REACTIONS OF RABBITS TO NON-HEMOLYTIC STREPTOCOCCI : I. GENERAL TUBERCULIN-LIKE HYPERSENSITIVENESS,ALLERGY, OR HYPERERGY FOLLOWING THE SECONDARY REACTION.

1. Accompanying and following the evolution of a secondary reaction in the skin of rabbits after inoculation with suitable doses of certain non-hemolytic streptococci there quickly develops a general state of hypersensitiveness or allergy towards these streptococci. 2. This state is made evident by ophthalmic reactions following corneal inoculations, by much increased reactivity of the skin following intracutaneous reinoculations, and by lethal reactions, resembling tuberculin shock, following intravenous inoculations. 3. In a given hypersensitive rabbit there is a rough parallelism in the intensities of these different kinds of reactions. 4. This type of hypersensitiveness or bacterial allergy does not follow primary intravenous inoculation of rabbits with comparable doses of the streptococci employed. 5. As the development of this type of hypersensitiveness or bacterial allergy seems to accompany the production of focal lesions of a certain intensity, it is probable that in these foci are produced the substances or conditions which lead to this type of bacterial allergy.     

THE EFFECT OF ANTECEDENT INFECTION AND IMMUNIZATION WITH STREPTOCOCCI UPON THE REACTIVITY OF RABBITS TO HORSE SERUM

1. The cutaneous responses of rabbits to small doses of horse serum intracutaneously is described. After an original injection of 0.1 cc. a secondary reaction often occurs about the 9th day, and tests at 3 day intervals with 0.001 cc. quantities indicate that general skin hypersensitivity is established at this time. Circulating precipitins for horse serum appear later. As the degree of sensitivity increases, lesions resulting from test doses reach a maximum development more quickly. 2. The reactivity of rabbits to horse serum is greatly increased by antecedent sensitization of the animals with repeated small intracutaneous inoculation of indifferent streptococci or immunization with large intravenous injections of either indifferent or hemolytic streptococci. Doses of indifferent streptococci precisely comparable to those producing greatly enhanced reactivity when given intracutaneously, increase reactivity to horse serum irregularly and to slight degree when injected intravenously. 3. Increases in reactivity (allergic irritability) are made evident by the occurrence of larger skin lesions at the site of the primary horse serum injections, the earlier development of more distinct secondary reactions in a higher percentage of animals, and by the more rapid appearance of skin sensitivity to the small test doses which may be first evidenced by delayed reactions at these injection sites. Accompanying these evidences of increased reactivity, there is an early appearance of blood serum precipitins in high titer. 4. There was no evidence of residual inflammation in any of the animals at the time reactivity to horse serum was tested. No attempt was made to determine the duration of this altered reactive capacity. It has been encountered in tests begun from 8 to 14 days after the last preliminary inoculation. 5. It is suggested that the increased reaction described is brought about by a persisting alteration in the functional activity of the reticulo-endothelial system.     

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.     

REACTION OF RABBITS TO STREPTOCOCCI: COMPARATIVE SENSITIZING EFFECT OF INTRACUTANEOUS AND INTRAVENOUS INOCULA IN MINUTE DOSES

1. Rabbits were rendered very hypersensitive by relatively small doses of green streptococci given intracutaneously, and somewhat less hypersensitive by similar doses of heat-killed vaccine prepared from hemolytic streptococci. 2. Animals receiving the same doses intravenously gave, upon subsequent testing, lesions slightly more marked than normal controls; but these lesions were qualitatively hard and nodular compared with the large edematous lesions in the cutaneously sensitized group. 3. There was no parallelism between the degree of cutaneous or ophthalmic hypersensitivity and agglutinin titer in the blood serum. 4. Bacterial hypersensitivity to whole streptococci appears to depend more upon previously induced focal infection than upon circulating antibodies.     

REACTIONS OF RABBITS TO NON-HEMOLYTIC STREPTOCOCCI : II. SKIN REACTIONS IN INTRAVENOUSLY IMMUNIZED ANIMALS.

