Chapter 21: Yersinia enterocolitica

• Potential Food Safety Hazard
• Control Measures
• FDA Guidelines
• Growth
• Heat Resistance
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• Y. enterocolitica and Y. pseudotuberculosis
• References

Contents

Potential Food Safety Hazard (Weagant et al., 1998)

Yersinia enterocolitica and bacteria that resemble it are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can grow at refrigeration temperatures (a strong argument for use of cold enrichment). Based on their biochemical heterogeneity and DNA relatedness, members of this group were separated into four species: Y. enterocolitica, Y. intermedia, Y. frederiksenii, and Y. kristensenii (Bercovier et al., 1980). Through additional revisions, the genus Yersinia has grown to include eleven species (Aleksic et al., 1987; Bercovier, 1980; Bercovier et al., 1984; Wauters et al., 1988), three of which are potentially pathogenic to humans: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Of these, Y. enterocolitica is most important as a cause of food-borne illness.

Y. enterocolitica strains and related species can be separated serologically into groups based on their heat-stable somatic antigens. Wauters (Wauters, 1981) described 54 serogroups for Y. enterocolitica and related species. Aleksic and Bockemuhl (1984) proposed simplifying this to 18 serogroups within the Y. enterocolitica species. Presently, pathogenic strains belonging to serogroups O:1, 2a, 3; O:2a,3; O:3; O:8; O:9; O:4,32; O:5,27; O:12,25; O:13a,13b; O:19; O:20; and O:21 have been identified. Therefore, pathogenic strains can belong to diverse serogroups. Serogroups that predominate in human illness are O:3, O:8, O:9, and O:5,27.

The association of human illness with consumption of Y. enterocolitica-contaminated food, animal wastes, and unchlorinated water is well documented (Aulisio et al., 1982; Aulisio, 1983). Refrigerated foods are potential vehicles because contamination is possible at the manufacturing site (Aulisio, 1982) or in the home (Aulisio, 1983). This organism may survive and grow during refrigerated storage.

A number of virulence tests have been proposed to distinguish potentially pathogenic Y. enterocolitica. Some strains of Y. enterocolitica and related species produce an in vitro heat-stable enterotoxin (ST) that can be detected by intragastric injection of cultural filtrates in suckling mice and is very similar to Escherichia coli ST (Boyce et al., 1979). However, Yersinia spp. produce ST only at temperatures below 30ºC. Many environmental strains of Yersinia produce this protein, whereas some otherwise fully virulent strains of Y. enterocolitica do not. The role of ST in the disease process of Yersinia remains uncertain.

Yersinia spp. that cause human yersiniosis carry a plasmid (41-48 Mdal) (Gemski et al., 1980; Kay et al., 1982; Zink et al., 1980) that is associated with a number of traits related to virulence: autoagglutination in certain media at 35-37ºC (Aulisio et al., 1983; Laird and Cavanaugh, 1980); inhibition of growth in calcium-deficient media (Gemski et al., 1980) and binding of crystal violet dye (Bhaduri et al., 1987) at 35-37ºC; increased resistance to normal human sera (Pai and DeStephano, 1982); production of a series of outer membrane proteins at 35-37ºC (Portnoy et al., 1981); ability to produce conjunctivitis in guinea pig or mouse (Sereny test) (Sereny, 1955; Zink et al., 1980); and lethality in adult and suckling mice by intraperitoneal (i.p.) injection of live organisms (Aulisio et al., 1983; Carter and Collins, 1974; Prpic et al., 1985; Robins-Brown and Prpic, 1985). The plasmid associated with virulence can be detected by gel electrophoresis or DNA colony hybridization (Hill et al., 1983). Recent evidence, however, indicates that presence of plasmid alone is not sufficient for the full expression of virulence in Yersinia (Heesemann et al., 1984; Portnoy and Martinez, 1985; Schiemann, 1989). The intensity of some plasmid-mediated virulence properties such as mouse lethality and conjunctivitis is variable, depending on the genes carried on the bacterial chromosome (Pai and DeStephano, 1982; Pierson and Falkow, 1990; Portnoy et al., 1981; Robins-Brown et al., 1989) and the serogroup, suggesting that chromosomal genes also contribute to Yersinia virulence.

Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture (Lee et al., 1977). However, strains that have lost other virulent properties retain HeLa invasiveness, because the invasive phenotype for mammalian cells is encoded by chromosomal loci. Two chromosomal genes of Y. enterocolitica, inv and ail, which encode the phenotype for mammalian cell invasion, have been identified (Miller and Falkow, 1988; Miller et al., 1989). Transfer of these genetic loci into E. coli confers the invasive phenotype to the E. coli host (Miller and Falkow, 1988). The inv gene allows high level Yersinia invasion of several tissue culture cell lines (Miller and Falkow, 1988). However, Southern blot analyses show that inv gene sequences are present on both tissue culture invasive and noninvasive isolates (Miller et al., 1989; Robins-Brown et al., 1989). Although this suggests that the inv gene in Y. enterocolitica may not be directly correlated with invasiveness, genetic evidence shows that inv genes are nonfunctional in the noninvasive isolates (Pierson and Falkow, 1990). The ail gene shows greater host specificity with regard to cell invasion and appears to be present only on pathogenic Yersinia. In disease-causing strains, all virulent Y. enterocolitica isolates were shown to be tissue culture-invasive and to carry the ail gene (Miller and Falkow, 1988; Portnoy et al., 1981). The ail locus, therefore, may be an essential chromosomal virulence factor in Y. enterocolitica (Miller et al., 1989; Robins-Brown et al., 1989).

Y. pseudotuberculosis is less ubiquitous than Y. enterocolitica, and although frequently associated with animals, has only rarely been isolated from soil, water, and foods (Fukushima et al., 1989; Tsubokura et al., 1989). Among Y. pseudotuberculosis strains there is little or no variation in biochemical reactions, except with the sugars melibiose, raffinose, and salicin. Serologically (based on a heat-stable somatic antigen), the Y. pseudotuberculosis strains are classified into six groups, each serogroup containing pathogenic strains. Gemski et al. (1980) reported that serogroup III strains harbor a 42-Mdal plasmid as do serogroup II strains that are lethal to adult mice. The association of yersiniosis in humans with the presence of a 42-Mdal plasmid in Y. pseudotuberculosis has been established (Schiemann and Wauters, 1992).

Virulence genes present on the chromosome of Y. pseudotuberculosis have also been identified (Isberg et al., 1987; Isberg and Falkow, 1985). The inv gene of Y. pseudotuberculosis is homologous with that of Y. enterocolitica, and encodes for an invasion factor for mammalian cells. Transfer of inv gene into E. coli K-12 resulted in the expression of the invasive phenotype in E. coli (Isberg and Falkow, 1985). The inv gene is thermoregulated (Isberg et al., 1988; Isberg, 1989); it encodes for a 103 Kdal protein, invasin, which binds to specific receptors on mammalian cells and facilitates the entry of Y. pseudotuberculosis into tissue (Isberg and Leong, 1988). Tests for Y. pseudotuberculosis virulence are not as abundant as those for Y. enterocolitica; however, tissue cell-invasive and plasmid-carrying isolates of Y. pseudotuberculosis may be identified by DNA colony hybridization.

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Control Measures

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FDA Guidelines

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Growth

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Heat Resistance

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Analytical Procedures

Contents

Food Sampling and Preparation of Sample Homogenate (Andrews and June, 1998)

The adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard.

The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the collector at the time of sampling by using the Investigations Operation Manual (FDA, 1993). Sampling and sample plans are discussed in detail in ICMSF (1986).

Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment.

Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. Whenever possible, avoid glass containers, which may break and contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Take care not to overfill bags or permit puncture by wire closure. Identify each sample unit (defined later) with a properly marked strip of masking tape. Do not use a felt pen on plastic because the ink might penetrate the container. Whenever possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile containers with the sample.

Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in pre-chilled containers.

Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4ºC and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4ºC until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection. Special conditions apply to the collection and storage of shucked, unfrozen shellfish and shell stock (APHA, 1985). Pack samples of shucked shellfish immediately in crushed ice (no temperature specified) until analyzed; keep shell stock above freezing but below 10ºC. Examine refrigerated shellfish and shell stock within 6 h of collection but in no case more than 24 h after collection. Further details on sample handling and shipment may be found in the Investigations Operation Manual (FDA, 1993) and the Laboratory Procedures Manual (FDA, 1989). The Investigations Operation Manual (FDA, 1993) contains sampling plans for various microorganisms. Some of those commonly used are presented here.

