trimethoprim: laboratory clinical studies · sulphonamides: para-aminobenzoic acid sulphonamides...

J. clin. Path. (1968), 21, 202-209

Trimethoprim: laboratory and clinical studiesJ. H. DARRELL, L. P. GARROD, AND PAMELA M. WATERWORTH

From the Department of Bacteriology, Royal Postgraduate Medical School, London

SYNOPSIS Trimethoprim is a synthetic compound which arrests the step in bacterial purine synthesisimmediately succeeding that interfered with by sulphonamides.

Acting alone it inhibits the growth of a wide range of bacteria in generally much lower concen-

trations than sulphonamides. Its action with a sulphonamide is strongly synergic and bactericidal.Studies are described of suitable cultural conditions, methods of testing sensitivity, and acquired

resistance.Some clinical results of its use are reported in respiratory tract infections, Gram-negative septi-

caemia, and urinary tract infections, and further therapeutic possibilities are discussed.

Trimethoprim (2,4,-diamino-5-(3', 4', 5'-trime-thoxybenzyl)-pyrimidine) is one of a series ofcompounds first fully described by Roth, Falco,Hitchings, and Bushby (1962) although their studywas begun 10 years earlier than this. They possessboth antimalarial and antibacterial activity, theformer being highest in pyrimethamine and thelatter in trimethoprim. This substance is a potentinhibitor of dihydrofolic acid reductase, which isresponsible for the conversion of dihydrofolic totetrahydrofolic acid, a stage in a sequence leadingto the synthesis of purines and ultimately of DNA.This stage immediately follows that blocked bysulphonamides:

Para-aminobenzoic acid

sulphonamides

Dihydrofolic acid

trimethoprim

Tetrahydrofolic acid

It is hence not surprising that trimethoprim andsulphonamides have been found to exert a stronglysynergic effect.Trimethoprim fortunately has a far lower affinity

for the corresponding mammalian enzyme, andthere has been very little evidence of interferenceReceived for publication 29 December 1967.

with folic acid metabolism following administrationto animals and man. The way was therefore clearfor exploiting what appeared to be a highly pro-mising therapeutic combination in the clinical field.Although a few investigators are now convinced ofits value, little has been published on the subject.Noall, Sewards, and Waterworth (1962) reportedthe successful treatment of a single case of Proteussepticaemia; another was described by Cooper andWald (1964). Schneider, Schwarzenberg, Caltan,Schlumberger, Amiel, and Mathe (1965) also studiedthetreatment of Proteus infections in theurinarytractand elsewhere. Thus for five years the only reportsof therapeutic use concerned infection by only oneorganism out of many which should be susceptible.Some contributions towards filling this gap haverecently been made by Csonka and Knight (1967),who describe the successful treatment of gonorrhoea,and by Drew, Hughes, and Jenkins (1967) andFowle, Drew, Hughes, and Cassell (1967) in briefcommunications on the treatment of bronchialinfections.So small a literature does scant justice to a new

chemotherapeutic agent of considerable interestand much wider promise. Moreover there is noreadily accessible publication giving a generalaccount of the properties of trimethoprim and of itsantibacterial activity as ascertainable in vitro. It isthe purpose of this paper to provide this, togetherwith some new observations and a short account ofclinical experience with the drug.The choice of a sulphonamide for in vitro studies

of combined action is almost immaterial. Sulphame-thoxazole (Gantanol) is regarded as the mostsuitable for clinical use, mainly because its curve of

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Trimethoprim: laboratory and clinical studies

blood concentrations and rate of elimination corre-spond most closely to those of trimethoprim. In thepresent experiments its near relative sulphafurazole(Gantrisin) was preferred for its higher solubility.

