Cultivation characteristics
Diphtheria is an aerobic or facultative anaerobic bacterium, the optimum temperature is 37℃, and the optimum PH is 7.2 ~ 7.8, and it grows well on the culture medium containing blood, serum or eggs. The colony is gray, smooth and round, and can absorb tellurium when growing and propagating on serum medium containing 0.033% potassium tellurite, and reduce it to metallic tellurium, making the colony black, which is the same as other Corynebacterium in this genus. And potassium tellurite can inhibit the growth of other bacteria in the sample, so potassium tellurite blood agar plate can be used as rod-shaped selection medium. According to the characteristics and biochemical reactions of diphtheria bacteria on this medium, diphtheria bacteria can be divided into three types: severe, intermediate and mild. The distribution of the three types of diphtheria bacteria is different, often in different regions and years, which is of epidemiological significance.
resist
The resistance to damp heat is not strong, and it is sensitive to general disinfectants. It died quickly after 60℃ 10 min or after boiling, and died after 1% carbolic acid 1 min, but its anti-drying, cold-resistant and sun-proof abilities were stronger than those of other spore-free bacteria, and it could survive for many days in daily necessities, food and clothes. This strain is sensitive to penicillin and common antibiotics.
pathogenicity
The pathogenic substance of this bacterium is mainly diphtheria toxin. Diphtheria toxin is a polypeptide chain with two disulfide bonds and its molecular weight is 62,000. After hydrolysis by protease, it can be divided into two fragments, A and B, which are still connected by disulfide bonds. B fragment, which has no enzyme activity, can bind to specific receptors on the surface of host susceptible cells, so that A fragment can enter the cells through translocation. Fragment A has enzymatic activity, which can hydrolyze oxidized nicotinamide adenine dinucleoside (NAD+) into nicotinamide and adenine ribose diphosphate (ADPR), and catalyze the valence binding of elongation factor -2 (EF-2) with ADPR, so that EF-2 loses its translocation activity, thus preventing the transfer of peptide -tRNA and mRNA from receptor to donor on ribosomes. Only lysogenic diphtheria bacillus carrying β -coryneform phage can produce exotoxin, because diphtheria toxin is protein encoded by β -coryneform phage toxin gene (tox+). The expression of tox+ gene is closely related to the inorganic iron content of bacteria. When the iron content is moderate, the tox+ gene is expressed, otherwise it is not expressed.
Diphtheria also produces some invasive substances, such as fungin similar to mycobacterium tuberculosis, which can destroy the mitochondrial membrane of cells and lead to respiratory inhibition and oxidative phosphorylation.
pathogenesis
Diphtheria is less invasive. After invading the upper respiratory mucosa, it only reproduces in superficial epithelial cells and generally does not cause bacteremia. When the local mucosa is damaged, such as measles, scarlet fever, whooping cough or upper respiratory tract infection, the invasion of diphtheria bacillus is enhanced. Exotoxin produced during the propagation of diphtheria Bacillus can not only cause local lesions, but also cause systemic toxic lesions, which is the main cause of disease. This toxin consists of two subunits, A and B, which are connected by disulfide bonds. Subunit B has no direct toxicity, but it has a receptor binding region and a translocation region. Subunit B can bind to the cell surface-specific receptor (membrane receptor pro-hHB-EGF), and after binding, subunit A can be transported to the host cytoplasm through the translocation region (figure 1).
