In vitro sensitivity of Hemophilus influenzae and Streptococcus pyogenes to co-trimoxazole W-D.
LEERS, fMD, PH D, FRCP[C], DIP BACT, CPH
Summary: The in vitro testing of Hemophilus influenzae and Streptococcus pyogenes for co-trimoxazole sensitivity requires certain "defined" media that have to be free of inhibitory substances. The use of Columbia agar base with Fildes extract for H. influenzae or of blood agar for S. pyogenes may produce "false-resistant" strains. The addition of thymidine phosphorylase in the form of gently lysed horse blood (2 to 10% ) does not remove all inhibitors in those tests, especially where "undefined" agar bases are used, and results in scanty growth of H. influenzae; the addition of more than 2% results in dark plates, making reading of sensitivities difficult. Fildes agar for testing H. influenzae may be made with enriched sheep or horse blood if the proper "defined" agar base is used. The use of Wellcotest or DST (Oxoid) agar is recommended with Fildes extract for H. influenzae or with blood for S. pyogenes for in vitro testing for co-trimoxazole sensitivity. The addition of thymidine phosphorylase in the form of 2% lysed horse blood does not interfere with reading. However, it results in scanty growth of H. influenzae. Proper inoculation of plates is important. The growth on the plates should be light, dense, but not confluent. Heavy growth may render some strains "false-resistant" even when defined media are used. Our results indicate that many of the previously reported resistant strains of H. influenzae and S. pyogenes may have been ''false-resistant" because of the use of "undefined" media. We believe that, in view of our results, respiratory infections may be treated with co-trimoxazole until bacteriologic studies prove that this treatment is contraindicated, since H. influenzae and S. pyogenes are usually found sensitive in vitro under proper conditions. Resume: La sensibilite in vitro c/Hemophilus influenzae et de Streptococcus pyogenes au co-trimoxazole Pour §tre valable, les epreuves de sensibilite de Hemophilus influenzae et de Streptococcus pyogenes au co-trimoxazole doivent utiliser des milieux "definis" qui sont exempts de
substances inhibitrices. L'emploi de gelose Columbia avec milieu de Fildes pour H. influenzae ou de gelose au sang pour S. pyogenes peut produire des souches "faussement resistantes". L'addition de phosphorylase de thimidine sous forme de sang de cheval legerement lyse (de 2 a 10 % ) n'elimine pas *The terms co-trimoxazole and
trimethoprim-sulfamethoxazole are synonymous. tChief, department of microbiology, The Wellesley Hospital, Toronto; assistant professor, department of medical microbiology, University of Toronto Reprint requests to: Dr. W-D. Leers, The Wellesley Hospital, 160 Wellesley St. E,
Toronto, Ont. M4Y U3
tous les inhibiteurs dans ces epreuves, surtout si on utilise des geloses "indefinies", et on n'en obtient qu'une pousse
mediocre de H. influenzae. Si on ajoute plus de 2 % on obtient des plaques foncees, qui rendent difficile I'interpretation de la sensibilite. La gelose de Fildes pour "tester" H. influenzae peut-etre faite avec du sang de mouton ou de cheval, si on utilise une gelose "definie" convenable. On conseille d'employer la gelose de Wellcotest ou la DST (Oxoid) avec le milieu de Fildes pour H. influenzae ou avec du sang pour S. pyogenes, en vue de I'etude de la sensibilite in vitro au co-trimoxazole. L'addition de phosphorylase de thimidine, sous forme de sang de cheval lyse a 2 %, ne gene pas la lecture. Mais on ne obtient une pousse mediocre de H. Influenzae. Ce qui est important, c'est I'inoculation adequate des plaques. La croissance sur les plaques doit §tre legere, dense, mais jamais confluente. Une pousse trop energique peut rendre certaines souches "faussement resistantes" meme si on a utilise des milieux definis. Notre experience indique que nombre de souches de H. influenzae et de S. pyogenes considerees auparavant comme resistantes, peuvent avoir ete "faussement resistantes", en raison de l'emploi de milieux "indefinis ". Considerant nos resultats, nous estimons qu'on peut traiter au co-trimoxazole les infections respiratoires, a moins que des epreuves bacteriologiques ne prouvent que ce traitement est contreindique. En effet, dans des conditions adequates, H. influenzae et S. pyogenes sont generalement sensibles in vitro.
