Observations On the Bacteriophage In Infections of The Urinary Tract

OBSERVATIONS ON THE BACTERIOPHAGE IN INFECTIONS OF THE URINARY TRACT A. P. KRUEGER, H . K. FABER

AND

E. W,: SCHULTZ

From the Departments of Pediatrics and Bacteriology and Experimental Pathology, Stanford university, California INTRODUCTION

Since d'Herelle's first observations on bacteriophagy, a large number of papers have appeared dealing with various phases of this phenomenon. Many of the studies have been directed towards a solution of the fundamental nature of bacteriophage, while others have aimed to elicit information relative to the role which the bacteriophage may play in health and in recovery from disease. The therapeutic possibilities of an agent normally present in the body and capable of adjusting itself to, and propagating at the expense of pathogenic bacteria are obviously worthy of investigation. Many researches have already been directed along these lines with varying degrees of success. A full presentation of the therapeutic results in various infections reported to date is beyond the scope of the present paper. Readers interested in these results are referred to recent reviews (d'Herelle, 1926 (1); Schultz, 1927 (2), and others) . We shall in our brief survey confine ourselves wholly to the literature dealing with the influence of the bacteriophage on the course of urinary infections. Soon after its discovery the bacteriophage was hailed, particularly in France, as a therapeutic agent of exceptional value in combatting certain types of infections of the urinary tract. Thus Beckerich and Hauduroy (1922) (3), reporting on 3 cases of puerperal pyelo-cystitis treated by subcutaneous injection of anti-coli bacteriophage, state that two of the patients recovered within forty-eight hours. The third failed to respond. Following this (1923) (4) they reported striking results in eleven addi397 THE JOURNAL OF UROLOGY, V OL. XXIII, NO.

4

398

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

tional cases of urinary-tract infection due to B. coli. Similarly encouraging observations have been recorded by Courcoux, Philibert and Cordey (1922) (5), Alphonsi (1924) (6), Arloing, Dufour, Bouvier and Sempe (1924) (7), Philibert (1924) (8), and by Pereira (1924) (9). During this period of investigation two points became obvious: (a) that instillations of bacteriophage suspensions into the bladder should be combined with subcutaneous injections, and (b) that favorable results may be expected only when the causal organism is found, by in vitro tests, to be fully susceptible to the action of the bacteriophage destined for therapeutic use. Marcuse (1924) (10) claims to have been able to cure experimental B. coli cystitis in guinea pigs by instillations into the bladder alone. The enthusiasm engendered by these clinical successes is reflected by the claims of Philibert and his associates at the Hopitaux de Paris. They consider every case of B. coli bacilluria, whether a pyelo-nephritis of pregnancy, a urinary calculus, a coli septicemia or a bacilluria secondary to intestinal disturbances, as in tabetics, amenable to bacteriophage treatment. The fact that no harmful effects seem to attend the administration of the bacteriophage, and the possibility that bacteriophage suspensions may possess the advantages of a vaccine, as well as the virtues of the lytic agent itself, added to this general enthusiasm. The successful treatment of staphylococcus cystitis by Nelson Barbosa (1923) (11) served to further establish a conviction as to the therapeutic value of the bacteriophage. Zdansky (1924, 1925) (12, 13) has also reported very favorably on bacteriophage therapy in chronic infections of the urinary tract. He recommends raw sewage as a good source of bacteriophages active for B. coli and points out that the following factors may contribute to occasional failures of this form of therapy in individual cases: (a) a too rapid dilution of the phage by urine, (b) an interference with the process of bacteriophagy by colloids in suspension, and (c) the rapid appearance of phage-resistant strains of B. coli. With these points in mind he gave his patients alkalies by mouth and resorted to a preliminary lavage before instilling the bacteriophage into the kidney pelves and bladder.

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

399

Urinary infections in children were investigated particularly by Lehndorff (1924) (14), Sickenga (1925) (15), and by Munter and Boenheim (1925) (16). Lehndorff has observed very good results on combining intravesical instillation of the bacteriophage with alkalinization of the urinary tract. On the other hand, Sickenaga, who was especially interested in the spontaneous appearance of bacteriophages during the course of B. coli pyelitis, found that while natural bacteriophages commonly make their appearance at some time during the course of the disease, their presence does not appear to alter the trend of the infection. It is noteworthy, however, that these naturally occurring bacteriophages were never of high virulence. Vaccine therapy when applied was not attended with as favorable results in the presence of a bacteriophage as in its absence. Munter and Boenheim in their series were not impressed by the possibilities of bacteriophage therapy, although they recommend that it be given further trial. Frisch (1925) (17), isolated particularly active races of anti-coli bacteriophage from sewage and employed them in the treatment of chronic cysto-pyelitis in females. He administered the phage by repeated instillations into the kidney pelves and bladder, and obtained a good proportion of cures. Dalsace (1926) (18), on the basis of experience with a fairly large series of B. coli and staphylococcus infections of the urinary tract, concludes that the bacteriophage is a dependable therapeutic agent in uncomplicated cases and is in every way preferable to vaccine therapy. Infections in which the mechanical integrity of the urinary tract was damaged yielded, however, less favorable results. Dalsace administered the bacteriophage in two to four hypodermic injections, two or three bladder instillations, and at the same time also by mouth. Ravina (1926) (19), feels that the bacteriophage should always be employed when the usual medical treatment of simple cases fails, every effort first being made to exclude tuberculous infection. Larkum (1926) (20) in a study of 109 urine specimens from 52 patients presenting symptoms of urinary infection found a bacteriophage associated with susceptible organisms in 25 per cent

400

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

of the cases; a bacteriophage associated with resistant strains in 9.6 per cent; a susceptible organism alone in 15.4 per cent; and a resistant organism alone in 48.1 per cent of the cases. From his observations he came to the conclusion that a bacteriophage frequently appears in the urine during the course of urinary tract infections and that it is almost a constant finding during acute stages. Larkum believes that either the cure or the development of a chronic urinary infection depends upon whether the bacteriophage or the bacterium gains ascendency. Four cases of pyelitis treated with bacteriophage by Larkum responded nicely both from the laboratory and clinical standpoint. Pockels (1927) (21) attempted to treat chronic urinary infections in children with commercially prepared bacteriophage tablets. 1 On the basis of his trials with these products, which may or may not contain bacteriophage, Pockels concludes that bacteriophage therapy is of no value in the treatment of pyuria. Rice (1928) (22) in a preliminary report on infections treated with bacteriophage and anti-virus filtrates included three cases of cystitis favorably influenced by the instillation of phage or antivirus into the bladder, neglecting to state which of these preparations was used. Cowie (1926) (23), who has also made a careful study of bacteriophage therapy in a small series of urinary infections, seems to have been favorably impressed with the results he has noted. Cowie administered 2.5 to 3.0 cc. of bacteriophage subcutaneously on alternate days for three doses. CLINICAL MATERIAL

The present studies were undertaken with the view of investigating further the occurrence of bacteriophage in urinary infec1 It is interesting to note that German commercial_firms are now pl,acing such preparations on the market. To anyone who has worked with bacteriophage, especially among a group of organisms as heterogeneous with reference to bact eriophage action as B. coli, the merits of such a commercial enterprise appear indeed questionab\e, not only because the bacteriophage does not withstand drying over a long period of time, but also because each given strain of B. coli must be individually matched with an active bacteriophage in order to fulfill the first requirement of bacteriophage therapy.

