C hair of Medical Biology, M icrobiology, V irology, and I mmunology

Chair of Medical Biology, Microbiology, Virology, and Immunology CORYNEBACTERIA. LISTERIA. Lecturer As. Prof.O.V. Pokryshko

CORYNEBACTERIUM Classification. The genus Corynebacterium comprises a species pathogenic for human beings and several species which are non-pathogenic for man and conditionally designated as diphtheroids. The majority of diphtheroids occurs in the external environment (water, soil, air), some of them are present as commensals in the human body.

Species Pathogenicity for humans and animals C. diphtheriae Pathogenic for humans, causes diphtheria C. pseudotuberculosis Pathogenic for sheep, goats, horses, and other warm-blooded animals, sometimes causes in fection in humans C. xerosis Non-pathogenic for humans, dwells on eye mucosa C. renale Induces pyelitis and cystitis in experimental animals and pyelonephritis in calves C. kulschen Parasitizes in the body of mice and rats C. pseudodiphtheriae Non-pathogenic for humans, dwells on the mucous membrane of the nasopharynx C. equi Detected in pneumonia in animals, weakly pathogenic for experimental animals C. bovis Causes mastitis in animals, found in milk

Causative Agent of Diphtheria. Extensive clinical, pathoanatomical, epidemiological, and experimental investigations preceded the discovery of the agent responsible for diphtheria. They paved the way for the discovery of the organism (E.Klebs, 1883), its isolation in pure culture (F. Loeffler, 1884), separation of the toxin (E. Roux and A. Yersin, 1888), antitoxin (E. Behring and S.Kitasato, 1890) and diphtheria toxoid (G. Ramon, 1923).

Morphology.Corynebacterium diphtheriae (L. coryna club) is a straight or slightly curved rod, 1-8 mcm in length and 0.3-0.8 mcm in breadth. The organism is pleomorphous and stains more intensely at its ends, which contain volutin granules (Babes-Ernst granules, metachromatin). C. diphtheriae frequently display terminal club-shaped swellings. Branched forms as well as short, almost coccal, forms sometimes occur. In smears the organisms are arranged at an angle and resemble spread-out fingers. They are Gram-positive and produce no spores, capsules, or flagella.

Corynebacterium diphtheriae, Gram’s technique

Corynebacterium diphtheriae, Neisser’s technique

C. diphtheriae, Loeffler’s technique

C. diphtheriae may change into cone-shaped, thread-like, fungi-like, and coccal forms. In old cultures the cytoplasm of the organisms acquires a zebra-like appearance with unequally stained stripes. On ultrathin sections the cell wall has two layers, an inner osmiophilic layer and an outer layer forming a microcapsule The cytoplasmatic membrane is composed of three layers. During maximum exotoxin liberation membrane structures are seen as 'organelles', ovals, and rings. The cytoplasm is granular. The nucleoid is filled with fine osmiophilic fibrils. The metachromatic granules appear as dense granular structures surrounded by a membrane. A correlation has been revealed between the development of the membrane and the production of exotoxin. The G-C content in DNA ranges from 51.8 to 60 per cent.

Cultivation. The causative agent of diphtheria is an aerobe or a facultative aerobe. The optimal temperature for growth is 37° C and the organism does not grow at temperatures Below 15 and above 40° C. The pH of medium is 7.2-7.6 The organism grows readily on media which contain protein (coagulated serum, blood agar, and serum agar) and on sugar broth. On Roux's (coagulated horse serum) and Loeffler's (three parts of ox serum and one part of sugar broth) media the organisms produce growth in 16-18 hours The growth resembles shagreen leather, and the colonies do not merge together. According to cultural and biological properties, three varieties of C.diphtheriae can be distinguished, gravis, mitis, and intermedius, which differ in a number of properties.

Corynebacteria of the gravis biovar produce large, rough (R-forms), rosette-like black or grey colonies on tellurite agar which contains defibrinated blood and potassium tellurite. The organisms ferment dextrin, starch, and glycogen and produce a pellicle and a granular deposit in meat broth. They are usually highly toxic with very marked invasive properties. Biovargravis

The colonies produced by corynebacteria of the mitis biovar on tellurite agar are dark, smooth (S-forms), and shining. Starch and glycogen are not fermented, and dextrin fermentation is not a constant property. The organisms cause haemolysis of all animal erythrocytes and produce diffuse turbidity in meat broth. Cultures of this biovar are usually less toxic and invasive than those of the gravis biovar. Biovarmitis