STUDIES IN VITRO

MATERIALS AND METHODS Trimethoprim is a colourlesscrystalline substance, only slightly soluble in cold water.For the experiments in vitro it was converted to theisethionate by adding sufficient normal isethionic acid tobring a suspension containing 10 mg per ml into solution.The lactate, now available for parenteral use, is moresoluble (> 25 mg/ml) and is suitable for laboratory work.The strains of bacteria used were mostly recent isolates

from clinical specimens in this department, but some hadbeen maintained for varying periods. The source ofstrains of a few species obtained from elsewhere is ac-knowledged later.For the determination of minimum inhibitory con-

centrations (MIC) the plate dilution method was used.Drug solutions to produce concentrations in the mediumdiffering by two-fold, and based on 1 ug/ml, were addedin a volume of 1 ml to 19 ml melted and cooled diagnosticsensitivity test (DST) agar (Oxoid) containing 5% lysedhorse blood, and areas on these plates were inoculatedwith a 2 mm loopful of diluted broth culture (dilution1/100 for most organisms). In later experiments a multipleinoculator (Steers, Foltz, and Graves, 1959) was used,permitting simultaneous inoculation with suspensions of32 bacteria. This admirable device enables such experi-ments to be set up in much less time than it takes later torecord them.

MIC OF TRIMETHOPRIM ALONE These results aregiven in Table I. It can be seen that trimethoprimhas a generally much higher activity than sulphona-mides, many of its MICs being well under 1 jug/mlwhereas that of sulphafurazole in exactly parallel

tests, even for fully sensitive species, was usuallyabove this level. Str. pyogenes and pneumoniae areconsistently sensitive, and even more so but withmore variation in Str. faecalis, in strong contrast toits high degree of resistance to sulphonamides.Staph. aureus is also consistently and highly sensi-tive, although none of these strains was inhibitedby less than 4 pg/ml of sulphafurazole and five ofthem only by 1024 sg/ml. H. influenzae is alsosensitive (but H. pertussis much more resistant).Neisseria spp., notably N. gonorrhoeae, are decidedlyless sensitive to trimethoprim than to sulphafura-zole: that this does not present synergy betweenthem will be seen later.Among enterobacteria Salmonella and Shigella

spp. are consistently sensitive, as are all species ofProteus to a slightly lesser degree. Klebsiella spp. arealso usually sensitive, but Ps. aeruginosa muchmore resistant.

MIC OF TRIMETHOPRIM + SULPHAFURAZOLE Thesewere determined in the same way, three series ofplates being inoculated, containing a suitable rangeof concentrations of each drug alone and of the twotogether in an appropriate ratio. It may be said atonce that in almost every experiment with anorganism sensitive to both drugs, synergy was de-monstrated in the sense that an independentlysubinhibitory concentration of one reduced theMIC of the other, commonly eight-fold but oftenmore. As an example, a strain of Str. pneumoniaewas inhibited by 32 ug/ml of sulphafurazole, andby 1 utg/ml trimethoprim acting alone but by 0-12/tg/ml in the presence of sulphafurazole in a 10:1ratio (i.e. actually 1-2 jig/ml). Thus there was aneight-fold potentiation of the action of trimethoprim

ILE IMIC OF TRIMETHOPRIM

Total Numbers Inhibited by (Ag/ml)No.

0-03 0-06 0-12 0-25 0-5 1 2 4 8 16 32 128

5 12 1107

2 11 9 3

14 3

7152

436

2

64212

Species

444

Staph. aureusStr. pyagenesStr. pneumoniaeStr. faecalisCl. welchiiN. gonorrhoeaeN. meningitidisN. catarrhalisH. influenzaeH. pertussisBacteroidesEsch. coliKlebsiella spp.Salmonella spp.Shigella spp.Pr. mirabilisPr. vulgarisPr. morganiPr. rettgeriPs. aeruginosa

No EndPoint

3

181414303

181310296121610141714101089

2 716

1 2

2 35

453

I102

2

2 24 4 3

6363

>4(1)

>64(1)

> 128(1)

> 128(2)> 32(2)

512(3)

3 12 332

2

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J. H. Darrell, L. P. Garrod, and Pamela M. Waterworth

by a concentration of sulphafurazole only 1/25thof its independent MIC.For the sake of brevity the results of many such

experiments are stated in a much condensed formin Table II, in terms of degrees of potentiation oftrimethoprim by sulphafurazole. These degrees varyamong different strains of a species according totheir sensitivity to the individual drugs. The degreeof potentiation is highest in N. gonorrhoeae, andvery high in all species of Proteus except Pr. rettgeri.It is four- to eight-fold for most strains of staphy-lococci, streptococci, pneumococci, H. influenzae,and Esch. coli. For some of these organisms, notablythe two last-named, potentiation would probablybe greater were the ratio used more nearly propor-tional to the MIC of the two drugs.

