WOUND INFECTION WITH PSEUDOMONAS MULTIVORANS

1188 The modified chi-square value for a given set of parameters is then defined as the sum over all entries in tableI of:

(observed - expected) 2 j( observed + 2) in Yneale .6s The best value is that which causes the modified chisquare to be minimised, and the equations leading to this result can only be solved on a computer. as

WOUND INFECTION WITH PSEUDOMONAS MULTIVORANS A WATER-BORNE CONTAMINANT OF DISINFECTANT SOLUTIONS

D. C.

J. BASSETT Cross-Infection Reference Laboratory, Central Public Health Laboratory, London N.W.9 K.

J. STOKES

W. R. G. THOMAS and Park Croydon Warlingham Group Pathological Laboratories, Mayday Hospital, Thornton Heath, Surrey Over a period of 5 weeks, Pseudomonas multivorans was isolated from infected operation wounds of nine patients in a hospital. In some cases the organism seemed to be a wound contaminant but in others it behaved as a pathogen. It was isolated from bottles of a 1 in 30 dilution of’ Savlon’ (chlorhexidine 0·05%, cetrimide 0·5%) in the hospital, and from samples of the piped water supply in the hospital and outside.

Sum ary

Introduction

Pseudomonas multivorans was described, but not named, by Morris and Roberts,l who investigated strains isolated from soil and river water samples in Trinidad. The name multivorans was proposed by Stanier et al.,2 who gave a description based on nineteen strains from Trinidad, Berkeley (California), and Bristol. The Bristol strains were isolated from postoperative urinary-tract infections in children3 and traced to contaminated 1 in 5000 chlorhexidine (’Hibitane’) which had been used to disinfect the bladder-irrigation reservoir in the operating-theatre. In the Birmingham area, Burdon and Whitby4 isolated an organism thought to be Ps. multivorans from 0-05% chlorhexidine, and in the same hospital recovered another pseudomonad from 1 in 30 ’Savlon ’ (chlorhexidine 0-05%, cetrimide 0.5%). They showed that pseudomonads could survive the bottle-washing procedure which was being used, and so contaminate freshly made solutions. No infection was found that could be attributed to these strains. We record here the isolation of Ps. multivorans from wound swabs taken from nine patients in a south London hospital, its origin, and pathogenicity.

still present on day 20 a wound swab was taken, an! Ps. multivorans was isolated after enrichment in cooked meat medium. Primary cultures seemed sterile and ni other organism was grown. The patient received a cours of trimethoprim and sulphamethoxazole tablets (’ Septrin’ and the wound healed satisfactorily. Case 2 A woman aged 71 with nail and plate inserted

fractured femur had a Thorntoi April 7. On day 18 afte: from the wound was noted a operation slight discharge A swab yielded Ps. multivorans only, on subculture o cooked-meat medium. On day 24, a further swab yieldec Ps. multivorans and Clostridium welchii. The pseudomonac was eliminated by treatment with trimethoprim anc sulphamethoxazole, but the anaerobe persisted. Secondary infection with staphylococci ensued, and failure to heal finally led to the removal of the nail and plate. a

on

Case 3 A Thornton nail and plate were applied to the fractured femur of a 73-year-old woman on April 16. On day 14 the wound was inflamed and on day 21 primary cultures from a wound swab showed both Ps. multivorans and Staphylococcus aureus in approximately equal numbers. A course of trimethoprim and sulphamethoxazole was followed by a satisfactory recovery. Case 4 An 89-year-old man with arteriosclerotic gangrene of the foot underwent a through-knee amputation of the leg on April 18. Treatment with tetracycline was started before the operation because of the patient’s chest condition, and after the operation the stump was treated with eusol soaks and topical polymyxin B, bacitracin, and neomycin. On day 18 infection of the wound was evident, and primary cultures of a wound swab yielded a heavy, pure growth of Ps. multivorans. The wound sloughed, and 5 weeks elapsed before the area became fit for skin grafting.

Case 5 A woman aged 73 with a fracture of the neck of femur had a Thompson prosthesis inserted on April 22. On day 8 there was a slight discharge from the wound. Ps. multivorans was isolated from the swab, on subculture of cooked-meat medium. After a course of trimethoprim and sulphamethoxazole tablets her wound healed and she was

discharged

on

day

29.

