- Email: [email protected]
Experimental Urolithiasis
THE JOURNAL OF UROLOGY
Vol. 68, No. 5, November 1952 Printed in U.S.A.
EXPERIMENTAL UROLITHIASIS V.
PREVENTION AND DISSOLUTION OF FOREIGN BODY CALCULI BY INFECTION WITH SALMONELLA ENTERITIDIS
C. W. VERMEULEN, C.R. HELSEY
AND
R. GOETZ
From the Department of Surgery, University of Illinois College of Medicine, Chicago. Ill.
In previous papers of this series on experimental urolithiasis a method has been presented for studying the process of stone formation as it occurs after introduction of a foreign body into the urinary bladder of the rat. 1 • 2 , 3 , 4 vVe have as yet reported no experiments investigating the influence of infection upon stone formation although we had noted, as was to be expected, that accidental infection with a urea-splitting organism tended to augment the process. In one of our experiments, however, a chance finding led us to suspect that infection may at times exert a pronounced inhibitory effect on stone formation. This observation was first made in a group of control rats from an unrelated experiment. The group was composed of 16 animals each with a foreign body in the bladder. When the experiment was completed, 6 weeks after the foreign bodies had been implanted, it was found that an accidental infection had developed in 5 of the animals. All of these infected rats failed to produce stones while the remaining animals, whose urines were sterile, developed the expected amount of stone upon the foreign bodies. The infecting organism was identified as Salmonella Enteritidis. The purpose of the present paper is to present further data showing that this organism has a pronounced inhibitory effect on stone formation. The experiments ,vere of two types, the first being concerned with prevention of stone formation and the second with the dissolution of previously formed stones. Prevention ·was investigated by introducing Salmonella enteritidis at the time the foreign body was implanted. Dissolution was attempted by introducing the infection only after the stone had first been allmved to form in sterile urine. EXPERIMENT I~( PREVENTION)
111ethods. Four successive groups of rats were employed in this experiment, using male animals of a single strain and weighing approximately 200 gm. Each group was treated in identical fashion, being divided into a subgroup whose This investigation was supported by a research grant from the National Institutes of Health, Public Health Service. Our thanks are due to Dr. Welton I. Taylor of the Bacteriology Department, University of Illinois, for his valuable advice and assistance in the identification of the organism used in this study. 1 Vermeulen, C. W., Grove, William J., Goetz, R., Ragins, H. D. and Correll, N. O.· J. Urol., 64: 541, 1950. 2 Grove, William J., Vermeulen, C. W., Goetz, R. and Ragins, H. D.: J. Urol., 64: 549, 1950. 3 Vermeulen, C. W., Ragins, H. D., Grove, William J. and Goetz, R.: J. Urol., 66: 1, 1951. 4 Vermeulen. C. W., Goetz, R., Ragins, H. D. and Grove, William J.: J. Urol., 66: 6, . 1951. 790
EXPERIMENTAL UROLITHIASIS. V
791
urine was to be infected with Salmonella enteritidis and a subgroup of control animals. Two operations, separated by an interval of 6 weeks, were performed on all animals. At the first operation, using aseptic technique, a foreign body was introduced into the bladder through a suprapubic cystotomy. The foreign body in each instance consisted of a flat rectangular piece of zinc weighing approximately 10 mg. In the subgroups to be infected, the organism was introduced by washing the foreign body in a Brewer's broth culture of Salmonella enteritidis immediately prior to its insertion in the bladder. To make strict comparison possible, the foreign body to be inserted in each control rat was previously washed in sterile Brewer's broth medium. The bladder was closed with a fine silk suture and the abdominal incision repaired. A stock diet of Purina Fox Chow Checkers and drinking water were available to the animals at all times. Normal rats on this diet do not form calculi spontaneously. However, as we have already shown, if a zinc foreign body be present, a magnesium ammonium phosphate calculus is rapidly produced around it. Six weeks after the initial operation the second operation was performed. The bladder was again exposed, and its urine aspirated with a sterile syringe and needle. After