Shigella spp.

• Potential Food Safety Hazard
• Control Measures
• FDA Guidelines
• Growth
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• Shigella
• References

Contents

Potential Food Safety Hazard

Shigellosis, although commonly regarded as waterborne, is also a food-borne disease restricted primarily to higher primates, including humans. It is usually spread among humans by food handlers with poor personal hygiene. Foods most often incriminated in the transmission have been potato salad, shellfish, raw vegetables, and Mexican dishes.

The genus Shigella consists of four species: S. dysenteriae (subgroup A), S. flexneri (subgroup B), S. boydii (subgroup C), and S. sonnei (subgroup D). Shigella organisms may be very difficult to distinguish biochemically from Escherichia coli. Brenner (1984) considers Shigella organisms and E. coli to be a single species, based on DNA homology. Nonetheless, Shigella species are Gram-negative, facultatively anaerobic, nonsporulating, nonmotile rods in the family Enterobacteriaceae. They do not decarboxylate lysine or ferment lactose within 2 d. They utilize glucose and other carbohydrates, producing acid but not gas. However, because of their affinity with E. coli, frequent exceptions may be encountered, e.g., some biotypes produce gas from glucose and mannitol. Neither citrate nor malonate is used as the sole carbon source for growth, and the organisms are inhibited by potassium cyanide (Andrews, 1998).

Contents

Control Measures

Hazards from Shigella can be prevented by preventing human waste contamination of water supplies and by improved personal hygiene for people who are ill or are carriers of Shigella and work in food operations (Ward et al., 1997).

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FDA Guidelines

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Growth

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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

Shigella

1. Equipment and materials
1. Same as for Salmonella, Chapter 17
2. Water baths maintained at 42 ± 0.2ºC and 44.0 ± 0.2ºC
3. Anaerobic jar with catalyst
2. Media

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

1. Shigella broth with novobiocin (M136)
2. Trypticase soy-yeast extract (TSYE) broth (M157)
3. MacConkey agar (M91)
4. Triple sugar iron (TSI) agar (M149)
5. Urea broth (M171)
6. Motility test medium (semisolid) (M103)
7. Potassium cyanide (KCN) broth (M126)
8. Malonate broth (M92)
9. Tryptone (tryptophane) broth, 1% (M164)
10. MR-VP broth (M104)
11. Christensen citrate agar (M39)
12. Veal infusion agar (M173)
13. Bromcresol purple broth (M26) supplemented with the following carbohydrates, each at a level of 0.5%: adonitol, salicin, rhamnose, glucose, inositol, lactose, mannitol, raffinose, sucrose, xylose, dulcitol, and glycerol.
14. Acetate agar (M3)
15. Mucate broth (M105)
16. Mucate control broth (M106)
17. Decarboxylase basal medium (lysine, Falkow) (M44)
18. Decarboxylase basal medium (ornithine) (M44)
3. Reagents and stains
1. Kovacs' reagent (R38)
2. Voges-Proskauer test reagents (R89)
3. 1 N Sodium hydroxide solution (R73)
4. 1 N Hydrochloric acid (R36)
5. Methyl red indicator (R44)
6. Physiological saline solution, 0.85% (sterile) (R63)
7. Novobiocin
8. Polyvalent Shigella antisera for groups A, A₁, B, C, C₁, C₂, D and Alkalescens-Dispar biotypes 1-4
9. Gram stain (R32)
4. Enrichment

Two approaches are provided for the recovery of Shigella. The first approach is a conventional culture method that involves the use of a specially formulated medium, Shigella broth. Novobiocin is added to provide a selective environment. Sample enrichments are incubated as described below, and streaked to MacConkey agar; typical colonies are confirmed as Shigella spp.

The second approach uses DNA hybridization. The enzyme DNA gyrase induces negative supercoiling into closed circular DNA. It has been reported, however, that novobiocin inhibits DNA gyrase (Sanzey, 1979). Thus, the use of novobiocin in Shigella broth may cause this medium to be incompatible with DNA hybridization for detecting Shigella. Because DNA hybridization can detect Shigella in the presence of overwhelming numbers of competitors, a selective agent such as novobiocin is not needed in the enrichment medium and may actually be counterproductive. Thus, the use of tryptic soy broth with yeast extract added (TSYE) to a final concentration of 0.6% is the recommended enrichment if DNA hybridization is being used.

