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The Pathogenicity of Cocci Isolated from the Urine1
THE PATHOGENICITY OF COCCI ISOLATED FROM THE URINE 1 MOE GOLDSTEIN Fellow in Pediatrics, The Mayo Foundation, Rochester, Minnesota
Staphylococcus aureus, Micrococcus (species not known), and Streptococcus faecalis are 3 gram-positive cocci which frequently are isolated from the urine (Cabot; Kretschmer; Young, Colston and Hill). It is a fundamental fact that the kidney will not excrete bacteria into the urine unless the renal parenchyma has been injured (Helmholz and Field) . Staphylococcus aureus is known to cause renal lesions almost invariably when it is injected intravenously. No experimental work has been done with Micrococcus or Streptococcus faecalis in order to show whether either of these organisms will injure the kidney when they are injected intravenously. It is not known, therefore, whether the presence of Micrococcus and Streptococcus faecalis in the vesical urine indicates that they are being excreted by the kidney or that they, as inhabitants of the lower part of the urinary tract, are not associated with an infection- of the upper part of the urinary tract. Since coccal infections of the urinary tract are almost always hematogenous in origin, it is important to know the source of the cocci that are found in the urine. Staphylococcus and Micrococcus are both mass-forming, gram-positive cocci; therefore, their differentiation may at times be difficult. Staphylococcus aureus is known to be pathogenic. If the Micrococcus is nonpathogenic, it would be of definite clinical importance to distinguish between these two by bacteriologic examination of the urine . This study was undertaken to determine the pathogenicity of Streptococcus faecalis and Micrococcus isolated from the uriqe. BACTERIOLOGY
Micrococci and staphylococci.- Micrococcus and Staphylococcus were isolated from the urine and identified chiefly by the morphologic characteristics which have been described by Luther Thompson. He furnished the cultures of these organisms for this study. Thompson studied 150 cultures of gram-positive, mass-forming cocci and found that mor1 Abridgment of thesis submitted to the Faculty of the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of M. S. in Pediatrics.
237
238
MOE GOLDSTEIN
phologically they tended to fall into three groups: Group 1 includes genus Micrococcus; group 2 includes a Staphylococcus which resembles Staphylococcus epidermidis but is not pathogenic, and group 3 includes Staphylococcus albus and Staphylococcus aureus. This group he would call Staphylococcus pyogenes. The organisms of group 1 were easily recognized by their greater size. The cocci appear in clusters and tetrads. The organisms of group 2 were larger, less regularly round, and varied more in size than those of group 3. The most helpful character in the identification of the organisms of this group was their tendency to form clusters so that one organism lay above another on the slide. The cocci in group 3 were arranged in flat plates, almost all of them in one plane. Within these planes, the cocci were so closely packed that their arrangement resembled a honeycomb. The outlines of individual cocci were distinct, owing to the distribution of the bacteria in one plane. These distinctions apply only to smears made from a 24 hour nutrient agar slant; they do not apply to smears made from broth or from pus. Streptococcus faecalis.-According to Sherman, Streptococcus faecalis is the predominating streptococcus in the human feces and has been known as the most characteristic streptococcus of the human intestine. He found that it also occurs in the feces of horses, cattle and other domestic animals. Streptococcus faecalis was named by Andrewes and Horder in 1906. Sherman classified this organism as an enterococcus, which he considered to be a clearly defined division of the streptococci, probably the most clearly marked subdivision of the whole group. Streptococcus faecalis shows great diversity with respect to fermentation tests but the fermentation of mannitol is considered very characteristic. It has a low minimal and high maximal temperature of growth and is resistant to a number of substances which are, in general, inhibitory to the growth of other streptococci. Andrewes and Horder found this organism in cases of endocarditis, cystitis, and other human infections. Houston and McCloy noted many types of infections in which the enterococcus appeared to be implicated. PROCEDURE
The organisms used in these experiments were the three common gram-positive cocci found in the urinary tract: Staphylococcus aureus, Micrococcus, and Streptococcus faecalis. These bacteria were obtained
PATHOGENICITY OF COCCI FROM URINE
239
in pure culture from the urine of patients and were then subcultured on a slant and kept in a refrigerator. The cultures were frequently transferred to a fresh slant. Twenty-four hours before any culture was to be used for injection, inoculation into a tube of dextrose brain broth medium was performed. All injections into animals were made with this culture. Young male rabbits weighing between 3 and 5 pounds (1.4 to 2.3 kg.) were used. The animal was first catheterized, the specimen of urine being collected in a sterile test tube for microscopic examination and culture. The culture containing the bacteria was then injected into the lateral ear vein of the animal. Forty-eight or 72 hours following injection, another catheterized specimen of urine was obtained in a sterile test tube for microscopic examination · and culture. At necropsy, specimens of urine for culture were obtained from the bladder by searing the surface of the bladder and drawing the urine into a sterile pipet. A complete necropsy was performed on all animals. All the organs were examined grossly for the presence of lesions. The kidneys alone were saved for microscopic examination. The kidneys were fixed in formalin and then sectioned according to the method advocated by Helmholz and Field. RESULTS
Staphylococcus aureus.- An injection of 0.5 cc. of a culture of Staphylococcus aureus was given to each of 11 rabbits. Five animals were killed 72 hours following injection; three of these had lesions of the kidney. Three animals were killed 96 hours following injection; two of these had lesions of the kidney. Two animals were killed 120 hours and one animal was killed 144 hours following injection, all three of these animals had renal lesions. In all, lesions of the kidney were produced in eight, or 73 per cent, of the 11 rabbits. Table 1 shows the type of renal lesions that occurred in the 8 rabbits. In 7 animals, the lesions were bilateral and in 1 animal a unilateral lesion was found. Seven rabbits had lesions in more than one portion of the kidney. In 1 animal, a single abscess, which was situated deep in the medulla, was the only lesion that could be found. Abscesses of the papilla, which were the most common type of lesion, occurred in 7 of the 8 rabbits. Pyelitis, which was the next most common lesion, was found in 6 of the 8 animals. Five animals had medullary abscesses and cortical abscesses were found in 5. Table 2 includes the urinary findings in the 11 animals which received
240
MOE GOLDSTEIN TABLE
1.-Type of renal lesions produced by Staphylococcus aureus TYPE OF LESION
STRAIN OF STAPHYLOCOCCUS AUREUS
Left side
--4
10 12 15 25 26
TABLE
+ + + + + -
-
-
in Animals which lesions were produced ...
