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Bacteriophage prevents destruction of skin grafts by Pseudomonas aeruginosa
209
Bacteriophage prevents destruction of skin grafts by Pseudomonas aeruginosa J. S. Soothill Department of Infection, The Medical School, Birmingham Hospital, UK
and The Bums Research Group, Birmingham
lnfection of split skin graffts inguinea-pigs by Pseudomonas aeruginosa 3719 destroys them, ana’ bacteriophage BS24, lytic for struin 3719, protects the graffs. 2%~ supports the vim that phage could be used to prevent infection of skin grafIs applied to the contaminakd woud5 of burned patienfs.
Bums (1994) 20, (3), 209-211
Introduction aemginusa frequently colonizes bumsl, and this Pseudomonus may lead to destruction of grafts, enlargement of wounds, and generalized, sometimes fatal, infection. Topical prophylactic agents are of limited value, and antibiotic treatment is often ineffective, partly because of bacterial resistance. Since Smith et a1.2*3showed that bacteriophages can be highly effective in the treatment of experimental Eschetichia coli infection, reappraisal is needed for their use in infections by antibiotic-resistant bacteria such as prophylaxis or treatment of Ps. aeruginosa infections in grafted burns. Therefore studies were made of the effect of Ps. aeruginosa infection on skin graft survival in guinea-pigs, and its control by an appropriate bacteriophage.
Materials
Accident
shaved area of skin on the left side of the back. A rectangle 2 cm x 1 cm was incised in the skin, and a partial thickness skin graft about 0.2 mm thick was removed using a Weck’s knife. A second slice of skin was removed to create a full thickness defect down to subcutaneous fat, comparable with that of an excised bum. 0.01 ml of bacteria, and/or phage, and/or control suspension was applied to the defect and the graft was then replaced in the defect and covered by a compression pack and then a large dressing. Grafts were blindly assessed 5 days later as ‘taken’ or ‘failed’. Typical examples are shown in Figures 1-3. Those that had partially taken were classed as taken or failed depending on whether more or less than half the graft had taken. Areas of graft were considered to have taken if they were covered with skin that was pink and blanched on pressure.
Statistical analysis This was by Fisher’s exact test (two-tailed).
and methods
Ps.awclgnzoSa strain 3719 was a clinical isolate from the Burns Unit, Birmingham Accident Hospita1 and was of known high virulente for mice’. Phages for Ps. aacginosa were isolated from sewage using the method described by Adams5, scaled up to use 15-litre volumes of sewage. The 169 isolateS obtained were tested to determine the minimum number of plaque-forming units (p.f.u) required to lyse, at 37”C, a shaken broth culture of Ps. aerugimsa, for which the starting inoculum was 105 colony-forming units (c.f.u.). The most effective phage, BS24, was selected for animal experiments. Methods for counting organisms and for production and purification of the phage and control suspensions and investigating their systemic toxicity to mice have been described by SoothilY. Local effects of phage and a control suspension on single uninfected guinea-pig skin grafts were assessed using the methods described below. Surgery Guinea-pigs (Dunkin Hartley strain) were anaesthetized with halothane. Seventy per cent ethanol was sprayed on a 0 1994 Butterworth-Heinemann 0305-4179/94/030209-03
Ltd
Figure 1. Appearance, 5 days after operation, graf% that took completely.
of a phage-treated
Bums (1994) Vol. 20/No. 3
210
TableI. Outcome of four guinea-pig grafts in the presence of 1.5 x 10~c.f.u. Ps. mginosa 3719 and of four control grafts With Ps. aeruginosa
Without Ps. aeruginosa
0 4
4 0
Took (no.) Failed (no.) P= 0.028.
Table 11. Outcome of graf& to 14 guinea-pigsin the presence of 6.0 x 10’ c.f.u.Ps.aeruginosa3719 and either 1.2 x 1O’p.f.u.phage Bs24 (seven guinea-pigs) or control suspension (seven guineapigs)
Took Failed
Phage treated
Controls
6 1
0 7
P= 0.0047.