It should be noted that the same sulphonamide-trimethoprim ratio was not employed in all tests.In the majority it was 9:1 but for N. gonorrhoeae itwas 1: 3, since this organism is much more sensitiveto sulphonamides than to trimethoprim. Forsulphonamide-resistant strains of Staph. aureus andProteus spp. it was respectively 2000:1 and 1000:1:these experiments were done simply to prove that asynergic effect is exerted when concentrations relatedto the MIC are combined, but it is recognized thatsuch sulphonamide concentrations are not attainablein vivo. The synergic effect on Ps. aeruginosa is pre-sumably only of academic interest, since highconcentrations of both drugs are required.

ACQUIRED RESISTANCE It is clearly important toknow whether bacteria are capable of becomingresistant to trimethoprim, whether alone or in thepresence of a sulphonamide.

This was first studied by serial transfer of a light

Species

inoculum on ditch plates, and little change in theMIC was produced even by 25 transfers. On theother hand, only five transfers of a heavy inoculumin rising concentrations in broth produced the con-siderable changes shown in Table III. Trimethoprimalone and trimethoprim + sulphafurazole (1:10)were added to the training medium, and after trainingthe MIC of both trimethoprim and of the mixturewas determined. In parallel experiments with sulpha-furazole alone no change in the MIC was observed.

TABLE IIIRESULTS OF TRAINING TO RESISTANCE BY TRANSFERS OF A

HEAVY INOCULUM IN BROTH

Fold Increase in MIC

Drug in training medium Trimethoprim Trimethoprim +Sulphonamide

Increase in resistance to Trimetho- Mixture Trimetho- Mixtureprim prim

Organisms sensitive to 128- 2-8 8-128 2-8both drugs" >1024

Organisms sensitive to 32- 32- 32- 32-trimethoprim only' >256 >128 128 256

'One strain each of Esch. coli, Pr. mirabilis, Pr. morgani and Pr.rettgeri2One strain each of Esch. coli, Pr. mirabilis, Pr. morgani, and Kleb-siella

It is at once evident that exposure to rising con-centrations of trimethoprim alone produces a largeincrease in resistance. It was also observed in doingMiles and Misra counts on agar containing differentconcentrations of trimethoprim that very few butnormal colonies of Esch. coli are obtained from aheavy inoculum on medium containing eight to 16times the MIC for the rest of the culture: these

TABLE IIDEGREE OF POTENTIATION OF THE ACTION OF TRIMETHOPRIM BY SULPHAFURAZOLE1

Range ofMIC (Ag/ml) Ratio Nos. of Strains Showing Fold Increases in Activity ofSulphafurazole: Trimethoprim in the Presence of Sulphonamide

Sulphafurazole Trimethoprim Trimethoprim --2 -4 -8 -16 -32 -64

Staph. aureusStaph. aureusStr. pyogenesStr. pneumoniaeN. gonorrhoeaeH. influenzaeEsch. coliKlebsiella spp.Salmonella spp.Shigella spp.Pr. mirabilisPr. vulgarisPr. morganiPr. rettgeriProteus spp.Ps. aeruginosa

4-8512-20481-162-128

0-5-80-5-644-328-6416-1284-164-164-162-3216-64>2000

256-1024

0-12-0-50-12-0-50-25-0-50 5-232-640-01-0-250-12-0-50-5-10-06-0-250-03-0 50-5-20-5-20 5-20-5-11-864-512

9:12000:1

9:19:11:39:19:19:19:19:19:19:19:19:1

1000:19:1

1 71 33 4

2

10 1486

10 41 2

513122261

25

1 24 10 1

4

327

223

13

8 1

3

4

"No potentiation was observed in tests of one strain of Str. pyogenes and two of H. influenzae.

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colonies yield progeny with uniform resistance ofthis degree. Bacterial populations may thus containa small minority either of resistant variants or ofcells capable of mutation to higher resistance.When the same organisms, originally sensitive to

both drugs, and trained to trimephoprim resistance,were tested with the mixture, only small increaseswere observed: the sulphonamide component washere evidently responsible for inhibition. Similarlywhen doubly sensitive organisms were trained toboth drugs only the same small increases in resis-tance to the mixture were obtained, and the increasein resistance to trimethoprim was less than aftertraining to this drug alone. Further progress inresistance is evidently blocked by the sulphonamide,sensitivity to which remains unchanged.When organisms were tested which were trimetho-

prim-sensitive but sulphonamide-resistant, bothforms of training produced large increases in resis-tance to both trimethoprim and the mixture.

