Saturday I February
SALMONELLA TYPHIMURIUM RESISTANT TO SILVER NITRATE, CHLORAMPHENICOL, AND AMPICILLIN A New Threat in Burn Units ?
GAIL LARKIN MCHUGH CYRUS C. HOPKINS
ROBERT C. MOELLERING MORTON N. SWARTZ
Departments of Medicine, Harvard Medical School and Massachusetts General Hospital (Infectious Disease Unit), Boston, Massachusetts 02114, U.S.A. A strain of Salmonella typhimurium appeared sequentially in three patients in a burn unit, and epidemiological study suggested the occurrence of person-to-person spread. This organism was responsible for both colonisation and invasive infection in these patients whose burn surfaces were receiving topical treatment with 0·5% silver nitrate (AgNO3) solution. The antibiotic and metal ion susceptibility pattern of this strain of S. typhimurium was unique and disturbing: resistant to silver nitrate, mercuric chloride, ampicillin, chloramphenicol, tetracycline, streptomycin, and sulphonamides. This pattern of multiple resistances could be transferred by invitro mating experiments to sensitive recipient strains of Escherichia coli and S. typhimurium. Further transfer of these resistances could be consumated between different strains of E. coli. A survey of other salmonella strains isolated from patients in this hospital without thermal burns did not reveal this pattern of resistance. Also, strains of S. typhimurium, isolated elsewhere and showing simultaneous resistance to both ampicillin and chloramphenicol, were not resistant to AgNO3 in vitro. The very real danger of this strain of S. typhimurium in burn units stems from its resistance to the two most effective antibiotics (ampicillin and chloramphenicol) available for systemic therapy; and this threat may be compounded through the selection effected by the widespread topical use of AgNO3 solutions and sulphonamide preparations on burned surfaces.
Summary
Introduction THE capacity of salmonella to exhibit new properties and to exploit the vulnerable host, particularly in the hospital setting, remains a continuing challenge in medicine. The sequential appearance, in three patients in the same burn unit, of a strain of S. typhimurium with unusual properties raised the possibility of person7901
I975
to-person spread. A report by MacGregor and Reinharthas suggested that nosocomial dissemination of salmonella is unlikely without a common source such as food or medication. Earlier, others indicated the contrary.2 A cause of particular concern is the resistance of the current strain to five antimicrobial agents, including chloramphenicol and ampicillin. Simultaneous resistance to these last two antibiotics has been described only recently among salmonellae.3-6 In addition, this present strain is highly resistant to AgN03 which had been used in topical therapy of burned surfaces. Resistance to AgN03 has been described only rarely among certain of the enterobacteriaceae, such as enterobacter and klebsiella strains, but not in salmonella.7-9 This resistance to AgN03 as well as to other antimicrobial agents in the current strain of salmonella is transferable to other members of the enterobacteriaceae. Silver resistance mediated by a conjugative plasmid has not previously been described.
Materials and Methods Bacterial Strains The multiply resistant salmonella strain used as donor in conjugation studies was the isolate obtained from the blood of case 1; and initially it was designated S, typhimurium (MGH). After demonstration of its conjugative resistance plasmid it was given the designation S. typhimurium (pMG101). Nine other salmonella strains isolated this past year from patients at the Massachusetts General Hospital were kindly provided by Dr Lawrence Kunz and were studied for sensitivity to AgNOa. Of these strains, two were S. typhimurium; two, S. heidelberg; and one each, S. newington, S. enteritidis, S. infantis, S. montevideo, and S. typhi. Ten additional strains of S. typhimurium isolated from an outbreak in Iran (" Tehran strains ") were studied for resistance to silver nitrate. Nine of these were ampicillin and chloramphenicol resistant; a tenth strain was only to ampicillin. They were kindly provided by Dr Clyde Thornsberry, of the Clinical Bacteriology Branch of the Center for Disease Control, Atlanta. In conjugation experiments with the Ag-resistant S. typhimurium from case 1 as donor, the salmonella recipient was a naladixic acid (NalR) resistant mutant of a leucine auxotroph of S. typhimurium leu-50 NaiR kindly provided by Dr K. E. Sanderson, Department of Biology, University of Calgary. In mating experiments between S. typhimurium (pMG101and E. coli, the recipient was E. coli C, F-, NAIR. In mating experiments between E. coli strains, E. coli C, P-; NalR and a rifampin-resistant aminoacid auxotroph, E. coli 3.?, F’, pro-, met-, RifR were
resistant
employed. The identity 50
was
of
transconjugants of S. typhimurium leudemonstrating growth of purified
confirmed by
E
236
agar 10 only in the presence of leucine ml.). Similarly, transconjugants of E. coli ’]53 were validated, after purification, by demonstrating growth on minimal agar only in the presence of both methionine (30 ,g. per ml.) and proline (50 Ag. per ml.). The conjugative plasmid in S. typhimurium has been given the designation pMG101. colonies
(80
on
minimal
gg. per
Antibiotic and Metal Ion
Sensitivity Antibiotic-susceptibility testing was performed by the single-disc technique described by Bauer et al.11 on Mueller-Hinton agar. Levels of resistance to drugs were determined by the agar-plate dilution technique in McConkey agar employing 10-2 dilution of 14-18 hour cultures with an inocula-replicating device similar to that of Steers et al.12
Levels of resistance to AgNOg were determined in TYE agar without added sodium similarly chloride (Bacto tryptone 10 g., yeast extract 5 g., agar 15 g. per litre). Levels of resistance to mercuric chloride (HgCl2) were determined on beef-heart infusion agar
(Difco). Conjugation Procedure Cultures of donor and recipient bacteria grown to midin TYE broth were mixed in a cell ratio of 1/1, incubated for 2 hours (unless otherwise specified) at 37 ° C, and then plated on to selective medium after appropriate dilution. The plates were incubated for 72 hours and the transconjugants were scored and purified. Antibiotic and metal resistance was then determined by the agar-plate dilution method. Most matings were performed on’Millipore’ filters to increase the number of transconjugants. 1 ml. quantities of log-phase cultures of donors and recipients were mixed and then passed through a sterile 0’45 jn millipore filter. The filter was then placed on a plain TYE plate and incubated overnight at 37 °C. The filter was then removed from the agar and shaken in 2 ml. of TYE broth to remove the bacteria from the filter. Appropriate dilutions were made, and then plated on to selective media and incubated at 37 °C for 72 hours. Plates containing AgNOg were incubated in the dark.