Figure 2. Appearance,5 days after operation, of a phage-treated
graft more than half of which took and was therefore classed as taken.
both failed. In a larger study, the grafts of four guinea-pigs failed after receiving 1.5 x 106 c.f.u. of Ps.aerccginosa,whereas those of four uninoculated controls ‘took’ (TableI). Thus Ps. aenrginosasignificantly inhibited graft take. Effects of phage on pseudomonas-infected grafts 1.2 x 10’ p.f.u. of phage BS24 significantly protected a group of seven grafts infected with 6.0 x 10’ c.f.u, of Ps.
aeru@nosa3719 (XddeII, Fipres 1,~) as compared with seven grafts treated with the phage-free control suspension (Figure 3). Effeds of phage and control suspension on uninfected grafts
The two guinea-pigs whose grafts were treated respectively with phage and with control suspension appeared healthy throughout the study. Both grafts had taken fully 5 days after the operations.
Discussion
Figure 3. Appearance of a control graft 5 days after operation,
classed as failed.
lhdts Invitroeffects of bacteriophages Of the 169 isolates of phage BS24 was the most active, 60 p.f.u. being the minimum dose capable of lysing a broth culture seeded with 10’ c.f.u. of Ps. aertcginosa3719. Effects of Ps. uerugitwtz on skin grafts
The grafts of two guinea-pigs which had received respectively 7.2 x 108 and 1.2 x 104 c.f.u. of Ps. LWL@KW3719
The results confirm the clinical evidente’ that infection with Ps. at=n@wsu results in failure of skin grafts. Application during grafting of phage, lytic for the ínfecting strain of Ps. UU@~ILJ~JZ, permitted graft survival. This is an in vivo confirmation of the previous demonstration that phage BS24 can penneate dead dermis, and prevent its destruction by Ps. aerugirwsa6. Excised skin defects without burning were used in these experiments, to minimize suffering. They are probably an adequate model for the defects produced when bums are excised before grafting. The application of phage at the time of grafting onto a pseudomonas-contaminated bed, as in this experiment, could be carried out on pseudomonas-infected patients. Although the phage/bacterium ratio in this experiment was high (20/1), ratios much higher than this might well be obtainable on freshly excised human bums, where numbers of bacteria are small. Some phages have been concentrated’ to lOlz per ml so favourable phage/bacterium ratios might be obtainable even in the presence of large bacterial numbers. The possibiiity that prophylactic agents can be applied to excised bum wounds in patients colonized by pseudomonas, before grafting,
is attractive
but none
of the agents
Soothill: Phage treatment of skin graft infections
currently available are suitable for the purpose as they themselves interfere with nraft take, and topical 1 antibiotics such as gentamicin would hasten the emergence of resistant strains. Purified suspensions of phage appear to have little toxicity and could be so applied. Since most phages are fairly strain specific the genera1 use of phage for this purpose would entail using a mixture of phages effective against the full range of strains of Ps. aemginosu.Since most strains can be typed using about 20 phages, the production
211
References
would
of such a mixture may wel1 be possible. The observational study is encouraging8. An appropriate phage (purified, and tested for toxicity on human keratinocytes in culture) proliferated looo-fold when applied, on filter paper discs, to a pseudomonas-infected human bum; when it was sprayed over the whole area, pseudomonas cultures became negative, and grafts took, but this was not necessarily the effect of phage, since antibiotics were also used. The results of the guinea-pig studies in this report support the need for appropriate controlled trials in human bums.
Acknowledgements 1would like to thank the following people for their help and
1 2
3
7
8
Jackson DM, Lowbury EJL, Topley E. Pseudonzmas pyoqanea in bums. Lancet 195 1; ii: 137-147. Smith HW, Huggins MB. Successful treatment of experimental Exhmchia coli infections in mice using phage: its general superiority over antibiotics. J Gen Microbiol 1982; 128: 307-318. Smith HW, Huggins MB, Shaw KM. The control of experimental Exherichia coli diarrhoea in calves by means of bacteriophages.] Gen Microbioll987; 133: 1111-1126. Soothill JS. Treatment of experimental infections of mice with bacteriophages. ] Med Microbiol 1992; 37: 258-261. Adams MH. Buctetiophages. New York: Interscience, 1959. Soothill JS, Lawrence JC, Ayliffe GAJ. The efficacy of phages in the prevention of the destruction of pig skin in vitro by Ps&omonas aeruginosa.Mtd .%i fhs 1988; 16: 1287-1288. Jarrell K, Kropinski AMB. The isolation and characterization of a lipopolysaccharide specific PsarAomonas amginosa bacteriophage. ] Gen Virol 1976; 33: 99-106. Soothill JS. Use of Bacteriophage in the Treatment of Infections. MD Thesis, University of London, 1993.