Organisms of the intermedius biovar are intermediate strains. They produce small (RS-forms) black colonies on tellurite agar. Starch and glycogen are not fermented. Growth in meat broth produces turbidity and a granular deposit. Biovarintermedius

Fermentative properties. All three biovars of C. diphtheriae do not coagulate milk, do not break down urea, produce no indole, and slowly produce hydrogen sulphide. They reduce nitrates to nitrites. Potassium tellurite is also reduced, and for this reason C. diphtheriae colonies grown on tellurite agar turn black or grey. Glucose and levulose are fermented whereas galactose, maltose, starch, dextrin, and glycerin fermentation is variable. Exposure to factors in the external environment renders the organisms incapable of carbohydrate fermentation.

C. diphtheriae

Antigenic structure. Eleven serovars of C. diphtheriae have been determined on the basis of the agglutination reaction. They all produce toxins which do not differ from each other and are neutralized completely by the standard diphtheria antitoxin. A number of authors have confirmed the presence of type-specific thermolabile surface protein antigens (K-antigens) and group-specific thermostable somatic polysaccharide antigens (O-antigens) in the diphtheria corynebacteria.

Toxin production. In broth cultures C. diphtheriae produce potent exotoxins (histotoxin, dermonecrotoxin, haemolysin). The toxigenicity of these organisms is linked with lysogeny (the presence of moderate phages-prophages in the toxigenic strains). The classical International standard strain, Park-Williams 8 exotoxin-producing strain, is also lysogenic and has retained the property of toxin production for over 85 years.

The genetic determinants of toxigenicity (tox+ genes) are located in the genome of the prophage, which is integrated with the C. diphtheriae nucleoid. Phage with tox+ genes

In the commercial production of diphtheria toxin for vaccine, the amount of iron present in the growth medium is critical. Good toxin production is obtained only at low concentrations of iron (2 mcmol/L). At concentrations aslow as 10 mcmol/L, toxin production becomes negligible. Evidence suggests that, normally, the bacterium forms are presser which prevents the expression of the phage tox+ gene, and that this represser is an iron-containing protein. Thus, when the concentration of iron is abnormally low, the complete represser is not formed, and the tox+ geneis transcribed, ultimately yielding toxin.

The diphtheria exotoxin is a complex of more than 20 antigens. It has been obtained in a crystalline form. The diphtheria toxin contains large amounts of amino-nitrogen and catalyses chemical reaction in the body. The toxigenic strains of C. diphtheriae are characterized by marked dehydrogenase activity, while the non-toxigenic strains do not possess such activity.

Diphtheria toxin is excreted from the bacterium as a single polypeptide chain of about 61,000 daltons with two disulfide bridges. Although highly toxic for cells or animals, the pure, intact toxin is inert in cell-free protein systems, even when NAD is present. Thus, the secreted toxin is actually a proenzyme which, in cell-free systems, must be activated before it can function as an enzyme. This activation is accomplished in two steps: (1) treatment with trypsm hydrolyzes a peptide bond between the disulfide-linked aminoacids; and (2) reduction of the disulfides to sulfhydryl groups using a reducing agent such as mercaptoethanol yields two smaller peptides, which have been designated fragment A (21,150 daltons) and fragment B (40,000 daltons).

Toxin’s structure

Fragment A is active in cleaving the nicotinamide moiety from NAD and in catalyzing the transfer of ADP-ribose from NAD to EF-2 when added to cell-free, protein-synthesizing systems, but it has no effect when given to animals or to intact HeLa cells. Thus, although fragment A is the activated enzyme (and hence contains all the toxic properties), it cannot get into intact cells.

Fragment B, on the other hand, has no enzymatic activity, but it is needed for attachment of the toxin to specific receptor sites on cells. Cells possess specific glycoprotein receptor sites for the diphtheria toxin, as suggested by the following observation: Rats and mice are over 1000 times more resistant to the intact toxin than are other susceptible animals, but their cell-free protein-synthesizing system is equally sensitive to the enzymatic action of fragment A. Moreover, toxin that is defective in its A fragment (and is, therefore, nontoxic) but retains a normal B fragment, will competitively inhibit the action of normal toxin on HeLa cells.

