CHAPTER V
BACTERIOLOGY
The bacteriological investigations which were carried out during the World War and the research in the period following showed the extreme variety of organisms which were concerned in the production of the various pneumonias or pulmonary inflammations. The results of cultural work on the secretions from the respiratory tract during life, together with the results of bacteriological examinations at necropsy, have shown that only extremely exhaustive investigation will define the bacterial flora and the relation of their components to the lesions in the lung. It was found that the various types of pulmonary inflammation were not produced by different organisms; rather, several different organisms apparently were each capable of producing practically all the varieties of inflammation which have been described. In general, however, the pneumococci produced a fibrinous type of inflammatory reaction, an exudate in which fibrin forms a prominent part, more often than other organisms, although varying amounts of fibrin were found in inflammations from which the streptococci, influenza bacilli, and rarely meningococci were isolated.
The streptococci, pneumococci, Friedlander's bacillus, Micrococcus catarrhalis, staphylococcus and meningococcus are conceded by practically all investigators to be the organisms which produced the pneumonic consolidation or pulmonary inflammation which was secondary to other acute respiratory diseases, principally measles and influenza. Certain members of the pneumococcus group, particularly Type I, probably Types II and III, produced primary pneumonia. Such pneumonia was most frequent following the mobilization of unseasoned troops, did not have a high mortality, and did not contribute much to the total of acute respiratory diseases of the war period.
No organism was proved to be the cause of the clinical disease, influenza, and in fact our ideas with reference to the etiology of this condition were perhaps more confused than clarified as the result of the numerous and varying reports of clinical and laboratory investigation. No new organisms were discovered until after the decline of the pandemic of influenza in the fall of 1918, though certain organisms were considered by some investigators to be new varieties because of their occurrence in diseases of a character not usually associated with bacteria of the same morphology, staining and cultural characteristics. Bacterium pneumosintes reported by Olitsky and Gates should be mentioned and the cultural methods used by these investigators should be utilized in future research to determine the etiology of clinical influenza with the idea that organisms of this character may be found to be responsible.1 The influenza bacillus, described by Pfeiffer, was frequently found but in so far as can be determined from published reports was not constantly present. If one read selected reports from stations widely separated throughout this and other countries and ignored other reports one would decide that the influenza bacillus
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was undoubtedly the proven cause of clinical influenza. On the other hand, one could likewise read as many reports from different stations also scattered and receive exactly the opposite impression. This suggests that the influenza bacillus was but another of the secondary invaders, which were extremely frequent in some stations and absent in others, yet at practically all stations bacteriologists isolated organisms which they considered to be influenza bacilli. Competent investigators studied bacterial flora at stations where this organism was found in abundance and at stations where it was seen relatively infrequently. In a careful perusal of the technique used for isolating the bacillus by the different workers it appears that the more thoroughly cultures were made, the more parts of the respiratory tract which were cultured, and the more care that was taken to provide a medium on which this organism grows, in other words, a selective medium, the greater was the number of cases from which the organism was isolated. It is impossible to decide from the various reports the ideal cultural medium for the isolation of this organism. Media containing hemoglobin appear to be essential, and the growth of the organism appears to be better in blood medium so heated as to destroy the normal complement of the serum. It is true, however, that high percentages were found by some investigators who used unchanged human blood.
The presence of colonies of streptococci or pneumococci appeared to hinder the growth of B. influenzae, while the presence of colonies of staphylococci apparently stimulated the growth. An inhibitory influence by pneumococci and streptococci possibly accounts in part for the failure to find B. influenzae in greater numbers, since the former organisms were practically always present in the cultures.
As the result of a review of the techniques used it is apparent that the failure to find the influenza bacillus does not prove that this organism was not present. At several places during the early part of the pandemic of the fall of 1918 B. influenzae was not found at all, in others rarely, and a change in technique resulted in securing high percentages of positive results.
In the histological examination of the tissues in the Army Medical Museum collection from the pneumonias secondary to clinical influenza it was found that small Gram-negative bacteria were present in practically every case which died after a short illness, the tissue of which was well fixed, whether or not cultures ante mortem and at necropsy had recorded their presence. They were not found in every case; but the fixation of the material in many instances was of such character as to preclude the possibility of finding any organism; in other cases advanced pulmonary lesions due to other organisms so filled the tissue that it was unreasonable to expect to find them by such examination. Of course, it is impossible to state that these Gram-negative bacteria were influenza bacilli. Morphologically they corresponded as they did also in their reactions to stains. This finding has little value in so far as it goes to prove the influenza bacillus an etiological factor in clinical influenza. It does show, however, that Gram-negative bacteria existed in the lung which were not found by the cultural examination. These small Gram-negative bacteria were seen most frequently along the smaller bronchioles, in the ductuli alveolares and in the atria; less frequently in the alveoli and rarely in the alveolar walls or peribronchial tissue.
