THE MODE OF ACTION OF SULFANILAMIDE ON STREPTOCOCCUS. II

The precise mode of therapeutic action of sulfanilamide on streptococcus can be arrived at only by considering the sum total of factors that inhibit or favor the natural growth of the microorganism under the experimental conditions that obtain, whether in vivo or in vitro. Too sweeping conclusions have hitherto been drawn from the study of a single variable factor, such as an unfavorable temperature or the absence or presence of peptone. We have attempted here to analyze the factors that have hitherto been recognized and some new ones, but particularly the relationship of these factors to one another. The result obtained on adding sulfanilamide to the streptococcus in the test tube is usually bacteriostasis and not complete destruction of even small numbers of bacteria. This is on the condition that the suspending medium is a favorable one for the growth of the microorganism; the more growth-promoting the medium is the less the bacteriostasis. If, on the other hand, the medium is too poor, or one that in itself inhibits growth, the addition of sulfanilamide may lead to sterilization of the culture. The conditions for growth of the streptococcus in the body of the rabbit or mouse, depend on the strain of bacteria used, but are on the whole favorable. Defence, however, in the form of phagocytosis by both polymorphonuclear and by mononuclear cells is attempted even in the susceptible animal. When sulfanilamide is used to treat such an animal, or when sulfanilamide-grown (inhibited) streptococci are employed, phagocytosis is pronounced, whether studied in the test tube or in the animal body. In the rabbit the delay by sulfanilamide and resultant increased phagocytosis by polymorphonuclears allows mononuclear cells to accumulate and recovery may result. Sulfanilamide not only does not completely destroy the streptococcus but does not even impair its innate virulence. It acts upon the streptococcus not only by inhibiting growth but by a temporary inhibition of hemotoxin formation, but only under certain conditions. The drug does not neutralize hemotoxin already formed. No significant effect of sulfanilamide on the formation of leucocidin or fibrinolysin by streptococcus has been evident in our experiments. Sulfanilamide differs in one important respect from other drugs that are destructive either in the test tube or actually in the body, for protozoa and bacteria. Protozoa fix or adsorb arsenicals and acriflavine that kill them variably in vitro and in vivo. Streptococci fix both gentian violet and acriflavine, which dyes have marked destructive action in the test tube but are less effective in vivo. Sulfanilamide is not diminished at all by contact in vitro with large masses of streptococci, nor does the action of this drug render the microorganism more capable than untreated cocci to adsorb gentian violet or acriflavine, or to be destroyed by these highly bactericidal substances.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : I. DEMONSTRATION OF A TYPE-SPECIFIC SUBSTANCE IN EXTRACTS OF STREPTOCOCCUS H?MOLYTICUS.

1. Hydrochloric acid extracts of Streptococcus hæmolyticus contain type-specific, as well as non-type-specific, substances. The precipitates formed by these crude extracts with homologous antibacterial serum are flocculent, while those obtained with heterologous serum are usually disc-like. 2. The type-specific substance may be detected by the use of anti-bacterial sera absorbed with heterologous strains of hemolytic streptococci. Such absorbed sera are type-specific: they are precipitated only by extracts of strains of the homologous type. 3. Any heterologous strain of hemolytic streptococcus absorbs the antibodies for all other heterologous strains, but homologous strains absorb type-specific antibodies as well. Numerous repeated absorptions with heterologous hemolytic streptococci tend to lower the titer of the type-specific antibody. A possible explanation of this fact is suggested. 4. Three strains did not yield a type-specific substance; and it seems probable that they had lost this function because of long continued cultivation in artificial media. 5. Classification based on the precipitin test with absorbed serum agrees with that previously determined by agglutination and protection tests. The method is, therefore, applicable to the problem of classification of the hemolytic streptococci.     

THE SKIN RESPONSE OF RABBITS TO NON-HEMOLYTIC STREPTOCOCCI : I. DESCRIPTION OF A SECONDARY REACTION OCCURRING LOCALLY AFTER INTRADERMAL INOCULATION.