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

401

tions and of studying the influence of bacteriophage therapy in such conditions. All cases of pyuria entering the Children's Ward at the Lane Hospital, Stanford University, during the period from October, 1927, to May, 1928, are included in the series studied. A number of patients from other wards of the hospital, and from the outpatient departments are also included in the group. We have had an opportunity, moreover, during the past summer to follow a number of cases in PaloAlto. The entire series is made up of 89 cases. (See table 5.) In studying each case the first thing which was always done was to determine whether the organism responsible for the infection was actually lysed by any one of several races 2 of bacteriophage in our possession. Only in the event that the responsible organism was completely lysed by a given race of bacteriophage was the resulting lysate employed as a potential therapeutic agent. These preliminary tests occasionally revealed organisms which resisted the action of all the bacteriophages in our collection and in such cases bacteriophage therapy was naturally not employed. Nevertheless, the data gathered from these studies are included because they tend to make clear certain points regarding the role of naturally occurring bacteriophage in urinary infections. METHODS

A. Method of determining the bacteriophage susceptibility of the causal organism

Specimens of urine procured sterilely from the bladder, and also, when possible, from the kidney pelves, were immediately seeded on Endo plates and into broth. 3 After incubation at 37°C. for twelve to eighteen hours, the cultures were examined for motility and stained according to Gram's method. When a 2 The term "race" is employed in this paper in the sense used by d'Herelle, to indicate bacteriophages isolated from different sources and possessing distinct properties. 3 By "broth" we refer throughout the paper to 1 per cent peptone, meat extract broth carefully adjusted to pH 7 .6. The agar, made with the same broth and containing 1 per cent agar, was also adjusted to pH 7 .6.

402

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

Gram-negative, actively motile rod, giving a red colony on the Endo plate was found, it was assumed for the time being to be B. coli, complete identification being reserved until the fermentation tests, set up at the same time, could be read'. The original broth culture of the Gram-negative organisms served as the source of material used in carrying out the bacteriophage-susceptibility test, which was performed with four different races of anti-coli bacteriophages, possessing more than usual potency and polyvalency. Five broth tubes were seeded with sufficient amounts of this bacterial suspension to render them slightly turbid. Each of the four tubes then received approximately 0.1 cc. of the respective bacteriophage suspensions, the fifth tube serving as a control. The contents of the tubes were mixed and two loopfuls transferred to an agar plate where the mixture was spread thoroughly over the surface. The entire set-up was then incubated at 25°C., a temperature at which the most satisfactory results were realized. Readings were made at the end of six, eighteen, twenty-four and forty-eight hours. The results, which ranged from complete lysis in broth, together with an absence of bacterial growth on agar, to a normal bacterial growth in both media, were expressed as 4 +, 3 +, 2 +, 1 +, 0, depending on the degree of bacterial lysis. In the event complete bacterial lysis occurred in an individual set-up, the lysed cultures were filtered at once through a Chamberland L3 candle and this filtrate was soon thereafter administered to the patient. In case only a partial lysis of the cultures followed, they were nevertheless filtered. This filtrate, however, was never employed therapeutically, but was added to a second freshly prepared culture of the organism, and these serial passages on the organism were repeated until complete lysis was obtained. This frequently occurred in the course of five or six passages.

B. Method of determining natural bacteriophage in urines The urine specimen was filtered through a Chamberland L3 candle immediately after making the cultures described above, since we were also interested in determining the natural occurrence of bacteriophages in urinary infections and the role which

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

403

these may play in spontaneous recovery, or in the formation of bacteriophage-resistant strains of bacteria encountered in chronic types of infection. One cubic centimeter of the urine filtrate was added to each broth tube containing an appropriate number of each of the following organisms: (a) causal bacterium; (b) a strain of Shiga dysentery bacillus known to be susceptible to several of our anti-coli bacteriophages; (c) a known bacteriophagesusceptible strain of B. coli; and, in the case of staphylococcus urinary infections, (d) a susceptible strain of Staph. aureus. After mixing the contents of the tubes, a drop of each mixture was spread out on agar plates. These set-ups were then incubated at 25°C. and read at the intervals stated above. In the absence of any demonstrable lytic action, the broth cultures were again filtered through a Chamberland L3 candle and passed once more on young cultures. At least four such serial passages were made before we considered the urine free of bacteriophages active for the organisms employed in the tests. THE INFLUENCE OF THE BACTERIOPHAGE ON THE COURSE OF URINARY INFECTIONS

A. General If we accept d'Herelle's view that spontaneous recovery from an infectious disease is brought about by the sudden activity of a natural bacteriophage, normally in symbiosis with members of the intestinal flora and capable of suddenly adapting itself to pathogenic invaders, we should then be able to demonstrate a direct correlation between the disappearance of the bacterium and the appearance of a bacteriophage highly active for it. Such a correlation has been said to exist in certain other infectious diseases, e.g., dysentery, typhoid and paratyphoid fevers, bubonic plague, avian typhosus and infections caused by staphylococci and streptococci. To quote d'Herelle (1926) (1): The observations made in bacillary dysentery, in the typhoid and paratyphoid fever, and in avian typhosis, show that the behavior of the intestinal bacteriophage toward a pathogenic bacterium which becomes implanted in the intestinal tract varies; being determined, (a) by the

404

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

conditions found in the intestine, and (b) by the hereditary characters of the bacteriophage and of the bacterium. The behavior of the bacteriophage may be summarized as follows: All of the conditions of medium and of characters working to the advantage of the bacteriophage, its virulence immediately increases for the invading pathogenic bacterium; the result being the immediate elimination of the bacterium before it can develop within the individual and cause any disturbance. The conditions being less favorable for the bacteriophage, its virulence increases less rapidly, bacteriophagy takes place after a longer latent period; the result being that after a few mild symptoms the disease is aborted. When the virulence of the bacteriophage is built up only after a considerable delay, the disease follows its course up to that time when the acquired virulence permits a complete bacteriophagy; the result being that at this time the symptoms of the disease regress, disappear, and the individual enters upon convalescence. When the virulence of the bacteriophage is enhanced very slowly and the delay in increased activity permits the bacterium to acquire a resistance, or when the bacterium at the time of invasion already has a resistance acquired outside of the body now invaded, a struggle takes place, the contending forces being the virulence of the bacteriophage on one hand, and the resistance of the bacterium on the other, and the condition of the patient reflects the ups and downs of this campaign. The result here may vary; if the conditions of environment and heredity favor the bacteriophage, the virulence permanently overcomes the resistance, complete bacteriophagy takes place; convalescence is established. If, on the contrary, the conditions favor the bacterium, it acquires a refractory stage, it is able to develop without hindrance; the individual dies. When, as a last possibility, the intestinal bacteriophage remains inert, the bacterium develops freely without any necessity for acquiring a resistance; the result being the death of the individual. The bacteriophage does not remain confined to the intestine; it passes into the circulation, from there into the tissues, and thence to the lesions where bacteria are to be found upon which it can exert its faculty of assimilation; the result being an in vivo bacteriophagy with the elimination of the invading microbe.

