Zbl. Bakt. Hyg., I.Abt. Orig. A 247,483-494 (1980)

Pharmaceutical Research Department, F.Hoffmann-La Roche & Co. Ltd., Basle, Switzer­land

Role of Thymidine for the Activity ofTrimethoprim, Sulfonamides

and their Combinations

Die Bedeutung des Thymidins fur die Wirkung von Trimethoprim, Sulfonamiden und deren Kombinationen

RUDOLF L. THEN

Received March 10, 1980

Abstract

Thymidine levels in urines from 14 patients suffering from severe chronic urinary tract infection were determined microbiologically. The concentrations found were < 10 ng/ml to 33 ng/ml and these do not seem to be any different from the concentrations determined in healthy persons. Results from growth kinetic experiments support the assumption of low thymidine levels in the urines of these patients and in most cases trimethoprim or co-trimoxazole were seen to act in a bactericidal manner in these urine samples. Various aspects of the role of exogenous and endogenous thymidine for the activity and bactericidal effect of trimethoprim, sulfonamides and their combinations, their mutual synergism, the importance of thymine auxotrophs, the problem of thymidine availability and its role in testing media are discussed.

Zusammenfassung

Thymidinkonzentrationen wurden mikrobiologisch in den Urinen von 14 Patienten mit schweren und chronischen Harnwegsinfektionen bestimmt. Die gefundenen Konzentra­tionen waren < 10 ng/ml bis 33 ng/ml, und sie scheinen nicht von denen bei Gesunden abzuweichen. Wachstumskinetische Untersuchungen in den Urinen dieser Patienten deu­teten ebenfalls auf niedrige Thymidinkonzentrationen hin, und in den meisten Fallen war eine bakterizide Wirkung von Trimethoprim oder Co-trimoxazol feststellbar. Auf die Rolle exogenen und endogenen Thymidins flir die Aktivitat und die bakterizide Wirkung von Trimethoprim, Sulfonamiden und deren Kombinationen, den wechselseitigen Synergismus, die Bedeutung thyminauxotropher Stamme, das Problem der Thymidinverfligbarkeit und seine Rolle bei der Sensibilitatsprlifung wird ausflihrlich eingegangen.

484 R.L.Then

Introduction

Sulfonamides as well as trimethoprim (TMP) specifically induce a detrimental shortage of tetrahydrofolate cofactors in susceptible bacteria and some other para­sitic organisms. The sulfonamide action is known to be overcome by p-amino­benzoic acid which competes for the active site of dihydropteroate synthetase. Apart from that, both the action of sulfonamides and TMP can be circumvented in a non-competitive way and to varying degrees by supplementing those compounds, whose synthesis is dependent on carbon one units delivered by tetrahydrofolate co­factors (Angehrn and Then, 1973 a, b; Then and Angehrn, 1973 a, b). The respective amino acids methionine, serine or glycine as well as purine compounds are not assumed to be crucial for the antibacterial action of antifolates. On the contrary, it was seen that their presence supports a bactericidal action of these drugs (Then and Angehrn, 1973 a, b; Angehrn and Then, 1973 a, b). These amino acids and purine compounds (also as nucleosides) are both available for bacteria in vivo and in vitro from the testing media usually used.

With thymidine and thymine, however, the situation seems much more com­plicated. The bactericidal effect of TMP and sulfonamides is well explained by the thymine starvation imposed (Angehrn and Then, 1973 a, b; Then and Angehrn, 1973 a, b; Amyes and Smith, 1974). The role of thymidine (and thymine) for the antibacterial and bactericidal action of these drugs in vivo, its influence on the synergism usually present between sulfonamides and TMP, its availability to fulfil the nutritional requirement of thymine auxotrophic mutants occasionally isolated, as well as its role in susceptibility testing have been disputed for a considerable time. To draw conclusions with respect to any of those questions, knowledge of thymidine levels in testing media and at the site of infection is required. Thymidine concen­trations in testing media have been investigated (Koch and Burchall, 1971; Then, 1977). Information for the in vivo situation is relatively easily to obtain for urine or blood samples and we have found low levels of thymidine (::; 0.021,Ltg/ml) in these fluids from healthy persons, applying a microbiological method (Nottebrock and Then, 1977). However, in patients, many of whom had renal calculi, and from whom thy- mutants had been isolated, Maskell et al. (1977) detected high concen­trations of a "thymine-like" substance in the urine, which would explain the pro·­pagation of these strains. In order to obtain some information on possible differ­ences in the thymidine levels of healthy persons and those suffering from destructive and infective renal diseases, we have now investigated some urine specimens ob·­tained from patients with severe DTIs, mostly chronic processes with abundant leucocyturia at the time of consulting the physician and taking urine samples. Thymidine concentrations discovered do not seem to exceed those in healthy per­sons. These findings will be discussed in connection with a more general overview of the role of thymidine and thymine for the action of TMP, sulfonamides as well as combinations thereof.

