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Grand rounds at the Washington University School of Medicine
J. &on.
Dis. 1966, Vol. 19, pp. 663-666. Pergamon Press Ltd. Printed in Great Britain
GRAND
ROUNDS AT THE WASHINGTON
UNIVERSITY
SCHOOL OF MEDICINE II.
PROTOPLASTS CASE
AND
PYELONEPHRITIS
HISTORY
Dr. Eduardo Slatopolsky (Fellow in Renal Division) : Mr. C., a 41-year-old white male school teacher, was first seen here in September 1961 for back pain, fever and chills. In 1943 he was discovered to have proteinuria and mild hypertension. In 1951 he was diagnosed as having gout. On his first admission here in 1961 he complained of weakness and urinary frequency. His urine at this time contained protein and white blood cells. A serum uric acid was 8.5 mg/lOO ml. An intravenous pyelogram showed changes consistent with chronic pyelonephritis. Later in 196 1 he was readmitted for chills, fever and back pain. His urinary sediment contained many white blood cells and a urine culture grew pseudomonas. The blood urea nitrogen (BUN) was 61 mg/lOO ml. During a short period, he received tetracycline, streptomycin and chloramphenicol without benefit and then improved after polymyxin therapy. He was relatively well until March 1964 when he developed nausea and extreme weakness. Again there was proteinuria with many white blood cells in the urine but the urine culture was negative. His BUN was 135 mg/lOO ml and a creatinine clearance was 4 ml/min. After hydration, he received a diet containing 4 g of salt and 40 g of protein per day. From April to September 1964 he was admitted six more times because of vomiting, weakness and congestive heart failure. His BUN ranged from 180 to 200 mg/lOO ml, his creatinine clearance was 1-2 ml/min and his hemoglobin usually was about 7 g/100 ml. He was subjected to hemodialysis 4 or 5 times. Renal transplantation was performed on 23 December 1964. Subsequently this patient’s renal function has been good and he has returned to work. Dr. Carl Moore: There were several reasons for asking Mr. C. to come here today. Four weeks after a renal transplant, he is doing well. In addition, he is a good example of the difficulty that may be encountered in the treatment of chronic pyelonephritis. Perhaps we should inspect his X-ray films before continuing. Dr. Tom Staple: The retrograde pyelogram in 1959 demonstrated moderate distortion of the renal calcyes. The kidneys were of normal size and an intravenous pyelogram afforded only poor visualization. During 1964 numerous chest films showed varying degrees of heart failure with enlargement of the heart, bilateral pleural effusions and at times prominent pulmonary vessels. In December 1964, prior to the transplant, the heart was smaller although the left ventricle remained enlarged and the pleural effusions had disappeared. 663
664
DR. D. P. EARLE DISCUSSION
Dr. Carl Moore: Recently two stimulating articles of potential practical importance have been published by GUZE and KALMANSON [ 1,2] relating to experimental pyelonephritis. Dr. Richard Krause, who has been interested in a somewhat related problem, will discuss the significance of protoplasts in chronic pyelonephritis. Dr. Richard Krause: Perhaps we should begin by posing this question. Are there in existence forms of bacteria, heretofore undetected because of their failure to grow on ordinary media? If such forms exist, what importance may they have in causing disease? We are familiar with the .usual forms of bacteria such as bacilli, cocci and spirochetes which owe their shapes to the rigid envelopes or cell walls which comprise about 20 per cent of the bacterial dry weight. These external cell walls are somewhat analogous to the outer walls of plant cells. Beneath the cell wall is a cytoplasmic membrane such as encases all mammalian cells. Some years ago, bacterial L-forms were discovered, the ‘L’ standing for the Lister Institute where they were first described. L-forms are obtained when the bacterial forms are grown in hypertonic nutrient agar to which penicillin has been added. Penicillin inhibits cell wall synthesis. Similarly, the external cell wall may disappear when bacteria are grown in a medium containing specific antibody to the bacteria and complement. L-forms once established will grow in a hypertonic medium of salt or sucrose and in the absence of penicillin as nondescript colonies so that colonies of L-forms of a streptococcus resemble colonies of L-forms of a staphylococcus. Protoplasts appear to have the same structure as Lforms but protoplasts are produced in a different fashion. The protoplast is a cytoplasmic body limited by a cytoplasmic membrane but in this instance the rigid outer cell wall has been dissolved by lysozyme or some other cell-wall-dissolving enzyme. Protoplasts, like L-forms, grow as minute colonies when cultured on