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

405

In urinary infections Larkum (1926) (20) was able to re.cover a lytic principle from a large percentage of urine specimens, particularly during the acute stage. He demonstrated that in many cases bacteriophages may be found in association with susceptible organisms one or more times during the course of the disease, but that neither the bacteriophage nor the susceptible bacteria are consistently present in all the specimens from a single individual. Larkum subscribes in general to the views which d'Herelle entertains relative to the role played by the bacteriophage in the phenomenon of recovery. Sickenga (1925) (15) found a bacteriophage constantly present in the urine in four out of 26 cases of B. coli pyelitis in children. The urine of twelve other patients contained a lytic principle at one time or another, while specimens from another group of ten failed to reveal the presence of any lytic principle. Sickenga, therefore, feels that the course of pyelitis is not modified by the presence of bacteriophages appearing spontaneously in the urine .

B. Experimental

In attempting to determine the influence of the bacteriophage on the course of urinary infections, periodic urine examinations of both acute all,d chronic cases were made, at short intervals. The following observations and deductions are based on a total of 167 urine specimens collected from 14 cases not treated with bacteriophage: 1. We were able to make frequent urine examinations in 4 cases of acute pyelitis, the first examination coming soon after the onset of the disease. In two of these cases the first examinations revealed only a pure culture of B. coli, without any evidence of the presence of a bacteriophage. In both cases specimens taken twenty-four hours later yielded bacterial cultures mixed with a weak bacteriophage. This persisted up to the time of convalescence, which in 1 case was four days after the onset, and seven days after the onset in the other. At the time of convalescence, however, the bacteriophages recovered from the urines were found to be highly virulent for the strain of B. coli originally isolated from the urine. At the same time the urine

406

A. P. KRUEGER, H . K. FABER AND E . W . SCHULTZ

was sterile. A temporary recrudescence i~ one of these patients six days after the urine had become sterile was marked by the appearance of a lysogenic strain of B. coli in the urine. Complete recovery, however, immediately followed this temporary flare-up. Two of the 4 cases were not seen as early in the disease as were the two just described. At the time of the first examinaDay I

Day II

Day III

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FIG.

1.

ACUTE PYELITIS.

• • • • •• ••\TT.--.-.-.-,. -.T_..r.-.,-:T;--•

VARIATION OF BACTERIOPHAGE OCCURRING IN URINE UNTREATED CASE (No. 16)

Virulence of Phage for causal B . coli. Virulence of Phage for stock B. coli. Virulence of Phage for Shiga dysentery. 4+ Complete lysis in broth. Sterile agar slant. 3+ Nearly complete lysis in broth. Confluent plaques on agar. 2+ Moderate lysis in broth. Many non-confluent plaques on agar. 1+ Slight lysis in broth. Occasional plaques on agar. 0 Normal bacterial growth in broth and on agar. * Urine filtrate completely lyses bacteria isolated on Day VII.

tion symbiotic relationships between a bacteriophage and the causal bacterium had already become established, and this symbiosis persisted until the onset of convalescence, when, as in the previous cases, the urines of both patients yielded bacteriophages capable of completely lysing cultures of the causal organism. During the interval between the first appearance of the

407

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

bacteriophage and the final disappearance of the causal bacterium in the urine, the balance of power between the two appears to shift for a period of time from one to the other. Figure 1 illustrates how the virulence of the natural bacteriophage in one of the cases changed during the course of the infection, while figure 2 illustrates the variation in the resistance of the causal organism J)a.y I 0

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ACUTE PYELITIS. VARIATION OF RESISTANCE OF CAUSAL UNTREATED CASE (No. 16)

Degree to which organism is lysed by urine filtrate. Degree to which organism is lysed by anti-coli Phage A. Degree to which organism is lysed by anti-coli Phage B . - 0 - 0 -0 -o Degree to which organism is lysed by anti-coil Phage C. Degree to which organism is lysed by anti-coli Phage D . Note: 0 Lysis of course corresponds to maximal resistance, while represents minimal resistance.

B.

COLI

0 0 0000° 0 0

4+

Lysis

to the natural bacteriophage and to stock bacteriophages. These observations tend to support the view that it is not merely the presence of a bacteriophage, but the virulence of a bacteriophage for the causal organism which is of primary importance. The fact that in these 4 cases the virulence of the naturally occurring bacteriophages was enhanced to the point of completely lysing the causal bacteria shortly before the organism disappeared

408

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

from the urine, suggests that the bacteriophage may possibly be connected in some way with recovery from acute urinary tract infection. The number of observations, however, is too small to make this more than a possibility, but it seems to us a point well worth further investigation. 2. In ten untreated cases of chronic urinary infection there appeared to be no definite relationship between the clinical condition of the patient and the presence or absence of a bacteriophage TABLE 1

Chronic pyelitis spontaneously occurring. Bacteriophage and clinical condition of patient. Untreated case (No. 22) ACTIVITY OF PHAGE IN URINE AS INDICATED BY LYTIC ACTION ON:

DAT

I II III

IV V VI VII VIII IX X XI XII

XIII XIV

Coli-Stock K-13

Shiga

Causal strain

2+ H 2+ 2+ 2+ H 2+ 3+ 2+ 3+ H

-

1+ H H 2+

-

1+ 2+ 2+ 1+ 2+ H

URINE SEDIMENT WHITE BLOOD CELLS PER HIGH POWER FIELD

60 40 35 50 45 65 50 20 30 40 25 40

60 50

GENERAL CONDITION

Good Poor Good Good Good Poor Good Good Good Good Good Poor Poor Fair

in the urine, nor was it possible to predict from the urine tests what the clinical trend of a given case would be. Table 1, prepared from a typical case, illustrates this point. The patient may at times appear improved despite the absence of a bacteriophage, and again may be in poorer clinical condition at times, although bacteriophage is present in the urine. These are, however, cases in which the bacteriophage naturally present never appears to be fully active, seldom producing more than a 3 + lysis, even when tested against highly susceptible laboratory

