Biology

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Vibrio Cholerae

Vibrio is one of the curved rod bacteria, prominent in the Medical Bacteriology. They are present in marine environment and surface waters worldwide.

Vibrio is a member of the family Vibrionaceae. The most important member of this genus is Vibrio cholerae, the causative agent of cholera. The term Vibrio is derived from Vibrare (Latin word) which means “to shake or vibrate” and the word Cholera is derived from Chole (Greek word) which means, “to bile”.

Morphology

Vibrio cholerae is gram negative, curved or comma shaped, (1.5um × 0.2 – 0.4um in size) non – capsulated. The organism is very actively motile with a single polar flagellum and the characteristic movement is called as darting motility. In stained smears of mucus flakes from acute cholera patients, the Vibriois seen
arranged in parallel rows. This was described by Robert Koch as “fish in stream” appearance.

Culture Characteristics

Vibrio cholera is strongly aerobic. It grows best in alkaline media with the optimum temperature 37°C and pH 8.2. It is nonhalophilic, therefore, cannot grow in media with a concentration of sodium chloride more than 7% (Figure 7.17). Some of the media in which Vibrio cholerae are cultivated are tabulated below
in Table 7.16.

Table 7.16: Colony morphology of Vibrio cholerae on various media

Enterotoxin

Vibrios multiplying on the intestinal epithelium produce an enterotoxin called Cholera toxin. It is also known as Choleragen (or CT). This toxin molecule is approximately 84,000 Dalton and consists of two major subunits namely A and B There is only one subunit in A (1A) whereas there are five subunits in B (5B) (Figure 7.18).

Mode of Action

• The B (binding) units of enterotoxin get attached to the GM₁ (Ganglioside membrane receptors I) on the surface of jejunal epithelial cells. (target cells).
• The A (active) subunits then enters the target cell and dissociates into 2 fragments, A₁ & A₂. The A₂  fragment links biologically active A₁ fragment to the B – subunit.
• The A₁ fragment causes prolonged activation of cellular adenylate cyclase which in turn accumulates CAMP in the target cell. This leads to outpouring of large quantities of water and electrolytes into small intestinal lumen. Thus, resulting in profuse watery diarrhea.

Pathogenesis

The pathogenic mechanism of Vibrio choleraeis discussed below in flowchart 7.7.
Source of Infection – contaminated water or food
Route of entry – fecal – oral route
Site of infection – small intestine
Incubation period – few hours to 5 days (usually 2 – 3 days)

Clinical Feature

Dehydration, anuria (absence of urine excretion), muscle cramps, hypokalemia (low blood potassium) & metabolic acidosis (low serum concentration of bicarbonates).

Laboratory Diagnosis

Specimen: Stool

Direct microscopy:

It is not a reliable method for rapid diagnosis, the characteristic darting motility of the vibrio can be observed under dark – field microscope.

Culture:

Stool sample is directly inoculated on MacConkey agar and TCBS agar. The plates are examined after overnight incubation at 37°C for typical colonies of Vibrio cholera, and the colonies are identified by gram staining and oxidasetest.

Prophylaxis

1. General Measures:

• Purification of water supplies
• Improvement of environment sanitation
• Infected patients should be isolated, and their excreta must be disinfected

2. Vaccines:

Two types of oral vaccines have been tried recently:

• Killed oral whole cell vaccines
• Live oral vaccines

Treatment

1. Oral Rehydrationtherapy:

The severe dehydration & salt depletion can be treated by oral rehydration therapy (as recommended by WHO).

2. Antibiotics:

It is of secondary importance, oral tetracycline was recommended for reducing the period of Vibrio excretion.

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Salmonella typhi of Bacteriology

The genus Salmonella consists of bacilli that parasites the intestines of vertebrates and human beings. It causes Enteric fever, which includes Typhoid and Paratyphoid fever. The most important species of the genus is Salmonella typhi which causes typhoid fever.

Morphology

Salmonellae are Gram – negative rods (1 – 3µm × 0.5 µm in size). They are motile with peritrichous flagella, non – capsulated and non – sporulated (Figure 7.14).

Cultural Characteristics

They are aerobic and facultative anaerobe, optimum temperature – 37°C and pH is 7 – 7.5. They grow on the following media and show the following characteristic colony morphology (Table 7.12).