1. Rabbits inoculated intradermally with certain strains of green streptococci have well marked lesions which, after reaching a maximum size in 24 to 48 hours and then beginning to retrogress, show in over 50 per cent of the animals a secondary increase in size and other signs of inflammation about 8 or 9 days after inoculation. 2. This secondary reaction may follow the inoculation of a variety of strains of green-producing streptococci from various sources, of indifferent streptococci, and occasionally of pneumococci. 3. The inoculation of hemolytic streptococci, staphylococci, Micrococcus catarrhalis, or Bacillus coli has not been followed by this secondary reaction. 4. The secondary reaction is not due to increased activity of the injected bacteria since the lesions are usually sterile at the time it occurs and since the secondary reaction occurs after inoculation of killed organisms as well as after that of living ones. 5. A second inoculation of green streptococci into a rabbit within 9 weeks of a first injection is followed by a primary but not by a secondary reaction. This inhibition of the secondary reaction is not specific; for the phenomenon of secondary reaction can be completely inhibited by previous inoculations of the rabbit with any living streptococcus and usually with pneumococci. Killed organisms are less effectual. 6. Preliminary injection with staphylococci, Micrococcus catarrhalis, and Bacillus coli has not prevented the appearance of a secondary reaction in response to subsequent injections of green streptococci. 7. The agent responsible for the secondary reaction has not as yet been determined; its nature is discussed. The phenomenon is possibly a form of allergic reaction.     

BACTERIOLOGY OF THE BLOOD AND JOINTS IN RHEUMATIC FEVER

1. During the Spring of 1928, 29 patients with acute rheumatic fever were subjected to blood cultures, of whom 9, or 31 per cent, yielded a streptococcus. During the Spring of 1929, 31 patients with acute rheumatic fever were studied by blood cultures, of whom 26, or 83.9 per cent, yielded a streptococcus. The higher percentage of positive cultures in the 1929 series appears to have been due to improved cultural methods. 2. Of the 35 strains of streptococci recovered from blood cultures, 33 have been classified as alpha streptococci (Str. viridans); one as a beta streptococcus (Str. hemolyticus); and one a gamma streptococcus (Str. anhemolyticus). Some of the viridans strains produced very little green on blood media. 3. Agglutination and absorption tests indicate that the strains of streptococcus viridans recovered from the blood of patients with rheumatic fever show a tendency to fall into specific biological groups. 4. In 7 patients with rheumatic fever who were subjected to cultures from affected joints, 5, or 71.4 per cent, yielded a streptococcus viridans. In 3 patients in whom green streptococci were recovered from both the blood and joint, agglutination and absorption tests proved the identity of the strains isolated from the two sources. 5. These findings corroborate those of previous investigators and make it difficult to escape the conclusion that rheumatic fever is a streptococcal infection usually of the alpha or viridans type. 6. The pathogenesis of rheumatic fever in respect to the joint lesions appears to be analogous to that of infectious arthritis and gonococcal arthritis. Bacterial allergy probably influences the clinical picture in all three conditions, but in each instance the joint manifestations are primarily dependent upon localization of bacteria in the joint, with subsequent infection.     

THE FIBRINOLYTIC ACTIVITY OF HEMOLYTIC STREPTOCOCCI

Broth cultures of hemolytic streptococci derived from patients are capable of rapidly liquefying normal human fibrin clot. The active fibrinolytic principle is also contained in sterile, cell-free filtrates of broth cultures. The degree of activity of filtrates parallels the activity of whole broth cultures sufficiently closely to indicate that large amounts of the fibrinolytic substance are freely excreted into the surrounding medium and pass readily through Berkefeld V, Seitz, and Chamberland filters. The occurrence of fibrinolysis is most strikingly observed when plasma or fibrinogen is mixed with active cultural material before clot formation is effected. Under the standard experimental conditions described, complete dissolution of human plasma clot (whole oxalated plasma + CaCl₂) occurs in about 10 minutes; complete dissolution of human fibrinogen clot (chemically isolated fibrinogen + thrombin) takes place in about 2 minutes. Titration of filtrate activity is recorded in Table IV. Twenty-eight strains of Streptococcus hemolyticus, isolated from patients suffering from various manifestations of streptococcus infection, have been tested for the capacity to liquefy fibrin clot. Broth cultures of all of the strains induced fibrinolysis. Of 18 strains of Streptococcus hemolyticus of animal origin, only three were capable of causing dissolution of clot. Completely negative results were obtained with 38 strains of other bacterial species. The list is presented on pages 492 and 493. The plasma of many patients recovered from acute hemolytic streptococcus infections, when clotted in the presence of active cultures, is highly resistant to fibrinolysis. Furthermore, serum, derived from patients whose plasma clot is resistant, often confers on normal plasma clot an antifibrinolytic property. One example of the resistance possessed by the blood of convalescent patients is presented in this report. A second paper, now in preparation, will give in detail a large number of observations on the relation of infection to the development of resistance to the fibrinolytic activity of hemolytic streptococci. In contrast to the susceptibility of normal human fibrin clot to liquefaction by active culture, normal rabbit fibrin clot is totally resistant to dissolution when tested under comparable conditions. The insusceptibility of rabbit fibrin clot is manifest provided the coagulum is composed of rabbit constituents. When human thrombin is used to clot rabbit plasma or fibrinogen in the presence of active cultures, fibrinolysis is not prohibited. The rôle of thrombin in determining the resistance or susceptibility of rabbit fibrin to dissolution offers a suggestive approach to problems relating to the underiving mechanism.     