It is well known that in vitro sensitivity
testing for sulfonamides
presents problems that are due to inhibitors found in the various media. l>* But this is also true of other antibiotics, for example
gentamicin, where the magnesium content and the agar base may influence the minimal inhibitory concentration.3'4 The inhibitory action of some media towards sulfonamides It has can be inactivated by addition of lysed horse blood.1 been shown that this inhibitory action is not due to p-aminobenzoic acid, which competes with the sulfonamides, but to thymidine phosphorylase.5 Thymidine phosphorylase acts on thymidine, which can be used by the bacteria to counteract the action of sulfonamides. Thymidine is present in a variety of bacteriologic media. The action of thymidine phosphorylase and its influence on in vitro sensitivity testing of co-trimoxazole has been studied thoroughly.6"8 Bushby showed conclusively that the presence of thymidine in bacteriologic media may give erroneous results when bacteria Addition of are tested for the action of co-trimoxazole.6'7'9 to lysed horse blood, which contains thymidine phosphorylase, bacteriologic media would render many resistant strains sensi¬ tive in vitro. Bushby7 emphasized the importance of "defined" media with a low thymidine content and the addition of lysed horse blood, prepared by gentle freezing and thawing as described by Harper and Cawston.1 CMA JOURNAL/JUNE 14, 1975/VOL. 112 59S
For some microorganisms that need highly enriched media the use of defined media for sensitivity testing may present problems. Hemophilus influenzae, for example, grows only on enriched media with essential factors. Sensitivity testing of these bacteria on enriched media for antimicrobial agents other than sulfonamides presents usually no problems.10 But these enrich¬ ed media may contain inhibitory substances such as thymidine, which may give erroneous results when testing H. influenzae and Streptococcus pyogenes for co-trimoxazole sensitivity. Because of apparent in vitro resistance of H. influenzae to co-trimoxazole, some investigators caution against using cotrimoxazole in patients with bacterial respiratory infections until the etiologie microorganism has been identified and tested for its sensitivity. The in vitro resistance to co-trimoxazole of H. the influenzae as described by those authors was up to 6,5% ofthan strains tested.11"13 Amies found in addition that less 20% of strains of S. pyogenes were sensitive to co-trimoxazole in 13 vitro.
On closer investigation of these reports and the bacteriologic methods of testing described, it is clear that many of the media used were not suited for sensitivity testing with co-
trimoxazole.1 l'! 4»15
In order to clarify the controversy about the in vitro sensitivity of H. influenzae and S. pyogenes, we undertook a study: (1) to find a bacteriologic medium that would support growth of H. influenzae and S. pyogenes and that would not interfere with the in vitro sensitivity testing to co-trimoxazole; and (2) to determine if some of the previous studies of in vitro resistant strains of H. influenzae and S. pyogenes may have been, in fact, reported erroneously as "false-resistant" strains
because of inhibitors in the media.
Materials and methods H. influenzae and S. pyogenes All strains were isolated from clinical specimens in our diagnostic hospital laboratory. Sensitivity testing H. influenzae was tested against ampicillin as well as against co-trimoxazole. Strains were inoculated on round 100 x 15-mm plastic plates by emulsifying approximately 1 mm3 in 1 ml of trypticase soy broth. The sensitivity plates were then seeded using a cotton-tipped swab.16 The plates were incubated for
1.25 ug of trimethoprim Mg of ampicillin (BBL).
Sensitivity discs (a) Paper discs contained 23.75 ug of sulfamethoxazole and 60S CMA JOURNAL/JUNE 14, 1975/VOL. 112
10
Thymidine phosphorylase (TP) Oxalated horse blood was prepared by gentle freezing and thawing, as described by Harper and Cawston.1 It was then added to the media to provide a final concentration of 2 to 10%.