Case 6 A 13-year-old girl was admitted to hospital for an osteotomy of the 1st metatarsal which was done on April 25. On day 18 she attended as an outpatient for the removal of her sutures. The plaster was replaced by a below-knee walking plaster. The wound was slightly infected: it was cleaned with savlon, which by this time was available only as an autoclaved solution. On day 32 the child was readmitted because the wound was grossly

Case-reports Case1 The injured index finger of a 50-year-old male inpatient was sutured on April 4, 1969. 13 days after the operation infection of the wound was evident. The wound was treated with eusol (chlorinated lime and boric acid solution) and tetracycline was given by mouth. Since infection was

Fig. 1-Case

6: osteotomy wound.

1189 infected

laboratory of the National Collection of Type Cultures.

yielded a heavy,

This showed very good agreement over a wide range of Stanier et al. proposed the name multivorans because of the exceptional nutritional versatility of the organism. The test strain utilised 34 out of 42 substrates tested as carbon or nitrogen sources; NCTC 10661 utilised 35. There were only 3 discrepancies, all with substrates variably utilised in Stanier’s series of 19 strains. Further biochemical tests, including fermentation tests in ammonium-salt sugars and D.N.A. base composition analysis, all confirmed the identity of the organism with Ps. multivorans. The type strain produced a non-diffusible yellow-green pigment, but pigment production is a variable feature of this species. Source of Infection Six of the cases were operated upon in the ortho-

(see figure). A wound swab taken at this time pure growth of Ps. multivorans on primary culture. Oral ampicillin and chloramphenicol were given, and local polymyxin B, bacitracin, and neomycin to the wound, but the organism was reisolated on day 35. Healing was slow, and the patient was discharged 69 days after

operati6n. Case 7 A perineal abscess in a 32-year-old man was drained in the accident department on April 27. A swab taken during the operation yielded Ps. multivorans only, after enrichment in cooked-meat medium. The lesion was treated with eusol and healed in 21 days. No systemic treatment was given.

Case8 A man aged 29 had an infected sebaceous cyst which was incised in the accident department on May 1. A swab of the cyst contents at that time yielded Ps. multivorans only, after subculture of cooked-meat medium. Healing of the wound was satisfactory on topical treatment with eusol. Case9 A man aged 54 had an infected sebaceous cyst which was incised on May 1, in the accident department. A swab from the lesion yielded a heavy pure growth of Ps. multivorans on primary culture. The lesion was treated with eusol and healed in 35 days. Identification The isolates from the patients’ wound swabs grew well on ordinary media: on blood-agar they produced greyish, mucoid, low convex colonies which showed some p-hasmolysis on primary isolation. There was no pigment production and no metallic sheen on mass growth. After 72 hours the colonies showed a dry and dimpled surface. They grew on MacConkey agar and on desoxycholate-citrate agar, but not on 0-1% or 0-03% cetrimide agar. Cultures on solid media remained viable for a few days only. The first suggestion that the isolates were similar was their distinctive antibiotic-sensitivity pattern. Filter-paper discs (’Multodiscs,’ Oxoid) were used for these tests. Each isolate displayed a narrow zone of inhibition round the chloramphenicol (10 g.) disc but showed resistance to all other antibiotics tested, including gentamicin (10 g.), carbenicillin (100 g.), and colistin (200 g.). They were, however, sensitive to sulphamethoxazole (50 g.) and to

trimethoprim (2-5 g.). Later, a second simple test of identity was obtained when it was found that antiserum against Ps. aeruginosa serotype 13 showed a cross-reaction with the organism. The organism was assigned to the genus Pseudomonas because of the following characteristics: motile, Gramnegative rods, growing well aerobically but very poorly anaerobically; oxidase and catalase positive; and oxidative metabolism of glucose. This generic determination was confirmed when electron microscopy showed polar, multitrichous flagellation. A colonial resemblance was noticed between this organism and Ps. pseudomallei, infection with which had recently been seen in a Pakistani patient in London.5,s In an attempt to elicit the Straus reaction with the isolate from case 1, male guineapigs were inoculated with 0.5 ml. of a 24-hour broth culture, by the intraperitoneal route. After 7 days there was some swelling of the testicles and the tunica vaginalis was slightly haemorrhagic, with two small nodules containing pus from which the pseudomonad was recovered. After smaller doses (30 million organisms), the pseudomonad was recoverable after 3 days, but not after 10 days; there were no visible changes in the testicles. These findings indicated that this was not Ps. pseudomallei, but an organism of lesser pathogenicity. Biochemical differences later confirmed this. Direct comparison of the isolate from case 1 with NCTC 10661, the type strain of Ps. multivorans, was done in the

tests.