THE CLASSIFICATION OF HEMOLYTIC STREPTOCOCCI

1. The hemolytic variety of streptococcus is homogeneous, consisting of members that are nearly identical. 2. This homogeneity is most strikingly displayed in the behavior of these streptococci in the complement fixation reaction, all the strains studied reacting in a nearly identical way with all the antisera.     

STUDIES ON THE RELATIONSHIP OF STREPTOCOCCUS HEMOLYTICUS TO THE RHEUMATIC PROCESS : III. OBSERVATIONS ON THE IMMUNOLOGICAL RESPONSES OF RHEUMATIC SUBJECTS TO HEMOLYTIC STREPTOCOCCUS

In the first two papers findings were presented which point to a close relationship between the incidence of rheumatic fever and the distribution of Streptococcus hemolyticus. The fact was emphasized that in the rheumatic subject a recrudescence of the disease process is usually preceded by pharyngeal infection with hemolytic streptococci. These organisms conspicuous in the throat flora during the period of infection preliminary to an attack of acute rheumatism fell into six antigenic groups and produced toxins which in 70 per cent were neutralized by a monovalent streptococcus antiserum. In the present study, four series of observations have been presented, demonstrating the development of immune bodies to hemolytic streptococcus during the course of rheumatic fever. The agglutination and complement fixation reactions of sera from patients with acute rheumatism suggest recent infection with streptococcus. Precipitin tests indicate that at the time of appearance of the rheumatic attack, individuals develop, in their blood, precipitins to the protein fractions of hemolytic streptococcus. That these precipitins may not be entirely specific is recognized from their cross-reactions with antigens of chemically related organisms. The studies made in association with E. W. Todd of England have demonstrated that at the onset of an attack of acute rheumatism, there occurs in each instance a rise in the antistreptolysin titer of the patient''s serum. This titer is much higher than that observed in normal subjects or in patients with bacterial infection other than hemolytic streptococcus. This presence of antistreptolysin with an N.D. of 0.005 cc. is considered strong evidence of recent infection by hemolytic streptococcus. In conclusion, the relationship between the incidence of hemolytic streptococcus and the geographical distribution of rheumatic fever, the relationship between the recrudescence in the rheumatic subject and infection of the throat with hemolytic streptococcus, the development of immune bodies for hemolytic streptococcus at the onset of the rheumatic attack and the apparently specific relationship of antistreptolysin formation to infection with hemolytic streptococcus,—together this combined evidence indicates that the infectious agent initiating the rheumatic process is Streptococcus hemolyticus.     

A COMPARATIVE METHOD FOR TESTING THE ENZYMES OF LIVING HEMOLYTIC STREPTOCOCCI : I. LIPASE.