Right side
- - - - --
+ + + + +
1 1
Papillary lesion
Pyelitis Right side
Left side --- -
+ + + + + + +
-
+ + + + +
6
Medullary lesion Right side
Left side
--
+ + + + +
Left side
- - - - - -+
+ + + +
5
7
Cortical lesion Right side
---
+ + + +
+ + + +
-
-
-
5
2.-Res-ults of microscopic examination of specimens of urine obtained by catheterization before and after injection of 0.5 cc. of a culture of Staphylococcus a-ure11s
STRAIN OF STAPHYLOCOCCUS
AUREUS
URINE OBTAINED BEFORE INJECTION
I
URINE OBTAINED FORTY-EIGHT HOURS AFTER INJECTION
Urine of eight rabbits in which lesions developed
I
1 1 4 10
12 15 25 26
Not examined Normal Normal Not examined Normal Normal Normal Normal
P us Pus Pus P us Not examined Pus Normal Many red blood cells; culture produced grampositive cocci
Urine of three rabbits in which lesions did not develop 4
10 15
Not examined Normal Normal
Not examined* Normal Normal
* Microscopic examination of vesical urine, which was obtained at necropsy did not disclose any abnormality; culture did not produce any growth.
an injection of a culture of Staphylococcus aureus. No abnormal elements were found in the urine of 2 of the 3 animals in which no lesions were produced. A culture of the urine, which was made at necropsy
PATHOGENICITY OF COCCI FROM URINE
241
also was negative. The urine of one of these 3 rabbits was not obtained prior to necropsy, but the vesical urine which was obtained at necropsy, was normal both on microscopic examination and on culture. In 6 of the 8 animals in which lesions of the kidney were produced, the urine was normal on microscopic examination prior to injection of the Staphylococcus aureus. The urine of two of the animals was not examined prior to the injection. Forty-eight hours following injection, the urine of 5 of the 8 rabbits contained pus. The urine of 1 rabbit did not contain any abnormal elements; the urine of one animal was not examined, and the urine of the remaining animal contained many red blood cells and a culture produced innumerable gram-positive cocci. In 4 of the 8 animals, the urine was examined at necropsy. It was obtained from the bladder by sterile methods. Three of the 4 cultures showed grampositive cocci. One culture did not produce any growth. Micrococcus.- Fourteen animals received injections of a culture of Micrococcus. Nine animals received an injection of 0.5 cc. of the culture; four of these animals were killed 72 hours following the injection, four were killed 96 hours following the injection and one was found dead 96 hours following the injection. Two cubic centimeters of the culture was injected into each of 5 animals; all of these rabbits were killed 124 hours following the injection. No renal lesions were produced in any of the 14 animals. Six strains of Micrococcus were used. Each of 4 strains was injected into 2 separate rabbits, and each of 2 strains was injected into 3 rabbits. Table 3 shows the urinary findings in the 14 animals which received an injection of a culture of Micrococcus. The urine of nine animals was examined before the injections were made. No abnormal elements were found in the urine of any of these animals. The urine of 6 of the 14 rabbits was examined 48 hours following injection and the urine of 8 of the 14 rabbits was examined 72 hours following injection. No abnormal elements were found in any of these specimens. In 11 instances the vesical urine was examined at necropsy. In 6 instances both microscopic examination and culture of the urine failed to disclose any evidence of infection. In 3 instances the culture of the urine gave negative results but microscopic examination was not performed. In the 2 instances in which a culture of the urine was not made, microscopic examination did not disclose any abnormal elements. Streptococcus jaecalis.- A culture of Streptococcus faecalis was in-
242
MOE GOLDSTEIN
jected into 19 rabbits. Each of 9 animals received 0.5 cc. of the culture. Two of these rabbits were killed 72 hours following injection, five, 96 hours following injection, and two, 120 hours after injection. Two cubic centimeters of the culture was injected into each of 8 animals. One of these rabbits was killed 48 hours following injection, one, 96 hours later, and six, 144 hours later. Each of 2 animals received an injection of 3 cc. of the culture. One of these rabbits was killed 48 hours later, and the other one, 96 hours later. Nine strains of Streptococcus faecalis were used. Each of 3 strains was injected into a separate rabbit, TABLE
3.-Results of microscopic examination and culture of urine of fourteen rabbits which received injections of a culture of micrococcus MICROSCOPIC EXAMINATION
STRAIN OF MICROCOCCUS
3 3 7 7 7 11 11 11
14 14 23 23 24 24
Urine obtained by catheterization before injection
Urine obtained by catheterization after injection
necropsy
Not examined Not examined Normal Normal Not examined Normal Normal Not examined Normal Not examined Normal Normal Normal Normal
Normal* Normal* Not examined Not examined Normal* Normal* Normalt Normal* Normal* Not examined Normalt Normalt Normalt Normal*
Not examined Not examined Normal Normal Normal Normal Normal Not examined Not examined Normal Normal Not examined Normal Normal
Urine obtained at
CULTURE OF URINE OBTAINED AT NECROPSY
Not made Not made Negative Negative Negative Negative Not made Negative Negative Negative Negative Not made Not made Negative
* Urine obtained
forty-eight hours after injection. t Urine obtained seventy-two hours after injection.
