Shigella dysenteriae

Dysentery is a clinical condition of multiple origin. It could be bacillary or amoebic in nature. Dysentery is characterised by frequent discharge of blood stained, mucopurulent stools. Infection with this organism often leads to ulceration of the intestinal epithelium.

Shigella  - gram-negative, facultatively anaerobic, rod-shaped, non motile, non-sporing, non capsulated - extremely pathogenic and causes severe dysentery.   Fimbriae may be present.

Shigella produce an exotoxin (Shiga toxin) which disrupts protein synthesis and produces endothelial damage. Infection with this organism often leads to ulceration of the intestinal epithelium. Shigella spread via fecal-oral and person-to-person transmission

Kiyoshi Shiga - Japanese physician and bacteriologist- In 1896, Shiga discovered and identified Shigella dysenteriae which caused dysentery in Japan, and the Shiga toxin which is produced by the bacteria. He conducted research on other diseases such as tuberculosis and trypanosomiasis.

Most individuals are infected with Shigellae when they ingest food or water contaminated with human fecal material. This results in Bacillary dysentery.

Shigella can survive upto 30 days in milk, eggs and cheese.

Bacillary dysentery is characterized by severe abdominal cramps and the frequent painful passage of low volume stools containing blood and pus

MORPHOLOGY

Shigella are short Gram -ve rods-non-sporing, non-motile-non-capsulated. Fimbriae are present only in S. flexneri

CULTURAL CHARACTERISTICS 

Aerobic and facultative anaerobes. Optimum temperature 37°C. They grow on ordinary media however less readily than other Enterobacteria.

Nutrient agar and Blood agar

On Nutrient agar and Blood agar, colony are smooth, circular convex greyish or colorless, translucent often 2-3 mm diameter.

MacConkey agar (MA)

On MA, colonies are pale and yellowish (non-lactose fermenting). 

Exception S. sonnei being late lactose fermenting,  become pink when incubation period is prolonged

Deoxycholate citrate agar (DCA)

DCA - excellent selective medium for isolation of Shigella from faeces. Colonies are pale and similar to though usually slightly smaller 1-1.5mm diameter and more translucent than those of Salmonella. They do not form black center.

Xylose lysine deoxycholate agar (XLD)

XLD - best selective media for Shigella - less inhibitory to S. dysenteriae and S. flexneri than DCA. Colonies are red and unlike those of most Salmonella without black centers.

 

Peptone water and Nutrient Broth

Good growth with uniform turbidity on incubation over night at 37°C. In some cases, especially fimbriated form a surface pellicle on longer incubation.

Selenite F-broth

Selenite F-broth enrich S. sonnei and S. flexneri but inhibitory to other Shigella.

[ XLD- Xylose lysine deoxycholate agar 

•        yeast extract

•        sodium chloride (NaCl)

•        xylose

•        lactose

•        sucrose

•        lysine

•        sodium thiosulfate

•        ferric ammonium citrate

•        phenol red

•        sodium deoxycholate

•        agar

•        water

sodium deoxycholate as the selective agent inhibitory to gram-positive micro-organisms. 

Xylose is fermented by practically all enterics except for the Shigella 

Lysine -Salmonella would ferment the xylose and exhaust the supply of xylose;  then, lysine is attacked via the enzyme lysine decarboxylase- creates alkaline pH – red colour (which mimics the Shigella reaction).

Sodium thiosulfate and ferric ammonium citrate in the medium, helps in the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers- Salmonellae

Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow- E. coli 

Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers- Salmonellae

Lysine decarboxylation  causes reversion to an alkaline condition and the color of the medium changes back to red- Salmonellae  

Salmonella Typhi – Red Colonies, Black Centers

Shigella sonnei – Red Colonies

Shigella flexneri – Red Colonies

Escherichia coli – Large, Flat, Yellow Colonies; some strains may be inhibited]

Antigenic structure of Shigella

Shigella are differentiated by their ‘O’ antigens into serotypes.