Materials and Methods

Urine samples: Urine samples were obtained frozen (- 20°C) from PD. Dr. med. von Riitte (Berne). After diagnosis of bacteruria, urine was collected before the onset of antimicrobial therapy. Most of the patients were then treated with co-trimoxazole and

Role of Thymidine 485

the urine samples under co-trimoxazole therapy were collected to perform growth kinetic experiments both with bacterial strains originally isolated from urines of these patients or with E. coli B.

Determination of thymidine and thymine: Frozen urine samples were thawed and leuco­cytes and debris removed by gentle centrifugation in the cold. The urine samples were subsequently sterilized by filtration (Millipore 0.22 ,um). All the samples were screened for the presence of antimicrobials in a cylinder assay with E. coli B, and occasionally with B. subtilis, in order to assure the absence of substances which interfere with the micro­biological assay of thymidine. The microbiological assay for thymidine was carried out on agar plates as described (Nottebrock and Then, 1977). The growth of the thymine auxotroph E. coli 15 T- was also checked in these samples. Inocula were made with washed cells of E. coli 15T- to prevent any transfer of thymidine. Plating after a 24 h incubation period on Wellcotest agar with and without the addition of thymidine assured that the organism grown was thy- and not a contaminant. Growth in different urine samples varied and was estimated by comparison with the growth obtained in samples after the addition of l,ug/ml thymidine, which is sufficient to fulfil the thymine requirement of E. coli 15T-. E. coli 15T- was also used to assay for thymidine plus thymine on agar plates. Levels of thymine of > 0.1 ,ug/ml would have been detectable in this way.

Growth kinetic experiments: In some instances growth of E. coli B or E. coli strains isolated from patient's urines was followed as described (Then and Angehrn, 1974b, 1975).

Results

Patients: Details of the patients studied are shown in Table 1. Many of them have a long history of chronic recurrent UTI and had courses with several anti­biotics and chemotherapeutics.

Thymidine concentrations: The microbiological determination of thymidine in urines of 14 patients revealed concentrations which seem not be different from those in 3 healthy persons and from those determined earlier (Nottebrock and Then, 1977). In most cases these concentrations of thymidine are at the fringe of the meth­od's sensitivity. The highest concentration determined was 0.033,ug/ml thymidine (Table 2). In all samples E. coli 15T- was able to grow to some extent (indicated by ± or + in Table 2), however, not as well as in the presence of added thymidine, indicating that the thymidine requirement of this low thymine requiring strain is not fully met.

Since the thymidine concentrations determined are too low to meet fully the thymine-requirement of E. coli 15T-, it was assumed that thymine as well as thy­midine may be present in these urines. A quantitative assay for thymine (+ thy­midine, the concentration of which should be negligible here) was therefore carried out on agar plates with E. coli 15T-. In no case was the concentration found to be higher than O.l,ug/ml which is the lowest level detectable in this way.