It seems reasonable to hope from these resultsthat treatment with the mixture of infections byorganisms sensitive to both drugs will not producea serious degree ofbacterial resistance. This has beenborne out by clinical experience so far: increasedresistance to trimethoprim has been seen only in twooriginally sulphonamide-resistant organisms (strainsof Klebsiella in which the MIC of trimethoprim rosefrom 02 to 2 and 0-06 to 16 tg/ml).

EFFECT OF COMPOSITION OF MEDIUM The action oftrimethoprim is not antagonized like that of sul-phonamides, by p-aminobenzoic acid, but otherconstituents in some culture media which may alsobe sulphonamide antagonists interfere with it. Onsuch media either no inhibition is produced by atrimethoprim disc (particularly in a culture of aKlebsiella) or an area equal to that of the inhibitionzone on a satisfactory medium is occupied bynumerous small colonies. Media vary in their con-tent of interfering substances: of those tested DSTagar (Oxoid) has a much lower content than bloodagar bases 1 or 2 (Oxoid).The addition of lysed horse blood, as for tests of

sulphonamide sensitivity, renders almost any me-dium suitable for tests of trimethoprim sensitivity.As little as 0 25% suffices for DST agar, but othermedia require much more, and an addition of 3 % isadvised. Whole blood is ineffective, unless lysis hasoccurred in it and the medium used requires only asmall addition of the neutralizing substance. Thissubstance is heat-labile, and a further addition oflysed blood must be made if chocolate agar is used.We have found that composition of the medium

affects the behaviour of Str. faecalis in a peculiarway. Twenty strains were inhibited by 0 06-0-25

ztg/ml trimethoprim on DST agar + 5 % lysed horseblood, but on blood agar base no. 1 with the sameaddition none were inhibited by 512 jig/ml.

ROUTINE SENSITIVITY TESTS Only a disc method isacceptable for routine use, but the definition of itsdetails presents difficulties. Ideally the test shouldindicate the degree of sensitivity to sulphonamide,trimethoprim, and the combination of the two, thelast being a measure of the degree of synergy. Thiscan be attempted as shown in Figure 1. On each ofthese plates the lower left disc contains sulphafura-zole, the lower right trimethoprim, and the upperthe same quantities of both drugs.

Synergy can be shown in this test in two ways. Thezone of inhibition produced by the combined discmay be larger, as in B and D, both being culturessensitive to both drugs. These plates also show it inthe second way, by a clearly enhanced effect wherethe zones produced by the individual drugs merge. Aand C are sulphonamide-resistant cultures, and thecombined drug inhibition zone is no larger than thetrimethoprim, but the latter extends in a remarkableway towards the sulphonamide disc, showing thatsulphonamide potentiates the action of trimetho-prim, even though the organism is sulphonamide-resistant. Unfortunately pictures of this kind dependon exact spacing between the two discs, and there isno means of knowing how far apart they should beto show the effect.

This three-disc test is nevertheless worth whilewhenever trimethoprim treatment is seriously con-templated, and it is consequently justifiable to devotea plate wholly to this test. On the other hand whenthe test is one of a series routinely applied, a singlecombined disc which may be one of a number con-taining antibiotics etc. applied to the same plate,should give an adequate indication of sensitivity.The decision on disc content for trimethoprim is

difficult, because some organisms are very highlysensitive (MIC 0-06 ,ug/ml or even less) and inhibi-tion zones are therefore inconveniently large. Yetorganisms with an MIC considerably higher thanthis cause infections known to be susceptible totreatment, and these must also be shown to be sensi-tive. The content of discs at present in use is 2 5 /Lgand this seems a satisfactory compromise; that ofsulphafurazole discs is 50 jAg. Users of discs con-taining both drugs in these amounts would do wellto familiarize themselves with the inhibition zonesproduced by them in cultures of (1) a trimethoprim-sensitive sulphonamide-resistant Pr. morgani; (2) astrain of Esch. coli sensitive to both drugs, in orderto have a standard on which to base their reportedfindings.The medium used must contain 5% lysed horse

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FIG. 1. Disc sensitivity tests. In each plate the bottom left disc contains sulphafurazole, 50 ,ug; the bottom righttrimethoprim, 2-5 ,ug; the top right the same amounts of bothA. Staph. aureus B. Proteus morgani C. Staph. aureus D. Esch. coli

blood. Inoculation should preferably be by floodingand draining, and the inoculum must not be tooheavy.