Paper accepted
25 October
1993.
advice: The staff of the Biomedical Services Department of Birmingham University, and of the Microbiology Group in
Aston University Department of Pharmaceutical Sciences. Dr J. C. Lawrence, Professor J. F. Soothill and the late Dr H. Williams Smith. This study was supported by a grant from the Wellcome
Trust.
Correspondence shouh be adresed to: Mr J. S. Soothill, Department of Manchester, Stopford Building, Oxford Road, Manchester Ml3 9PL, UK.
of Pathological Sciences, University
~TH INTERNATIONAL SEMINARON WOUND HEALINGAND WOUND MOVEMENT CHICAGO, ILLINOIS, USA OCTOBER
8-9,1994
T’he International Bum Foundation is sponsoring the 7th annual two day International Symposium on Wound Healing & Wound Management, October 8-9, 1994 in Chicago. This is a Saturday and Sunday preceding the Clinical Congress of the American College of Surgeons. rhis meeting is designed to assemble some intemationally recognized experts in wound healing md wound management to discuss the state of the art in these topics. The in-depth program wil1cover basic and clinical research and application to patient care. Potential topics include: principles of wound healing synthetic slcin epithelial cel1 cultures wound assessment wound hormones pressure sores
growth factors biosynthetic dressings cultured dermis and epidermal tissues immune response & wound healing prostaglandin’s in wound healing wound contracturo
For further infomation please contact: Dr John A. Boswick, FACS., Course Directer, Merndional Bum Foundation, 2005 Franklin Street, #355, Denver, Colorado 80205, USA. Iel: (303) 839 1694 or Fax: (303) 839 1695.
Bacteriophage prevents destruction of skin grafts by Pseudomonas aeruginosa J. S. Soothill Department of Infection, The Medical School, Birmingham Hospital, UK
and The Bums Research Group, Birmingham
lnfection of split skin graffts inguinea-pigs by Pseudomonas aeruginosa 3719 destroys them, ana’ bacteriophage BS24, lytic for struin 3719, protects the graffs. 2%~ supports the vim that phage could be used to prevent infection of skin grafIs applied to the contaminakd woud5 of burned patienfs.
Bums (1994) 20, (3), 209-211
Introduction aemginusa frequently colonizes bumsl, and this Pseudomonus may lead to destruction of grafts, enlargement of wounds, and generalized, sometimes fatal, infection. Topical prophylactic agents are of limited value, and antibiotic treatment is often ineffective, partly because of bacterial resistance. Since Smith et a1.2*3showed that bacteriophages can be highly effective in the treatment of experimental Eschetichia coli infection, reappraisal is needed for their use in infections by antibiotic-resistant bacteria such as prophylaxis or treatment of Ps. aeruginosa infections in grafted burns. Therefore studies were made of the effect of Ps. aeruginosa infection on skin graft survival in guinea-pigs, and its control by an appropriate bacteriophage.
Materials
Accident
shaved area of skin on the left side of the back. A rectangle 2 cm x 1 cm was incised in the skin, and a partial thickness skin graft about 0.2 mm thick was removed using a Weck’s knife. A second slice of skin was removed to create a full thickness defect down to subcutaneous fat, comparable with that of an excised bum. 0.01 ml of bacteria, and/or phage, and/or control suspension was applied to the defect and the graft was then replaced in the defect and covered by a compression pack and then a large dressing. Grafts were blindly assessed 5 days later as ‘taken’ or ‘failed’. Typical examples are shown in Figures 1-3. Those that had partially taken were classed as taken or failed depending on whether more or less than half the graft had taken. Areas of graft were considered to have taken if they were covered with skin that was pink and blanched on pressure.
