THE OCCURRENCE AND VIRULENCE OF PNEUMOCOCCI IN THE CIRCULATING BLOOD DURING LOBAR PNEUMONIA AND THE SUSCEPTIBILITY OF PNEUMOCOCCUS STRAINS TO UNIVALENT ANTIPNEUMOCOCCUS SERUM

Studies of the bacteriology of the blood were made in thirty-seven cases of lobar pneumonia. The pneumococcus was isolated from the blood in approximately 50 per cent. of the cases studied. The course of infection in individuals with pneumococcus in the blood was more severe than in those in which no organism could be cultivated from the blood. 77 per cent. of the patients with positive blood cultures died, and 79 per cent. of patients with negative blood cultures recovered. In fatal instances of pneumonia, where the pneumococcus was found in the blood, the number of organisms per cubic centimeter of blood was very high in the last stage of the disease. In individuals dying of pneumonia without blood infection, the disease was characterized by a rapid spread of the local process in the lungs. It is not unlikely that the symptoms of collapse, developing on the fifth or sixth day of lobar pneumonia, are often the expression of serious invasion of the blood by the pneumococcus. In other instances, they mark an extension of the local process in the lungs. Strains of pneumococcus isolated from the blood of patients with lobar pneumonia were usually of high animal virulence. In a few instances where the organism isolated from the blood was of low virulence for animals, the patients recovered. The protective power of a univalent antipneumococcus serum was tested against nineteen strains of typical pneumococcus and against four strains of closely allied organisms. The serum manifested some degree of protection against twelve out of nineteen strains of typical pneumococci. No protection was observed against the atypical organisms. In eight instances the degree of protection obtained was high, in three low, and in one there occurred only a prolongation of the period of life of the inoculated animal

PROTEOLYTIC ENZYMES AND ANTI-ENZYMES OF NORMAL AND PATHOLOGICAL CEREBRO-SPINAL FLUIDS

The experiments which have been presented show that the spinal fluid occupies a unique position among the fluids which accumulate in serous cavities of the body. It contains normally neither proteolytic enzyme nor anti-enzyme, whereas blood serum, from which it is derived, exhibits both enzymotic and anti-enzymotic activity. In the blood anti-enzyme greatly predominates over enzyme, so that proteolysis does not occur, unless the anti-enzymotic power of the serum has been destroyed by the addition of acid. In pathological conditions both enzyme and anti-enzyme may make their appearance in the spinal fluid. With inflammations of other serous cavities of the body the anti-enzyme of the exuded serum as a rule preponderates over and restrains the activity of the proteolytic enzyme freed from leucocytes. On the other hand, in infection of the meninges with Diplococcus lanceolatus and with Streptococcus mucosus free proteolytic enzyme has been present in considerable amount in four of five fluids which have been tested. Free proteolytic enzyme has not been observed in the spinal fluid in cases of epidemic meningitis. The cases which have been studied demonstrate that in epidemic meningitis some anti-enzymotic action may be present in the early stages of the disease; but it tends to disappear rapidly so that anti-enzyme seems to be constantly at a low ebb. It is possible that the absence of anti-enzyme in normal spinal fluid, and the tendency for it to disappear so much more rapidly than in other inflammatory exudates, may explain in part the severity of acute meningeal infections. Non-inflammatory transudates into the subdural spaces differ from inflammatory exudates in that the inhibitory element of the blood serum accumulates, and this accumulation suggests an interference with the elimination of the antibody from the spinal fluid. Such interference is not evident in so-called serous meningitis. In content of anti-enzyme the spinal fluid of chronic conditions, such as tuberculous meningitis, apparently occupies an intermediate position between acute inflammation and serous effusion, and five of seven tuberculous fluids which were tested exhibited various degrees of anti-enzymotic action. Variations in content of enzyme and anti-enzyme, noted above, may depend upon the rapidity with which the fluid, carrying the elements mentioned, enters the spinal cavity, as well as upon the rate of their elimination from the spinal fluid. Subdural injection of large quantities of anti-meningitis serum (horse's blood serum) does not increase the anti-enzymotic activity of fluids withdrawn twenty-four hours after its injection; disappearance of anti-enzyme being caused by rapid elimination of serum from the spinal fluid

