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A Study of Concentration as a Factor in the Antiseptic Properties of Dog’s Urine
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A STUDY OF CONCENTRATION AS A FACTOR IN THE ANTISEPTIC PROPERTIES OF DOG'S URINE EDWIN C. WHITE From the Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland
The antiseptic properties of dog's urine were first observed in this laboratory by Davis and Hain (1). They summarized their findings as follows: Normal dog's urine, obtained by aseptic catheterization, shows a distinct antiseptic action which is particularly marked against Staphylococcus aureus and entirely inert against at least one strain of Staphylococcus albus. This antiseptic action is not constant for all dogs, nor for the same dogs on all_occasions. Dog's urine, inoculated with Bacillus coli and incubated may become sterile in as short a period as six hours. The antiseptic action bears no relationship to the hydrogen ion concentration, nor is it influenced by the action of various extractives on the urine. Of those specimens of urine which act as suitable culture media, some may be rendered unsuitable by passage through a Berkefeld filter.
In 1920 Hain (2) showed the striking effect of variation of diet and water intake on the antiseptic properties of dog's urine. Byfeeding dogs large amounts of meat and restricting the amount of water allowed the urine became, in all cases, bactericidal toward Bact. coli, although bactericidal towards Staph. aureus only occasionally. When the amount of meat was decreased and the amount of water increased, the incidence of bactericidal effect decreased, and when the animals were fed on a diet of bread and milk the bactericidal effect against both organisms disappeared entirely. Two possible explanations of these results suggest themselves. First, the mere concentration of the urine may be a factor, with501 THE JOURNAL OF UROLOGY, VOL, XXII, N O,
5
502
EDWIN C. WHITE
out regard to the particular substances responsible for the concentration. High protein diet results in the elimination of large amounts of nitrogenous end products of metabolism, principally urea, in the urine. Secondly, the bactericidal properties may be due to some bactericidal substance or substances resulting from the metabolism of food high in protein. Although these substances may themselves be present only in small amounts, their concentration may parallel the total concentration until they reach a point at which they can exert bactericidal action. The present paper records the results of the investigation of the first of these possibilities. It should be stated that Davis and Hain (loc. cit.) suggested the possibility of the relationship between bactericidal property and specific gravity, but did not investigate it. METHOD
Dogs were placed on a diet consisting exclusively of liver. Each animal received about 1½ pounds per day, without reference to body weight. Water was withheld at night and not given until after catheterization next morning; otherwise, the dogs were allowed as much water as they would take. In this way less protein and more water was allowed than in Rain's experiments, as it was desired to obtain for purposes of comparison some samples that showed bactericidal effect and some that did not. The animals were catheterized aseptically as described by Davis and Hain. As dog's urine is often very cloudy, sterility controls were made by transferring 0.1 cc. to nutrient broth and examining for growth after proper incubation. To 1 cc. of urine was added a loopful of a twenty-four-hour broth culture of the organism. After twenty-four hours' incubation 0.1 cc. was transferred to 5 cc. of nutrient broth, which in turn was incubated and examined for growth after twenty-four and forty-eight hours. The freezing point of each sample was determined by means of a Beckmann thermometer in the usual way. The depression thus noted is a more rational measure of total concentration
ANTISEPTIC PROPERTIES OF DOG'S URINE
503
than is specific gravity, for it measures the osmotic pressure of the solution. The studies of A. Fischer (3) show that the bacterial cell consists of an outer supporting wall against which presses an inner membrane of protoplasm, the interior of the cell being filled with a solution. When the bacterium is placed in a solution hypertonic with reference to the cell contents, one and perhaps two processes will come into play. First, water will pass out from the cell to the stronger solution and as the interior solution thus decreases in volume the membrane will shrink away from the supporting wall. This shrinking process is called plasmolysis. If it proceeds far enough before osmotic equilibrium is reached between the solutions on both sides of the membrane, the ability of the cell to reproduce is interferred with and the organism may even be killed. The membrane of protoplasm is not ideally semi-permeable, but is permeable to some dissolved substances as well as to water. If the bacterium is placed in the hypertonic solution of a substance to which its membrane is permeable, water will pass out and simultaneously the dissolved substance will pass into the weaker solution within the cell. As the second of these processes goes on the osmotic pressure of the solution within the cell increases and osmotic equilibrium may be established before enough water has been lost to cause plasmolysis. Indeed, the fact that a bacterium cannot be plasmolyzed by solutions of a particular substance is taken by Fischer as evidence that its membrane is permeable to that substance. In the case of such diffusible substances the bacterial processes may be disturbed because of the high concentration reached in the interior of the cells. It may even cause the death of the organism by precipitating or "salting out" substances necessary for life. Guillemard (4) determined the highest concentration of certain salts permitting bacterial growth in bouillon. For Bact. coli this was 1.1 molar, for staphylococci 3.5 molar, when sodium chloride was used. The findings of this author, together with the high resistance of staphylococci to the bactericidal action of dog's urine, as compared to the resistance of Bact. coli, provided a
504
E DWI N C. WHI TE
