ANTISERUM AND ANTIBIOTIC IN THE PROPHYLAXIS OF BURNS AGAINST PSEUDOMONAS AERUGINOSA

Transcription

1 ANTISERUM AND ANTIBIOTIC IN THE PROPHYLAXIS OF BURNS AGAINST PSEUDOMONAS AERUGINOSA By R. J. JONES, B.Sc., Ph.D., D. McG. JACKSON, M.D., F.R.C.S. and E. J. L. LowmlRy, D.M., F.C.Path. From the Medical Research Council Industrial Injuries and Burns Research Unit Birmingham Accident Hospital THE pathogenic role of Pseudomonas aeruginosa (pyocyanea) in patients with burns has been widely reported (Colebrook, Duncan, and Ross, I948 ; Jackson, Lowbury, and Topley, i951 ; Liedberg, Reiss, and Artz, I954 ; Markley et al., I957 ; Tumbusch et al., I96I ; Decoulx et al., I964 ; Sevitt, 1964 ; Teplitz et al., 1964). Both local and general effects are described, but particular emphasis has been placed on invasive infection, often with septicmmia, which is a common cause of death in severely burned patients. At the same time it is recognised thatps, aeruginosa is a weak" opportunistic" pathogen, causing clinical infection only in subjects rendered susceptible by such conditions as leukmmia, hypogammaglobulinremia, and burns. Local application of polymyxin has been shown to have some prophylactic and therapeutic value (Jackson et al., I95I ; Cason and Lowbury, 196o). Although systemic treatment with the polymyxins, including colistin, has been found effective in the treatment of some infections with Ps. aeruginosa (e.g., MacMillan et al., 1962 ; Speirs, Selwyn, and Nicholson, I963), their use in the treatment of severely burned patients is reported to have little if any value (Rabin et al., I96I ; Tumbusch et al., I96I ; Kefalides et al., 1964). Some promising results, however, were obtained in the protection of mice against Ps. aeruginosa by gamma globulin (Rosenthal, Millican, and Rust, I957), and a greater protective effect was obtained with specific antiserum (Millican and Rust, 196o). In this paper we describe experiments on the pathogenic role of Ps. aeruginosa and other bacteria in burns of mice and on the relative value of antiserum and of colistin (polymyxin E) methane sulphonate in prophylaxis. We include a brief report on the use of colistin methane sulphonate in prophylaxis and treatment of infection with Ps. aeruginosa in human burns. INFECTION AND PROPHYLAXIS IN MICE MATERIALS AND METHODS Burning.--Albino mice weighing approximately 3 o g. were ana:sthetised with ether and burned over an estimated 6 per cent. of the body surface by application for ten seconds of a brass block heated to IOO C. in water ; this caused a full skin thickness burn with low mortality. The area selected for the burn was depilated on the previous day by plucking under light ether ana:sthesia. The extent of the burn was estimated from the formula of Benedict (I93O-3I). Mice were kept in individual polystyrene cages with perforated lids (Jones, 1964). Infection.--Three hours after burning, the burned surface was inoculated with o'i ml. of a saline suspension containing approximately 4oo million viable organisms prepared from overnight cultures on nutrient agar ; the bacteria were spread over the whole burned area with a wire loop. The following strains of Ps. aeruginosa and of other bacterial species, isolated from human burns and from other sources, were 43

2 44 BRITISH JOURNAL OF PLASTIC SURGERY used: Ps. aeruginosa (8687), Proteus mirabilis (M 892), Escherichia coli (M 894), Klebsiella aerogenes (1838 b), Serratia marcescens (M 895), Staphylococcus aureus (3542), and coagulase-negative micrococcus (M 893). In further experiments, which are still in progress, strains of Ps. aeruginosa with different phage types have been studied (numbers 4054, 4o73, 4369, 4048, 4468, 4065, 4o34, and 41o9). Prophylaxis.