THE PRESENCE OF PROTECTIVE SUBSTANCES IN HUMAN SERUM DURING LOBAR PNEUMONIA

These experiments demonstrate that protective substances are usually present in the blood of patients recovering from lobar pneumonia. As a rule, the appearance of protective bodies in the blood, when demonstrable, coincides rather sharply with the period of critical fall in temperature and the disappearance of symptoms. These substances are not present in the blood in any measurable degree before the crisis, but afterward they may reach a considerable concentration. In certain instances, protective substances either become evident some time after the occurrence of the crisis, or cannot be demonstrated at any period of the disease. Experiments in which it is possible to test serum against an homologous strain of pneumococcus yield in the majority of cases evidence of the presence of protective bodies; whereas in those in which stock cultures are used, the serum, as a rule, shows no protection. The development of specific protective substances in the serum of patients with lobar pneumonia suggests that these bodies may play a part in the mechanism of recovery

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : VII. ALLERGIC REACTIONS WITH STRAINS FROM ERYSIPELAS.

Rabbits immunized with filtrates of cultures of hemolytic streptococcus from erysipelas show cutaneous allergy. Two periods of allergy have been observed, an early and a late phase. The earliest reactions occurring in the first period of allergy can be neutralized with erysipelas immune sera. The rash of scarlet fever and the Dick reaction are apparently allergic reactions to products of Streptococcus scarlatinæ

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : IV. THE OCCURRENCE OF STREPTOCOCCUS SCARLATINÆ IN CONVALESCENCE AND IN THE COMPLICATIONS OF SCARLET FEVER.

1. A group of streptococci which agglutinate with scarlatinal immune sera has been isolated from the throats of 65 per cent of cases of scarlet fever during the 1st week of the disease. 2. Strains of hemolytic streptococci belonging to this group have been isolated from the throats of patients at the termination of quarantine (30 days). 3. Hemolytic streptococci are found most frequently in the throat in convalescent patients in whom the tonsillar inflammation has not entirely subsided. 4. The complications occurring in scarlet fever may be due to the original scarlatinal strain or may be the result of a secondary infection with pyogenic strains of streptococci

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : VI. BIOLOGY OF HEMOLYTIC STREPTOCOCCUS: ANTIGENIC RELATIONSHIPS BETWEEN STRAINS OF THE SCARLATINAL AND ERYSIPELAS GROUPS.