Growth kinetic experiments: Supplementary growth kinetic experiments were carried out in some instances to correlate the observed bacteriostatic or bactericidal effect of known or unknown drug concentrations with the thymidine levels found. The viable cell count of E. coli B was followed up for 24 h in urine samples from a healthy volunteer with known amounts of added TMP or sulfamethoxazole (SMZ). The same strain was inoculated into urine samples from patients taken 4 days after withdrawal of co-trimoxazole (the samples still containing high amounts of active drug, as assayed in the cylinder test). Two patients' samples, taken on day 3 under co-trimoxazole therapy, were also inoculated with those E. coli strains

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Role of Thymidine 487

Table 2. Thymidine concentrations determined microbiologically and growth of E. coli 15 T­in urines from patients collected before onset of antibacterial therapy

Patients Growth of E. coli 15 T- Thymidine (,ug/ml)

W.A. + 0.03 B.E. ± 0.033 K.W. ++ < 0.010 W.R. + < 0.010 A.F. ++ 0.010

H.K. + < 0.010 H.T. ++ < 0.010 K.P. ++ 0.010 S.S. ++ < 0.010 H.R. + 0.020

A.M. + < 0.010 M.M. + < 0.020 G.G. + 0.010 M.Z. + < 0.010

T.L.'> ± 0.012 J.F.'> + 0.025 F.H.'> ++ 0.029

,> = healthy volunteers

which were isolated from these patients before therapy. Very similar results were obtained in all instances. They are not described here in detail, since they merely confirmed previous findings, but can be summarized as follows: 1. With 2-100,Ltg/ml TMP or SMZ, alone or in combination, a moderate decrease

in the viable cell count after an 8 h period is observed in most experiments (90 to 99.9%); the same holds for urine samples containing unknown drug concentra­tions.

2. Bactericidal effects are less pronounced with high drug concentrations (either alone or combined) than with lower concentrations.

3. Reduction of the viable cell count begins after lag phases of 1 to several hours. 4. There is no obvious correlation between a static effect and the thymidine level

found (which is very low in any case), probably because the drug level is of major importance here.

5. The three different strains of E. coli used exhibited much the same behaviour.

Discussion

The data presented here as well as some other important aspects will be discussed in some detail below in order to give a brief survey of our present understanding of the in vivo action of TMP, sulfonamides and their combinations.

Exogenous thymidine: The antibacterial and bactericidal action of TMP and sulfonamides is inevitably connected with the level of "antagonists" present at the site of action (that means in the milieu surrounding an infective organism in vivo).

488 R.L. Then

For p-aminobenzoic acid, e.g. it is known that certain levels, obtained e.g. by food intake, greatly impair the antiparasitic and especially antimalarial effect of sulfon­amides (Perone, 1977; Sandhu et aI., 1976).

For thymidine, the most important antagonist for both TMP and sulfonamides, the effect of exogenously supplied nucleoside (e.g. by oral administration or food intake) has not been studied much. It seems to be negligible, as thymidine is rapidly incorporated into DNA and metabolized. Efforts to demonstrate an antagonistic effect of orally applied thymidine in animals were unsuccessful (Bushby, 1973). This is in accordance with our own experience.

This failure, however, may not be proof for the absence of any interfering effects of exogenously given thymidine, since mice and rats already exhibit relatively high concentrations of thymidine in blood (Nottebrock and Then, 1977), and further increasing these levels will not cause any changes. A certain antagonistic effect of thymidine (100-200 mg/kg) in mice on the antiviral activity of 5,6-dihydro-6-aza­thymidine was, however, observed (Renis and Eidson, 1979). Antagonistic effects of exogenously administered thymidine may therefore be detected more easily in ani­mals which do not normally contain such high levels as rodents.

Endogenous thymidine: The concentration and role of endogenous thymidine, however, either normally present at certain low levels or generated by destructive processes either in white blood cells or bacteria, and present in pus, has been the subject of many articles and disputed for quite some time.

Thymidine concentrations in urine from patients suffering from severe urinary tract infections were found to be low in all cases. A cautious conclusion may there­fore be drawn, namely that thymidine levels in urine do not significantly change during infective processes of the urinary tract and that the concentrations found do not seem to be any different from those in healthy persons. Even if the patients studied were a non representative collection, it must be assumed that high concen­trations of thymidine (> 0.05,ug/ml) or thymine rarely occur. Urine from these patients also certainly did not contain thymine concentrations in excess of O.l,ug/ml.