Table 7.12: Colony morphology of Salmonella typhi

Pathogenicity

Salmonella typhi causes typhoid fever and its pathogenesis is discussed in flowchart 7.6.

Source of infection – food, feces, fingers, flies
Route of entry – faecal oralroute (ingestion)
Incubation period – 7 – 14 days

Clinical Manifestations

• The illness is usually gradual, with headache, malaise (feeling of discomfort), an2orexia (loss of appetite), coated tongue, abdominal discomfort with either constipation or diarrhea.
• Hepatosplenomegaly (enlargement of liver and spleen), step ladder pyrexia (continuous fever) and rose – spots (during 2^2nd or 3^rd week).

Laboratory Diagnosis

Specimens:

Blood, stool and urine are the clinical samples collected from typhoid patients. The selection of relevant specimen depends upon duration of illness, which is very important for diagnosis (Table 7.13 & Figure 7.15).

Table 7.13: Specimen collection for typhoid

The bacteriological diagnosis of enteric fever consists of the following methods, which are:

• Isolation of the bacilli
• Demonstration of antibodies

Isolation of the bacilli

The typhoid bacilli are isolatedfrom the following clinical specimens which are tabulated (Table 7.14).

Table 7.14: Isolation method of typhoid bacilli from various clinical speciments.

Demonstration of Antibodies:

Slide – agglutination: The isolate is identified by slide agglutination with ‘O’ and ‘H’ antisera.

Widal Test:

It is an agglutination test for detection of agglutinins ‘H’ and ‘O’ in patients with enteric fever. Salmonella antibodies start appearing in the serum at the end of 1^st week and rise sharply during the 3^rd week of enteric fever.

Prophylaxis

Various types of vaccine and their doses are given in Table 7.15.

Table 7.15: Various types of vaccine and their doses.

Treatment and Control Measures

• Antibacterial therapy has been very effective in the treatment of patients.
• Ampicillin, amoxicillin and cotrimoxazole are useful in the treatment of typhoid fever.
• At present, ciprofloxacin is the drug of choice.
• Typhoid fever can be effectively controlled by sanitary measures for disposal of sewage, clean water supply and supervision of food processing and handling.

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Medical Shigella dysenteriae

The genus Shigellaare exclusively parasites of human intestine and other primates. Shigella dysenteriae is the causative agent of bacillary dysentery or shigellosis in humans. It is a diarrheal illness which is characterized by frequent passage of bloodstained mucopurulent stools.

The four important species of the genus Shigella are: Shigella dysenteriae, Shigella flexneri, Shigella sonnei and Shigella boydii.

Morphology

Shigella are short, Gram negative rods (0.5µm × 1-3 µm in size). They are non – motile, non – sporing and non – capsulated (Figure 7.12).

Cultural Characteristics

• They are aerobes and facultative anaerobes. Optimum temperature is 37°C and optimum pH – 7.4.
• They can be grown on the following media and show the characteristic colony morphology (Table 7.10 & Figure 7.13)

Table 7.10: Colony morphology of Shigella

Toxins

Shigella dysenteriae produces toxins, which is of 3 types, namely, endotoxin, exotoxin and verocytotoxin. The mode of action of these toxins is illustrated in the Table 7.11.

Table 7.11: Various toxins of Shigelladysenteriae

Pathogenesis

The pathogenic mechanism of Shigella dysenteriaeis discussed below in flowchart 7.5.

Source of Infection – Patient or carriers
Route of entry – faecal – oral route
Site of infection – Large intestine
Incubation Period – Less than 48 hours (1-7 days)
Mode of transmission – Food, finger, faeces and flies

Clinical Manifestations

• Frequent passage of loose, scanty faeces containing blood and mucus.
• Abdominal cramps and tenesmus (straining to defecate).
• Fever and vomiting.
• Hemolytic uremic syndrome (It is a condition caused by the abnormal destruction of red blood cells)

Laboratory Diagnosis

Specimens:
Fresh stool is collected.

Direct Microscopy:
Saline and Lugol’s iodine preparation of faeces show large number of pus cells, and erythrocytes.

