Br. J. exp). Path. (1975) 56, 99
THE GROWTH OF SOME MYCOBACTERIA ON CULTURED HUMAN TISSUES D. A. J. TYRRELL, S. L. McLAUCHLAN AND C. S. GOODWIN From the Clinical Research Centre and Northwick Park Hospital, Watford Road, Harrow, Middlesex HAI 3 UJ
Receive(I for publication June 11, 1974
Summary.-M. ulcerans grows in close association with the surface of cultured human foetal and adult tissue and produces necrosis. M. tuberculosis grows also but M. xenopi grows little or not at all. The two first named seem to stick well to the tissue surface and the last does not. These results are discussed in relationship to the pathogenesis of Buruli ulcer.
A STRIKING and characteristic disease is caused by Mycobacterium ulcerans. This is variously named and occurs in many parts of the world. It was first recognized in Australia (MacCallum et al., 1948) and is called Buruli ulcer in Uganda. It appears to be due to organisms being introduced under the skin from the immediate environment (Uganda Buruli Group, 1971; Lancet, 1972). The organisms are found in association with the strands of fibrous tissue and give rise to characteristic painless subcutaneous nodules. The ulcer occurs when there is extensive necrosis of the subcutaneous fat and this finally breaks through the overlying skin (Uganda Buruli Group, 1970). The organism grows slowly on bacteriological media and requires a temperature of 330 (Clancey, 1964; MacCallum et al., 1948). We wished to study some of the elements of the pathological process in vitro and for this purpose we used the method of organ culture in plastic petri dishes. It was possible to keep tissues alive for weeks and, by microscopy of fixed and stained sections, we have studied the growth of the organisms and their effect on the tissues.
School. 31. xenopi and Ml. tuberculosis (BCG)
were standard laboratory strains. The 1. ulcerans was known to be pathogenic for mice. Culture. -All organisms were grown on
Lowenstein and Jensen medium and suspended Care was taken that egg medium was not removed with the bacteria. Smooth suspensions were matched to Wellcome opacity tube no. 4. Cultures were inoculated by dropping a total of 0-2 ml of suspension on to the tissue in each dish. The organisms were shown to grow freely in liquid medium in the presence of the mixture of antibiotics used in the organ cultures. in sterile physiological saline.
Tissues Human foetal tissue was obtained fromn Dr H. E. M. Kay, Tissue Bank, Royal Marsden Hospital. Adult fat, muscle and fibrous tissue were obtained incidentally at operations involving the anterior abdominal wall. Organ culture Preparation. -Tissues were dissected with as little handling as possible. Pieces of tissue approximately 0-2 cm3 were cut with a scalpel using a rocking motion and were placed on sterile stainless steel mesh triangles (mesh 50 holes per inch) in 50 mm Nunclon petri dishes; 2-5 ml of medium was added to each petri dish. The medium was Liebowitz L15, supplemented with 0.2% bovine plasma albumin, 100 u/mI of penicillin and either 100 ,ug/ml or 5 ug/ml of colistin sulphate. Medium was changed every 3 or 4 days. The cultuires were incubated in air at 330.
MATERIALS AND METHODS
Bacteria Strains. MJ. ulcerans was obtained from Dr John Stanford, Middlesex Hospital Medical
Harvesting and fixation The tissue and mesh triangles were fixed in formol saline (10% formalin in saline).
I 00)
D). A. J. TYRRELL, S. L. McLAUCHLAN AND C. S. GOODWIN
The tissutes wvere sectioned and stained with lhaernatoxylin and eosin, Ziehl-Neelsen and Triff stains.