These are classified into 4 structures or subgroups based on a combination of biochemical and serological characteristics.

Shigella dysenteriae (Sub groupA):

1)These are mannitol non-fermenting, consists of 12 serotypes.

2)Shigella dysenteriae type-1 forms exotoxin – Shiga toxin

3)3 types of toxic activity have been demonstrated in Shigella culture filtrates.( Neurotoxicity, enterotoxicity, and cytotoxicity/verotoxin)

Shigella flexneri (Subgroup B)

Named after Flexner, who first time described first of the mannitol fermenting Shigella from Phillipines (1900).

Based on type specific and group specific antigen, they have been classified into six serotypes (1-6) and several subtypes

Shigella flexneri - belongs to group B. 

S. flexneri infections can usually be treated with antibiotics, although some strains have become resistant.

Shigella boydii (Subgroup C):

Boyd first described this strain from India (1931).

S. boydii isolates -18 serotypes have been identified.

S. boydii is the most genetically divergent species of the genus Shigella

Shigella sonnei (Subgroup D):

Isolated by Danish bacteriologist Carl Olaf Sonne (1915) in Germany.

Ferment lactose and sucrose late, indole negative.

Causes mildest form of bacillary dysentery.

Shigella sonnei together with Shigella flexneri, is responsible for 90% of shigellosis cases. 

Sub group A	Subgroup B	Subgroup C	Subgroup D
Shigella dysenteriae	Shigella flexneri - Flexner from Philippines (1900).	Shigella boydii -Boyd from India (1931)	Shigella sonnei- Carl Olaf Sonne (1915) in Germany.
Mannitol non-fermenting	Mannitol fermenting	Mannitol fermenting	Ferment mannitol, also ferment lactose and sucrose late
12 serotypes	six serotypes (1-6) and several subtypes	18 serotypes have been identified.	Shigella sonnei together with Shigella flexneri- 90% of Shigellosis cases
Type-1 forms Shiga toxin- exotoxin- Neurotoxicity, enterotoxicity & cytotoxicity/verotoxin	Usually treated with antibiotics, although some strains have become resistant.	Most genetically divergent species of the genus Shigella	Mildest form of bacillary dysentery.

Resistance

1) Shigella are killed at 56°C in one hour and by 1% phenol in 30 minutes.

2) In ice they last for 1-6 months.

3) They remain viable in moist environment.

4) In faeces they die within few hours due acidity produced by growth of coliforms.

Biochemical tests of Shigella

Carbohydrate utilization:

1)Most strains utilize sugar to produce acid but not gas though some strain S. flexneri and S. boydii form gas.

2)Glucose is fermented by almost all strains.

3)Lactose is not fermented within 24 hrs.

4) S. sonnei and some strains of S. dysenteriae produce acid from lactose after prolonged incubation.

5)Mannitol fermentation - differentiates Group A strain (which do not ferment mannitol) from group B, C and D, most strains of which ferment it.

6) Sucrose is not fermented except S. sonnei and some strains of S. flexneri.

Methyl red test: +ve

VP test: -ve

Reduce nitrate to nitrite

Catalase +ve

Indole -ve

Citrate -ve

H2S -ve

Urease -ve

KCN growth -ve

Gelatin not liquified.

Decarboxylation test:

Group A, B and C fail to decarboxylate lysine and ornithine.

S. sonnei decarboxylate ornithine but not lysine

Epidemiology

• Human beings are the only natural hosts

• Transmission through

•        Contaminated water & food

•        Contaminated fingers, flies, food/faeces, fomites (door handles, water taps, lavatory seats) - Four “F” s

•        In young male homosexuals, through gay bowel syndrome

•        Low infective dose – as low as 100 Bacilli

Pathogenicity of Shigella dysenteriae

•        Shigella cause disease by invading and replicating in cells lining the colon.

•        Adhere to the cells, invade, replicate intracellularly and spread cell-to-cell.