opening the bladder, the stones were removed and weighed upon an analytical balance. In some instances where large stones had developed, small "daughter" stones ·were also found associated with them. These "daughter" stones have not been included in the stone ·weights recorded in the accompanying tables. Whenever a sufficient volume of urine was aspirated, its pH was determined with a Beckman pH meter. Cultures of the urine were then made on blood agar plates and in Brnwer's anaerobic media. The bacterial cultures were examined 48 hours after inoculation. In the infected group, Salmonella enteritidis was recovered in pure culture from the urine of all surviving rats. If the urine of any of the control animals was found to have become accidentally infected, such animals were excluded from the series. Strict comparison could therefore be made between stone growth in animals whose urine was infected with Salmonella enteritidis and controls ·whose urine had remained sterile throughout the experiment. A quantitative study was made of the urinary excretion of magnesium, calcium, phosphorus, and ammonia nitrogen in order to determine if Salmonella infection inhibits stone formation by altering the concentration of any of these substances. Four weeks from the original operation urine was collected for 3 consecutive days from 8 rats of the sterile group and from an equal number of the infected group and the appropriate determinations made. The ammonia nitrogen was estimated by a photometric modification of Folin and Bell's method. 5 The methods for the other chemical determinations are described in previous papers. Results. The degree of stone formation in the sterile controls was in marked contrast to that found in the infected animals. In the controls the incidence and average weight of stone was found to correspond with the results obtained in 5 Levinson, S. A. and MacFate, R. P.: Clinical Laborntory Diagnosis. Philadelphia: Lea and Febiger, 1943, p. 426.
792
C. W. VERMEULEN, C. R. HELSEY AND R. GOETZ
previous experiments. On the other hand, the absence of stone formation in the infected animals was most striking. The results obtained from the four experimental groups are shown in table 1. If the figures for all four groups be combined, it is seen that stones formed in 37 of 42 rats with sterile urine but in only 2 of 54 rats whose urine was infected with Salmonella enteritidis. The average weight of calculus deposit in the control animals was 52.9 milligrams but only 1.8 milligrams in the infected animals. The photograph (fig. 1) clearly demonstrates this difference. Statistical analysis of these results shows that the possibility of their occurrence by chance alone is much less than 1 in 100 in the first three groups and less than 5 in 100 in the fourth group. TABLE
1. Prevention of calculi by Salmonella infection NUMBER OF RATS
EXPERIMENTAL GROUPS
I
RATS WITH CALCULUS WT. OVER 10 MG.
AVER. WEIGHT OF CALCULUS FOR WHOLE GROUP
I mgm.
1
Control Salmonella
2
Control Salmonella
---
7 0
34.3 1.0
15 15
11
1
34.3 3.5
15 1
84.6 0.9
4 0
39.4 0.0
37 2
52.9 ± 7.4 (S.E.) 1.8 ± 0.82
7 5 ---
- - ---
I
3
Control Salmonella
15 28
4
Control Salmonella
5 6
I
ALL GROUPS cm.iBTKED
Control Salmonella
I I
42 54
Statistical comparison all groups combined: Stone weight, control vs. Salmonella - t = 7.813.
In table 2 the average daily volume and chemical composition of urine from the control and infected rats are compared. It was found that the urine volume was somewhat greater in the infected rats and was accompanied by a corresponding dilution of calcium, magnesium, and phosphorus, while the ammonia nitrogen remained unchanged. We do not believe that these chemical findings explain the failure of calculus formation in the presence of Salmonella infection. The infection also failed to produce any significant change in the urinary pH as judged by the values obtained at the end of the experiment. The average for the controls was 6.70 while that for the infected rats was 6.72. It should be emphasized that the Salmonella infection appeared to remain limited to the urinary tract. No systemic disturbance was apparent. Representative histological sections of the bladder wall of a control and an infected animal are shown in the photomicrographs (fig. 2). In both sections the mucosa is intact and of normal thickness, with a few chronic inflammatory cells in the submucosa.