each of 5 strains was injected into 2 separate rabbits and 1 strain was injected into 6 rabbits. No lesions of the kidney were found in 12 of the 19 animals (63 per cent). Lesions of the kidney were produced in 7 of the 19 rabbits (37 per cent). Lesions were produced in one (11 per cent) of the 9 animals that received an injection of 0.5 cc. of the culture, in 4 (50 per cent) of the 8 animals that received an injection of 2 cc. and in both animals (100 per cent) that received an injection of 3 cc. of the culture. Lesions were caused by 3 different strains of Streptococcus faecalis. One strain produced lesions in 5 of the 6 animals into which it was injected, 1 strain
243
P ATHOGENICITY OF COCCI FROM URINE
produced lesions in 1 of the 2 animals into which it was injected and 1 strain produced lesions in the 1 animal into which it had been injected. Table 4 summarizes the types of renal lesions that occurred in 7 rabbits. In 5 animals, the lesions were bilateral; in two, the lesion was unilateral. Three rabbits had lesions in more than one portion of the kidney. In 3 animals, the pathologic findings were confined to the pelvis of the kidney, and in 1 animal they were confined to the cortex of the kidney. Pyelitis which was the most common lesion, occurred in 5 of the 7 rabbits. Abscess of the medulla, which was the next most common lesion, was found in 3 of the 7 animals. Papillary abscesses occurred in 2 animals, and cortical abscess, in one. TABLE
4.-Type of renal lesions produced by Streptococcus f aecalis TYPE OF LESION
STRAIN OF
Pyelitis
STREPTOCOCCUS
Papillary lesion
Medullary lesion
Cortical lesion
FAECALIS
Right side
13 17 21 21 21 21 21 Animals in which lesions were produced ...
Left side
Right side
Left side
Right side
Left side
--- --- --- ---
-
+ + + +
+ + + +
-
5
-
-
-
-
+
+ +
+
-
+
+ +
-
2
-
-
-
3
Left side ---
---
-
Right side
-
-
-
-
-
+ -
-
1
In table 5 are summarized the urinary findings in the 7 animals in which lesions were produced and in the 12 rabbits in which no lesions were produced. No abnormal elements were found in the urine of 5 of the 7 rabbits examined before injection of Streptococcus faecalis. The urine of 2 rabbits was not obtained. Forty-eight hours following injection, the urine of 1 rabbit did not contain any abnormal elements, the urine of 5 rabbits contained pus, and the urine of 1 rabbit contained gross blood, probably owing to the trauma caused by catheterization. Three of the 48-hour specimens represented urine obtained from the bladder at necropsy; two of these contained pus but one was normal. In the 12 rabbits in which no lesion was produced, no abnormal elements
244
MOE GOLDSTEIN
were found in the urine prior to the injection; 48 hours after the injection the urine of 8 rabbits was examined and was found to be normal. At necropsy, the vesical urine of 6 rabbits was normal both on microscopic examination and on culture. The urine of one rabbit was normal on TABLE
5.- Results of microscopic examination and culture of urine of rabbits which received injections of a culture of Streptococcus f aecalis MICROSCOPIC EXAMINATION
STRAIN OF STREPTO-
coccus
FAECALIS
Urine obtai!'ed by cathete:i~ab
I Urine obtained by catbeterization I forty-eight hours after injection
Urine obtained from bladder at
CULTURE OF URINE OBTAINED AT NECROPSY
necropsy
Urine of seven rabbits in which lesions developed
13 17 21 21 21 21 21
Not examined Not examined Normal Normal Normal Normal Normal
Pus Normal Pus Gross blood Pus* Pust Normal; culture produced gram-positive cocci
Urine of twelve rabbits in which lesions did not develop
2 2 6 6
13
Normal Normal Normal Normal Normal Normal Normal Normal Normal
Normal Not examined Not examined Normal Not examined Normal Normal Normal Not examined
16
Normal
Normal
20 21
Normal Normal
Normal Normal
8 8 9 9
Not examined Normal Normal Normal Normal Normal Not examined Normal Many white blood cells Many white blood cells Not examined Normal
Not made Negative Negative Negative Negative Negative Negative Negative Not made Not made Not made Not made
* Urine obtained at necropsy, forty-eight hours after injection. t Urine obtained at necropsy, ninety-six hours after injection.
microscopic examination; culture of the urine was not made; the urine of another rabbit showed no growth on culture; microscopic examination was not made. There was no urine in the bladder of the 2 rabbits. The urine of 2 rabbits contained pus but a culture of the urine was not made.
PATHOGENICITY OF COCCI FROM URINE
245
DESCRIPTION OF PATHOLOGIC FINDINGS
Typical focal interstitial suppurative nephritis was produced by the Staphylococcus aureus. The lesions contained many polymorphonuclear cells and the blood vessels were dilated at the periphery of the abscesses. In some places, the blood vessels had ruptured and extravasation of blood had occurred. In many regions, the abscesses could
FIG. L Lesions produced by injection of Staphylococcus aureus; necrotic lesion in renal cortex; abscess in medulla and pus in pelvis (X7).
be seen breaking through the wall of the tubules and the lumina of many tubules were filled with pus, cellular debris, and red blood cells. Certain abscesses were wedge-shaped, the base of the wedge lying in the cortex and the apex extending into the medulla (fig. 1). In the center of these wedge-shaped abscesses were pale necrotic tubules which were surrounded by a zone of marked polymorphonuclear infiltration. Coccal thrombi
246
MOE GOLDSTEIN
FIG. 2. Large amount of pus in renal pelvis following injection of Staphylococcus aureus (X9).
FIG. 3. Pyelitis produced by injection of Streptococcus faecalis (X85)
could be seen lying within the lumina of some of the collecting tubules in the papilla of the kidney.
