Arthur W. Frisch

Specific Inhibition of Bacteriophage by Bacterial Extracts

There is as yet no final explanation for the specificity involved in bacteriophage action. Burnet 1 claims on the basis of experiments made with the Salmonella group of organisms that the action, in a general way, parallels the serologic definition of the heat stable factors. However, he recognized that several observations made by himself and others are not compatible with this point of view. One of these is the failure to demonstrate inhibition of phage action by means of extracts of bacteria containing specific carbohydrate material.∗ In our experiments positive results were obtained using saline extracts prepared according to the method of Furth and Landsteiner, 3 except that no alkali was employed. The 2 phages employed, antidysenteriae Shiga and anti-B. paratyphosus B were derived from chicken stool filtrates. They were specific in their reactions when tested against these 2 organisms; the anti-B. paratyphosus B phage, however, acted also on B. aertrycke. The tests were made by mixing serial dilutions of phage in a volume of 0.5 cc. with 0.5 cc. of a 1% solution of the extracts previously run through a Berkefeld filter and incubating the mixture overnight at 37° and 24 hours in the ice-chest. To each of the tubes were added 4 cc. of broth and a few drops of a young suspension of the homologous or test organism. Readings were made on the basis of the amount of growth. The signs ftr, tr, ±, +, +, ±, indicate increasing degrees of turbidity; the letters cl. indicate complete clearing. It is seen from Table I that the action of the phage against B. dysenteriae Shiga was inhibited by saline extracts of the homologous organism but only to a slight degree by a similar product derived from B. aertrycke.

Further Observations on Specific Inhibition of Bacteriophage Action

Recently we described1 specific inhibition of bacteriophage by bacterial extracts (B. dysenteriae Shiga and B. paratyphosus B). To test further the specificity of the inhibition, experiments were made with phages and extracts of organisms within the salmonella group, namely, B. paratyphosus B and B. suipestifer. Both phages were derived from chicken stool filtrates and were specific in their reactions on the 2 cultures. The extracts were dissolved in saline with the aid of weak alkali and heat, neutralized, diluted with saline to make a 1-200 solution, and passed through Seitz filters. Suitable dilutions of the 2 phages in beef extract broth were prepared and mixed with equal quantities of the filtered bacillary extracts. Two tests were made with these mixtures; one after incubation overnight at 37°∗ and another following an additional interval of 12 hours at the same temperature. One-half cc. was removed from each one of the tubes, diluted with 4.5 cc. of beef extract broth and the homologous test organism was added. The course of lysis in both tests showed that the action of anti-B. suipestifer phage was inhibited by its homologous extract, but only slightly by an extract of B. paratyphosus B.; on the other hand, the extract of B. suipestifer but weakly influenced the action of anti B. paratyphosus B. phage which was inhibited to a considerable degree by the B. paratyphosus B. extract. The effects were pronounced in both tests but the results were somewhat better in the mixtures incubated for the longer period. Furthermore, the course of lysis of the anti-B. suipestifer phage was normal when placed in contact with extracts of B. stanley, B. aertryck, and B. dysenteriae (Shiga). Other experiments were made to determine the relationship between phage titre and inhibition by extracts of various organisms acted on by a related phage.

Clumping of Extracellular Encapsulated Pneumococci in Sputum for Control of Serum Therapy