Subunit A is toxic and can inactivate intracellular elongation factor -2 (EF-2). EF-2 is an essential enzyme for the translocation reaction of peptide chain synthesis. Due to its inactivation, the peptide chain being synthesized on the ribosome "receptor" can not be translocated to the ribosome "donor", so that aminoacyl -tRNA can not be combined with ribosomes, the peptide chain extension reaction stops, and the target cell dies because it cannot synthesize protein, so diphtheria toxin has a direct lethal effect on mammalian cells. Bacteria cause mucosal epithelial cell necrosis, vasodilation, massive fibrin exudation and local tissue leukocyte infiltration. The strong toxic effect of exotoxin on cells aggravates local inflammation and necrosis, and a large amount of exudative fibrin condenses with necrotic cells, white blood cells and bacteria, covering the damaged mucosal surface to form a special lesion of this disease, that is, pseudomembrane. The pseudomembrane is generally grayish white, which can be yellow or dirty when mixed with infection and black when bleeding. First thin and then thick, with neat edges, not easy to fall off, and bleeding spots can be seen when hard peeling. The pseudomembrane formation site and surrounding tissues showed mild congestion and swelling. The pseudomembrane formed in the part of larynx, trachea and bronchus covered by columnar epithelium is not closely adhered to mucosa, and it is easy to fall off and cause suffocation. Exotoxin is absorbed locally, causing systemic toxemia symptoms. The amount of toxin absorbed will change with the position and range of the false membrane. The pharynx absorbs the most toxins, followed by tonsils, larynx and trachea. The more extensive the false membrane, the greater the toxin absorption and the more serious the condition. Toxins can be neutralized by antitoxin when adsorbed on the cell surface, but cannot be neutralized by antitoxin after entering the cell. Therefore, early and full application of antitoxin is emphasized in clinic. Exotoxin can cause systemic lesions after binding with various tissues and cells. Among them, the myocardium and peripheral nerves are more prominent. The heart is often enlarged in the early stage, and the myocardium is often turbid, swollen and steatosis. In the later stage, multifocal vitreous degeneration, myocardial necrosis and monocyte infiltration may occur, and the conduction bundle may also be involved. Finally, the heart may have connective tissue hyperplasia and occasionally thrombosis. Neuropathy is more common in peripheral nerves, myelin sheath is often fatty and nerve axis is broken. Both sensory nerves and motor nerves can be involved, but motor nerves are the main ones, and the ninth and tenth cranial nerves are the most easily involved. Injured nerves rarely die, so diphtheria paralysis can almost be recovered. The kidney can be turbid and swollen, and the epithelial cells of renal tubules fall off. Adrenal gland may be congested: degeneration or bleeding. Hepatocytes can be steatosis and hepatic lobules can have central necrosis.
Transmission routing
It is mainly transmitted by respiratory droplets. It can also spread through contaminated hands, toys, stationery, utensils and handkerchiefs. Occasionally, it is reported that contaminated milk has caused an epidemic. It can also be infected through damaged skin and mucous membranes.
Clinical manifestations of early symptoms
The incubation period is 1 ~ 7 days, usually 2 ~ 4 days. According to the location of false membrane and the severity of poisoning symptoms, it can be divided into the following categories:
(1) Pharyngeal diphtheria is the most common, accounting for about 80% of the incidence.
(1). When diphtheria without pseudomembrane is prevalent, some patients only have upper respiratory symptoms, such as sore throat, and the symptoms of systemic poisoning are mild. There is only slight inflammation in the pharynx. Tonsils can be swollen, but no false membrane is formed, or only a small amount of fibrin oozes out, and bacterial culture is positive. Such patients are easy to be misdiagnosed and missed.
(2) localized pharyngeal diphtheria
① Pharyngeal diphtheria pseudomembrane is confined to the palatal arch, uvula and other places, with mild symptoms.
② The false membrane of tonsil diphtheria is confined to one or both tonsils. Slow onset, mild and moderate fever, general malaise, fatigue, loss of appetite and mild sore throat. The tonsils are congested and slightly swollen, and the pseudomembrane is punctate at first, and then fused into pieces. Submandibular lymph nodes may be swollen and slightly painful.
(3) Poisonous pharyngeal diphtheria is mainly transformed from localized type and disseminated type. Most of them are accompanied by mixed infection. If not treated in time, the mortality rate is extremely high.
This kind of symptoms: the pseudomembrane is mostly black due to bleeding, the tonsils and pharynx are highly swollen, blocking the pharynx, or necrotizing to form ulcers, with a special rotten smell. Enlargement of cervical lymph nodes and edema of surrounding soft tissues make the neck thicker (bovine neck). Symptoms of systemic poisoning are serious, including high fever, shortness of breath, cyanosis of mouth and lips, rapid pulse and arrhythmia.