Rabbits immunized intravenously with living culture or nucleoproteins of non-hemolytic streptococci react to subsequent intracutaneous inoculations with homologous streptococci with smaller and harder lesions than are shown by normal animals similarly inoculated; and they do not develop the general manifestations of hypersensitiveness such as are shown by animals previously inoculated into the tissues with the same cultures. A rabbit may react to intracutaneous inoculation with non-hemolytic streptococci in one of four ways, depending on whether it is normal, hypersensitive, immune or cachectic. Most normal animals show a secondary reaction about 10 days after inoculation with suitable strains of non-hemolytic streptococci; hypersensitive, allergic, or hyperergic animals show much larger lesions than do normals with the corresponding doses of the same streptococci, and practically never show secondary reactions; immune animals show smaller and harder early lesions and usually do not have secondary reactions if they are fairly well immunized. Cachectic animals show very soft and rapidly fading primary reactions and no secondary reactions.     

THE QUESTION OF SENSITIZATION OF JOINTS WITH NON-HEMOLYTIC STREPTOCOCCI

It was impossible to demonstrate a condition of specific joint sensitization to non-hemolytic streptococci by first injecting the joints of rabbits with small doses of killed non-hemolytic streptococci, or with extracts of these organisms, and subsequently inoculating the rabbits intravenously with homologous living bacteria. Joints so treated were no more liable to involvement than were other untreated joints of the same animals.     

EXPERIMENTAL INFECTION OF THE SKIN IN THE HAMSTER SIMULATING HUMAN IMPETIGO : III. INTERACTION BETWEEN STAPHYLOCOCCI AND GROUP A STREPTOCOCCI

The interaction between staphylococci and Group A beta hemolytic streptococci in mixed lesions was investigated in an experimental impetigo model. A strain of staphylococcus of phage Type 71, which has been shown in vitro to produce a bacteriocin for streptococci and other Gram-positive organisms, eliminates or reduces Group A streptococci in mixed lesions. In contrast, staphylococcal strains of phage Types 75 and 81, which do not produce a demonstrable bacteriocin in vitro, exhibit no such effect. Some variation was noted in the in vivo response of two different streptococcal M Types to the bactericidal effect of phage Type 71 staphylococci. Bacterial antagonism is more pronounced when staphylococci and streptococci are injected simultaneously into animals than when staphylococci are superimposed on preexisting streptococcal lesions. Marked variations were found in the numbers of viable streptococci (colony-forming units) recovered from individual lesions containing identical mixtures of streptococci and phage Type 71 staphylococci. The frequency of a demonstrable bactericidal effect was related to the number of streptococci injected. With small inocula of streptococci, the tendency towards an all-or-none effect was particularly striking. No evidence of selection of streptococcal or staphylococcal mutants which might explain this phenomenon was obtained. These observations suggest that the bactericidal effect of phage Type 71 staphylococci on other Gram-positive organisms, previously demonstrated in vitro, appears to operate also in vivo.     

A CLASSIFICATION OF NON-HEMOLYTIC STREPTOCOCCI

1. No connection can be demonstrated between grouping of non-hemolytic streptococci based upon fermentation reactions, and grouping based upon immunological reactions. 2. A classification of non-hemolytic streptococci can be effected by studying the complement fixation reactions between the streptococci and their antisera. 3. The arrangement of the streptococci in such a classification depends on the fact that two diverse elements are present in the group. Some strains partake entirely of one of these elements, some entirely of the other, while other strains partake of both. 4. The arrangement is further determined by the fact that among the strains composed of one element there are differences in complexity, some strains being made up of many molecules or features, others having much simpler structure. This gives rise to an inverse ratio between the fixing capacity of a serum and the capacity of the corresponding antigen to be fixed.     

TWO SEROLOGICAL TYPES OF GROUP B HEMOLYTIC STREPTOCOCCI WITH RELATED,BUT NOT IDENTICAL,TYPE-SPECIFIC SUBSTANCES

1. Among group B hemolytic streptococci one serological type previously described as homogeneous has been shown, instead, to contain two closely related types, distinguishable by reciprocal absorption experiments. These streptococci are designated Types Ia and Ib. 2. Homologous organisms in each case absorb all antibodies from their respective antisera, while organisms of the heterologous related type absorb only the antibody responsible for the cross reactions. Group B streptococci of other types do not absorb the antibodies responsible for the cross reactions between these two related types. The precipitin reaction and passive protection tests in mice were employed in this analysis. 3. The type-specific substance of Type Ia is a polysaccharide. Preliminary study indicates that this is also true of Type Ib. While no data are available concerning the chemical relationships of these substances, it seems probable that the two types elaborate polysaccharides, related chemically as well as serologically.     