RESULTS
TABLE I. Sunmmary of Results of Inoculatiny M. ulcerans into Organ Cultures of Human Foetal Tissues P'reseicie (of Tissue
Skinl
Time in days 1() 20
Mycobacteria
Necrosis
++ ++
The first experiment was intended to determine if M. ulcerans would multiply 40 -t- -t A in organ culture of human tissues. A Muscle 10 + -1 20 human foetus of 16 weeks was a con+j + 40 venient source of sterile tissue, and Lunig 10 N+ cultures of skin, muscle, lung and kidney 20 ++ + -I ND ND described. as 40 inoculated were set up and 10 The medium remained clear but the tissue Kidney +I-A + 20 of inoculated cultures became bulky and A40 ++4 gelatinous compared with uninoculated Bacterial growth is graded arbitrarily. Comcontrol cultures. The results of microwith Fig. 1. Uninoculated cultures showed scopic examination are summarized in pare some degeneration which increased( during cultivaTable I. Masses of organisms were seen tion. The necrosis is graded by comparison with these. The grade + + + means no intact cells on the surface of the tissue fragments seen an(l gross swelling and disorganization of the amount the in 1, Fig. be seen as can and, fibrous strand. of these increased with time, indicating ND = not (lone. + A
FIG. 1. Growth of M. ulcerans on the surface of (b) after 40 days. Z.N. x 48.
an organ culture of muscle
(a) after 10 days,
THE GROWTH OF SOMIE MYCOBACTERIA ON CULTURED IIUMAN TISSUES
10]
FIG. 2. (a) Necrosis of peripheral cells of Ml. ulceran8 infected culture of human foetal lunig. The surface is at the top of the picture and the relatively normal inner tissue below. control culture of human foetal lung. H. & E. x 450.
clearly that the bacteria were multiplying. Organisms appeared to grow most rapidly and extensively on muscle, and less readily on lung and kidney. On skin, they were attached particularly to the fibrous tissue rather than to the epithelium. The tissue in the control cultures showed some deterioration after long periods of incubation but it was quite clear that there was much more cell death in inoculated cultures. Furthermore, necrosis was seen mainly in the outer rim of tissue while the inside of the fragment remained normal. Cells showed karyorrhexis or pyknosis, and the fibrous stroma became structureless (Fig. 2). This was most obvious in lung and in the tissue harvested at 10 days. Liver cells were also cultured but died rapidly and organisms were not seen. These
(b) Uninfected
experiments showed thiat the organisms can multiply in association with living tissue and that it seems to have a toxic effect in certain cells.
Other mycobacteria and adult tissue In order to extend these studies, Mi. ulcerans and other species of bacteria were added to cultures of human adult striated muscle, fascia and subcutaneous fat. After 3 weeks of culture the results were as shown in Table II. It seemed that organisms had grown on the muscle and fibrous tissue but not on the fat. We wondered if the bacteria had failed to grow because the tissue did not provide the right nutrients or because the organisms did not stick firmly to the surface. Further experiments were therefore made in which cultures which had been inocu-
102)
D. A. J. TYRRELL, S. L. McLAUCHLAN ANI) C. S. GOODWIN
TABLE II. Summary of Results of Inoculating Mycobacteria into Organ Cultures of Human Adult Tissues (3 Weeks Incubation) Presence of bacteria in culltures inoculated with Tissue AMuscle Fat Fibrous tis,iue (fascia)
M. ulcorans +
lkI. tuberculosis M. (BCG) xenopi 0 ++
0
0
+
+
0 0
survival of the dermal epithelium. Muiscle is not much involved in the human disease but this might be due to its higher temperature. Nevertheless, in infected mice large numbers of organisms grow in a narrow band under the skin and the adjacent muscle becomes necrotic (Dodge, 1964). None of our experiments prove the existence of a toxin and in other experiments, not reporti-d here, we were unable to demonstrate a toxic effect with extracts of infected LowensteinJensen slopes. It seems likely that it is some feature of the postulated toxin, or of its effect on tissue or white cells, which explains the lack of cellular infiltration in the human disease. Further laboratory experiments will be needed to confirm and elaborate this hypothesis. There might be an enzyme which produced the changes in the fibrous stroma.
