly hemolytic L. seeligeri unless resolved by the CAMP test.

After all other results are available, the serotyping of Listeria isolates becomes meaningful. Biochemical, serological, and pathogenicity data are summarized in Tables 15-4 to 15-6. All data collection must be completed before species are determined.

For bacteriophage typing of L. monocytogenes isolates, contact R.E. Haymond, FDA, Denver, CO 80225. To identify Listeria isolates (with known hemolytic reactions) by their fatty acid profiles, contact L.L. English, FDA, Baltimore, MD 21201.

10. Enumeration (optional)

Contents

Other analytical procedures

• Listeria monocytogenes in Milk and Dairy Products: Selective Enrichment and Isolation Method (AOAC, 1995a)
• Listeria Species: Biochemical Identification Method (Vitek GPI and GNI) (AOAC, 1995b)
• Listeria in Dairy Products, Seafoods, and Meats: Colorimetric Deoxyribonucleic Acid-hybridization Method (GENE-TRAK Listeria Assay) (AOAC, 1995c)
• Listeria monocytogenes in Dairy Products, Seafoods, and Meats: Colorimetric Monoclonal Enzyme-linked Immunosorbent Assay Method (AOAC, 1995d)
• Listeria in Foods, Colorimetric Polyclonal Enzyme Immunoassay Screening Method (TECRA Listeria Visual Immunoassay) (AOAC, 1995e)
• Listeria Species: Biochemical Identification Method (MICRO-ID Listeria) (AOAC, 1995f)

Contents

Commercial Test Products

Contents

Other analytical procedures

• Listeria monocytogenes in Milk and Dairy Products: Selective Enrichment and Isolation Method (AOAC, 1995a)
• Listeria Species: Biochemical Identification Method (Vitek GPI and GNI) (AOAC, 1995b)
• Listeria in Dairy Products, Seafoods, and Meats: Colorimetric Deoxyribonucleic Acid-hybridization Method (GENE-TRAK Listeria Assay) (AOAC, 1995c)
• Listeria monocytogenes in Dairy Products, Seafoods, and Meats: Colorimetric Monoclonal Enzyme-linked Immunosorbent Assay Method (AOAC, 1995d)
• Listeria in Foods, Colorimetric Polyclonal Enzyme Immunoassay Screening Method (TECRA Listeria Visual Immunoassay) (AOAC, 1995e)
• Listeria Species: Biochemical Identification Method (MICRO-ID Listeria) (AOAC, 1995f)

Contents

References

Alden, M.J., L. Marconi, J. Hogan, I.G. Rosen, and R. Johnson. 1990. A chemiluminescent DNA probe assay for the identification of Listeria monocytogenes from culture plates. Abstr. ICAAC 1990:109.

AOAC. 1995a. Listeria monocytogenes in milk and dairy products: Selective enrichment and isolation method. Sec. 17.10.01, Method 993.12. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), p. 94a-98. AOAC International, Gaithersburg, MD.

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