BACTERICIDAL ACTION The action of sulphona-mides is purely bacteristatic, and this was confirmedwith the strains of bacteria and in the culture mediaused in the experiments now to be briefly described.

Broth containing lysed horse blood and eithertrimethoprim or sulphafurazole or both was inocu-lated with one of the following organisms and asmall loopful was spread over one fourth of a platehourly up to seven hours and after 24 hours ofincubation. Trimethoprim alone sometimes causeda substantial fall in the viable count (from innumer-able to few colonies) but trimethoprim (1 fLg/ml) +

sulphafurazole (10 Hg/ml) sterilized an inoculum ofPr. morgani in five hours, of Pr. rettgeri in six hours,and almost sterilized one of Esch. coli in five hours.A similar but slower effect was seen in a test withStaph. aureus. An elaborate experiment demon-strating bactericidal synergy against N. gonorrhoeaeis reported elsewhere (Garrod and Waterworth,1968).The bactericidal effect exerted by the combination

may well be responsible for the rapid improvementoftem seen following its clinical use.

SOME CLINICAL OBSERVATIONS

Trimethoprim has now been used in the treatmentof about 60 cases in this hospital. The following

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brief account of results is intended only to indicatesome types of infection in which its use appears tous to merit further study.

CHRONIC BRONCHITIS The combination of trime-thoprim and sulphonamide has been most extensivelyused in this hospital in the treatment of infectiveexacerbations in chronic bronchitics. Most had beenpreviously treated with ampicillin and tetracyclines,some with chloramphenicol also. In the majority,trimethoprim produced a good initial response withstrikingly rapid reduction in the purulence of thesputum. In some cases loss of the initial pathogenwas followed by colonization by resistant organismssuch as Pseudomonas aeruginosa or Candida species,an example of the characteristic tendency of bron-chitics to re-infection.

T.S., a man aged 73, a known bronchitic for 16years, required hospital admission nearly every winter.Ampicillin and tetracyclines had been used on numerousoccasions and he had also been given steroids. Admittedin an acute exacerbation in September 1966, he wasproducing copious purulent sputum. Treated initiallywith ampicillin 1-5 g daily (intramuscularly and orally)and physiotherapy, his general condition improved buthe continued to cough up large volumes of green purulentsputum. Cultures showed that from the onset the in-fecting organism had been Klebsiellapneumoniae resistantto ampicillin.

His treatment was changed to trimethoprim 250 mgand Gantanol 500 mg twice daily, with a dramaticreduction in the amount and degree of purulence of thesputum. After five days' treatment the patient complainedof inability to cough up sputum; this was thought to bebecause there was none to cough up. A small mucoidspecimen obtained at this time yielded a Candida speciesonly.

In this and other patients the effect of this treat-ment appeared superior than that of any antibioticin eliminating infection for the time being. Organ-isms eliminated in other patients include pneu-mococci and H. influenzae. A controlled trial of thetreatment is now in progress.

STAPHYLOCOCCAL PNEUMONIA R.D., a man aged 59years, a chronic bronchitic with cor pulmonale, hepresented with purulent bloodstained sputum due to astaphylococcal pneumonia. Before admission he had hadtwo weeks' treatment with tetracyclines, followed by oneweek of chloramphenicol.The organism isolated on admission was resistaiit to

benzylpenicillin, streptomycin, tetracycline, chloram-phenicol, erythromycin, and lincomycin. Treatment wasstarted with cephaloridine but was changed after twodays to cloxacillin plus kanamycin because of the patient'scritical condition.