logical basis for investigating the possible relation between osmotic pressure, as measured by depression of freezing point , and bactericidal action in dog's urine. T his relation is shown in TABLE 1
Relation between depression of freezing point, in degrees Centigrade, and bactericidal action of dog's urine BACT. COLI ( STRAIN 1)
Depressions at which organism was killed
3.95 3.71 4.57 3.67 4.32 3.67 3.02 3.35 3.07 3.43 3.54 3.15 2.97 2.92 4.03 3.04 2.69 3.57
STAPH. AUREUS
D epressions at which organism survived
3.07 3.58 3.18 1.33 0.75 2.42 2.27 1.89 2.25 2.39 2.13 1.95 2.33 1.98 2.02 2.44 3.22 2.45
Depressions at which organism was killed
3.54 3.15 2.97 2.92 4.03 2.69 2. 45
Depressions at which organism survived
3.95 3.71 4.57 3.67 3.58 3.18 1.33 0.75 2.42 3.02 2.27 1.89 3.35 3.07 2.25 2.39 3.43 2.13 1.95 2.33 1.98 2.02 2.44 3.22 3.04 3.57
table 1. The organisms were all isolated from cases of urinary infection. Inspection of this table brings out several significant facts. The relative infrequency with which Staph. aureus was killed is striking and confirms the findings of Hain and Davis. There is no discernible relation between depression of freezing point and
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ANTISEPTIC PROPERTIES OF DOG'S URINE
505
bactericidal action against this organism. The greatest depression noted, 4.57°, as well as the smallest, both failed to kill it. At points in between these values, the organism sometimes was killed and sometimes survived. Thus, at 3.15°, it was killed in one case, while in another sample, at 3.18°, practically the same value, it grew. Again at 3.54°, it was killed once, but survived in another sample at 3.57°. These two values are practically identical. There is no minimum above which the organism was always killed. It is clear that as far as this particular strain of Staphylococcus is concerned, osmotic pressure bears no relation to bactericidal action, the explanation of which must be sought in some other agency. The facts with reference to the strain of Bact. coli used are different. It is seen at a glance that in general those urine samples with the greatest depressions of freezing point killed the organism while those with the smaller depressions failed to kill. The urine with the greatest depression observed, 4.57°, killed and that with the smallest depression, 0.75°, failed to kill. There is one point in between these extremes, 3.07°, at which the organism was killed once and survived once. The smallest depression at which killing occurred was 2.69°, the great depression permitting survival was 3.58°. Of eighteen samples that killed fifteen showed a depression of 3.0° or more and seventeen showed a depression greater than 2.9°. Of eighteen samples that failed to kill, fourteen showed a depression less than 3.0°, sixteen showed a depression less than 3.2°, and seventeen a depression less than 3.5°. Stated in another way, all samples with a depression greater than 3.58° killed this strain of Bact. coli and with one exception (3.58° once failed) all samples with a depression of 3.3° or more also killed all organisms. These facts focus attention upon the critical range of osmotic pressure corresponding to depressions of 3.0° to 3.5°. It was therefore of interest to see what effect could be produced on this strain of Bact. coli by solutions, originally favorable culture media, whose osmotic pressure was gradually increased through the range met with in dog's urine, by addition of varying amounts of urea and sodium chloride. These two substances were chosen
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EDWIN C. WHITE
because they are the chief constituents in point of percentage in urine and to them and especially to urea is due the greatest part of the total osmotic pressure. Moreover, a large amount of urea is present in the urine of animals on high protein diet. To prepare the salt solution it was only necessary to add the desired amounts of salt to ordinary infusion broth and sterilize by autoclaving. This method, of course, could not be used for urea solutions because urea is hydrolyzed when its solutions are heated above 100°. Consequently the desired amounts of urea were weighed into sterile tubes, heated to 100° for one hour and added to the broth. Fortunately, these urea broths were sterile and TABLE 2
Effect of addition of sodium chloride to infusion broth, pH 6.0, on bactericidal action DEPRESSION OF FREEZING POINT
2.37 2.61 2.96 3.20 3.50 3.62 3.77 4.01
BACT . COLI (STRAIN 1)
STAPH . AUREUB
+ + + +
+ + + + + + + +
0 0 0 0
+ indicates survival; 0 indicates sterilization. remained so for several months, but in subsequent attempts this method always failed to sterilize the urea, as shown by bacterial growth in the broth. Another series of sterile urea broths were later prepared in the following manner. Fifty per cent aqueous solution of urea was sterilized by Berkefeld filtration and varying amounts of this solution were added to a beef extract, peptone, salt, sodium-phosphate solution, containing such amounts of these four constituents as would make their final concentration 0.3, 0.5, 1.0, and 0.1 per cent, respectively. Both the infusion broth and extract broth were titrated to pH 6.0, which represents about the average reaction of dog's urine examined in this study, the highest being 6.4 and the lowest 5.4°. With these solutions prepared and their freezing points deter-
507
ANTISEPTIC PROPERTIES OF DOG'S URINE
mined, their effect on bacteria was studied by precisely the same method as was used in the case of dog's urine, viz. inoculation of 1 cc. portions with a loopful of culture, transfer of 0.1 cc. to broth after twenty-four hours' incubation, and observation after twenty-four and forty-eight hours. The results are shown in table 2. The smallest depression at which Bact. coli was killed was somewhere between 3.2° and 3.5°, corresponding to 5 to 6 per cent of salt. This is very striking for it represents just about the zone above which dog's urine, with one exception, never failed to kill. TABLE 3
Effect of addition of sodium chloride to infusion broth, pH 6.0, on bactericidal action APPROXIMATE SALT
ADDED
DEPRESSION OF FREEZING POINT
BACT. COLI (STRAIN 1)
STAPH. AUREUS
3. 19 3.35 3.49 3.60 3.78 3.84
+
+ + + + + +
per cent
5.0 5.2 5.4 5.6 5.8 6.0
+ indicates
0 0 0 0 0
survival; 0 indicates sterilization.
