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BAM: Shigella

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January 2001

Bacteriological Analytical Manual
Chapter 6
Shigella

Authors: Wallace H. Andrews and Andrew Jacobson

Revision History:

  • February 2013: Section D1: Enrichment of Shigella sonnei modified; use of waterbaths for incubation modified to use of forced air incubators
  • June 2012: Table 1 restored
  • May 2000 and October 2000: chapter revised

Contents

Shigellosis, although commonly regarded as waterborne, is also a foodborne 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 (1) 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 days. They utilize glucose and other carbohydrates, producing acid but not gas. However, because of their affinity to 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.

  1.  Equipment and Materials

    1. Same as for Salmonella, Chapter 5
    2. Forced air incubators, maintained at 42.0 ± 0.3°C and 44.0 ± 0.3°C
    3. Anaerobic jar
    4. Anaerobic gas generating pouch/sachet
    5. Anaerobic indicator
  2. Media

    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)

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  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 reagents(R32)

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  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 biocehmically and serologically 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 (3). 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, insert anaerobic gas generating pouch/sachet (use number recommended by the anaerobic jar manufacturer, according to the volume of the jar), insert an anaerobic indicator, and tighten the lid. Incubate jars at 44.0°C in a forced air incubator for 20 h. Agitate enrichment culture suspension and streak on a MacConkey agar plate. Incubate 20 h at 35°C.
      2. Enrichment of other Shigella species. Proceed as above, but use novobiocin at 3.0 µg/ml and incubate anaerobically at 42.0°C in a forced air incubator.
    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 and 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 35-37°C.

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  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 35°C 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 indole formation, discard positive cultures from 44.0°C enrichment. All suspicious isolates from 42°C enrichment may be either positive or negative and consequently should be retained.

    2. DNA hybridization method.

      Proceed as described in Chapter 24.

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  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 days), KCN, malonate, citrate, and salicin; positive for methyl red. Pick isolates having positive reactions for Shigella to veal infusion agar slants. Use antisera for identification of serotype or compare with physiological behavior of the 32 serotypes presented in Table 1. 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 tend to be positive in at least one of the reactions (Table 2) (2).

     

    Table 1. Biochemical reactions of serotypes of Shigella(a)
    Subgroup
    and serotype
    Mannitol%+Dulcitol%+Xylose%+Rhamnose%+Raffinose%+Glycerol%+Indole%+Ornithine
    decarboxylase
    %+
    Subgroup A
    S. dysenteriae
    100000+or(+)10000
    2000+980(+)or+98+1000
    300000(+)or+10000
    400000(+)or+10000
    50+or(+)100000+or(+)10000
    600000–or(+)3800
    7000(+)or+9000+1000
    800+or(+)9680+or(+)100+1000
    900000+or(+)10000
    1000+10000000
    Subgroup B
    S. flexneri
    1+95000D890–or+350
    2+99000D770–or+440
    3+9800D12D880+or–880
    4+9900D23D820+or–550
    400D71–or+4830+980
    5+9900SD720+950
    6+>99D80460D8800
    6(b)+100D86D7500+or(+)10000
    6(b)_0+or(+)100000(+)10000
    Subgroup C
    S. boydii
    1+1001+or(+)9700(+)or+9600
    2+1001000+or(+)10000
    3+100D75D8600+or(+)9100
    4+99–or(+)28000+or(+)10000
    5+1000(+)9400D61+1000
    6+or(+)100(+)or+100+10000(+)or+10000
    7+1000+9800(+)or+98+1000
    8+1000+9400(+)or+10000
    9+9500D800(+)or –82+1000
    10+94+100D8400(+)or+10000
    11+100–or(+)34+or(+)10000(+)or+100+1000
    12+100–or(+)14000–or+1400
    13+1000000(+)or–63+100+100
    14–or+290(+)or+10000+or(+)10000
    15+900000(+)or–64+1000
    Subgroup D
    S. sonnei
    +9911+or(+)98D84D460+>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 [+, (+), –].

    b Some S. flexneri 6 cultures (the Newcastle and Manchester biotypes) produce gas from fermentable substrates; other shigellae are anaerogenic.

    NOTE: In this table percentages of + and (+) reactions are combined.

    Ref: Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed., pp.146-147. Elsevier, New York. Reproduced with permission.

     

    Table 2. Reactions of Shigella and Escherichia coli in acetate, citrate, and mucate mediaa,b
    Genera and speciesSodium acetate%+(%+)Christensen's
    citrate
    %+(%+)Sodium mucate%+(%+)
    S. dysenteriae000000
    S. flexneri000000
    S. boydii000000
    S. sonnei0000D6.4(30.3)
    E. coli+ or (+)83.8(9.7)D15.8(18.4)+91.6(1.4)
    Alkalescens-Dispar biotypes+ or (+)89.6(4.7)D75(12.5)D29.5(27.9)
    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 [+,(+), −]. bFrom ref. 2. Reproduced with permission.
  7.  Serological characterization

    Suspend growth from 24 h veal infusion agar 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.

    RectangleSuspensionPolyvalent antiserumSaline
     A  A1  B  C  C1  C2  D A - D
    1++        
    2+ +       
    3+  +      
    4+   +     
    5+    +    
    6+     +   
    7+      +  
    8+       + 
    9+        +

    Mix contents of each rectangle with a 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.

 References

  1. Brenner, D.J. 1984. Family I. Enterobacteriaceae, pp.408-420. In: Bergey's Manual of Systematic Bacteriology, Vol. 1. N.R. Krieg (ed). Williams & Wilkins, Baltimore.
  2. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.
  3. Mehlman, I.J., Romero, A., and Wentz, B.A. 1985. Improved Enrichment for Recovery of Shigella sonnei from Foods. J. Assoc. Off. Anal. Chem. 68(3):552-555.
  4. Sanzey, B. 1979. Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase. J. Bacteriol. 136:40-47.

Hypertext Source: Shigella, Bacteriological Analytical Manual, 8th Edition, Revision A, 8. Chapter 6.