793
EXPERIMENTAL UROLITHIASIS. V
We wondered if the inhibitory effect of Salmonella infection ,vould persist for periods longer than 6 weeks. Accordingly, at the time of the second operation, the bare zinc foreign bodies from six of the infected rats were replaced in their bladders. These six animals were then allowed to live for an additional nine weeks and at the end of this time the Salmonella enteritidis was again recovered. In no instance had stone formation occurred.
Fm. L (Experiment 1) Photograph of bladder contents of control and Salmonella infected rats. It is seen that in sterile control rats foreign bodies are encr1sed in stone while jn jnfected rats foreign bodies are almost completely free of calculous deposit. TABLE
2. Average values on analysis of urine ----------- - - - - - -
!
URE\TE COLLECTED
I
3
DAYS FRO\i 8 RATS IN EA.CH GROUP
\ . o lume
. Calcmm
lVIagne.
I P h osphorus
cc./diem
mg.%
mg.%
mg.%
15.8 20.1
21.2
33.6
125
17.7
29.1
105
1
pH
AT OPERATION
Ammonia N
-----------,--- --- ~1---Sterile control .. Salmonella infected.
!
-----------------'
2 1
! ----- -
-
--
mg.%
43 44
6. 7 (3± rats) 6.72 (45 rnts)
EXPERIMENT II-(DISSOL UTION)
Nfethods. Having established that Salmonella enteritidis is capable of preventing foreign body stone formation, an experiment was now designed to study the effect of delaying such infection until stones had already formed. Rats were again used, and at the first operation a standard zinc foreign body ,vas introduced into the bladder by the usual aseptic technique. Six weeks later the bladders were reopened after aspiration of the urine for culture. In the instances where stones had formed they were removed, weighed and re-implanted. In one half of the rats the stones, prior to their re-implantation, were washed in a Brewer's broth culture of Salmonella enteritidis. In the remaining animals ·which were to serve as controls, the stones were simply washed in sterile Brewer's broth before re-implantation. It will be noted that in the group to be infected
794
C. W. VERMEULEN, C. R. HELSEY AND R. GOETZ
with Salmonella, the stones were considerably larger than in those whose urine was to remain sterile. This provided a severe test of the "dissolving power" of the infection in rats whose stones had been rapidly growing up to the time when the infection was introduced. Four weeks after the second operation the bladder was again exposed. The urine was aspirated for culture and the stone removed and weighed. The change which occurred in the weight of the stones during the four weeks of Salmonella infection could then be compared with that occurring in the control animals. It,
Fm. 2. A, photomicrograph (X 132) of bladder of rat with sterile urine. B, photomicrograph (X 132) of bladder of rat with Salmonella infection.
should be stated that only such animals are included in the control group whose urine was proved to be sterile throughout the experiment. Likewise, the Salmonella group includes only those animals which yielded a sterile urine immediately prior to the introduction of the Salmonella and from which, at the end of the experiment, Salmonella was recovered in pure culture. Resillts. The results indicate that Salmonella enteritidis, already shown to be capable of inhibiting stone formation, is further able to effect a reversal of the process if the introduction of the infection is delayed until stones have already formed. As can be seen in table 3 and figure 3, the stones in the control group irr
795
EXPERIMENTAL UROLITHIASIS. V
most cases continued to grow. In the other group the stones decreased in size after the Salmonella infection was introduced. In the 11 animals of the control group, one stone lost weight, 3 stones remained essentially unchanged, while in the remaining 7 animals the stones continued to grow. For the entire group of controls the stones showed an average increase in weight of 50 mg. On the other hand, after the infection was introduced into the bladder of the 12 animals of the second group, only one stone gained weight, another remained unchanged, TABLE
3. Dissolution of calculi by Salmonella infection
AV. INITIAL
NO. OF RATS
PROCEDURE AT RE.IMPLANTATION
STONE WT.
I
AV. STONE WT. 4 WKS. LATER
mg.
11 12
42
No infection introduced Salmonella introduced
108
AV. CHANGE IN WT. OF STONE
mg.
mg.