logarithmic phase
Results FIRST CASE
A 32-year-old White male was admitted to the Massachusetts General Hospital on Oct. 3, 1973, with 3rd and 4th degree electrical burns of his left arm, flank, and both legs, involving 30% of his body-surface area. The patient had never travelled outside of the United States and had lived all his life in the north-eastern part of the country. His general health had been excellent, and there was no prior history of diarrhoea. Immediately after admission he underwent surgical removal of eschar, and therapy with parenteral penicillin and topical 0’5 % AgN03 was begun. Between Oct. 5 and 12, he underwent further debridement and then total amputation of both legs when they became non-viable. Parenteral therapy with gentaInitial micin was begun because of persistent fever. bum-wound cultures showed E. coli, herellea, Pseudomonas maltophilia, and enterococci. By Oct. 16, however, a strain of S. typhimurium which was resistant to tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides was isolated from his burn sites. A stool culture obtained on Oct. 24 also yielded S. typhimurium with a similar pattern of antibiotic resistance. On Oct. 26 treatment with tobramycin was begun because of persistent bactersemia due to a gentamicin-resistant strain of herellea. Subtotal gastrectomy for massive upper gastrointestinal bleeding was performed on Oct. 27. Bumwound cultures during this period consistently revealed S. typhimurium despite topical therapy with AgN03 and
mafenide (’ Sulfamylon’) and parenteral administration of tobramycin, to which the organism was sensitive in vitro. By Oct. 29 blood-cultures began to grow EnteroIntravenous therapy with bacter sp. and enterococci. penicillin was added. On Nov. 3, S. typhimurium was isolated from blood-cultures, and bactersemia due to this organism persisted until death. The antibiotic-sensitivity pattern of the S. typhimurium isolated from the patient’s blood was identical with that isolated from the burned surfaces. The same strain of S. typhimurium was isolated from sputum specimens between Nov. 13 and 19, but there was no clinical evidence of pneumonia. The isolate of S. typhimurium from his blood was susceptible to trimethoprim-sulphamethoxazole by in-vitro testing. Thus, despite persistent ileus, the patient was begun on trimethoprim-sulphamethoxazole therapy by nasogastric tube on Nov. 19. Serum-sulphonamide levels varied between 1’2 and 2’5 mg. per 100 ml. on doses of trimethoprim (320 mg.) and sulphamethoxazole (1600 mg.) every 12 Treatment with intravenous carbenicillin and hours. gentamicin was also begun when Pseudomonas aeruginosa appeared in blood-cultures. On this treatment, the pseudomonas bacteraemia cleared, but S. typhimurium was repeatedly isolated from blood-cultures. Hypotension and oliguria developed, and the patient died on Nov. 25, 1973. Post-mortem examination revealed acute peritonitis with subhepatic and left subphrenic abscesses, acute mediastinitis,. and acute left pleuritis. In addition, there was acute fibrinous pericarditis with a purulent pericardial effusion totalling 600 ml. Cultures taken from these purulent collections grew a mixture of gram-negative organisms, including S. typhimurium, and enterococci. Post-mortem culture of heart blood grew only S. typhimurium with a colony-count of 100 per ml. SECOND CASE
A 55-year-old woman entered the Massachusetts General Hospital on Oct. 15, 1973, because of thirddegree burns involving 35 % of her body-surface area, including her abdomen, chest, and face. She was placed in a room next to case 1 and was treated with topical 0’5 % AgN03 to her trunk and povidone-iodine (’Betadine’) ointment to her face and neck. Parenteral penicillin was also begun. On Oct. 19 she underwent primary excision of the burned areas of her chest and abdominal wall and S. typhiapplication of split-thickness skin grafts. resistant to murium tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides, were first isolated from her burn wounds on Oct. 23. Over the subsequent 6 days the same strain was isolated from cultures of blood, sputum, and urine. A stool-culture was not obtained. On Oct. 29 massive upper-gastrointestinal bleeding occurred, and this was controlled by angiography and intra-arterial vasopressin. However, despite cessation of bleeding, severe hypotension ensued, and the patient died on Oct. 29, 1973. The only blood-culture (obtained from an arterial cannula in a non-burned area on Oct. 26) showed S. typhimurium and Herellea sp. Necropsy revealed bilateral pulmonary cedema, gastric stress ulcers, and tracheo-bronchitis. Post-mortem blood and lung cultures yielded S. typhimurium. THIRD CASE
A 20-year-old White male was admitted to the Massachusetts General Hospital on July 31, 1973, following an airplane crash. He had suffered third-degree burns over 80% of his body-surface area, sparing only his right shoulder, part of his right arm, and a portion of his head. He also suffered a compound fracture of the right tibia. He was placed in a room next to that occupied by case 1, and treatment with parenteral penicillin and topical mafenide, followed by topical AgNOs, was begun. On
237
Aug. 3 the patient underwent bilateral above-the-knee He had numerous surgical procedures for debridement and skin grafting of his burns. During the first 3 months of his stay in hospital his course was com-
amputation.
plicated by upper-gastrointestinal bleeding, numerous bouts of bacterial pneumonia, burn-wound infection, and intermittent bacterxmia due to enterococci, Klebsiella pneumoniae, and Enterobacter sp. He received courses of treatment with numerous antibiotics, including oxacillin, gentamicin, chloramphenicol, kanamycin, and tobramycin. On Nov. 19, 1973, S. typhimurium, resistant to tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides, was isolated from his bum Nov. 18 revealed the same organism. Topical povidone-iodine ointment was added to the topical silver-nitrate therapy. Woundcultures continued to yield S. typhimurium, and on Nov. 27, 1973, he was begun on therapy with trimethoprim (320 mg.) and sulphamethoxazole (1600 mg.) orally every 12 hours; the frequency of these doses was increased to every 6 hours on Dec. 1. On this regimen the serumsulphonamide level was 3-0 mg. per 100 ml. Trimethoprim-sulphamethoxazole therapy was stopped on Dec. 5. Wound-cultures thereafter did not yield salmonella, but S. typhimurium persisted in his stool-cultures. He subwounds.
A stool-culture obtained
on
sequently developed disseminated candidiasis, and, despite therapy with amphotericin B, he became oliguric and azotmic, and died on Dec. 11, 1973. Necropsy revealed bilateral bronchopneumonia, and evidence of candida endocarditis with fungal abscesses in multiple organs. Post-mortem blood, brain, kidney, and lung cultures were negative for S. typhimurium. EPIDEMIOLOGICAL OBSERVATIONS
The three patients infected with the multiply resistant strain of S. typhimurium in this study were housed in three adjacent rooms (A, B, C) in a ten-room isolation wing of the Massachusetts General Hospital. Case 1 initially resided in room A, and was transferred to room B after a surgical procedure on Oct. 15, 1973. At this point, case 2 was admitted to room A. Case 3 was housed in room C throughout his hospital stay. There were seven patients on the wing at the beginning of the salmonella outbreak, gradually dwindling to two by the time of the death of case 3. Their illnesses ranged from relatively minor injuries to severe and complicated burns. For all patients, food was brought from a central kitchen, elsewhere in the hospital, on separate servingtrays, portioned on to individual dishes by kitchen aides on the floor, and served to the patients by aides or nurses. While most of the patients were served in this way, the three infected patients received little, if any, of this prepared food because of the severity of their illnesses. Individual beverages and water were kept separate for each patient. Eggs were not included in the diets of these patients. There were no other agents of common contact among these patients. The remaining seven patients in the wing never became colonised or infected with salmonella, and this organism was not identified anywhere else in the hospital. With the first isolate of S. typhimurium from case 1, strict isolation procedures were enforced in the unit. The remaining patients on the floor and all staff, including nursing and kitchen personnel, were also cultured ; none had salmonella in their stools. When salmonella was isolated from case 3 in November, the unit was closed to further admissions.