The interpretation of the results in these experiments is difficult on account of the variations in the physical capacity of different strains of hemolytic streptococcus to agglutinate and absorb agglutinin. Even when the antigens for agglutination and absorption are standardized and sera are carefully titrated variations in agglutination occur with different strains and sera. Variations may occur because of differences in the titer of the immune sera employed. In a large series of reactions with the same sera and strains, errors in interpretation depending on these factors can be eliminated because strains which agglutinate poorly will consistently give weak reaction with all sera, and weak sera will agglutinate all strains poorly. after such variations See PDF for Structure in agglutination are eliminated, if a strain varies grossly in agglutination with different sera, or the titer of different sera varies with the same strain as in Tables I to III, variations which occur must represent antigenic dissimilarities. We have previously commented on the dissimilarity of the individual strains in the scarlatinal and erysipelas groups. Although the strains within each of the groups are related they are seldom identical. This individuality of the strain was indicated by the fact that absorption by heterologous strains failed to absorb the agglutinin for the strain homologous with the serum. This strain individuality is again apparent in Tables I to III. Doses of heterologous strains equivalent to the unit absorptive dose which has already been defined, fail to remove the agglutinin for the homologous strain. This dose is sufficient however, to absorb the agglutinin for practically all heterologous strains. It appears from this observation that the strain specificity dominates the group specificity. Between the unit absorptive dose and a dose which is approximately 0.2 to 0.3 of this unit, is a zone of great variation in absorption and agglutination. Below this zone absorption is complete for few strains. The differences in titer of various strains and absorbed sera in the zone where variations occur are very definite. For example in Table II, Strain E IX agglutinates well in Serum II absorbed with Strains E I and E III but poorly in serum absorbed with Strain E II. Numerous similar examples may be found. In a few instances heterologous strains agglutinate nearly as well as the homologous strains in these absorbed sera. Such strains probably resemble the homologous strains closely. These resemblances are not confined to the erysipelas strains but occur among the scarlet strains which agglutinate in erysipelas sera. Apparently it is impo—ssible to distinguish such scarlatinal strains from erysipelas strains by absorption of agglutinin. The relationship shown by these absorption tests is additional evidence in favor of the mosaic nature of the antigen. Strains of streptococcus of the erysipelas groups are apparently composed of several agglutinogenic fractions. If the antigen of a strain is made up of the fractions A, B, C, D, and E, serum for that strain would contain A, B, C, D, and E agglutinins. If this serum were absorbed with the homologous strain or an identical heterologous strain the agglutinin would be completely absorbed. On the other hand a qualitative or quantitative difference between the two strains would be apparent in the absorption and agglutination reactions. Such differences are evident in Tables I to III. This serum would agglutinate any strain containing any one or several of the fractions, but absorption of the serum by such a strain would leave fractions of the agglutinin in the serum. This absorbed serum would agglutinate the homologous strain. Heterologous strains would agglutinate if fractions remained in the serum corresponding to their antigenic components. Hence the reactions in the absorbed serum would depend on the qualitative and quantitative relationships between the component fractions in the serum, the absorbing strain, and the strains agglutinated. This conception of multiple antigens is not new in bacteriology. Durham (3) explained the reactions in the colon-typhoid group of bacilli on this basis. More recently Durand and Sedallian (4), and Andrewes, Derick, and Swift (5) have expressed the opinion that the agglutination reactions with hemolytic streptococcus can only be accounted for in this way. In many respects the reactions observed with the colon-typhoid group of Gram-negative bacilli resemble those experienced with the erysipelas and scarlatinal groups of hemolytic streptococcus. Apparently we have exhausted the possibilities of studying these groups further by agglutination and absorption. Our knowledge regarding the specificity and relationship of the antigenic fractions must come from the study of fractions isolated and refined by chemical methods

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : III. AGGLUTINATION AND ABSORPTION OF AGGLUTININ WITH STREPTOCOCCUS SCARLATINÆ.

1. Strains of hemolytic streptococci from cases of scarlet fever occurring in New York, San Francisco, Chicago, Baltimore, and Copenhagen, Denmark, all interagglutinate with immune sera prepared with these strains. 2. Sera prepared with these strains do not agglutinate pyogenic streptococci or strains isolated from cases of septic sore throat. 3. The strains obtained from the throats of patients from an epidemic of scarlet fever and the strain from the milk responsible for this epidemic fall into the scarlatinal group according to these agglutination tests. 4. Absorption tests can be carried out with these strains and sera under proper conditions. 5. A group of hemolytic streptococci biologically distinct from streptococci from other sources than scarlet fever is constantly associated with scarlatina. They constitute a group of closely related streptococci which may be identified by agglutination tests

ANTIBLASTIC IMMUNITY

1. Antipneumococcus serum possesses the power of inhibiting for a certain period of time the multiplication of pneumococci. 2. It also has the capacity of inhibiting the proteolytic and glycolytc functions of pneumococci. 3. This power is acquired for the first time or appears in increased amounts in human serum at the time of crisis in lobar pneumonia. 4. The retardation of bacterial growth is thought to be dependent upon the inhibition of metabolic function due to the presence of anti-enzymotic substances in antipneumococcus serum. To this phenomenon we have applied the term antiblastic immunity