92
+so
50
-58
Statistical comparison-change in weight of stone: t = 4.734 CHANGE
Mgm. increase
IN
WEIGHT
OF
STONES
200
150 ~gress1on
Continued growth
100
in
sterile
by_
salmonella infection
controls
150 Mgm.
decrease
Fm. 3. (Experiment 2-Dissolution). Graph showing changes in weights of individual stones in 2 series of rats in last 4 weeks of experiment. Gain or loss in weight of stone is indicated for each rat by length of bar above or below zero base line.
while the remaining 10 all lost weight significantly. Dissolution of stone was complete in 4 instances leaving the zinc foreign body bare in the bladder. For the entire group of rats infected with Salmonella, the stones showed an average weight loss of 58 mg., which is in sharp contrast to the average gain of 50 mg. in the controls. DISCUSSION
Infection is ordinarily considered to be an important factor in the formation and growth of urinary calculi. The evidence for such a relationship seems to be
796
C. W. VERMEULEN, C. R. HELSBY AND R. GOETZ
quite satisfactory when the infection is of the urea-splitting variety. The increased alkalinity of the urine along with the increased concentration of urinary ammonia satisfactorily explains the rapid development of stones in the presence of this type of infection. Animal experiments have also confirmed the clinical impression that infection with urea-splitting organisms augments stone formation. 6 • 7 The possibility that infection could ever inhibit stone formation does not seem to have been seriously considered. The data presented here indicate to us that under the experimental conditions used, such a possibility does indeed exist. It is true that an occasional stone developed in the first experiment despite the presence of Salmonella infection but when the infected animals as a group are compared with the uninfected controls a striking difference is evident. This conclusion is strengthened by the results of the second experiment where Salmonella infection was found to be capable of dissolving stones that had previously been formed in sterile urine. It is our belief that the infection does not act by altering the surface characteristics of the foreign body. It would be difficult to believe that dissolution of calculi would occur if this were so, for the foreign body in the dissolution experiments was completely encased in the stone. Apparently the infection acts not on the foreign body but on the stone itself, either its crystalline or matrix component. At the present time an explanation of the action of Salmonella infection is not available. It is evident that the infection does not act by producing acidification of the urine, for no difference in the urinary pH could be detected. Likewise chemical study of the urine has failed to show any significant change in the concentrations of the ionic components of the stone. Histologic study of the bladders of the infected rats has not shown any great change in the mucosa; actually, remarkably little local or general inflammatory reaction was found. One might speculate that Salmonella infection acts upon the crystalline component of the stone despite the negative evidence already mentioned. It may be that a chemical agent is produced, or some urinary colloidal change occurs that leads to an increase in solubility of the crystalloid. It might even be postulated that the growing organisms act as colloidal particles which bind to themselves the crystalline material that would otherwise lead to the formation of a stone. Then again, it is conceivable that Salmonella growing in the urine produces an enzyme which destroys or in some way alters the material composing the stone matrix or binding substance so that a stone cannot be formed. Experiments in vitro have so far failed completely when attempts were made to dissolve stones in urine infected with Salmonella. ·we do not wish to suggest in this paper that our findings have any immediate clinical application; but a knowledge of the mechanism by which Salmonella infection prevents and dissolves stones might well throw further light on the problem of urolithiasis. Other experiments are therefore in progress using this and other similar organisms. 6 7
Hager, B. H. and Magath, T. B.: J. A. M.A., 85: 1352, 1925. Davalos, H. A., Jr.: J. Ural., 49: 639, 1943.
EXPERIMENTAL UROLITHIASIS. V
797
SUMMARY
In a continuing study of stone formation as it occurs on foreign bodies in the bladders of rats, it was found that infection with Salmonella enteritidis is able to prevent stones that would otherwise occur on the foreign bodies. An additional experiment demonstrated that if the infection is introduced only after stones have already formed, the infection is capable of producing regression and sometimes complete dissolution of such stones. The mechanism by which the infection produces stone inhibition is as yet unknown. No change was detected in either the urinary pH or the urinary excretion of the crystalloid components of the stone.