--Two forms of prophylaxis against Ps. aeruginosa were studied: (I) intramuscular injection of colistin (" colomycin ") methane sulphonate at three dosage levels corresponding on the basis of body weight approximately to 3, i2, and 48 million units daily in human adult patients (1,75o units, 7,0oo units, and 28#0o units daily) ; two injections were given daily over a period of ten days, the first dose being injected immediately before the application of the bacterial suspension. (2) Injection of an antiserum prepared by immunising rabbits with a formolised suspension ofps. aeruginosa (8687) in the manner described elsewhere for preparation of staphylococcal antisera (Jones and Lowbury, 1963). Two intraperitoneal injections of antiserum were given, one immediately after burning and three hours before application of the bacterial suspension, and one on the sixth day. Two dosage levels of antiserum were used (z and o'oz ml.) ; the titre of agglutinin of the antiserum was I in 2o48. Experiments.--Groups of six or twelve burned mice were used ; in each experiment a control group of uninfected burned mice was included. Daily cultures were obtained with moist swabs from all burns. Swabs were inoculated on blood agar and on cetrimide agar (Lowbury and Collins, I955), and examined for Ps. aeruginosa and other organisms in the manner used for examination of human burns (Jackson et al., 1951). Mice were weighed with a beam scale balance and their skin temperature adjacent to the burn was measured with a skin thermometer daily. The animals were handled with polyethylene gloves at all times ; to prevent cross-infection a clean pair of gloves was worn for the examination of each group of mice. The pan of the balance and the heat sensor probe were disinfected between groups of mice by wiping with o. 5 per cent. chlorhexidine in alcohol. Necropsies were made on mice which died. Heart blood was taken through seared areas and cultures were made in broth, on blood agar and on cetrimide agar. RESULTS Mortality from Infection of Burns.--The mortality of mice with uninfected and with infected burns is shown in Table I. Whereas uninfected mice had a mortality of only 5"1 per cent., those infected with Ps. aeruginosa and Proteus mirabilis showed 71 and 54 per cent. mortality respectively. The mean survival time of mice which died with Ps. aeruginosa infection was 6"2 ±o.18 days, while that of mice which died with Proteus mirabilis infection was 2-8 ±0"27 days ; all but one of the former showed Is. aeruginosa and all of the latter showed Proteus mirabilis in cultures of heart blood taken at autopsy. In contrast with Ps. aeruginosa and Proteus mirabilis, none of the other bacteria was associated with a mortality rate significantly greater than that of the burned but uninfected mice of the control series. Experiments in progress have shown that strains of Ps aeruginosa with different phage types cause widely varying mortality when inoculated on the burns of mice (Table II). The mice which died with Ps. aeruginosa infection looked ill from the second or third day ; in most animals there was a purulent conjunctival discharge which yielded a pure culture ofps. aeruginosa. At autopsy the kidneys of mice which died with Ps. aeruginosa infection and

3 THE PROPHYLAXIS OF BURNS 45 TABLE I Mortality of Mice after 6 per cent. Burn Bacteria Inoculated on Burn Number of Mice --Di---~--~! Percentage u~t Deaths Mean Survival Time of Mice which Died (Days) Blood Cultures at Autopsy of Mice which Died yielding same Organisms as those Inoculated on Burn Significance of Difference between Death Rates of Infected and Uninfected Mice X e P. None 1'3 o Pseudomonas aeruginosa 30 I2 71 "4 6' <o.ooi Proteus mirabilis 13 II 54"2 2"8 L-o-27 I3 i3.6 ' ~o.ooi Escherichia coli 0 I2 O Klebsiella aerogenes 2 IO "5 I Serratia marcescens 2 IO 16.6 IO.5 I Staphylococcus aureus 2 16 II'I 6"0 0!Micrococcus " 5 I 0 TABLE II Mortality of Burned Mice infected with different Phage Types of Ps. aeruginosa / Num~be; of~aic~e I ;ercentage- - Ps. ;eruginosa Serial Number Phage Type of [----/----! of in Heart Blood of Strain Strain I Died Survived J Deaths at Autopsy 4o54 4o73 B 4 B 12 ~ 14 7 L 4 5 i, 77 '7 58' B 13 B 11 ] 7 6 [ '3 5 o.o B io 6 6 5o.o B8 ~ ; 41'6 5 4o34 41o 9 B 5 B 9, I I I 25-o I survived longer than five days showed multiple abscesses of the kidneys (Figs. I and 2) ; these yielded pure cultures of Ps. aeruginosa ; renal abscesses were not found in mice which died from Proteus infection or after infection with other organisms. Mice which died from infection with Ps. aeruginosa lost weight progressively from about the third day, while those which survived, whether infected with Ps. aeruginosa or not, maintained their weight (see Fig. 3)- Figure 4 shows the mean skin temperature of mice on the first eight days after burning. Mice which were infected with