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

409

strains, and generally is appreciably less active against the causal strain. Why the natural bacteriophage fails to become virulent under these conditions is not altogether clear. The virulence of such bacteriophages may often be greatly enhanced when passed serially on the causal strain in vitro. It is known that bacteriophages in the laboratory when exposed for long periods of time to resistant organisms in secondary cultures tend to lose their virulence and this may be the mechanism which, under natural conditions, tends to keep down the virulence in the chronic infections. Strains of B. coli isolated from these chronic cases in general possess a greater resistance to test bacteriophages than do bacteria isolated from acute cases. This is in harmony with d'Herelle's view of the mechanism by which chronic infections become established. He believes that invading organisms coming in contact with a relatively weak natural bacteriophage acquire as a result a more or less well defined immunity to bacteriophage action and this enables them to become more firmly established in the host. Only a highly active bacteriophage, according to d'Herelle, will then serve to dislodge them. STUDIES ON THE VALUE OF THE BACTERIOPHAGE AS A THERAPEUTIC AGENT

The difficulties in determining the actual value of any agent used in the treatment of urinary infections is generally recognized. It is, moreover, well known that acute cases of pyel'itis and cystitis often recover spontaneously. We have, therefore, not been so much interested in the acute cases as in chronic cases, so far as attempts to evaluate the therapeutic efficiency of the bacteriophage are concerned. Larkum (1926) (20) has reported phage-susceptible strains of B. coli in 83.3 per cent of the acute cases, as contrasted with 17.9 per cent in the chronic cases, while our figures for the same groups are 92 and 46 per cent respectively. In our own series, besides the ten untreated cases of chronic urinary infection harboring phage-resistant bacteria and followed in our attempt to ascertain the effect of naturally occurring bacteriophage on the course of the disease, there were 32 other chronic cases

>f. I-'

0

TABLE 2

Illustrating the results of Phage therapy in isolated cases K. S., female, twenty-eight years of age, admitted November 23, 1927, with diagnosis of bilateral chronic pyelitis of one year's dur_a.tion; observed five months. URINE CULTURE

Day

1*

3

5 7

{~ {~ {~ {~

14 BC

Organism

B. coli B. coli B. coli B. col'i

PHAGE IN URINE FILTRATE

Kl3

Il3

Or

REACTION OF INFECTING ORGANISM TO STOCK PHAGES

A

B

.

C

D

WHITE BLOOD CELLS PER HIGH POWER FIELD IN URINARY SEDIMENT

Day

-- -- -- -- -- -- -- -3+ + 2+ + 3+ 4+ 1 3+ + 2+ + 3+ 4+ + +

-

-

B. coV Sterile

2+

-

-

-

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-

-

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-

- -

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+ +

3+ 4+ 4+ 3+ 3+ 4+

3

Source

R L

10

5

R 10 L 10

5

R L

10 10

No culture No culture

7

R L

0

No culture

14

3+ 3+ 2+ 4+ No culture

TREATMENT

CLINICAL COURSE

Day

Material

3 Phage D 5 Phage D

;d

~

t;,:j

~

Route

SC-KP-M SC-KP-M

!>

J=d Very marked improvement following treatment. Last seen May 1, 1928, and had been symptom-free since last treatment

1

~

~

~

t;,:j

!;,:I

§ t;,:j

~

Bl O cc.

Ul

4+, complete lysis ; 3+, nearly complete lysis; 2+, moderate lysis; 1+, slight lysis; O, no lysis; K13, stock B. coli; !13, stock Shiga dysentery; Or, organism isolated from uri_ne; Bl, bladder instillation; KP, kidney pelvis instillation; SC, subcutaneous injection; M, oral administration; 0cc, occasional; Rand L, right and left kidney pelves; A, B, C, D, test phages; BC, bladder culture. * Date November 24, 1927.

c::1

0 ~

~ N

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

411

infected with strains of B. coli incapable of being completely lysed by any of our stock bacteriophages. None of these 42 cases, of course, received bacteriophage therapy. It is quite evident, therefore, that in dealing with chronic urinary infections one is far more often confronted with bacteriophage-resistant organisms, a situation which renders the study of bacteriophage therapy difficult at the outset. However, if one has at his disposal a sufficient number of highly virulent, more or less polyvalent anti-coli bacteriophages, studies of this sort are not altogether hopeless. It is known that the resistance of the causal organism may vary some from day to day, and if one assumes that contact of the organism with a fully active bacteriophage should, theoretically at least, lead to a therapeutic result, it should then be possible to fulfill the required conditions at some time during the course of the disease. A rapid method for testing phage-susceptibility has not yet been developed, nor has any investigator succeeded in isolating a bacteriophage of such polyvalency and virulence for different strains of B. coli as to enable one to easily meet these most trying conditions. Without these important aids it becomes necessary in treating chronic cases to approach the ideal as closely as possible, so far as the use of bacteriophage suspensions is concerned, by employing pooled suspensions containing as many different races of anti-coli bacteriophages as happen to be available. To this end we pooled ten or more anti-coli bacteriophages listed in the laboratory as separate races. This polyvalent suspension was tested for lytic action on the bacteria isolated from the urine, and, if complete lysis ensued, was administered to the patient as follows: (a) subcutaneous injections of three doses varying from 1.5 to 3.0 cc. at intervals of twenty-four hours; (b) instillations of the bacteriophage suspensions into the kidney pelves once weekly and into the bladder twice to three times weekly over periods of from one to four weeks. The instillations in the later members of our series were always preceded by a preliminary lavage with an alkaline physiological saline solution. This was done to remove pus and tissue debris,

412

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

which may tend to adsorb the bacteriophage after it is introduced. Moreover, with the beginning of bacteriophage therapy, the urine of the patient was alkalinized by oral administration of the usual alkalies, and all forms of urinary antiseptics were at once discontinued. Sixteen cases were treated with these pooled bacteriophage suspensions, while nineteen others were treated in the same manner, except that they received suspensions prepared by propagating individual races of bacteriophage on the bacterial strains isolated from the patient. These two groups were followed carefully from the bacteriological as well as the clinical standpoint. We should state at once that in our total series we observed in only a few instances what seemed to be spectacular recoveries, such as those reported in chronic cases by other investigators. Four of our patients may possibly fall into this category. Since the completion of our original series we have had five other cases which seem to fall into this group, but these were not followed up bacteriologically. Table 2 illustrates the striking results which were realized in one of the 4 cases referred to. The significance of such observations would be more obvious were they of more regular occurrence in treated cases. Twenty-five of the chronic cases treated failed to show a strikingly rapid recovery. The progress in these cases was much more gradual and cannot be definitely laid to the effect of phage therapy. A typical example of this group is patient A.B. (Table 3). This case on the 169th day after original entry to outpatient service, harbored in the left kidney pelvis a strain of B. coli which was completely lysed by all of the bacteriophages against which it was tested, whereas the strain isolated .from the right kidney pelvis was only partially lysed by these bacteriophages. On the same day Phage D was administered subcutaneously and also instilled into the kidney pelves and bladder. Specimens taken fourteen days later (183rd day) showed the left kidney to be free of both the organisms and the bacteriophage, while a specimen taken from the right kidney gave a positive culture of B. coli and showed the presence of a lytic principle.