However, though the phage type remained the same

(type 84) staphylococci from subsequent sputum speci-mens were found to be resistant to methicillin andcephaloridine. Treatment was started with trimethoprimn250 mg and Gantanol 500 mg twice daily, previouslyused in this patient as an outpatient against his infectivebronchitis. The pneumonia rapidly resolved and foursubsequent sputum specimens were either sterile oryielded coagulase-negative staphylococci only.

This case not only shows the efficacy of trimetho-prim in adverse clinical circumstances, but suggeststhat it is worthy of trial in staphylococcal infectionsof other kinds.

GRAM-NEGATIVE SEPTICAEMLA E.K., a coloured womanaged 32, had a caesarean section for prolonged labourwith a breech presentation performed in Sierra Leone.Fever up to 103°F with rigors occurred two days later. Alaparotomy was performed and a haematoma evacuatedfrom the left broad ligament. Before transfer to Hammer-smith Hospital the patient had been given benzylpeni-cillin and streptomycin, followed by chloramphenicol,then ampicillin and finally intravenous tetracyclines. Onadmission she was found to have bacteraemia with astrain of Escherichia coli (non-lactose fermenting variant)resistant to penicillin, ampicillin, streptomycin, tetracy-clines, chloramphenicol, and also to cephaloridine, andsensitive only to kanamycin and polymyxins.The chart (Fig. 2) shows that blood cultures re-

mained positive with this same organism over fourweeks, in spite of treatment with benzylpenicillin 12mega units daily intravenously together with kanamycin1 g daily, followed by polymyxin B sulphate 1-5 megaunits daily intravenously.During the course of polymyxin treatment a laparo-

tomy was performed and a large abscess cavity in the leftbroad ligament was drained. The patient suffered acardiac arrest in the immediate postoperative period.Following the operation high fever and rigors withpositive blood cultures recurred. This was felt to indicatea persisting focus of infection, but this could not belocalized and the patient's condition did not warrant afurther exploratory operation. Kanamycin and polymyxinB, the only antibiotics active against the organism, weregiven again, this time the polymyxin in the form of themethane-sulphonate. There was again no response,though the organism remained fully sensitive to bothantibiotics. Treatment was then changed to trimethoprim250 mg three times daily with Sulphatriad 6 g daily, bothgiven by mouth. Vomiting was controlled with phenergan.The patient responded dramatically, her temperaturesettling to normal over three days and the blood culturebecoming negative. No further surgery was required.

T.M., a man aged 55, had a congenital aortic valvelesion, a murmur first being noticed at the age of 11 years.Four days before admission dental filling and scaling wascarried out under oral penicillin cover. Two days later hedeveloped fever and rigors. Bacterial endocarditis wasdiagnosed and after blood cultures had been prepared,treatment was started with intramuscular benzylpenicillinand streptomycin. Following transfer to Hammersmith

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it l l 1 71 1 11112 128EE1EE1 7 14 21 28 35 42

DAYS OF ILLNESS

Hospital the same treatment was continued by theintravenous route until all blood cultures were found tocontain Enterobacter aerogenes, resistant to benzylpeni-cillin, ampicillin, and cephaloridine, but sensitive tostreptomycin, tetracyclines, chloramphenicol, kanamycin,and polymyxins. Treatment was changed to benzylpeni-cillin, cephaloridine, and kanamycin to cover the possi-bility of an underlying undiscovered streptococcalinfection, for instance with a faecal streptococcus. Such acombination is likely to be bactericidal, a necessity intreating endocarditis. During the next week all bottles of13 blood cultures grew the same organism after overnightincubation in spite of antibiotic treatment. In the face ofthis heavy persisting bacteraemia, surgical excision of thevalve and prosthetic replacement was considered butrejected. No extracardiac focus to account for the coli-form septicaemia could be found. Polymyxin B was nowadded to the antibiotic regime. The next three bloodcultures gave growth from only one of the three bottles,the Robertson's cooked meat medium. All antibioticswere now discontinued except polymyxin, and trimetho-prim 250 mg and Gantanol 500 mg were given twicedaily with this. The patient's condition continued todeteriorate and he died of the haemodynamic complica-tions of gross acute endocarditis. However, three bloodcultures taken after the institution of trimethoprimtherapy yielded no growth after three weeks' incubationand only a single colony of Enterobacter was grown fromthe heart valve removed at necropsy. It appears thattrimethoprim plus sulphonamide added to polymyxinproved more effective than either kanamycin or polymy-xin alone in spite of the fact that the infecting organismshowed the normal level of sensitivity to both drugs

(minimum bactericidal concentration: polymyxin 0-25pg/ml, kanamycin between 2 and 4 ,&g/ml).