1. Conventional culture method
1. Enrichment of Shigella sonnei. Aseptically weigh 25 g sample into 225 ml Shigella broth to which novobiocin (0.5 µg/ml) has been added. Hold suspension 10 min at room temperature and shake periodically. Pour supernatant into sterile 500 ml Erlenmeyer flask. Adjust pH, if necessary, to 7.0 ± 0.2 with sterile 1 N NaOH or 1 N HCl. Place flask in anaerobic jar with fresh catalyst, insert GasPak and activate by adding water. Because of high humidity within jar, heat catalyst as recommended after each use. Incubate jars in 44ºC water bath for 20 h. Agitate suspension and streak on MacConkey agar plates. Incubate 20 h at 35ºC.
2. Enrichment of other Shigella species. Proceed as above, but use novobiocin at 3 µg/ml and incubate anaerobically in 42.0ºC water bath.

2. DNA hybridization method

Aseptically weigh 25 g sample into 225 ml TSYE. Hold suspension 10 min at room temperature and shake periodically. Pour supernatant into sterile 500 ml Erlenmeyer flask. Adjust pH, if necessary, to 7.0 ± 0.2 with sterile 1 N NaOH or 1 N HCl. Incubate sample enrichment 20-24 h at 35-37ºC.

5. Isolation of Shigella species
1. Conventional culture method

Examine MacConkey agar plates. Shigella colonies are slightly pink and translucent, with or without rough edges. Inoculate suspicious colonies into the following media: glucose broth, TSI agar slant, lysine decarboxylase broth, motility agar, and tryptone. Incubate at 35ºC for 48 h, but examine at 20 h. Discard all cultures showing motility, H₂S, gas formation, lysine decarboxylation, and fermentation of sucrose or lactose. With respect to formation of indole, discard positive cultures from 44ºC enrichment.

All suspicious isolates from 42ºC enrichment may be either positive or negative and consequently should be retained.

1. DNA hybridization method. Proceed as described in Merker (1998), Chapter 24.

6. Physiological characterization

Perform Gram stain and inoculate cultures giving satisfactory screening reactions to the other recommended biochemicals. The characteristics of Shigella are summarized as follows: Gram-negative rods; negative for H₂S, urease, glucose (gas), motility, lysine decarboxylase, sucrose, adonitol, inositol, lactose (2 d), KCN, malonate, citrate, and salicin; positive for methyl red. Use antisera for identification of serotype or compare with physiological behavior of the 32 serotypes presented in Table 18-2. If serotype cannot be identified by these tests, two explanations are possible: 1) Several provisional serotypes have not been accepted by an international commission on the taxonomy of Shigella species. Resolve by referral to the U.S. Centers for Disease Control and Prevention (CDC), Atlanta, GA, or to the World Health Organization (WHO), Shigella spp. Reference Laboratories. 2) The cultures may be E. coli. Proper interpretation of the mucate and acetate reactions should help. Shigella species tend to be negative in all these reactions, whereas anaerogenic E. coli tends to be positive in at least one of the reactions (Table 18-3) (Ewing, 1986).

7. Serological characterization

Suspend growth from 24 h veal infusion slant in 3 ml 0.85% saline to McFarland Turbidity Standard No. 5. Mark nine 3 x 1 cm rectangles on clear glass petri dish with wax pencil. Add drops of suspension, antisera, and saline in accordance with the following protocol.