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

---J'

413

At this time another instillation of the same race of bacteriophage into both kidney pelves was made. Tests run with the strain of B. coli obtained from the right kidney on the 183rd day indicated that the organism had developed almost complete resistance to the Phage D injected on the 169th and 183rd days, but was now susceptible to Phage B. A suspension of Phage B was, therefore, instilled into the bladder and kidney pelves and injected hypodermically at the next visit (191st day). Examination of table 3 shows that we had again failed on our attempt to use a "matched" bacteriophage, for the culture taken on the 191st day consisted of a strain of B. coli totally resistant to Phage B . We arbitrarily chose Phage D for instillation at the next clinic visit of the patient (199th day) and by chance picked a race capable of completely lysing the bacterial strain isolated from the urine at that particular time. This same bacteriophage was again employed in the treatment seven days later (206th day) with the final result that entirely sterile specimens were obtained from both kidney pelves on the 213th day. There was marked subjective and objective improvement with only occasional leucocytes in the kidney specimens at the time of the last visit.

The pooled mixtures were employed in 16 cases with, in the main, definitely better results than those which attended our attempts to use individual carefully matched races of bacteriophages in nineteen other chronic cases. Repeated instillations appear in general to be beneficial although this is quite at variance with the general practice in bacteriophage therapy. It is ordinary held that bacteriophage treatment is either immediately and strikingly successful or else fails entirely. This is often the case, but we have seen a number of patients who gave no evidence of yielding to this form of therapy during the earlier part of the treatment, but who nevertheless gradually improved and finally attained a complete clinical and bacteriological cure. That the bacteriophage may have played some role in these successful outcomes over and above the factor of lavage, is perhaps possible since these were almost without exception chronic cases previously treated by lavage and instillations of antiseptics for comparatively long periods of time. Six of the thirty-five chronic cases treated failed entirely to

TABLE 3

Illustrating lack of rapid response to Phage thera_py A. B., female, fifty-one years of age; diagnosis of bilateral chronic pyelitis of six years duration; observed 11 months URINE CULTURE

Day

7BC R L 70 R L 140 169 BC R L 176 183 R L 191 R L 199 R L

Organism

PHAGE IN URINE FILTRATE

K 13

-

113

- --

Or

REACTION OF INFECTING ORGANISM TO STOCK PHAGES

-

A

-

B

C --

B. coli Sterile B. coli

D

Day

140 B. B. B. B. B. B. B. B.

coli coli coli coli coli coli coli coli

B. coli Sterile

169

-

-

-

3+ 3+ 3+ 4+ 4+

-

-

-

-

4+ H 176 183

+ + + +

2+ 3+ 2+ 4+ 4+ 4+ 4+ 191 - 2+ 2+ 199 3+ - 4+ + 206 0 No culture

B. coli B. coli

3+ 3+ 3+ 3+ 3+ 3+

B. coli Sterile

-

-

4+

2+ 0

-

-

- a+ - -

3+ 3+

-

. '

-

4+ 4+ No culture

?'"

WHITE BLOOD CELLS PER HIGH POWER F I ELD IN URINARY SEDIMENT

-- -1 70

213

H"-f--' H"--

TREATMENT CLINICAL COURSE

Source

Day

Material

Bl 40 R 1 L 10 R 10 L 10 R 20 L 15 Bl 10 R 10 L occ R 3 L 1 R L 1 R 5 L 1 Rocc. L occ.

1 to 169 169 183 191 199 206 213

Mere. and AgNOa

KP-Bl

Phage D Phage D P hage B Phage D Phage D Phage D

KP-Bl-Sc KP-Bl-Sc KP-Bl-Sc KP-Bl-Sc KP-Bl-Sc KP-BI-Sc

;d

i

Route

§ Jo Slight improvement Much improvement Marked subjective and objective improvement

~ pl

~

t".l ~

§ Dismissed on 215th day after entry to service. Last heard from Octo14, 1928* when she was in good health. No complaints

be~

~

~ Ul

0

:::r:

~

N

206 R L

213 R

B. coli Sterile

4+ - -

3+ 2+1-13+14+ No culture -

Sterile Sterile

-

-

-

No culture No culture

- 4+, complete lysis; 3+, nearly complete lysis; 2+, moderate lysis; 1+, slight lysis; O, no lysis; Bl, bladder instillation; KP, kidney pelvis instillation; Sc, subcutaneous injection; 0cc, occasional; R and L, right and left kidney pelves; K13, stock L

B. coli; !13, stock Shiga dysentery; Or, organisms isolated from patient's urine; A, B, C, D, test phages employed; BC, bladder culture. * Five months after dismissal.

b:l

>

C'.l

~

::d .... 0

~

>

Q l?'.f

.... z ....

z>zj l?'.f

C'.l

"3 0

.... z Ul 0

>zj

c::

::d ....

~

"3

~ ~

~

~

Cl1

416

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

improve during or immediately after bacteriophage therapy. Two of these received the pooled anti-col_i bacteriophages, and four the matched bacteriophages. All six patients were infected with strains of B. coli which varied considerably in bacteriophage susceptibility from time to time, but which nevertheless were fully susceptible at one time or another during the period of treatment. THE INFLUENCE OF ALKALINIZATION OF THE COURSE OF ACUTE PYELITIS

In view of Sickenga's finding (1925) (15) that the only drug which modified the course of an acute pyelitis is sodium citrate, the effect of alkalinizing the urine on the spontaneous development of virulent bacteriophages was studied. Drugs such as sodium bicarbonate, sodium citrate, citro carbonates, etc., were administered by mouth to eight patients suffering from acute pyelitis. Twelve hour urine specimens were examined for bacteriophage according to the usual technic. Five of these patients reacted unfavorably to the alkalinization and were subsequently placed on other medication. During the period in which they were excreting alkaline urine not one of them developed a race of bacteriophage capable of completely lysing the causal organism isolated from the urine. The bacteriophages which did appear spontaneously behaved in a ma~ner entirely similar to those already described in untreated acute cases (fig. 1). Three of this series of eight patients recovered rapidly. Interestingly enough the urine of each patient contained at recovery a race of bacteriophage which completely lysed the B. coli strain originally isolated. In all 3 cases the bacteriophage developed full activity very shortly after the urine became alkaline, and the urine quite promptly became free of bacteria. It appears, therefore, that alkalinization of the urine may facilitate the natural occurrence of bacteriophagy, just as an alkaline medium does in vitro, but it is not clear from this small series just how important either alkalinization or the bacteriophage, or the two together may be in the recovery of the average acute case. The results are at any rate suggestive and indicate that this point may deserve further study.