Three other patients with septicaemia illustratean apparent defect of this treatment. A Bacteroidessepticaemia with a liver abscess subsequent to per-foration of the caecum was uncontrolled, and intwo patients with E. coli septicaemia complicatingseptic abortion this organism was eliminated butreplaced in the blood by a Bacteroides.

URINARY TRACT INFECTIONS The following are typicalexamples of success and partial failure.

R.S., a man aged 57, a staff member, developed anacute urinary infection and was given ampicillin with nosymptomatic improvement after three days. The treat-ment was changed to sulphonamides. Bacteriologyshowed the infection to be due to a Klebsiella speciesresistant to ampicillin and partially resistant to sulphona-mides, which also failed to produce symptomatic reliefin three days. Within 48 hours of starting trimethoprim250 mg and Gantanol 500 mg twice daily urinary symp-toms disappeared and the urine was shown to be sterile.

J.B., a woman aged 45, a case of pyelonephritis, pre-sented with a mixed infection with E. coli and K. aero-genes. Both organisms were resistant to sulphonamidebut sensitive to trimethoprim. Before this was knownempirical treatment was started with the mixture, trime-thoprim 250 mg and Gantanol 500 nmg. The E. coli waseliminated but a significant growth of Klebsiella persistedand the patient's symptoms remained unchanged. Duringthe course of treatment, which lasted seven days, the

I.

0

L&J

la

cza: le11~--

- 11x

BloodCultures

FIG. 2. Chart of laterstages ofan Esch. colisepticaemia (case E.K.)

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Klebsiella became more resistant to trimethoprim (MICbefore 0-12 pg; after, 2 pg/ml). This patient was subse-quently given both polymyxin and gentamicin withouteffect. A laparotomy showed that she had uretericobstruction on the right. When this was relieved the uri-nary infection subsided but recurred four months later.

The case demonstrates the well known difficultyof eradicating urinary infections complicated byobstruction and urinary stasis, using chemothera-peutic agents alone.These cases also suggest that sulphonamide

resistance is not a bar to success from combinedtreatment. (The appearances in Fig. 1 are proofthat a sulphonamide can contribute to the action oftrimethoprim despite such resistance.)

DISCUSSION

The object of this paper is to call wider attention toa drug which we believe to represent a major dis-covery and to have far-reaching therapeutic possi-bilities. Independently it is very highly active againstmany species of bacteria, much more so in terms ofMIC than sulphonamides. Its combined action withthem is strongly synergic, and differs from that ofsulphonamides in being bactericidal. The rapid anddecisive therapeutic effect often observed may thusbe accounted for.Apart from infections discussed here, gonorrhoea

has recentlybeen shown to be susceptible to treatmentby Csonka and Knight (1967). Among those we havestudied enterobacterial infections (E. coli, Proteusand Klebsiella) bulk largely. The place of trimetho-prim in treating urinary infections caused by theseorganisms remains to be further defined. We wouldurge that this treatment be considered early, if not

indeed preferred in the first instance to the use ofantibiotics, for treating this form of septicaemia.Naturally much further experience is necessary: forexample, no one, so far as we are aware, has attemp-ted this treatment in bacteriaemic shock (for whichthe intravenous preparation of trimethoprim and ofthe sulphonamide used would evidently be advisable).H. influenzae is sensitive, as are other bacteria con-cerned in bronchial infections, and this is anotherfield meriting further study.Among Gram-positive coccal infections, those due

to group A streptococci and pneumococci stand inno need of any new form of treatment, althoughaccording to findings in vitro they should respondwell. Staphylococcal infections represent an almostentirely unexplored field in which gratifying resultsmight well be obtained, particularly if the strainis sulphonamide-sensitive.

We are indebted to the Wellcome Foundation for suppliesof the drug for therapeutic use, for sensitivity test discs,and for much information and useful discussions, and toour clinical colleagues for permission to refer theirpatients.

REFERENCES

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