PATHOGENICITY O:F COCCI FROM URINE
247
In many of the kidneys, the pelves were filled with pus and the subepithelial tissues were infiltrated with polymorphonuclear leukocytes and lymphocytes (fig. 2). The renal lesions produced by the Streptococcus faecalis were far less severe than were those produced by the Staphylococcus aureus. In a few kidneys, pyelitis was the only lesion present (fig. 3). In other kidneys focal interstitial suppuration was present in the medulla and papilla (fig. 4). These were typical abscesses with dilated capillaries at the periphery of the lesions. The abscesses could be seen breaking through the lumina of the tubules and filling them with pus. In only 1
FIG. 4. Abscess of renal papilla produced by injection of Streptococcus faecalis (X30).
kidney was there an abscess of the cortex. This lesion, however, was of such chronicity that it was questionable whether it had been produced by the injection of the culture of Streptococcus faecalis. COMMENT
The results of the experiments with Staphylococcus aureus bear out the conclusions of previous investigators regarding the pathogenicity of this organism. Lesions of the kidney were produced in 8 (73 per cent) of 11 animals which received an injection of 0.5 cc. of a culture of Staphylococcus aureus. Helmholz and Field have produced lesions of the kidney
248
MOE GOLDSTEIN
in 100 per cent of animals, but they injected 2 cc. of a 24-hour culture of Staphylococcus aureus. It is of interest that abscesses were more common in the papilla of the kidney than they were in the cortex. Abscesses of the cortex are usually mentioned as the most frequent type of lesion in staphylococcic infections of the kidney. The lesions produced by Staphylococcus aureus were far more intense than those produced by Streptococcus faecalis. The pathologic findings in 1 rabbit (number 792) deserve some emphasis. This animal was killed 120 hours after injection of 0.5 cc._of
FIG.
5. Solitary abscess of renal medulla produced by injection of Staphylococcus aureus
(X4½).
a culture of Staphylococcus aureus. Microscopic examination of the kidneys disclosed a single abscess, deep in the medulla of one kidney (figs. 5 and 6). No other lesions were found in several sections which were studied. The abscess that was found might easily have been overlooked if many gross sections of the kidney had not been made. One must therefore stress the importance of examining many sections of the kidney before coming to any conclusion as to the presence or absence of pathologic lesions. In contrast to the constancy with which lesions are produced in the
PATHOGENICITY OF COCCI FROM URINE
249
kidney after intravenous injection of Staphylococcus aureus is the complete absence of renal lesions after intravenous injection of Micrococcus. Although the original amount of the culture of Micrococcus that was injected into 14 rabbits was increased fourfold as compared to the Staphylococcus aureus, necropsy did not disclose any lesions and examination of the urine did not reveal pus or organisms. The striking difference in the two groups of gram-positive, massforming cocci, when injected into the blood stream of rabbits, is of definite clinical importance. It suggests that if the Micrococcus, which is found
FrG. 6. Solitary abscess of renal meduJ!a; same section as shown in figure 5 (X40)
normally in the urethra, is also present in the vesical urine it does not necessarily indicate the presence of infection of the upper part of the urinary tract; whereas the presence of Staphylococcus aureus in the urine usually indicates infection of the upper part of the urinary tract. In the presence of mass-forming, gram-positive cocci in the urine, it is, therefore, of utmost importance to know whether one is dealing with Micrococcus or Staphylococcus aureus. The mere finding of grampositive cocci in the stained smear of the urine will not distinguish between these two organisms. Cultures of the urine should be made, whenever possible, and stained smears should be made from 24-hour
250
MOE GOLDSTEIN
slant cultures. In this way it is possible to identify these organisms by the technique advocated by Thompson. Renal lesions were produced in 7 of 19 rabbits (37 per cent) which received injections of a culture of Streptococcus faecalis. A larger dose of Streptococcus faecalis than of Staphylococcus aureus was required to produce renal lesions. The renal lesions produced by Streptococcus faecalis were far less severe than the lesions produced by Staphylococcus aureus. The presence of Streptococcus faecalis in cases of pyuria may therefore be considered of etiologic importance. CONCLUSIONS
The almost constant presence of renal lesions after intravenous injection of the Staphylococcus aureus as compared with their complete absence after injections of Micrococcus seems to indicate that the finding of Staphylococcus aureus in the urine is of great clinical significance while the finding of Micrococcus is of little, if any, significance. When mass-forming, gram-positive cocci are found in the urine, it is of clinical importance to determine their nature. The frequency of renal lesions after intravenous injection of Streptococcus faecalis seems to indicate that its presence in the urine is of pathologic significance. REFERENCES ANDREWES, F. W., AND HoRDER, T. F.: A study of the streptococci pathogenic for man. Lancet. 2: 708-713, 775-782, 852-855, 1906. CABOT, HUGH: Infections of the kidney. In: Cabot, Hugh: Modern urology. Ed. 3, Philadelphia, Lea and Febiger, Vol. 2, Chapt. 12, 1936, pp. 510-569. HELMHOLZ, H. F., AND FIELD, RUTHS.: The kidney: a filter for bacteria. IL The effect of diuresis on the excretion of bacteria by the kidney. Am. Jour. Dis. Child. 29: 506-512, 1925. HELMHOLZ, H. F., AND FIELD, RUTH S.: The kidney: a filter for bacteria. IV. The effect of clamping the renal artery or renal vein on the passage of bacteria into the urine. Am. Jour. Dis. Child. 29: 645-649, 1925. HOUSTON, THOMAS, AND McCLOY, J. M.: The relation of the enterococcus to "trench fever" and allied conditions. Lancet. 2: 632-637, 1916. KRETSCHMER, H. L.: The treatment of pyelitis. Surg., Gynec., and Obst. 33: 632-641, 1921. SHERMAN, J.M.: The streptococci. Bact. Rev.1: 3-97, 1937. THOMPSON, LUTHER: Morphologic and cultural study of staphylococci with special reference to source. Am. Jour. Clin. Path. 2: 125-134, 1932. YOUNG, H. H., COLSTON, J. A. C., AND HILL, J. H.: Infections in the genito-urinary tract, and complications: further advances in the treatment. Jour. Am. Med. Assn. 98: 715-722 (Feb. 27) 1932.