paedic theatre, whereas the abscess and the cysts were incised in the accident department. These two departments did not share staff, equipment, or accommodation. The orthopaedic patients had occupied various wards, and their operations had been done by various surgeons. The minor any ward.

cases

had not been admitted to

reasonable to suppose that all the patients had the pseudomonad from the hospital environwould mean that the minor cases were which ment, contaminated with the organism at the time when their lesions were incised. Solutions used to disinfect the skin were clearly suspect. Povidone-iodine had been used to disinfect the skin of two of the orthopxdic cases, but in all the others a chlorhexidine-containing solution had been used for preoperative skin preparation. The delay between surgery and the isolation of the pseudomonad was such that any of the first five orthopaedic cases might have been infected during It

was

acquired

postoperative procedures. Investigation was begun in the accident department and the orthopaedic theatre. 3 Winchester quart bottles of 1 in 30 dilution of savlon in the accident department all yielded Ps. multivorans. In the theatre Ps. multivorans was isolated from a pint bottle of 1 in 30 savlon, but not from 2 Winchester quarts of the same disinfectant. These recoveries were made by the inoculation of 50 ml. volumes of glucose broth with approximately 1 ml. of the samples. Ps. multivorans was recovered next from stock 1 in 30 savlon in the pharmacy, so water samples were examined to trace the source of the contaminant. Waters taken in the hospital were treated with 1/30 v/v autoclaved savlon hospital concentrate (chlorhexidine gluconate 1.5%, cetrimide 15%). After incubation at 30°C for 24 hours, 2 ml. of the mixture was added to 50 ml. of 0-1% glucose broth: this subculture was incubated for 24 hours before plating on solid media. Water samples from the public-health department were examined by savlon treatment of the 100 ml. MacConkey broth cultures used in routine testing for Escherichia coli, in the same manner. Samples of water from the hospital, from a neighbouring hospital, and from the surrounding district were examined. Ps. multivorans was isolated from two cold taps in the hospital, from the water-softening plant, and from the mains to the hospital’s header tank. In the pharmacy of the neighbouring hospital, the same organism was found in 0-02% hibitane, in the outflow from the deioniser, and in the storage tank of the still. From the surrounding area that shared the same water

1190

supply, 5 out of 125 samples yielded Ps. multivorans isolates, indistinguishable from the earlier cultures. Resistance

to

Chemical Disinfection recovered from bottles of savlon

Ps. multivorans was in the hospital, but it proved unexpectedly difficult to demonstrate a comparable degree of resistance in the laboratory. When tests were done on broth cultures of the strains that had been isolated from 1 in 30 savlon in the hospital, the minimum inhibitory concentration (M.iC.) of Savlon was 1 in 320, and the organisms did not survive in a 1 in 30 dilution. Occasionally, organisms survived in 1 in 30 savlon when large inocula from solid media were used. Such inocula did not disperse evenly in the disinfectant but remained in visible aggregates which may have helped to protect the organisms. The surviving organisms had an increased resistance to savlon, but reverted to sensitivity when cubcultured on to nutrient media. For example, in one experiment viable counts were made on two suspensions of the organism-a suspension of organisms surviving in 1 in 30 savlon, and a subculture of the first on to blood-agar. Replicate counts were made on nutrient agar plates and on plates containing various concentrations of savlon in nutrient agar. On 1 in 300 savlon agar the first suspension gave a count which was 89% of the count on nutrient agar; in contrast, only 6% of the organisms taken from subculture formed colonies on 1 in 300 savlon agar, and only 47% on 1 in 1200 savlon agar. The resistance of the organism was influenced by the temperature of incubation, and was greater at room temperature or at 30°C than at 37°C. The rate of growth, on the other hand, was greatest at 37°C. Cultures that had been incubated for 24-72 hours provided more resistant inocula for M.LC. tests than either logarithmic-phase or aged cultures. These were, however, minor differences, and there remained a great discrepancy between the resistance of the organ-

Fig. 2-Multiplication of Ps. multivorans in 1 in 30 dilution of savlon hospital concentrate. 0 mark the counts obtained. After each count the disinfectant was replaced by twice the volume of a fresh solution: the lower points (.) reflect the resultant two-fold dilution of the culture.

ism in culture and that of the

organism

in the

hospital

environment.

