III. THE FATE OF INFLUENZA BACILLI INTRODUCED INTO THE UPPER AIR PASSAGES By Arthur L. Bloomfield (From the Biological Division of the Medical Clinic, The Johns Hopkins University and Hospital) [From The Johns Hopkins Hospital Bulletin, Vol. XXXI, No. 349, March, 1920] III. THE FATE OF INFLUENZA BACILLI INTRODUCED INTO THE UPPER AIR PASSAGES By Arthur L. Bloomfield {From the Biological Division of the Medical CUnic, The Johns Hopkins University and Hospital) In previous papers1 we reported observations on the fate of certain bacteria after their introduction into various parts of the upper air passages. This work represented a portion of a systematic study of one phase of infection, namely, the period from the arrival of the organism at the mouth or nose until it is eliminated or until disease is produced. Sarcina lutea, B. coli, and Staph, albus were studied. It was found that these bacteria were rapidly disposed of; Sarcina was promptly destroyed by the saliva, and B. coli and Staph, albus were washed away by mechanical processes without showing any tendency to colonize. The present report deals with a similar study of the hemoglobinophilic bacteria (B. influenzal). No attempt was made to produce disease, and our purpose was simply to study the fate of the organisms and the method of their disposal. LITERATURE Several records of inoculations of human beings with B. influenzal are found in the recent literature. These experi- ments were all made on the hypothesis that the organisms used might be the cause of epidemic influenza and in the attempt to demonstrate this fact by the production of disease. Rosenau2 introduced strains of influenza bacilli freshly iso- lated from cases of epidemic influenza into the throats of volunteers without producing any local or general disease. Sellards and Sturm,3 in the course of a study of hemophilic bacilli isolated from cases of measles, sprayed a saline emul- sion of five strains upon the mucous membranes of the eye, (D nose and throat of four volunteers. Cultures made at three- day intervals for a period of two weeks were uniformly nega- tive, and no local or general symptoms were produced. Sellards reports no observations made during the first three days after inoculation. Wahl, White and Lyall * found that the applica- tion to the mucous membrane of the nares and naso-pharynx of a saline emulsion of strains from cases of epidemic influenza failed to produce any abnormal symptoms in five healthy men. These workers were unable to recover B. influenzal from the nose after 48 hours except in one case, but found the organisms present in the naso-pharynx for two weeks or longer. In some cases the bacteria disappeared after a few days to return later. In summary, then, the above experiments, while indicating the general trend of events, give no detailed infor- mation about the immediate and exact fate of the influenza bacillus. METHODS Strains.—Until the needs of the recent epidemic stimulated the development of media satisfactory for the growth of B. influenzal, little success had been met with in the study of the finer details of its natural history or in the differentia- tion of strains by biological or other methods. All small Gram-negative hemophilic bacilli isolated from the respiratory passages were placed together in one group. Recent work, however, particularly that of Rivers,5 suggests that all the organisms previously included under one head are by no means identical, but that they represent various groups differing in essential biological characteristics. It is therefore impossible to be certain now of the exact nature of the original organism of Pfeiffer as well as of the nature and identity of the " in- fluenza bacilli" which were so prominent in the cases of epidemic disease in 1918. Three strains were employed in the present work. They were all isolated from the throats of healthy men,* and conformed to all the usual criteria of the influenza bacillus group. Media.—In view of the difficulty of growing influenza bacilli under unfavorable conditions, the utmost care was used in the preparation of the media. Fresh 2 per cent meat infusion * We are indebted to Dr. Rivers for these strains. (2) agar (pH 7.3 to 7.5) to which 1 per cent of fresh defibrinated [85] rabbit's blood was added, or the sodium oleate hemoglobin agar of Avery were used in all the work. The medium was always fresh and moist, and each lot was tested by inoculation with the various strains. It was considered unsatisfactory unless the colonies were large and reached their maximum growth in from 24 to 36 hours. Inoculations.