Diphtheria bacteria (Corynebacterium diphtheriae)

2021-08-19 01:41 PM

Diphtheria bacteria are aerobic bacteria. Grows well in normal culture, but grows well and quickly in blood and serum

In 1826, Klebs observed and described the diphtheria bacillus, one year later in 1884 Loeffler isolated this bacterium, then Roux and Yersin discovered the exotoxin (1888). Roux prepared antitoxin serum to cure disease (1894) and Ramon prepared diphtheria toxoid to prevent disease (1924). Diphtheria bacteria belong to the family Corynebacteriaceae. This family includes the genera Corynebacterium, Listeria, Erysipelothrix, mostly non-pathogenic, parasitic in soil, animals, and humans, a few causing diseases in humans.

Shape

Diphtheria bacteria have a size of 0.5 -1x 2 - 8 µm, straight or slightly curved rod shape, rounded ends, and often bulge larger than the body, making the bacteria club-shaped. Bacteria can form a barrier or form the letter H, V, X, Y ... Bacteria are not mobile, do not have a shell, do not produce spores. Diphtheria bacillus picks up Gram-positive color, but with prolonged bleaching, it easily loses its purple color. When staining bacteria with methods such as Albert or Neisser, there will be chromophore particles (volutin particles) that are blackened differently from the color of the bacterial body.

Viral Culture 

Diphtheria bacteria are aerobic bacteria. Grows well in normal culture, but grows well and quickly in blood and serum. A suitable temperature is 37oC, suitable pH 7.6 - 8.

In Loeffler serum coagulation medium, egg medium, bacteria grow quickly, 10-18 hours later they form small, round, convex, evenly bordered colonies with light gray color.

In media containing 0.3% potassium tellurite such as Mac-Leod medium, Schroer's medium, diphtheria bacteria grow into black or dark gray colonies depending on the type.

In blood agar: bacteria grow to form colonies with regular margins, high center, milky white color, and hemolytic rings around the colonies depending on the type. Based on the ability to hemolysis, people distinguish 3 types: gravis, mitis and intermedius

In broth the bacteria cause slight turbidity, forming particles that stick to the tube wall and appear film on the surface of the medium.

Biochemical properties

 Fermented and not vaporized sugars: glucose, galactose, not fermenting sucrose, and lactose.        

 To distinguish between true diphtheria bacteria and pseudodiphtheria bacilli (Corynebacterium hoffmani, Corynebacterium xerosis), people rely on the table of biological and chemical properties below:

Table of basic biochemical properties

Bacteria

Glucose

Saccharose

Starch fermentation

Hemolysis

Corynebacterium diphteriae gravis

+

-

+

-

Corynebacterium diphteriae mitis

+

-

-

+

Corynebacterium diphteriae intermedius

+

-

-

-

 Corynebacterium hoffmani

-

-

-

-

 Corynebacterium xerosis

+

+

+

-

 Resistance abilities

Bacteria are sensitive to heat, at 56oC die within 5 minutes. In the pseudomembranous region and when attached to toys and clothing, bacteria survive for a long time at normal temperatures. Under dry and cold conditions, diphtheria bacteria are more resistant than other non-spore bacteria. Common disinfectants kill bacteria quickly after 1 minute.

Possibility to produce toxins

Diphtheria bacilli produce exotoxins when lysed with phage b. The presence of prophage gave bacteria genes capable of synthesizing toxins. Their toxin formation also depends on a number of other factors such as Fe ++ concentration in the environment, osmotic pressure, amino acid concentration, an appropriate source of N2 and C.

The nature of diphtheria exotoxin is a heat-labile protein, a strong toxin, 1mg of toxin can kill 1,000 guinea pigs weighing 250g after 96 hours. If used with 0.3-0.4% formol at a temperature of 40oC after 1 month, the diphtheria toxoid turns into an antitoxin that can be used to make a vaccine against diphtheria.

Antigen structure

Toxic antigens: Toxins isolated from all diphtheria strains show immunological similarity, i.e. form a single resistant toxin, which ensures the success of the vaccine in prevention. sick.

Bacterial antigens: Surveys showed that polysaccharide antigens on the surface of bacteria in all 3 types of gravis, mitis, intermedius were not different while pseudo diphtheria bacilli did not have this antigen.

Possibility to cause disease in humans

Diphtheria bacillus is the cause of diphtheria in humans, most commonly in children, aged 2-7 years. The disease occurs all over the world, occurs all year round, but is more common in the cold season. The disease is mainly transmitted through the respiratory tract when the child speaks, coughs or sneezes. In addition, it can be transmitted indirectly through toys and clothes of children.

Diphtheria is an acute infectious and toxic disease that can cause epidemics and has a relatively high mortality rate. The disease has 2 main manifestations: causing pseudomembranous membranes in the throat with lymphadenopathy in the neck and systemic toxicity.