1. A method is presented for the quantitative comparison under various conditions of the activity of the lipolytic enzyme of the hemolytic steptococcus. The speed of acid production as shown in the color change from pH 8.0 to 7.2, when the living streptococcus is suspended in association with ethyl butyrate, is considered to be indicative of the amount of ferment elaborated by the organism. 2. The lipolytic action is a function of living, actively growing organisms such as are present in 4 to 8 hour cultures. 3. The speed of the lipolytic action is approximately in linear proportion to the concentration of the organisms. 4. The lipolytic action is most rapid at 37.5°C., slower at 50°, and absent at 62°. 5. The organisms are partially destroyed and the lipolytic action is markedly delayed by previous heating to 55°C. for 10 minutes. Both the organisms and the lipolytic activity are completely destroyed by continued contact with this temperature for 30 minutes. 6. Increasing the virulence of the organism for rabbits by repeated animal passage does not increase the lipolytic action. 7. The predilection of the hemolytic streptococcus for the subcutaneous fat in local streptococcus infection associated with extensive superficial gangrene cannot be explained on the basis of an increase of lipolytic ferment in the organisms recovered from these cases over that of heterologous strains of streptococci.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : III. CHEMICAL AND IMMUNOLOGICAL PROPERTIES OF THE SPECIES-SPECIFIC SUBSTANCE.

1. The chemical and immunological characteristics of the species-specific substance (C) of Streptococcus hæmolyticus are considered. (a) It seems to be a carbohydrate because considerably purified preparations of C resisted prolonged tryptic and peptic digestion and were negative for the ordinary protein color tests but gave positive Molisch reactions to the limit of the precipitin titer. One such "purified" lot, however, had 4.2 per cent nitrogen and only 28 per cent reducing sugars on hydrolysis. Whether the nitrogen was due to impurities or was combined in the C substance itself, as is true of the Type I pneumococcus specific polysaccharide, cannot be stated without more material. (b) The C substance forms precipitates with antibacterial sera prepared against heterologous, as well as against homologous hemolytic streptococci. These precipitates are typical discs like those formed by type-specific carbohydrates of other species of bacteria. C does not precipitate antinucleoprotein sera. (c) While there is only slight direct evidence that the C substance is not antigenic, there is considerable indirect proof that this is the case. It probably is a haptene in the sense of Landsteiner. 2. A discussion is included of the chemical and immunological relationships of all the serologically active substances so far identified in extracts of the hemolytic streptococcus.     

OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCOCCI : I. THE ROLE OF VARIOUS AGENTS IN THE PRODUCTION OF THE HEMOLYSINS

The production of oxygen-stable hemolysin in growing and resting Group A streptococci has been induced by RNA, by detergents, and by mammalian blood serum proteins, in the presence of glucose, Mg⁺⁺, and cysteine. Of the serum proteins, albumin and α lipoprotein could act as inducers. In the case of both these serum proteins treatment with trypsin did not affect the capacity to induce hemolysin production, but removal of the bound lipids by alcohol-ether or chloroform-methanol destroyed this property. In comparisons of the conditions of production and of activity between the hemolysin produced by RNA on one hand and albumin and detergents on the other, some data indicated similarities among the hemolysins, and others, differences. The similarities included similar degrees of temperature dependence for production and equal degrees of inhibition by serum β lipoprotein. Differences found among these hemolysins included differences between, the rate of production of the RNA hemolysin from that of albumin or detergent hemolysin by both resting and growing streptococci, and the failure of utilization of glucosamine as an energy source for the production of albumin hemolysin, in contrast with that of RNA hemolysin. The fact that the data have in some cases indicated similarities and in other cases differences among the hemolysins raises the question of whether these are different molecular species, or a single hemolysin synthesized by the streptococci via different pathways of metabolism, or complexes of a single hemolytic moiety with various molecular carriers.     

A CONTRIBUTION TO THE BACTERIOLOGY OF RHEUMATIC FEVER

The conclusions I would draw from this work are merely those stated in a former paper. 1. The results obtained by injections of streptococci are different from those produced by Micrococcus rheumaticus. 2. Micrococcus rheumaticus cannot be regarded as an attenuated streptococcus, nor acute rheumatism as an attenuated streptococcal pyæmia. 3. In uncomplicated cases of acute rheumatism the organism may not be found in the blood or in the joint exudates.     