These low concentrations found are in contrast to much higher values of a "thymine-like" substance, found in patients studied by Maskell and co-workers (1977). At present the question of whether these high concentrations are strictly connected with the diagnosis in these patients (nearly all had renal calculi) or whether the method of determination (microbiological vs. isotachophoresis) is in­volved in some way must be left open. The results of the growth kinetic experiments carried out here, however, agree well with the microbiologically determined thy­midine levels. In most cases a relatively short lag before the onset of killing was observed. High thymidine (and thymine) concentrations present would have com­pletely prevented any bactericidal effect, especially at high drug concentrations (Then, 1976).

It was seen that urines from both patients and healthy persons to some extent supported growth of thy- strains. Since very low thymidine concentrations were found, it could be assumed that a certain amount of thymine is also present. Rough estimations, however, showed that concentrations of thymine are below O.l,ug/mi.

It may be mentioned here that estimation of thymidine levels by the turbidity obtained by growing thymine auxotrophs in urine will not yield very accurate results, since under limiting thymine a great part of the cellmass observed is probably dead cells and filaments, contributing to optical density. Viable cell counts will therefore give more reliable results.

Role of Thymidine 489

Dynamics of thymidine levels: From the data reported here and from earlier data (Then and Angehrn, 1974 b, 1975) it could be concluded that both in heatlhy persons and in patients with severe infections of the urinary tract low concentrations of thymine and thymidine are usually present in urine; these levels would not interfere much with a bactericidal action of antifols.

In vivo, however, it is more likely that thymidine which is used up by growing bacteria, incorporation into DNA or metabolism, is continuously replaced. Hence conclusions based on thymidine levels determined in urine samples (or other probes) may not be adequate and in vitro growth kinetic experiments in batch cultures may not necessarily reflect the in vivo situation. Although it is possible to simulate more realistic conditions with respect to changing drug concentration (Greenwood and O'Grady, 1976), more appropriate models simulating varying concentrations of thymidine or maintaining limiting concentrations have not been used. Experiments with limiting concentrations of thymidine in continuous culture would be of basic interest at least. For this reason it is certainly not wise to completely rely on the results obtained in the way described, as is often done, since it is obvious that the correlation with in vivo conditions may be poor.

Moreover, conditions in urine itself are probably of little interest, since the im­portant site, where we want to eradicate bacteria, is mostly not urine but tissue (Naumann, 1978). If bacteria are present only in the lumen, it is known that they are easily abolished. Information on thymidine levels in tissues, however, is almost totally lacking, because of problems in determining these levels. This necessarily leads to the question of how meaningful and valid experiments in urine are, espe­cially with respect to the action of drugs like sulfonamides or TMP. Because of this lack of basic knowledge the question of whether these drugs are bacteriostatic or bactericidal should not be overemphasized.

Thymidine and bactericidal activity of TMP, or TAIP-sulfonamide combinations: Coming back to the finding that low levels of thymidine usually seem to be present in patients' urine which would favour a bactericidal action of TMP or of its com­binations, one must answer the question of why in some instances bacteriostatic ac­tion is observed. It is important here to remember that due to the mode of action (Angehrn and Then, 1973 b) high concentrations of the drug lead to a reduction in the rate of inactivation, which will ultimately result in a merely bacteriostatic action, especially if thymidine is present, even in low amounts (Then, 1976). Hence the question whether these drugs act in a bacteriostatic or a bactericidal manner not only depends on the presence of thymidine, but also on the drug concentration.

The data obtained here as well as earlier data support the view that co-trimoxazole has a bactericidal potential during therapy. Indirect evidence to further support this assumption was recently provided by Kirven and Thornsberry (1978). They found that only those strains of H. influenzae present in asymptomatic carriers were eradicated by co-trimoxazole for which the drug was bactericidal in vitro. Those, in which only a bacteriostatic action was observed were not cleared. It is not quite clear why only 31 % of the CDC-strains of H. influenzae were influenced in a bactericidal way, but it must be assumed that H. influenzae responds to thy­midine in the same way as other species do. The clearance of carriers from those strains of H. influenzae seems therefore to be due to the killing effect of co-trimox­azole.