1. Sampling plans
1. Aerobic plate counts, total coliforms, fecal coliforms, Escherichia coli (including enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp., Campylobacter spp., Yersinia spp., Bacillus cereus, and Clostridium perfringens
1. Sample collection. From any lot of food, collect ten 8 ounce (227 g) subsamples (or retail packages) at random. Do not break or cut larger retail packages to obtain an 8 ounce (227 g) subsample. Collect the intact retail unit as the subsample even if it is larger than 8 ounce (227 g).
2. Sample analysis. Analyze samples as indicated in current compliance programs.
2. Equipment and materials
1. Mechanical blender. Several types are available. Use blender that has several operating speeds or rheostat. The term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at 10,000-12,000 rpm or with equivalent shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring blender, or equivalent, meets these requirements.
2. Sterile glass or metal high-speed blender jar. 1000 ml, with cover, resistant to autoclaving for 60 min at 121ºC.
3. Balance, with weights. 2000 g capacity, sensitivity of 0.1 g.
4. Sterile beakers. 250 ml, low-form, covered with aluminum foil.
5. Sterile graduated pipets. 1.0 and 10.0 ml.
6. Butterfield's phosphate-buffered dilution water (R11). Sterilized in bottles to yield final volume of 90 ± 1 ml.
7. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue depressors. For sample handling.
3. Receipt of samples
1. The official food sample is collected by the FDA inspector or investigator. As soon as the sample arrives at the laboratory, the analyst should note its general physical condition. If the sample cannot be analyzed immediately, it should be stored as described later. Whether the sample is to be analyzed for regulatory purposes, for investigation of a foodborne illness outbreak, or for a bacteriological survey, strict adherence to the recommendations described here is essential.
2. Condition of sampling container. Check sampling containers for gross physical defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture marks. If sample units were collected in plastic bottles, check bottles for fractures and loose lids. If plastic bags were used for sampling, be certain that twist wires did not puncture surrounding bags. Any cross-contamination resulting from one or more of above defects would invalidate the sample, and the collecting district should be notified (see 3-e, below).
3. Labeling and records. Be certain that each sample is accompanied by a completed copy of the Collection Report (Form FD-464) and officially sealed with tape (FD-415a) bearing the sample number, collecting official's name, and date. Assign each sample unit an individual unit number and analyze as a discrete unit unless the sample is composited as described previously in this chapter.
4. Adherence to sampling plan. Most foods are collected under a specifically designed sampling plan in one of several ongoing compliance programs. Foods to be examined for Salmonella, however, are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. Depending on the food and the type of analysis to be performed, determine whether the food has been sampled according to the most appropriate sampling plan.
5. Storage. If possible, examine samples immediately upon receipt. If analysis must be postponed, however, store frozen samples at -20°C until examination. Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. Store nonperishable, canned, or low-moisture foods at room temperature until analysis.
6. Notification of collecting district. If a sample fails to meet the above criteria and is therefore not analyzed, notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced.
4. Thawing

Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5ºC within 18 h. If rapid thawing is desired, thaw the sample at less than 45ºC for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath.

5. Mixing

Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. If contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test.

6. Weighing

Tare high-speed blender jar; then aseptically and accurately (± 0.1 g) weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Total volume in blender must completely cover blades.

7. Blending and diluting of samples requiring enumeration of microorganisms
1. All foods other than nut meat halves and larger pieces, and nut meal. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and blend 2 min. This results in a dilution of 10^-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25 times in 30 cm (1 foot) arc in 7 s. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media.
2. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into sterile screw-cap jar. Add 50 ml diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10⁰ dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.
3. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add 90 ml of diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10^-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.

Contents

Y. enterocolitica and Y. pseudotuberculosis

1. Equipment and materials
1. Incubators, maintained at 10 ± 1ºC and 35-37ºC
2. Blender, Waring or equivalent, 8000 rpm, with 500 ml-1 liter jar
3. Sterile glass or plastic petri dishes, 15 x 100 mm
4. Microscope, light 900X and illuminator
5. Sterile syringes, 1 ml; 26-27 gauge needle
6. Disposable borosilicate tubes, 10 x 75 mm; 13 x 100 mm
7. Wire racks to accommodate 13 x 100 mm tubes
8. Vortex mixer
9. Mouse racks and mouse cages
10. Laminar flow animal isolator
11. Anesthetizing jar
12. CO₂, compressed
13. Minitek (BBL, Division of Bioquest, Cockeysville, MD). System includes disk dispenser, humidor, pipet and tips, Minitek 20-well plates, and color comparator.
2. Media

Codes, e.g., "M27" refer to media recipes in the FDA Bacteriological Analytical Manual (Merker, 1998).