SOURCE AND SIGNIFICANCE OF STREPTOCOCCI IN MARKET MILK

The principal source of streptococci in milk is the cow''s udder. The udder streptococci fall into two broad groups; those of the larger group agree in cultural characters and agglutination affinities with mastitis streptococci; the smaller group is composed of low acid-producing streptococci. The streptococci of the latter group produce clear zones of hemolysis about surface and deep colonies in horse blood agar plates. They attack dextrose, lactose, saccharose, and maltose, but do not ferment raffinose, inulin, mannite, or salicin. Acid production in dextrose by the members of this group is about the same as that produced by human streptococci under the same conditions. The limiting hydrogen ion concentration for these pleomorphic udder streptococci in dextrose serum bouillon is within the limits of the limiting hydrogen ion concentration observed by Avery and Cullen for human streptococci. All the streptococci from the vagina, saliva, skin, and feces have been non-hemolytic. Those from the saliva form a heterogeneous aggregation in which individuals fermenting raffinose, inulin, and mannite predominate. From the skin a characteristic streptococcus has been found. It produces acid in dextrose, lactose, saccharose, maltose, raffinose, mannite, and salicin, but fails to acidulate media containing inulin. The fecal streptococci are characterized by the formation of large amounts of acid in dextrose, lactose, saccharose, maltose, raffinose, inulin, and salicin. Mannite is not fermented. Neither the fecal nor the skin streptococci have been isolated from the bottled milk with any great frequency.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : II. CHEMICAL AND IMMUNOLOGICAL PROPERTIES OF THE PROTEIN FRACTIONS.

The chemical and immunological characteristics of the type-specific substance (M) of Streptococcus hæmolyticus are considered. 1. A summary of the evidence for the protein nature of this substance follows: (a) It is precipiated by the usual protein precipitants such as, dilute alcohol, dilute acetic acid, and picric acid. (b) It contains 14 per cent protein nitrogen after considerable purification. (c) It is progressively destroyed by removal of the NH₂ group by treatment with nitrous acid. (d) It is completely and readily digested by trypsin and by pepsin. 2. "Purified" extracts react in relatively high dilution with homologous antibacterial sera, but do not precipitate most heterologous antibacterial sera or sera potent in non-type-specific antibodies for the group reactive nucleoprotein P or for the species-specific probable carbohydrate C. Attempts to immunize rabbits with the type-specific protein have been unsuccessful, with simple salt solution extracts of streptococci as well as with purified solutions. This protein seems, therefore, to have the characteristics of a haptene. The type-specific substance (M) is contrasted with the so called nucleoprotein (P) which shows group relationships with nucleoproteins of related species and is the only fraction of hemolytic streptococcus extracts so far obtained which, after separation from the bacterial cell, is a true antigen leading to antibody production when injected into rabbits. The occurrence of another non-type-specific protein (Y) is suggested by occasional cross-reactions of purified M with certain antibacterial sera. Since it has not been separated from extracts containing the type-specific M, little is known of it either chemically or serologically. The cross-reaction disappears on tryptic or peptic digestion of the extract. The fact that such extracts do not show cross-reactions with anti-P sera is evidence that this non-type-specific protein is not P.     

THE EFFECT OF A POLYSACCHARIDE-SPLITTING ENZYME ON STREPTOCOCCAL INFECTION

1. Confirming the observations of other experimenters, it has been found that group A hemolytic streptococci produce a capsule containing a polysaccharide which is similar to, if not identical with, certain high molecular weight sugars found in the mammalian body. 2. Leech extract possesses a powerful enzyme capable of splitting one of the linkages in this polysaccharide and of decapsulating group A and group C hemolytic streptococci in vitro and in vivo. 3. Mice and guinea pigs can be protected from intraperitoneal infection with a virulent group C streptococcus by the intraperitoneal administration of leech extract. In contrast there is little protective action of leech extract in mice infected with group A hemolytic streptococci. 4. The protective effect of leech extract against streptococcal group C infection is probably due to the removal of the capsule in vivo. 5. The capsule of mouse virulent group C streptococci plays a major rôle in the virulence of that microorganism, while the capsule of certain mouse virulent group A streptococci plays little, if any, rôle in virulence, at least when the infection is intraperitoneal in the mouse.     

BIOLOGICAL AND PHYSICAL PROPERTIES OF THE HEMOTOXIN OF STREPTOCOCCI

The hemotoxin of streptococcus is a labile substance affected by centrifugation or shaking. It is adsorbed by various organic and inorganic substances. Hemotoxin is produced within a wide range of hydrogen ion concentrations. It is neither in nor on the bacterial cell but is free in the culture medium. It is probably not an enzyme. There are at least two substances which are essential to the medium for the elaboration of hemotoxin, one of which is phosphorus; the other is a substance of unknown composition. The unknown component is present in small quantities in unfiltered muscle infusion, but is more abundantly supplied by blood serum and kidney infusion. This substance is not an albumin, globulin, primary or secondary proteose, metaprotein, or peptone of the medium or enriching fluid. It is water-soluble, is destroyed by boiling in alkaline solution and by prolonged heating, and is removed to a considerable extent by passage through a diatomaceous filter.     