Culture:
For inoculation, it is best to use mucus flakes (if present in the specimen) on MacConkey agar and SS agar. After overnight incubation at 37°C, the plates are observed for characteristic colonies, which is confirmed by Grams staining and biochemical reactions.

Treatment and Prevention

• Uncomplicated shigellosis is a self – limiting condition that usually recovers spontaneously.
• In acute cases, oral rehydration therapy (ORT) is done.
• In all severe cases, the choice of antibiotic should be based on the sensitivity of prevailing strain.
• Many strains are sensitive to Nalidixic acid and Norfloxacin.
• Improving personal and environmental sanitation.
• The detection and treatment of patients and carriers.

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Clostridium tetani of Medical Bacteriology

The genus Clostridium consists of anaerobic, spore forming Gram positive bacilli. The spores are wider than the bacterial bodies, giving the bacillus a swollen appearance resembling a spindle. The name Clostridium is derived from the word ‘kluster’ (a spindle).

Most species are saprophytes found in soil, water and decomposing plant and animal matter. Some of the pathogens are normal flora of intestinal tract of human and animals.

The genus Clostridium includes bacteria that causes 3 major diseases of human – Tetanus, gas gangrene and food poisoning. Clostridium pathogenicity is mainly due to production of a powerful exotoxin.

Clostridium of medical importance may be classified based on diseases they produce, which is given the Table (7.7).

Table 7.7: Clostridium sp, causing pathogenic diseases.

Morphology

They are Gram positive spore forming rods. The spores are spherical and terminal in position giving a drumstick appearance. They are motile and non – capsulated.

Culture Characteristics

• They are obligate anaerobes, optimum temperature is 37°C and pH is 7.4.
• It grows on ordinary media, but growth is enhanced by addition of blood and serum. Clostridia tetani grows on the following media and show the characteristic colony morphology (Table 7.8).

Table 7.8: Colony characteristics of Clostridium tetani

Toxins

Clostridium tetani produces two distinct toxins namely,

• Tetanolysis (haemolysin)
• Tetanospasmin (neurotoxin)

Tetanolysis

• Heat labile and oxygen labile toxin.
• It lysis erythrocytes and also acts as neurotoxin.

Tetanospasmin

• It is heat labile and oxygen stable powerful neurotoxin.
• It is protein in nature. consisting of a large polypeptide chain (93,000 Dalton) and a smaller polypeptide chain (52,000 Dalton) joined by a disulphide bond.
• Mode of Action: Tetanospasminis a neurotoxin, which blocks the release of inhibitory neurotransmitters (glycine and gamma – amino butyric acid) across the synaptic junction.
• The toxin acts presynaptically, the abolition of spinal inhibition causes uncontrolled spread of impulses in CNS (Central Nerves System).

This results in muscle rigidity and spasms (due to the simultaneous contraction of agonists and antagonists, in the absence of reciprocal inhibition (Figure 7.11).

Pathogenesis

Clostridium tetani is the causative organism of tetanus or lock jaw disease. pathogenesis of Clostridium tetani was discussed in detail in flowchart 7.4.

Source of infection – Soil, dust, faeces.
Route of entry – Through wound
Incubation period – 6 – 12 days

Clinical Feature

It includes, pain and tingling at the site of wound, Lock jaw ortrismus (It is reduced opening of the jaws), Risus sardonicus (mouth kept slightly open), Dysphasia (impairment of the ability to speak or to understand language) and acute asphyxia.

Laboratory Diagnosis

Specimens:
Wound swab, exudates or tissue from wound.

Microscopy:
Gram staining shows Gram positive bacilli with drumstick appearance.

Culture:
The clinical specimen is inoculated on blood agar and incubated at 37°C for 24-48 hours under anaerobic conditions. The colonies are confirmed by gram staining, where it shows gram positive bacilli with drumstick appearance.

Treatment

Tetanus patients are treated in special isolated units, to protect them from noise and light which may provoke convulsions. The spasm can be controlled by diazepam (0.1 – 0.2 mg/kg) injection. Antibiotic therapy with pencilin or metroniadazole should be done for a week or more.