Rectangle	Suspension	Polyvalent antiserum								Saline
		A	A1	B	C	C1	C2	D	A-D
1	+	+
2	+		+
3	+			+
4	+				+
5	+					+
6	+						+
7	+							+
8	+								+
9	+									+

Mix content of each rectangle with needle, taking care that no mixing between rectangles occurs. Rock petri dish 3-4 min to accelerate agglutination. Read extent of agglutination as follows: 0 = no agglutination; 1+ = barely detectable agglutination; 2+ = agglutination with 50% clearing; 3+ = agglutination with 75% clearing; 4+ = visible floc with suspending fluid totally cleared. Re-examine suspension in monovalent sera belonging to each polyvalent in which a distinct positive reaction (2+, 3+, 4+) has occurred. In the event of a negative reaction, heat suspension in steamer 30 min to hydrolyze interfering capsular antigen. Re-examine in polyvalent, and, if positive, in corresponding monovalent sera. Because of tentative serotypes, a negative reaction may occur with the available sera. Consequently, it is advised that cultures retrieved from an outbreak and suspect foods giving Shigella-like reactions in physiological tests be referred to the CDC or to a WHO Shigella laboratory for confirmation.


Table 18-2. Biochemical reactions of serotypes of Shigella^a

Subgroup and serotype		Mannitol	%+	Dulcitol	%+	Xylose	%+	Rhamnose	%+
Subgroup A S. dysenteriae
1	-		0	-	0	-	0	-	0
2	-		0	-	0	-	0	+	98
3	-		0	-	0	-	0	-	0
4	-		0	-	0	-	0	-	0
5	-		0	+ or (+)	100	-	0	-	0
6	-		0	-	0	-	0	-	0
7	-		0	-	0	-	0	(+) or +	90
8	-		0	-	0	+ or (+)	96	-	8
9	-		0	-	0	-	0	-	0
10	-		0	-	0	+	100	-	0
Subgroup B S. flexneri
1	+		95	-	0	-	0	-	0
2	+		99	-	0	-	0	-	0
3	+		98	-	0	-	0	D	12
4	+		99	-	0	-	0	D	23
4	-		0	-	0	D	71	- or +	48
5	+		99	-	0	-	0	-	5
6b	+		>99	D	80	-	4	-	6
6b	+		100	D	86	D	75	-	0
6	-		0	+ or (+)	100	-	0	-	0
Subgroup C S. boydii
1	+		100	-	1	+ or (+)	97	-	0
2	+		100	-	1	-	0	-	0
3	+		100	D	75	D	86	-	0
4	+		99	- or (+)	28	-	0	-	0
5	+		100	-	0	(+)	94	-	0
6	+ or (+)		100	(+) or +	100	+	100	-	0
7	+		100	-	0	+	98	-	0
8	+		100	-	0	+	94	-	0
9	+		95	-	0	-	0	D	80
10	+		94	+	100	D	84	-	0
11	+		100	- or (+)	34	+ or (+)	100	-	0
12	+		100	- or (+)	14	-	0	-	0
13	+		100	-	0	-	0	-	0
14	- or +		29	-	0	(+) or +	100	-	0
15	+		90	-	0	-	0	-	0
Subgroup D S. sonnei	+		99	-	1	-	1	+ or (+)	98


 
 

Subgroup and serotype	Raffinose	%+	Glycerol	%+	Indole	%+	Ornithine decarboxylase	%+
Subgroup A S. dysenteriae
1	-	0	+ or (+)	100	-	0	-	0
2	-	0	(+) or +	98	+	100	-	0
3	-	0	(+) or +	100	-	0	-	0
4	-	0	(+) or +	100	-	0	-	0
5	-	0	+ or (+)	100	-	0	-	0
6	-	0	- or (+)	38	-	0	-	0
7	-	0	-	0	+	100	-	0
8	-	0	+ or (+)	100	+	100	-	0
9	-	0	+ or (+)	100	-	0	-	0
10	-	0	-	0	-	0	-	0
Subgroup B S. flexneri
1	D	89	-	0	- or +	35	-	0
2	D	77	-	0	- or +	44	-	0
3	D	88	-	0	+ or -	88	-	0
4	D	82	-	0	+ or -	55	-	0
4	-	3	-	0	+	98	-	0
5	D	72	-	0	+	95	-	0
6b	-	0	D	88	-	0	-	0
6b	-	0	+ or (+)	100	-	0	-	0
6	-	0	(+)	100	-	0	-	0
Subgroup C S. boydii
1	-	0	(+) or +	96	-	0	-	0
2	-	0	+ or (+)	100	-	0	-	0
3	-	0	+ or (+)	91	-	0	-	0
4	-	0	+ or (+)	100	-	0	-	0
5	-	0	D	61	+	100	-	0
6	-	0	(+) or +	100	-	0	-	0
7	-	0	(+) or +	98	+	100	-	0
8	-	0	(+) or +	100	-	0	-	0
9	-	0	(+) or -	82	+	100	-	0
10	-	0	(+) or +	100	-	0	-	0
11	-	0	(+) or +	100	+	100	-	0
12	-	0	- or +	14	-	0	-	0
13	-	0	(+) or -	63	+	100	+	100
14	-		+ or (+)	100	-	0	-	0
15	-	0	(+) or -	64	+	100	-	0
Subgroup D S. sonnei	D	84	D	46	-	0	+	>99