(4) The disseminated pseudomembrane of pharyngeal diphtheria extends from tonsil to uvula, soft palate, posterior pharyngeal wall, nasopharynx or larynx. The pseudomembrane is gray or yellow-white, with clear boundaries and severe swelling of surrounding tissues. Bilateral tonsils are swollen, even filling the pharynx, which leads to dyspnea. The cervical lymph nodes are swollen and there is edema around them.
Such symptoms: severe systemic poisoning symptoms, such as high fever, fatigue, anorexia, sore throat, etc. ; Severe cases can lead to circulatory failure.
(2) Nasal diphtheria is relatively rare, more common in infants. Systemic symptoms are mild or absent, such as nasal congestion, nosebleed, exfoliation of epidermis around nostrils or superficial ulceration soup, and white false membrane in nasal vestibule.
(3) Laryngeal diphtheria Laryngeal diphtheria accounts for about 20%. A few are primary, and about 3/4 are caused by the downward spread of diphtheria in the pharynx. Primary laryngeal diphtheria toxin is less absorbed, and the symptoms of systemic poisoning are not serious. Secondary laryngeal diphtheria often occurs on the basis of pharyngeal diphtheria, accompanied by clinical manifestations of laryngeal diphtheria, and the symptoms of systemic poisoning are serious.
However, a few false membranes extend to trachea and bronchus, which can cause different degrees of hard resistance. Symptoms are severe dry cough, hoarseness, even loss of voice and shortness of breath. In severe cases, cyanosis may occur, and the knife may suffocate and die.
(4) Diphtheria in other parts can invade the conjunctiva, ears, vulva, umbilical cord and skin injury of newborns, producing false membranes and purulent secretions. Diphtheria in eyes, ears and vulva is mostly secondary. Skin diphtheria is more common in tropical areas and has a long course of disease. Skin lesions often last for a long time, and melanosis may occur after healing.
Such symptoms: symptoms of systemic poisoning, but peripheral nerve paralysis may occur.
diagnose
Patients suspected of diphtheria in clinic should be treated with antitoxin and antibiotics immediately without waiting for the test results. However, the first case of diphtheria should be confirmed by microbiological examination.
(a) direct staining microscopy
Take the exudate from the edge of the false membrane with a cotton swab, smear it, stain it with Nessler's or Meilan, and examine the Corynebacterium containing metachromatic granules under the microscope. Combined with clinical symptoms, a preliminary diagnosis can be made. Diagnosis must pass bacterial culture and toxicity test.
(2) Coagulated serum cotton swab culture method
Put the cotton swab stained with horse or bovine serum in a sterile test tube and sterilize it with 8 ~ 10 lb steam for 20 ~ 30 minutes to coagulate the serum. Coagulated serum cotton swabs were used to take throat samples of patients, which were cultured at 37℃ for 8 ~ 10 hours and then smeared directly for microscopic examination. This method can be used for rapid culture diagnosis during a large number of examinations.
(3) Training inspection
Cotton test materials were inoculated on egg slant or Loew's serum coagulation slant medium and potassium tellurite plate medium, and cultured at 37℃. When typical gray suspected colonies grow on the inclined plane or plate, they are picked and transplanted to the inclined plane of eggs or serum for isolation and culture for further morphological staining or toxicity test identification.
(4) Toxicity test
1. Two guinea pigs weighing 250g were selected, and one of them was intraperitoneally injected with 250 ~ 500 units of diphtheria antitoxin 12 hours before the test as a control. Then compare. Then, 2 ml of 48-hour culture solution was injected subcutaneously. If the guinea pigs injected with antitoxin die within 2 ~ 4 days, while the control guinea pigs survive, it is proved that the tested strain is toxic diphtheria Bacillus.
2.e- plate test: Stick a sterile filter paper strip soaked with diphtheria antitoxin on an agar plate containing 20% horse serum, then inoculate the bacteria to be detected along the vertical direction of the filter paper strip, and inoculate the known virus-producing strains and non-virus-producing strains as control. After 48 hours of culture at 37℃, if the strain to be tested produces diphtheria exotoxin, a white precipitation line will appear at the junction of the filter paper strip and the marked growth fungus moss.