FURTHER STUDIES WITH TOXIC SERUM EXTRACTS OF HEMOLYTIC STREPTOCOCCI

1. The same Streptococcus hemolyticus organisms may be subjected to extraction six times in 2 days with untreated inactivated serum with no loss in potency of the later extracts when the organisms are kept frozen solid during the night between the extractions. 2. The serum extract toxins of hemolytic streptococci can be preserved without deterioration for at least 6 months if kept frozen solid. 3. No toxins stronger than those containing 10 units per cc. for mice have been prepared. Reasons for thinking that this is due to the saturation of the serum with the toxin at this point are given. 4. Half saturation with (NH₄)₂SO₄ precipitates out practically all of the hemotoxin in a preparation. 5. Serum extracts were made from strains of hemolytic streptococci other than the Gay strain and attempts were made to correlate the virulence and toxin production from each strain. No such correlation could be established. 6. The principal pathologic finding in mice inoculated with the streptococcus serum extract toxin is a marked degeneration of the tubular epithelium of the kidney.     

STUDIES UPON MINUTE HEMOLYTIC STREPTOCOCCI : II. THE DISTRIBUTION OF MINUTE HEMOLYTIC STREPTOCOCCI IN NORMAL AND DISEASED HUMAN BEINGS

1. Minute beta hemolytic streptococci were found to occur from one-third to one-half as frequently in normal individuals as do ordinary beta hemolytic streptococci. 2. They were rarely isolated from the rhinopharynges of individuals suffering from chronic disease. 3. In acute respiratory tract infection other than that due to the ordinary beta hemolytic streptococcus the incidence of minute streptococci was slightly higher than in normal individuals. 4. In acute streptococcal infections, scarlet fever and acute tonsillitis, for example, the incidence of minute hemolytic streptococci did not significantly vary from the incidence found in normal human beings. 5. Minute beta hemolytic streptococci were found in the throats of 33 out of 42 patients ill with glomerular nephritis and in 25 out of 59 patients who were suffering from the various manifestations of rheumatic fever. 6. In glomerular nephritis and rheumatic infection the minute beta hemolytic streptococci were isolated from the throats of more patients than were the ordinary beta hemolytic streptococci.     

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : II. ANTIGENIC RELATIONSHIPS BETWEEN STRAINS OF STREPTOCOCCUS H?MOLYTICUS ISOLATED FROM SCARLET FEVER.

1. Hemolytic streptococcus has been found in 100 per cent of the throats of patients with scarlet fever during the 1st week of the disease. 2. The average length of time that these organisms are present in the throat varies from 10 to 20 days. 3. No morphological or cultural characteristics peculiar to the hemolytic streptococcus from scarlet fever can be demonstrated. 4. Ten immune sera have been prepared from different strains of scarlet fever streptococci and each of the sera agglutinated more than 80 per cent of the strains isolated from scarlatinal throats. On the other hand, scarlatinal streptococci are not agglutinated by immune sera prepared from hemolytic streptococci isolated from other pathological sources. 5. Serum from patients convalescent from scarlet fever agglutinates weakly or not at all the homologous strain of hemolytic streptococcus. 6. The specificity of the agglutination reaction of scarlatinal streptococci is confirmed by absorption experiments. 7. Scarlatinal antistreptococcic serum affords some degree of protection against virulent scarlet fever streptococci but has no protective power against hemolytic streptococci from other diseases. 8. In a small epidemic of scarlet fever a healthy carrier of hemolytic streptococcus was detected; the organism carried was identical in its serological reactions with strains of hemolytic streptococci isolated from active cases of scarlet fever. 9. In a study of a number of contacts with a case of scarlet fever, in only one instance was a scarlatinal type of hemolytic streptococcus recovered from the throat.     