In summary, the usual series of events leading to toxin action is as follows: (1) the toxin binds to specific receptor sites on susceptible cells; (2) the toxin enters the cell (perhaps through a phagocytic vesicle that can then fuse with a lysosome), and lysosomal proteases hydrolyze the toxin into fragments A and B; and (3) reduction of the disulfide bridges (perhaps by glutathione) releases fragment A from fragment B; and (4) fragment A can then enzymatically inactivate EF-2.

The diphtheria toxin is unstable, and is destroyed easily by exposure to heat, light, and oxygen of the air, but is relatively resistant to super-sonic vibrations. The toxin is transformed into the toxoid by mixture with 0.3-0.4 per cent formalin and maintenance at 38-40° C for a period of 3 or 4 weeks. The toxoid is more resistant to physical and chemical factors than the toxin.

Because diphtheria toxin is effective against many cells, the use of tissue cultures provides a model for studying its mode of action. Early studies reported that, although toxin had no effect on the respiration of HeLa cells (human cervical carcinoma tissue culture cells), all protein synthesis stopped about 1 to 1.5 hours after the addition of the toxin. Surprisingly, dialyzed, cell-free, protein-synthesizing systems were entirely insensitive to the action of the toxin, unless oxidized nicotinamide-adeninedinucleotide (NAD) was added to the reaction.

Subsequent research has shown that the toxin possesses enzymatic activity that cleaves nicotinamide from NAD and then catalyzes the ADP-ribosylation of elongation factor 2 (EF-2). EF-2 is required for the translocase reaction of polypeptide synthesis, in which the ribosome is moved to the next codon on the mRNA after the peptide bond is formed to the most recent aminoacid to be added to the chain. When EF-2 is inactivated by the addition of ADP-ribose, the ribosome is frozen, and protein synthesis stops. Insofar as is known, EF-2 from all eucaryotic cells (those studied include vertebrate, invertebrate, wheat, and yeast) is inactivated in the presence of diphtheria toxin and NAD, whereas the corresponding factor, EF-G (which occurs in bacteria), or the analogous factor from mitochondria, is not affected. The ADP-ribose is transferred to a histidine modified residue on the EF-2 molecule. This modified aminoacid (commonly called diphtheramide) does not exist in bacterial or mitochondnal elongation factors.

C. diphtheriae also contain bacteriocines (corynecines) which provide these organisms with certain selective advantages.

Resistance. C. diphtheriae are relatively resistant to harmful environmental factors. They survive for one year on coagulated serum, for two months at room temperature, and for several days on children's toys. Corynebacteria remain viable in the membranes of diphtheria patients for long periods, particularly when the membranes are not exposed to light. The organisms are killed by a temperature of 60° C and by a 1 percent phenol solution in 10 minutes.

Pathogenicity for animals. Animals do not naturally acquire diphtheria. Although, virulent diphtheria organisms were found to be present in horses, cows, and dogs, the epidemiological significance of animals in diphtheria is negligible. Among the laboratory animals, guinea pigs and rabbits are most susceptible to the disease.

Inoculation of these animals with a culture or toxin gives rise to typical manifestations of a toxinfection and the appearance of inflammation, oedema, and necrosis at the site of inoculation. The internal organs become congested, particularly the adrenals in which haemorrhages occur. Toxic reactions in animal

Pathogenesis

Pathogenesis and disease in man. Patients suffering from the disease and carriers are the sources of infection in diphtheria. The disease is transmitted by an air-droplet route, and sometimes with dust particles. Transmission by various objects (toys, dishes, books, towels, handkerchiefs, etc.) and foodstuffs (milk, cold dishes, etc.) contaminated with C. diphtheriae is also possible. Carriers play an essential part in the epidemiology of diphtheria. The carrier state averages from 3 to 5 per cent among convalescents and healthy individuals. Diphtheria is most prevalent in autumn. This is due to the fact that children are more crowded in the autumn months and that body resistance is reduced by a drop in temperature.

Histotoxin plays the principal role in the pathogenesis of diphtheria. It blocks protein synthesis in the cells of mammals and inactivates transferase, the enzyme responsible for the formation of the polypeptide chain. C. diphtheriae penetrate into the blood and tissues of sick humans and infected animals. The diffusion factor due to which these organisms are capable of invasion is formed of a complex of K-antigen and lipids of the wall of bacterial cells. The lipids contain corynemicolic and corynemicolenic acids, the cord factor (trehalose dimicolate), and mannose and inositol phosphatides. The cord factor causes the death of mice, destroys mitochondria, and disturbs the processes of respiration and phosphorylation. The necrotic factor, alpha-glutaric acid, and haemolysin are considered to be factors of invasiveness.