The exact mode of interpatient transfer of salmonella could not be determined. However, it was felt more likely that transmission of the S. typhimurium was from patient to patient in adjacent rooms via members of the staff, rather than from a contaminated common source, since such a source could not be identified despite an intensive search. Care of these patients was compared retrospectively with a small case control group of similar but salmonella-free patients on the same floor. No significant differences in therapy, particularly of local care, were observed between these groups. RESISTANCE AMONG SALMONELLA
The
antibiotic-susceptibility patterns of the S. typhimurium strains isolated from the three cases were identical on testing by the Kirby-Bauer method 11; but they differed in susceptibility to kanamycin from the multiply resistant Tehran strains (table I). One of the isolates from Tehran differed from the others in its susceptibility to both tetracycline and chloramphenicol. The unique feature in the three isolates of S. typhimurium from the three present cases was their high level of resistance to AgN03 in contrast to the susceptibility of other salmonella strains (of various serotypes) and of E. coli strains (table 11). These figures do not give a true reflection of the frequency of AgN03resistant salmonella since the three isolates undoubtedly represent the same strain. Furthermore, this strain was the initial one in which susceptibility to AgNO3 was examined, since it had been isolated from a patient receiving that agent topically. TRANSFER OF RESISTANCE
Resistance to AgN03 (and HgCl2) as well as resisfive drugs was co-transferable to another strain of S. typhimurium, leu-SO NaIR, or to E. coli C. tance to
TABLE OF
I-ANTIBIOTIC SUSCEPTIBILITY PATTERNS (KIRBY-BAUER) AND TEHRAN STRAINS OF SALMONELLA TYPHIMURIUM
MGH
Ap = ampicillin. Tc = tetracycline. Cm chloramphenicol. Sm streptomycin. Km=kanamycin. Gm = gentamicin. Cl=colistin. Cp cephalothin. S susceptible. R resistant. =
=
=
TABLE
=
=
II-SUSCEPTIBILITY TO SILVER NITRATE OF SALMONELLA AND E. COLI
inhibitory concentration determined by the
*
M.LC.
t
agar dilution method. These 9 strains were isolated in 1973. 2 of them were S. typhimurium; the other 7 belonged to a variety of serotypes (see Materials and
is the minimum
Methods).
238 TABLE
*
Abbreviations
as
t Matings carried
t Matings carried
IV-FREQUENCY
OF TRANSFER OF ANTIBIOTIC RESISTANCE
in table out out
i. Also, Su=sulphonamides. overnight in broth. Frequency expressed as number of transconjugants by millipore filter procedure (Materials and Methods).
Further transfer could then be effected from E. coli C into another E. coli strain, J53. The properties of the donor and recipient strains in these transfer experiments are summarised in table III. In addition, the S. typhimurium (MGH) was resistant to sulphathiazole (300 Ag.) by the disc technique. The frequency of resistance transfer was very low with conventional 2hour mating in broth and was between 10-6 and 10-5 in overnight matings (table iv). The highest frequency of transfer (3-6 X 10-2) was observed in E. coli to E. coli matings on millipore filters. Resistance to sulphonamides was transferred as well as resistance to the antibiotics listed in table in. For all matings, control plates (either donor cells only on media containing the counter-selecting antibiotic, or recipient cells only on media containing the antibiotic for selection: tetracycline or chloramphenicol) failed to show growth. TABLE III-ANTIMICROBIAL SUSCEPTIBILITY MATING STRAINS AND
*
Abbreviations
(M.I.C.)
PATTERNS OF
TRANSCONJUGANTS
as
in table
i.
The spontaneous occurrence of resistance to AgN03 occurred in the recipient strains, E. coli C and E. coli 153, at a frequency of less than 1 X 10-9. Using AgN03 for selection in matings between E. coli C (pMG101) and E. coli 153, transconjugants could be isolated which were simultaneously resistant to AgNO3 and to HgCl2, Ap, Cm, Tc, and Sm as well.
Discussion Most outbreaks of salmonellosis are generally believed to result from contact with a common vehicle such as contaminated food or water."-14 Although person-to-person spread may occur, especially in children, most hospital and institutional outbreaks have been related to transmission of organisms via a common vehicle. Indeed, MacGregor and Reinhart have concluded on the basis of a study of eight hospital inpatients with acute salmonellosis that person-to-person spread of salmonella in the hospital setting is difficult to establish without an intermediary common vehicle.1
per donor at end of
mating.
The findings in the present small outbreak do not suggest a common source. Admittedly this cluster of cases occurred in a special setting where person-toperson transfer may have occurred not only via the usual oral route but also by contamination of large denuded burned areas. The exact method by which the index case acquired his infection with salmonella is not known, since admission stool-cultures were not obtained. Nonetheless, it seems most likely that he was an intestinal carrier of this strain of S. typhimurium, and his wound infection resulted from autoBoth secondary infection with contaminated faeces. cases were burn patients in rooms adjacent to the index case. The lack of other cases of salmonellosis (among patients with burns or other illnesses) due to a similar organism on this floor, despite the sharing of a common central food and water source, and the lack of isolates from other inpatients in other parts of the hospital make a common vehicle unlikely. Moreover, environmental and personnel cultures were constantly negative for S. typhimurium. Thus, it seems most reasonable to conclude that patient-to-patient transmission did occur, despite the vigorous precautions taken to prevent such an event following isolation of the organism from the index case. This is particularly disturbing because of the apparent virulence, as well as the antibiotic resistance, of the strains involved in this outbreak. Two of the three patients developed bacteraemia, and in none of the patients was the organism eradicated despite antimicrobial therapy with drugs to which the salmonella exhibited in-vitro susceptibility. However, all three colonised patients had suffered severe burns, and it is entirely possible that local host factors were responsible, in large part, for both the apparent virulence of the strain and the poor therapeutic results. Multiple antibiotic resistance among salmonella has recently emerged as a significant medical problem. The strains involved have belonged predominantly to S. typhi. In the extensive epidemic of typhoid fever which began in Mexico in 1972, most strains carried transmissible multiple-resistance factors conferring resistance to chloramphenicol, tetracycline, streptomycin, and sulphonamides.3.4 In addition, in the course of the outbreak seven unusual strains were isolated bearing resistance to ampicillin as well as to the four other drugs.3 These posed a genuine potential threat, since chloramphenicol and ampicillin have been the nrst-line. drugs for treatment of invasive salmonella infections. A single isolate was resistant also to kanamycin, and was shown to harbour two transmissible plasmids: one carrying resistance to Cm, Tc, Sm, and Su; the second, to Ap and Km. Among the six other isolates, a second plasmid (carrying resistance only to
239
