Corynebacteria

The genus Corynebacterium is comprised of many species, but Corynebacterium diphtheriae has the most important pathogenic properties. C. diphtheriae is the agent of diphtheria, a serious throat infection and a systemic, toxic disease. If they have an opportunity to colonize in the throat, virulent strains of this organism not only damage the local tissue (causing formation of a pseudomembrane), but they produce a powerful exotoxin that disseminates through the body from the site of its production in the upper respiratory tract. When this toxin reaches the cells of the myocardium, adrenal cortex, or other vital organs, it has very damaging effects. The systemic effect of toxin is the primary cause of death in those patients with diphtheria who are not promptly recognized and treated. In rare cases, the skin rather than throat is affected, but all toxic disease manifestations are the same. The disease is controlled by maintaining active immunization with diphtheria toxoid (purified toxin treated so that it is no longer toxic but remains immunogenic).

Early clinical and laboratory recognition of diphtheria infection developing in the throat is critical because prompt treatment with antitoxin (antibody that neutralizes the toxin) and an appropriate antimicrobial agent are required for patient recovery. In the laboratory, the microbiologist must distinguish C. diphtheriae from other corynebacteria that are harmless members of the normal flora but usually present in throat specimens. Identification must be made as rapidly as possible, for the laboratory report is essential for clinical decisions. In patients with decreased immune function (referred to as immunocompromised patients), corynebacteria other than C. diphtheriae may cause disease by invading the weakened host to produce bacteremia and pneumonia. In spite of widespread immunization in the United States, occasional sporadic outbreaks of both pharyngeal and skin diphtheria occur. In the 1990s, more than 150,000 cases and 5,200 deaths were reported in the former Soviet Union, primarily among adults who were not vaccinated as children.

Corynebacteria are gram-positive, nonmotile, nonsporing bacilli that, like staphylococci, are widely distributed on our bodies. Nonpathogenic species are often called diphtheroids because their microscopic morphology resembles that of C. diphtheriae. These rods often contain granules that stain irregularly (they are said to be metachromatic) and give the organisms a beaded or clubbed appearance. Pairs or small groups characteristically fall into patterns that look like Chinese letters, or like Vs and Ys. Usually, C. diphtheriae is longer, thinner, and more beaded in appearance than diphtheroids, which are generally short and thick by comparison. This differentiation can be very difficult to make in examining a stained throat smear and cannot be relied on for accurate diagnosis.

In culture, corynebacteria are not highly fastidious. They grow well aerobically on nutrient media. When diphtheria is suspected, the primary isolation media used for throat swabs include those that are selective and differential for C. diphtheriae and also blood agar. Loeffler’s serum medium is commonly used for direct inoculation and transport of the swab to the laboratory. This is a firm coagulated serum medium containing nutrient broth, prepared as a tubed slant. Many of the normal throat flora organisms do not grow on Loeffler’s medium, so it is somewhat selective. In addition, when C. diphtheriae grows on this medium its microscopic morphology is characteristic. A methylene-blue-stained smear reveals thin, club-shaped bacilli and reddish-purple metachromatic granules. This appearance can lead to a rapid presumptive diagnosis of diphtheria. Blood agar to which potassium tellurite has been added constitutes a good selective and differential medium for primary isolation of C. diphtheriae. The tellurite not only suppresses many other throat flora, but it is metabolized by C. diphtheriae with resulting blackening of its colonial growth. Thus the organism is differentiated from others that can grow on the agar medium. The use of blood agar in the initial battery assures the recovery of corynebacteria, as well as other pathogenic bacterial species that might be present, and differentiates those that are hemolytic.

The biochemical differentiation of C. diphtheriae from other corynebacteria is based on carbohydrate fermentations. Demonstration of toxin production is essential in reporting identification of a strain of C. diphtheriae, for not all strains are toxigenic. Tests for virulence, that is, toxigenicity, are made either in experimental animals (rabbits or guinea pigs) or by an in vitro method (Elek test). In the Elek test, antitoxin strips are placed on agar plates to detect toxin produced by strains of C. diphtheriae growing on the medium.

Purpose	To identify corynebacteria in smears and cultures
Materials	Blood agar plate Blood tellurite plate Tubed phenol red glucose broth Tubed phenol red maltose broth Tubed phenol red sucrose broth Prepared Gram- and methylene-blue-stained smears of C. diphtheriae Loeffler’s slant cultures of Corynebacterium xerosis and Corynebacterium pseudodiphtheriticum Nutrient agar slant culture of Escherichia coli

Procedures

1. Prepare a Gram stain and a methylene blue stain from either one of the Corynebacterium cultures. Read and compare these with the Gram- and methylene-blue-stained smear of C. diphtheriae, recording your observations under Results.
2. Inoculate a blood agar plate with either one of the Corynebacterium cultures. Streak for isolation.
3. Divide the blood tellurite plate into two parts with your marker. Inoculate one side of the plate with a Corynebacterium species, the other side with E. coli.
4. Inoculate the C. xerosis culture into each of the three carbohydrate broths. Repeat with the culture of C. pseudodiphtheriticum.
5. Incubate all plate and tube cultures at 35°C for 24 hours.
6. Examine your cultures and record your observations.

Results

1. Illustrate the microscopic morphology of corynebacteria:
   
   
   
2. Describe the appearance of a Corynebacterium species on blood agar.
3. Describe the appearance of E. coli and of a Corynebacterium species on blood tellurite agar.
4. Complete the following table.