1. Peptone sorbitol bile broth (PSBB) (M120)
2. MacConkey agar (M91) (use mixed bile salts; BBL Mac agar and DIFCO Mac CS are acceptable)
3. Celfsulodin-irgasan-novobiocin (CIN) agar (M35)
4. Bromcresol purple broth (M26) supplemented individually with the following carbohydrates, each at 0.5%: mannitol, sorbitol, cellobiose, adonitol, inositol, sucrose, rhamnose, raffinose, melibiose, salicin, xylose, and trehalose
5. Christensen's urea agar (M40) (plated media or slants)
6. Phenylalanine deaminase agar (M123) (plated media or slants)
7. Motility test medium (M103). Add 5 ml of 1% 2,3,5-triphenyl tetrazolium chloride per liter before autoclaving.
8. Tryptone broth, 1% (M164)
9. MR-VP broth (M104)
10. Simmons citrate agar (M138)
11. Veal infusion broth (M173)
12. Bile esculin agar (M18)
13. Anaerobic egg yolk agar (M12)
14. Minitek enteric and nonfermenter broth (BBL)
15. Trypticase (tryptic) soy agar (TSA) (M152)
16. Lysine arginine iron agar (LAIA) (M86)
17. Decarboxylase basal medium (Falkow) (M44) supplemented individually with 0.5% arginine, 0.5% lysine, or 0.5% ornithine
18. Congo Red-brain heart infusion agarose (CRBHO) (M41)
19. Irgasan-ticarcillin-chlorate (ITC) broth (M67)
20. Pyrazinamidase agar slants (M131)
21. PMP broth (M125)
22. b-D-glucosidase test (see instructions at end of chapter)
3. Reagents
1. Gram stain reagents (R32)
2. Voges-Proskauer (VP) test reagents (R89)
3. Ferric chloride, 10% in distilled water (R25)
4. Oxidase test reagent (R54)
5. Saline, 0.5% (sterile) (R66)
6. Kovacs' reagent (R38)
7. 0.5% Potassium hydroxide in 0.5% NaCl, freshly prepared
8. Minitek biochemical discs for the following substrates: esculin, V-P, H₂S-indole, citrate, lysine, arginine, ornithine, mannitol, sorbitol, cellobiose, adonitol, inositol, sucrose, rhamnose, raffinose, melibiose, salicin, xylose, and trehalose
9. Mineral oil, heavy grade, sterile (R46)
10. API 20E system
11. 1% Ferrous ammonium sulfate
4. Enrichment

The following simplified procedure for isolating Yersinia from food, water, and environmental samples is recommended:

1. Analyze samples promptly after receipt, or refrigerate at 4ºC. (Freezing of samples before analysis is not recommended, although Yersinia have been recovered from frozen products.) Aseptically weigh 25 g sample into 225 ml PSBB. Homogenize 30 s and incubate at 10ºC for 10 d.
2. If high levels of Yersinia are suspected in product, spread-plate 0.1 ml on MacConkey agar (Doyle et al., 1981; Weissfeld and Sonnenwirth, 1982) and 0.1 ml on CIN agar (Schiemann, 1982; Schiemann and Wauters, 1992) before incubating broth. Also transfer 1 ml homogenate to 9 ml 0.5% KOH in 0.5% saline (Aulisio et al., 1980), mix for several s, and spread-plate 0.1 ml on MacConkey and CIN agars. Incubate agar plates at 30ºC for 1 d. Transfer colonies from one of these plates onto sterile Whatman 541 filter paper for direct examination for Yersinia virulence gene by DNA colony hybridization (Hill et al., 1983) (see also Merker (1998), Chapter 24). If high levels of Yersinia contamination are not suspected, omit this step.
3. On day 10, remove enrichment broth from incubator and mix well. Transfer 0.1 ml enrichment to 1 ml 0.5% KOH in 0.5% saline and mix for 5-10 s (Aulisio et al., 1980). Successively streak one loopful to MacConkey plate and one loopful to CIN plate. Transfer additional 0.1 ml enrichment to 1 ml 0.5% saline and mix 5-10 s before streaking, as above. Incubate agar plates at 30ºC for 1 d.
4. Optional. An alternative enrichment technique (Wauters et al., 1988), although unconfirmed, gives promising preliminary results. Aseptically weigh 25 g sample into 225 ml ITC broth. Homogenize for 30 s and incubate at room temperature (RT) for 2 d. Continue as in 4-b, above.