ampicillin) was transmissible in a few, and was nonconjugative in several, but in the latter could be mobilised by the accompanying transferable plasmids.4 In a study of the antimicrobial resistance of 2246 strains of salmonella isolated from humans, 33 (1’5%) 19 of were found to be resistant to chloramphenicol 5 these were strains of S. typhi carrying the same transmissible antibiotic-resistance pattern as those found Of the remaining 14, 8 in the Mexican outbreak. were resistant to both chloramphenicol and ampicillin as well as at least three other antimicrobial agents. These salmonella serotypes included S. derby, S. infantis, S. java, S. senftenberg, and S. worthingtonbut not S. typhimurium, the serotype responsible for the infections in the burn unit in this hospital. Only 2 of the strains (S. infantis and S. java) were shown to contain transmissible plasmids which bore resistance to both ampicillin and chloramphenicol. No information is reported as to metal ion resistance in these two strains. An extensive study of S. typhimurium isolates from farm animals and poultry in the Netherlands revealed that 0’1-0-5% of strains, during the period 1966-69, were simultaneously resistant to both chloramphenicol and ampicillin, as well as to tetracycline.6 An outbreak due to S. typhimurium strains resistant to Cm, Ap, Tc, Sm, and Km has recently occurred in Tehran. We have had an opportunity to test these strains and they are all sensitive to AgNOs, in contrast to the isolates from our burn unit. Transmissible plasmids in a variety of gram-negative bacilli, including E. coli, S. typhimurium, klebsiella, serratia, and pseudomonas, have been shown to mediate mercury resistance.15-11 The basis for this resistance has been shown to involve the volatilisation of mercury from the medium as metallic Hgj’--l’ Resistance to arsenic compounds (arsenate, arsenite, and arsenic trioxide) has been shown to be transmissible between strains of E. coli by a plasmid which also carries resistance to tetracycline and streptomycin, but not to
mercury.19
Other instances of resistance to metal ions (mercury or silver) among bacteria have been reported, but without demonstration of their mediation by transmissible
plasmids.7,2O The use of topical AgN03 in the management of burn wounds has been widespread since its value in the prophylaxis against colonisation and invasive infection of extensive burns was first demonstrated et al.21 The wound flora of patients who have been treated with silver-nitrate dressings differs from that of patients treated in a different fashion. A small percentage of strains isolated from the burn surface of patients undergoing treatment with silver nitrate have proved to be resistant to this compound on in-vitro testing. These few resistant strains have belonged mainly to the enterobacteriaceae,7 and there have also been very rare strains of pseudomonas.8 No silver-resistant strains of salmonella have been reported. Some silver-resistant strains of klebsiella and enterobacter have been isolated from burn wounds treated with silver-nitrate compresses.8.9 Among 168 strains of klebsiella and 119 strains of enterobacter isolated from patients at this hospital, 5
by Moyer
of the former and 6 of the latter had M.i.c.s for AgNO3 of 2-5 mM or greater.22 These strains all showed
resistance. Altnbugh the majority of strains were isolated from patients receiving topical AgNO3, 3 were obtained from the urine or sputum of patients who were not being treated for burns. Attempts to transfer silver resistance from these strains to a variety of recipients (E. coli, enterobacter, klebsiella) using AgN03 for selection have been unsuccessful. Additional attempts to mobilise a putative nonconjugative Ag-resistance plasmid by the introduction of another conjugative plasmid (RPI) likewise have failed. Two isolates of Enterobacter cloacae resistant to silver sulphadiazine and to silver benzoate (but not to silver nitrate) have recently been reported.23 Although these strains were shown to contain conjugative plasmids bearing resistance to kanamycin and carbenicillin, their resistance to silver sulphadiazine was not transferable. Attempts to transfer silverresistance from highly resistant strains of E. coli and klebsiella to silver-sensitive strains of E. coli have been reported as unsuccessful by Gravens et al.8 The present isolates of S. typhimurium are unique in the multiplicity of their drug resistances, their resistance to AgNOs, and their carriage of a conjugative plasmid responsible for these properties. The origin of these strains is unclear, but the extensive clinical use of topical silver-nitrate dressings in a burn unit doubtless served as an effective means of selection. The combination of the intrinsic pathogenicity of salmonella, the resistance to five of the commonly used antimicrobial agents, and resistance to the most widely employed topical agents (AgNO3 and sulphonamides) presents a potential hazard in a burn unit. Although a common source for this outbreak has not been excluded, the distinct possibility of person-to-person spread exists and emphasises the need for special care in this setting.
multiple antibiotic
We thank Dr George A. Jacoby, Jr., for his many helpful conversations and critical advice. This investigation was supported, in part, by U.S. Public Health Service grant no. 5 T01 AI00215-12. Requests for reprints should be addressed to M. N. S., Infectious Disease Unit, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114, U.S.A. REFERENCES 1. MacGregor, R. R., Reinhart, J. Lancet, 1973, ii, 1001. 2. Datta, N., Pridie, R. B., Anderson, E. S. J. Hyg., Camb. 1960, 58, 229. 3. Olarte, J., Galindo, E. Antimicrob. Ag. Chemother. 1973, 4, 597. 4. Datta, N., Olarte, J. ibid. 1974, 5, 310. 5. Bissett, M. L., Abbott, S. L., Wood, R. M. ibid. p. 161. 6. Guinee, P. A. M. Ann. N.Y. Acad. Sci. 1971, 182, 40. 7. Lowbury, E. J. L., Jackson, D. M. Lancet, 1968, i, 654. 8. Gravens, D. L., Margraf, H. W., Gravens, C. K., Thomerson, J. E., Butcher, H. R. Archs Surg., Chicago, 1969, 99, 454. 9. Cason, J. S., Jackson, D. M., Lowbury, E. J. L., Ricketts, C. R. Br. med. J. 1966, ii, 1288. 10. Davis, B. D., Mingioli, E. S. J. Bact. 1950, 60, 17. 11. Bauer, A. L., Kirby, W. M. M., Sherris, J. C., Turck, M. Am. J. clin. Path. 1966, 45, 493. 12. Steers, E., Foltz, E. L., Graves, B. S. Antibiot. Chemother. 1959, 9, 307. 13. Schroeder, S. A., Aserkoff, B., Brachman, P. S. New Engl. J. Med. 1968, 279, 674. 14. Hook, E. W., Kaye, D. (editors). Proceedings of the National Conference on Salmonella. U.S. publ. Hlth Serv. Publ. 1965, 15. 16. 17.
no. 1262, p. 47. Smith, D. H. Science, 1967, 156, 1114. Komura, I., Izaki, K. J. Biochem., Tokyo, 1971, 70, 885. Schottel, J., Mandal, A., Clark, D., Silver, S., Hedges, R. Nature, 1974, 251, 335.
240 HL-A MATCHING AND KIDNEY-GRAFT
SURVIVAL
Methods were done in one of laboratories. The cross-match was done with our routine lymphocytotoxicity technique but with prolonged incubation (45 rather than 30 minutes) to increase the sensitivity. It is considered positive if there are more than 10% killed cells. HL-A typing was carried out with workshop sera or with antisera checked against workshop sera. We do not use a standard set, but the typing is monitored by All laboratories frequent exchange of cells and sera. use lymphocytotoxicity typing while two centres also type using complement fixation on platelets. For evaluation of match grade the following antigens were considered: HL-A1, 2, 3, 9, 10, and 11, and W19 and 28 in the LA series ; and HL-A5, 7, 8, 12, 13, 14, 17, and 27, and W5, 10, 15, 16, 18, 21, and 22 in the FOUR series. The survival curves are presented as actuarial cumulative graft survival, but the statistical comparisons are based on direct graft survival.
HL-A
only
Scandiatransplant Report
*
follow-up of 523 kidney transplants with grafts from full-house donors shows a significant (P
SALMONELLA TYPHIMURIUM RESISTANT TO SILVER NITRATE, CHLORAMPHENICOL, AND AMPICILLIN A New Threat in Burn Units ?