4 46 BRITISH JOURNAL OF PLASTIC SURGERY FIG. I Autopsy appearance of a mouse which died on ninth day after infection of burn with Ps. aeruginosa ; the kidneys show multiple abscesses. FIG. 2 Stained section of kidney from burned mouse which died with Ps. aeruginosa infection (see Fig. I), showing a superficial abscess.

5 THE PROPHYLAXIS OF BURNS 47 Ps. aeruginosa and died showed a higher mean temperature than the control animals on days one to six, but after this the temperature fell steeply In mice which survived after infection with Ps. aeruginosa the mean temperature in the first eight days was similar to that of the uninfected mice. Prophylaxis against Ps. aeruginosa Infection with Colistin.mProphylaxis with colistin methane sulphonate did not reduce appreciably the mortality from Grams O 28 MEAN WEIGHT OF BURNED MICE: INFECTED WITH Ps. AERUGINOSA. o C MEAN SKIN TEMPERATURE OF BURNED MICE ;,, I l I I l ~ ; ~ l l l l DAYS AFTER BURNING Mice which survived ctfter protection with antiserum. - - Mice not protected with (antiserum which died v o x DAYS AFTER BURNING o Uninfected controts x Mice which survived after infection with Ps. cmruqinosca. = - Mice which died offer infection with Ps. aeruqinosa. FIG. 3 FIG. 4 Fig. 3.--The mean weight (grams) after burning of mice successfully protected with antiselum against Ps. aeruginosa and of mice which were unprotected and died. Fig. 4.--The mean skin temperature (cc.) of uninfected burned mice, of burned mice which died with Ps. acruginosa infection, and of burned mice which were infected with Ps. aeruginosa and survived. Ps. aeruginosa infection, but when given at the highest dosage it delayed the onset of symptoms and lengthened the period of survival (see Table III). Swabs from the surfaces of the burn showed a reduction and sometimes a transient disappearance ofps. aeruginosa when the highest dosage of colistin was given. The minimal inhibitory concentration of colistin methane sulphonate against the strain ofps. aeruginosa in this experiment was r6 units (r "3 Fg.) per millilitre. Prophylaxis against Ps. aeruginosa Infection with an Antiserum.--Mice treated with antiserum prepared against Ps. aeruginosa were protected ; even when the lowest dosage was used all the mice receiving this treatment survived (Table IV). Ps. aeruginosa was not removed from the surface of the burn by this treatment ; prophylactic action of antiserum was presumably attributable to the failure of Ps. aeruginosa to invade the tissues and blood stream of protected mice. Local Effects of Baeteria in Burns.raThe burns of the uninfected mice appeared dry and had a dull yellow appearance. By contrast, burns of the mice infected with

6 48 BRITISH JOURNAL OF PLASTIC SURGERY i None TABLE III Survival of Mice after 6 per cent. Burn with Ps. aeruginosa Infection: Prophylaxis by Colistin Methane Sulphonate Prophylactic I Number of Mice Mean Survival [ Treatment J Percentage Time of Mice ] (Dose of Colistin which Died Methane Sulphonate) Died Survived] i Deaths of (Days) i 1,75o (units/day) i 7,000 (units/day) 28,000 (units/day) I " o " '6 6"2 6"6 6"7 io.25 Positive Blood Cultures at Autopsy on Mice which Died TABLE IV Survival of Mice after 6 per cent. Burn with Ps. aeruginosa Infection: Prophylaxis by Antiserum Prophylactic Treatment Number of Mice Died Survived Percentage of Deaths Mean Survival Time of Mice which Died (Days) Positive Blood Cultures at Autopsy on Mice which Died None "4 6'2 29 Normal rabbit serum I "3 4'5 9 Antiserum (2 2 ml.) o ii 0... Antiserum (2 o'o2 ml.) o Ps. aeruginosa