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In the early cases, they were found frequently in the terminal bronchioles without admixture of other organisms, while, in addition, in the bronchi near the hilus streptococci and pneumococci usually were seen. It was in the bronchioles also that bacteriologists found them most frequently in cultures. They were less apt to be present in cultures from definite consolidations involving lobules or larger portions of the lung. They were rarely found in the pleura or in the heart's blood. During life they were found most frequently in swabs from the posterior nasopharynx, much less frequently in the sputum. Organisms of identical morphology were found more frequently in smears from the nasopharynx and sputum than they were in the cultures.
The results in smears and culture plainly indicate that in order to determine the prevalence of the different organisms in the respiratory tract, all parts of this tract must be cultured and the media used for each part must be of such variety as to include those which have been found to favor the growth of each and every organism described. Unless this is done and each organism in the resulting cultures identified and worked out serologically with prepared immune serum and with the serum from the patient, dependable results will not be secured.
Investigations which have been undertaken during the last four decades have been scarcely adequate to show the prevalence of the influenza bacillus. Numerous reports, useful in determining the incidence of the organism during epidemics and in the preepidemic periods, have been accumulated and reviewed by Zinsser 2 and by McCloud, Ritchie, and Dottridge.3 Certain investigations of similar character, some intentionally made, others not, appear in the reports of the Army camps in the United States. Admitting that the cultural results are only relatively accurate and realizing the relative paucity of the material examined, it still appears that the influenza bacilli or organisms of similar morphology, staining reactions, and cultural characteristics are found in greater frequency during epidemic periods and were particularly frequent during the epidemic of the fall of 1918 in this country. It is further evident from bacterio- logical investigations that this organism increased both in the unaffected and in the affected population at the time of the influenza pandemic and gradually decreased after it. In fact, in many of the places where it would appear from reviewing the work that the best and most exhaustive bacterial research was made, this organism showed an increase until it was universally prevalent in those affected with clinical influenza. No other organisms increased to such an extent. Of course, we must ignore pneumococci of Group IV, organisms present in a very large proportion if not all normal mouths, practically as prevalent as the colon bacillus in the rectum.
In spite of this great prevalence, in the large number of cultures obtained of this organism the strains isolated did not show common immunological reactions. In fact, these small Gram-negative bacteria, culturally influenza bacilli, were immunologically of very many different strains. At one and the same place it could scarcely be said that there was a predominant type in so far as could be determined by serological reactions. Serological reactions are generally con- sidered to be the most accurate method of separation of bacterial varieties, and thus the fact that no common strain was found is against the probability of the influenza bacillus being the cause of the pandemic disease.
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A relatively small amount of work was done in testing strains for agglutination and complement fixation, using the serum of the persons affected with the clinical disease. Some investigators reported positive reactions in relatively low dilutions, others failed to find any reactions even with the sera of persons from whom the strains were isolated.
The experiments with influenza vaccines in the effort to protect from the clinical disease indicate that the vaccines so far utilized did not excite the production of protective bodies. Such a result has no influence either for or against the etiologic relationship of the influenza bacillus to the disease, as the methods of preparation of vaccine, the dosage, and the methods of giving it were various and may not have been of such nature as to yield the maximum in protective substances, while vaccines have been found of little value in protection against many diseases whose etiology is well known.
The influenza bacillus produces toxic substances of two varieties, one of which is found in culture media and appears to be at least in part a soluble toxin against which antitoxin can be produced. Extracts of the bodies of influenza bacilli yield a poisonous substance against which an antitoxin apparently can not be prepared. An antitoxin against the substance produced in cultures apparently has some protective action in experimental animals against infection by the influenza bacillus. The symptoms produced by the injection of the toxin were, in general, a congestion of the viscera and in some animals a relatively extreme prostration. While these symptoms are in accord with the lesions seen in the lungs in influenza and in experimental lesions with the influenza bacillus they are not dissimilar to reactions to the toxins of other organisms.