GAIL LARKIN MCHUGH CYRUS C. HOPKINS
ROBERT C. MOELLERING MORTON N. SWARTZ
Departments of Medicine, Harvard Medical School and Massachusetts General Hospital (Infectious Disease Unit), Boston, Massachusetts 02114, U.S.A. A strain of Salmonella typhimurium appeared sequentially in three patients in a burn unit, and epidemiological study suggested the occurrence of person-to-person spread. This organism was responsible for both colonisation and invasive infection in these patients whose burn surfaces were receiving topical treatment with 0·5% silver nitrate (AgNO3) solution. The antibiotic and metal ion susceptibility pattern of this strain of S. typhimurium was unique and disturbing: resistant to silver nitrate, mercuric chloride, ampicillin, chloramphenicol, tetracycline, streptomycin, and sulphonamides. This pattern of multiple resistances could be transferred by invitro mating experiments to sensitive recipient strains of Escherichia coli and S. typhimurium. Further transfer of these resistances could be consumated between different strains of E. coli. A survey of other salmonella strains isolated from patients in this hospital without thermal burns did not reveal this pattern of resistance. Also, strains of S. typhimurium, isolated elsewhere and showing simultaneous resistance to both ampicillin and chloramphenicol, were not resistant to AgNO3 in vitro. The very real danger of this strain of S. typhimurium in burn units stems from its resistance to the two most effective antibiotics (ampicillin and chloramphenicol) available for systemic therapy; and this threat may be compounded through the selection effected by the widespread topical use of AgNO3 solutions and sulphonamide preparations on burned surfaces.
Summary
Introduction THE capacity of salmonella to exhibit new properties and to exploit the vulnerable host, particularly in the hospital setting, remains a continuing challenge in medicine. The sequential appearance, in three patients in the same burn unit, of a strain of S. typhimurium with unusual properties raised the possibility of person7901
I975
to-person spread. A report by MacGregor and Reinharthas suggested that nosocomial dissemination of salmonella is unlikely without a common source such as food or medication. Earlier, others indicated the contrary.2 A cause of particular concern is the resistance of the current strain to five antimicrobial agents, including chloramphenicol and ampicillin. Simultaneous resistance to these last two antibiotics has been described only recently among salmonellae.3-6 In addition, this present strain is highly resistant to AgN03 which had been used in topical therapy of burned surfaces. Resistance to AgN03 has been described only rarely among certain of the enterobacteriaceae, such as enterobacter and klebsiella strains, but not in salmonella.7-9 This resistance to AgN03 as well as to other antimicrobial agents in the current strain of salmonella is transferable to other members of the enterobacteriaceae. Silver resistance mediated by a conjugative plasmid has not previously been described.
Materials and Methods Bacterial Strains The multiply resistant salmonella strain used as donor in conjugation studies was the isolate obtained from the blood of case 1; and initially it was designated S, typhimurium (MGH). After demonstration of its conjugative resistance plasmid it was given the designation S. typhimurium (pMG101). Nine other salmonella strains isolated this past year from patients at the Massachusetts General Hospital were kindly provided by Dr Lawrence Kunz and were studied for sensitivity to AgNOa. Of these strains, two were S. typhimurium; two, S. heidelberg; and one each, S. newington, S. enteritidis, S. infantis, S. montevideo, and S. typhi. Ten additional strains of S. typhimurium isolated from an outbreak in Iran (" Tehran strains ") were studied for resistance to silver nitrate. Nine of these were ampicillin and chloramphenicol resistant; a tenth strain was only to ampicillin. They were kindly provided by Dr Clyde Thornsberry, of the Clinical Bacteriology Branch of the Center for Disease Control, Atlanta. In conjugation experiments with the Ag-resistant S. typhimurium from case 1 as donor, the salmonella recipient was a naladixic acid (NalR) resistant mutant of a leucine auxotroph of S. typhimurium leu-50 NaiR kindly provided by Dr K. E. Sanderson, Department of Biology, University of Calgary. In mating experiments between S. typhimurium (pMG101and E. coli, the recipient was E. coli C, F-, NAIR. In mating experiments between E. coli strains, E. coli C, P-; NalR and a rifampin-resistant aminoacid auxotroph, E. coli 3.?, F’, pro-, met-, RifR were
resistant
employed. The identity 50
was
of
transconjugants of S. typhimurium leudemonstrating growth of purified
confirmed by
E
236
agar 10 only in the presence of leucine ml.). Similarly, transconjugants of E. coli ’]53 were validated, after purification, by demonstrating growth on minimal agar only in the presence of both methionine (30 ,g. per ml.) and proline (50 Ag. per ml.). The conjugative plasmid in S. typhimurium has been given the designation pMG101. colonies
(80
on
minimal
gg. per
Antibiotic and Metal Ion
Sensitivity Antibiotic-susceptibility testing was performed by the single-disc technique described by Bauer et al.11 on Mueller-Hinton agar. Levels of resistance to drugs were determined by the agar-plate dilution technique in McConkey agar employing 10-2 dilution of 14-18 hour cultures with an inocula-replicating device similar to that of Steers et al.12
Levels of resistance to AgNOg were determined in TYE agar without added sodium similarly chloride (Bacto tryptone 10 g., yeast extract 5 g., agar 15 g. per litre). Levels of resistance to mercuric chloride (HgCl2) were determined on beef-heart infusion agar
(Difco). Conjugation Procedure Cultures of donor and recipient bacteria grown to midin TYE broth were mixed in a cell ratio of 1/1, incubated for 2 hours (unless otherwise specified) at 37 ° C, and then plated on to selective medium after appropriate dilution. The plates were incubated for 72 hours and the transconjugants were scored and purified. Antibiotic and metal resistance was then determined by the agar-plate dilution method. Most matings were performed on’Millipore’ filters to increase the number of transconjugants. 1 ml. quantities of log-phase cultures of donors and recipients were mixed and then passed through a sterile 0’45 jn millipore filter. The filter was then placed on a plain TYE plate and incubated overnight at 37 °C. The filter was then removed from the agar and shaken in 2 ml. of TYE broth to remove the bacteria from the filter. Appropriate dilutions were made, and then plated on to selective media and incubated at 37 °C for 72 hours. Plates containing AgNOg were incubated in the dark.
