Lane Barksdale

VIRULENCE, TOXINOGENY, AND LYSOGENY IN CORYNEBACTERIUM DIPHTHERIAE*

About 15 years ago in Japan my associates and I developed an interest in certain unorthodox manifestations of the disease diphtheria. This led to a study of equally unorthodox diphtheria bacilli. Since then we have continukd to explore the biology of Corynebacterium diphtheriae. This pursuit has been principally as (1) a study of properties of diphtheria bacilli that may be expressed in the animal host, for example, virulence and toxicity; and (2) an inquiry into the origin of these same attributes in the lysogenic, toxinogenic diphtheria bacillus.

Pyrazine Carboxylamidase Activity in Corynebacterium

A total of 124 strains representing Corynebacterium diphtheriae (mitis, gravis, intermedius, and ulcerans) and 12 strains of the closely related Corynebacterium pseudotuberculosis (Corynebacterium ovis) were found to be pyrazinamidase negative (Pyz-). By contrast, a large number of strains representing various corynebacterial species, nocardias, bacterionemas, actinomyces, and rothias were pyrazinamidase positive (Pyz+). The usefulness and possible implications of this finding are discussed, and a rapid test for pyrazinamidase activity is described.

Spheroidal bodies and globi of human leprosy

From the plasma and/or buffy coats of 80% of 38 cases of (tuberculoid and lepromatous) leprosy have been isolated in pure culture a group of spheroidal organisms (spheroidal bodies of leprosy, SPBL) showing on various media a versatility of differention ranging from naked protoplasts to globi containing acid-fast rods. The acid-fastness of the latter, like the unique acid-fastness of leprosy bacilli from lepromatous leprosy, can be extracted with C5H5 N. Inoculation of chick embryos with SPBL elicits the nodular response evoked by homogenates of lepromatous tissue. From these nodules SPBL can be recovered in pure culture. SPBL appears to be the long sought etiologic agent of leprosy.

Immunobiology of Diphtheria

Between 1821 and 1826 Pierre Fidele Bretonneau (1826) established the clinical entity diphtheria. Its hallmark was the pseudomembrane or diphtheritic membrane. By 1869 Trendelenberg had shown that pseudomembranes could be produced in rabbits and in pigeons following the injection of pseudomembranous material from human cases of diphtheria. Bretonneau conceived of diphtheria as a disease of singular etiology comparable in its uniqueness to the causes of measles and of variola. This Unitarian view was soon lost to some students of the disease. In methyleneblue-stained smears of material taken from pseudomembranes, more than one shape of bacterium could be observed. In fact, Klebs (1875) found diphtheria from one locality to be caused by a fungus, Microsporon diphthericum, and later he described a bacillary type of diphtheria from another locality (Klebs, 1883). When Friedrich Loeffler tackled the problem of the etiology of diphtheria, he accepted the concept of a single etiology as promulgated by Bretonneau. Working in Koch’s laboratory, he designed a program for direct microscopic examination of methylene-bluestained material from pseudomembranes, cultivation of such materials on solid media, and inoculation of animals with bacterial cultures (Loeffler, 1884). This remarkable investigator was the first to distinguish the probable role of streptococci in diphtheria associated with scarlatinal infections from that of occasional chains of streptococci (normal flora) found associated with frank cases of diphtheria.* He was able to demonstrate diphtheria bacilli in smears of only 13 out of 22 cases of clinical diphtheria. From six of these he isolated Corynebacterium diphtheriae. He isolated diphtheria bacilli from one normal child. Because of the lack of a one-to-one correlation in these findings, he was not adamant in claiming to have established the etiology of the disease. But he had in fact clearly established its etiology. From the time of Loeffler, it has been evident that harboring diphtheria bacilli is not synonymous with diphtheria and that Koch’s postulates cannot be fulfilled in every case of diphtheria. Loeffler’s experimental infections in animals led to the discovery that diphtheria bacilli tended to remain at the site of injection, although autopsy revealed damage to organs far from that site. Roux and Yersin (1888) demonstrated that the connection between diphtheria bacilli at one site and damage to distant internal organs was a soluble poison, diphtherial toxin. These observations have given rise to the concept that diphtheria bacilli always remain at the superficial site of colonization, the pseudomembrane or the cutaneous lesion.* Further, since Roux and Yersin pointed out that toxin itself could be responsible for many of the signs of the disease, in the legend of diphtheria there has been a tendency to confuse intoxication with infection. The discovery of antibodies capable of neutralizing the toxicity of diphtherial toxin (Behring and Kitasato, 1890) and their apparent effects in reducing case fatality rates in epidemics of diphtheria obscured for many the need for investigating antibacterial immunity toward C. diphtheriae. As a result, textbook presentations of the immunology of diphtheria are often confused. In fact, it has been implied that immunization with toxoid has got rid of diphtheria bacilli in some societies. Bonventre (1975) has called this sort of assertion a manifestation of arrogance and has suggested that there are still many questions about diphtheria which remain unanswered. The discussion of the immunology of diphtheria that follows indicates a number of aspects of the interaction of diphtheria bacilli and human beings which merit further study.

Nitrate Reductase Activities in Lysogenic and Nonlysogenic Strains of Corynebacterium diphtheriae and Related Species

Corynebacterium diphtheriae and closely related corynebacteria are gram-positive, pleomorphic, facultatively anaerobic bacteria which have been classified (in part) according to their capacity to reduce or not reduce nitrates to nitrites. An investigation of the presence or absence of nitrate reductase activity in C. diphtheriae, in particular, and in C. belfanti, C. ulcerans, C. ovis, C. hofmanni, and C. xerosis, in general, indicates that (i) the control of the synthesis of the enzyme is a readily mutable property, (ii) enzyme synthesis occurs under anaerobic conditions, and (iii) maximal activity is associated with the “pellet” or “membrane” fraction. Additional evidence indicates that the gene N-red is not on the phage chromosome and, therefore, is not linked to the gene governing the synthesis of diphtherial toxin (tox) as previously claimed.

Glucose-1-Phosphate-Induced Accumulation of Intracellular Starch: A Distinguishing Feature of Certain Corynebacteria

Fifty-seven bacterial strains, including corynebacteria, “plant pathogenic corynebacteria,” mycobacteria, and nocardias, were examined (aerobically and anaerobically) for their capacity to produce a glucan phosphorylase (α-1,4-glucan:orthophosphate glucosyl transferase, EC 2.4.1.1), which synthesizes an iodinophilic starch-like polysaccharide from glucose-1-phosphate. Only Corynebacterium diphtheriae, C. belfanti, C. ulcerans, C. ovis, C. kutscheri, C. minutissimum, and two (out of eight) strains of C. renale yielded positive results. The presence of glucose-1-phosphate-induced starch-like material appears to be a valid additional means of distinguishing most true corynebacteria from mycobacteria and nocardias.