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

417

EXPERIMENTS WITH PURIFIED BACTERIOPHAGE SUSPENSIONS

Finally, in our attempt to evaluate bacteriophage therapy in urinary infections we employed bacteriophage suspensions containing a minimum of associated bacterial protein, and prepared according to both the methods described by Arnold and Weiss (1925) (24). These preparations showed an actual bacteriophage content slightly less than that of the ordinary unpurified suspensions. With these suspensions we treated six chronic cases of pyelo-cystitis. Four patients received the pooled bacteriophage suspensions and two received "matched" bacteriophage · suspensions. In other respects the procedure was the same as in the case of patients treated with the usual suspensions. In none of this group of cases treated with protein-free lysates did we observe the least effect on the course of the infection, while all but one of the 6 cases treated subsequently with the same bacteriophages, but in unpurified suspensions, responded quite well. A typical example of this group is patient S. T. (table 4). In this patient the first dose of the "purified" bacteriophage suspension served to establish the lytic principle in the kidney pelves, but its presence there did not result in any subjective or objective improvement in the patient's condition. Two subcutaneous injections, six bladder instillations and three instillations into the kidney pelves, as well as six doses of the bacteriophage by mouth, all failed to induce a change in the clinical course of the disease. On the twenty-second day after entry, however, we abandoned the use of the "purified" suspensions and began the administration of the usual pooled unpurified bacteriophage suspension. Four subcutaneous injections, together with a similar number of bacteriophage instillations into the bladder and kidney pelves, were administered from the 22nd to the 30th day. On the 28th day the kidney urine was found sterile, with only an occasional pus cell, and the patient's general condition was greatly improved.

The sequence and general character of the results in four of the other 5 cases parallel those of the case just described. The reason for the apparent difference in the therapeutic effects of "purified" and unpurified bacteriophage suspensions in this series

TABLE 4

Purified and unpurified bacteriophages as therapeutic agents S. T., female, forty-three years of age; diagnosis, bilateral pyelitis of twenty-four years duration; observed 30 days URINE CULTURE

Day

Organism

PHAGE IN URINE F ILTRATE

Kl3

1 R L

4BC 8 R L 11 R L 13 R

L 14BC 22 R L 25 R L 28

B. coli B. coli B . coli

B. coli B. coli B. coli B. coli

2+

Il3

-

-

Or

REACTION OF INFECTING ORGAN ISM TO STOCK PHAGES

-- -

-

A

-

B

4+ 4+ 4+ 4+ 4+ 2+

4+ 3+ 2+ 4+ 4+ 3+ 2+ 4+

-

-··-

4+ 3+ 2+ 2+ + 4+ 3+ 3+ 2+ +

B. col~ 4+ 2+ 2+ 3+ + B. coli 4+ 2+ 2+ 3+ + Specimen lpst

C

-

·-

-

D

Day

1

---·-·

4+ 4+ 4+

-

4+ 11 4+ 13 4+ 4+ 22

..

-

. ..

-

-

-

4 8

4+ 25 4+ 28

B. coli B. coli

4+ 3+ 2+ 4+ 2+ 4+ 3+ 2+ 4+ 2+

---- .

4+ 4+ 30

B. coli B. coli

4+ 2+ + 4+ 2+ ·+

-

4+ 4+

4+ 4+

+ +

I>

WHITE BLOOD CELLS PER HIGH POWER F IELD IN URINARY SEDIMENT

-

Source

R L Bl R L R L R L R L R L

10

20 70 30 30 20 30 40 25 30 30 3 10

Rocc. L occ. Rocc. L occ.

~ I-'

00

TREATMENT

CLIN ICAL COURSB

Day

-

4 6

8 11

13 17 22 25 28 30

Material

Pur. Ph A Pur. Ph A Pur. PhD Pur. PhD Pur. PhD Pur. PhD Old PhD Old PhD Old PhD Old PhD

~

Route

SC-Bl-M-KP SC-Bl-M-KP Bl-M-KP Bl-M Bl-M-KP Bl-M SC-Bl-KP SC-Bl-KP SC-Bl-KP SC-Bl-KP

!"d trl 0 trl

No improvement

Jd

Clinical condition poor N o improvement

~

Slight improvement Marked improvement Last seen May 21, 1928* and had been symptom free since last treatment

Letter received September 5, 1928

!11

~

td trl

~

§ trl

~ Ul 0

:::r: q

~ ~

_I

R L 30

Sterile Sterile

4+ - -4+ - -- --···

No culture No culture

No culture 2+ - No culture 2+ - 4+, complete lysis; a+, nearly complete lysis; 2+, moderate

R L

Patient had no complaints

Sterile Sterile

lysis; 1+, slight lysis ; O, no lysis; BI, bladder instillation; KP, kidney pelvis instillation; SC, subcutaneous injection; 0cc, occasional; M, oral administration; R and L, right and left kidney pelves; Kl3, stock B. coli; Il3, stock Shiga dysentery; Or, organisms isolated from patient's urine; A, B, C, D, test phages employed; BC, bladder culture. * Fift.y second day after admission.

t.d

> ~

(".l

l=d .....

0

~

;,..

~

.....

z ..... z',:l trJ

(".l

>-3 ..... 0

z

"(/).

0

',:l

~

l=d

~ ~

~

(".l

>-3 ],j::>.

.....

~

420

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

of cases is not altogether clear. cussed below.

Certain possibilities are dis-

DISCUSSION

In clinical studies on the therapeutic value of any agent considerable care must be exercised in interpreting the results obtained. It is quite obvious that our limited series of cases precludes the drawing of any sweeping conclusions as to the efficacy of the bacteriophage in treating urinary tract infections. The prompt recovery which followed the administration of the bacteriophage in a few cases, together with the fact that spontaneous recovery may, at times at least, be clearly associated with the appearance of a highly active bacteriophage, tempts one to the belief that the bacteriophage may under certain conditions play an important role in the phenomenon of recovery. It is, however, impossible to say from a small series of observations just how generally such a mechanism comes into play in recovery. Whatever enthusiasm one may have aroused as a result of the more or less spectacular recoveries in individual cases is offset by the clear-cut failures, or doubtful results which may be observed side by side with the successful outcomes. At the same time there is an additional difficulty precluding the acceptance of unsuccessful results at their face value; namely, the operation of factors which may tend to prevent a normal sequence of events in the process of recovery from infection. Before one is permitted to draw conclusions from these cases it therefore becomes necessary to first probe into the possible causes of failure . This we have not been able to do to any great extent, but certain possibilities have nevertheless suggested themselves. Among them are such factors as the reaction of the urine; the presence of adsorbing colloids; of bacterial protein lysates and of antibodies against the bacteriophage itself, etc. A factor which may be of considerable importance and one which has not been studied in connection with the treatment of chronic urinary infections; in which the causal organism is of ten in symbiosis with a weak natural bacteriophage (Sickenga, 1925 (15), Larkum, 1926 (20), our own observations), is the

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

421

possible appearance of antiphagic antibodies in the blood and tissues of chronic cases. Such antibodies have been found to TABLE 5