—As in the previous work, healthy individuals presenting no unusual abnormality of the upper air passages were used. The whole 24-hour growth from an agar slant was collected on a loop and deposited on the desired site—the tongue, nasal septum, naso-pharynx or tonsil crypt. Cultures were made at various intervals by scraping the site of inocula- tion with a platinum loop or (in the case of the naso-pharynx) by means of the usual cotton swab. The material collected was spread with a glass rod over two plates. It was regarded as essential that the spread of colonies be discreet; plates covered by a confluent mass of growth are useless in work of this sort. The cultures were studied after from 24 to 48 hours, and in every case the diagnosis of B. influenzal was established by isolation of the organism in pure culture. Great care was taken in estimating the quantitative relations. The site of inoculation was watched for signs of reaction. In no case was there any change in the appearance of the mucosa, or any constitutional reaction. EXPERIMENTS Exp. I.—Fate of influenza bacilli swabbed on the tongue. The bacteria were placed on the tongue, and cultures were made from the tongue and from the pharynx at various intervals. The results are summarized in Table I. Summary.—Influenza bacilli swabbed on the tongue in large amounts were promptly spread over the mouth cavity. The organisms disappeared both from the tongue and pharynx very rgyi rapidly. After two hours the number recovered was much re- duced, and after 24 hours only a very few organisms were obtained in two out of four cases. Control cultures before inoculation yielded a very few colonies from the pharynx in two cases. The general result of this experiment is that in 24 to 48 hours the inoculated organisms have disappeared or at least been reduced to the number " normally " present in the pharynx. There was no local or general reaction following inoculation. (3) CO TABLE I.—FATE OF B. INFLUENZAE SWABBED ON THE TONGUE Date Procedure Number of colonies of B. influenzae per plate recovered from tongue and pharynx Name Tongue Pharynx inoculation After 10 min. After 2 hrs. After 1 day After 2 days After 3 days After 4 days After 10 min. After 2 hrs. After 1 day After 2 days After 3 days After 4 days. Tongue Pharynx M. J. C. B. Nov. ' 0. Nov. 10. Nov. 17. Nov. 17. One slant strain 55 B. swabbed on anterior half of tongue. One slant strain 55 B. swabbed on anterior half of tongue. One slant Btrain 63 swabbed on anterior half of tongue. One slant strain 33 swabbed on anterior half of tongue. CO* 00 00 00 A good many colonies B. infl. A very few cols. B. infl. 150 cols. B. infl. A few cols. B.infl. A very few colonies B. infl. No B. infl. found. No B. infl. No B. infl. No B. infl. No B. infl. A good many colonies B. infl. 00 A few cols. B.infl. Many cols. B. infl. No B. infl. 150 cols. B. infl. A few cols. B. infl. A few cols. B. infl. No B. infl. 20 cols. B. infl. No B. infl. No B. infl. No B. infl. No B. infl. No B. infl. No B. infl. No B. infl. No B. infl. A few cols. B. infl. A few cols. B. infl. No B. infl. No B. infl. * oo = innumerable. TABLE II.—THE FATE OF INFLUENZA BACILLI INTRODUCED INTO THE NOSE Date Procedure Number of colonies of B. influenzae per plate recovered from nose and pharynx Control culture before Name Nose Pharynx inoculation After 10 min. After 2 hrs. After 1 day After 2 days After. 10 min. After 2 hrs. After 1 day After 2 days After 4 days After 8 days Nose Pharynix C. s. St. Ch. B. Nov. 11. Nov. 12. Nov. 13. Nov. 18. Nov. 19. One slant strain 56 B. swabbed on left nasal septum. One slant strain 55 B. swabbed on left nasal septum. One slant strain 55 B. swabbed on left nasal septum. One slant strain 63 swabbed on right nasal septum. One slant strain 33 swabbed on left nasal septum. 00 00 00 00 oo 00 200 cols. B. infl. 200 cols. B. infl. 50 cols. B. infl Several hun-dred cols. B. infl. 1000 cols. B. infl. No B. infl. 20 cols. B. infl. No B. infl. No B. infl. No B. infl. No B. infl. No B. infl. ...... A few cols. B. infl. No B. infl. No B. infl. About 20 cols. B. infl. NoB. infl. No B. infl. A few cols. B.infl. No B. infl. No B. infl. No B. infl. No B. infl. About 50 cols. B. infl.* No B. infl. No B. infl. No B. infl. No B. infl. NoB. infl. No B. infl. No B. infl. A few cols. B. infl. No B. infl. No B. infl. A few cols. B. infl. No B. infl. •This strain was not 55 B. (See Table V.) oo TABLE III.