Staph. aureus, on the other hand, was not killed by the solution at any depression within the range usually encountered in dog's urine. Table 3 shows the results with a new salt broth series in which the intervals were made smaller. This series shows that killing of Bact. coli begins at a depression of about 3.3°. As in table 2, Staph. aureus was not killed by any member of the series. As the observations recorded in table 3 were made two weeks later than those in table 2, the two experiments serve as definite checks on each other and show that the results were not due to any fortuitous circumstance at the time of the first experiment. Table 4 shows the effect of adding urea to infusion broth. The results are practically identical with those in the salt series.
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EDWIN C. WHITE
Staph. aureus was not killed by any member of the series, but Bact. coli was killed at a depression of about 3.3°. This represents the addition of approximately 10 per cent of urea. Tests with urea, using precisely the same solutions as in the first experiments, were repeated one month later and five weeks later. During this interval the strain of Ba~t. coli had become somewhat more resistant to concentration effects for in both these later tests the organisms survived a depression of 3.53°, but were killed at 3.63°. From these results it is clear that the concentration reached in dog's urine is not of itself sufficiently high, by virtue either of TABLE 4
Effect of addition of urea to infusion broth, pH 6.0, on bactericidal acti on APPROXIMATE UREA ADDED
DEPRESSION OF FREEZING POINT
BACT. COLI (STRAIN 1)
STAPH. AUREUS
+ + + +
+ + + + + + + +
per cent
8 .5 9.0 9 .5 10.0 10.5 11.0 11 .5 12.0
2.75 2.94 3.11 3.24 3.34 3.53 3.63 3.77
0 0 0 0
+ indicates survival; 0 indicates sterilization. sodium chloride or urea, to kill Staph. aureus. When this organism is killed by dog's urine, as it occasionally is, some other agency must be at work. As regards the particular strain of Bact. coli used, the concentration reached in dog's urine is in many cases of itself sufficiently high to account for the sterilization. In other samples which killed Bact. coli, the concentration is definitely not high enough to cause sterilization and here too some other factor must be the cause. It is possible that in the most concentrated samples both the unknown factor and the osmotic effect are at work simultaneously, although in those cases it is not necessary to postulate anything beyond mere concentration to explain these findings.
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ANTISEPTIC PROPERTIES OF DOG'S URINE
Some weeks later another series of tests were carried out with this same strain of Bact. coli and three other strains, all isolated from urinary infections. The four strains were subjected to the TABLE 5
Second series showing relation between depression of freezing point of dog's urine and bactericidal action BACT. COLI
DEPRESION OF FREEZING POINT
Strain 1
Strain 2
Strain 3
Str ain 4
+ + + +
+ + + +
0 0 0 0
0 0 0 0
+ + + + +
+ + + + + +
1.57 2.27 2.57 3.20 3.50 3.86 3.86 4.29
0 0 0
0 0
+ indicates survival; 0 indicates sterilization. TABLE 6
Effect of addition of urea to beef extract broth, pH 6.0, on bactericidal action APPROXIMATE UREA ADDED
BACT. COLI
DEPRESSION OF FREEZING POINT
Strain 1
Strain 2
Strain 3
Strain 4
+ + +* +* +* +* +*
+ + + +* +* +* +* +*
+ + +* +* +* +* +*
per cent
5 6 7 8 9 10 11
12
2.27 2.57 2.88 3.22 3.55 3.87 4.16 4.47
+ + +* +* +* 0 0 0
0
0
+
indicates growth in broth. 0 indicat es sterilization. +* indicates inhibition of growth in broth, but growth in transfer.
action of eight samples of dog's urine and also to the action of urea broth as before. The results in dog's urine are shown in table 5, and the behavior of the organisms tested at the same time in beef extract broth containing urea is shown in table 6.
510
EDWIN C. WHITE
Table 5 shows a striking parallelism between the depression of freezing point and bactericidal action. The four least concentrated samples killed none of the four strains, the three next most concentrated killed two, three and four strains, respectively, and the most concentrated sample killed all four. But while all four strains were killed in dog's urine at a depression of 3.86°, table 6 shows that the last three strains were not killed rin urea-salt broth1 when this depression was considerably exceeded, viz. at 4.16°. Strain 3 was not killed in broth even at 4.47°. As far as these last three strains are concerned it may be definitely concluded that when they are killed by dog's urine the factor responsible is not mere concentration. Strain 1, however, was killed by urea-salt broth at this time at a depression somewhere betwen 3.55° and 3.87°; hence, in the three samples of dog's urine showing depression of 3.86°, 3.86° and 4.29° the concentration alone is sufficient to account for the sterilization, although there may also have been present in these samples the other, at present unknown factor. APPLICATION OF THESE RESULTS TO HUMAN URINE
Hewlett, Gilbert and Wickett (5) administered urea to men in amounts sufficient to raise the blood urea to levels found in uremia. The doses averaged about 100 grams, being given either at once or divided over intervals. As the subjects were made very ill by these amounts they certainly represent the maximum amounts that could be considered with the idea of concentrating the urine so as to secure a bactericidal effect. The highest total urea concentration found in the urine after ingestion of these large doses was 6.4 per cent. Schill and Kunze (6) injected 10 to 15 cc. of "100 per cent" urea solution intravenously in humans. (This is presumably 1: 1 solution, which is approximately saturated.) T he total nitrogen in the urine reached a maximum of 2.62 per cent, in 1 case. If all this was present as urea the concentration would have been only about 5.2 1 This broth contained a constant amount of sodium chloride, 1 per cent, in addition to the increasing amounts of urea.