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.     

Bacterial contamination of three-way taps--one Australian hospital's experience.

The incidence and types of organisms present in the vacant ports of 424 three-way taps were studied. The taps were used in 50 children in ICU. Twenty-five (6%) of the taps showed organism growth of one to three colonies from swabs taken of one or more vacant ports. The organisms cultured were staphylococci, klebsiella, streptococci, bacilli and fungi. While there was no marked difference in the growth rate of organisms in three-way taps used in intravenous, intra-arterial and central venous lines, growth rate increased with duration of use.     

TRANSMISSION OF RESPIRATORY ANAPHYLAXIS (ASTHMA) FROM MOTHER TO OFFSPRING

Rabbits immunized intravenously with living culture or nucleoproteins of non-hemolytic streptococci react to subsequent intracutaneous inoculations with homologous streptococci with smaller and harder lesions than are shown by normal animals similarly inoculated; and they do not develop the general manifestations of hypersensitiveness such as are shown by animals previously inoculated into the tissues with the same cultures. A rabbit may react to intracutaneous inoculation with non-hemolytic streptococci in one of four ways, depending on whether it is normal, hypersensitive, immune or cachectic. Most normal animals show a secondary reaction about 10 days after inoculation with suitable strains of non-hemolytic streptococci; hypersensitive, allergic, or hyperergic animals show much larger lesions than do normals with the corresponding doses of the same streptococci, and practically never show secondary reactions; immune animals show smaller and harder early lesions and usually do not have secondary reactions if they are fairly well immunized. Cachectic animals show very soft and rapidly fading primary reactions and no secondary reactions.     

UDDER INFECTION WITH STREPTOCOCCI OF THE SCARLET FEVER TYPE : III. THE INFLUENCE OF MILK ON THE GROWTH OF SCARLET FEVER STREPTOCOCCI.

The experiments indicate that milk filtered through a Berkefeld candle V or heated at 58°C. for 20 minutes when added to blood agar plate cultures interferes with the development of colonies of the scarlet fever streptococcus. The observed inhibition is proportional to the amount of milk. When the approximate milk dilution in the Petri dish is 1:20 or 1:25 growth of the organisms is completely suppressed or only a small proportion of non-hemolytic colonies develop. As the amount of milk is decreased the colonies become larger and their hemolytic zones more pronounced, although even when the final dilution of milk reaches 1:100 or 1:125 only colonies easily mistaken for the narrow zoned bovine streptococci appear. The effects upon the surviving organisms would appear to be transient since both the non-hemolytic colonies and those with small zones manifest the original hemolytic properties when transferred to other media. When scarlet fever streptococci are added in small quantities to milk heated at 58°C. for 20 minutes and incubated growth is inhibited. If the period is prolonged the streptococci are killed. On refrigeration of such mixtures some of the streptoccci are killed but others survive the test period.     

THE DIFFERENTIATION OF STREPTOCOCCI BY MEANS OF FERMENTATIVE TESTS

If we glance again at the classification proposed by Andrewes and Horder we are struck at once by the fact that no hard and fast differences in fermentative properties characterize the various groups, and that the authors prefer to have recourse to the most frequent habitat of the organisms and to chemical tests, rather than to confine themselves to the latter method alone. From what has been said earlier in this paper, it becomes clear at once that the results gained by these authors are not convincing, for in their work they have failed to use the most favorable medium for growth of the organisms. And thus it can hardly be doubted that different reactions might have been obtained, under the conditions adopted by us. How otherwise could the fact be explained that of thirty-four of their pneumococci only eight fermented inulin. We had a similar experience while using the plain broth which led us to reject it as an unfavorable medium. Further, all our pneumococci fermented lactose with great rapidity. Andrewes and Horder report that eight of their series left lactose unchanged. Taking our own tests into consideration we find that working with but a small number of carbohydrates we were able to find six different varieties of streptococci among only 33 pathogenic strains. Which of the various substances is to decide us in the grouping of the organisms? Would we not find even greater variations from the most common type (see No. 4 in table) if we were to extend our tests over a larger series of chemical agents? These questions are difficult to answer and only extended experimentation with a great many streptococci and many media will clear up the doubtful points. In concluding it may be stated: 1. Streptococci vary considerably in their ability to produce acid from various carbohydrates. 2. Chemical tests of this kind should be made only in the media which are most favorable for the growth of the organisms. 3. Our results gave us six groups of streptococci, when tested upon dextrose, levulose, galactose, maltose, saccharose, lactose, inulin, dextrin and mannite, viz.: Those fermenting (1) all; (2) all but mannite; (3) all but inulin; (4) all but inulin and mannite; (5) all but inulin and lactose; and (6) all but inulin, mannite and saccharose. 4. In view of the comparatively small number of streptococci employed we are hardly warranted in making a definite classification. Perhaps a larger series of tests upon the media employed will enable us to divide streptococci into distinct classes characterized by certain fixed fermentative properties.     