and with Proteus mirabilis appeared moist, red, and inflamed, the former sometimes showing hmmorrhagic areas; local inflammatory changes were also found in mice infected with Serratia marcescens (perhaps because of its proteolytic action) and, to a smaller extent, in mice infected with E. coli and with Staph. aureus. Route of Inoculation of Antiserum.BPreliminary experiments in which mice were given prophylactic antiserum (I.O ml.) by intramuscular injection have shown that protective action by this route was comparable with that of intraperitoneal injection, provided that the serum was given in divided doses of ml. on four successive days ; when I ml. was given in one dose on the first day the prophylactic effect was less good than in mice given I ml. antiserum by the intraperitoneal route. Cross-protection with Antisera prepared against different Phage Types of Ps. aeruginosa.bstudies in progress show that antisera prepared against known serotypes of Ps. aeruginosa do not protect (or give only limited protection to) burned mice infected with other serotypes of Ps. aeruginosa ; antiserum protects mice against infection with strains of the same serotype even when the phage type is different. A polyvalent antiserum will clearly be required for prophylaxis against Ps. aeruginosa in burned patients ; the types selected for this purpose will be determined in the light of cross-protection and of the range of types isolated from infected burns.

7 THE PROPHYLAXIS OF BURNS 49 PROPHYLAXIS AND THERAPY OF BURNED PATIENTS WITH COLISTIN METHANE SULPHONATE PROPHYLAXIS A small controlled trial was made on patients admitted to the Burns Unit with burns of more than 35 per cent. of the body surface. Eligible patients over I year of age were allocated alternately to groups for treatment with (x) intramuscular colisti (" colomycin ") methane sulphonate (adult dosage, 3 million units six-hourly), oral cloxacillin (adult dosage, I g. six-hourly), and intramuscular kanamycin (adult dosage, x g. on alternate days) ; and (2) crystalline penicillin G, 5o%ooo units twelve-hourly. Patients were treated and examined for bacteria by methods described elsewhere (Jackson et al., I951 ; Bull and Jackson, I952 ; Lowbury, I96o). Blood cultures were taken, when possible, on days z, 4, 7, x4, and 2I ; also when the patients' temperatures rose to Io3 F. or higher. Table V shows the burn flora of patients in the trial. The incidence of infection with Staph. aureus was significantly smaller in patients treated with the combination of antibiotics, but Ps. aeruginosa was present more often in the burns of those treated with the combined antibiotics than in the control series treated with penicillin. TABLE V Prophylactic Trial of Systemic Chemotherapy for Severely Burned Patients : Bacteriology of Burns Bacteria Burns Colonised with Bacteria* in Patients Treated Systemically with : Combined Cloxacillin, Colistin Methane Sulphonate and Kanamycin (74 Burns in II Patients) Staph. aureus 21 (28%) Strep. pyogenes o (o %) Ps. aeruginosa 50 (68%) Proteus 27 (36 %) Coliform bacilli 46 (62 %) Crystalline Penicillin (75 Burns in io Patients) 58 (77%) 5 (6.6%) 33 (44 % ) 33 (44%) 57 (76%) Significance Tests X 2 P. 33 "7 <o.ooi 7 '5 <o.oi * Shown by heavy, moderate or scanty growth on solid medium inoculated with swabs. Systemic Invasion.--Of the ten patients treated with penicillin, five developed septicaemia (four with Ps. aeruginosa in pure or mixed culture) ; six patients, including those with Ps. aeruginosa septica~mia, died. Two of the eleven patients treated with cloxacillin, colistin methane sulphonate, and kanamycin developed septicremia, both with Ps. aeruginosa and both with a fatal outcome ; five other patients in this group died from other causes. In the smaller incidence of and death from pseudomonas septica~mia and the higher incidence of Ps. aeruginosa in burns among patients treated with colistin, the results in this trial are similar to those in a previous trial of systemic prophylaxis with four antibiotics, including polymyxin B (Cason and Lowbury, I96o) ; both studies suggest that prophylaxis with these antibiotics, though ineffective in preventing the colonisation of burns with Ps. aeruginosa, may have a limited value in preventing invasion of the blood stream. ID