Statistical analysis No attempt was made to evaluate the difference in results because of the small number of microorganisms
statistically tested. Blood
Sheep or horse blood prepared by the Fildes digest method is referred to as "acid-lysed blood". The "gently" frozen and thawed horse blood in concentrations of 2 to 10%, which contains the thymidine phosphorylase, is referred to as "gently lysed blood" or "thymidine phosphorylase (TP)". Results and discussion
Hemophilus influenzae In our diagnostic hospital laboratory we use routinely Fildes medium with Columbia base agar (CAB) for isolation and sensitivity testing for H. influenzae. So far we have encountered only one strain that was resistant to ampicillin but many that
resistant to co-trimoxazole. Also, strains of S. pyogenes tested on horse blood agar with CAB were found to be resistant to co-trimoxazole in our routine diagnostic laboratory. The studies of Bushby6'7'9 and others8'18 convinced us that our results might not have been correct because of the Table I.Bacteriologic culture media were
Supplier or Abbreviation
Medium
manufacturer
CAB
DST
18 to 24 hours at 35°C in 5 to 10% CO*. As an alternative method for inoculation, selected colonies were suspended in 4 ml of Levinthal broth and incubated for 6 hours. A dilution of 1:100 of this suspension was then seeded with a swab as above. Although determination of minimal inhibitory concentration (MIC) would have contributed to this study, time did not allow for this procedure.
Media The bacteriologic culture media and their abbreviated symbois used in the text are described in Table I. H. influenzae was grown on a variety of agar bases with Fildes digest:17 1 g of pepsin is dissolved in 150 ml of saline. Fifty millilitres of sheep blood or horse blood and 6 ml of pure concentrated HCI are added. The extract is incubated for 1 to 24 hours at 50°C and the pH adjusted to 7.4 with 20% NaOH. Then 0.5 ml of 0.25% chloroform is added and the mixture shaken vigorously. The extract is heated for 20 minutes at 56°C to remove the chloroform and added to agar for a final concentration of 5%. S. pyogenes was grown on 5% sheep or horse blood agar using different agar bases.17
(Difco). (b) Paper discs contained
WT MH Fildes
SB HB (oxal)
HB (def) LB TP
Thymidine phosphorylase prepared by gently freezing and thawing of horse blood
(1) (2) (3) (4) (5) (6) (7) (8) (9)
(9)
Baltimore Biological Laboratories, Baltimore, Maryland Oxoid Ltd., supplied by Medox Co., Ottawa Burroughs-Wellcome, Montreal Supplied by Canlab, Toronto
CowanandSteel17
Woodlyn Farms, Guelph, Ont. Connaught Laboratories Limited, Diagnostic Division, Willowdale, Ont. Trypticase soy broth with 5% Fildes enrichment Harper and Cawston1
inhibitory effect of some of our media on the action of co-trimoxazole on these bacteria. We therefore tested our Fildes medium, which was prepared with acid-lysed sheep blood and CAB. As recommended by Bushby9 we added 10% gently lysed horse blood prepared according to the method of Harper and Cawston1 by freezing and thawing, which contains the TP. It must be appreciated that H. influenzae grows aerobically only in the presence of the X factor (hemin) and the V factor, a coenzyme, nicotinamide adenine dinucleotide, which are suppli¬ ed by the addition of acid-hydrolysed sheep or horse blood. Therefore//. influenzae will not grow on nonenriched "defined" media such as Wellcotest agar (WT), Oxoid sensitivity agar (DST) or Mueller-Hinton sensitivity agar (MH). When the gently lysed blood (TP) is added to the various agar bases the H. influenzae does not grow, indicating that this additive does not contain the necessary growth factors. We also compared acid-lysed horse blood with acid-lysed sheep blood in the Fildes medium to see if the results of the sensitivity testing would produce any difference. Table 11 shows the result with 16 strains of H. influenzae. There is no significant difference between acid-lysed sheep and horse blood as enrichment for the Fildes medium. Also the addition of TP did not make any difference. We assumed that the acid-lysed horse blood did not contain TP because it might have been destroyed during the process of preparation. The gently lysed horse blood, containing TP, has to be lysed very carefully by freezing and thawing in order to preserve the enzyme.1 Howev¬ er, we did not study this question further. The Fildes medium with 5% acid-lysed blood and 10% gently lysed blood (TP) resulted in a very dark medium, which made Table II.Sensitivity of 16 strains of H. influenzae to co-trimoxazole Fildes medium with CAB and sheep or horse blood