One of the few indications where it is generally accepted that bactericidal drugs are superior to bacteriostatic ones and where a cure is dependent on complete elimi-

490 R.L.Then

nation of the last cell is bacterial endocarditis. Though co-trimoxazole is not the drug of choice and relatively little experience exists, definite cures in 9 out of 13 cases of bacterial endocarditis, which are well documented, are encouraging. In these cases co-trimoxazole was used after failure of several antibiotics. The causative agents were E. coli, Staphylococcus aureus, Coxiella burnetti, Pseudomonas cepa­cia, Pseudomonas maltophilia and Streptococcus viridans. In another 4 out of 7 cases, cures were obtained, improvement in 2 cases and 1 failure, all less well docu­mented, however.

Thymidine and synergism: There has been some confusion about the influence of thymidine on synergism between TMP and sulfonamides. Since thymidine re­verses part of the antibacterial action both of TMP and sulfonamide, higher con­centrations are required to obtain an MIC (or IC5Q etc., but not MBC, since suffi­cient thymidine will abolish any bactericidal effect, which is due to thymineless death). Synergism is present, though at a higher drug level. This has been shown earlier (Then, 1973) and recently also in a medium containing pus (Edmunds, 1978 a, b; Lacey and Stokes, 1978). The concentrations needed to demonstrate synergism in the presence of thymidine are therefore higher than in its absence, but these are obtainable in vivo and hence synergism can be assumed to be present in vivo even at sites where thymidine should be.

Data from animal experiments also give useful information here. We and others have shown that in blood and urine from rats and mice relatively high levels of thymidine are present. Nevertheless, sulfonamides, generally considered as bac­teriostatic drugs, show good activity, TMP is less active (probably due to its shorter half-life), but together, there is pronounced synergy despite these high thymidine levels (Boehni, 1969; Grunberg et aI., 1970).

Thymidine auxotrophs: We have judged the occurrence of thy- auxotrophs to be a rare event and of little practical importance (Then and Angehrn, 1974a). This seems to be still relevant, though apparently more thy- auxotrophs are isolated at present. It remains open whether this is simply due to increased awareness, to in­creased drug consumption or to a true increase, which, however, would be difficult to conceive of.

The findings presented here and those of Maskell et al. (1977) as well as others show that some thymine auxotrophs are able to survive at least in the patient. Since they derive from strains which are pathogenic and which possess the immuno­genic properties required for pathogenicity, thy- strains are of course pathogenic and resistant to co-trimoxazole. We have assumed that these thymine auxotrophs, which can be selected easily by TMP in vitro, are rarely isolated ex vivo since they lack sufficient thymidine and/or are hampered by the deficiency in general and do not survive for long periods.

However, thymine auxotrophs, as other auxotrophs, are not so rarely found among reisolates after several passages through man or animals (Kenyon, 1978; Borderon and Horodniceanu, 1978; Crawford et aI., 1977) even in the absence of any selecting drug. In one of the 16 patients studied by Maskell et al. (1977) there seems also to be no strict correlation between the thy- strain isolated and the intake of co-trimoxazole, as the strain was isolated 5 months after co-trimoxazole had been withdrawn. Some background level of thymine auxotrophs isolated from pa­tients under co-trimoxazole, but for which co-trimoxazole was not the selecting agent, has therefore also to be considered.

Again, to allow the growth of thy- strains, thymidine would be required and

Role of Thymidine 491

thymine seems of lesser importance. Mutants defective in thymidylate synthetase require high levels of thymine to fulfil their thymidylate requirement, since utiliza­tion of thymine is restricted by the low level of deoxyribose-1-phosphate normally present. A second mutation in ribose metabolism, which leads to higher levels of deoxyribose-1-phosphate, and which occurs relatively frequently, can, however, increase the extent of thymine utilization.