STUDIES ON STREPTOCOCCAL FIBRINOLYSIS : V. THE IN VITRO PRODUCTION OF FIBRINOLYSIN BY VARIOUS GROUPS AND TYPES OF BETA HEMOLYTIC STREPTOCOCCI; RELATIONSHIP TO ANTIFIBRINOLYSIN PRODUCTION

A method for the measurement of fibrinolysin production by beta hemolytic streptococci is described. The test was shown to be highly accurate in that repeated determinations showed only small variations. A study of 766 strains of beta hemolytic streptococci isolated from normal soldiers and patients with respiratory disease showed that fibrinolysin was produced by Lancefield groups A, C, and G, and, in addition, by a few strains of groups B and F. Group A streptococci produced more fibrinolysin on the average than the other groups. The median titers were 117 for group A, 61 for group C, and 20 for group G streptococci. In a study of 388 typed group A streptococci from different subjects the fibrinolytic capacity of an organism was shown to be related to the serological type. The importance of this observation in relation to the role of streptococcal fibrinolysis in infections is discussed. Finally, it was demonstrated that strains of streptococci which produced large amounts of fibrinolysin were capable of stimulating antifibrinolysin formation in patients whereas strains that produced small amounts only occasionally caused antibody formation.     

Parenteral sparfloxacin compared with ceftriaxone in treatment of experimental endocarditis due to penicillin-susceptible and -resistant streptococci.

A new, investigational, parenteral form of sparfloxacin was compared with ceftriaxone in the treatment of experimental endocarditis caused by either of three penicillin-susceptible streptococci or one penicillin-resistant streptococcus. Both drugs have prolonged half-lives in serum, allowing single daily administration to humans. Sparfloxacin had relatively low MICs (0.25 to 0.5 mg/liter) for all four organisms and was also greater than or equal to eight times more effective than the other quinolones against 21 additional streptococcal isolates recovered from patients with bacteremia. Ceftriaxone MICs were 0.032 to 0.064 mg/liter for the penicillin-susceptible strains and 2 mg/liter for the resistant isolate. Both antibiotics resulted in moderate bacterial killing in vitro. Rats with catheter-induced aortic vegetations were inoculated with 10(7) CFU of the test organisms. Antibiotic treatment was started 48 h later and lasted either 3 or 5 days. The drugs were injected at doses which mimicked the kinetics in human serum produced by one intravenous injection of 400 mg of sparfloxacin (i.e., the daily dose expected to be given to human adults) and 2 g of ceftriaxone. Both antibiotics significantly decreased the bacterial densities in the vegetations. However, sparfloxacin was slower than ceftriaxone in its ability to eradicate valvular infection caused by penicillin-susceptible bacteria. While this difference was quite marked after 3 days of therapy, it tended to vanish when treatment was prolonged to 5 days. In contrast, sparfloxacin was very effective against the penicillin-resistant isolate, an organism against which ceftriaxone therapy failed in vivo. No sparfloxacin-resistant mutant was selected during therapy. Thus, in the present experimental setting, this new, investigational, parenteral form of sparfloxacin was effective against severe infections caused by both penicillin-susceptible and penicillin-resistant streptococci.     

STUDIES UPON MINUTE HEMOLYTIC STREPTOCOCCI : I. THE ISOLATION AND CULTURAL CHARACTERISTICS OF MINUTE BETA HEMOLYTIC STREPTOCOCCI

The cultural and biochemical characteristics of a group of minute hitherto undescribed beta hemolytic streptococci from human sources have been recorded and upon the basis of these cultural reactions it is suggested that these organisms may represent new species of the genus streptococcus.     