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10 5 2 BRITISH JOURNAL OF PLASTIC SURGERY THERAPY The conduct of a therapeutic trial for burned patients with Ps. aeruginosa septicaemia presents exceptional difficulties, due to the relative infrequency of cases, the wide range in severity of the burns, and the fact that septicaemia is often not diagnosed until a stage when the patient is moribund. Table VI summarises the principal findings in a pilot study of ten burned patients with Ps. aeruginosa septicaemia who were admitted to this hospital betwen May 1962 and June The diagnosis of these cases was made on the clinical criteria described below, and confirmed with positive blood cultures (Table VI). Ps. aeruginosa was isolated in blood cultures incubated for twenty-four or forty-eight hours from all of the patients, and in eight patients (all except B.B. and C.F.) it was present in both culture bottles inoculated. Prineipal Diagnostie Criteria.--The presence of septicaemia in these cases was suspected from the signs of general illness (being apathetic, immobile, irritable, and disinterested in people, food, and toys), and a pyrexia of over lo3 F. A pyrexia of over lo3 F. was the commonest indication for taking a blood culture ; it was an arbitrary figure which had become accepted in the Unit as " serious " during the last few years. All ten patients had a temperature above lo2.8 F. ; in seven of them it was over lo4 F., and in one it reached lo6.4 F. The second commonest feature of the illness, and one which we now regard as diagnostically significant, was the presence of cerebral symptoms. Of the ten patients in our study, two had convulsions, one was semi-conscious with a stiff neck and temporary blindness (due probably to encephalitis or meningitis), one had hallucinations, and three were disorientated, confused, and drowsy ; of the remaining three, one was urmmic and two became comatose before death. A third sign suggestive of septicaemia--the rapid development of anaemia--was evident in four cases. Cases missed by these Criteria.--A study was made of the autopsy cases for the same period (May 1962 to June I964), and seven additional cases were found in which Ps. aeruginosa was cultured from the spleen although no positive blood culture had been obtained in life; we regard these as septicaemias missed by the diagnostic criteria described above. Moreover, since Ps. aeruginosa could be grown at autopsy in only three of the six fatal cases with positive cultures in Table VI, the actual number of missed cases may be greater. The reason why these seven cases were missed can be summarised as follows :-- I. In three cases the temperature never reached lo3 F., and blood cultures were not taken. The highest temperatures were lo2 F., IOI F., and 99.6 F. The first was a child of 3 years with a 73 per cent. burn ; she had convulsions and a terminal fall of temperature to 97 F. The other two patients were over 7o years with a 2o per cent. and a 4 per cent. burn; both had paraplegia; one had an ascending pyelonephritis and the other severe anaemia and agranulocytosis. 2. One case had a pyrexia of lo3 F. on ten occasions, but three blood cultures were negative. This child of 5 years with a 70 per cent. burn should perhaps have been diagnosed by her hallucinations and the rapid development of severe anaemia. The other three cases were treated with colistin methane sulphonate, although not diagnosed as septicaemia. 3. Two cases had pyrexia over lo3 F. with negative blood cultures: one (4 years and 38 per cent.) was treated with colistin, kanamycin, and cloxacillin prophylactically, became unconscious and rapidly anaemic, and developed ecthymatous ulcers; the other (3 years and 20 per cent.) was treated terminally for a profuse Ps. aeruginosa bowel infection.