Analysis of clinical material GROUP

---

NUMBER OF CASES

TYPE OF URINARY TRACT INFECTION

PHAGE SUSCEPTIBILITY OF CAUSAL ORGANISM

EXPERIMENTAL PROCEDURE

---

A

10

Chronic infection

Phage resistant

B

32

C

4

Chronic infection Acute infection

Phage resistant Phage susceptible

D

8

Acute infection

E

16*

Chronic infection

7 phage SUSceptible; 1 phage resistant Phage susceptible

F

19t

Chronic infection

Phage susceptible

-4

Untreated. Followed for evidence as to the influence of natural,ly occurring phage on the course of chronic infection Untreated. Transferred for symptomatic therapy Untreated. Followed for evidence as to the influence of naturally occurring phage on the course of acute infection Untreated. Followed for effect of alkalinization on bacteriophagy in urine Treated with bacteriophage (pooled suspensions): 2 complete failures, 3 rapid and striking recoveries probably due to phage action, 11 gradual recoveries; not clearly attributable to phage Treated with bacteriophage (matched individual races): 4 complete failures, 1 rapid and striking recovery probably due to phage action, 14 gradual recoveries not clearly attributable to phage

* Four of these cases were first treated with purified protein free phage (pooled suspension) . Four complete failures . All subsequently recovered when treated with non-purified phage (pooled). t Two of these cases were first treated with purified protein free phage (matched individual races). Two complete failures, 1 recovered on subsequent treatment with matched non-purified phage, while one failed to respond.

make their appearance in the blood of animals injected with bacteriophage suspensions (Bordet and Ciuca, 1921 (25);

422

A. P. KRUEGER, H . K. FABER AND E. W. SCHULTZ

d'Herelle and Eliava, 1921 (26); and many others). Moreover, Sonnenschein (1925) (27), Katsu (1925) (28) and d'Herelle (1928) (29) have recently noted that the antiphagic antibodies may appear naturally in the blood of patients suffering from infectious diseases. It is conceivable that a certain percentage at least of therapeutic failures may be due in part to the inhibiting action of these antibodies. This is a question which should be studied carefully in the future. Anti anti-phagic antibodies have been developed by Fabry and van Beneden (30) and there is a theoretical possibility that some clinical use may be found for them in temporarily overcoming the inhibiting influence of natural antiphagic antibodies. Theoretically at least, bacteriophage suspensions may exercise a therapeutic effect in two different ways; (a) by direct action of the lytic principle on the causal microbe, and (b) by a stimulating action which the dissolved bacterial proteins in the suspension may exercise on the defense mechanism of the host. The first mechanism, when it operates effectively, is presumably the one primarily responsible for the immediate recoveries associated with a complete disappearance of the causal microbe and its replacement by a highly active bacteriophage. It is a mechanism which we seek to put into effective action when we administer a bacteriophage suspension as a therapeutic measure and it must perforce operate with sweeping effectiveness when it strikes, if it is not to fail entirely in bringing about a cure. A failure to bring about complete, or nearly complete, sterilization of the diseased tissue or organ, results in the ascendancy of organisms resistant to this particular bacteriophage and a continuation of the disease process. The burden is now placed wholly upon the anti-microbic defense mechanism of the host, unless a more effective bacteriophage is introduced or develops in the infected individual. In cases which do not clear up promptly, that is, within twenty-four to forty-eight hours after the administration of the bacteriophage, and which clear up only after a number of treatments, it naturally becomes a matter of some doubt as to the role played by the bacteriophage itself in recovery. In these instances it is conceivable, however, that some therapeutic effect

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

423

may have been realized from the bacterial proteins still in the suspension. While the quantity of original unchanged bacterial proteins in completely lysed bacterial suspensions is generally small (Osumi, 1924 (31); Marshall, 1925 (32) and others), it has been recently shown by Jungeblut and Schultz (1929) (33) that the bacteriophage produces a specific denaturization of the bacterial proteins and that these denaturized proteins are capable of sensitizing tissues. If we remember that in most chronic urinary infections the bacterium is often found in association with a weak bacteriophage, and that possibly in consequence of this the tissues have become naturally sensitized to similar denaturization products, it becomes apparent how the products of bacteriophage action, arising from lysis of the causal organism, may be of value in themselves. Our failure to get results with "purified" bacteriophage suspensions may hinge in part on this point. These suspensions were free of detectable protein, and consequently lacked the specific desentitizing and immunizing action, which seems to reside in the unpurified suspension. This may not, however, represent the only difference, for theoretically this should not in any way preclude actual lysis of the bacteria in the tissues. Granting the presence of definite bacteriophagy in vivo, it still does not necessarily follow that the by-products of bacterial lysis actually come into intimate relationship with tissue cells in sufficient concentration to influence them. This would seem to be especially true in the urinary tract, where the products of lysis are apt to be greatly diluted and washed away. Some advantages would seem, therefore, to be afforded by the instillation of larger concentrations of these products in order to activate local tissue response. That the natural, unaltered bacterial proteins are not of primary importance in this connection seems to be reflected by the therapeutic failures which have followed the use of bacterial vaccines (Sickenga, 1925 (15); Dalsace, 1926 (18) and others). There can be no doubt that a satisfactory answer to the question of the role which the bacteriophage plays directly, and the byproducts of bacterial lysis play indirectly, in the phenomenon of recovery from urinary tract infections, can only come from a

424

A. P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

much more exhaustive study than we have been able to make. We are offering the results of our experience thus far with this form of therapy for what they may be worth. The suggestions offered may be of some value to those who are interested in carrying out similar studies on a more extensive scale. SUMMARY

The data obtained by periodic examinations of urine specimens from 8 cases of acute urinary infection treated with alkalies by mouth indicate that alkalinization, when effective, may act in part by facilitating enhancement of the virulence of bacteriophages naturally occurring in the urine, probably byproviding the optimal reaction for bacteriophagy. Of a total of 12 cases of acute urinary tract infection, only one, or 8 per cent, harbored a strain of B. coli resistant to the bacteriophages in our possession, whereas among 77 patients suffering from chronic infections of the urinary tract, 42, or 54 per cent, were infected with bacteriophage-resistant organisms. Thirtyfive chronic cases harbored bacteria capable of being completely lysed by one or more races of bacteriophage. Sixteen of these patients were treated with pooled bacteriophage suspensions, while 19 received "matched" individual races of bacteriophage. In both groups the bacteriophage suspensions employed were found by preliminary tests to be fully active on the causal organism and were administered by subcutaneous injections and by instillations into the bladder and kidney pelves. In only 3 cases among the 16 receiving pooled suspensions was a prompt recovery noted; 2 patients failed to respond at all to the treatment, and 11 gradually recovered. Among the 19 patients to whom "matched" individual races of bacteriophage were administered there occurred only one prompt recovery, 4 total failures and 14 gradual recoveries. There were then 25 patients out of 35 treated cases in whom recovery was gradual and not clearly attributable to the bacteriophage itself; 4 instances of immediate and striking response and 6 complete failures. The use of purified protein-free bacteriophage in 6 cases of chronic urinary infection failed entirely to elicit any therapeutic