511
ANTISEPTIC PROPERTIES OF DOG'S URINE
per cent. Addis and Foster (7) found that administration of 30 grams of urea by mouth led to a maximum urea concentration in urine of 4.85 per cent, while administration of 10 grams of sodium chloride under similar conditions caused the salt concentration in the urine to reach a maximum of 2.25 per cent. The last figure is osmotically equivalent to no more than 4.5 per cent of urea, even assuming complete dissociation of the salt. TABLE 7
Ejfect of addition of urea to human urine on antiseptic action
URINE SAMPLE
MA E< M -< A :e! A
;;i-.
pH
C-< o, M
NUMBER
~p "'"
-- --
1
5 2
--
5.4
~
OM 0
BACT. COLI
z"' 0 0
MZ M
aJ
"'" "'"'
o, M M ._ A
"'"
Strain 1
Strain 2
Strain 3
Strain 4
--
per cent
ll
0 1 2 3 4 5
1.82 2 .07 2.32 2.62 2.92 3.19
0
1.67 1.95 2.27 2.57 2.85 3. 12
1
2
.
2 3 4 5
+ + +
sl sl sl
+ + + +
sl
0
+ + + + + + + + + + + +
+ + +
sl sl sl
+ + +
sl sl
0
+ + + + + + + + + + + +
+ + + + +
sl
+ + + +
sl
0
+ + + + + + + + + + + +
+ +
sl sl sl sl
+ + +
sl sl
0
+ + + + + + + + + + + +
+
= growth as heavy as control. sl = markedly lighter growth than control. 0 = no apparent growth.
In view of these findings a few experiments have been included in the present study in which sterile solid urea was added to sterile human urine in increasing amounts so as to bring the total up to the amounts observed by Hewlett, Gilbert and Wickett. The sterile solid urea was obtained by adding measured amounts of sterile 50 per cent urea solution (Berkefeld filtered) to sterile test tubes and evaporating to dryness at body temperature in a high vacuum. The proper volume of urine was then added.
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EDWIN C. WHITE
The total urea may be estimated to be at least 1 to 1.5 per cent more than the added urea. Sterility controls were made by adding 1 cc. of urine to 10 cc. of broth and incubating. Controls made by inoculating the urine separately with each of the four . strains showed that the urine without addition of urea was a very favorable culture medium; as evidenced by heavy growth in twenty-four hours. The results are shown in table 7. Under each strain two columns are shown. In the first column is noted the appearance of the inoculated urine, after twenty-four hours' incubation, as compared with the control simultaneously inoculated but without addition of urea. In the second column is shown the result of transfer of 0.1 cc. of inoculated urine, after standing twenty-four hours in the incubator, to 10 cc. of broth, and reading the broth tubes after forty-eight hours. It is seen from this table that although sterilization was not effected in any case, even though the urine was made very concentrated, yet in many cases there was definite retardation of growth at concentrations perhaps attainable in human urine. The idea suggests itself that concentrated urine, by retarding to some extent the growth of the organism, may in this way act as an adjuvant to drugs used in the therapy of urinary infections. SUMMARY
This study confirms the findings of Davis and Hain that dog's urine is frequently bactericidal towards Bact. coli and only occasionally bactericidal towards Staph. aureus. Dog's urine is much more concentrated than human urine. The former frequently shows a freezing point depression of 3.5°or more, even increasing to 4.5° at times, and this without special attempts to concentrate the urine by dietary measures. Human urine, on the other hand, usually shows a depression of less than 2.0°, and rarely as great as 3.0°. Comparative tests were made in which Staph. aureus and Bact. coli were subjected to the bactericidal action of dog's urine and to that of nutrient broth containing increasing amounts of sodium
ANTISEPTIC PROPERTIES OF DOG'S URINE
513
chloride and urea. The depression of the freezing point was determined for the urine and the broths. Staph. aureus when killed at all by dog's urine, was killed at concentrations even lower than those which fail to kill this organism in salt or urea broth. Hence concentration is not a factor in the bactericidal action towards Staph. aureus. In the case of three out of four strains of Bact. coli studied in this way, the concentration of the dog's urine was in no case sufficient to account for the sterilization observed. One strain of Bact. coli was killed by dog's urine in many cases, at concentrations which also killed when reached in broth by addition of salt of urea. In other instances the same strain was killed by dog's urine at concentrations which failed to kill when reached in broth. It is therefore concluded that at least two factors may contribute to the bactericidal action of dog's urine towards Bact. coli. One of these factors is yet undetermined; the other is, in some cases, mere concentration. In those cases where concentration alone is sufficient to account for sterilization, the other unknown factor may also be active at the same time. Sterile salt and sterile urea were added to human urine so as to bring it up to the concentrations practically attainable. In no case did this urine become sterile when inoculated with Bact. coli and incubated for twenty-four hours. In some cases there was, however, marked retardation of growth as compared with growth in the original urine similarly inoculated. This finding suggests the possibility that concentrated urine may aid the action of drugs used in urinary infections. REFERENCES (1) (2) (3) (4) (5) (6) (7)
DAVIS AND HAIN : Journ. Urol., 1918, ii, 309. HAIN: Ibid, 1920, iv, 177. FISCHER, A.: Vorlesungen uber Bakteriologie, 2 t e. Aufl., 1903. GuILLEMARD: Comptes rend. de la Societe de Biologie, 1909, !xvii, 538. HEWLETT, GILBERT AND WICKETT: Arch. Int. Med., 1916, xviii, 636. SCHILL AND KuNzE : Wiener Arch. fur innere Medizin, 1925, x, 329. ADDIS AND FOSTER : Arch. Int. Med., 1924, xxxiv, 462.