lated as described and allowed to incubate for 3 h were washed by vigorously pipetting about 5 ml of Hanks' saline over the tissue. The tissue was fixed and stained and it was noted that M. ulcerans and M. tuberculosis had stuck to the fibrous tissue and muscle but not to the fat. On the other hand M. xenopi had not stuck to any of the tissues. The failure of this organism to grow could well have been due to failure to adhere. We thank Mr A. G. Cox for human In other stimilar studies, M. ulcerans tissue and Dr J. Stanford for the culture adhered to foetal tissue. of M. ulcerans. We thank Mr C. Sowter for technical assistance, Dr D. Rubenstein DISCUSSION for help in initial experiments and ProWe would like to be able to explain fessor M. Hutt for helpful discussions. the pathogenicity of M. ulcerans on the We thank Mr J. B. Clark for the photobasis of its behaviour in the laboratory. micrographs. Obviously the fact that it divides slowly and is inhibited by higher temperatures REFERENCES would explain why it causes lesions which J. K. (1964) Mycobacterial Skiii Ulcers develop slowly and affect mainly super- CLANCEY, in Ugandla: Description of a New Mycobacterium ficial tissues. Both in organ culture and in (AlMjcobacteriumn buruli). J. Path. Bact., 88, 175. vivo the organism remains extracellular. DODcIE, 0. G. (1964) Mycobacterial Skin Ulcers in Uganda: Histopathological and Experimental AsIt is tempting to believe that it grows pects. J. Path. Bact., 88. 167. in association with fibrous tissue under LANCET (1972) Whence Buru1i? Lanicet, i, 363. P., TOLHURST. J. C., BUCKLE, G. & the skin because it can attach to and MACCALLUM, SIssoNs, H. A. (1948) A New Mycobacterial grow well on such tissue, and that it Infection in Man. J. Path. Bact., 60, 93. does not grow on fat cells because it UGANDA BURULI GROUP (1 970) Clinical Featuires an(I Treatment of Pre-ulcerative Buruli Lesions cannot stick to them; nevertheless, it (Mycobacteriurn ulcerans Infection). Br. mied. can kill adjacent cells such as fat by 1., ii, 390. means of the toxin which it seems to UGANDA BURULI GROUP (1971) Epidemiology of Mycobacterium ulcerans Infection (Buruli Ulcer) produce. Foetal skin epithelium is relaat Kinyara, Uganda. Trans. R. Soc. trop. Med. tively unaffected and this agrees with the Hyg., 65, 763.
THE GROWTH OF SOME MYCOBACTERIA ON CULTURED HUMAN TISSUES D. A. J. TYRRELL, S. L. McLAUCHLAN AND C. S. GOODWIN From the Clinical Research Centre and Northwick Park Hospital, Watford Road, Harrow, Middlesex HAI 3 UJ
Receive(I for publication June 11, 1974
Summary.-M. ulcerans grows in close association with the surface of cultured human foetal and adult tissue and produces necrosis. M. tuberculosis grows also but M. xenopi grows little or not at all. The two first named seem to stick well to the tissue surface and the last does not. These results are discussed in relationship to the pathogenesis of Buruli ulcer.
A STRIKING and characteristic disease is caused by Mycobacterium ulcerans. This is variously named and occurs in many parts of the world. It was first recognized in Australia (MacCallum et al., 1948) and is called Buruli ulcer in Uganda. It appears to be due to organisms being introduced under the skin from the immediate environment (Uganda Buruli Group, 1971; Lancet, 1972). The organisms are found in association with the strands of fibrous tissue and give rise to characteristic painless subcutaneous nodules. The ulcer occurs when there is extensive necrosis of the subcutaneous fat and this finally breaks through the overlying skin (Uganda Buruli Group, 1970). The organism grows slowly on bacteriological media and requires a temperature of 330 (Clancey, 1964; MacCallum et al., 1948). We wished to study some of the elements of the pathological process in vitro and for this purpose we used the method of organ culture in plastic petri dishes. It was possible to keep tissues alive for weeks and, by microscopy of fixed and stained sections, we have studied the growth of the organisms and their effect on the tissues.
School. 31. xenopi and Ml. tuberculosis (BCG)
were standard laboratory strains. The 1. ulcerans was known to be pathogenic for mice. Culture. -All organisms were grown on
Lowenstein and Jensen medium and suspended Care was taken that egg medium was not removed with the bacteria. Smooth suspensions were matched to Wellcome opacity tube no. 4. Cultures were inoculated by dropping a total of 0-2 ml of suspension on to the tissue in each dish. The organisms were shown to grow freely in liquid medium in the presence of the mixture of antibiotics used in the organ cultures. in sterile physiological saline.