In a previous report 1 it was shown that clumping of encapsulated extracellular diplococci in pneumonic sputum could either occur spontaneously or be induced by the intravenous administration of specific horse- or rabbit-serum. Further evidence that antibody is responsible for the clumping has been obtained from a patient infected with type 8 pneumococci. Nine hours after the intravenous injection of 160,000 units of rabbit serum the clumped pneumococci in the sputum showed “Quellung” in preparations to which methylene blue alone had been added. At the present time, clumping has been induced after the administration of Types 1, 2, 3, 5, 7, and 8 serum. Spontaneous clumping, now believed to represent evidence of developing active immunity, has been observed in patients infected with Types 1, 2, 3, 4, 5, 7, 8, 12, 18, 19, 24 and 25 pneumococci. The induction and maintenance of clumping has a direct bearing upon the outcome of the pneumonia, as shown in Table I. Sixty-nine roentgenographically proved cases of pneumonia were followed from the sputum by the method previously described. 2 All of the patients received either horse or rabbit antipneumococcic serum; 10 were given sulfanilamide† in addition to serum. The 10 patients who either failed to develop or to maintain clumping, expired. In 47 of the 53 serum-treated patients who recovered, clumping in the sputum was induced and maintained throughout the acute stages of the disease. The remaining 6 recovered cases showed clumping of extracellular pneumococci in specimens of sputum obtained before serum therapy was instituted. Although clumping was induced and maintained in 6 patients, they failed to recover. One (Type 2) in the authors opinion, died of asphyxia. Another (Type 2) was convalescing and expired shortly after an attempted aspiration of an interlobar empyema.

Note on Absorption of Phage by Heat-Killed Bacilli

Investigations on the Salmonella were carried out employing the method recently described by us 1 for studying the specificity of absorption, particularly from polyvalent phages, by heat-killed bacilli. In principle the method depends on the fact that the absorption of phage by heat-killed bacilli is largely irreversible and consequently the residual phage may be studied both qualitatively and quantitatively without removing the absorbing organisms. In general, it was possible with this procedure to differentiate some of the following groups, for instance, the suipestifer group which absorbed specifically the homologous phage and also the suipestifer fraction 1 of the polyvalent anti-paratyphosus A phage. However, some of the suipestifer strains, particularly those of the European type, were characterized by a capacity to absorb actively also other fractions, for example, the paratyphosus B fraction of the polyvalent anti-paratyphosus A phage when B. aertrycke (of the paratyphosus B group) was used to test for residual phage. (Table I.) This same type of suipestifer absorbed the anti-paratyphosus B phage to a moderate degree, but less intensely than the paratyphosus B group of organisms, while other suipestifer strains showed no absorption.∗ The paratyphosus A group of organisms and also some of the European suipestifers employed were the most active absorbers of the anti-paratyphosus A phage when the homologous strain was the test organism. The distinction between these could be made readily since the paratyphosus A strains failed to absorb actively the anti-suipestifer phage. In contrast to the ease with which differences within the suipestifers could be demonstrated, was the failure to distinguish the typhosus-enteritidis group from the aertrycke-paratyphosus B group, since in the several combinations of phages and test organisms available, both groups absorbed equally well. (Presumably this observation is in harmony with the findings recorded by Furth and Landsteiner, 2 that some anti-sera of the 2 groups gave weak to moderate precipitin reactions also with the heterologous extract.)

SPECIES SPECIFIC BLOOD GROUPS IN CYNOPITHECUS NIGER (CELEBES APE).

Summary Specific isoantigens, designated Cn1 and Cn2, have been detected on erythrocytes of the Celebes ape, thus defining 3 types of individuals, Cn1, Cn2, and Cn1,2. Methods have been developed which may prove useful in detection of isoantigens among other primate species.

New Heat-Stable Agglutinogens in the Suipestifer Group

Observations on absorption of phage by heat-killed bacilli particularly those on the typhosus-paratyphosus B group and some strains of suipestifer suggested that further investigations along this line might yield information revealing new relationships in this highly complex group of organisms. 1 , 2 , 3 We here report the existence of differences in the suipestifer group first detected by characteristic phage absorption effects and confirmed by subsequent immunization and experiments on agglutinin absorption. The observations were made with a phage derived from chicken stool filtrates and propagated with a particular American strain of suipestifer, No. 26∗ in our collection. This phage lysed to a high titer numerous smooth cultures of the biochemically different types, namely American, Kunzendorf and one out of 4 strains of the Hirschfeld type, the other 3 being comparatively resistant. As already reported, 1 , 3 the strain Glässer-voldagsen was completely resistant even to the action of undiluted phage. We have since found one culture, No. 92 (American∗) in our collection which was poorly lysed but differed from other suipestifer strains in failing to absorb actively from the suipestifer 26 phage, particularly from some passages. Somewhat similar effects were observed with strains 80 and Glässer-voldagsen. Curiously enough, the same cultures, 80 and 92 were 2 of several strains, mostly of the Kunzendorf type, which on repeated occasions absorbed small quantities of the phage for paratyphosus B in contrast to the other suipestifer strains which did not at all absorb. These effects are indicated in Table I. In view of these results, it seemed desirable to determine whether serological characteristics could be found to correspond more or less with the qualitative differences shown in phage absorption.