a suitable hypertonic medium. Biologically, L-forms and protoplasts appear to have the same type of structure. Protoplasts might arise in the host either because of antibiotic treatment or because the cell walls are removed by lysosome or other cell-wall-dissolving enzymes within white blood cells or cells of other tissues. Dr. Guze’s experiments indicate that one can culture protoplasts from experimental animals with special techniques but not with ordinary bacteriological media. Let me give an illustration of his experimental pyelonephritis. Rats on day-l were On day-2 they were begun on therapy given 4 x lOa Streptococcus fecalis organisms. with 200,000 units of penicillin. This amounts to about 2000 units of penicillin per gram of body weight in the rat, clearly a large dose. The penicillin is continued for 14 days. At the end of 14 days, kidneys from infected rats are sterile when cultured on ordinary media. Cultures of rat kidneys killed on day-28, two weeks after discontinuance of penicillin, are sterile on usual media, but on hypertonic media one finds approximately 103 L-colonies per gram of kidney tissue. Under appropriate conditions these can revert back to the parent strain of Streptococcus fecalis. In infected rats, not treated with penicillin, culture of the kidney on day-28 yields about lo5 organisms on usual media and about lo5 protoplast colonies per gram of kidney when cultured on special media. In this experimental renal infection in rats, it is evident that despite penicillin treatment, there persists a bacterial form which hitherto was regarded as a
Grand Rounds at the Washington
University School of Medicine-II
665
curiosity in the laboratory. One could suppose that the protoplasts might be unrelated to the original infecting agent since protoplasts of different bacteria look alike and also because it is difficult to obtain by culture sufficient numbers of protoplasts to permit the usual identification tests. In our own studies, we appear to be recovering L-forms of group A streptococci in certain experimental infections. If the usual bacterial form of this organism is grown in about 30 ml of broth, the bacterial pellet collected by centrifugation yields an extractable soluble protein which will react in a precipitation test with an antiserum of the specific Lancefield type to which the organism belongs. Thus a type 12 group A streptococcus can be identified with appropriate typing antisera. One cannot readily identify the L-forms in this fashion, but they can be identified by an immunofluorescent technique whereby type specific Lancefield antisera are coupled with a fluorescent dye, which then reacts with L-form colonies. Such a method is extremely rapid and does not require the growth of large quantities of the slow growing L-forms. L-colonies of a specific group A streptococcus when coated and stained with type specific fluorescent antibody have no regular shape. This ability to identify L-colonies as being derived from specific bacterial types provides a useful advance in our knowledge of L-forms. The importance of Dr. Guze’s study relates to this question; are we inducing Lforms in patients who are being treated with penicillin and if so can the L-forms persist in tissues? Dr. Guze suggests that the kidney, an organ with variable osmality might be a fertile soil and permit survival of these fragile bodies. In our own studies, we have found that L-forms are stable in solutions of spermine or other proteins rich in basic amino acids. The in vitro concentration of spermine for cultivation of L-forms probably does not differ greatly from the concentration that may exist in certain body cells. Perhaps the L-forms exist in a state of intracellular symbiosis. In such a state are they capable of producing toxins? In this regard, it may be relevant that L-colonies of P-hemolytic streptococci can produce M-protein, a toxic substance, Finally one should ask, do L-forms or protoplasts produce disease? This cannot be answered. Much effort has been directed to culturing L-forms from blood or other body fluids of patients with acute rheumatic fever and other diseases of obscure etiology. There is currently speculation that the collagen disorders might result from an as yet undetected infection by an unrecognized bacterium or by some atypical form of a known bacterium. Presently this is speculation but it has been fostered by the disenchantment that has grown out of the immunologic approach to the pathogenesis of these disorders. Dr. Carl Moore: I am interested that you have ignored another aspect of Dr. experiments in which he first administered bacteria, then penicillin which synthesis of bacterial membranes, and subsequently gave another antibiotic erythromycin to inhibit synthesis of bacterial protein so that he was apparently rid the rat kidney of L-forms.