5. Isolation of Yersinia

Examine MacConkey agar plates after 1 d incubation. Reject red or mucoid colonies. Select small (1-2 mm diameter) flat, colorless, or pale pink colonies. Examine CIN plates after 1 d incubation. Select small (1-2 mm diameter) colonies having deep red center with sharp border surrounded by clear colorless zone with entire edge. Inoculate each selected colony into LAIA slant (Weagant, 1982), Christensen's urea agar plate or slant, and bile esculin agar plate or slant by stabbing with inoculation needle. Incubate 48 h at RT. Isolates giving alkaline slant and acid butt, no gas and no H₂S (KA--) reaction in LAIA, which are also urease-positive, are presumptive Yersinia. Discard cultures that produce H₂S and/or any gas in LAIA or are urease-negative. Give preference to typical isolates that fail to hydrolyze (blacken) esculin.

6. Identification

Using growth from LAIA slant, streak culture to one plate of anaerobic egg yolk (AEY) agar and incubate at RT. Use growth on AEY to check culture purity, lipase reaction (at 2-5 d), oxidase test, Gram stain, and inoculum for biochemical tests. From colonies on AEY, inoculate the following biochemical test media and incubate all at RT for 3 d (except one motility test medium and one MR-VP broth, which are incubated at 35-37ºC for 24 h).

1. Decarboxylase basal medium (Falkow) (M44), supplemented with each of 0.5% lysine, arginine, or ornithine; overlay with sterile mineral oil
2. Phenylalanine deaminase agar (M123)
3. Motility test medium (semisolid) (M103), 22-26ºC and 35-37ºC
4. Tryptone broth (M164)
5. Indole test (see instructions at end of chapter)
6. MR-VP broth (M104). RT for autoagglutination test (see 8-b, below), followed by V-P test (48 h) (see instructions at end of chapter); 35-37ºC for autoagglutination test (see 8-a)
7. Bromcresol purple broth (M26) with 0.5% of the following filter-sterilized carbohydrates: mannitol, sorbitol, cellobiose, adonitol, inositol, sucrose, rhamnose, raffinose, melibiose, salicin, trehalose, and xylose
8. Simmons citrate agar (M138)
9. Veal infusion broth (M173)
10. In lieu of Nos. 1,2,4,7, and 8, Minitek (BBL) Minikit biochemical test kit can be used with biochemical disks for esculin, V-P, H₂S, indole, citrate, phenylalanine deaminase, lysine, arginine, ornithine, mannitol, sorbitol, cellobiose, adonitol, inositol, sucrose, rhamnose, raffinose, melibiose, salicin, trehalose, and xylose. Set up and inoculate according to manufacturer's instructions. Incubate in humidor at RT for 48 h.
11. Use API 20E system for biochemical identification of Yersinia. Follow manufacturer's instructions.
12. Pyrazinamidase agar slants (48 h) (see instructions at end of chapter)
13. b-D-glucosidase test (30ºC, 24 h) (see instructions at end of chapter)
14. Lipase test. Positive reaction is indicated by oily, iridescent, pearl-like colony surrounded by precipitation ring and outer clearing zone.

7. Interpretation

Yersinia are oxidase-negative, Gram-negative rods. Use Tables 21-3 and 21-4 to identify species and biotype of Yersinia isolates. Currently only strains of Y. enterocolitica biotypes 1B, 2, 3, 4, and 5 are known to be pathogenic. These biotypes and Y. enterocolitica biotype 6 and Y. kristensenii do not rapidly (within 24 h) hydrolyze esculin or ferment salicin (Tables 21-3 and 21-4). However, Y. enterocolitica biotype 6 and Y. kristensenii are relatively rare; they can be distinguished by failure to ferment sucrose, and they are pyrazinamidase-positive (Kandolo and Wauters, 1985). Hold Y. enterocolitica isolates which are within biotypes 1B, 2, 3, 4, and 5 for further pathogenicity tests.