Thymidine availability: Though thymidine mostly strongly antagonizes the bac­tericidal activity of TMP, sulfonamides or their combinations, there are exceptions. No influence by either thymine or thymidine on the activity of TMP or of sulfona­mides was seen in the family Neisseriaceae (Dr. Bushby, personal communication; own observations). This is probably due to the lack of thymidine converting enzymes in neisseriae (Jyssum, 1974; Jyssum and Bgvre, 1974). B. fragilis seems to require higher levels of thymidine for an antagonistic effect than other pathogens (Then and Angehrn, 1979). Also plasmodia seem to be unable to utilize exogenous thymine or thymidine (Ferone, 1977). This makes it obvious that even if thymine or thymidine is present, it does not impair activity in all instances.

In fact, the situation is even more complicated. Even if thymine or thymidine are present, especially at low levels « 1 ,ug/ml) it seems that they are not necessarily available for many organisms. It has been known for some time that certain pyri­midines and especially their nucleosides (e.g. uridine) strongly influence the avail­ability of exogenous thymine and also to some extent thymidine in prototrophs as well as in auxotrophs (Freifelder, 1965; Behki and Lesley, 1973; Boyle and Jones, 1970; Then and Angehrn, 1975). These effects also playa role in the thymineless state imposed by TMP or other inhibitors of tetrahydrofolate synthesis (own ex­periments; Amyes and Smith, 1978 a, b). This also suggests that thymine is of little importance as an antagonizing agent in vivo. Although these effects mainly affect the availability of thymine, the availability of thymidine may also be affected by other mechanisms, e. g. by impaired uptake due to chromosomal mutation in the gene controlling nucleoside transport (McKeown et al., 1976) . In contrast to many prototrophs and thy- mutants, however, Streptococcus faecalis is able to use low levels of thymine and of thymidine (Andrew, 1973).

In general thymidine is very efficiently utilized in strains in the thymineless state and concentrations of 1,ug/ml or less are sufficient to support growth. However, there are examples, where thymidine as well is poorly utilized and very high con­centrations are required (Kelln and Warren, 1973). It must be considered a possi­bility therefore, that for some (or for many) bacteria thymidine concentrationsoc­curring in body fluids are too low to interfere significantly with the thymineless state imposed by antifols.

Finally it may be recalled that, though many bacteria respond to the thymineless state (either induced by starvation of thy- strains, or by inhibition of thymidylate synthesis in prototrophs in the absence of thymine) with loss of viability, the sen­sitivity to thymine starvation varies. There are strains and species which are more or less resistant to the thymineless state (that means do not or not readily undergo thymineless death (Cummings and Mondale, 1967; Ephrati-Elizur et al., 1974; Maisch and Wachsman, 1972).

Thymidine in testing media: From all the information presently available it seems reasonable to use testing media which are devoid of thymidine, as is usually recom­mended and done, though this has been questioned (Stokes and Lacey, 1978). Not only would the use of thymidine containing media greatly impair or invalidate MIC

492 R.L.Then

determinations but this would also not seem to reflect conditions at the site of infection (as far as identical conditions in general can be provided by any medium used for susceptibility testing).

Conclusions: To conclude then, this whole range of possibilities which exists in different pathogens to react to a challenge by TMP or its sulfonamide combinations makes it nearly impossible to draw general conclusions. The answer to what will happen in vivo to a certain bacterium under therapy e.g. with TMP or co-trimoxa­zole depends on the drug concentration present at the site of infection, the duration of exposure, the level of thymine or thymidine, the pathogen and its genetically determined ability to mutate, take up and utilize thymine or thymidine or to react to the induced thymineless state. Despite all these precautions the low failure rate under co-trimoxazole and the rare occurrence of thy- strains under therapy support the assumption that thymine and thymidine seem of relatively low importance for the drug's action in vivo and hence their role should not be overstated, although more information on this is certainly of interest.

Acknowledgements: We thank PD Dr. med. B. von Rutte, Berne, for providing us with urine samples and all the required data of the patients.