Vol. 68, No. 5, November 1952 Printed in U.S.A.
EXPERIMENTAL UROLITHIASIS V.
PREVENTION AND DISSOLUTION OF FOREIGN BODY CALCULI BY INFECTION WITH SALMONELLA ENTERITIDIS
C. W. VERMEULEN, C.R. HELSEY
AND
R. GOETZ
From the Department of Surgery, University of Illinois College of Medicine, Chicago. Ill.
In previous papers of this series on experimental urolithiasis a method has been presented for studying the process of stone formation as it occurs after introduction of a foreign body into the urinary bladder of the rat. 1 • 2 , 3 , 4 vVe have as yet reported no experiments investigating the influence of infection upon stone formation although we had noted, as was to be expected, that accidental infection with a urea-splitting organism tended to augment the process. In one of our experiments, however, a chance finding led us to suspect that infection may at times exert a pronounced inhibitory effect on stone formation. This observation was first made in a group of control rats from an unrelated experiment. The group was composed of 16 animals each with a foreign body in the bladder. When the experiment was completed, 6 weeks after the foreign bodies had been implanted, it was found that an accidental infection had developed in 5 of the animals. All of these infected rats failed to produce stones while the remaining animals, whose urines were sterile, developed the expected amount of stone upon the foreign bodies. The infecting organism was identified as Salmonella Enteritidis. The purpose of the present paper is to present further data showing that this organism has a pronounced inhibitory effect on stone formation. The experiments ,vere of two types, the first being concerned with prevention of stone formation and the second with the dissolution of previously formed stones. Prevention ·was investigated by introducing Salmonella enteritidis at the time the foreign body was implanted. Dissolution was attempted by introducing the infection only after the stone had first been allmved to form in sterile urine. EXPERIMENT I~( PREVENTION)
111ethods. Four successive groups of rats were employed in this experiment, using male animals of a single strain and weighing approximately 200 gm. Each group was treated in identical fashion, being divided into a subgroup whose This investigation was supported by a research grant from the National Institutes of Health, Public Health Service. Our thanks are due to Dr. Welton I. Taylor of the Bacteriology Department, University of Illinois, for his valuable advice and assistance in the identification of the organism used in this study. 1 Vermeulen, C. W., Grove, William J., Goetz, R., Ragins, H. D. and Correll, N. O.· J. Urol., 64: 541, 1950. 2 Grove, William J., Vermeulen, C. W., Goetz, R. and Ragins, H. D.: J. Urol., 64: 549, 1950. 3 Vermeulen, C. W., Ragins, H. D., Grove, William J. and Goetz, R.: J. Urol., 66: 1, 1951. 4 Vermeulen. C. W., Goetz, R., Ragins, H. D. and Grove, William J.: J. Urol., 66: 6, . 1951. 790
EXPERIMENTAL UROLITHIASIS. V
791
urine was to be infected with Salmonella enteritidis and a subgroup of control animals. Two operations, separated by an interval of 6 weeks, were performed on all animals. At the first operation, using aseptic technique, a foreign body was introduced into the bladder through a suprapubic cystotomy. The foreign body in each instance consisted of a flat rectangular piece of zinc weighing approximately 10 mg. In the subgroups to be infected, the organism was introduced by washing the foreign body in a Brewer's broth culture of Salmonella enteritidis immediately prior to its insertion in the bladder. To make strict comparison possible, the foreign body to be inserted in each control rat was previously washed in sterile Brewer's broth medium. The bladder was closed with a fine silk suture and the abdominal incision repaired. A stock diet of Purina Fox Chow Checkers and drinking water were available to the animals at all times. Normal rats on this diet do not form calculi spontaneously. However, as we have already shown, if a zinc foreign body be present, a magnesium ammonium phosphate calculus is rapidly produced around it. Six weeks after the initial operation the second operation was performed. The bladder was again exposed, and its urine aspirated with a sterile syringe and needle. After opening the bladder, the stones were