11 THE PROPHYLAXIS OF BURNS In the last case, a blood culture was not taken in spite of pyrexia of lo3 F. on five occasions : this error was possibly due to preoccupation with a bleeding ulcer in a child of I year and with a 29 per cent. burn--a complication from which she eventually died. To summarise : Of the seven cases, three were not suspected because of insufficient pyrexia ; the other four had a pyrexia over io3 F. ; but three of the four had negative blood cultures, and the blood of the fourth was not examined. In the report of Tumbusch et al. (I96I), infection of burns with Ps. aeruginosa was in some respects different from the infections with this organism in our series. FIG. 5 Case D. B. This rash which was reddish-brown, flat, and non-irritating appeared on the fifth day after burning ; this was also two days after the first positive blood culture with Ps. aeruginosa and one day after colistine methane sulphonate was started. This picture was taken on the sixth day, and the rash disappeared completely in the next two to three days. I. Tumbusch describes a rapid course (twelve to 36 hours), with no survivors. Our fatal cases died in the first twelve days (mean four days) after the first positive blood culture. The patients who recovered had negative blood cultures (three to ten days, mean six days) after they were first positive : they were possibly cured in an even shorter time. The duration of septicmmia--from taking the first positive culture until cure or death--was usually less than a week, but septicaemia may have been present some days before the first positive culture. 2. Hypopyrexia was a characteristic feature in the series of Tumbusch et al. Two out of our ten patients had temperatures of 95 F. on the day they died, but both of these had previous pyrexias above lo3 F.

12 54 BRITISH JOURNAL OF PLASTIC SURGERY 3. Disoftentation was mild or absent in the series of Tumbusch et al. All of our cases had cerebral symptoms (see above). 4. Ileus, which Tumbusch found to be a characteristic feature of pseudomonas septicmmia, occurred in only one of our patients, who had peritonitis. Skin lesions were present in a few of our cases--small red papules (one case), ecthymatous ulcers (two cases), and, in a fourth case, a transient rash (Fig. 5). Results of Colistin Methane Sulphonate Therapy.--Four of the patients (M. A., N. T., L. G., and C. F.) received no colistin or only the first dose, because death occurred soon after the diagnosis of septica:mia was made; one (C. F.) had previously been receiving prophylaxis with polymyxin B. Of the two other patients who died, one (D. B.) had lost Is. aeruginosa from the blood stream and from the burns after very high dosage of colistin, but his temperature had risen to lo6.4 F. on the day when the course of antibiotic treatment was started ; although he lived for a week after this, irreparable damage had apparently occurred before treatment was started. Meningitis was found at necropsy, but very scanty Ps. aeruginosa were isolated from pus. (More recently a 3-year-old boy with a 16 per cent. burn had his burn cleared of Ps. aeruginosa by colistin given systemically in a dosage of 15 million units daily for five days.) The other patient who died (D. W.) had very extensive burns (8o per cent.); she died while receiving 4 million units of colistin methane sulphonate daily and while the blood levels of the antibiotic (64 to 128 per miuilitre) were found to be in the same range as its minimal inhibitory concentration for the infecting organism. The four patients who survived were all receiving colistin methane sulphonate ; Ps. aeruginosa persisted in the burns of all these patients, but it was removed from the blood stream, as shown by later blood culture. It is hard to draw firm conclusions from this study. Although the survivors were all treated with colistin, while three of the six fatal cases received no course of antibiotic treatment, the latter included patients with the higher expectation of mortality on the basis of age and area of burn (Bull and Fisher, 1954) ; one patient (D. B.) who died, however, had quite a small burn (8 per cent.). The limitations of chemotherapy were strikingly illustrated in the patient D. B. from whom Ps. aeruginosa was eliminated by very large doses of colistin, but who died nevertheless from the damage caused by the organism before treatment was started. Possibly very early treatment is the secret of success : the patients D. B. and E. B. l~ad their burns colonised on the first and second days after burning, and both had a positive blood culture on the third day ; colistin, started on the fourth day, apparently cured one and cleared the burn ofps. aeruginosa in the other before he died of cerebral damage due to hyperpyrexia. Sensitivity of Ps. aeruginosa to Colistin.