TYPE-SPECIFIC PROTECTION AND IMMUNITY FOLLOWING INTRANASAL INOCULATION OF MONKEYS WITH GROUP A HEMOLYTIC STREPTOCOCCI

1. Nasopharyngeal carrier states of several weeks to several months duration were induced in the Macaca mulatta by the intranasal inoculation with matt strains of group A streptococci. 2. Following such a successful inoculation with a particular type of group A streptococcus, the animal was usually resistant to reimplantation with that same type for several months to a year or more, although reimplantation with a heterologous type could generally be easily effected. 3. This resistance was shown to be closely correlated with the antibodies directed toward the type-specific M antigen, not toward the T antigen of the strains employed. 4. A majority (83.8 per cent) of the animals in which intranasal inoculation was followed by successful implantation developed significant increases in the antistreptolysin O titres of their sera; and in a limited number of instances, type-specific agglutinins and bacteriostatic antibodies were demonstrable in the animals'' sera following successful implantation. 5. Nasopharyngeal carrier states could not be induced with the glossy, avirulent variants of group A streptococci; these animals, moreover, failed to show antibody responses and were susceptible to implantation with the matt variants of the homologous glossy strains. 6. The findings are in accord with the known facts regarding immunity to group A streptococci gained through experiments on rodents; and the possible relationship of these observations to the problem of type-specific immunity in human beings is discussed.     

THE FORMATION OF METHEMOGLOBIN BY STREPTOCOCCUS VIRIDANS

Cultures of Streptococcus viridans when brought into contact with red blood corpuscles have the power of transforming oxyhemoglobin into methemoglobin. The reaction occurs only in the presence of living streptococci when they are able to carry on their metabolic activities. The intensity of the reaction runs roughly parallel with the period of growth and multiplication of the bacteria and gradually diminishes and disappears as growth ceases. There is no apparent relation between the activity of a given strain of Streptococcus viridans in producing methemoglobin and its source or virulence. If the streptococci are suspended in salt solution they are unable to change oxyhemoglobin into methemoglobin unless some nutrient substance is present. Of the various nutrient substances tested dextrose is the most efficient in enabling the organisms to bring about the reaction. The reaction does not occur in the absence of oxygen, and is retarded by an excess of oxygen. Substances which tend to reduce the metabolic activities of the bacteria to a minimum exert an inhibitory action on methemoglobin formation. While not definitely proving it to be so, the results obtained in the above experiments strongly support the supposition that the reaction is not due to injurious substances produced by the bacteria or to products arising from the decomposition of the nutrient material present, but rather to the metabolic activities of the bacteria themselves when they are surrounded by environmental conditions which render growth and multiplication possible. The exact chemical nature of the change of oxyhemoglobin to methemoglobin is not known, but it is probably an oxidation process or a combination of reduction and oxidation processes, as pointed out by Heubner. As Cole has shown, the action of aminophenol is of great interest in this connection, in that it acts like a catalytic agent in being able to transform much more hemoglobin into methemoglobin than would be possible if the reaction were a simple molecular one. The metabolic activities of bacteria are largely in the nature of oxidation and reduction processes. The transformation of oxyhemoglobin into methemoglobin by streptococci of the viridans type, therefore, may be analogous to the action of such substances as aminophenol, and the reaction may be due to the active oxidation and reduction processes occurring in the neighborhood of the bacterial cells. The failure of the reaction to occur in the absence of oxygen and its retardation in the presence of an excess of oxygen, both with streptococci and with pneumococci (Cole) would seem to support this theory. Such results, however, may be due to the abnormal conditions surrounding the bacteria with consequent inhibition of their metabolic activities. Cole concluded as the result of his study of methemoglobin formation by pneumococci that since bacteria may injure red blood cells apparently by disturbances in oxidation in the immediate neighborhood of the organisms rather than by the production of a definite toxin, it is possible that bacteria may injure other tissue cells in a like manner and that the pathological effects produced by these bacteria may be explained on this basis. The experimental results recorded above have shown that the formation of methemoglobin by Streptococcus viridans in no way differs from its formation by pneumococci, and they lend support to the theory that bacteria may be injurious to tissues because of the disturbances in oxidation brought about by the metabolic activities of the organisms, especially those associated with growth and multiplication. It is believed that this theory may be particularly applicable to the pathological effects caused by Streptococcus vindans because the lesions produced by it, whether single or multiple, both in man and in experimental animals, are prone to be localized and associated with the actual presence of the streptococci in the lesions.