Pathogenesis

Diphtheria bacteria enter the respiratory tract, reside in the pharynx and pharynx mucosa, grow in the upper respiratory tract, and secrete exotoxins. On the one hand, bacteria and toxins cause local ulcers to form a gray-white pseudo-membrane that adheres tightly to the mucosa, peels off, causes bleeding, and is put into insoluble water. Pseudo-plaques usually appear first in the pharynx and then spread up the nasal passages or down the trachea. Laryngeal diphtheria is particularly serious because it causes difficulty breathing. On the other hand, blood-borne exotoxins affect the nervous system, causing phenomena such as paralysis of the palate, eye muscles, extremities and damage to the adrenal glands. arrhythmia, heart failure...

There is also diphtheria in the skin or in the wounds, where pseudomembranous membranes are also formed, but the dispersion of the toxin is usually mild, causing no significant symptoms.

Immune

The virulence of diphtheria bacteria depends on a single toxin type, so the acquired immunity after the underlying disease is antitoxin. Newborns gain temporary immunity from maternal antitoxins that cross the placenta, this passive immunity lasts only a few months. Most children, especially 1 to 7 years old, are very susceptible to diphtheria bacteria. In the process of growing up, the body will create active immunity on its own, due to illness or mild infection, due to living near healthy people carrying bacteria. Currently in our country, the older the children, the less likely they are to get the disease, and the adults almost do not get diphtheria.

To assess the immune status of diphtheria toxin, the Shick reaction is used: 0.1 ml of diphtheria toxin is injected intradermally in the front of the arm. Positive reaction, after 24 - 48 hours, the injection site emerges a red, hard halo with a diameter of 1-2cm, the local inflammatory response peaks within 5 days and then fades. This proves that the toxin is not neutralized by antitoxin, the body can absorb diphtheria bacteria. If the reaction is negative, then there is no reaction at the injection site, which means that the toxin has been neutralized by antitoxin. However, it is necessary to simultaneously inject the same amount of toxin into the control hand but distilled at 60oC for 15 minutes to destroy the effect of the toxin, the control party will have no reaction or if it does, it will disappear very quickly during the treatment. A truly positive Shick response is associated with an inflammatory response that persists for several days.

Diagnose

The only method is to isolate the bacteria. Specimens are pseudomembranous membranes or throat swabs at the site of the lesion.

Direct survey of the patient

Make Gram stain, Albert stain to detect bacteria. If you see a typical bacterial shape, it will give preliminary results so that the clinical direction can be treated promptly.

Culture, isolation, identification

Inoculate the specimens into suitable media such as egg medium, tellurite medium at 37oC for 18-24 hours, then select typical colonies to investigate morphological, biochemical and toxicological properties. of diphtheria bacteria by one of the following methods:

Neutralization reaction in rabbit skin

Shave the rabbit's ribs using a pencil to divide the small squares. Intradermal injection of 0.2ml of test diphtheria broth for 48 hours into 1 cell (at the same time injecting 0.2ml of toxic diphtheria broth as a control into the adjacent cell), 5-7 hours after intravenous injection of 1,000 units diphtheria antitoxin. Immediately then intradermal injection into another cell 0.2 ml of test broth. Read results after 48 - 72 hours. If the test bacteria produce toxins, in the first injection plot, a necrotic nodule appears 5 - 10 mm, surrounded by a red area of ​​10-15 mm like the control plot, while in the second injection plot, only a pink nodule 10-15mm.

Eleck's reaction

Place a strip of blotting paper containing diphtheria antitoxin in the center of the bottom of an agar plate containing 20% ​​horse or calf serum. The test bacteria were inoculated perpendicular to the piece of paper. Antitoxin diffuses into the environment when met with bacterial toxins will form white precipitates in agar as well as virulent diphtheria strains test.

Co-agglutination reaction

Culture the test diphtheria bacteria on egg medium at 37oC overnight, then add 1ml of sterile phosphate buffer (PBS) (pH: 7.2) for 1 hour, then remove PBS for centrifugation. , separate the supernatant for the determination of diphtheria exotoxins using a slide divided into 3 cells:

Box 1: Mix test diphtheria supernatant with SAD-affixed staphylococci (Serum Anti Diphterie).

Box 2: Consists of rabbit serum-bound staphylococci with diphtheria supernatant.

Box 3: Consists of SAD-bound staphylococci with PBS buffer.

Cells 2, 3 are 2 negative control cells. Gently shake the slide for 1 minute and read the result after 15 - 30 minutes. The reaction is positive when in the test plot (cell 1) there are agglutination particles visible to the naked eye or with a magnifying glass, and in the 2 control plots there is no agglutination.

Prevention and treatment

Prevention

Diphtheria spreads mainly through the respiratory tract, so it is necessary to thoroughly isolate and treat sick and healthy carriers. The current best preventive measure is a systemic diphtheria toxoid vaccine given to children under 1 year of age to induce basal immunity. Repeat vaccination after 1 year and 5 years to strengthen immunity. Currently, our country is using the mixed vaccine DTC (Diphterie-Tetanie-Cough).

Treatment

As a rule:

Neutralize diphtheria toxoid by prompt injection of diphtheria antitoxin (SAD).

Kill diphtheria bacteria with antibiotics such as penicillin, erythromycin...

Resolve patient asphyxiation by administering oxygen or tracheostomy.