logarithmic phase
Results FIRST CASE
A 32-year-old White male was admitted to the Massachusetts General Hospital on Oct. 3, 1973, with 3rd and 4th degree electrical burns of his left arm, flank, and both legs, involving 30% of his body-surface area. The patient had never travelled outside of the United States and had lived all his life in the north-eastern part of the country. His general health had been excellent, and there was no prior history of diarrhoea. Immediately after admission he underwent surgical removal of eschar, and therapy with parenteral penicillin and topical 0’5 % AgN03 was begun. Between Oct. 5 and 12, he underwent further debridement and then total amputation of both legs when they became non-viable. Parenteral therapy with gentaInitial micin was begun because of persistent fever. bum-wound cultures showed E. coli, herellea, Pseudomonas maltophilia, and enterococci. By Oct. 16, however, a strain of S. typhimurium which was resistant to tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides was isolated from his burn sites. A stool culture obtained on Oct. 24 also yielded S. typhimurium with a similar pattern of antibiotic resistance. On Oct. 26 treatment with tobramycin was begun because of persistent bactersemia due to a gentamicin-resistant strain of herellea. Subtotal gastrectomy for massive upper gastrointestinal bleeding was performed on Oct. 27. Bumwound cultures during this period consistently revealed S. typhimurium despite topical therapy with AgN03 and
mafenide (’ Sulfamylon’) and parenteral administration of tobramycin, to which the organism was sensitive in vitro. By Oct. 29 blood-cultures began to grow EnteroIntravenous therapy with bacter sp. and enterococci. penicillin was added. On Nov. 3, S. typhimurium was isolated from blood-cultures, and bactersemia due to this organism persisted until death. The antibiotic-sensitivity pattern of the S. typhimurium isolated from the patient’s blood was identical with that isolated from the burned surfaces. The same strain of S. typhimurium was isolated from sputum specimens between Nov. 13 and 19, but there was no clinical evidence of pneumonia. The isolate of S. typhimurium from his blood was susceptible to trimethoprim-sulphamethoxazole by in-vitro testing. Thus, despite persistent ileus, the patient was begun on trimethoprim-sulphamethoxazole therapy by nasogastric tube on Nov. 19. Serum-sulphonamide levels varied between 1’2 and 2’5 mg. per 100 ml. on doses of trimethoprim (320 mg.) and sulphamethoxazole (1600 mg.) every 12 Treatment with intravenous carbenicillin and hours. gentamicin was also begun when Pseudomonas aeruginosa appeared in blood-cultures. On this treatment, the pseudomonas bacteraemia cleared, but S. typhimurium was repeatedly isolated from blood-cultures. Hypotension and oliguria developed, and the patient died on Nov. 25, 1973. Post-mortem examination revealed acute peritonitis with subhepatic and left subphrenic abscesses, acute mediastinitis,. and acute left pleuritis. In addition, there was acute fibrinous pericarditis with a purulent pericardial effusion totalling 600 ml. Cultures taken from these purulent collections grew a mixture of gram-negative organisms, including S. typhimurium, and enterococci. Post-mortem culture of heart blood grew only S. typhimurium with a colony-count of 100 per ml. SECOND CASE
A 55-year-old woman entered the Massachusetts General Hospital on Oct. 15, 1973, because of thirddegree burns involving 35 % of her body-surface area, including her abdomen, chest, and face. She was placed in a room next to case 1 and was treated with topical 0’5 % AgN03 to her trunk and povidone-iodine (’Betadine’) ointment to her face and neck. Parenteral penicillin was also begun. On Oct. 19 she underwent primary excision of the burned areas of her chest and abdominal wall and S. typhiapplication of split-thickness skin grafts. resistant to murium tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides, were first isolated from her burn wounds on Oct. 23. Over the subsequent 6 days the same strain was isolated from cultures of blood, sputum, and urine. A stool-culture was not obtained. On Oct. 29 massive upper-gastrointestinal bleeding occurred, and this was controlled by angiography and intra-arterial vasopressin. However, despite cessation of bleeding, severe hypotension ensued, and the patient died on Oct. 29, 1973. The only blood-culture (obtained from an arterial cannula in a non-burned area on Oct. 26) showed S. typhimurium and Herellea sp. Necropsy revealed bilateral pulmonary cedema, gastric stress ulcers, and tracheo-bronchitis. Post-mortem blood and lung cultures yielded S. typhimurium. THIRD CASE
A 20-year-old White male was admitted to the Massachusetts General Hospital on July 31, 1973, following an airplane crash. He had suffered third-degree burns over 80% of his body-surface area, sparing only his right shoulder, part of his right arm, and a portion of his head. He also suffered a compound fracture of the right tibia. He was placed in a room next to that occupied by case 1, and treatment with parenteral penicillin and topical mafenide, followed by topical AgNOs, was begun. On
237
Aug. 3 the patient underwent bilateral above-the-knee He had numerous surgical procedures for debridement and skin grafting of his burns. During the first 3 months of his stay in hospital his course was com-
amputation.
plicated by upper-gastrointestinal bleeding, numerous bouts of bacterial pneumonia, burn-wound infection, and intermittent bacterxmia due to enterococci, Klebsiella pneumoniae, and Enterobacter sp. He received courses of treatment with numerous antibiotics, including oxacillin, gentamicin, chloramphenicol, kanamycin, and tobramycin. On Nov. 19, 1973, S. typhimurium, resistant to tetracycline, chloramphenicol, ampicillin, streptomycin, and sulphonamides, was isolated from his bum Nov. 18 revealed the same organism. Topical povidone-iodine ointment was added to the topical silver-nitrate therapy. Woundcultures continued to yield S. typhimurium, and on Nov. 27, 1973, he was begun on therapy with trimethoprim (320 mg.) and sulphamethoxazole (1600 mg.) orally every 12 hours; the frequency of these doses was increased to every 6 hours on Dec. 1. On this regimen the serumsulphonamide level was 3-0 mg. per 100 ml. Trimethoprim-sulphamethoxazole therapy was stopped on Dec. 5. Wound-cultures thereafter did not yield salmonella, but S. typhimurium persisted in his stool-cultures. He subwounds.
A stool-culture obtained
on
sequently developed disseminated candidiasis, and, despite therapy with amphotericin B, he became oliguric and azotmic, and died on Dec. 11, 1973. Necropsy revealed bilateral bronchopneumonia, and evidence of candida endocarditis with fungal abscesses in multiple organs. Post-mortem blood, brain, kidney, and lung cultures were negative for S. typhimurium. EPIDEMIOLOGICAL OBSERVATIONS
The three patients infected with the multiply resistant strain of S. typhimurium in this study were housed in three adjacent rooms (A, B, C) in a ten-room isolation wing of the Massachusetts General Hospital. Case 1 initially resided in room A, and was transferred to room B after a surgical procedure on Oct. 15, 1973. At this point, case 2 was admitted to room A. Case 3 was housed in room C throughout his hospital stay. There were seven patients on the wing at the beginning of the salmonella outbreak, gradually dwindling to two by the time of the death of case 3. Their illnesses ranged from relatively minor injuries to severe and complicated burns. For all patients, food was brought from a central kitchen, elsewhere in the hospital, on separate servingtrays, portioned on to individual dishes by kitchen aides on the floor, and served to the patients by aides or nurses. While most of the patients were served in this way, the three infected patients received little, if any, of this prepared food because of the severity of their illnesses. Individual beverages and water were kept separate for each patient. Eggs were not included in the diets of these patients. There were no other agents of common contact among these patients. The remaining seven patients in the wing never became colonised or infected with salmonella, and this organism was not identified anywhere else in the hospital. With the first isolate of S. typhimurium from case 1, strict isolation procedures were enforced in the unit. The remaining patients on the floor and all staff, including nursing and kitchen personnel, were also cultured ; none had salmonella in their stools. When salmonella was isolated from case 3 in November, the unit was closed to further admissions.