Staphylococcus aureus, Micrococcus (species not known), and Streptococcus faecalis are 3 gram-positive cocci which frequently are isolated from the urine (Cabot; Kretschmer; Young, Colston and Hill). It is a fundamental fact that the kidney will not excrete bacteria into the urine unless the renal parenchyma has been injured (Helmholz and Field) . Staphylococcus aureus is known to cause renal lesions almost invariably when it is injected intravenously. No experimental work has been done with Micrococcus or Streptococcus faecalis in order to show whether either of these organisms will injure the kidney when they are injected intravenously. It is not known, therefore, whether the presence of Micrococcus and Streptococcus faecalis in the vesical urine indicates that they are being excreted by the kidney or that they, as inhabitants of the lower part of the urinary tract, are not associated with an infection- of the upper part of the urinary tract. Since coccal infections of the urinary tract are almost always hematogenous in origin, it is important to know the source of the cocci that are found in the urine. Staphylococcus and Micrococcus are both mass-forming, gram-positive cocci; therefore, their differentiation may at times be difficult. Staphylococcus aureus is known to be pathogenic. If the Micrococcus is nonpathogenic, it would be of definite clinical importance to distinguish between these two by bacteriologic examination of the urine . This study was undertaken to determine the pathogenicity of Streptococcus faecalis and Micrococcus isolated from the uriqe. BACTERIOLOGY
Micrococci and staphylococci.- Micrococcus and Staphylococcus were isolated from the urine and identified chiefly by the morphologic characteristics which have been described by Luther Thompson. He furnished the cultures of these organisms for this study. Thompson studied 150 cultures of gram-positive, mass-forming cocci and found that mor1 Abridgment of thesis submitted to the Faculty of the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of M. S. in Pediatrics.
237
238
MOE GOLDSTEIN
phologically they tended to fall into three groups: Group 1 includes genus Micrococcus; group 2 includes a Staphylococcus which resembles Staphylococcus epidermidis but is not pathogenic, and group 3 includes Staphylococcus albus and Staphylococcus aureus. This group he would call Staphylococcus pyogenes. The organisms of group 1 were easily recognized by their greater size. The cocci appear in clusters and tetrads. The organisms of group 2 were larger, less regularly round, and varied more in size than those of group 3. The most helpful character in the identification of the organisms of this group was their tendency to form clusters so that one organism lay above another on the slide. The cocci in group 3 were arranged in flat plates, almost all of them in one plane. Within these planes, the cocci were so closely packed that their arrangement resembled a honeycomb. The outlines of individual cocci were distinct, owing to the distribution of the bacteria in one plane. These distinctions apply only to smears made from a 24 hour nutrient agar slant; they do not apply to smears made from broth or from pus. Streptococcus faecalis.-According to Sherman, Streptococcus faecalis is the predominating streptococcus in the human feces and has been known as the most characteristic streptococcus of the human intestine. He found that it also occurs in the feces of horses, cattle and other domestic animals. Streptococcus faecalis was named by Andrewes and Horder in 1906. Sherman classified this organism as an enterococcus, which he considered to be a clearly defined division of the streptococci, probably the most clearly marked subdivision of the whole group. Streptococcus faecalis shows great diversity with respect to fermentation tests but the fermentation of mannitol is considered very characteristic. It has a low minimal and high maximal temperature of growth and is resistant to a number of substances which are, in general, inhibitory to the growth of other streptococci. Andrewes and Horder found this organism in cases of endocarditis, cystitis, and other human infections. Houston and McCloy noted many types of infections in which the enterococcus appeared to be implicated. PROCEDURE
The organisms used in these experiments were the three common gram-positive cocci found in the urinary tract: Staphylococcus aureus, Micrococcus, and Streptococcus faecalis. These bacteria were obtained
PATHOGENICITY OF COCCI FROM URINE
239
in pure culture from the urine of patients and were then subcultured on a slant and kept in a refrigerator. The cultures were frequently transferred to a fresh slant. Twenty-four hours before any culture was to be used for injection, inoculation into a tube of dextrose brain broth medium was performed. All injections into animals were made with this culture. Young male rabbits weighing between 3 and 5 pounds (1.4 to 2.3 kg.) were used. The animal was first catheterized, the specimen of urine being collected in a sterile test tube for microscopic examination and culture. The culture containing the bacteria was then injected into the lateral ear vein of the animal. Forty-eight or 72 hours following injection, another catheterized specimen of urine was obtained in a sterile test tube for microscopic examination · and culture. At necropsy, specimens of urine for culture were obtained from the bladder by searing the surface of the bladder and drawing the urine into a sterile pipet. A complete necropsy was performed on all animals. All the organs were examined grossly for the presence of lesions. The kidneys alone were saved for microscopic examination. The kidneys were fixed in formalin and then sectioned according to the method advocated by Helmholz and Field. RESULTS
Staphylococcus aureus.- An injection of 0.5 cc. of a culture of Staphylococcus aureus was given to each of 11 rabbits. Five animals were killed 72 hours following injection; three of these had lesions of the kidney. Three animals were killed 96 hours following injection; two of these had lesions of the kidney. Two animals were killed 120 hours and one animal was killed 144 hours following injection, all three of these animals had renal lesions. In all, lesions of the kidney were produced in eight, or 73 per cent, of the 11 rabbits. Table 1 shows the type of renal lesions that occurred in the 8 rabbits. In 7 animals, the lesions were bilateral and in 1 animal a unilateral lesion was found. Seven rabbits had lesions in more than one portion of the kidney. In 1 animal, a single abscess, which was situated deep in the medulla, was the only lesion that could be found. Abscesses of the papilla, which were the most common type of lesion, occurred in 7 of the 8 rabbits. Pyelitis, which was the next most common lesion, was found in 6 of the 8 animals. Five animals had medullary abscesses and cortical abscesses were found in 5. Table 2 includes the urinary findings in the 11 animals which received
240
MOE GOLDSTEIN TABLE
1.-Type of renal lesions produced by Staphylococcus aureus TYPE OF LESION
STRAIN OF STAPHYLOCOCCUS AUREUS
Left side
--4
10 12 15 25 26
TABLE
+ + + + + -
-
-
in Animals which lesions were produced ...