—FATE OF INFLUENZA BACILLI INTRODUCED INTO TONSIL CRYPTS Procedure Number of colonies per plat e of B. influenzae recovered from crypt and pharynx Control culture before inoeulation Crypt Pharynx After 10 min. After 2 hrs. After 1 day After 2 days After 4 days After 6 days After 8 days After 10 min. After 2 hrs. After 1 day After 2 days After 4 days Several* hundred cols. B. infl. After 6 days After 8 days Crypt No B. infl. NoB. infl. NoB. infl. Pharynx One loop strain 33 placed in a tonsil cr.vpt. One loop strain 63 placed in a tonsil crypt. One loop strain 55 B. placed in a tonsil crypt. 300 cols. B.infl. About 100 cols. B. infl. 10 cols. B.infl. 00 A few cols. B. infl. No B. infl. No B. infl. 100* cols. B. infl. 200* cols. B.infl. NoB. infl. No B. infl. Many cols. B. infl. Several hundred cols. B. infl. Many cols. B. infl. Many cols. B.infl. Several hundred cols. B. infl. A few cols. B. infl. No B. infl. No B. infl. Several* hundred cols. B. infl. About* 100 cols. B. infl. About* 100 cols. B. infl. No B. infl. A few cols. B. infl. No B. infl. 63. (See Table V.) Exp. II.—Fate of influenza bacilli introduced into the nose. The bacteria were deposited on the nasal septum behind the vestibule and cultures were made at various intervals from the nose and pharynx. The results are summarized in Table II. Summary.—Large numbers of B. influenzae swabbed on the nasal septum disappeared rapidly from the site of inoculation. None could be recovered after 48 hours. Simultaneous cultures from the pharynx in three of five cases yielded a few colonies of B. influenzae in from two hours to 24 hours after inoculation. In one case (S.) 50 colonies were recovered from the pharynx in the 48-hour culture, but this organism was found to be a different strain from that introduced (see Table V). Exp. III.—Fate of influenza bacilli introduced into tonsil crypts. The bacteria were introduced into a tonsil crypt with a platinum loop and cultures were made at various intervals from the crypt and from the pharynx. The results are summarized in Table III. Summary.—Influenza bacilli introduced into tonsil crypts could not be recovered from the site of inoculation after 24 hours in two of three cases. In the third case influenza bacilli were recovered after two and also after four days, but they were found to be different strains from those introduced (see Table V). In this case influenza bacilli were also recovered from the pharynx several days after inoculation, but the strain was not only different from that introduced, but it was also different from that obtained from the crypt after two and after four days (see Table V). Exp. IV.—Fate of influenza bacilli introduced into the naso- pharynx. Influenza bacilli were swabbed on the naso-pharynx and cultures were made at various intervals. The results are sum- marized in Table IV. Summary.—Influenza bacilli swabbed in large amounts on the naso-pharynx disappeared very rapidly. After two hours very few organisms could be recovered from the site of inoculation. In one case, influenza bacilli recovered after one and after four days were different strains from those introduced (see Table V). DISCUSSION The general result of these experiments seems to indicate two facts: First, that several strains belonging to the group of so-called influenza bacilli swabbed in large amounts on the normal mucous membranes of the upper air passages failed to colonize and disappeared in about a day; and secondly, that in no case did they produce any demonstrable local lesion or general reaction. (7) Name W. R. Date Dec. 1. Dec. 1. TABLE IV.—FATE OF INFLUENZA BACILLI INTRODUCED INTO THE NASO-PHARYNX Procedure One slant of strain 33 swabbed on naso-pharynx. One slant of strain 33 swabbed on naso-pharynx. Number of colonies per plate recovered from naso-pharynx After 10 min. After 2 hrs. 200 cols. B. infl. 10 cols. B. infl. After 1 day No B. infl. A very few* cols. B. infl. After 2 days No B. infl. After 3 days 4 cols. B. infl. Control culture before inoculation * Not the strain which was introduced. (See Table V.) No B. infl. No B. infl. TABLE V.—COMPARISON OF CHARACTERISTICS OF STRAINS OF INFLUENZA BACILLI INTRODUCED AND RECOVERED IN VARIOUS CASES Name Characteristics of Morphology Staining Hemoglobinophilia Hemolysis Indol formation Nitrite formation Agglutina-tion with stock serum Sp. Ha. R. Strain introduced (55 B). Strain recovered from throat 48 hrs. after inoculation. Strain introduced (63). Strain recovered from throat 4 days after inoculation. Strain recovered from crypt 4 days after inoculation. Strain introduced (33). Strain recovered from throat 24 hrs. after inoculation. Small regular bacilli. Small regular bacilli. Small regular bacilli. Small regular bacilli. Large thick bacilli. Small regular bacilli. Very large numerous threads. Gram-neg. Gram-neg. Gram-neg. Gram-neg. Gram-neg. ' Gram-neg. Gram-neg. Gram-neg. Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Hemoglobinophilic Non-hemolytic. Non-hemolytic. N on-hemolytic. Non-hemoly t i c. Hemolytic. Non-hemolytic. Non-hemolytic. Non-hemolytic. Marked. None. None. Marked. Marked. Marked. None. None. Marked. Marked. Marked. Marked. Marked. Marked. Marked. Marked. + 0 0 0 0 0 0 aft it inoculation. 0 TABLE VI.—EFFECT OF SALIVA ON INFLUENZA BACILLI Date pH. of saliva Strain of B. infl. Time of culture after inoculation 0.5 c. c. broth and growth from one slant B. infl. 0.5 c. c. saliva and growth from one slant B. infl. Dec. 6. Dec. 6. 7.0 7.0 7.3 7.3 6.4 55 B. 33 63 33 65 B. Immediately. After 2 hrs. After 24 hrs. Immediately. After 2 hrs. After 24 hrs. Immediately. After 2 hrs. After 24 hrs. Immediately. After 2 hrs. After 24 hrs. Immediately. After 2 hrs. After 24 hrs. Innumerable cols. B. influenza?. Innumerable cols. B. influenzae. Innumerable cols. B. influenzae. (About same number as in previous culture.) About 50 cols. " mouth bacteria" per plate + innumerable cols. B. influenzae. About 50 cols, "mouth bacteria" per plate + innumerable cols. B. influenza?. About 300 cols. " mouth bacteria." No B. influenzae. About 100 cols. " mouth bacteria " per plate + innumerable cols B. influenzas. About 75 cols. '' mouth bacteria " per plate + innumerable cols. B. influenzae. About 500 cols. " mouth bacteria " per plate. No B. influenzae. i About 300 cols, "mouth bacteria" per plate + innumerable B. influenzae. About 300 cols. " mouth bacteria" per plate + innumerable B. influenzae. About 300cols, "mouth bacteria" per plate. No B. influenzae. About 50 cols, "mouth bacteria" per plate + innumerable B. influenzae. About 75 cols, "mouth bacteria" per plate + innumerable B. influenzae. About 500 cols. " mouth bacteria " per plate. No B. influenzae. About 100 cols, "mouth bacteria" per plate + innumerable B. influenzae. About 76 cols, "mouth bacteria" per plate-f innumerable B. influenzae. About 100 cols. " mouth bacteria " per plate. No B. influenzae. Dec. 3. Dec. 3. Dec. 4. [ST] In five instances influenza bacilli were recovered at periods later than 24 hours after inoculation. It seemed of importance to determine whether these strains were identical with those introduced or whether the strain introduced had disappeared and another had supplanted it. Biological differential methods worked out by Dr. Eivers were applied by him to the study of these cultures. In every case it was possible to show that the strain introduced had been replaced by organisms possessing different characteristics (see Table V). It seems desirable to bring these findings into relation with the known facts about the presence of influenza bacilli in the throats of healthy people. Winchell and Stillman * review the recent literature on this subject and report careful studies of their own on ;i series of individuals over a period of eight months. It seems quite certain that hemophilic bacilli may be found in from 40 to 80 per cent of various groups of healthy people. The exact seat of bacterial multiplication in the pharynx and the length of time over which any given strain persists are however un- known. Our experiments suggest that the free surfaces of the normal mucous membranes present a relatively unfavorable environment for these bacilli to colonize upon. It may be that their source in certain cases is a focus, such as a chronic sinusitis, bronchitis, or bronchiectasis, or an acute infection of the respiratory tract either in the same or in another individual from which they are discharged into the open pharyngeal cavity. It should be emphasized that all studies on the per- sistence of influenza bacilli in the throat are of uncertain significance in the absence of proof that the strains recovered from the same individual at various times are identical. The final solution of this question must await the development of complete and accurate methods of differentiating the various strains of hemophilic bacilli. Inasmuch as the organisms employed in our experiments had been grown on artificial media for several generations the objection may be raised that their failure to colonize was due to alteration in their virulence by growth outside the body. This objection cannot be answered and the experiments are presented with this in mind. (10) THE MECHANISM OF DISPOSAL OF B. INFLUENZA rg7] As in the case of the organisms previously studied an attempt was made to analyze the various factors which might be re- sponsible for the disposal of B. influenzal. 