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A STUDY OF CONCENTRATION AS A FACTOR IN THE ANTISEPTIC PROPERTIES OF DOG'S URINE EDWIN C. WHITE From the Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland
The antiseptic properties of dog's urine were first observed in this laboratory by Davis and Hain (1). They summarized their findings as follows: Normal dog's urine, obtained by aseptic catheterization, shows a distinct antiseptic action which is particularly marked against Staphylococcus aureus and entirely inert against at least one strain of Staphylococcus albus. This antiseptic action is not constant for all dogs, nor for the same dogs on all_occasions. Dog's urine, inoculated with Bacillus coli and incubated may become sterile in as short a period as six hours. The antiseptic action bears no relationship to the hydrogen ion concentration, nor is it influenced by the action of various extractives on the urine. Of those specimens of urine which act as suitable culture media, some may be rendered unsuitable by passage through a Berkefeld filter.
In 1920 Hain (2) showed the striking effect of variation of diet and water intake on the antiseptic properties of dog's urine. Byfeeding dogs large amounts of meat and restricting the amount of water allowed the urine became, in all cases, bactericidal toward Bact. coli, although bactericidal towards Staph. aureus only occasionally. When the amount of meat was decreased and the amount of water increased, the incidence of bactericidal effect decreased, and when the animals were fed on a diet of bread and milk the bactericidal effect against both organisms disappeared entirely. Two possible explanations of these results suggest themselves. First, the mere concentration of the urine may be a factor, with501 THE JOURNAL OF UROLOGY, VOL, XXII, N O,
5
502
EDWIN C. WHITE
out regard to the particular substances responsible for the concentration. High protein diet results in the elimination of large amounts of nitrogenous end products of metabolism, principally urea, in the urine. Secondly, the bactericidal properties may be due to some bactericidal substance or substances resulting from the metabolism of food high in protein. Although these substances may themselves be present only in small amounts, their concentration may parallel the total concentration until they reach a point at which they can exert bactericidal action. The present paper records the results of the investigation of the first of these possibilities. It should be stated that Davis and Hain (loc. cit.) suggested the possibility of the relationship between bactericidal property and specific gravity, but did not investigate it. METHOD
Dogs were placed on a diet consisting exclusively of liver. Each animal received about 1½ pounds per day, without reference to body weight. Water was withheld at night and not given until after catheterization next morning; otherwise, the dogs were allowed as much water as they would take. In this way less protein and more water was allowed than in Rain's experiments, as it was desired to obtain for purposes of comparison some samples that showed bactericidal effect and some that did not. The animals were catheterized aseptically as described by Davis and Hain. As dog's urine is often very cloudy, sterility controls were made by transferring 0.1 cc. to nutrient broth and examining for growth after proper incubation. To 1 cc. of urine was added a loopful of a twenty-four-hour broth culture of the organism. After twenty-four hours' incubation 0.1 cc. was transferred to 5 cc. of nutrient broth, which in turn was incubated and examined for growth after twenty-four and forty-eight hours. The freezing point of each sample was determined by means of a Beckmann thermometer in the usual way. The depression thus noted is a more rational measure of total concentration
ANTISEPTIC PROPERTIES OF DOG'S URINE
503
than is specific gravity, for it measures the osmotic pressure of the solution. The studies of A. Fischer (3) show that the bacterial cell consists of an outer supporting wall against which presses an inner membrane of protoplasm, the interior of the cell being filled with a solution. When the bacterium is placed in a solution hypertonic with reference to the cell contents, one and perhaps two processes will come into play. First, water will pass out from the cell to the stronger solution and as the interior solution thus decreases in volume the membrane will shrink away from the supporting wall. This shrinking process is called plasmolysis. If it proceeds far enough before osmotic equilibrium is reached between the solutions on both sides of the membrane, the ability of the cell to reproduce is interferred with and the organism may even be killed. The membrane of protoplasm is not ideally semi-permeable, but is permeable to some dissolved substances as well as to water. If the bacterium is placed in the hypertonic solution of a substance to which its membrane is permeable, water will pass out and simultaneously the dissolved substance will pass into the weaker solution within the cell. As the second of these processes goes on the osmotic pressure of the solution within the cell increases and osmotic equilibrium may be established before enough water has been lost to cause plasmolysis. Indeed, the fact that a bacterium cannot be plasmolyzed by solutions of a particular substance is taken by Fischer as evidence that its membrane is permeable to that substance. In the case of such diffusible substances the bacterial processes may be disturbed because of the high concentration reached in the interior of the cells. It may even cause the death of the organism by precipitating or "salting out" substances necessary for life. Guillemard (4) determined the highest concentration of certain salts permitting bacterial growth in bouillon. For Bact. coli this was 1.1 molar, for staphylococci 3.5 molar, when sodium chloride was used. The findings of this author, together with the high resistance of staphylococci to the bactericidal action of dog's urine, as compared to the resistance of Bact. coli, provided a
504
E DWI N C. WHI TE