Guze’s inhibits such as able to
Dr. Krause: The evidence presented in the article in the Journal of the American Medical Association is not entirely clear on this point. Dr. Carl Moore: Whenever I have expressed my enthusiasm in Dr. Guze’s work to the house officers, they have evinced skepticism partly because human kidney infections
DR. D. P. EARLE
666
are usually due to gram-negative rather than gram-positive organisms such as the Strep. fecalis. Yet, I believe his observations may have practical therapeutic applications. Dr. Krause: I think the fact that there may be undetected renal infection may be greatly significant. Just because the experimental model used Streptococcus fecalis does not mean that one might not devise a similar model for gram-negative organisms. Dr. Carl Moore: I am intrigued with the idea of tandem antibiotic to interfere with formation of cell wall, another duction within a bacterium.
antibiotic therapy-one to inhibit protein pro-
Dr. William Sly: I am certain that cultures of E. coli in hypertonic the presence of penicillin will produce a comparable organism.
sucrose and in
Dr. Harry Fozzard: Is the hypertonic solution employed for growth or L-forms similar to the tonicity existing in the renal medulla?
of protoplasts
Dr. Krause: The difficulty is that there is no certainty about the optimal substrate composition of the medium for cultivation of these curious bacterial forms. One can reduce the salt concentration to less than one per cent in the presence of spermine and even lower if magnesium ions are added. Dr. Sly:
How stable are protoplasts
in serum?
Dr. Krause: They are not particularly from serum alone for, at most, an hour. Dr. Reiselbach: Dr. Krause:
Will L-forms
stable in serum.
persist in high concentrations
We can recover
L-forms
of urea?
I don’t know.
Dr. Cederbaum: It bothers me that such an unhardy form of bacterium can cause infection. Also if it reproduces so slowly, will any of the usual antibiotics provide effective therapy ? for ridding itself of Dr. Krause: The body ought to have ample opportunity protoplasts yet this may be a paradox analogous in part to the situation in so-called For a long period, an etiologic agent was missed because primary atypical pneumonia. it could not be cultivated on ordinary bacteriological media. It was only when special techniques were employed that Eaton’s agent was cultivated. Postscript: transplanted
Seven months later the patient kidney is excellent.
is alive and well. Renal function
in the
REFERENCES 1. 2.
GUZE, L. B. and KALMANSON,C. M.: Persistence of bacteria in ‘protoplast’ forln after apparent cure of pyelonephritis in rats, Science 143, 1340, 1964. KALMANSON,G. M. and GUZE, L. B.: Role of protoplasts in pathogenesis of pyelonephritis, J. Am. med. Ass. 190, 1107, 1964.
Dis. 1966, Vol. 19, pp. 663-666. Pergamon Press Ltd. Printed in Great Britain
GRAND
ROUNDS AT THE WASHINGTON
UNIVERSITY
SCHOOL OF MEDICINE II.
PROTOPLASTS CASE
AND
PYELONEPHRITIS
HISTORY
Dr. Eduardo Slatopolsky (Fellow in Renal Division) : Mr. C., a 41-year-old white male school teacher, was first seen here in September 1961 for back pain, fever and chills. In 1943 he was discovered to have proteinuria and mild hypertension. In 1951 he was diagnosed as having gout. On his first admission here in 1961 he complained of weakness and urinary frequency. His urine at this time contained protein and white blood cells. A serum uric acid was 8.5 mg/lOO ml. An intravenous pyelogram showed changes consistent with chronic pyelonephritis. Later in 196 1 he was readmitted for chills, fever and back pain. His urinary sediment contained many white blood cells and a urine culture grew pseudomonas. The blood urea nitrogen (BUN) was 61 mg/lOO ml. During a short period, he received tetracycline, streptomycin and chloramphenicol without benefit and then improved after polymyxin therapy. He was relatively well until March 1964 when he developed nausea and extreme weakness. Again there was proteinuria with many white blood cells in the urine but the urine culture was negative. His BUN was 135 mg/lOO ml and a creatinine clearance was 4 ml/min. After hydration, he received a diet containing 4 g of salt and 40 g of protein per day. From April to September 1964 he was admitted six more times because of vomiting, weakness and congestive heart failure. His BUN ranged from 180 to 200 mg/lOO ml, his creatinine clearance was 1-2 ml/min and his hemoglobin usually was about 7 g/100 ml. He was subjected to hemodialysis 4 or 5 times. Renal transplantation was performed on 23 December 1964. Subsequently this patient’s renal function has been good and he has returned to work. Dr. Carl Moore: There were several reasons for asking Mr. C. to come here today. Four weeks after a renal transplant, he is doing well. In addition, he is a good example of the difficulty that may be encountered in the treatment of chronic pyelonephritis. Perhaps we should inspect his X-ray films before continuing. Dr. Tom Staple: The retrograde pyelogram in 1959 demonstrated moderate distortion of the renal calcyes. The kidneys were of normal size and an intravenous pyelogram afforded only poor visualization. During 1964 numerous chest films showed varying degrees of heart failure with enlargement of the heart, bilateral pleural effusions and at times prominent pulmonary vessels. In December 1964, prior to the transplant, the heart was smaller although the left ventricle remained enlarged and the pleural effusions had disappeared. 663