1. The Antagonistic Action of Other Bacteria in the Mouth and Throat.—No direct experimental method was available for determining to what extent the growth and multiplication of B. influenzal in the throat is prevented by bacteria already present. It is known that B. influenzal grows well on artificial media in symbiosis with many other bacteria under proper quantitative relations. It may, however, be rapidly overgrown by an excess of other bacteria which in smaller numbers would favor its growth. In the case of nasal inoculations other bacteria can hardly play an important part since the nasal mucosa is practically free of organisms. 2. The Effect of the Mouth Secretions.—An attempt was made to reproduce mouth conditions by testing the effect of saliva on influenza bacilli in the test tube. Fresh saliva was centrifuged at high speed for a few minutes to remove gross particles. Small amounts of the resulting clear fluid were placed in test tubes and heavily inoculated with B. influenzal. The entire growth from an agar slant was suspended in 0.5 c. c. of saliva, and control tubes of plain broth were similarly inocu- lated. Cultures were made at various intervals on oleate hemoglobin agar plates. Three salivas covering the normal range of reaction (pH 6.4 to pH 7.3), and three strains of [89] influenza bacilli were employed. The results are summarized in Table VI. This experiment shows the following points. Influenza bacilli suspended in plain meat infusion broth were viable at the end of 24 hours. From saliva inoculated with the same amount of culture living influenza bacilli were recovered after two hours, but not after 24 hours. The proportion of mouth bacteria in the saliva to the inoculated influenza bacilli was about 1 to 10,000 at the beginning of the experiment. At the end of 24 hours there was only a slight increase in the number of mouth bacteria, whereas no influenza bacilli grew. The experiment seems therefore to indicate that while saliva exer- (ii) cises no immediate destructive effect on B. influenzae, it is an unfavorable medium for the growth of these bacteria and that they do not remain viable in this medium for as long as 24 hours. This result was constant with salivas of different pH. The probable explanation, therefore, of the rapid disappear- ance of B. influenza when introduced into the normal upper air passages involves both the action of a medium unfavorable for the growth of these organisms, together with the mechani- cal flushing processes constantly at work in these regions. The question naturally arises in this connection as to what makes possible the tremendous growth of influenza bacilli in disease conditions, such as acute sinusitis, pharyngitis, laryn- gitis, pneumonia, measles, influenza, etc. A possible explana- tion which still lacks final proof is that these acute processes may alter the environment in such a way that the organisms take hold and grow rapidly at the seat of disease. SUMMARY 1. Three strains of influenza bacilli introduced in large amounts into the normal upper air passages disappeared very rapidly—within from one to two days. In no case was a carrier state produced. 2. In no case did any local or general pathological process result from such inoculation. 3. In five instances influenza bacilli isolated later than 24 hours after inoculation were shown to be different strains from those introduced. 4. Influenza bacilli were no longer viable after being sus- pended in saliva for 24 hours at 37°C. 5. The rapid disappearance of influenza bacilli from the upper air passages is probably due to the combination of an unfavorable environment with the mechanical flushing pro- cesses at work in these regions. 6. The question of the persistence of influenza bacilli in normal throats cannot be finally settled until we possess accu- rate methods for differentiating various strains of hemophilic bacteria. (12) REFERENCES 1. Bloomfield, A. L.: Johns Hopkins Hosp. Bull., 1919, XXX, 317; ibid., 1920, XXXI, 14. 2. Rosenau, M. J. (quoted by J. P. Leake): Boston Med. and Surg. Jour., 1919, CLXXI, 675. 3. Sellards, A. W., and Sturm, E.: Johns Hopkins Hosp. Bull., 1919, XXX, 331. 4. Wahl, H. R., White, Geo. B., and Lyall, H. W.: Jour. Infect. Dis., 1919, XXV, 419. 5. Rivers, T. M.: Johns Hopkins Hosp. Bull., 1920, XXXI, February. 6. Winchell, A. I., and Stillman, E. G.: Jour. Exp. Med., 1919, XXX, 497. (131 O-V"-.""":'*-