Epithelial cell binding of group A streptococci by lipoteichoic acid on fimbriae denuded of M protein

Group A streptococci were treated with various enzymatic and chemical agents in an attempt to dissociate the type-specific M protein from intact surface "fimbriae." Mild peptic digestion at pH 5.8, which was previously shown to extract serologically active M antigen from intact streptococci had little visible effect on the fimbriae even though virtually all of the M protein was removed as demonstrated by (a) increased susceptibility to phagocytosis, (b) lack of opsonic effect of homologous M antibody on the treated streptococci, and (c) loss of HCl- extractable M protein. These fimbriated streptococci which lacked M protein adhered to human oral mucosal cells equally as well as untreated, fimbriated organisms which retained their M protein. Removal of both fimbriae and M protein by digesting organisms with HCL at pH 2.0 at 94 degrees C. or with trypsin abolished their ability to bind mucosal cells. Electron microscopy of streptococci bound to epithelial cells demonstrated fimbriae radiating from the surface of the organisms to the membrane of the epithelial cells. It is apparent, therefore, that the determinants of streptococcal fimbriae involved in resistance to phagocytosis can be dissociated from those involved in epithelial cell binding. These results are consistent with our previous studies which suggested that fatty acids ester linked with glycerol teichoic acid rather than M protein of streptococci binds the organisms to epithelial cells.     

THE RóLE OF STREPTOCOCCI IN EXPERIMENTAL POLIOMYELITIS OF THE MONKEY

It is the opinion of Bull, that the streptococci recovered from poliomyelitic tissues, while having no etiological or pathological relationship to the virus of poliomyelitis, occur as secondary invaders in the disease. Smillie and Amoss indicated that the bacteria may be agonal invaders. The results of the experiments reported in this paper point to another source of the streptococci. They occur as contaminants which are introduced into the cultures during the process of grinding tissues. The source of the streptococcus may therefore be the air of the place in which the cultures are made. We have come to this conclusion because first, the tissues of which cultures yielded streptococci were derived from a number of monkeys with experimental poliomyelitis still in a vigorous state. Secondly, when the tissues were ground bacteria were noted much more frequently in their cultures than in those in which fragments of the same brains were used. Thirdly, microorganisms occurred more often in cultures made in the routine laboratory than in a special room where asepsis was carried to the extreme of a major surgical operation on man. Fourthly, streptococci were obtained from the air of the places where cultures were made. Finally, there is no correlation between the cultures of two portions of the same brain. The streptococci occurred in some cultures in pure growth and in others admixed with other ordinary species of bacteria. The latter were often found, in turn, in pure culture and what applies to streptococci, as mentioned in the preceding paragraph, applies equally to the staphylococci, diphtheroids, spore-bearing rods, and other miscellaneous, familiar microorganisms. We could not determine that there exists any etiological relation of the streptococci to poliomyelitis. The fermentation reactions of the microorganisms obtained from the air, from non-poliomyelitic and poliomyelitic monkey brains indicate that bacteria from any of these sources are markedly different. So also with the serological reactions of agglutination and precipitation. Furthermore no agglutination was observed when the serum of monkeys convalescent from experimental poliomyelitis was mixed with any of the streptococci recovered or those received directly or indirectly from Rosenow. Moreover, the intracerebral injection with cultures, irrespective of their source, induced in rabbits a purulent type of meningoencephalitis, often associated with streptococcic septicemia. This result is at marked variance with any known effects of the true filtrable virus of poliomyelitis in man and in the monkey.     

THE BACTERIAL INDUCTION OF HOMOGRAFT SENSITIVITY : I. EFFECTS OF SENSITIZATION WITH GROUP A STREPTOCOCCI

Heat-killed Group A hemolytic streptococci can induce in guinea pigs a state of altered reactivity to skin homografts which is indistinguishable from that which results from sensitization with homologous tissues. Challenge of suitably prepared recipients with first-set skin homografts obtained from unrelated randomly selected donors elicits white graft reactions or accelerated rejection of such grafts. The gross and histologic appearance of these grafts is identical with that observed in similar reactions obtained in guinea pigs sensitized with homologous tissues. The ability of Group A hemolytic streptococci to induce homograft sensitivity in the guinea pig is a property shared by Types 4, 5, 6, 11, 12, 14, and 49 of Group A streptococci.