Tissues Human foetal tissue was obtained fromn Dr H. E. M. Kay, Tissue Bank, Royal Marsden Hospital. Adult fat, muscle and fibrous tissue were obtained incidentally at operations involving the anterior abdominal wall. Organ culture Preparation. -Tissues were dissected with as little handling as possible. Pieces of tissue approximately 0-2 cm3 were cut with a scalpel using a rocking motion and were placed on sterile stainless steel mesh triangles (mesh 50 holes per inch) in 50 mm Nunclon petri dishes; 2-5 ml of medium was added to each petri dish. The medium was Liebowitz L15, supplemented with 0.2% bovine plasma albumin, 100 u/mI of penicillin and either 100 ,ug/ml or 5 ug/ml of colistin sulphate. Medium was changed every 3 or 4 days. The cultuires were incubated in air at 330.
MATERIALS AND METHODS
Bacteria Strains. MJ. ulcerans was obtained from Dr John Stanford, Middlesex Hospital Medical
Harvesting and fixation The tissue and mesh triangles were fixed in formol saline (10% formalin in saline).
I 00)
D). A. J. TYRRELL, S. L. McLAUCHLAN AND C. S. GOODWIN
The tissutes wvere sectioned and stained with lhaernatoxylin and eosin, Ziehl-Neelsen and Triff stains.
RESULTS
TABLE I. Sunmmary of Results of Inoculatiny M. ulcerans into Organ Cultures of Human Foetal Tissues P'reseicie (of Tissue
Skinl
Time in days 1() 20
Mycobacteria
Necrosis
++ ++
The first experiment was intended to determine if M. ulcerans would multiply 40 -t- -t A in organ culture of human tissues. A Muscle 10 + -1 20 human foetus of 16 weeks was a con+j + 40 venient source of sterile tissue, and Lunig 10 N+ cultures of skin, muscle, lung and kidney 20 ++ + -I ND ND described. as 40 inoculated were set up and 10 The medium remained clear but the tissue Kidney +I-A + 20 of inoculated cultures became bulky and A40 ++4 gelatinous compared with uninoculated Bacterial growth is graded arbitrarily. Comcontrol cultures. The results of microwith Fig. 1. Uninoculated cultures showed scopic examination are summarized in pare some degeneration which increased( during cultivaTable I. Masses of organisms were seen tion. The necrosis is graded by comparison with these. The grade + + + means no intact cells on the surface of the tissue fragments seen an(l gross swelling and disorganization of the amount the in 1, Fig. be seen as can and, fibrous strand. of these increased with time, indicating ND = not (lone. + A
FIG. 1. Growth of M. ulcerans on the surface of (b) after 40 days. Z.N. x 48.
an organ culture of muscle
(a) after 10 days,
THE GROWTH OF SOMIE MYCOBACTERIA ON CULTURED IIUMAN TISSUES
10]
FIG. 2. (a) Necrosis of peripheral cells of Ml. ulceran8 infected culture of human foetal lunig. The surface is at the top of the picture and the relatively normal inner tissue below. control culture of human foetal lung. H. & E. x 450.
clearly that the bacteria were multiplying. Organisms appeared to grow most rapidly and extensively on muscle, and less readily on lung and kidney. On skin, they were attached particularly to the fibrous tissue rather than to the epithelium. The tissue in the control cultures showed some deterioration after long periods of incubation but it was quite clear that there was much more cell death in inoculated cultures. Furthermore, necrosis was seen mainly in the outer rim of tissue while the inside of the fragment remained normal. Cells showed karyorrhexis or pyknosis, and the fibrous stroma became structureless (Fig. 2). This was most obvious in lung and in the tissue harvested at 10 days. Liver cells were also cultured but died rapidly and organisms were not seen. These
(b) Uninfected
experiments showed thiat the organisms can multiply in association with living tissue and that it seems to have a toxic effect in certain cells.