8. Pathogenicity testing
1. Autoagglutination test. The MR-VP tube incubated at RT for 24 h should show some turbidity from bacterial growth. The 35-37ºC MR-VP should show agglutination (clumping) of bacteria along walls and/or bottom of tube with clear supernatant fluid. Isolates giving this result are presumptive positive for the virulence plasmid. Any other pattern for autoagglutination at these two temperatures is considered negative.
2. Freezing cultures. Plasmids that determine traits related to pathogenicity of Yersinia can be spontaneously lost during culture above 30ºC or with lengthy culture and passage below 30ºC in the laboratory. It is important, therefore, to immediately freeze presumptive positive cultures to protect plasmid content. Inoculate into veal infusion broth and incubate 48 h at RT. Add 10% sterile glycerol (e.g., 0.3 ml in 3 ml veal infusion broth) and freeze immediately. Storage at -70ºC is recommended.
3. Low calcium response Congo Red agarose virulence test. Inoculate test organism into BHI broth. Incubate overnight at 25-27ºC. Make decimal dilutions in physiologic saline to obtain 10 cells/ml (10^-6) Spread-plate 0.1 ml of appropriate dilution on each of two Congo Red agarose plates. Incubate one at 35ºC and one at 25ºC.

Examine at 24 and 48 h. Presumptive plasmid-bearing Y. enterocolitica will appear as pinpoint, round, convex, red, opaque colonies. Plasmidless Y. enterocolitica will appear as large, irregular, flat, translucent colonies.

4. Crystal violet binding test (Bhaduri et al., 1987. This rapid screening test differentiates potentially virulent Y. enterocolitica cultures. Grow suspect cultures 18 h at 22-26ºC in BHI broth with shaking. Dilute each culture to 1,000 cells/ml in physiological saline. Spread-plate 0.1 ml of each culture to each of two BHI agar plates. Incubate one plate at 25ºC and the other at 37ºC for 30 h. Gently flood each plate with 8 ml of 85 µg/ml crystal violet (CV) solution for 2 min and decant the CV. Observe colonies for binding of CV. Plasmid-containing colonies grown at 37ºC will bind CV, but not when grown at 25ºC. Plasmidless colonies grown at either temperature should not bind CV.
5. DNA colony hybridization. Currently, three oligonucleotide probes are available from FDA's Center for Food Safety and Applied Nutrition for the detection of virulence factors in Yersinia. The probe INV-3 is specific only for the inv gene of Y. pseudotuberculosis. All INV-3 reactive isolates of Y. pseudotuberculosis examined are also invasive for HeLa cells. The PF13 oligo probe is specific only for the invasion gene, ail, of Y. enterocolitica. Comparison of colony and Southern blot analysis using PF13 vs. HeLa cell invasion studies shows close correlation between probe and cell invasion. The oligo probe, SP12, developed under FDA contract by the Maryland Center for Vaccine Development (Miliotis et al., 1989; Robins-Brown et al., 1989), is specific for the 41-48 Mdal virulence plasmid in both Y. enterocolitica and Y. pseudotuberculosis. For more information on probes and protocols, see Merker (1988), Chapter 24.
6. Intraperitoneal infection of adult mice pretreated with iron dextran and desferrioxamine B. A positive result from any test (8, a-e above) is presumptive evidence of pathogenicity and should be confirmed by a biological test. Inoculate presumptively virulent Y. enterocolitica isolates into BHI broth and incubate overnight at RT with agitation. This will result in broth cultures at approximately 10⁹ bacterial cells per ml. Make decimal dilutions in sterile physiologic saline to use for mouse infections. Spread-plate 0.1 ml of appropriate dilutions (usually 10⁶) to two plates each of TSAYE and CRBHO. Incubate TSAYE at RT for 48 h and CRBHO at 35ºC for 24 h. Count TSAYE colonies to determine inoculum level and CRBHO for ratio of plasmid to non-plasmid cells in inoculum.