•        They first attach to and invade the M cells located in Peyer patches

•        Shigella lyse the phagocytic vacuole and replicate in the host cell cytoplasm- cause necrosis of epithelial cells – superficial ulcers

• Cell-to-cell passage: The bacteria are propelled through the cytoplasm to adjacent cells, with the rearrangement of actin filaments in the host cells

• Shigella survive phagocytosis by inducing programmed cell death (apoptosis).

• This process releases IL-1β- attracts polymorphonuclear leukocytes into the infected tissues.
• This destabilizes the integrity of the intestinal wall and allows the bacteria to reach the deeper epithelial cells.

 

S. dysenteriae strains produce an exotoxin, Shiga similar to the verotoxin of E. coli O157:H7.

The toxin primarily

Ø  damages the intestinal epithelium;

Ø  also damage glomerular endothelial cells, resulting in renal failure (HUS)

 

The clinical features of Shigella dysenteriae type 1 infection includes:

1) toxemia, sometimes bacteremia and severe dysentery leading to marked dehydration and protein loss

2) Inflammation and ulceration of the large intestine

3) Hemorrhage, abdominal pain and high fever

4) Death from circulatory collapse or kidney failure

 

Shigella can cause

v Shigellosis

v Other complications

v  Rectal prolapse

v Toxic megacolon

v Hemolytic-uremic syndrome  (HUS)

 Shigellosis is characterized by:

1)Abdominal cramps    2)Diarrhea    3)Fever     4)Bloody stools

5)The clinical symptoms of the disease appear 1 to 3 days after the bacteria are ingested. Lasts for 5-7 days, recovery maybe in two weeks also (patient is contagious in this period).

6) The first sign of infection is profuse watery diarrhea which is mediated by an enterotoxin

7) Invasion of the colonic mucosa by the bacteria – result in  lower abdominal cramps and tenesmus (straining to defecate- feeling to defecate even if bowels are empty), with abundant pus and blood in the stool.

8)Abundant neutrophils, erythrocytes, and mucus are found in the stool.

9)Infection is generally self-limited, although antibiotic treatment is recommended to reduce the risk of secondary spread to family members and other contacts.

10)Asymptomatic colonization of the organism in the colon develops in a small number of patients – acts as a persistent reservoir for infection.

 

Complications of Shigellosis

          In some cases, Shigellae also cause inflammation of the lining of the rectum (proctitis) or rectal prolapse.

          In rare cases (more commonly in S. dysenteriae infection), “toxic megacolon” -a deadly complication - colon becomes paralyzed, preventing bowel movements - abdominal pain and swelling, fever, weakness, and disorientation.

          Untreated, the colon may rupture and cause peritonitis, a life-threatening condition requiring emergency surgery.

 

Hemolytic Uremic Syndrome (HUS)

1) Hemolytic-uremic syndrome (HUS) is a group of blood disorders primarily in children, characterized by HUS triad -acute kidney failure, destruction of red blood cells, and low platelets – due to Shiga toxin

2)HUS can occur after S. dysenteriae type 1 infection.

3)Convulsions in children

4)It is usually complicated by severe dysentery, intravascular volume depletion, and cardiovascular collapse; has a higher morbidity and mortality rate than E. coli associated HUS

 Shiga toxin enters the bloodstream and attacks endothelial cells throughout the body, damages the lining of small blood vessels and triggers tiny blood clots resulting in HUS triad

Ø  Microangiopathic Hemolytic Anemia: The destruction of red blood cells as they are forced through damaged, narrowed vessels, leading to anemia and fatigue.

Ø  Thrombocytopenia: A low platelet count, as platelets are rapidly consumed trying to repair the damaged vessel walls.

Ø  Acute Kidney Injury (AKI): Impaired kidney function (azotemia) caused by clots clogging the small blood vessels in the kidneys.