Other mycobacteria and adult tissue In order to extend these studies, Mi. ulcerans and other species of bacteria were added to cultures of human adult striated muscle, fascia and subcutaneous fat. After 3 weeks of culture the results were as shown in Table II. It seemed that organisms had grown on the muscle and fibrous tissue but not on the fat. We wondered if the bacteria had failed to grow because the tissue did not provide the right nutrients or because the organisms did not stick firmly to the surface. Further experiments were therefore made in which cultures which had been inocu-
102)
D. A. J. TYRRELL, S. L. McLAUCHLAN ANI) C. S. GOODWIN
TABLE II. Summary of Results of Inoculating Mycobacteria into Organ Cultures of Human Adult Tissues (3 Weeks Incubation) Presence of bacteria in culltures inoculated with Tissue AMuscle Fat Fibrous tis,iue (fascia)
M. ulcorans +
lkI. tuberculosis M. (BCG) xenopi 0 ++
0
0
+
+
0 0
survival of the dermal epithelium. Muiscle is not much involved in the human disease but this might be due to its higher temperature. Nevertheless, in infected mice large numbers of organisms grow in a narrow band under the skin and the adjacent muscle becomes necrotic (Dodge, 1964). None of our experiments prove the existence of a toxin and in other experiments, not reporti-d here, we were unable to demonstrate a toxic effect with extracts of infected LowensteinJensen slopes. It seems likely that it is some feature of the postulated toxin, or of its effect on tissue or white cells, which explains the lack of cellular infiltration in the human disease. Further laboratory experiments will be needed to confirm and elaborate this hypothesis. There might be an enzyme which produced the changes in the fibrous stroma.
lated as described and allowed to incubate for 3 h were washed by vigorously pipetting about 5 ml of Hanks' saline over the tissue. The tissue was fixed and stained and it was noted that M. ulcerans and M. tuberculosis had stuck to the fibrous tissue and muscle but not to the fat. On the other hand M. xenopi had not stuck to any of the tissues. The failure of this organism to grow could well have been due to failure to adhere. We thank Mr A. G. Cox for human In other stimilar studies, M. ulcerans tissue and Dr J. Stanford for the culture adhered to foetal tissue. of M. ulcerans. We thank Mr C. Sowter for technical assistance, Dr D. Rubenstein DISCUSSION for help in initial experiments and ProWe would like to be able to explain fessor M. Hutt for helpful discussions. the pathogenicity of M. ulcerans on the We thank Mr J. B. Clark for the photobasis of its behaviour in the laboratory. micrographs. Obviously the fact that it divides slowly and is inhibited by higher temperatures REFERENCES would explain why it causes lesions which J. K. (1964) Mycobacterial Skiii Ulcers develop slowly and affect mainly super- CLANCEY, in Ugandla: Description of a New Mycobacterium ficial tissues. Both in organ culture and in (AlMjcobacteriumn buruli). J. Path. Bact., 88, 175. vivo the organism remains extracellular. DODcIE, 0. G. (1964) Mycobacterial Skin Ulcers in Uganda: Histopathological and Experimental AsIt is tempting to believe that it grows pects. J. Path. Bact., 88. 167. in association with fibrous tissue under LANCET (1972) Whence Buru1i? Lanicet, i, 363. P., TOLHURST. J. C., BUCKLE, G. & the skin because it can attach to and MACCALLUM, SIssoNs, H. A. (1948) A New Mycobacterial grow well on such tissue, and that it Infection in Man. J. Path. Bact., 60, 93. does not grow on fat cells because it UGANDA BURULI GROUP (1 970) Clinical Featuires an(I Treatment of Pre-ulcerative Buruli Lesions cannot stick to them; nevertheless, it (Mycobacteriurn ulcerans Infection). Br. mied. can kill adjacent cells such as fat by 1., ii, 390. means of the toxin which it seems to UGANDA BURULI GROUP (1971) Epidemiology of Mycobacterium ulcerans Infection (Buruli Ulcer) produce. Foetal skin epithelium is relaat Kinyara, Uganda. Trans. R. Soc. trop. Med. tively unaffected and this agrees with the Hyg., 65, 763.