Polyvalency Demonstrated by Antiphages

A general procedure was described for the study of the specificity of absorption of bacteriophage by heat-killed bacilli. 1 , 2 , 3 Polyvalent phages derived from chicken stool filtrates and propagated either against a strain of paratyphosus A or enteritidis were shown to consist of at least 2 prominent qualitatively different fractions, the one selective for suipestifer strains and another for the typhosus-paratyphosus B group. This effect was readily demonstrated by testing the quality of the residual phage with several serologically different sensitive organisms. The question naturally presented itself as to whether or not antiphages may be employed to indicate the presence in a phage of the several fractions. Accordingly 4 series of rabbits were injected intravenously with the 2 polyvalent phages for paratyphosus A and for enteritidis and also with the presumably monovalent paratyphosus B and suipestifer phages. After 4 injections of 2 cc. each, potent antiphages, practically lacking in agglutinins were readily obtained in all instances except in the case of the suipestifer phage. Experiments demonstrating the production of 2 antibodies corresponding to the fractions in the polyvalent phages are given in Table I. The results show that the antisera for the paratyphosus A and enteritidis phages contain potent antibodies which neutralize the action of the typhosus-paratyphosus B fraction of the polyvalent enteritidis phage (Table I A, aertrycke of the paratyphosus B group, as test organism); both antisera also possess antibodies for the suipestifer fraction but to varying degrees of activity. Although the antiphage for paratyphosus B contains just as potent neutralizing antibodies as the other 2 antiphages, the former has no inhibiting action whatever on the same phage when suipestifer is used as the test organism. (Table IB.)

Observations on Phage Inhibition by Bacillary Extracts

The specific inhibition of bacteriophage by bacillary extracts recently described by us 1 , 2 and confirmed by Kligler and Olitski, 3 Burnet, 4 and Gough and Burnet 5 may be employed to study the chemical properties of these complex cellular products with the aid of various manipulations such as fractionation and hydrolysis. In a previous communication, 2 the observation was made that crude saline extracts of B. aertrycke boiled in N/2 alkali, still retained their phage inhibiting capacity and, consequently, it seemed desirable to study in detail the sensitivity of these substances to alkaline and acid treatment before attempting purification. For this purpose, a crude saline extract of B. aertrycke was employed and tested for phage inhibition before and after boiling in N/2 sodium hydroxide for 5 minutes∗ and subsequent neutralization. Such alkali treated extracts showed marked increase in phage inhibiting capacity although they gave a precipitin titre that was slightly lower than that of the untreated material. This observation, therefore, indicated that the degree of inhibition of phage did not run parallel to the precipitin titre of the crude extract. Striking confirmation of this view was found when these extracts were subjected to mild hydrolysis in N/15 and N/30 hydrochloric acid at 80° C. This procedure was adopted because boiling for 5 minutes or longer in N/2 acid completely destroyed both the phage inhibiting reaction and also the capacity to react with precipitins. A crude extract of B. aertrycke was dissolved in saline, passed through a Seitz filter and then treated with N/5 hydrochloric acid in sufficient quantity to produce a final concentration of N/15 acid and held at 80°C. Samples were removed at various intervals, neutralized, brought to a dilution of 1:500, centrifuged to remove the precipitates, and sterilized by heating for one hour at 80° C.