One d before infection, inject Swiss Webster adult mice i.p. with 0.2 ml physiologic saline solution containing 25 mg/ml each of iron dextran (Fermenta Animal Health Co., Kansas City, MO 64153) and desferrioxamine B (Desferal mesylate, Ciba Geigy, Greensboro, NC 27409). Inject 0.1 ml of decimally diluted bacterial cells i.p. to each of five mice per dilution. Observe mice for 7 d. If possible, maintain infected mice in a laminar flow isolator. Deaths occurring within 7 d, especially preceded by signs of illness, are specific for Y. enterocolitica virulence and are used to calculate LD₅₀ titer by method of Reed and Muench (Reed and Muench, 1938) as outlined in Table 21-5. Calculated LD₅₀ titer of less than 10⁴ cell is typical of virulent Y. enterocolitica regardless of biotype or serotype. A screening test may be performed by inoculating five pretreated mice at the 10^-4 dilution only. A virulent Yersinia culture will kill at least 4 of 5 mice.

7. Invasiveness. An in vitro HeLa cell assay is available for screening Yersinia isolates for invasive potential (Miliotis, 1991; Miliotis and Feng, 1992). Acridine orange is used to stain infected HeLa cell monolayers, which are then examined under fluorescence microscope for the presence of intracellular Yersinia (Miliotis, 1991; Miliotis and Feng, 1992). This in vitro staining technique can be used to determine invasiveness in both Y. enterocolitica and Y. pseudotuberculosis (Feng, 1992).

9. Yersinia pseudotuberculosis

Generally, all Y. pseudotuberculosis strains are biochemically homogeneous except for production of acid from melibiose, raffinose, and salicin. Y. pseudotuberculosis heat-stable somatic antigens are also used to subgroup the species. At present there are six serogroups represented by Roman numerals I-VI. Serogroups I, II, III, and IV have subtypes, but antiserum to one serogroup type will cross-react with the subtype strain and vice versa. Strains belonging to serogroups II and III are lethal when fed to adult mice even though these strains do not elaborate lipase. HeLa cell-invasive strains are esculin-positive, which is contrary to findings with Y. enterocolitica. Y. pseudotuberculosis strains harbor a 41-48 Mdal plasmid and will autoagglutinate at 37ºC. Association of yersiniosis in humans with the presence of a plasmid has been established (Schiemann and Wauters, 1992).

1. Enrichment. Aseptically weigh 25 g sample into 225 ml PMP broth (Fukushima et al., 1984). Homogenize for 30 s and incubate at 4ºC for 3 weeks. At 1, 2, and 3 weeks, mix enrichment well. Transfer 0.1 ml enrichment to 1 ml 0.5% KOH in 0.5% NaCl and mix for 5-10 s. Successively streak one loopful to MacConkey agar plate and one loopful to CIN agar plate. Streak one additional loopful directly from enrichment broth to one MacConkey and one CIN agar plate. Incubate agars at RT.
2. Isolation and identification. Continue as in 5-8, above, noting biochemical differences (Table 21-3). Notably, Y. pseudotuberculosis strains are ornithine-, sorbitol-, and sucrose-negative.

Instructions for Yersinia Identification Tests

Phenylalanine deaminase agar test: Add 2-3 drops 10% ferric chloride solution to growth on agar slant. Development of green color is positive test.

Indole test: Add 0.2-0.3 ml Kovacs' reagent. Development of deep red color on surface of broth is positive test.

V-P test: Add 0.6 ml "-naphthol and shake well. Add 0.2 ml 40% KOH solution with creatine and shake. Read results after 4 h. Development of pink-to-ruby red color in medium is positive test.

Pyrazinamidase test: After growth of culture on slanted pyrazinamidase agar at RT, flood 1 ml of 1% freshly prepared ferrous ammonium sulfate over slant. Development of pink color within 15 min is positive test, indicating presence of pyrazinoic acid formed by pyrazinamidase enzyme.

b-D-Glucosidase test: Add 0.1 g 4-nitrophenyl-b-D-glucopyranoside to 100 ml 0.666 M NaH₂PO₄ (pH 6). Dissolve; filter-sterilize. Emulsify culture in physiologic saline to McFarland Turbidity Standard No. 3. Add 0.75 ml of culture to 0.25 ml of test medium. Incubate at 30ºC overnight. A distinct yellow color indicates a positive reaction.