Statistical analysis This was by Fisher’s exact test (two-tailed).
and methods
Ps.awclgnzoSa strain 3719 was a clinical isolate from the Burns Unit, Birmingham Accident Hospita1 and was of known high virulente for mice’. Phages for Ps. aacginosa were isolated from sewage using the method described by Adams5, scaled up to use 15-litre volumes of sewage. The 169 isolateS obtained were tested to determine the minimum number of plaque-forming units (p.f.u) required to lyse, at 37”C, a shaken broth culture of Ps. aerugimsa, for which the starting inoculum was 105 colony-forming units (c.f.u.). The most effective phage, BS24, was selected for animal experiments. Methods for counting organisms and for production and purification of the phage and control suspensions and investigating their systemic toxicity to mice have been described by SoothilY. Local effects of phage and a control suspension on single uninfected guinea-pig skin grafts were assessed using the methods described below. Surgery Guinea-pigs (Dunkin Hartley strain) were anaesthetized with halothane. Seventy per cent ethanol was sprayed on a 0 1994 Butterworth-Heinemann 0305-4179/94/030209-03
Ltd
Figure 1. Appearance, 5 days after operation, graf% that took completely.
of a phage-treated
Bums (1994) Vol. 20/No. 3
210
TableI. Outcome of four guinea-pig grafts in the presence of 1.5 x 10~c.f.u. Ps. mginosa 3719 and of four control grafts With Ps. aeruginosa
Without Ps. aeruginosa
0 4
4 0
Took (no.) Failed (no.) P= 0.028.
Table 11. Outcome of graf& to 14 guinea-pigsin the presence of 6.0 x 10’ c.f.u.Ps.aeruginosa3719 and either 1.2 x 1O’p.f.u.phage Bs24 (seven guinea-pigs) or control suspension (seven guineapigs)
Took Failed
Phage treated
Controls
6 1
0 7
P= 0.0047.
Figure 2. Appearance,5 days after operation, of a phage-treated
graft more than half of which took and was therefore classed as taken.
both failed. In a larger study, the grafts of four guinea-pigs failed after receiving 1.5 x 106 c.f.u. of Ps.aerccginosa,whereas those of four uninoculated controls ‘took’ (TableI). Thus Ps. aenrginosasignificantly inhibited graft take. Effects of phage on pseudomonas-infected grafts 1.2 x 10’ p.f.u. of phage BS24 significantly protected a group of seven grafts infected with 6.0 x 10’ c.f.u, of Ps.
aeru@nosa3719 (XddeII, Fipres 1,~) as compared with seven grafts treated with the phage-free control suspension (Figure 3). Effeds of phage and control suspension on uninfected grafts
The two guinea-pigs whose grafts were treated respectively with phage and with control suspension appeared healthy throughout the study. Both grafts had taken fully 5 days after the operations.
Discussion
Figure 3. Appearance of a control graft 5 days after operation,
classed as failed.
lhdts Invitroeffects of bacteriophages Of the 169 isolates of phage BS24 was the most active, 60 p.f.u. being the minimum dose capable of lysing a broth culture seeded with 10’ c.f.u. of Ps. aertcginosa3719. Effects of Ps. uerugitwtz on skin grafts
The grafts of two guinea-pigs which had received respectively 7.2 x 108 and 1.2 x 104 c.f.u. of Ps. LWL@KW3719
The results confirm the clinical evidente’ that infection with Ps. at=n@wsu results in failure of skin grafts. Application during grafting of phage, lytic for the ínfecting strain of Ps. UU@~ILJ~JZ, permitted graft survival. This is an in vivo confirmation of the previous demonstration that phage BS24 can penneate dead dermis, and prevent its destruction by Ps. aerugirwsa6. Excised skin defects without burning were used in these experiments, to minimize suffering. They are probably an adequate model for the defects produced when bums are excised before grafting. The application of phage at the time of grafting onto a pseudomonas-contaminated bed, as in this experiment, could be carried out on pseudomonas-infected patients. Although the phage/bacterium ratio in this experiment was high (20/1), ratios much higher than this might well be obtainable on freshly excised human bums, where numbers of bacteria are small. Some phages have been concentrated’ to lOlz per ml so favourable phage/bacterium ratios might be obtainable even in the presence of large bacterial numbers. The possibiiity that prophylactic agents can be applied to excised bum wounds in patients colonized by pseudomonas, before grafting,
is attractive
but none
of the agents
Soothill: Phage treatment of skin graft infections
currently available are suitable for the purpose as they themselves interfere with nraft take, and topical 1 antibiotics such as gentamicin would hasten the emergence of resistant strains. Purified suspensions of phage appear to have little toxicity and could be so applied. Since most phages are fairly strain specific the genera1 use of phage for this purpose would entail using a mixture of phages effective against the full range of strains of Ps. aemginosu.Since most strains can be typed using about 20 phages, the production
211
References
would
of such a mixture may wel1 be possible. The observational study is encouraging8. An appropriate phage (purified, and tested for toxicity on human keratinocytes in culture) proliferated looo-fold when applied, on filter paper discs, to a pseudomonas-infected human bum; when it was sprayed over the whole area, pseudomonas cultures became negative, and grafts took, but this was not necessarily the effect of phage, since antibiotics were also used. The results of the guinea-pig studies in this report support the need for appropriate controlled trials in human bums.
