Jorgen M. Birkeland

THE ROLE OF HYALURONIDASE IN HEMOLYTIC STREPTOCOCCAL INFECTION

Certain pathogenic bacteria, including strains of staphylococci, pneumococci, the gas gangrene organisms, and hemolytic streptococci, have been reported as producing hyaluronidase. Accordingly, it has been postulated that the enzyme affords these bacteria a greater degree of invasiveness and, as a result, an enhanced virulence. As reported in the literature, the results of efforts made to uncover what relationship, if any, exists between hyaluronidase production and virulence of the organisms have been conflicting. We have been able to find but one systematic investigation of hyaluronidase production by hemolytic streptococci and its relationship to virulence. In an examination of 376 strains of hemolytic streptococci, Crowleyl reported that most Lancefield groups C and G strains showed hyaluronidase production but that only types 4 and 2 2 of the group A strains tested produced the enzyme. She also reported that there was no significant difference in the frequency with which strains isolated from severe and slight infections and from normal individuals were found to produce hyaluronidase. In our investigation of hemolytic streptococcal hyaluronidase activity, we used a beef-heart infusion culture medium containing neopeptone, dextrose, and disodium hydrogen phosphate. The hyaluronidase content of 18-hour cultures was assayed by the turbidimetric test procedure.2, We found that a large majority of the human strains of streptococci recently obtained by nasopharyngeal or throat swabs produced hyaluronidase. Streptococcal strains obtained from clinically normal individuals consistently gave hyahronidase titers of less than 1 turbidimetric reducing unit per cc. of culture media, while those isolated from patients with streptococcal infections gave hyaluronidase titers ranging from 2 to 100 TRU per cc. Furthermore, the higher hyaluronidase titers seemed to be associated with the more clinically severe infections. I n ascending order of hyaluronidase production, such typical infections were septic sore throat, ulcers, scarlet fever, abscesses, streptococcal pneumonia, and acute otitis media. We next examined 9 group A stock laboratory strains which had been Griffith typed. These did not include types 4 and 2 2 , which had previously been reported to produce hyaluronidase. Six of the 9 strains were found to produce the enzyme in hyaluronate-free media. They were 2 strains each of types 10 and 11 and 1 strain each of types 5 and 9. The 3 nonproducing organisms were 2 strains of type 2 and 1 of type 9. Since hemolytic streptococci in the body presumably have hyaluronic acid as a substrate, it was decided to investigate the effect of culturing the 9 stock strains in infusion media containing crude hyaluronate. After 10

Selective Bacteriostatic Action of Sodium Lauryl Sulfate and of “Dreft.”

The bacteriostatic action of various soaps has undergone a considerable amount of study by many investigators. Recently, a somewhat related class of substances, the sodium alkyl sulfates, has been synthesized. These substances have also been made use of as detergents in such preparations as “Dreft,” “Drene” and other commercial products. The alkyl sulfates are half-esters of sulfuric acid. In this respect they differ from soaps which are combinations of fatty acids and alkalis. In both cases, however, the alkyl chain probably determines its bactericidal effectiveness. Sodium lauryl sulfate is a sodium alkyl sulfate containing 12 carbon atoms. The similarity of these substances to soaps suggested the possibility that similar bacteriostatic action might be exhibited by them. Accordingly, sodium lauryl sulfate as such, and “Dreft” which contains a large amount of sodium lauryl sulfate were tested for their bacteriostatic action. Twenty-one strains of Gram positive bacteria, 20 strains of Gram negative bacteria and 6 strains of common molds were used in these studies. In general, all tests were made on Difco standard nutrient agar to which the test substances had been added in varying amounts so as to make concentrations of 1.0% sodium lauryl sulfate and of 1.0, 0.5, 0.25, and 0.1% “Dreft”. The plates were streaked with heavy suspensions of the organisms and then incubated at appropriate temperatures for 48 hours. The effects of sodium lauryl sulfate and of “Dreft” on the growth of the 41 strains of bacteria and 6 strains of molds studied in this investigation are shown in Table I. It is evident that the Gram positive organisms were prevented from growing, whereas the Gram negative bacteria grew as freely as, on the nutrient agar controls. There were 3 exceptions to the latter statement: Vibrio comma, Alcaligenes fecalis, and Neisseria catarrhalis were definitely inhibited in their growth.

Effect of Intravenous Injection of Tuberculin on Leucocytes of Normal and Tuberculous Rabbits.

Summary Following the intravenous injection of P.P.D. into tuberculous rabbits there is a decrease in the total number of white cells and lymphocytes. The maximum decrease occurs in 6 to 9 hours and is followed by a gradual return to normal. There is also frequently a delayed increase in neutrophiles.

Hyaluronic Acid Utilization by Hemolytic Streptococci in Relation to Possible Hyaluronidase Function in Pathogenesis

Summary Hemolytic streptococcal strains, virulent for chick embryos, were found able to utilize hyaluronic acid for respiration to a greater extent than relatively avirulent strains. With the micro-organisms tested, it appears that the hydrolytic products and not hyaluronate itself are utilized in respiration, such hydrolysis occurring presumably through the agency of hyaluronidase. Pathogenic bacteria frequently found associated with group A streptococci in vivo had a synergistic relationship with the latter in utilizing hyaluronate as a substrate for energy. On the basis of these findings, a mechanism of pathogenesis by hemolytic streptococci alone and with associated agents of infection was proposed. Certain clinical findings, interpreted as evidence for the existence of such a process, were cited. It is a pleasure to acknowledge the assistance of Miss Clara Boyd.

Growth of Newcastle disease virus in the embryonated egg.

Summary 1. Data are presented to support the contention that the growth of Newcastle disease virus in the developing chick embryo occurs in cycles of 4 hr. The apparent 4-hr latent period in the production of virus following the inoculation of an infective dose does not correspond to the lag phase in the multiplication of bacterial cells, but rather indicates an intercellular development of virus particles that cannot be determined by any of the present technics for measuring viral activity. The maximum adsorption of virus onto susceptible cells occurs within 10 minutes after its introduction into the embryonated egg. 2. The existence of an unequal development of infective and hemagglutinating titers during the growth of Newcastle disease virus is described and an attempted explanation for this discrepancy is made on the hypothesis that the hemagglutination unit results from a maturation of the infectious particles and cannot therefore be detected early in its growth.

Effect of temperature and humidity on nasal flora of mice.

Summary The count of the normal bacterial flora of the nasal cavity was markedly reduced when mice were maintained under low humidity for three weeks, whereas no significant change was observed under high humidity. The reduction was noted mainly in the gram positive organisms.