The exact mode of interpatient transfer of salmonella could not be determined. However, it was felt more likely that transmission of the S. typhimurium was from patient to patient in adjacent rooms via members of the staff, rather than from a contaminated common source, since such a source could not be identified despite an intensive search. Care of these patients was compared retrospectively with a small case control group of similar but salmonella-free patients on the same floor. No significant differences in therapy, particularly of local care, were observed between these groups. RESISTANCE AMONG SALMONELLA
The
antibiotic-susceptibility patterns of the S. typhimurium strains isolated from the three cases were identical on testing by the Kirby-Bauer method 11; but they differed in susceptibility to kanamycin from the multiply resistant Tehran strains (table I). One of the isolates from Tehran differed from the others in its susceptibility to both tetracycline and chloramphenicol. The unique feature in the three isolates of S. typhimurium from the three present cases was their high level of resistance to AgN03 in contrast to the susceptibility of other salmonella strains (of various serotypes) and of E. coli strains (table 11). These figures do not give a true reflection of the frequency of AgN03resistant salmonella since the three isolates undoubtedly represent the same strain. Furthermore, this strain was the initial one in which susceptibility to AgNO3 was examined, since it had been isolated from a patient receiving that agent topically. TRANSFER OF RESISTANCE
Resistance to AgN03 (and HgCl2) as well as resisfive drugs was co-transferable to another strain of S. typhimurium, leu-SO NaIR, or to E. coli C. tance to
TABLE OF
I-ANTIBIOTIC SUSCEPTIBILITY PATTERNS (KIRBY-BAUER) AND TEHRAN STRAINS OF SALMONELLA TYPHIMURIUM
MGH
Ap = ampicillin. Tc = tetracycline. Cm chloramphenicol. Sm streptomycin. Km=kanamycin. Gm = gentamicin. Cl=colistin. Cp cephalothin. S susceptible. R resistant. =
=
=
TABLE
=
=
II-SUSCEPTIBILITY TO SILVER NITRATE OF SALMONELLA AND E. COLI
inhibitory concentration determined by the
*
M.LC.
t
agar dilution method. These 9 strains were isolated in 1973. 2 of them were S. typhimurium; the other 7 belonged to a variety of serotypes (see Materials and
is the minimum
Methods).
238 TABLE
*
Abbreviations
as
t Matings carried
t Matings carried
IV-FREQUENCY
OF TRANSFER OF ANTIBIOTIC RESISTANCE
in table out out
i. Also, Su=sulphonamides. overnight in broth. Frequency expressed as number of transconjugants by millipore filter procedure (Materials and Methods).
Further transfer could then be effected from E. coli C into another E. coli strain, J53. The properties of the donor and recipient strains in these transfer experiments are summarised in table III. In addition, the S. typhimurium (MGH) was resistant to sulphathiazole (300 Ag.) by the disc technique. The frequency of resistance transfer was very low with conventional 2hour mating in broth and was between 10-6 and 10-5 in overnight matings (table iv). The highest frequency of transfer (3-6 X 10-2) was observed in E. coli to E. coli matings on millipore filters. Resistance to sulphonamides was transferred as well as resistance to the antibiotics listed in table in. For all matings, control plates (either donor cells only on media containing the counter-selecting antibiotic, or recipient cells only on media containing the antibiotic for selection: tetracycline or chloramphenicol) failed to show growth. TABLE III-ANTIMICROBIAL SUSCEPTIBILITY MATING STRAINS AND
*
Abbreviations
(M.I.C.)
PATTERNS OF
TRANSCONJUGANTS
as
in table
i.
The spontaneous occurrence of resistance to AgN03 occurred in the recipient strains, E. coli C and E. coli 153, at a frequency of less than 1 X 10-9. Using AgN03 for selection in matings between E. coli C (pMG101) and E. coli 153, transconjugants could be isolated which were simultaneously resistant to AgNO3 and to HgCl2, Ap, Cm, Tc, and Sm as well.
Discussion Most outbreaks of salmonellosis are generally believed to result from contact with a common vehicle such as contaminated food or water."-14 Although person-to-person spread may occur, especially in children, most hospital and institutional outbreaks have been related to transmission of organisms via a common vehicle. Indeed, MacGregor and Reinhart have concluded on the basis of a study of eight hospital inpatients with acute salmonellosis that person-to-person spread of salmonella in the hospital setting is difficult to establish without an intermediary common vehicle.1
per donor at end of
mating.
The findings in the present small outbreak do not suggest a common source. Admittedly this cluster of cases occurred in a special setting where person-toperson transfer may have occurred not only via the usual oral route but also by contamination of large denuded burned areas. The exact method by which the index case acquired his infection with salmonella is not known, since admission stool-cultures were not obtained. Nonetheless, it seems most likely that he was an intestinal carrier of this strain of S. typhimurium, and his wound infection resulted from autoBoth secondary infection with contaminated faeces. cases were burn patients in rooms adjacent to the index case. The lack of other cases of salmonellosis (among patients with burns or other illnesses) due to a similar organism on this floor, despite the sharing of a common central food and water source, and the lack of isolates from other inpatients in other parts of the hospital make a common vehicle unlikely. Moreover, environmental and personnel cultures were constantly negative for S. typhimurium. Thus, it seems most reasonable to conclude that patient-to-patient transmission did occur, despite the vigorous precautions taken to prevent such an event following isolation of the organism from the index case. This is particularly disturbing because of the apparent virulence, as well as the antibiotic resistance, of the strains involved in this outbreak. Two of the three patients developed bacteraemia, and in none of the patients was the organism eradicated despite antimicrobial therapy with drugs to which the salmonella exhibited in-vitro susceptibility. However, all three colonised patients had suffered severe burns, and it is entirely possible that local host factors were responsible, in large part, for both the apparent virulence of the strain and the poor therapeutic results. Multiple antibiotic resistance among salmonella has recently emerged as a significant medical problem. The strains involved have belonged predominantly to S. typhi. In the extensive epidemic of typhoid fever which began in Mexico in 1972, most strains carried transmissible multiple-resistance factors conferring resistance to chloramphenicol, tetracycline, streptomycin, and sulphonamides.3.4 In addition, in the course of the outbreak seven unusual strains were isolated bearing resistance to ampicillin as well as to the four other drugs.3 These posed a genuine potential threat, since chloramphenicol and ampicillin have been the nrst-line. drugs for treatment of invasive salmonella infections. A single isolate was resistant also to kanamycin, and was shown to harbour two transmissible plasmids: one carrying resistance to Cm, Tc, Sm, and Su; the second, to Ap and Km. Among the six other isolates, a second plasmid (carrying resistance only to
239
