Shigella on MacConkey agar: Introduction, Morphology, Culture Characteristics, Pathogenesis, Lab Diagnosis and Treatment

Shigella on MacConkey agar

Shigella

Shigella boydii colony characteristics on MacConkey agar are non-lactose fermenter colonies as shown above picture.

Introduction of Shigella

 Shigella is a genus of the Enterobacteriaceae family of bacteria and causing an infectious disease called Shigellosis. Shigella is named after the Japanese microbiologist Kiyoshi Shiga who isolated the first member of the group in 1896 from epidemic dysentery in Japan which was then called Shigella shiga and is now called S. dysenteriae. Most who are infected with Shigella develop diarrhea, fever, and stomach cramps starting a day or two after they are exposed to the bacteria. Shigellosis usually resolves in 5 to 7 days. Some people who are infected may have no symptoms at all, but may still pass the Shigella bacteria to others. The spread of these organisms can be stopped by frequent and careful handwashing with soap and taking other hygiene measures because of being a mode of infection feco-oral route.

Morphology

Shigellae are short, Gram-negative rods measuring about 1-3 µm X 0.5  µm. They are non-motile, non-encapsulated, non-sporing, and non-acid fast.

Culture characteristics

They are aerobes and facultative anaerobes and can grow on ordinary media like nutrient agar. The optimal temperature and pH for growth are 37°C and 7.4 respectively.  But they can grow at a temperature range of 10 to 40°C.

MacConkey agar: Colonies are non-lactose fermenting (except S. sonnei) large, circular, convex, smooth, and translucent.

Deoxycholate citrate agar (DCA): Colonies are colorless (non-lactose fermenting) except in the case of S. sonnei which forms pink colonies due to late lactose fermentation.

Xylose lysine deoxycholate (XLD) agar: Colonies are red  due to the organism has ability to decarboxylate  lysine without black centers

Salmonella-Shigella(SS) agar: It is a highly selective medium for the isolation of Salmonella and Shigella. Colorless colonies with no blackening whereas Salmonella colonies are colorless with black centers due to their ability to produce hydrogen sulfide.

Heaktoen Enteric Agar (HEA): They give green to blue-green colonies.

Selenite F broth ( enrichment medium): Sodium selenite of this medium inhibits coliform bacilli while permitting salmonellae and shigellae to grow. This is recommended medium for the isolation of these organisms from feces.

Resistance

They are killed at 56°C in an hour and by 1 % phenol in 30 minutes. They remain viable in water and ice for 1-6 months. Chlorination or boiling of water and pasteurization of milk is effective and destroys the bacilli.

Antigenic Structures

They possess a large number of antigens and they are-

  • somatic (O) antigens
  • Capsular(K) antigen ( some strains only)
  • Fimbrial antigens.

Classification

Scientific classification

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Shigella

There are four species or serogroups with multiple serotypes –

A. Shigella dysenterae: 12 Serotypes

B. Shigella flexneri: 6 serotypes

C. Shigella boydii: 18 serotypes  and

D. Shigella sonnei : 1 sreotype

On Basis of nannitol Fermentation-

  1. Non-mannitol-fermenters: Shigella dysenteriae
  2.  Mannitol-fermenters:  Shigella flexneri,  Shigella boydii and Shigella sonnei

Toxins

  • Endotoxin
  • Exotoxin
  • Verotoxin

 Habitat and Transmission

Shigella species are found only in the human intestinal tract. Carriers of pathogenic strains can excrete the organism up to two weeks after infection and occasionally for longer periods. They are killed by drying and are transmitted by the fecal-oral route. The highest incidence of Shigellosis occurs in areas of poor sanitation and where water supplies are polluted. Factors Contributing Spread-Spread is always from a human resource and generally involves one of the five Fs i.e. food, fingers,  feces, flies, and fomites. This is in contrast to salmonellae, which are often spread to humans from infected animals. Transmission-fecal-oral transmission is the main path of Shigella infection. Other modes of transmission include ingestion of contaminated food or water, contact with infected objects, or sexual contact. Outbreaks of Shigella infection are common in places where sanitation is poor.

Pathogenesis 

The species of this genus cause a serious illness known as dysentery /shigellosis, which is an acute diarrheal disease characterized by the passage of pus, blood, or mucous through the stool. Infection mainly occurs because of the ingestion of contaminated food or water. The incubation period is 12-48 hours but may vary between 1-7 days. Through the ingestion, the bacilli will reach the large intestine of humans. The multiplication occurs in the epithelial cells of the large intestine. Then the bacteria spreads to adjacent cells and to the lamina propria (which is a thin layer of loose connective tissue which lies beneath the epithelium) where the colonization occurs. After the growth and multiplication, it starts to produce toxins. The lamina propria and submucosa develop an acute inflammatory reaction with the formation of abscess on the mucosal surface along with capillary thrombosis by the production of toxins. The necrosed epithelium becomes soft and sloughed out and causing superficial ulcers and bleeding. The toxin produced by the shigella bacteria has both enterotoxic effect and a neurotoxic effect. Thus their combined action leads to severe diarrhea, polyneuritis, coma, and meningitis.