removed and weighed upon an analytical balance. In some instances where large stones had developed, small "daughter" stones ·were also found associated with them. These "daughter" stones have not been included in the stone ·weights recorded in the accompanying tables. Whenever a sufficient volume of urine was aspirated, its pH was determined with a Beckman pH meter. Cultures of the urine were then made on blood agar plates and in Brnwer's anaerobic media. The bacterial cultures were examined 48 hours after inoculation. In the infected group, Salmonella enteritidis was recovered in pure culture from the urine of all surviving rats. If the urine of any of the control animals was found to have become accidentally infected, such animals were excluded from the series. Strict comparison could therefore be made between stone growth in animals whose urine was infected with Salmonella enteritidis and controls ·whose urine had remained sterile throughout the experiment. A quantitative study was made of the urinary excretion of magnesium, calcium, phosphorus, and ammonia nitrogen in order to determine if Salmonella infection inhibits stone formation by altering the concentration of any of these substances. Four weeks from the original operation urine was collected for 3 consecutive days from 8 rats of the sterile group and from an equal number of the infected group and the appropriate determinations made. The ammonia nitrogen was estimated by a photometric modification of Folin and Bell's method. 5 The methods for the other chemical determinations are described in previous papers. Results. The degree of stone formation in the sterile controls was in marked contrast to that found in the infected animals. In the controls the incidence and average weight of stone was found to correspond with the results obtained in 5 Levinson, S. A. and MacFate, R. P.: Clinical Laborntory Diagnosis. Philadelphia: Lea and Febiger, 1943, p. 426.
792
C. W. VERMEULEN, C. R. HELSEY AND R. GOETZ
previous experiments. On the other hand, the absence of stone formation in the infected animals was most striking. The results obtained from the four experimental groups are shown in table 1. If the figures for all four groups be combined, it is seen that stones formed in 37 of 42 rats with sterile urine but in only 2 of 54 rats whose urine was infected with Salmonella enteritidis. The average weight of calculus deposit in the control animals was 52.9 milligrams but only 1.8 milligrams in the infected animals. The photograph (fig. 1) clearly demonstrates this difference. Statistical analysis of these results shows that the possibility of their occurrence by chance alone is much less than 1 in 100 in the first three groups and less than 5 in 100 in the fourth group. TABLE
1. Prevention of calculi by Salmonella infection NUMBER OF RATS
EXPERIMENTAL GROUPS
I
RATS WITH CALCULUS WT. OVER 10 MG.
AVER. WEIGHT OF CALCULUS FOR WHOLE GROUP
I mgm.
1
Control Salmonella
2
Control Salmonella
---
7 0
34.3 1.0
15 15
11
1
34.3 3.5
15 1
84.6 0.9
4 0
39.4 0.0
37 2
52.9 ± 7.4 (S.E.) 1.8 ± 0.82
7 5 ---
- - ---
I
3
Control Salmonella
15 28
4
Control Salmonella
5 6
I
ALL GROUPS cm.iBTKED
Control Salmonella
I I
42 54
Statistical comparison all groups combined: Stone weight, control vs. Salmonella - t = 7.813.
In table 2 the average daily volume and chemical composition of urine from the control and infected rats are compared. It was found that the urine volume was somewhat greater in the infected rats and was accompanied by a corresponding dilution of calcium, magnesium, and phosphorus, while the ammonia nitrogen remained unchanged. We do not believe that these chemical findings explain the failure of calculus formation in the presence of Salmonella infection. The infection also failed to produce any significant change in the urinary pH as judged by the values obtained at the end of the experiment. The average for the controls was 6.70 while that for the infected rats was 6.72. It should be emphasized that the Salmonella infection appeared to remain limited to the urinary tract. No systemic disturbance was apparent. Representative histological sections of the bladder wall of a control and an infected animal are shown in the photomicrographs (fig. 2). In both sections the mucosa is intact and of normal thickness, with a few chronic inflammatory cells in the submucosa.