Right side
- - - - --
+ + + + +
1 1
Papillary lesion
Pyelitis Right side
Left side --- -
+ + + + + + +
-
+ + + + +
6
Medullary lesion Right side
Left side
--
+ + + + +
Left side
- - - - - -+
+ + + +
5
7
Cortical lesion Right side
---
+ + + +
+ + + +
-
-
-
5
2.-Res-ults of microscopic examination of specimens of urine obtained by catheterization before and after injection of 0.5 cc. of a culture of Staphylococcus a-ure11s
STRAIN OF STAPHYLOCOCCUS
AUREUS
URINE OBTAINED BEFORE INJECTION
I
URINE OBTAINED FORTY-EIGHT HOURS AFTER INJECTION
Urine of eight rabbits in which lesions developed
I
1 1 4 10
12 15 25 26
Not examined Normal Normal Not examined Normal Normal Normal Normal
P us Pus Pus P us Not examined Pus Normal Many red blood cells; culture produced grampositive cocci
Urine of three rabbits in which lesions did not develop 4
10 15
Not examined Normal Normal
Not examined* Normal Normal
* Microscopic examination of vesical urine, which was obtained at necropsy did not disclose any abnormality; culture did not produce any growth.
an injection of a culture of Staphylococcus aureus. No abnormal elements were found in the urine of 2 of the 3 animals in which no lesions were produced. A culture of the urine, which was made at necropsy
PATHOGENICITY OF COCCI FROM URINE
241
also was negative. The urine of one of these 3 rabbits was not obtained prior to necropsy, but the vesical urine which was obtained at necropsy, was normal both on microscopic examination and on culture. In 6 of the 8 animals in which lesions of the kidney were produced, the urine was normal on microscopic examination prior to injection of the Staphylococcus aureus. The urine of two of the animals was not examined prior to the injection. Forty-eight hours following injection, the urine of 5 of the 8 rabbits contained pus. The urine of 1 rabbit did not contain any abnormal elements; the urine of one animal was not examined, and the urine of the remaining animal contained many red blood cells and a culture produced innumerable gram-positive cocci. In 4 of the 8 animals, the urine was examined at necropsy. It was obtained from the bladder by sterile methods. Three of the 4 cultures showed grampositive cocci. One culture did not produce any growth. Micrococcus.- Fourteen animals received injections of a culture of Micrococcus. Nine animals received an injection of 0.5 cc. of the culture; four of these animals were killed 72 hours following the injection, four were killed 96 hours following the injection and one was found dead 96 hours following the injection. Two cubic centimeters of the culture was injected into each of 5 animals; all of these rabbits were killed 124 hours following the injection. No renal lesions were produced in any of the 14 animals. Six strains of Micrococcus were used. Each of 4 strains was injected into 2 separate rabbits, and each of 2 strains was injected into 3 rabbits. Table 3 shows the urinary findings in the 14 animals which received an injection of a culture of Micrococcus. The urine of nine animals was examined before the injections were made. No abnormal elements were found in the urine of any of these animals. The urine of 6 of the 14 rabbits was examined 48 hours following injection and the urine of 8 of the 14 rabbits was examined 72 hours following injection. No abnormal elements were found in any of these specimens. In 11 instances the vesical urine was examined at necropsy. In 6 instances both microscopic examination and culture of the urine failed to disclose any evidence of infection. In 3 instances the culture of the urine gave negative results but microscopic examination was not performed. In the 2 instances in which a culture of the urine was not made, microscopic examination did not disclose any abnormal elements. Streptococcus jaecalis.- A culture of Streptococcus faecalis was in-
242
MOE GOLDSTEIN
jected into 19 rabbits. Each of 9 animals received 0.5 cc. of the culture. Two of these rabbits were killed 72 hours following injection, five, 96 hours following injection, and two, 120 hours after injection. Two cubic centimeters of the culture was injected into each of 8 animals. One of these rabbits was killed 48 hours following injection, one, 96 hours later, and six, 144 hours later. Each of 2 animals received an injection of 3 cc. of the culture. One of these rabbits was killed 48 hours later, and the other one, 96 hours later. Nine strains of Streptococcus faecalis were used. Each of 3 strains was injected into a separate rabbit, TABLE
3.-Results of microscopic examination and culture of urine of fourteen rabbits which received injections of a culture of micrococcus MICROSCOPIC EXAMINATION
STRAIN OF MICROCOCCUS
3 3 7 7 7 11 11 11
14 14 23 23 24 24
Urine obtained by catheterization before injection
Urine obtained by catheterization after injection
necropsy
Not examined Not examined Normal Normal Not examined Normal Normal Not examined Normal Not examined Normal Normal Normal Normal
Normal* Normal* Not examined Not examined Normal* Normal* Normalt Normal* Normal* Not examined Normalt Normalt Normalt Normal*
Not examined Not examined Normal Normal Normal Normal Normal Not examined Not examined Normal Normal Not examined Normal Normal
Urine obtained at
CULTURE OF URINE OBTAINED AT NECROPSY
Not made Not made Negative Negative Negative Negative Not made Negative Negative Negative Negative Not made Not made Negative
* Urine obtained
forty-eight hours after injection. t Urine obtained seventy-two hours after injection.