Clinical Syndromes (Shigellosis)

  • Ranges from asymptomatic infection to severe bacillary dysentery
  • Two-stage disease: watery diarrhea changing to dysentery with frequent small stools with blood and mucus, tenesmus, cramps, fever
  •  Early-stage:- Watery diarrhea attributed to the enterotoxic activity of Shiga toxin
  • Fever attributed to the neurotoxic activity of the toxin

Clinical Features

Following are the clinical features of  Shigellosis-

  • abdominal pain,
  • tenesmus,
  • watery diarrhea, and/or dysentery (multiple scanty, bloody, mucoid stools)
  • abdominal tenderness,
  • fever,
  • vomiting,
  • dehydration,
  • and convulsions.

Epidemiology

Shigellosis is a major cause of the diarrheal disease (developing nations). The major cause of bacillary dysentery (severe second stage form of shigellosis). Leading cause of infant diarrhea and mortality (death) in developing countries. They occur naturally in higher primates. Spread from human to human via the fecal-oral route. Less frequently, transmission by ingestion of contaminated food or water. Outbreaks usually occur in close communities; Secondary transmission occurs frequently. Low infectious dose (10-100 bacilli) with 1-3 day incubation period. Carriage of the organism persists for approximately one month following convalescence.

Laboratory Diagnosis

Diagnosis depends upon isolating the organisms, Shigella from feces.

Specimen: Collection of fresh stool

Transport: Specimens should be transported immediately otherwise, use a transport medium like Sach’s buffered glycerol saline.

Note: Avoid the use of alkaline transport medium because it is inhibitory for shigellae ( but useful for Vibrios).

Direct Microscopy of feces

Saline and iodine wet mount: Fields are suggestive for pus cells, red blood cells, and absence of parasites.

Culture of specimens

MacConkey agar: Colonies are non-lactose fermenting (except S. sonnei) large, circular, convex, smooth, and translucent.

Deoxycholate citrate agar (DCA): Colonies are colorless (non-lactose fermenting) except in the case of S. sonnei which forms pink colonies due to late lactose fermentation.

Xylose lysine deoxycholate (XLD) agar: Colonies are red  due to the organism has ability to decarboxylate  lysine without black centers

Salmonella-Shigella(SS) agar: It is a highly selective medium for the isolation of Salmonella and Shigella. Colorless colonies with no blackening whereas Salmonella colonies are colorless with black centers due to their ability to produce hydrogen sulfide.

Heaktoen Enteric Agar (HEA): They give green to blue-green colonies.

Shigella biochemical tests

Shigella species biochemical tests-

TSI test: Red/yellow, no production of hydrogen sulfide and gas formation

SIM test: The indole test is negative, non-motile, and has no production of hydrogen sulfide.

Urease test: The urea hydrolyzation test is negative.

Citrate test: The citrate utilization test is also negative as shown above image.

Slide Agglutination Test

Identification of Shigella is conformed by slide agglutination test using polyvalent and monovalent antisera. Then type-specific antisera belonging to subgroups A, B, or C is used for the agglutination test.

Colicin Typing

It is used for subgroup D (S. sonnei) strains.

Differences between Amoebic dysentery and bacillary dysentery

Amoebic dysentery

  • Trophozoites of Entamoeba histolytica
  • Cyst of E. histolytica
  • RBCs in clumps
  • Macrophages
  • pus cells
  • CL crystals

Bacillary dysentery

  • Non-motile bacteria
  • Shigella
  • Plenty pus cells
  • RBCs

Treatment

Tetracycline and chloramphenicol is the drug of choice against the Shigella dysenteriae. The treatment should be continued for 5-7 days.

Prevention

It may prevent by following ways-

  • Using pure water supply
  • Maintaining personal hygiene
  • Proper disposal of sewage
  • Controlling of insects ( flies).

Further Readings

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. Topley & Wilsons Principle of Bacteriology, Virology and immunology Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  9. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
  10. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update
  11. https://www.cdc.gov/shigella/index.html#:~:text=Shigellosis
  12. https://www.mayoclinic.org/diseases-conditions/shigella/symptoms-causes/syc
  13. https://www.ncbi.nlm.nih.gov/books/NBK8038/
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