VARIETIES OF PNEUMOCOCCUS AND THEIR RELATION TO LOBAR PNEUMONIA

A study of pneumococci isolated from individuals suffering from lobar pneumonia has shown that the majority of these organisms fall into definite biological groups. These groups have been arbitrarily numbered from I to IV. The first three groups consist of organisms which within the group are closely related to each other by certain immunological reactions; i. e., protection and agglutination. Extensive study has failed to reveal crossing in either of these reactions between members of separate groups. The fourth group is formed of a series of independent varieties which cannot be definitely related to one another by the immune reactions employed. Up to the present time we have observed no tendency of these organisms to lose their specific characters, nor have we observed a change of one type into another. These groups vary in their pathogenicity for human beings, and in the order of their virulence are as follows: group III, group II, group I, group IV. The degree of protective power developed in the sera of animals immunized against members of the different groups varies inversely with the virulence and with the amount of capsular development. This, however, applies only to tests of passive immunity. The highly virulent groups give as good active immunity as those of lower virulence, if not better. In view of these constant differential characters of the pneumococcus, it was deemed advisable to study the pneumococci occurring in normal sputum. It has been commonly assumed that infection in pneumonia is autogenic, and occurs from the invasion of the lungs by a pneumococcus habitually carried in the mouth. If this is so, we should find the same types in the normal mouth as occur during the disease. Examination of a series of normal individuals showed this not to be the case. In no instance was an organism found which could be grouped with any of the fixed types of pneumococcus. All exhibited the same characters as those organisms obtained from lobar pneumonia which belong to group IV. Inasmuch as organisms belonging to this group are of low virulence, and are responsible in our experience for only 20 per cent. of the cases of pneumonia, it is at once manifest that the majority and more virulent cases of pneumonia are due to organisms which are not found in normal mouths. To gain further evidence of this difference, a study has been made of convalescents from pneumonia who had been infected by typical organisms. During the period of recovery these typical organisms are supplanted by the type which occurs in normal mouths. The period of disappearance of the typical varieties has varied. The shortest time in which disappearance has occurred has been twelve days, and the longest period in which typical organisms have been carried has been ninety days. In the latter instance the patient was lost sight of, so that he may well have carried the virulent form for a longer period of time. In general, when typical organisms persist for a long time, there is delay in the healing of the lung lesion. If recovery is prompt, as a rule the virulent types disappear rapidly. We have said that the virulent types do not occur in normal mouths. There are exceptions to this observation. In a number of instances organisms belonging to the typical groups have been isolated from the mouth sputum of healthy individuals. So far this has occurred only in individuals intimately in contact with cases of lobar pneumonia. Wherever typical organisms have been obtained under such circumstances, the type has always corresponded to that with which the case of pneumonia was infected. Such individuals, therefore, become infected with virulent types of pneumococcus by contact, and may be regarded as healthy carriers of disease-producing types. This study makes it probable that the majority of cases of pneumonia are dependent upon either direct or indirect contact with a previous case. Mere infection of the mouth by virulent types is by no means sufficient to cause the disease. In order to invade the lungs, these virulent types must find the circumstances favorable, or a suitable condition must arise during the period when they are harbored in the mouth. Comparative study of certain strains of pneumococci received from South Africa suggests that new groups of parasitic organisms develop only during the period of high racial susceptibility. A like condition of affairs is brought about when a group of hitherto unexposed individuals is brought into contact with an infectious microörganism. The development of racial immunity soon limits the number of new types which may arise. The suggestion is made that strictly parasitic races of microorganisms are pure lines and have established themselves as parasites during a period of high racial susceptibility

THE ELABORATION OF SPECIFIC SOLUBLE SUBSTANCE BY PNEUMOCOCCUS DURING GROWTH

1. A specifically reacting substance of bacterial origin is present in the cell-free fluids of young cultures of pneumococcus. This substance is present when the organisms are growing at their maximum rate and undergoing little or no cell death, and consequently its. presence is not dependent upon cell disintegration but represents the extrusion of bacterial substance by the living organism. 2. The blood and urine of rabbits experimentally infected with pneumococcus contain a similar specific soluble substance during the early hours of the infectious process. 3. Human beings suffering from lobar pneumonia have in their blood and more frequently in their urine a specific soluble substance of pneumococcus origin. The amount of this substance present in the urine varies in different individuals and the presence of a large amount is of unfavorable prognostic import. This specific precipitin reaction in the urine is of diagnostic value. 4. Rabbits injected with soluble pneumococcus material continue to excrete this substance for a considerable period of time. 5. The specifically soluble substance obtained from bacterial cultures and from the urine during infection is not destroyed by boiling, by precipitation with alcohol, acetone, or ether, or by trypsin digestion. 6. Studies are in progress at this time on the degree of toxicity and on the antigenic properties of the substance