on
CAB with acid-lysed horse blood + TP 10%
reading the sensitivities very difficult. We therefore had to use a lower concentration of TP. We again prepared Fildes medium with acid-lysed horse and sheep blood (Table III). The TP was prepared in the form of oxalated or defibrinated horse blood because we wanted to see if this would make any difference. The gently lysed horse blood was added to give a 2% concentration, which resulted in plates that could easily be read. The addition of 2% gently lysed horse blood (TP) resulted in a higher number of sensitive strains than when we tested without TP. Whether oxalated or defibrinated blood was used made no difference, nor whether acid-lysed horse or sheep blood was used as Fildes enrichment. From the foregoing it seems obvious that there were some inhibitory substances in the medium, either in the agar base or in the acid-lysed blood. We therefore decided to try different "defined" agar bases instead of the "undefined" CAB for preparing the Fildes agar. The term "defined" means that the medium has a very low thymidine content.6 The term "undefin¬ ed" means that the thymidine content is not known. The results of these experiments are shown in Table IV. Acid-lysed sheep blood was used throughout. Nineteen strains were tested but some of the WT plates without TP were contaminated so that only seven strains could be compared. The data confirm the results of the previous experiment, in which it
was shown that the addition of TP rendered some "resistant" strains sensitive to co-trimoxazole. When a "defined" agar base (DST) was used, however, all but one of the strains were found sensitive without the additional TP and all were sensitive when 2% TP was added. If Fildes agar was prepared with WT all strains were found sensitive, regardless of the addition of TP. The same experiment was performed using the Fildes agar prepared with acid-lysed horse blood instead of sheep blood (Table IV). The number of resistant strains with or without TP in CAB differs by only one. In regard to the DST agar base there is no difference with or without TP, and WT without TP produces one resistant strain. If these results are compared no significant difference is apparent whether acid-lysed sheep or horse blood is used as enrichment, for all agar bases tested. However, the "undefined" CAB with the Fildes extract contains inhibitors that seem to be only partially inactivated by the addition of gently lysed horse blood (TP). It seems, therefore, that the acid-lysed horse and sheep blood, which contain the X and V factors, do not contain inhibitors that interfere with the action of co-trimoxazole on H. influenzae in vitro. The inhibitors are in the "undefined" agar
14
Table IV.Sensitivity of 19 strains of H. influenzae on Fildes medium with acid-lysed sheep or horse blood and different agar bases and 2% TP
Table III.Sensitivity of 28 strains of H. influenzae on Fildes medium with CAB and 2 % TP made from oxalated or defibrinated blood Media Number of strains Resistant
Sensitive
Medium
CAB CAB with acid-lysed sheep blood 11
17
CAB + TP
CAB with acid-lysed sheep blood + TP (oxal.) 15
13
DST
13
DST 4- TP
18
WT
13
WT + TP
blood + TP
15 acid-lysed sheep CAB with acid-lysed horse blood 10 CAB with acid-lysed horse blood + TP (oxal.) 15 CAB with acid-lysed horse blood + TP (defib.)15
CAB with
(defib.)
13 *Some plates contaminated
CMA JOURNAL/JUNE
14, 1975/VOL. 112 61S
base, in our case the CAB. However, it may be possible that 2% lysed horse blood (TP) may not be present in sufficient quantity to inactivate the inhibitors in the "undefined" CAB. Therefore, our next set of experiments included the addition of the gently lysed horse blood (TP) as 2, 4 or 6%. Four of 10 strains were sensitive and 6 were resistant. The 6% TP produced the same difficulty in reading the results that we found with the 10% preparation. The Fildes agar already contains 5% of acid-lysed blood, so that a very dark plate is produced if another 6 or 10% of gently lysed blood is added. The results of this experiment are interesting. The addition of TP, regardless of concentration, made no difference on the in vitro sensitivity of H. influenzae to co-trimoxazole. We then compared different defined agar bases without the addition of TP and included the MH in
our series (Table V). With WT or DST agar base all but one strain in DST agar are sensitive, thus confirming the previous results. A surprise is the result with the MH. One half of the strains are resistant, the same proportion as with the CAB. Three of the five strains
correspond.