logical basis for investigating the possible relation between osmotic pressure, as measured by depression of freezing point , and bactericidal action in dog's urine. T his relation is shown in TABLE 1
Relation between depression of freezing point, in degrees Centigrade, and bactericidal action of dog's urine BACT. COLI ( STRAIN 1)
Depressions at which organism was killed
3.95 3.71 4.57 3.67 4.32 3.67 3.02 3.35 3.07 3.43 3.54 3.15 2.97 2.92 4.03 3.04 2.69 3.57
STAPH. AUREUS
D epressions at which organism survived
3.07 3.58 3.18 1.33 0.75 2.42 2.27 1.89 2.25 2.39 2.13 1.95 2.33 1.98 2.02 2.44 3.22 2.45
Depressions at which organism was killed
3.54 3.15 2.97 2.92 4.03 2.69 2. 45
Depressions at which organism survived
3.95 3.71 4.57 3.67 3.58 3.18 1.33 0.75 2.42 3.02 2.27 1.89 3.35 3.07 2.25 2.39 3.43 2.13 1.95 2.33 1.98 2.02 2.44 3.22 3.04 3.57
table 1. The organisms were all isolated from cases of urinary infection. Inspection of this table brings out several significant facts. The relative infrequency with which Staph. aureus was killed is striking and confirms the findings of Hain and Davis. There is no discernible relation between depression of freezing point and
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ANTISEPTIC PROPERTIES OF DOG'S URINE
505
bactericidal action against this organism. The greatest depression noted, 4.57°, as well as the smallest, both failed to kill it. At points in between these values, the organism sometimes was killed and sometimes survived. Thus, at 3.15°, it was killed in one case, while in another sample, at 3.18°, practically the same value, it grew. Again at 3.54°, it was killed once, but survived in another sample at 3.57°. These two values are practically identical. There is no minimum above which the organism was always killed. It is clear that as far as this particular strain of Staphylococcus is concerned, osmotic pressure bears no relation to bactericidal action, the explanation of which must be sought in some other agency. The facts with reference to the strain of Bact. coli used are different. It is seen at a glance that in general those urine samples with the greatest depressions of freezing point killed the organism while those with the smaller depressions failed to kill. The urine with the greatest depression observed, 4.57°, killed and that with the smallest depression, 0.75°, failed to kill. There is one point in between these extremes, 3.07°, at which the organism was killed once and survived once. The smallest depression at which killing occurred was 2.69°, the great depression permitting survival was 3.58°. Of eighteen samples that killed fifteen showed a depression of 3.0° or more and seventeen showed a depression greater than 2.9°. Of eighteen samples that failed to kill, fourteen showed a depression less than 3.0°, sixteen showed a depression less than 3.2°, and seventeen a depression less than 3.5°. Stated in another way, all samples with a depression greater than 3.58° killed this strain of Bact. coli and with one exception (3.58° once failed) all samples with a depression of 3.3° or more also killed all organisms. These facts focus attention upon the critical range of osmotic pressure corresponding to depressions of 3.0° to 3.5°. It was therefore of interest to see what effect could be produced on this strain of Bact. coli by solutions, originally favorable culture media, whose osmotic pressure was gradually increased through the range met with in dog's urine, by addition of varying amounts of urea and sodium chloride. These two substances were chosen
506
EDWIN C. WHITE
because they are the chief constituents in point of percentage in urine and to them and especially to urea is due the greatest part of the total osmotic pressure. Moreover, a large amount of urea is present in the urine of animals on high protein diet. To prepare the salt solution it was only necessary to add the desired amounts of salt to ordinary infusion broth and sterilize by autoclaving. This method, of course, could not be used for urea solutions because urea is hydrolyzed when its solutions are heated above 100°. Consequently the desired amounts of urea were weighed into sterile tubes, heated to 100° for one hour and added to the broth. Fortunately, these urea broths were sterile and TABLE 2
Effect of addition of sodium chloride to infusion broth, pH 6.0, on bactericidal action DEPRESSION OF FREEZING POINT
2.37 2.61 2.96 3.20 3.50 3.62 3.77 4.01
BACT . COLI (STRAIN 1)
STAPH . AUREUB
+ + + +
+ + + + + + + +
0 0 0 0
+ indicates survival; 0 indicates sterilization. remained so for several months, but in subsequent attempts this method always failed to sterilize the urea, as shown by bacterial growth in the broth. Another series of sterile urea broths were later prepared in the following manner. Fifty per cent aqueous solution of urea was sterilized by Berkefeld filtration and varying amounts of this solution were added to a beef extract, peptone, salt, sodium-phosphate solution, containing such amounts of these four constituents as would make their final concentration 0.3, 0.5, 1.0, and 0.1 per cent, respectively. Both the infusion broth and extract broth were titrated to pH 6.0, which represents about the average reaction of dog's urine examined in this study, the highest being 6.4 and the lowest 5.4°. With these solutions prepared and their freezing points deter-
507
ANTISEPTIC PROPERTIES OF DOG'S URINE
mined, their effect on bacteria was studied by precisely the same method as was used in the case of dog's urine, viz. inoculation of 1 cc. portions with a loopful of culture, transfer of 0.1 cc. to broth after twenty-four hours' incubation, and observation after twenty-four and forty-eight hours. The results are shown in table 2. The smallest depression at which Bact. coli was killed was somewhere between 3.2° and 3.5°, corresponding to 5 to 6 per cent of salt. This is very striking for it represents just about the zone above which dog's urine, with one exception, never failed to kill. TABLE 3
Effect of addition of sodium chloride to infusion broth, pH 6.0, on bactericidal action APPROXIMATE SALT
ADDED
DEPRESSION OF FREEZING POINT
BACT. COLI (STRAIN 1)
STAPH. AUREUS
3. 19 3.35 3.49 3.60 3.78 3.84
+
+ + + + + +
per cent
5.0 5.2 5.4 5.6 5.8 6.0
+ indicates
0 0 0 0 0
survival; 0 indicates sterilization.