ampicillin) was transmissible in a few, and was nonconjugative in several, but in the latter could be mobilised by the accompanying transferable plasmids.4 In a study of the antimicrobial resistance of 2246 strains of salmonella isolated from humans, 33 (1’5%) 19 of were found to be resistant to chloramphenicol 5 these were strains of S. typhi carrying the same transmissible antibiotic-resistance pattern as those found Of the remaining 14, 8 in the Mexican outbreak. were resistant to both chloramphenicol and ampicillin as well as at least three other antimicrobial agents. These salmonella serotypes included S. derby, S. infantis, S. java, S. senftenberg, and S. worthingtonbut not S. typhimurium, the serotype responsible for the infections in the burn unit in this hospital. Only 2 of the strains (S. infantis and S. java) were shown to contain transmissible plasmids which bore resistance to both ampicillin and chloramphenicol. No information is reported as to metal ion resistance in these two strains. An extensive study of S. typhimurium isolates from farm animals and poultry in the Netherlands revealed that 0’1-0-5% of strains, during the period 1966-69, were simultaneously resistant to both chloramphenicol and ampicillin, as well as to tetracycline.6 An outbreak due to S. typhimurium strains resistant to Cm, Ap, Tc, Sm, and Km has recently occurred in Tehran. We have had an opportunity to test these strains and they are all sensitive to AgNOs, in contrast to the isolates from our burn unit. Transmissible plasmids in a variety of gram-negative bacilli, including E. coli, S. typhimurium, klebsiella, serratia, and pseudomonas, have been shown to mediate mercury resistance.15-11 The basis for this resistance has been shown to involve the volatilisation of mercury from the medium as metallic Hgj’--l’ Resistance to arsenic compounds (arsenate, arsenite, and arsenic trioxide) has been shown to be transmissible between strains of E. coli by a plasmid which also carries resistance to tetracycline and streptomycin, but not to
mercury.19
Other instances of resistance to metal ions (mercury or silver) among bacteria have been reported, but without demonstration of their mediation by transmissible
plasmids.7,2O The use of topical AgN03 in the management of burn wounds has been widespread since its value in the prophylaxis against colonisation and invasive infection of extensive burns was first demonstrated et al.21 The wound flora of patients who have been treated with silver-nitrate dressings differs from that of patients treated in a different fashion. A small percentage of strains isolated from the burn surface of patients undergoing treatment with silver nitrate have proved to be resistant to this compound on in-vitro testing. These few resistant strains have belonged mainly to the enterobacteriaceae,7 and there have also been very rare strains of pseudomonas.8 No silver-resistant strains of salmonella have been reported. Some silver-resistant strains of klebsiella and enterobacter have been isolated from burn wounds treated with silver-nitrate compresses.8.9 Among 168 strains of klebsiella and 119 strains of enterobacter isolated from patients at this hospital, 5
by Moyer
of the former and 6 of the latter had M.i.c.s for AgNO3 of 2-5 mM or greater.22 These strains all showed
resistance. Altnbugh the majority of strains were isolated from patients receiving topical AgNO3, 3 were obtained from the urine or sputum of patients who were not being treated for burns. Attempts to transfer silver resistance from these strains to a variety of recipients (E. coli, enterobacter, klebsiella) using AgN03 for selection have been unsuccessful. Additional attempts to mobilise a putative nonconjugative Ag-resistance plasmid by the introduction of another conjugative plasmid (RPI) likewise have failed. Two isolates of Enterobacter cloacae resistant to silver sulphadiazine and to silver benzoate (but not to silver nitrate) have recently been reported.23 Although these strains were shown to contain conjugative plasmids bearing resistance to kanamycin and carbenicillin, their resistance to silver sulphadiazine was not transferable. Attempts to transfer silverresistance from highly resistant strains of E. coli and klebsiella to silver-sensitive strains of E. coli have been reported as unsuccessful by Gravens et al.8 The present isolates of S. typhimurium are unique in the multiplicity of their drug resistances, their resistance to AgNOs, and their carriage of a conjugative plasmid responsible for these properties. The origin of these strains is unclear, but the extensive clinical use of topical silver-nitrate dressings in a burn unit doubtless served as an effective means of selection. The combination of the intrinsic pathogenicity of salmonella, the resistance to five of the commonly used antimicrobial agents, and resistance to the most widely employed topical agents (AgNO3 and sulphonamides) presents a potential hazard in a burn unit. Although a common source for this outbreak has not been excluded, the distinct possibility of person-to-person spread exists and emphasises the need for special care in this setting.
multiple antibiotic
We thank Dr George A. Jacoby, Jr., for his many helpful conversations and critical advice. This investigation was supported, in part, by U.S. Public Health Service grant no. 5 T01 AI00215-12. Requests for reprints should be addressed to M. N. S., Infectious Disease Unit, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114, U.S.A. REFERENCES 1. MacGregor, R. R., Reinhart, J. Lancet, 1973, ii, 1001. 2. Datta, N., Pridie, R. B., Anderson, E. S. J. Hyg., Camb. 1960, 58, 229. 3. Olarte, J., Galindo, E. Antimicrob. Ag. Chemother. 1973, 4, 597. 4. Datta, N., Olarte, J. ibid. 1974, 5, 310. 5. Bissett, M. L., Abbott, S. L., Wood, R. M. ibid. p. 161. 6. Guinee, P. A. M. Ann. N.Y. Acad. Sci. 1971, 182, 40. 7. Lowbury, E. J. L., Jackson, D. M. Lancet, 1968, i, 654. 8. Gravens, D. L., Margraf, H. W., Gravens, C. K., Thomerson, J. E., Butcher, H. R. Archs Surg., Chicago, 1969, 99, 454. 9. Cason, J. S., Jackson, D. M., Lowbury, E. J. L., Ricketts, C. R. Br. med. J. 1966, ii, 1288. 10. Davis, B. D., Mingioli, E. S. J. Bact. 1950, 60, 17. 11. Bauer, A. L., Kirby, W. M. M., Sherris, J. C., Turck, M. Am. J. clin. Path. 1966, 45, 493. 12. Steers, E., Foltz, E. L., Graves, B. S. Antibiot. Chemother. 1959, 9, 307. 13. Schroeder, S. A., Aserkoff, B., Brachman, P. S. New Engl. J. Med. 1968, 279, 674. 14. Hook, E. W., Kaye, D. (editors). Proceedings of the National Conference on Salmonella. U.S. publ. Hlth Serv. Publ. 1965, 15. 16. 17.
no. 1262, p. 47. Smith, D. H. Science, 1967, 156, 1114. Komura, I., Izaki, K. J. Biochem., Tokyo, 1971, 70, 885. Schottel, J., Mandal, A., Clark, D., Silver, S., Hedges, R. Nature, 1974, 251, 335.
240 HL-A MATCHING AND KIDNEY-GRAFT
SURVIVAL
Methods were done in one of laboratories. The cross-match was done with our routine lymphocytotoxicity technique but with prolonged incubation (45 rather than 30 minutes) to increase the sensitivity. It is considered positive if there are more than 10% killed cells. HL-A typing was carried out with workshop sera or with antisera checked against workshop sera. We do not use a standard set, but the typing is monitored by All laboratories frequent exchange of cells and sera. use lymphocytotoxicity typing while two centres also type using complement fixation on platelets. For evaluation of match grade the following antigens were considered: HL-A1, 2, 3, 9, 10, and 11, and W19 and 28 in the LA series ; and HL-A5, 7, 8, 12, 13, 14, 17, and 27, and W5, 10, 15, 16, 18, 21, and 22 in the FOUR series. The survival curves are presented as actuarial cumulative graft survival, but the statistical comparisons are based on direct graft survival.
HL-A
only
Scandiatransplant Report
*
follow-up of 523 kidney transplants with grafts from full-house donors shows a significant (P