The virulence of this group of Gram-negative bacteria, morphologically and culturally the influenza bacillus, is very variable. Some strains appear virulent from the outset and retain their virulence, others can have their virulence raised by animal passage, while some strains apparently possess little virulence for the experimental animals and it has not been possible by animal passage to raise this virulence to any great degree. The possibilities of variants must be kept in mind as a possible explanation of changes in virulence not only among strains at a given time, as was seen in the numerous isolations of the influenza bacillus, but also those changes which sometimes occur in a virus independently of efforts to raise that virulence by animal passage.
There is thus no explanation, based on incontrovertible facts, of the variations in virulence and pathogenicity of the small Gram-negative organisms which are morphologically and culturally influenza bacilli. The variations in serological reactions suggest that we are dealing here with a group some members of which have much greater virulence than others in the production of disease. Like the pneumococcus group some of these Gram-negative bacilli may be analo- gous to the Group IV pneumococci, some of which appear to have extreme invasiveness and pathogenicity, while others appear little more than saprophytes.
Bacteriological studies on this group have not as yet reached that state of perfection which allow conclusions to be made; furthermore, the evidence presented by one group of workers may be the exact opposite of that of another. Difficulties in cultivation, isolation, and maintenance of the organisms, their low virulence in experimental animals, and their lack of resistance to deleterious
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influences present in the culture media or environment, make the group an exceedingly difficult one to handle, which, up to date, has yet to be so handled as to permit definite conclusions as the result of investigations.
Inoculation experiments have been disappointing. Suggestive results have been obtained with filtered sputum and with cultures in both man and animals, yet the majority of results were absolutely negative. Many volunteers have been inoculated and suggestive results obtained in very few. Blake and Cecil,4 using monkeys as experimental animals, proved that B. influenzae would produce pneumonia of a more or less characteristic variety. In their work on human beings Cecil and Steffen' report the production of influenza-like attacks of mild character by the use of one culture while another, though apparently from an infection of considerable intensity, did not result in a recognizable reaction. Bacterium pneumosintes of Olitsky and Gates is on no better footing in so far as proof of its connection with pandemic influenza or clinical influenza is concerned.
Organisms of the pneumococcus group are present in the cultures of a very large proportion of cases of the pneumonias following influenza. Organisms of Group IV preponderated to a marked degree, while organisms of Types 1, 11, and III and II atypical varied markedly from camp to camp and station to station. In some camps a considerable number of Type I infections were found, in others Type II showed a larger proportion than the average and the same was true of Type III. Type I produced typical lobar consolidations, but following influenza the consolidations rarely had the typical lobar distribution. Much more frequently many lobules were involved, but the consolidation was not uniform, though the appearances of the tissues in a given lobule showed considerable uniformity and presented some one of the stages characteristic of lobar pneumonia.
Type II pneumococcus produced lobular pneumonia and spreading broncho- pneumonia and occasionally was found scattered throughout the lung in cases in which it was difficult to find any definite consolidations, there being present heavy soggy lungs with general thickening of the tissues and but little exudate in the alveoli except serum. Occasionally cases were seen with an intense hemorrhagic bronchitis and a leucocytic infiltration of the alveolar walls throughout the lung, with more or less serous exudate in the alveoli but with no consolidation; a pulmonary septicemic condition causing death with extreme rapidity.
Type III pneumococcus produced spreading bronchopneumonia, and lobular pneumonia in which fibrin formation was not a prominent feature. The consolidations were boggy and moist but not particularly bloody, while the exudate on the cut surface strung out like mucin from the knife edge. Occasionally small amounts of fibrin were seen but rarely was hepatization present.
Group IV pneumococci occasionally produced typical consolidations with lobar distribution, but following influenza and measles, bronchopneuinonia and lobular pneumonia were the types of distribution most commonly found.
Streptococcus viridans, or organisms so classified, produced lobular and bronchopneumonia and usually caused considerable inflammatory reaction in the interstitial tissues. In the streptococcus group the bacteriological results
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were not always of such a character as to allow accurate differentiation of the bacteria. Media requirements for this differentiation as between the viridans and the hemolytic streptococcus are such that unless one knows the exact technique used by the bacteriologist one is apt to doubt the diagnosis. Traces of sugar in media used to bring out the characteristic reaction on blood media of the hemolytic streptococcus will lead to an erroneous interpretation so that, in the absence of a definite statement as to the technique, including the method of preparation of the media used, bacteriological investigations do not justify any statement of a positive nature as to exactly what type of pulmonary inflammation resulted from infection with this group of organisms. These difficulties must be remedied before worth-while conclusions can be reached and few of the published reports give sufficient data. Streptococcus hemolyticus certainly produced the majority if not all of the lesions which we have described as lymphangitis of the pulmonary interstitial tissues and was responsible for many of the hemorrhagic lesions where considerable amounts of blood escaped into the pulmonary tissues. It was also responsible for a large proportion of the empyemas and for the extensions into the mediastinum.