664
DR. D. P. EARLE DISCUSSION
Dr. Carl Moore: Recently two stimulating articles of potential practical importance have been published by GUZE and KALMANSON [ 1,2] relating to experimental pyelonephritis. Dr. Richard Krause, who has been interested in a somewhat related problem, will discuss the significance of protoplasts in chronic pyelonephritis. Dr. Richard Krause: Perhaps we should begin by posing this question. Are there in existence forms of bacteria, heretofore undetected because of their failure to grow on ordinary media? If such forms exist, what importance may they have in causing disease? We are familiar with the .usual forms of bacteria such as bacilli, cocci and spirochetes which owe their shapes to the rigid envelopes or cell walls which comprise about 20 per cent of the bacterial dry weight. These external cell walls are somewhat analogous to the outer walls of plant cells. Beneath the cell wall is a cytoplasmic membrane such as encases all mammalian cells. Some years ago, bacterial L-forms were discovered, the ‘L’ standing for the Lister Institute where they were first described. L-forms are obtained when the bacterial forms are grown in hypertonic nutrient agar to which penicillin has been added. Penicillin inhibits cell wall synthesis. Similarly, the external cell wall may disappear when bacteria are grown in a medium containing specific antibody to the bacteria and complement. L-forms once established will grow in a hypertonic medium of salt or sucrose and in the absence of penicillin as nondescript colonies so that colonies of L-forms of a streptococcus resemble colonies of L-forms of a staphylococcus. Protoplasts appear to have the same structure as Lforms but protoplasts are produced in a different fashion. The protoplast is a cytoplasmic body limited by a cytoplasmic membrane but in this instance the rigid outer cell wall has been dissolved by lysozyme or some other cell-wall-dissolving enzyme. Protoplasts, like L-forms, grow as minute colonies when cultured on a suitable hypertonic medium. Biologically, L-forms and protoplasts appear to have the same type of structure. Protoplasts might arise in the host either because of antibiotic treatment or because the cell walls are removed by lysosome or other cell-wall-dissolving enzymes within white blood cells or cells of other tissues. Dr. Guze’s experiments indicate that one can culture protoplasts from experimental animals with special techniques but not with ordinary bacteriological media. Let me give an illustration of his experimental pyelonephritis. Rats on day-l were On day-2 they were begun on therapy given 4 x lOa Streptococcus fecalis organisms. with 200,000 units of penicillin. This amounts to about 2000 units of penicillin per gram of body weight in the rat, clearly a large dose. The penicillin is continued for 14 days. At the end of 14 days, kidneys from infected rats are sterile when cultured on ordinary media. Cultures of rat kidneys killed on day-28, two weeks after discontinuance of penicillin, are sterile on usual media, but on hypertonic media one finds approximately 103 L-colonies per gram of kidney tissue. Under appropriate conditions these can revert back to the parent strain of Streptococcus fecalis. In infected rats, not treated with penicillin, culture of the kidney on day-28 yields about lo5 organisms on usual media and about lo5 protoplast colonies per gram of kidney when cultured on special media. In this experimental renal infection in rats, it is evident that despite penicillin treatment, there persists a bacterial form which hitherto was regarded as a
Grand Rounds at the Washington
University School of Medicine-II
665
curiosity in the laboratory. One could suppose that the protoplasts might be unrelated to the original infecting agent since protoplasts of different bacteria look alike and also because it is difficult to obtain by culture sufficient numbers of protoplasts to permit the usual identification tests. In our own studies, we appear to be recovering L-forms of group A streptococci in certain experimental infections. If the usual bacterial form of this organism is grown in about 30 ml of broth, the bacterial pellet collected by centrifugation yields an extractable soluble protein which will react in a precipitation test with an antiserum of the specific Lancefield type to which the organism belongs. Thus a type 12 group A streptococcus can be identified with appropriate typing antisera. One cannot readily identify the L-forms in this fashion, but they can be identified by an immunofluorescent technique whereby type specific