Clinical studies and experiments on animals have provided evidence of the influence of pathogenic staphylococci and streptococci, on the development of diphtheria, the infection becoming more severe in the presence of these organisms. Hypersensitivity to C. diphtheriae and to the products of their metabolism is of definite significance in the pathogenesis of diphtheria.

In man, membranes containing a large number of C. diphtheriae and other bacteria are formed at the site of entry of the causative agent (pharynx, nose, trachea, eye conjunctiva, skin, vulva, vagina, and wounds). The toxin produces diphtheria! inflammation and necrosis in the mucous membranes or skin. On being absorbed, the toxin affects the nerve cells, cardiac muscle, and parenchymatous organs and causes severe toxaemia. Deep changes take place in the cardiac muscle, vessels, adrenals, and in the central and peripheral nervous systems.

According to the site of the lesion, faucial diphtheria and diphtheritic croup occur most frequently, and nasal diphtheria somewhat less frequently. The incidence of diphtheria of the eyes, ears, genital organs, and skin is relatively rare. Faucial diphtheria constitutes more than 90 per cent of all the diphtherial cases, and nasal diphtheria takes the second place.

Faucial diphtheria

Nasal diphtheria Diphtheritic croup

Immunity following diphtheria depends mainly on the antitoxin content in the blood However, a definite role of the antibacterial component, associated with phagocytosis and the presence of opsonins, agglutinins, precipitins, and complement-fixing substances cannot be ruled out. Therefore, immunity produced by diphtheria is anti-infectious (anti-toxic and antibacterial) in character.

Schick test. This test is used for detecting the presence of antitoxin in children's blood. The toxin is injected intracutaneously into the forearm in a 0 2 ml volume which is equivalent to 1/40 DLM for guinea pigs. A positive reaction, which indicates susceptibility to the disease, is manifested by an erythematous swelling measuring 2 cm in diameter which appears at the site of injection in 24-48 hours. The Schick test is positive when the blood contains either no antitoxin or not more than 0.005 units per millilitre of blood serum. A negative Schick reaction indicates, to a certain degree, insusceptibility to diphtheria.

In view of the fact that the diphtheria exotoxin produces a state of sensitization and causes the development of severe reaction in many children, it is advisable to restrict the application of the Schick test and conduct it with great care. Children from 1 to 4 years old are most susceptible to diphtheria. A relative increase of the incidence of the disease among individuals 15years of age and older has been noted in recent years. Diphtheria leaves a less stable immunity than do other children's diseases (measles, whooping cough). Diphtheria reinfection occurs in 6-7 per cent of the cases.

Laboratory diagnosis.Discharges from the pharynx, nose, and, some-times, from the vulva, eyes, and skin are collected with a sterile cotton-wool swab for examination. The material under test is seeded on special media, e. g. coagulated serum, Clauberg's II medium, blood-tellurite agar, serum-tellurite agar, etc. Smears are examined under the microscope after 12-24-48 hours' growth, and preliminary diagnosis is made on the basis of microscopic findings. C. diphtheriae does not always occur in its typical form. Short rods arranged not at a particular angle but in disorder and containing few granules are found in a number of cases. Diagnostic errors are made most frequently when investigations are confined to microscopical examination.

Other bacterial species and non-pathogenic corynebacteria which are morphologically identical with the diphtheria organisms maybe mistaken for the diphtheria corynebacteria. It must also be borne in mind that formation of volutin granules is variable, and therefore, this is not an absolute property. For this reason, contemporary laboratory diagnosis comprises isolation of the pure culture and its identification by cultural, biochemical, serological and toxigenic properties.

The toxigenic and non-toxigenic strains of diphtheria corynebacteria are differentiated either by subcutaneous or intracutaneous infection of guinea pigs, or by the agar precipitation method, the latter being relatively simple and may be carried out in any laboratory. It is based on the ability of the diphtheria toxin to react with the antitoxin and produce a precipitate resembling arrow-tendrils.

The agar precipitation method

The agglutination reaction with patient's sera (similar to the Widal reaction) is employed as an auxiliary and retrospective method. It is performed with 5 serovars of C. diphtheriae; the reaction is considered positive beginning from 1 :50-1 :100 dilutions of serum. To detect the sources of infection, the isolated cultures are subject to phagotyping. There are 19 known phage types.

C hair of Medical Biology, M icrobiology, V irology, and I mmunology