Prophylaxis

It is done by the following methods, which are as follows.

a. Surgical prophylaxis:

It aims at removal of foreign body, blood clots and damaged tissue in order to prevent anaerobic conditions favorable for the
germination of spores.

b. Immunoprophylaxis:

Tetanus is a preventable disease. Immune prophylaxis is of 3 types, which is given in the (Table 7.9).
Table 7.9: Immunization for tetanus.

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Corynebacterium diphtheriae

Several species of the genus Corynebacterium are normal flora of skin, upper respiratory tract (URT), urogenital and intestinal tract. The most important member of the genus is C. diphtheriae the causative agent of diphtheria, a localized inflammation of the throat with greyish white pseudomembrane and a generalized toxemia due to the secretion and dissemination of a highly potent toxin.

The name Corynebacterium diphtheria is derived from Greek word ‘Coryne’ – “Club shaped swellings” or “Knotted rod” ‘Diphthera’ – Leather.

Morphology

They are Gram positive slender rods, pleomorphic club shape or coryneform bacterium Non – motile, non – sporing and non – capsulated (Figure 7.9 a & b).

The bacilli are arranged in a characteristic fashion in angular fashion resembling the letters V or L. This has been called Chinese letter or cuneiform arrangement (Figure 7.10).

They are club shaped due to the presence of metachromatic granules at one or both ends. These granules are composed of polymetaphosphates and represent energy storage depots.

Cultural Characteristics

• They are aerobic and facultative anaerobe. Optimum temperature is 37°C and pH 7.2.
• They grow on the following media and show the characteristic colony morphology (Table 7.5).

Table 7.5: Colony Morphology of Corynebacterium diphtheriae on cultural media

Toxin

• The pathogenicity is due to production of a very powerful exotoxin by virulent strains of diphtheria bacilli.
• The toxigenicity of diphtheria bacillus depends on the presence of a tox⁺ gene which can be transferred from one bacterium to another by lysogenic bacteriophages, of which beta phage is the most important.

Properties

The diphtheria toxin is a heat – labile protein and has a molecular weight of about 62,000 Dalton. It consists of two fragments

• Fragment A (24,000 Dalton) – It has all enzymatic activity.
• Fragment B (38,000 Dalton) – It is responsible for binding the toxin to the target cells.

Mode of Action

The toxin acts by inhibiting protein synthesis, specifically fragment A inhibits polypeptide chain elongation in the presence of NAD by inactivating the elongation factor (EF – 2) the toxin has special affinity for myocardium, adrenal gland and nerve endings.

Pathogenicity

Source of infection – Airborne droplets
Route of entry – Upper respiratory tract
Incubation period – 3 – 4 days

Site of infection – Faucial (nasal, otitis, conjunctival, laryngeal, genital) diphtheria is most commonly seen in children of 2-10 years.

Faucial diphtheria is the most common type. The infection is confined to humans only. The toxin has both local (flowchart 7.3) as well as systemic effects.

Flowchart 7.3: Localized effect of diphtheria toxin

Systemic effects

The toxin diffuses into the blood stream and causes toxemia. It has got affinity for cardiac muscle, adrenal and nerve endings. It acts on the cells of these tissues.

Clinical Manifestations

1. Laryngeal obstruction, asphyxia (it is a condition of severe deficient supply of oxygen, causing suffocation).
2. Diphtheritic myocarditis (inflammation of heart muscle), polyneuropathy (damage of multiple peripheral nerves), paralysis of palatine (the top part of the inside of the mouth) and ciliary muscles.
3. Degenerative changes in adrenal glands, kidney and liver may occur.

Specimen:
Two swabs from the lesions are collected. One swab is used for smear preparationand other swab for inoculation on culturemedia.

Direct microscopy:
Smears are stained with both Gram stain and Albert stain.

• Gram Staining – Gram positive slender rods were observed.
• Albert staining – Club shaped with metachromatic granules were observed.

Culture:
The swabis inoculated on Loeffler’s serum slope, after overnight incubation at 37°C, the plates were observed for characteristic colonies, which are identified by gram staining.

Prophylaxis

Diphtheria can be controlled by immunization. Three methods of immunization are available (Table 7.6).

Table 7.6: Immunization for diphtheria

Treatment

The specific treatment for diphtheria consists of administration of antitoxin with dose of 20,000-100,000 units of ADS intramuscularly and antibiotic therapy using penicillin.