 

 Lab diagnosis

Sample

• Fresh stool sample
• Rectal swab
• Serum

Laboratory Diagnosis

Microscopy

• Gram negative, rods, along with pus cells and RBC's.
• Non-motile, non-capsulated, non-spore forming

  

Biochemical Tests

• Oxidase negative
• Catalase positive

 

Cultural Characters

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

Deoxycholate citrate agar (DCA): Colorless colonies , (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 without black centers.

Salmonella-Shigella (SS) agar: Colorless colonies with no blackening

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

 

              MacConkey agar                              Deoxycholate citrate agar (DCA)              

  

                   SS Agar                                                                        XLD Agar

Serological Diagnosis

• Using monovalent and polyvalent anti-sera
• For serotyping- to know the strain and not for lab diagnosis

Treatment

• Uncomplicated Shigellosis – self-limiting – rest & rehydration must- no antibiotics for adults.
• For infants & children – oral rehydration to be ensured
• Perform antibiotic sensitivity test
• Fluoroquinolones such as ciprofloxacin or cotrimoxazole
• The treatment should be continued for 5-7 days.

�    Postinfection carriage is generally less than 3-4 weeks.

�    Mild cramps and diarrhea may continue for many days to weeks after treatment of shigellosis.

PREVENTION AND CONTROL

• No vaccine currently available.
• Personal & environmental hygiene

Prevention by

1) Use of safe drinking water

2) Chlorination of water source

3) Strict handwashing

4) Refrigeration and proper preparation and cooking of food

5) Food handlers must be treated with antibiotics and should not be involved in food preparation as long as stool cultures are positive for Shigella infection.

 

Cell disruption technique by repeated freezing and thawing

 

Aim
To demonstrate the extent of cell disruption by freezing and thawing. 

Principle
Microorganisms are protected by extremely tough cell walls. In order to release their cellular contents during downstream processing a number of methods are available for cell disruption. Cell disruption can be done by physical or chemical methods.

 Repeated freezing and thawing is a physical cell disruption method used to break cells and release intracellular materials. The technique works on the principle that ice crystals are formed during freezing and their melting during thawing damage the cell membrane and cell wall, leading to cell lysis. During freezing treatment, water inside cells forms ice crystals which expand and puncture cellular structures during thawing. Rapid melting causes osmotic shock and membrane rupture.  Repeated cycles increase the extent of cell disruption. The intracellular contents comes out which can be easily separated by centrifugation. By plating microbial suspension after centrifugation the number of viable cells can be obtained.

 It is crucial that cell disruption methods do not denature labile materials present in the cell. Repeated freezing and thawing is a simple, inexpensive, and chemical-free cell disruption technique that efficiently releases intracellular biomolecules while preserving many heat-sensitive and biologically active compounds. It is commonly used for laboratory-scale extraction of biomolecules from microbial and animal cells.

Materials  required
1. Overnight grown culture of E.coli

2. Sterile distilled water
3. Sterile saline
4. Nutrient Agar
5.Routine Microbiological facilities

 

Procedure:
1.10 ml of distilled water and saline were taken in sterile test tubes.
2. Test tubes were inoculated with 0.5 ml of overnight broth culture of E coli.

3. One test tube each of distilled water and saline were kept at 5°C.
4. The second pair of test tube with distilled water and saline was  kept at room temperature for next 1hour. This process was repeated for 4 times
5. 0.1 ml sample were taken from each tube and proper dilution was plated.
6. The plates were incubated at 37 °C overnight and colonies were counted.

Result

A fall in cell count was obtained in tubes subjected to freezing and thawing

 

 

 

Methanogenesis

Methanogenesis, or biomethanation, is a form of anaerobic respiration that uses carbon as the terminal electron acceptor, resulting in the production of methane by the reduction of CO₂ to CH₄. H₂ is commonly used as electron donor however, formate, CO₂ and even certain organic compounds such as alcohol may also be used as electron donors.

Methanogenesis (methane production) is characteristic to a group of obligate anaerobic archaea (archaebacteria) called the methanogens (e.g., Methanobacterium, Methanobrevibacter, Methanococcus, Methanogenium, Methanospirillum, Methanomicrobium, etc.).