each of 5 strains was injected into 2 separate rabbits and 1 strain was injected into 6 rabbits. No lesions of the kidney were found in 12 of the 19 animals (63 per cent). Lesions of the kidney were produced in 7 of the 19 rabbits (37 per cent). Lesions were produced in one (11 per cent) of the 9 animals that received an injection of 0.5 cc. of the culture, in 4 (50 per cent) of the 8 animals that received an injection of 2 cc. and in both animals (100 per cent) that received an injection of 3 cc. of the culture. Lesions were caused by 3 different strains of Streptococcus faecalis. One strain produced lesions in 5 of the 6 animals into which it was injected, 1 strain
243
P ATHOGENICITY OF COCCI FROM URINE
produced lesions in 1 of the 2 animals into which it was injected and 1 strain produced lesions in the 1 animal into which it had been injected. Table 4 summarizes the types of renal lesions that occurred in 7 rabbits. In 5 animals, the lesions were bilateral; in two, the lesion was unilateral. Three rabbits had lesions in more than one portion of the kidney. In 3 animals, the pathologic findings were confined to the pelvis of the kidney, and in 1 animal they were confined to the cortex of the kidney. Pyelitis which was the most common lesion, occurred in 5 of the 7 rabbits. Abscess of the medulla, which was the next most common lesion, was found in 3 of the 7 animals. Papillary abscesses occurred in 2 animals, and cortical abscess, in one. TABLE
4.-Type of renal lesions produced by Streptococcus f aecalis TYPE OF LESION
STRAIN OF
Pyelitis
STREPTOCOCCUS
Papillary lesion
Medullary lesion
Cortical lesion
FAECALIS
Right side
13 17 21 21 21 21 21 Animals in which lesions were produced ...
Left side
Right side
Left side
Right side
Left side
--- --- --- ---
-
+ + + +
+ + + +
-
5
-
-
-
-
+
+ +
+
-
+
+ +
-
2
-
-
-
3
Left side ---
---
-
Right side
-
-
-
-
-
+ -
-
1
In table 5 are summarized the urinary findings in the 7 animals in which lesions were produced and in the 12 rabbits in which no lesions were produced. No abnormal elements were found in the urine of 5 of the 7 rabbits examined before injection of Streptococcus faecalis. The urine of 2 rabbits was not obtained. Forty-eight hours following injection, the urine of 1 rabbit did not contain any abnormal elements, the urine of 5 rabbits contained pus, and the urine of 1 rabbit contained gross blood, probably owing to the trauma caused by catheterization. Three of the 48-hour specimens represented urine obtained from the bladder at necropsy; two of these contained pus but one was normal. In the 12 rabbits in which no lesion was produced, no abnormal elements
244
MOE GOLDSTEIN
were found in the urine prior to the injection; 48 hours after the injection the urine of 8 rabbits was examined and was found to be normal. At necropsy, the vesical urine of 6 rabbits was normal both on microscopic examination and on culture. The urine of one rabbit was normal on TABLE
5.- Results of microscopic examination and culture of urine of rabbits which received injections of a culture of Streptococcus f aecalis MICROSCOPIC EXAMINATION
STRAIN OF STREPTO-
coccus
FAECALIS
Urine obtai!'ed by cathete:i~ab
I Urine obtained by catbeterization I forty-eight hours after injection
Urine obtained from bladder at
CULTURE OF URINE OBTAINED AT NECROPSY
necropsy
Urine of seven rabbits in which lesions developed
13 17 21 21 21 21 21
Not examined Not examined Normal Normal Normal Normal Normal
Pus Normal Pus Gross blood Pus* Pust Normal; culture produced gram-positive cocci
Urine of twelve rabbits in which lesions did not develop
2 2 6 6
13
Normal Normal Normal Normal Normal Normal Normal Normal Normal
Normal Not examined Not examined Normal Not examined Normal Normal Normal Not examined
16
Normal
Normal
20 21
Normal Normal
Normal Normal
8 8 9 9
Not examined Normal Normal Normal Normal Normal Not examined Normal Many white blood cells Many white blood cells Not examined Normal
Not made Negative Negative Negative Negative Negative Negative Negative Not made Not made Not made Not made
* Urine obtained at necropsy, forty-eight hours after injection. t Urine obtained at necropsy, ninety-six hours after injection.
microscopic examination; culture of the urine was not made; the urine of another rabbit showed no growth on culture; microscopic examination was not made. There was no urine in the bladder of the 2 rabbits. The urine of 2 rabbits contained pus but a culture of the urine was not made.
PATHOGENICITY OF COCCI FROM URINE
245
DESCRIPTION OF PATHOLOGIC FINDINGS
Typical focal interstitial suppurative nephritis was produced by the Staphylococcus aureus. The lesions contained many polymorphonuclear cells and the blood vessels were dilated at the periphery of the abscesses. In some places, the blood vessels had ruptured and extravasation of blood had occurred. In many regions, the abscesses could
FIG. L Lesions produced by injection of Staphylococcus aureus; necrotic lesion in renal cortex; abscess in medulla and pus in pelvis (X7).
be seen breaking through the wall of the tubules and the lumina of many tubules were filled with pus, cellular debris, and red blood cells. Certain abscesses were wedge-shaped, the base of the wedge lying in the cortex and the apex extending into the medulla (fig. 1). In the center of these wedge-shaped abscesses were pale necrotic tubules which were surrounded by a zone of marked polymorphonuclear infiltration. Coccal thrombi
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MOE GOLDSTEIN
FIG. 2. Large amount of pus in renal pelvis following injection of Staphylococcus aureus (X9).
FIG. 3. Pyelitis produced by injection of Streptococcus faecalis (X85)
could be seen lying within the lumina of some of the collecting tubules in the papilla of the kidney.