References

Amyes, S. G.B. and J. T.Smith: Trimethoprim action and its analogy with thymine star­vation. Antimicrob. Agents Chemother. 5 (1974) 169-178

Amyes, S. G. B. and J. T. Smith: Trimethoprim antagonists: effect of uri dine on thymine and thymidine uptake in mineral salts medium. J. Antimicrob. Chemother. 4 (1978 a) 415-419

Amyes, S. G. B. and J. T. Smith: Trimethoprim antagonists: effect of uri dine in laboratory media. J. Antimicrob. Chemother. 4 (1978b) 421-429

Andrew, M.H.E.: Use of trimethoprim to obtain thymine-requiring mutants of Strepto­coccus faecalis. J. gen. Microbio!. 74 (1973) 195-199

Angehrn, P. and R. Then: Nature of trimethoprim-induced death in Escherichia coli. Arzneimitt.-Forsch.23 (1973 a) 447-451

Angehrn, P. and R. Then: Investigations on the mode of action of the combination sulfa­methoxazole/trimethoprim. Chemotherapy 19 (1973 b) 1-10

Behki, R.M. and S.M.Lesley: Effect of uri dine on the incorporation of thymine and thy­midine in Escherichia coli. Canad. ]. Microbio!' 19 (1973) 485-490

Boehni, E.: Vergleichende bakteriologische Untersuchungen mit der Kombination Tri­methoprim/Sulfamethoxazol in vitro und in vivo. Chemotherapy Supp!. ad vo!' 14 (1969) 1-21

Borderon, E. and T. Horodniceanu: Metabolically deficient dwarf-colony mutants of Esche­richia coli: deficiency and resistance to antibiotics of strains isolated from urine culture. J. clin. Microbio!. 8 (1978) 629-634

Boyle, J. V. and M. E. Jones: Effects of ribonucleosides on thymidine incorporation: selec­tive reversal of the inhibition of deoxyribonucleic acid synthesis in thymineless auxo­trophs of Escherichia coli. J. Bact. 104 (1970) 264-271

Bushby, S. R. M.: Trimethoprim-Sulfamethoxazole: In vitro microbiological aspects. J. in­fect. Dis. Supp!. 128 (1973) 442-462

Crawford, G., J.S.Knapp, J.Hale, and K.K.Holmes: Asymptomatic gonorrhea in men caused by gonococci with unique nutritional requirements. Science 196 (1977) 1352-1353

Cummings, D. J. and L. Mondale: Thymineless death in Escherichia coli: strain specificity. J. Bact. 93 (1967) 1917-1924

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Edmunds, P. N.: Synergy between sulfonamides and trimethoprim in the presence of pus. ]. clin. Path. 31 (1978a) 162-164

Edmunds, P. N.: Synergy between sulfonamides and trimethoprim in the presence of pus. ]. clin. Path. 31 (1978b) 701

Ephrati-Elizur, E., D. Yosuv, E.Shmueli, and A.Horowitz: Thymineless death in Bacillus subtilis: Correlation between cell lysis and deoxyribonucleic acid breakdown. J. Bact. 119 (1974) 36-43

Ferone, R.: Folate metabolism in malaria. Bull. WId Hlth Org. 55 (1977) 291-298 Freifelder, D.: Technique for starvation of Escherichia coli of thymine. ]. Bact. 90 (1965)

1153-1154 Greenwood, D. and F. O'Grady: Activity and interaction of trimethoprim and sulfamethox­

azole against Escherichia coli. J. din. Path. 29 (1976) 162-166

Grunberg, E., H. N. Prince, and W. F. de Lorenzo: The in vivo effect of folinic acid (citro­vorum factor) on the potentiation of the antibacterial activity of sulfisoxazole by tri­methoprim. J. clin. Pharmacol. 10 (1970) 231-234

Jyssum, S.: Search for thymidine phosphorylase, nucleoside deoxyribosyl-transferase and thymidine kinase in genus Neisseria. Acta path. microbiol. scand. Sect. B 82 (1974) 53-56

Jyssum, S. and K. Bovre: Search for thymidine phosphorylase, nucleoside deoxyribosyl transferase and thymidine kinase in Moraxella, Acinetobacter and allied bacteria. Acta path. microbiol. scand. Sect. B 82 (1974) 57-66

Kelln, R.A. and R.A.J. Warren: Obligate thymidine auxotrophs of Pseudomonas acido­vorans. J. Bact. 113 (1973) 510-511