^a+, 90% or more positive in 1 or 2 days; -, 90% or more negative; + or -, majority positive; - or +, majority negative; (+) delayed positive; D, different reactions [+, (+), -].
^bSome S. flexneri 6 cultures (the Newcastle and Manchester biotypes) produce gas from fermentable substrates; other Shigella are anaerogenic. NOTE: In this table percentage of + and (+) reactions are combined. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Pp. 146-147. Elsevier, New York.

Table 18-3. Reactions of Shigella and Escherichia coli in acetate, citrate, and mucate media^a,b

Genera and species	Sodium acetate	%+	(%+)	Christensen's  Citrate	%+	(%+)	Sodium Mucate	%+	(%+)
S. dysenteriae	-	0	0	-	0	0	-	0	0
S. flexneri	-	0	0	-	0	0	-	0	0
S. boydii	-	0	0	-	0	0	-	0	0
S. sonnei	-	0	0	-	0	0	D	6.4	(30.3)
E. coli	+ or (+)	83.8	(9.7)	D	15.8	(18.4)	+	91.6	(1.4)
Alkalescens-Dispar biotypes	+ or (+)	89.6	(4.7)	D	75	(12.5)	D	29.5	(27.9)

^a+, 90% or more positive in 1 or 2 days; -, 90% or more negative; (+) delayed positive; D, different reactions [+, (+), -].
^bFrom Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Pp. 146-147. Elsevier, New York.

Contents

References

Andrews, W.H. 1998. Shigella, Ch. 6. In Food and Drug Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). R.L. Merker (Ed.). AOAC International, Gaithersburg, MD.

Andrews, W.H., and June, G.A. 1998. Food sampling and preparation of sample homogenate, Ch. 1. In Food and Drug Administration Bacteriological Analytical Manual , 8th ed. (revision A), (CD-ROM version). R.L. Merker (Ed.). AOAC International, Gaithersburg, MD.

Brenner, D.J. 1984. Family I. Enterobacteriaceae. In Bergey's Manual of Systematic Bacteriology, Vol. 1. N.R. Krieg (Ed.), p.408-420. Williams & Wilkins, Baltimore.

Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.

FDA. 1998. Bacterial pathogen growth. Appendix 4. In Fish and Fishery Products Hazards and Controls Guide, 2^nd ed., p. 241-244. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC.

Fehlhaber, K. 1981. Untersuchungen über lebensmittelhygienisch bedeutsame Eigenschaften von Shigellen. Arch. Exper. Vet. Med. (Leipzig) 35(6):955-964.

Jyhshiun, L., In, S.L., Frey, J., Slonczewski, J.L., and Foster, J.W. 1995. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177(14):4097-4104.

Merker, R.L. (Ed.). 1998. Media and Reagents, Appendix 3. In Food and Drug Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). AOAC International, Gaithersburg, MD.

Sanzey, B. 1979. Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase. J. Bacteriol. 136:40-47.

Ward, D., Bernard, D., Collette, R., Kraemer, D., Hart, K., Price, R., and Otwell, S. (Eds.) 1997. Hazards Found in Seafoods, Appendix III. In HACCP: Hazard Analysis and Critical Control Point Training Curriculum, 2^nd ed., p. 173-188. UNC-SG-96-02. North Carolina Sea Grant, Raleigh, NC.