experiment included only a small number of different media bases and compared the various agar bases with and without the additions of thymidine phosphorylase (Table VI). As expected, more than one half of the strains of H. influenzae were resistant to co-trimoxazole when tested on Fildes medium with CAB. This result changed somewhat when Since this last
strains, in our next experiment we used only 4% TPF with four
was added, but still about one third of the strains were found to be resistant in vitro. DST agar base showed two strains resistant without the addition of TPF. The MH showed one strain resistant regardless of the presence of TP. With WT all strains tested were sensitive, regardless of the addition of TP. Therefore the incorporation of WT into the Fildes medium gives results for testing H. influenzae for co-trimoxazole sensitivity that are unequivocal. Addition of gently lysed horse blood (TP) to Fildes media made reading of sensitivities difficult. It also resulted in scanty growth of H. influenzae. We think that the gently lysed blood contains inhibitors to the V factor, since the addition of a "staphylococcus streak" allowed heavy growth on the medium around the staphylococcus colonies. We also compared two methods of inoculation. One method was to use a freshly emulsified suspension of organisms from the agar plate. The other method was to use a 1:100 dilution of a
TP
Table VI.Sensitivity of 24 strains of H. influenzae on Fildes medium with different agar bases and 4 % TP, comparing two different
inoculating techniques
62S CMA JOURNAL/JUNE 14, 1975/VOL. 112
6-hour growth of actively multiplying organisms in Levinthal broth. The latter method produced a slightly less dense nonconfluent growth than the first method. The results are shown in Table VI. There are some minor, nonsignificant differences with the CAB and the DST without TP. All other results are the same.
Streptococcus pyogenes We wanted to find out if TP would have
an
effect
on
the
sensitivity of S. pyogenes to co-trimoxazole when grown on sheep or horse blood agar. All 15 strains became resistant. Blood agar made from sheep or horse blood produced the same results, rendering all strains resistant. The addition of TP did not alter this pattern. This is in contrast to the same experiment with H. influenzae (Table III), in which one third of the strains
found sensitive and the addition of TP rendered at least of the resistant strains sensitive. The result is in contrast, also, to previous reports, in which it is postulated that the addition of 0.25 to 3% gently lysed horse blood would render almost any medium suitable for sensitivity testing with were some
trimethoprim.2 then
We tested 30 strains of S. pyogenes on horse blood agar with different agar bases. As already mentioned, Amies found more than 80% of S. pyogenes in vitro resistant to co-trimox¬ azole.13 He used a tryptose blood agar base with 5% sheep blood as sensitivity medium. Our results of testing the in vitro sensitivity of 30 strains of S. pyogenes to co-trimoxazole on different blood agar bases are Table V.Sensitivities of 10 strains of H. influenzae on Fildes medium with different agar bases
Medium
Number of strains Sensitive Resistant
CAB
5
DST
9
WT
10
MH
5
Table VII.Sensitivity of 30 strains of S. pyogenes on horse blood agar with different agar bases and 4 % TP, comparing two different
inoculating techniques
shown in Table VII. Columbia blood agar base rendered all but one strain resistant to co-trimoxazole, and 2 out of 30 were sensitive with the addition of TP. Wellcotest blood agar base showed all 30 strains sensitive and DST blood agar base rendered one strain resistant. The four resistant strains on MH showed in vitro sensitivity if TP was added. These results indicate that the undefined Columbia blood agar base is unreliable for testing S. pyogenes for co-trimoxazole sensitivity, regardless of the presence of TP. After incubation the WT turns light brown, which makes the reading of S. pyogenes hemolysis at times difficult. However, there was never doubt as to the in vitro sensitivity of the organism on this account. As with H. infiuenzae, we tested two different methods of inoculation. The first method is fast, using a freshly emulsified suspension. The second, time-consuming method is a 6-hour broth culture used in a dilution of 1:100. The latter method produces lighter growth than the first one. Some of the strains on CAB that were found to be resistant with the "fast inoculation method" were found to be sensitive with the 6-hour incubation method. The addition of TP did not change the pattern. On the other hand, the 6-hour incubation method rendered all strains sensitive when tested on the other three blood-agar base media. It may be concluded that a lighter inoculum with actively multiplying S. pyogenes produces a higher degree of in vitro sensitivity when testing for co-trimoxazole sensitivity. These results indicate that S. pyogenes is sensitive to co-trimoxazole in vitro if proper agar bases and a light inoculum are used for the preparation of the blood agar. The high incidence of resistant S. pyogenes as reported previously may indeed be due to the culture media used.1 . The technical assistance of Mrs. Kay Arthurs, chief technologist, and Mr. William Tennant, charge technologist, is gratefully acknowledged.