BACTERIOPHAGE IN INFECTIONS OF URINARY TRACT

425

response, while all but one of these patients subsequently recovered during a course of treatment with ordinary non-purified bacteriophage. The general trend of our results, therefore, leaves us in no position to state with any accuracy how much, if any, value may be attributable to the bacteriophage as a therapeutic agent in chronic urinary infection. Our limited series of cases would preclude any far reaching conclusions although one point is obvious; namely, that in our hands the bacteriophage treatment of urinary infections has failed to realize the brilliant results reported by several previous investigators. The probability that bacteriophagy in vivo may be greatly influenced by various factors which may eventually be brought under control, should be borne in mind in future studies. REFERENCES (1) D'HERELLE, F: The Bacteriophage and its Behavior. The Williams & Wilkins Company, 1926. (2) SCHULTZ, E.W. : The bacteriophage: its prophylactic and therapeutic value. California and Western Med., 1927, xxvii, no. 4. (3) BECKERICH, A., AND HAUDUROY, P.: Le bacteriophage dans le traitement de la fievre typhoide. Compt. rend. Soc. de biol., 1922, lxxxvi, 168. (4) BECKERICH, A., AND HAURDUROY, P . : Le traitement des infections urinaires a colibacilles par le bacteriophage de d'Herelle. Bull. med . Par., 1923, xxxvii, 273. (5) CouRcoux, PHILIBERT AND CORDEY: Un cas de pyelionephrite gravidique traitee par le bacteriophage de d'Herelle. Bull. et Mem de la Soc. Med. Hop. de Paris, 1922, xlvi, 1151. (6) ALPHONsI, M. : Guerison rapide de deux cas de pyelonephrite gravidique traites par le bacteriophage de d'Herelle. Bull. de la Soc. d 'Obstet. et de Gynecol. de Paris, 1924, xiii, 573. (7) ARLOING, F ., DUFOUR, BouvIER AND SEMPE : Traitement de la pyelonephrite par le bacteriophage de d'Herelle. Compt. rend. Soc . med . des Hop, de Lyon, May 13, 1924. (8) PHILIBERT, A.: Les appijcations therapeutiques du bacteriophage. Clinique et Lab., Par., January 20, 1924. (9) PEREIRA, 0.: 0 phenomeno de d'Herelle e as infeccoes pelos bacilos coli e dysentericos. These Porto Alegre, Lib. do Globo, 1924. (10) MARcusE, K . : Grundlagen und Aufgaben der Lysintherapie (d'H erelle's Bakteriophagen) . Deutsche. Med . Wchnschr., 1924, 1, 334. (11) NELSON BARBOSA: Ac<;ao curativa do bacteriofago estaphylococcico. Brazil-med. Rio de Jan., 1923, xxxvii, 297. (12) ZDANSKY, E.: Gewinnung von ubertragbaren Lysinen (Bakteriophagen) fur therapeutische Zwecke. Wiener. kl. Wchnschr., 1924, xx, 501.

426

A, P. KRUEGER, H. K. FABER AND E. W. SCHULTZ

(13) ZDANSKY, E. : Versuche einer bakteriophagentherapie bei Koli-infektionen der abfiihrenden Harnwege. Wiener Arch. f. inner. Med., 1925, xi, 533. (14) LEHNDORFF, H . : Therapeutische Anwendung des bakteriophagen Lysins (Twort-d' Herelle) bei Kinderkrankheiten . Wien . Med. Wchnschr., 1924, xxi, 1051. (15) SICKENGA, F . N . : The bacteriophage in pyelitis of children . Nederlandsch. Maandschr. f. G,e neeck ., Amsterdam, 1925, xiii, 141. (16) MUNTER, H ., AND BoENHEiM, C . : Uber therapeutische Versuche mit bakteriophagen Lysin bei Kindern und Saiiglingen. Zeitschr. f. Kinderheilk., 1925, xxxix, 388. (17) FRISCH, B.: Zur Behandlung der Koliinfektion des Harntraktes mit Bakteriophagen. Wien. Klin. Wchnschr., 1925, xxxviii, 839. (18) DALSACE, R . : Applications du Bacteriophage de d'Herelle a la cure des infectionsurinaires. Jr. d'Urol., 1926, xxi, 123. (19) RAVINA, A.: Traitement des infections urinaires par le bacteriophage de d'Herelle . Presse med., 1926, xxxiv, 548. (20) LARKUM, N. W. : Bacteriophagy in urinary infection. Part I. The incidence of bacteriophage and of Bacillus coli susceptible to dissolution by the bacteriophage in urines. Presentation of cases of renal infection in which bacteriophage was used therapeutically. Jour. Bacteriol., 1926, xii, 203. (21) PocKELS, W. : Die Bakteriophagen-Therapie in der Kinderheilkunde. Monatschr. f. Kinderheilk. , 1927, xxxv, 229. (22) RICE, T. B . : Infections treated with bacteriophage and antivirus filtrates . Indiana State Med. Ass. Jr., March, 1928, 21- 93. (23) Cowrn, D . MURRAY: Observations on the Bacteriophage. Ann. Clin. Med., 1926, v, 57 . (24) ARNOLD, L., AND WEISS, E.: Isolation of bacteriophage free from bacterial proteins. Jour . Infect. Dis., 1925, xxxvii, 411. (25) BoRDET, J ., AND CrncA, M .: Specificite de l'autolyse microbienne transmissible. Compt. rend. Soc. de biol., 1921, lxxxiv, 278 . (26) d'HERELLE, F ., AND ELIAVA, G . : Sur le serum antibacteriophage . Compt. rend. Soc . de biol., 1921, lxxxiv, 719 . (27) SoNNENSCHEIN, C . : Der Nachweis antibacteriophage-Serumwirkung. Deut. Med. Wchnschr ., 1925, Ii, 1434. (28) KATsu, S.: Antibacteriophage Wirkj.ngen im menschenserum. Deut . Med. Wchnschr., 1925, li, 1896. (29) d'HERELLE, F. : Lane Lectures, Stanford University, California, October, 1928. (30) FABRY, P ., AND VAN BENEDEN, J .: Serum antilytique et antiserum antiantilytique. Compt. rend. Soc. de biol., 1924, xc, 111. (31) OsUMI, S. : Serologisch,e Studien mit einem Bacteriophagen. Zeitsch,r. f. Immunitatsforsch., 1924, xl, 261. (32) MARSHALL, M. S.: Observations on d'Herelle's bacteriophage. Jour. Infect . Dis ., 1925, xxxvii, 126. (33) JUNGEBLUT, C. W., AND ScHULTz, E.W . : Studies on the sensitizing properties of the bacteriophage. Jour. Exper. Med., 1929, xlix, 127.