793
EXPERIMENTAL UROLITHIASIS. V
We wondered if the inhibitory effect of Salmonella infection ,vould persist for periods longer than 6 weeks. Accordingly, at the time of the second operation, the bare zinc foreign bodies from six of the infected rats were replaced in their bladders. These six animals were then allowed to live for an additional nine weeks and at the end of this time the Salmonella enteritidis was again recovered. In no instance had stone formation occurred.
Fm. L (Experiment 1) Photograph of bladder contents of control and Salmonella infected rats. It is seen that in sterile control rats foreign bodies are encr1sed in stone while jn jnfected rats foreign bodies are almost completely free of calculous deposit. TABLE
2. Average values on analysis of urine ----------- - - - - - -
!
URE\TE COLLECTED
I
3
DAYS FRO\i 8 RATS IN EA.CH GROUP
\ . o lume
. Calcmm
lVIagne.
I P h osphorus
cc./diem
mg.%
mg.%
mg.%
15.8 20.1
21.2
33.6
125
17.7
29.1
105
1
pH
AT OPERATION
Ammonia N
-----------,--- --- ~1---Sterile control .. Salmonella infected.
!
-----------------'
2 1
! ----- -
-
--
mg.%
43 44
6. 7 (3± rats) 6.72 (45 rnts)
EXPERIMENT II-(DISSOL UTION)
Nfethods. Having established that Salmonella enteritidis is capable of preventing foreign body stone formation, an experiment was now designed to study the effect of delaying such infection until stones had already formed. Rats were again used, and at the first operation a standard zinc foreign body ,vas introduced into the bladder by the usual aseptic technique. Six weeks later the bladders were reopened after aspiration of the urine for culture. In the instances where stones had formed they were removed, weighed and re-implanted. In one half of the rats the stones, prior to their re-implantation, were washed in a Brewer's broth culture of Salmonella enteritidis. In the remaining animals ·which were to serve as controls, the stones were simply washed in sterile Brewer's broth before re-implantation. It will be noted that in the group to be infected
794
C. W. VERMEULEN, C. R. HELSEY AND R. GOETZ
with Salmonella, the stones were considerably larger than in those whose urine was to remain sterile. This provided a severe test of the "dissolving power" of the infection in rats whose stones had been rapidly growing up to the time when the infection was introduced. Four weeks after the second operation the bladder was again exposed. The urine was aspirated for culture and the stone removed and weighed. The change which occurred in the weight of the stones during the four weeks of Salmonella infection could then be compared with that occurring in the control animals. It,
Fm. 2. A, photomicrograph (X 132) of bladder of rat with sterile urine. B, photomicrograph (X 132) of bladder of rat with Salmonella infection.
should be stated that only such animals are included in the control group whose urine was proved to be sterile throughout the experiment. Likewise, the Salmonella group includes only those animals which yielded a sterile urine immediately prior to the introduction of the Salmonella and from which, at the end of the experiment, Salmonella was recovered in pure culture. Resillts. The results indicate that Salmonella enteritidis, already shown to be capable of inhibiting stone formation, is further able to effect a reversal of the process if the introduction of the infection is delayed until stones have already formed. As can be seen in table 3 and figure 3, the stones in the control group irr
795
EXPERIMENTAL UROLITHIASIS. V
most cases continued to grow. In the other group the stones decreased in size after the Salmonella infection was introduced. In the 11 animals of the control group, one stone lost weight, 3 stones remained essentially unchanged, while in the remaining 7 animals the stones continued to grow. For the entire group of controls the stones showed an average increase in weight of 50 mg. On the other hand, after the infection was introduced into the bladder of the 12 animals of the second group, only one stone gained weight, another remained unchanged, TABLE
3. Dissolution of calculi by Salmonella infection
AV. INITIAL
NO. OF RATS
PROCEDURE AT RE.IMPLANTATION
STONE WT.
I
AV. STONE WT. 4 WKS. LATER
mg.
11 12
42
No infection introduced Salmonella introduced
108
AV. CHANGE IN WT. OF STONE
mg.
mg.