Acknowledgements 1would like to thank the following people for their help and
1 2
3
7
8
Jackson DM, Lowbury EJL, Topley E. Pseudonzmas pyoqanea in bums. Lancet 195 1; ii: 137-147. Smith HW, Huggins MB. Successful treatment of experimental Exhmchia coli infections in mice using phage: its general superiority over antibiotics. J Gen Microbiol 1982; 128: 307-318. Smith HW, Huggins MB, Shaw KM. The control of experimental Exherichia coli diarrhoea in calves by means of bacteriophages.] Gen Microbioll987; 133: 1111-1126. Soothill JS. Treatment of experimental infections of mice with bacteriophages. ] Med Microbiol 1992; 37: 258-261. Adams MH. Buctetiophages. New York: Interscience, 1959. Soothill JS, Lawrence JC, Ayliffe GAJ. The efficacy of phages in the prevention of the destruction of pig skin in vitro by Ps&omonas aeruginosa.Mtd .%i fhs 1988; 16: 1287-1288. Jarrell K, Kropinski AMB. The isolation and characterization of a lipopolysaccharide specific PsarAomonas amginosa bacteriophage. ] Gen Virol 1976; 33: 99-106. Soothill JS. Use of Bacteriophage in the Treatment of Infections. MD Thesis, University of London, 1993.
Paper accepted
25 October
1993.
advice: The staff of the Biomedical Services Department of Birmingham University, and of the Microbiology Group in
Aston University Department of Pharmaceutical Sciences. Dr J. C. Lawrence, Professor J. F. Soothill and the late Dr H. Williams Smith. This study was supported by a grant from the Wellcome
Trust.
Correspondence shouh be adresed to: Mr J. S. Soothill, Department of Manchester, Stopford Building, Oxford Road, Manchester Ml3 9PL, UK.
of Pathological Sciences, University
~TH INTERNATIONAL SEMINARON WOUND HEALINGAND WOUND MOVEMENT CHICAGO, ILLINOIS, USA OCTOBER
8-9,1994
T’he International Bum Foundation is sponsoring the 7th annual two day International Symposium on Wound Healing & Wound Management, October 8-9, 1994 in Chicago. This is a Saturday and Sunday preceding the Clinical Congress of the American College of Surgeons. rhis meeting is designed to assemble some intemationally recognized experts in wound healing md wound management to discuss the state of the art in these topics. The in-depth program wil1cover basic and clinical research and application to patient care. Potential topics include: principles of wound healing synthetic slcin epithelial cel1 cultures wound assessment wound hormones pressure sores
growth factors biosynthetic dressings cultured dermis and epidermal tissues immune response & wound healing prostaglandin’s in wound healing wound contracturo
For further infomation please contact: Dr John A. Boswick, FACS., Course Directer, Merndional Bum Foundation, 2005 Franklin Street, #355, Denver, Colorado 80205, USA. Iel: (303) 839 1694 or Fax: (303) 839 1695.