All of the organisms above mentioned, Group IV pneumococci being considered as one organism, occasionally produce infection in the peritoneum, pleura, meninges, and pericardium. Whether these infections represent a marked increase in virulence whereby the infection extends readily, the tissues offering little resistance to its progress, or whether these lesions are the result of a pathological accident whereby large numbers of organisms gained the circulation in the form of small emboli which blocked capillaries and involved serous surfaces, is not known. Some cases appear best explained in one way, some in another. It does appear, however, that virulence and invasiveness account for at least part of the cases of polyserositis. Of the pneumococci, Type II appears to be most likely to produce such reaction, though at some camps many cases were reported from which organisms of Group IV were isolated.
Staphylococci frequently were found in the pulmonary tissues and in some camps were responsible for some of the pneumonic lesions present. Like other organisms there appeared to be virulent strains as lesions due to it were reported at some camps and not in others. It is an easily cultivated organism so that variations in technique should not interfere with its recognition, but many bacteriologists chose to ignore its presence in the cultures, considering it of little importance.
Micrococcus catarrhalis was occasionally found in the lungs, more particularly along the bronchi, but its pathogenic significance in these cases was not determined with sufficient clarity to make possible any statement with regard to its pathogenicity in the lungs.
Authentic reports of pneumonias of the lobular and spreading bronchopneumonic variety, caused by members of the meningococcus group are on record and it seems fairly definitely proven that this organism occasionally produced such lesions.
The bacillus of Friedlander was occasionally found and its lesions, except for the bacteria present, were scarcely to be distinguished from those due to
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pneumococcus Type III. It occasionally was present in considerable numbers in some camps, but as a whole it was not often reported as a cause of inflammatory lesions in the lung.
Summarizing, one may say that the influenza bacillus has more evidence behind it to prove it the cause of pandemic influenza than any other organism, but that the proof is not final and convincing and awaits a great deal of further work, and that during a pandemic period. Its occurrence and frequency during interepidemic periods, particularly during the seasons of the increased prevalence of respiratory diseases, must be determined, as well as its incidence during the general epidemics or pandemics which occasionally visit us.
It is evident that more thorough bacteriological work must be done as practically every type of media known must be used and all parts of the tissues must be cultivated at practically all stages of the disease by competent bacteriologists associated with competent pathologists, so that the organisms and the lesions may be properly correlated. Because of the large variety of flora present in the respiratory tract, there is no other alternative than to investigate every organism that can be cultivated as some one, morphologically indistinguishable from the others and similar in cultural characteristics may be responsible. No absolute dependence can be placed on the results of bacteriological investigation during the pandemic of influenza of 1918, except that they showed the large numbers of different bacteria concerned in the production of the pulmonary inflammation and of the presence in one and the same lung of lesions due to different organisms.
REFERENCES
(1) Olitsky, P. K., and Gates, F. L.: Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients. Journal of Experimental Medicine, Baltimore, 1921, xxxiii, 125, 361, 375, and 713; ibid., 1921, xxxiv, 1; ibid., 1922, xxxv, 1, 553, and 813; 1922, xxxvi, 685.
(2) Zinsser, Hans: The Etiology and Epidemiology of Influenza. Medicine, 1922, i, 213-309.
(3) McLeod, J. W., Ritchie, A. G., and Dottridge, C. A.: Incidence of Infections with Pfeiffer's Bacillus before, during, and after the 1918 Epidemic. Quarterly Journal Medicine, Oxford, 1920-21, 327-338.
(4) Blake, F. G., and Cecil, R. L : Studies on Experimental Pneumonia. Experimental Streptococcus Hemolyticus Pneumonia in Monkeys. Journal of Experimental Medicine, Baltimore, 1920, xxxii, 1; xxxi, 403-1920.
(5) Cecil, R. L., and Steffen, G. I.: Acute Respiratory Infection in Man Following Inoculation with Virulent Bacillus Influenzae. Journal of Infectious Diseases, Chicago, 1921, xxviii, 201-225.