We thank Miss Elizabeth Mmmo for the diligent preparation of the manuscript. References I. HARPER GJ, CAWSTON WC: The in vitro determination of the sulphonamide sensitivity of bacteria. I Pathol Bacteriol 57: 59, 1945 2. DARRELL JH, GARROD LP, WATERWORTH PM: Trimethoprim: laboratory and clinical studies. I C/in Pathol 21: 202. 1968 3. DUNCAN IB: Susceptibility of 1500 isolates of Pseudomonas aeruginosa to gentamicin, carbenicillin, colissin, and polymyxin B. Antio,icrob Agents Chemother 5: 9, 1974 4. GARROD LP, WATERWORTH PM: Effect of medium composition on the apparent sensitivity of Pseudomonas aeruginosa to gentamicin. I (liii Pathol 22: 534, 1969 5. BUSHBY SRM, HITCHINGS GH: Trimethoprim. a suiphonamide potentiator. Br I Chemother 33: 72, 1968 6. BUSHBY SRM: Sensitivity testing with trimethoprim-sulfamethoxazole. MedJAust(suppl) 1:10.1973 7. Idem: Trimethoprim-sulfamethoxazole: in vitro microbiological aspects. I Infect Dis 128 (suppl): 442, 1973 8. KOCH AE, BURCHALL ii: Reversal of the antimicrobial activity of trimethoprim by thymidine in commercially prepared media. Appi Microbial 22: 812, 1971 9. BUSHBY SRM: Combined antibacterial action in vitro of trime.hoprim and sulphonamides. The in vitro nature of synergy. Postgrod Med 1 45 (suppl): 10, 1969 10. WILLIAMS JD, ANDREWS J: Sensitivity of Haemophilus influenzae to antibiotics. Br Med J 1: 34, 1974 I. MAY JR. DAVIES J: Resistance of Haemophilus influcnzac to trimethoprim. Br MedJ 3: 376, 1972 12. BURNS MW, DEVITT L: Trimethoprim-sulfamethoxazole. Problems in treating lower respiratory tract infections. Med I Aust (suppl) 1: 62, 973 13. AMIES CR: Trimethoprim-sulfameshoxazole: in vitro sensitivity of 1000 clinical isolates. Can Med Assoc J 110: 336, 1974 14. BUSHBY SRM: Haemophilus influenzae apparently resistant to trimethoprim. Br Med 1 3: 50, 1973 15. BUSHBY SRM, BUSHBY MB: Haemophilus influenzae apparently resistant to trimethoprim. Presented at 8th Internat Congress Chemother, Athens, 1973 16. BAUER AW, KIRBY WMM, SHERRIS JC, et al: Antibiotic susceptibility testing by a standardized disk method. An, I C/in Pathol 45: 493, 1966 17. COWAN ST. STEEL KJ: Manual for the identification of Medical Bacteria. Cambridge U Pr, 1966 18. WATERWORTH PM, DAVIES J: Practical aspects of testing sensitivity to trimethoprim and sulfonamide. Postgrad Med 1 45 (suppl): 21, 1969
Synergy of trirnethoprim-sulfamethoxazole S.RM. BUSHBY, PH D
The basis for the synergy of the antibacterial drugs trimethoprim (TMP) and sulfamethoxazole (SMX) is well documented.1'2 Both drugs interfere with the biosynthesis of the folate coenzymes and thus ultimately affect the biosynthesis of proteins and nucleic acids. The sulfonamide acts as a competitor for para-aminobenzoic acid (PAB) in the formation of dihydropteroic acid by dihydropteroate synthetase.3 This acid is condensed with glutamic acid to form dihydrofolic acid, which is then reduced to tetrahydrofolic acid by dihydrofolate reductase. This reduction is essential in the formation of the coenzymes and it is at this step that TMP acts through binding with the dihydrofolate reductase.4 SMX and TMP therefore act in the same biochemical pathway and the enhancement of activity from their simultaneous administration is due to their actions being sequential. The blocking of the enzyme dihydropteroate synthetase by Reprint requests to: Dr. S.R.M. Bushby, Weilcome Research Laboratories, 3030 Cornwallis Rd., Research Triangle Park, NC 27709, USA