92
+so
50
-58
Statistical comparison-change in weight of stone: t = 4.734 CHANGE
Mgm. increase
IN
WEIGHT
OF
STONES
200
150 ~gress1on
Continued growth
100
in
sterile
by_
salmonella infection
controls
150 Mgm.
decrease
Fm. 3. (Experiment 2-Dissolution). Graph showing changes in weights of individual stones in 2 series of rats in last 4 weeks of experiment. Gain or loss in weight of stone is indicated for each rat by length of bar above or below zero base line.
while the remaining 10 all lost weight significantly. Dissolution of stone was complete in 4 instances leaving the zinc foreign body bare in the bladder. For the entire group of rats infected with Salmonella, the stones showed an average weight loss of 58 mg., which is in sharp contrast to the average gain of 50 mg. in the controls. DISCUSSION
Infection is ordinarily considered to be an important factor in the formation and growth of urinary calculi. The evidence for such a relationship seems to be
796
C. W. VERMEULEN, C. R. HELSBY AND R. GOETZ
quite satisfactory when the infection is of the urea-splitting variety. The increased alkalinity of the urine along with the increased concentration of urinary ammonia satisfactorily explains the rapid development of stones in the presence of this type of infection. Animal experiments have also confirmed the clinical impression that infection with urea-splitting organisms augments stone formation. 6 • 7 The possibility that infection could ever inhibit stone formation does not seem to have been seriously considered. The data presented here indicate to us that under the experimental conditions used, such a possibility does indeed exist. It is true that an occasional stone developed in the first experiment despite the presence of Salmonella infection but when the infected animals as a group are compared with the uninfected controls a striking difference is evident. This conclusion is strengthened by the results of the second experiment where Salmonella infection was found to be capable of dissolving stones that had previously been formed in sterile urine. It is our belief that the infection does not act by altering the surface characteristics of the foreign body. It would be difficult to believe that dissolution of calculi would occur if this were so, for the foreign body in the dissolution experiments was completely encased in the stone. Apparently the infection acts not on the foreign body but on the stone itself, either its crystalline or matrix component. At the present time an explanation of the action of Salmonella infection is not available. It is evident that the infection does not act by producing acidification of the urine, for no difference in the urinary pH could be detected. Likewise chemical study of the urine has failed to show any significant change in the concentrations of the ionic components of the stone. Histologic study of the bladders of the infected rats has not shown any great change in the mucosa; actually, remarkably little local or general inflammatory reaction was found. One might speculate that Salmonella infection acts upon the crystalline component of the stone despite the negative evidence already mentioned. It may be that a chemical agent is produced, or some urinary colloidal change occurs that leads to an increase in solubility of the crystalloid. It might even be postulated that the growing organisms act as colloidal particles which bind to themselves the crystalline material that would otherwise lead to the formation of a stone. Then again, it is conceivable that Salmonella growing in the urine produces an enzyme which destroys or in some way alters the material composing the stone matrix or binding substance so that a stone cannot be formed. Experiments in vitro have so far failed completely when attempts were made to dissolve stones in urine infected with Salmonella. ·we do not wish to suggest in this paper that our findings have any immediate clinical application; but a knowledge of the mechanism by which Salmonella infection prevents and dissolves stones might well throw further light on the problem of urolithiasis. Other experiments are therefore in progress using this and other similar organisms. 6 7
Hager, B. H. and Magath, T. B.: J. A. M.A., 85: 1352, 1925. Davalos, H. A., Jr.: J. Ural., 49: 639, 1943.
EXPERIMENTAL UROLITHIASIS. V
797
SUMMARY
In a continuing study of stone formation as it occurs on foreign bodies in the bladders of rats, it was found that infection with Salmonella enteritidis is able to prevent stones that would otherwise occur on the foreign bodies. An additional experiment demonstrated that if the infection is introduced only after stones have already formed, the infection is capable of producing regression and sometimes complete dissolution of such stones. The mechanism by which the infection produces stone inhibition is as yet unknown. No change was detected in either the urinary pH or the urinary excretion of the crystalloid components of the stone.