Staph. aureus, on the other hand, was not killed by the solution at any depression within the range usually encountered in dog's urine. Table 3 shows the results with a new salt broth series in which the intervals were made smaller. This series shows that killing of Bact. coli begins at a depression of about 3.3°. As in table 2, Staph. aureus was not killed by any member of the series. As the observations recorded in table 3 were made two weeks later than those in table 2, the two experiments serve as definite checks on each other and show that the results were not due to any fortuitous circumstance at the time of the first experiment. Table 4 shows the effect of adding urea to infusion broth. The results are practically identical with those in the salt series.
508
EDWIN C. WHITE
Staph. aureus was not killed by any member of the series, but Bact. coli was killed at a depression of about 3.3°. This represents the addition of approximately 10 per cent of urea. Tests with urea, using precisely the same solutions as in the first experiments, were repeated one month later and five weeks later. During this interval the strain of Ba~t. coli had become somewhat more resistant to concentration effects for in both these later tests the organisms survived a depression of 3.53°, but were killed at 3.63°. From these results it is clear that the concentration reached in dog's urine is not of itself sufficiently high, by virtue either of TABLE 4
Effect of addition of urea to infusion broth, pH 6.0, on bactericidal acti on APPROXIMATE UREA ADDED
DEPRESSION OF FREEZING POINT
BACT. COLI (STRAIN 1)
STAPH. AUREUS
+ + + +
+ + + + + + + +
per cent
8 .5 9.0 9 .5 10.0 10.5 11.0 11 .5 12.0
2.75 2.94 3.11 3.24 3.34 3.53 3.63 3.77
0 0 0 0
+ indicates survival; 0 indicates sterilization. sodium chloride or urea, to kill Staph. aureus. When this organism is killed by dog's urine, as it occasionally is, some other agency must be at work. As regards the particular strain of Bact. coli used, the concentration reached in dog's urine is in many cases of itself sufficiently high to account for the sterilization. In other samples which killed Bact. coli, the concentration is definitely not high enough to cause sterilization and here too some other factor must be the cause. It is possible that in the most concentrated samples both the unknown factor and the osmotic effect are at work simultaneously, although in those cases it is not necessary to postulate anything beyond mere concentration to explain these findings.
----------
--
509
ANTISEPTIC PROPERTIES OF DOG'S URINE
Some weeks later another series of tests were carried out with this same strain of Bact. coli and three other strains, all isolated from urinary infections. The four strains were subjected to the TABLE 5
Second series showing relation between depression of freezing point of dog's urine and bactericidal action BACT. COLI
DEPRESION OF FREEZING POINT
Strain 1
Strain 2
Strain 3
Str ain 4
+ + + +
+ + + +
0 0 0 0
0 0 0 0
+ + + + +
+ + + + + +
1.57 2.27 2.57 3.20 3.50 3.86 3.86 4.29
0 0 0
0 0
+ indicates survival; 0 indicates sterilization. TABLE 6
Effect of addition of urea to beef extract broth, pH 6.0, on bactericidal action APPROXIMATE UREA ADDED
BACT. COLI
DEPRESSION OF FREEZING POINT
Strain 1
Strain 2
Strain 3
Strain 4
+ + +* +* +* +* +*
+ + + +* +* +* +* +*
+ + +* +* +* +* +*
per cent
5 6 7 8 9 10 11
12
2.27 2.57 2.88 3.22 3.55 3.87 4.16 4.47
+ + +* +* +* 0 0 0
0
0
+
indicates growth in broth. 0 indicat es sterilization. +* indicates inhibition of growth in broth, but growth in transfer.
action of eight samples of dog's urine and also to the action of urea broth as before. The results in dog's urine are shown in table 5, and the behavior of the organisms tested at the same time in beef extract broth containing urea is shown in table 6.
510
EDWIN C. WHITE
Table 5 shows a striking parallelism between the depression of freezing point and bactericidal action. The four least concentrated samples killed none of the four strains, the three next most concentrated killed two, three and four strains, respectively, and the most concentrated sample killed all four. But while all four strains were killed in dog's urine at a depression of 3.86°, table 6 shows that the last three strains were not killed rin urea-salt broth1 when this depression was considerably exceeded, viz. at 4.16°. Strain 3 was not killed in broth even at 4.47°. As far as these last three strains are concerned it may be definitely concluded that when they are killed by dog's urine the factor responsible is not mere concentration. Strain 1, however, was killed by urea-salt broth at this time at a depression somewhere betwen 3.55° and 3.87°; hence, in the three samples of dog's urine showing depression of 3.86°, 3.86° and 4.29° the concentration alone is sufficient to account for the sterilization, although there may also have been present in these samples the other, at present unknown factor. APPLICATION OF THESE RESULTS TO HUMAN URINE
Hewlett, Gilbert and Wickett (5) administered urea to men in amounts sufficient to raise the blood urea to levels found in uremia. The doses averaged about 100 grams, being given either at once or divided over intervals. As the subjects were made very ill by these amounts they certainly represent the maximum amounts that could be considered with the idea of concentrating the urine so as to secure a bactericidal effect. The highest total urea concentration found in the urine after ingestion of these large doses was 6.4 per cent. Schill and Kunze (6) injected 10 to 15 cc. of "100 per cent" urea solution intravenously in humans. (This is presumably 1: 1 solution, which is approximately saturated.) T he total nitrogen in the urine reached a maximum of 2.62 per cent, in 1 case. If all this was present as urea the concentration would have been only about 5.2 1 This broth contained a constant amount of sodium chloride, 1 per cent, in addition to the increasing amounts of urea.