Methanogenesis involves the anaerobic conversion of carbon compounds to methane and typically follows four steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. During hydrolysis, complex organic matter is broken down, followed by acidogenesis to produce volatile fatty acids. Acetogenesis converts these fatty acids into acetate. Finally, methanogens (archaea) utilize acetate, CO₂, and hydrogen gas to produce methane. 

The reduction of CO₂ to methane can be summarised in the following way

1. Hydrolysis:

Complex organic matter (proteins, carbohydrates, lipids) are broken down into simpler, smaller molecules, such as sugars, fatty acids and amino acids, by hydrolytic bacteria e.g., Clostridium spp.

2. Acidogenesis:

Acidogenic bacteria further break down the simpler organic compounds into volatile fatty acids (VFAs), alcohols, H₂, and CO₂. e.g., Bacteroides, Lactobacillus. 

3. Acetogenesis:

Acetogenic bacteria convert the volatile fatty acids, alcohols and other products from the previous steps into acetate, carbondioxide, and hydrogen gas. Acetogenic bacteria include Syntrophomonas, Syntrophobacter etc.

    Acetogenic bacteria occur in syntrophy with methanogens — hydrogen must be kept at low levels for the reaction to proceed efficiently.

4. Methanogenesis:

This is the final step, carried out by methanogenic archaea. They consume the acetate, CO₂, and H₂ produced in the earlier stages to generate methane. 

There are three primary types of methanogenesis, depending on the methanogens and the substrates they use:

• Hydrogenotrophic methanogenesis: Methanogens reduce CO₂ with hydrogen gas (H₂) to produce methane. eg., Methanobacterium 

            CO₂ + H₂ → CH₄ + H₂O (Hydrogenotrophic pathway)

• Aceticlastic methanogenesis: Methanogens cleave acetate directly into methane and CO₂. eg., Methanosarcina and Methanotricha

            CH₃COOH → CH₄ + CO₂ (Aceticlastic pathway)

• Methylotrophic methanogenesis: Methanogens convert methylated compounds, such as methanol or methylamines, into methane. eg., Methanomethylovorans 

            Methyl compounds → CH₄ (Methylotrophic pathway)

Ecological Role of Methanogens:

• • Key players in anaerobic ecosystems – they remove end products like H₂ and acetate, allowing upstream fermentative and syntrophic processes to continue.

  • Major contributors to global methane emissions, influencing climate change.

  • Used in biogas production for renewable energy (methane as fuel).

The process of methanogenesis is crucial for the degradation of organic matter in anaerobic environments, such as wetlands, animal digestive tracts, and anaerobic digesters used in waste treatment. Methanogenesis is the primary pathway that breaks down organic matter in landfills (can release large volumes of methane into the atmosphere if left uncontrolled). It can be used to treat organic waste and to produce useful compounds. Biogenic methane can be collected and used as a sustainable alternative to fossil fuels.

The production of methane is an important and widespread form of microbial metabolism, and in most environments, it is the final step in the decomposition of biomass. During the decay process, electron acceptors (such as oxygen, ferric iron, sulfate, and nitrate) become depleted, while hydrogen (H₂), carbon dioxide, and light organic compounds produced by fermentation accumulate. Without methanogenesis, a great deal of carbon (in the form of fermentation products) would accumulate in anaerobic environments.

Methanogenesis also occurs in the guts of humans and other animals, especially ruminants. In the rumen, anaerobic organisms, including methanogens, digest cellulose into forms usable by the animal. Without these microorganisms, animals such as cattle would not be able to consume grass. The useful products of methanogenesis are absorbed by the gut. Methane is released from the animal mainly by belching.The average cow emits around 250 liters of methane per day. 

Methane is one of the earth’s most important greenhouse gases, with a global warming potential 25 times greater than carbon dioxide. Therefore, the methane produced by methanogenesis in livestock contributes to global warming.