PATHOGENICITY O:F COCCI FROM URINE
247
In many of the kidneys, the pelves were filled with pus and the subepithelial tissues were infiltrated with polymorphonuclear leukocytes and lymphocytes (fig. 2). The renal lesions produced by the Streptococcus faecalis were far less severe than were those produced by the Staphylococcus aureus. In a few kidneys, pyelitis was the only lesion present (fig. 3). In other kidneys focal interstitial suppuration was present in the medulla and papilla (fig. 4). These were typical abscesses with dilated capillaries at the periphery of the lesions. The abscesses could be seen breaking through the lumina of the tubules and filling them with pus. In only 1
FIG. 4. Abscess of renal papilla produced by injection of Streptococcus faecalis (X30).
kidney was there an abscess of the cortex. This lesion, however, was of such chronicity that it was questionable whether it had been produced by the injection of the culture of Streptococcus faecalis. COMMENT
The results of the experiments with Staphylococcus aureus bear out the conclusions of previous investigators regarding the pathogenicity of this organism. Lesions of the kidney were produced in 8 (73 per cent) of 11 animals which received an injection of 0.5 cc. of a culture of Staphylococcus aureus. Helmholz and Field have produced lesions of the kidney
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MOE GOLDSTEIN
in 100 per cent of animals, but they injected 2 cc. of a 24-hour culture of Staphylococcus aureus. It is of interest that abscesses were more common in the papilla of the kidney than they were in the cortex. Abscesses of the cortex are usually mentioned as the most frequent type of lesion in staphylococcic infections of the kidney. The lesions produced by Staphylococcus aureus were far more intense than those produced by Streptococcus faecalis. The pathologic findings in 1 rabbit (number 792) deserve some emphasis. This animal was killed 120 hours after injection of 0.5 cc._of
FIG.
5. Solitary abscess of renal medulla produced by injection of Staphylococcus aureus
(X4½).
a culture of Staphylococcus aureus. Microscopic examination of the kidneys disclosed a single abscess, deep in the medulla of one kidney (figs. 5 and 6). No other lesions were found in several sections which were studied. The abscess that was found might easily have been overlooked if many gross sections of the kidney had not been made. One must therefore stress the importance of examining many sections of the kidney before coming to any conclusion as to the presence or absence of pathologic lesions. In contrast to the constancy with which lesions are produced in the
PATHOGENICITY OF COCCI FROM URINE
249
kidney after intravenous injection of Staphylococcus aureus is the complete absence of renal lesions after intravenous injection of Micrococcus. Although the original amount of the culture of Micrococcus that was injected into 14 rabbits was increased fourfold as compared to the Staphylococcus aureus, necropsy did not disclose any lesions and examination of the urine did not reveal pus or organisms. The striking difference in the two groups of gram-positive, massforming cocci, when injected into the blood stream of rabbits, is of definite clinical importance. It suggests that if the Micrococcus, which is found
FrG. 6. Solitary abscess of renal meduJ!a; same section as shown in figure 5 (X40)
normally in the urethra, is also present in the vesical urine it does not necessarily indicate the presence of infection of the upper part of the urinary tract; whereas the presence of Staphylococcus aureus in the urine usually indicates infection of the upper part of the urinary tract. In the presence of mass-forming, gram-positive cocci in the urine, it is, therefore, of utmost importance to know whether one is dealing with Micrococcus or Staphylococcus aureus. The mere finding of grampositive cocci in the stained smear of the urine will not distinguish between these two organisms. Cultures of the urine should be made, whenever possible, and stained smears should be made from 24-hour
250
MOE GOLDSTEIN
slant cultures. In this way it is possible to identify these organisms by the technique advocated by Thompson. Renal lesions were produced in 7 of 19 rabbits (37 per cent) which received injections of a culture of Streptococcus faecalis. A larger dose of Streptococcus faecalis than of Staphylococcus aureus was required to produce renal lesions. The renal lesions produced by Streptococcus faecalis were far less severe than the lesions produced by Staphylococcus aureus. The presence of Streptococcus faecalis in cases of pyuria may therefore be considered of etiologic importance. CONCLUSIONS
The almost constant presence of renal lesions after intravenous injection of the Staphylococcus aureus as compared with their complete absence after injections of Micrococcus seems to indicate that the finding of Staphylococcus aureus in the urine is of great clinical significance while the finding of Micrococcus is of little, if any, significance. When mass-forming, gram-positive cocci are found in the urine, it is of clinical importance to determine their nature. The frequency of renal lesions after intravenous injection of Streptococcus faecalis seems to indicate that its presence in the urine is of pathologic significance. REFERENCES ANDREWES, F. W., AND HoRDER, T. F.: A study of the streptococci pathogenic for man. Lancet. 2: 708-713, 775-782, 852-855, 1906. CABOT, HUGH: Infections of the kidney. In: Cabot, Hugh: Modern urology. Ed. 3, Philadelphia, Lea and Febiger, Vol. 2, Chapt. 12, 1936, pp. 510-569. HELMHOLZ, H. F., AND FIELD, RUTHS.: The kidney: a filter for bacteria. IL The effect of diuresis on the excretion of bacteria by the kidney. Am. Jour. Dis. Child. 29: 506-512, 1925. HELMHOLZ, H. F., AND FIELD, RUTH S.: The kidney: a filter for bacteria. IV. The effect of clamping the renal artery or renal vein on the passage of bacteria into the urine. Am. Jour. Dis. Child. 29: 645-649, 1925. HOUSTON, THOMAS, AND McCLOY, J. M.: The relation of the enterococcus to "trench fever" and allied conditions. Lancet. 2: 632-637, 1916. KRETSCHMER, H. L.: The treatment of pyelitis. Surg., Gynec., and Obst. 33: 632-641, 1921. SHERMAN, J.M.: The streptococci. Bact. Rev.1: 3-97, 1937. THOMPSON, LUTHER: Morphologic and cultural study of staphylococci with special reference to source. Am. Jour. Clin. Path. 2: 125-134, 1932. YOUNG, H. H., COLSTON, J. A. C., AND HILL, J. H.: Infections in the genito-urinary tract, and complications: further advances in the treatment. Jour. Am. Med. Assn. 98: 715-722 (Feb. 27) 1932.