Multiplication of the organism in a high concentration of savlon was first obtained in a mixture of 1 part of savlon hospital concentrate and 29 parts of 1 % peptone in distilled water. When this was inoculated with organisms surviving in 1 in 30 savlon and incubated for 4 weeks at room temperature in the dark the number of viable organisms rose from an initial 10 per ml. to 25 104 per ml. At this point the pseudomonads were deposited by centrifugation. The supernatant savlon-peptone mixture was decanted and replaced by twice the volume of freshly prepared 1 in 30 savlon in distilled water. Incubation at room temperature in the dark continued, and multiplication continued through this and two further changes of disinfectant performed in the same manner (fig. 2). The final viable count obtained after a total of 8 weeks was 4.1 x 107 organisms per ml. Thus Ps. multivorans not only survived but multiplied in 1 in 30 savlon under these conditions. Again, there was reversion to the former level of sensitivity when subcultures were made on to nutrient medium. The resistance of the cultures of Ps. multivorans to other disinfectants was tested in the Disinfection Reference Laboratory. M.l.c. determinations showed that these strains were less resistant than Ps. teruginosa NCTC 6749 to a variety of phenolic disinfectants. They were, however, more resistant to a disinfectant containing picloxidine and benzalkonium chloride (’Resiguard’), with an M.I.C. four times greater than that against the standard test organism.7 Discussion

Ps. multivorans

variable part in the wound (cases 7-9) the organism must be regarded as a simple contaminant of the lesions; in one case this contamination was gross, but there was no evidence that healing was materially delayed, even in the absence of specific treatment. At the other extreme (case 6), the organism was evidently introduced at the time of operation and no antibacterial treatment was given for 32 days. It was then isolated in pure culture from a grossly infected wound. The healing of this wound was greatly delayed. Ps. multivorans must be regarded as a potential pathogen in the context of wound infection, although it showed little or no invasiveness either in the patients or in the animal experiments described. The pseudomonad was present in the tap water supplied to the hospital and the surrounding district, and it seems likely that it reached the patients’ wounds in a disinfectant solution. The organism was recovered from several bottles of 1 in 30 savlon in the departments of the hospital that were associated with the infections. This disinfectant was used for skin preparation before minor surgery and in the postoperative management of other and more extensive surgical wounds. Although Ps. multivorans multiplies in 1 in 30 savlon slowly and under rather restricted conditions, we noted an increase of bacterial concentration to over 107 conditions simulating the organisms per ml., under " of a disinfectant stock bottle. repeated " topping up The manufacturer of chlorhexidine-containing disin-

infections. At

played

a

one extreme

1191 fectants emphasises that certain precautions are necessary to prevent contamination of these products. Containers must be carefully cleaned before they are refilled, any stock solutions (i.e., any dilute solutions that are not for immediate use) should contain either 7% v/v ethanol or 4% of isopropanol, and contact between disinfectant and corks or cork cap-liners must be avoided. The hospital decided to issue chlorhexidinecontaining disinfectants only as heat-sterilised solutions in small-capacity containers. This step was taken in view of the likelihood of continued introduction of the organism by way of the water supply, and the ability of the organism to multiply in the disinfectant. It should be a sufficient precaution against contamina-’ tion provided serious abuses are avoided, such as the disinfectant for prolonged retention of bottles of dilute occasional use and the refilling or " topping up " of such bottles in the wards or departments. In 12 months since this precaution was taken there has been no further incident of infection with this organism. We thank Mr. 1. J. MacQueen and Mr. E. C. Lewis for permission to refer to the patients in their care; Dr. M. T. Parker for his valuable advice on the problem; Dr. S. P. Lapage and Mr. L. R. Hill for the identification of the organism; Mrs. Isobel M. Maurer for certain of the tests on resistance to disinfection ; Mrs. Diana Martin for the Ps. ceruginosa typing sera, and Miss Ann M. Field for the electron microscopy. The photograph was taken by Mr. C. J. Grummitt. Requests for reprints should be addressed to D. C. J. B. REFERENCES 1. 2.

3. 4. 5. 6. 7.