Kenyon, J. E.: Neisseria gonorrhoeae: Acquisition of auxotrophy in the mouse. Curro Microbiol. 1 (1978) 253-256

Kirven, L. A. and C. Thornsberry: Minimum bactericidal concentration of sulfamethox­azole-trimethoprim for Haemophilus in(luenzae: Correlation with prophylaxis. Anti­microb. Agents Chemother. 14 (1978) 731-736

Koch, A. E. and J. J. Burchall: Reversal of the antimicrobial activity of trimethoprim by thymidine in commercially prepared media. Appl. Microbial. 22 (1971) 812-817

Lacey, R. W. and A. Stokes: Synergy between sulfonamides and trimethoprim in the pres­ence of pus. ]. clin. Path. 31 (1978) 700-701

Maisch, F. W. and J. T. Wachsman: Properties of thymineless strains of Bacillus megate­rium. Appl. Microbial. 24 (1972) 717-720

Maskell,R., O.A.Okubadeio, R.H.Payne, and L.Pead: Human infections with thymine­requiring bacteria. ]. med. Microbiol. 11 (1977) 33-45

McKeown, M., M. Kahn, and P. Hanawalt: Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport. J. Bact. 126 (1976) 814-822

Naumann, P.: The value of antibiotic levels in tissue and in urine in the treatment of urinary tract infection. J. Antimicrob. Chemother. 4 (1978) 9-17

Nottebrock, H. and R. Then: Thymidine concentrations in serum and urine of different animal species and man. Biochem. Pharmacol. 26 (1977) 2175-2179

Renis, H.E. and E.E.Eidson: Activities of 5,6-dihydro-5-azathymidine against Herpes simplex virus infections in mice. Antimicrob. Agents Chemother. 15 (1979) 213-219

Sandhu, D.K., R.S.Sandhu, Z. U.Khan, and V.N.Damodaran: Conditional virulence of a p-aminobenzoic acid-requiring mutant of Aspergillus fumigatus. Infect. Immun. 13 (1976) 527-532

Stokes, A. and R. W. Lacey: Effect of thymidine on activity of trimethoprim and sulfa­methoxazole. J. clin. Path. 31 (1978) 165-171

Then, R.: Influence of sulfonamide antagonists on the synergism of sulfamethoxazole/tri­methoprim in Escherichia coli. Zbl. Bakt. Hyg., LAbt. Orig. A 225 (1973) 34-41

Then, R.: Thymidine and the assessment of co-trimoxazole action in liquid media. In Chemotherapy (Williams, J.D. and A. M. Geddes, Eds.), Vol. 2, pp. 67-70. Plenum Pub­lishing Corp., New York (1976)

494 R.L. Then

Then, R.: Thymidine content in commercially prepared media. Zbl. Bakt. Hyg., 1.Abt. Orig. A 237 (1977) 372-377

Then, R. and P.Angehrn: Sulfonamide-induced "thymineless death" in Escherichia coli. J. gen. Microbiol. 76 (1973 a) 255-263

Then, R. and P.Angehrn: Effects of trimethoprim on Escherichia coli under limited nutri­tion. Arzneimitt.-Forsch. 23 (1973 b) 451-455

Then, R. and P.Angehrn: Co-trimoxazole resistance. Brit. med. J. I (1974a) 78-79 Then, R. and P.Angehrn: The biochemical basis of the antimicrobial action of sulfon­

amides and trimethoprim in vivo. 1. Action of sulfonamides and trimethoprim in blood and urine. Biochem. Pharmacol. 23 (1974b) 2977-2982

Then, R. and P.Angehrn: The biochemical basis of the antimicrobial action of sulfon­amides and trimethoprim in vivo. II. Experiments on the limited thymine and thymidine availability in blood and urine. Biochem. Pharmacol. 24 (1975) 1003-1006

Then, R. and P. Angehrn: Low trimethoprim susceptibility of anaerobic bacteria due to insensitive dihydrofolate reductases. Antimicrob. Agents Chemother. 15 (1979) 1-6

Dr. Rudolf L. Then, F.Hoffmann-La Roche & Co. Ltd., Grenzacherstr. 124, CH-4002 Basle, Switzerland