Lancefield antisera are coupled with a fluorescent dye, which then reacts with L-form colonies. Such a method is extremely rapid and does not require the growth of large quantities of the slow growing L-forms. L-colonies of a specific group A streptococcus when coated and stained with type specific fluorescent antibody have no regular shape. This ability to identify L-colonies as being derived from specific bacterial types provides a useful advance in our knowledge of L-forms. The importance of Dr. Guze’s study relates to this question; are we inducing Lforms in patients who are being treated with penicillin and if so can the L-forms persist in tissues? Dr. Guze suggests that the kidney, an organ with variable osmality might be a fertile soil and permit survival of these fragile bodies. In our own studies, we have found that L-forms are stable in solutions of spermine or other proteins rich in basic amino acids. The in vitro concentration of spermine for cultivation of L-forms probably does not differ greatly from the concentration that may exist in certain body cells. Perhaps the L-forms exist in a state of intracellular symbiosis. In such a state are they capable of producing toxins? In this regard, it may be relevant that L-colonies of P-hemolytic streptococci can produce M-protein, a toxic substance, Finally one should ask, do L-forms or protoplasts produce disease? This cannot be answered. Much effort has been directed to culturing L-forms from blood or other body fluids of patients with acute rheumatic fever and other diseases of obscure etiology. There is currently speculation that the collagen disorders might result from an as yet undetected infection by an unrecognized bacterium or by some atypical form of a known bacterium. Presently this is speculation but it has been fostered by the disenchantment that has grown out of the immunologic approach to the pathogenesis of these disorders. Dr. Carl Moore: I am interested that you have ignored another aspect of Dr. experiments in which he first administered bacteria, then penicillin which synthesis of bacterial membranes, and subsequently gave another antibiotic erythromycin to inhibit synthesis of bacterial protein so that he was apparently rid the rat kidney of L-forms.
Guze’s inhibits such as able to
Dr. Krause: The evidence presented in the article in the Journal of the American Medical Association is not entirely clear on this point. Dr. Carl Moore: Whenever I have expressed my enthusiasm in Dr. Guze’s work to the house officers, they have evinced skepticism partly because human kidney infections
DR. D. P. EARLE
666
are usually due to gram-negative rather than gram-positive organisms such as the Strep. fecalis. Yet, I believe his observations may have practical therapeutic applications. Dr. Krause: I think the fact that there may be undetected renal infection may be greatly significant. Just because the experimental model used Streptococcus fecalis does not mean that one might not devise a similar model for gram-negative organisms. Dr. Carl Moore: I am intrigued with the idea of tandem antibiotic to interfere with formation of cell wall, another duction within a bacterium.
antibiotic therapy-one to inhibit protein pro-
Dr. William Sly: I am certain that cultures of E. coli in hypertonic the presence of penicillin will produce a comparable organism.
sucrose and in
Dr. Harry Fozzard: Is the hypertonic solution employed for growth or L-forms similar to the tonicity existing in the renal medulla?
of protoplasts
Dr. Krause: The difficulty is that there is no certainty about the optimal substrate composition of the medium for cultivation of these curious bacterial forms. One can reduce the salt concentration to less than one per cent in the presence of spermine and even lower if magnesium ions are added. Dr. Sly:
How stable are protoplasts
in serum?
Dr. Krause: They are not particularly from serum alone for, at most, an hour. Dr. Reiselbach: Dr. Krause:
Will L-forms
stable in serum.
persist in high concentrations
We can recover
L-forms
of urea?
I don’t know.
Dr. Cederbaum: It bothers me that such an unhardy form of bacterium can cause infection. Also if it reproduces so slowly, will any of the usual antibiotics provide effective therapy ? for ridding itself of Dr. Krause: The body ought to have ample opportunity protoplasts yet this may be a paradox analogous in part to the situation in so-called For a long period, an etiologic agent was missed because primary atypical pneumonia. it could not be cultivated on ordinary bacteriological media. It was only when special techniques were employed that Eaton’s agent was cultivated. Postscript: transplanted
Seven months later the patient kidney is excellent.
is alive and well. Renal function
in the
REFERENCES 1. 2.
GUZE, L. B. and KALMANSON,C. M.: Persistence of bacteria in ‘protoplast’ forln after apparent cure of pyelonephritis in rats, Science 143, 1340, 1964. KALMANSON,G. M. and GUZE, L. B.: Role of protoplasts in pathogenesis of pyelonephritis, J. Am. med. Ass. 190, 1107, 1964.