Morris, M. B., Roberts, J. B. Nature, Lond. 1959, 183, 1538. Stanier, R. Y., Palleroni, N. J., Doudoroff, M. J. gen. Microbiol. 1966, 43, 247. Mitchell, R. G., Hayward, A. C. Lancet, 1966, i, 793. Burdon, D. W., Whitby, J. L. Br. med. J. 1967, ii, 153. Stokes, K. J., McCarthy, S. J. med. Lab. Technol. 1969, 26, 199. Morrison, I. M. Proc. R. Soc. Med. 1970, 63, 289. Maurer, I. M. Personal communication.

SKIN-REACTIVE SOLUBLE ANTIGEN FROM INTESTINAL CANCER-CELLMEMBRANES AND RELATIONSHIP TO CARCINOEMBRYONIC ANTIGENS A. HOLLINSHEAD

D. GLEW

B. BUNNAG

Laboratoryfor Virus and Cancer Research, George Washington University Medical Center, Washington, D.C. 20037, U.S.A. P. GOLD Division of Clinical Immunology, McGill University Clinic, Montreal General Hospital, Montreal 109, Quebec, Canada R. HERBERMAN

Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Summary

Delayed hypersensitivity reactions in seventeen of nineteen patients with the colon and rectum were elicited by

carcinoma of soluble fraction obtained from the membranes of autologous tumour cells. Negative reactions were obtained with comparable fractions obtained from normal cells. Skin-reactive antigen was also detected in the digestive-tract cells of first-trimester and second-trimester fetuses. Carcinoembryonic antigen

(C.E.A.) was detected in many of the fractions producing positive skin reactions. The skin-reactive tumour antigen appeared to be closely related, and possibly identical, to the C.E.A. but, although found in comparable fractions, it may well be distinct from the

C.E.A.

Introduction

Preliminary studies have been made of certain fractions of partially separated soluble-membrane antigens from animal and human cancer cells. Such antigens appear to afford protection to tumour-cell challenge in animals or to elicit a specific cutaneous

hypersensitivity reaction in

man, whereas similar fractions of soluble-membrane antigens of an equal protein content from normal tissue do not elicit such

responses. 1-3 In the present

study, skin tests were performed on with carcinoma of the rectum and colon in an patients to detect attempt cellular-immunity (delayed-hypersensitivity) reactions to tumour antigens. Delayedhypersensitivity reactions to antigens on human tumour cells have been reported by several investigators.4-9 All of these studies were performed with cell extracts containing surface membranes or other particulate material. The tumour specificity of the observed skin reactions has not been conclusively demonstrated; some reactions have been obtained with control extracts as well as with tumour-cell extracts. In this study soluble extracts were prepared from whole cells or from membrane extracts, and this allowed more definitive demonstration of tumour specificity. It was also possible to compare the soluble membrane antigens with the carcinoembryonic antigen (C.E.A.) of Gold. Gold et al. have shown that certain c.E.A.s of the human digestive system are found only in adenocarcinomas arising from the entodermally derived digestive-system epithelium and in embryonic and fetal gut, pancreas, and liver tissue during the first two trimesters of pregnancy.10-12 This c.E.A. appears to be a glycoprotein closely associated with the cell surfacemembrane.13 70% of patients with non-metastatic cancers of the digestive system have anti-c.E.A. antibodies in their sera as measured by agglutination of

erythrocytes chemically coupled

to

C.E.A.

Antibody

titres did not correlate, however, with the clinical status of the patient. In addition to these studies by Gold, he has ’recently described the presence of the C.E.A. antigens in the blood-stream; in blood tests, by blindscreening of 200 patients with various diseases, the antigen was easily detectable in the 35 patients with colon and rectal cancer.14 Materials and Methods

Preparation of Soluble-membrane Antigens Fetal digestive tracts were obtained from first, second, and third trimester therapeutic abortions, from mothers with no history of any drugs which might induce chromosomal aberrations in the fetal tissue. The fetuses were obtained either by aspiration or by surgical removal from the intact uterus after hysterotomy. In each patient at the time of operation rectal and colonic cancer and normal tissue were obtained to be skin-tested. The fetal gut, adult carcinoma, and normal tissues were washed for ten minutes at 37°C five times in physiological saline solution. The cells were teased into suspension and counted. Membrane extractions were performed by the procedure des-