--Ps. aeruginosa shows a narrow range of sensitivity to the polymyxins, including colistin, and resistance is not found to emerge in patients during treatment or in the wards where the antibiotic is used (e.g., Jackson et al., 1951 ; Kagan et al., 1951; Sherwood, Delage and Herman, ; Jawetz, 1956 ; Graber, Tumbusch and Vogel, o ; MacMillan et al., 1962). The minimal inhibitory concentration (M.I.C.) of colistin methane sulphonate against strains ofps. aeruginosa was assessed by a tube dilution test ; the inoculum of bacteria was o.o2 ml. of eighteen-hour broth culture diluted I in IOO. Seventy-three strains ofps. aeruginosa from patients in the Burns Unit were tested ; the M.I.C. of colistin methane sulphonate of these strains fell within the range of 16 to 128 units per millilitre.

13 THE PROPHYLAXIS OF BURNS 55 Blood Levels of Collstin.--Patients who were under treatment with colistin methane sulphonate were tested for serum levels of the antibiotic by a tube dilution method ; the sensitive test organism was a strain ofescherichia coli kindly provided by Dr A. T. Wallace. Levels varying between 32 and z28 units of colistin methane sulphonate were found in the serum of patients taken about three hours after intramuscular iniection of the antibiotic ; the highest level obtained in adults receiving 48 million or Iz million units a day was Iz8 units per millilitre ; this was about twice the M.I.C. of the antibiotic against the infecting organism. DISCUSSION In the pilot study of colistin methane sulphonate, prophylactic use of the drug in combination with cloxacillin and kanamycin failed to prevent colonisation of burns with Ps. aeruginosa, except possibly in a child of 3 years with 8o per cent. burns who received I2 million units of the antibiotic daily for a week, during which Ps. aeruginosa did not appear in the burns. The incidence of Ps. aeruginosa was actually greater in the burns of patients treated with the combination of antibiotics and colistin methane sulphonate than in those treated with penicillin. When colistin methane sulphonate was used for therapy ofps. aeruginosa infection, the only burns from which the organism was removed were those of a child of I5 months and one of 3 years who received 7 million and 2o million units a day respectively. Unfortunately, therapy for septicmmia, even with such high dosage, is apt to fail because the diagnosis is often made when the patient is moribund, or when the organism has already caused irreparable damage. In spite of these limitations, however, colistin seems to have helped some of our patients to resist infection. In the prophylactic trial there were fewer cases of Ps. aeruginosa septicmmia in patients treated with an intermediate dosage of I2 million units (adult dosage) of colistin methane sulphonate daily than in those treated with penicillin ; of the ten patients with Ps. aeruginosa in our series, the four who survived were treated with colistin, while only three out of the six who died were treated with a polymyxin (two of them with colistin). It seems likely that treatment with colistin, even at dosages too small to suppress Ps. aeruginosa in the burns, can sometimes prevent invasion of the tissues and the blood stream, but the prophylactic and therapeutic effects of such treatment in patients with burns appear to be very limited ; more studies are required with colistin administered at very high dosage. Our preliminary experiments with mice showed the importance of some strains ofps. aeruginosa and, to a smaller extent, of Proteus as invasive and lethal pathogens of burns in these animals ; they also showed the value of a specific antiserum in preventing fatal infection with Ps. aeruginosa. Prophylactic treatment with colistin methane sulphonate, given at doses proportional to those used in man, had little prophylactic value, though the highest dosage (corresponding to 48 