Table 21-3. Biochemical characteristics^a of Yersinia species.^d

Reaction	Y. pestis	Y. pseudo-tuberculosis	Y. enterocolitica	Y. intermediab	Y. frederiksenii	Y. kristensenii	Y. aldovae	Y. rohdei	Y. mollaretii	Y. bercovieri	Y. ruckeri
Lysine	-	-	-	-	-	-	-	-	-	-	-
Arginine	-	-	-	-	-	-	-	-	-	-	-
Ornithine	-	-	+c	+	+	+	+	+	+	+	+
Motility at RT 22-26ºC	-	+	+	+	+	+	+	+	+	+	+
35-37ºC	-	-	-	-	-	-	-	-	-	-	-
Urea	-	+	+	+	+	+	+	+	+	+	-
Phenylalanine deaminase	-	-	-	-	-	-	-	-	-	-	-
Mannitol	+	+	+	+	+	+	+	+	+	+	+
Sorbitol	+/-	-	+	+	+	+	+	+	+	+	-
Cellobiose	-	-	+	+	+	+	-	+	+	+	-
Adonitol	-	-	-	-	-	-	-	-	-	-	-
Inositol	-	-	+/-(+)	+/-(+)	+/-(+)	+/-(+)	+	-	+/-	-
Sucrose	-	-	+c	+	+	-	-	+	+	+	-
Rhamnose	-	+	-	+	+	-	+	-	-	-	-
Raffinose	-	+/-	-	+	-	-	-	+/-	-	-	-
Melibiose	-	+/-	-	+	-	-	-	+/-	-	-	-
Simmons citrate	-	-	-	+/-	+/-	-	-	+	-	-	+
Voges-Proskauer	-	-	+/-(+)	+	+	-	+	-	-	-	-
Indole	-	-	+/-	+	+	+/-	-	-	-	-	-
Salicin	+/-	+/-	+/-	+	+	-(+/-)	-	-	+/-	(+)
Esculin	+	+	+/-	+	+	-	+	-	(+)	(+)/-	-
Lipase	-	-	+/-	+/-	+/-	+/-	+/-	-	-	-
Pyrazinamidase	-	-	+/-	+	+	+	+	+	+	+

^a+ = positive after 3 d, (+) = positive after 7 d.
^bSome strains of Y. intermedia are negative for either Simmons citrate, rhamnose, and melibiose, or raffinose and Simmons citrate.
^cSome biotype 5 strains are negative.
^dFrom Aleksic et al. (1987), Bercovier et al. (1980), Bercovier et al. (1984), and Wauters et al. (1988).

Table 21-4. Biotype scheme^a for Y. enterocolitica.

Biochemical test	Reaction for biotypesb
	1A	1B	2	3		4		5			6
Lipase	+	+	-	-		-		-			-
Esculin/salicin (24 h)	+/-	-	-	-		-		-			-
Indole	+	+	(+)	-		-		-			-
Xylose	+	+	+	+		-		V			+
Trehalose	+	+	+	+		+		-			+
Pyrazinamidase	+	-	-	-		-		-			+
Beta-D-Glucosidase	+	-	-	-		-		-			-
Voges-Proskauer	+	+	+	+/-c		+		(+)			-

^aBased on Wauters (1981).
^b( ) = Delayed reaction; V = variable reactions.
^cBiotype of serotype O:3 found in Japan.

Table 21-5. Calculation of LD₅₀, by Reed-Muench method^d

A	B	C	D	E		F	G		H
Dilution	Bacterial cells/mL	Cells/mouse Log10	No. of mice		Cumulative valuesa  No. of mice				% Mortalityb
			Dead	Live		Dead	Live
100	3 x 108	7.477	3	0		15	0		100
10-1	3 x 107	6.477	3	0		12	0		100
10-2	3 x 106	5.477	3	0		9	0		100
10-3	3 x 105	4.477	3	0		6	0		100
10-4	3 x 104	3.477	2	1		3	1		75
10-5	3 x 103	2.477	1	2		1	3		25c
10-6	3 x 102	1.477	0	3		0	6		0
10-7	3 x 101	0.447	0	3		0	9		0

^aCumulative values in columns F and G are determined by adding values vertically in columns D and E. respectively.
^b% Mortality (H) is determined by dividing the number from column F by the total of the number from column F and column G for each dilution.
^cThe 50% endpoint lies between l0^-4 and 10^-5 dilutions.
^d(Reed and Muench, 1938)



Contents

References

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