511
ANTISEPTIC PROPERTIES OF DOG'S URINE
per cent. Addis and Foster (7) found that administration of 30 grams of urea by mouth led to a maximum urea concentration in urine of 4.85 per cent, while administration of 10 grams of sodium chloride under similar conditions caused the salt concentration in the urine to reach a maximum of 2.25 per cent. The last figure is osmotically equivalent to no more than 4.5 per cent of urea, even assuming complete dissociation of the salt. TABLE 7
Ejfect of addition of urea to human urine on antiseptic action
URINE SAMPLE
MA E< M -< A :e! A
;;i-.
pH
C-< o, M
NUMBER
~p "'"
-- --
1
5 2
--
5.4
~
OM 0
BACT. COLI
z"' 0 0
MZ M
aJ
"'" "'"'
o, M M ._ A
"'"
Strain 1
Strain 2
Strain 3
Strain 4
--
per cent
ll
0 1 2 3 4 5
1.82 2 .07 2.32 2.62 2.92 3.19
0
1.67 1.95 2.27 2.57 2.85 3. 12
1
2
.
2 3 4 5
+ + +
sl sl sl
+ + + +
sl
0
+ + + + + + + + + + + +
+ + +
sl sl sl
+ + +
sl sl
0
+ + + + + + + + + + + +
+ + + + +
sl
+ + + +
sl
0
+ + + + + + + + + + + +
+ +
sl sl sl sl
+ + +
sl sl
0
+ + + + + + + + + + + +
+
= growth as heavy as control. sl = markedly lighter growth than control. 0 = no apparent growth.
In view of these findings a few experiments have been included in the present study in which sterile solid urea was added to sterile human urine in increasing amounts so as to bring the total up to the amounts observed by Hewlett, Gilbert and Wickett. The sterile solid urea was obtained by adding measured amounts of sterile 50 per cent urea solution (Berkefeld filtered) to sterile test tubes and evaporating to dryness at body temperature in a high vacuum. The proper volume of urine was then added.
512
EDWIN C. WHITE
The total urea may be estimated to be at least 1 to 1.5 per cent more than the added urea. Sterility controls were made by adding 1 cc. of urine to 10 cc. of broth and incubating. Controls made by inoculating the urine separately with each of the four . strains showed that the urine without addition of urea was a very favorable culture medium; as evidenced by heavy growth in twenty-four hours. The results are shown in table 7. Under each strain two columns are shown. In the first column is noted the appearance of the inoculated urine, after twenty-four hours' incubation, as compared with the control simultaneously inoculated but without addition of urea. In the second column is shown the result of transfer of 0.1 cc. of inoculated urine, after standing twenty-four hours in the incubator, to 10 cc. of broth, and reading the broth tubes after forty-eight hours. It is seen from this table that although sterilization was not effected in any case, even though the urine was made very concentrated, yet in many cases there was definite retardation of growth at concentrations perhaps attainable in human urine. The idea suggests itself that concentrated urine, by retarding to some extent the growth of the organism, may in this way act as an adjuvant to drugs used in the therapy of urinary infections. SUMMARY
This study confirms the findings of Davis and Hain that dog's urine is frequently bactericidal towards Bact. coli and only occasionally bactericidal towards Staph. aureus. Dog's urine is much more concentrated than human urine. The former frequently shows a freezing point depression of 3.5°or more, even increasing to 4.5° at times, and this without special attempts to concentrate the urine by dietary measures. Human urine, on the other hand, usually shows a depression of less than 2.0°, and rarely as great as 3.0°. Comparative tests were made in which Staph. aureus and Bact. coli were subjected to the bactericidal action of dog's urine and to that of nutrient broth containing increasing amounts of sodium
ANTISEPTIC PROPERTIES OF DOG'S URINE
513
chloride and urea. The depression of the freezing point was determined for the urine and the broths. Staph. aureus when killed at all by dog's urine, was killed at concentrations even lower than those which fail to kill this organism in salt or urea broth. Hence concentration is not a factor in the bactericidal action towards Staph. aureus. In the case of three out of four strains of Bact. coli studied in this way, the concentration of the dog's urine was in no case sufficient to account for the sterilization observed. One strain of Bact. coli was killed by dog's urine in many cases, at concentrations which also killed when reached in broth by addition of salt of urea. In other instances the same strain was killed by dog's urine at concentrations which failed to kill when reached in broth. It is therefore concluded that at least two factors may contribute to the bactericidal action of dog's urine towards Bact. coli. One of these factors is yet undetermined; the other is, in some cases, mere concentration. In those cases where concentration alone is sufficient to account for sterilization, the other unknown factor may also be active at the same time. Sterile salt and sterile urea were added to human urine so as to bring it up to the concentrations practically attainable. In no case did this urine become sterile when inoculated with Bact. coli and incubated for twenty-four hours. In some cases there was, however, marked retardation of growth as compared with growth in the original urine similarly inoculated. This finding suggests the possibility that concentrated urine may aid the action of drugs used in urinary infections. REFERENCES (1) (2) (3) (4) (5) (6) (7)
DAVIS AND HAIN : Journ. Urol., 1918, ii, 309. HAIN: Ibid, 1920, iv, 177. FISCHER, A.: Vorlesungen uber Bakteriologie, 2 t e. Aufl., 1903. GuILLEMARD: Comptes rend. de la Societe de Biologie, 1909, !xvii, 538. HEWLETT, GILBERT AND WICKETT: Arch. Int. Med., 1916, xviii, 636. SCHILL AND KuNzE : Wiener Arch. fur innere Medizin, 1925, x, 329. ADDIS AND FOSTER : Arch. Int. Med., 1924, xxxiv, 462.