Shigella Serotyping: Introduction, Principle, Test Requirements, Test Procedure, Result-Interpretation, and Keynotes

Shigella polyvalent A, B and C2 for Shigella dysenteriae, Shigella flexneri, and Shigella boydii

Introduction of Shigella Serotyping

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Shigella Serotyping helps to differentiate serotypes of Shigella. Serotypes refer to separate groups within a species of microorganisms that all share a similar property. More specifically, each serotype has the same number of antigens on its surfaces. For example, Shigella flexneri serotype 1, 2, 2a, S. sonnei, and S. dysenteriae type 1. Serotypes are differentiated on the basis of agglutination tests and used to help with the identification of Salmonella, Shigella, Vibrio cholerae, Haemophilus influenzae, etc. Serotyping of Shigella is performed on the basis of these bacteria having the somatic O antigen. There are many different serotypes that can be identified using the type-specific monovalent antisera.

Fig. Bacillary dysentery stool findings

Difco Shigella antisera available for use in the referral laboratory are as follows-

Polygroup A
Polygroup A1
Polygroup B
Polygroup C1
Polygroup C2
Polygroup C3
Polygroup D

Fig. Shigella boydii growth on MacConkey agar

Principle of Shigella Serotyping

Shigella Serotyping works on the principle of agglutination. When a particulate antigen (agglutinogen) combines with its antibody (agglutinin) in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Agglutination is the aggregation of already insoluble particles or cells into larger clumps. Interaction between the antibody (Ab) and particulate antigen results in visible clumping called agglutination.

Test Requirements for Shigella Serotyping

Shigella serotyping needs the following requirements-

Test organism ( pure colony)
Antisera (serogroups)
Sterile, clean, and grease-free glass slides
Normal saline
Inoculating loop/ Sterile mixing sticks
Bunsen burner
Gloves
Waste bin
Control strains

Test Procedure of Shigella Serotyping

(Slide agglutination of live organisms)

All materials have to be at room temperature at the time of testing.
Dispense two 5-10µl volumes of sterile 0.85% saline solution(saline) onto a carefully cleaned microscope slide. The slide may be partitioned using a chinagraph pencil. With a platinum wire of disposable inoculation loop take one 1-2 colonies of live organisms from a fresh culture on nutrient agar or similar and emulsify into each drop of saline to produce distinct and uniform turbidity.
Place a drop (30-40 µl ) of antiserum onto one of the emulsified isolates and onto the other a drop (30-40 µl ) of saline as a control. Note: do not allow the organism to contaminate the antiserum dropper bottle.
Mix the reagent by tilting the slide back and forth for 60 seconds while viewing under indirect light against a dark background.
distinct clumping or agglutination within this period, without clumping in the saline control (auto-agglutination), should be regarded as a positive result. Weak agglutination should record as negative.

Slide agglutination of heat-treated organisms

If the biochemical feature is as of Shigella but gives no agglutination then it is necessary to heat the suspension of the bacteria in saline and heat it to 100 °C for one hour. Repeat the slide agglutination tests as described above with suspension after spinning down, and taking the sediment.

Result and Interpretation of Shigella Serotyping

Agglutination : Positive

No agglutination: Negative

Polygroup A reacting with S. dysenteriae 1-7
Polygroup A1 reacting with S. dysenterae 8ab, 8ac,9,10
Polygroup B reacting with S. flexnerii 1-7
Polygroup C1 reacting with S. boydii 1-7
Polygroup C2 reacting with S. boydii 8-11
Polygroup C3 reacting with S. boydii 12-18
Polygroup D reacting with S. sonnei phase i and ii

Fig. Shigella polyvalent A, B and C2 for Shigella dysenteriae, Shigella flexneri, and Shigella boydii

Quality Control (QC): It is recommended that quality control should be performed with at least one organism to demonstrate a positive reaction and at least one organism to demonstrate a negative reaction. Do not use the product if the reactions with the control organisms are incorrect. Check for signs of deterioration. Do not use reagents if they are contaminated or cloudy.

Fig. Shigella serotype-Positive

Keynotes on Shigella Serotyping

Salmonella, Shigella, Vibrio cholerae, and Haemophilus influenzae are the most common bacteria in that serotyping is performed.
Serotyping works on the principle of agglutination.
Only do agglutination on pure colonies, which biochemically are the suspected organism. If the specimen consists of multiple strains, the serotype may not be correctly identified.
Nutrient agar is best for the agglutination tests.
Serotyping is recommended from non-selective media such as MHA, Blood agar, or TSI.
In the case of Shigella, taking growth from the KIA agar for routine slide agglutination test heating is not required.

Fig. Shigella flexneri colony morphology on Salmonella-Shigella agar, XLD agar, and biochemical reactions in TSI, SIM, citrate, and urea agar

Fig. Shigella boydii colony morphology on XLD agar

Fig. Salmonella Typhi (black colony) and Shigella boydii growth on XLD agar

Further Readings on Shigella Serotyping

https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/762019/TP_3i4.pdf
https://www.frontiersin.org/articles/10.3389/fmicb.2019.02554/full
https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/lam.12690
http://legacy.bd.com/ds/technicalCenter/inserts/8085884(05).pdf
https://www.bd.com/documents/brochures/microbiology-solutions/DS_SR_Difco-antiserum-solutions_BR_EN.pdf
http://legacy.bd.com/ds/technicalCenter/inserts/8085884(05).pdf
https://antimicrobialresistance.dk/CustomerData/Files/Folders/6-pdf-protocols/58_23-10-gfn-shigellaserotypification-final-29-06-10.pdf
https://bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-1-74

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