million units daily in man) delayed death and caused the transient removal ofps. aeruginosa from some burns. From the results of our studies, as from those of Millican and Rust (I96o), it would seem that an antiserum to Ps. aeruginosa offers better prospects of successful prophylaxis against infection with the organism in burned subjects than the available antibiotics provide. It might be questioned whether antisera are likely to have any protective value in the light of our earlier findings of very high titres of antibody to Ps. aeruginosa in burned patients with septicmmia (Fox and Lowbury, I953). These higher titres, however, were found in the later stages of the patients' illnesses; the protective value of treatment with antisera in mice may be due to the sudden elevation

14 5 6 BRITISH JOURNAL OF PLASTIC SURGERY of antibody levels soon after the burning injury, at a time when the animals' resistance is lowered by the secretion of corticosteroid hormones and the reduction in circulating gamma globulin. It is probable, too, that antibody levels are lower in patients who succumb to infection than in those who are resistant to it ; titres of antibodies to Staph. aureus were found to be significantly lower in burned patients who died than in those who survived (Jones and Lowbury, I963), and deaths from septiczemia have been found to vary inversely with titre of antibodies to Ps. aeruginosa (Kefalides et al., I964). Further studies are planned, including an attempt to assess the value of specific antisera and possibly the value of combined use of antiserum and colistin in prophylaxis and therapy of patients with severe burns. SUMMARY Small (6 per cent.) full-skin-thickness burns in mice caused a mortality of 5"1 per cent. In mice infected by application of cultures ofps. aeruginosa and Proteus mirabilis to such burns the mortality was 71 and 54 per cent. respectively ; the mice died with septica~mia. By contrast, infection with strains of E. coli, Klebsiella aerogenes, Serratia marcescens, Staph. aureus, and a micrococcus was not associated with higher mortality rates than those of the control animals. Different phage types of Ps. aeruginosa showed a wide range of virulence, and some caused no increase in mortality of burned mice. Intraperitoneal and intramuscular injection of an antiserum prepared by immunising rabbits with Ps. aeruginosa prevented the death of burned mice infected with the immunising strain of Ps. aeruginosa. Intramuscular injection of colistin methane sulphonate in large dosage (28,000 units daily for ten days) delayed but did not prevent death from septic~emia in burned mice infected with Ps. aeruginosa; smaller doses had no apparent prophylactic effect. In a small controlled trial of colistin methane sulphonate (combined with cloxacillin and kanamycin) on patients with severe burns, the frequency ofps. aeruginosa colonisation of burns was significantly higher in the treated patients than in the controls ; there were, however, fewer cases with Ps. aeruginosa septica.'mia (2-11) in the treated group than in the control series (4-1o). A pilot study of treatment with colistin (" colomycin ") methane sulphonate was made on ten patients with Ps. aeruginosa septica~mia. Of the four patients who survived, all had received a course of colistin, while only two of the six who died received such a course ; five of the latter, however, died because they were moribund or irreparably damaged by infection when the diagnosis of septic~emia was made. In three patients who received a very high dosage of colistin, two for therapy and one for prophylaxis, Ps. aeruginosa, was, respectively, eliminated when present and not acquired by burns at a time when the organisms were expected to appear. We wish to thank Mr A. Jones and Mrs A. Kidson, A.LM.L.T., for technical assistance, Dr S. Sevitt for information from autopsy records, Dr M. T. Parker for different serotype strains of Ps. aeruginosa, and Messrs Pharmax Ltd. for a supply of colistin methane sulphonate used in part of this study. REFERENCES BENEDICT, F. G. (193o-31). YaleJ. Biol. Ivied., 4, 385 BULL, J. P., and FISHER, A. J. (1954). Ann. Surg., x39, 269. BULL, J. P., and JACKSON, D. M. (x952). Brit. med. ft., x, Io18, lo78. CASON, J. S., and LowBuRY, E. J. L. (196o). Lancet, z, 5Ol.

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