sulfonamides and of dihydrofolate reductase by TMP is competitive, and the amount of inhibitor needed to reduce the biosynthesis of dihydropteroic acid or tetrahydrofolic acid to levels below those essential for growth depends on the amount of substrate present. Because the sulfonamide acts before TMP in the biosynthesis, its role in the dual action is merely to reduce the amount of dihydrofolic acid against which TMP competes. If the competition between TMP and dihydrofolic acid were linear, then the effects of the dual action would be no more than additive, but because the competition by TMP increases relatively with decreases in dihydrofolic acid, the effects are synergic.5 Another factor that will also affect the dual action is that TMP affects not only the de novo synthesis of tetrahydrofolic acid but also its recycling. In the biosynthesis of thymine, at the stage at which uridylate is reduced to thymidylate, tetrahydrofolic acid is reoxidized to dihydrofolic acid, which is then returned to the tetrahydrofolate pooi by the action of dihydrofolate reductase; in the presence of TMP this recycling is inhibited and in conjunction with the partial cut-off CMA JOURNAL/JUNE 14, 1975/VOL. 112
63S
LEERS, fMD, PH D, FRCP[C], DIP BACT, CPH
Summary: The in vitro testing of Hemophilus influenzae and Streptococcus pyogenes for co-trimoxazole sensitivity requires certain "defined" media that have to be free of inhibitory substances. The use of Columbia agar base with Fildes extract for H. influenzae or of blood agar for S. pyogenes may produce "false-resistant" strains. The addition of thymidine phosphorylase in the form of gently lysed horse blood (2 to 10% ) does not remove all inhibitors in those tests, especially where "undefined" agar bases are used, and results in scanty growth of H. influenzae; the addition of more than 2% results in dark plates, making reading of sensitivities difficult. Fildes agar for testing H. influenzae may be made with enriched sheep or horse blood if the proper "defined" agar base is used. The use of Wellcotest or DST (Oxoid) agar is recommended with Fildes extract for H. influenzae or with blood for S. pyogenes for in vitro testing for co-trimoxazole sensitivity. The addition of thymidine phosphorylase in the form of 2% lysed horse blood does not interfere with reading. However, it results in scanty growth of H. influenzae. Proper inoculation of plates is important. The growth on the plates should be light, dense, but not confluent. Heavy growth may render some strains "false-resistant" even when defined media are used. Our results indicate that many of the previously reported resistant strains of H. influenzae and S. pyogenes may have been ''false-resistant" because of the use of "undefined" media. We believe that, in view of our results, respiratory infections may be treated with co-trimoxazole until bacteriologic studies prove that this treatment is contraindicated, since H. influenzae and S. pyogenes are usually found sensitive in vitro under proper conditions. Resume: La sensibilite in vitro c/Hemophilus influenzae et de Streptococcus pyogenes au co-trimoxazole Pour §tre valable, les epreuves de sensibilite de Hemophilus influenzae et de Streptococcus pyogenes au co-trimoxazole doivent utiliser des milieux "definis" qui sont exempts de
substances inhibitrices. L'emploi de gelose Columbia avec milieu de Fildes pour H. influenzae ou de gelose au sang pour S. pyogenes peut produire des souches "faussement resistantes". L'addition de phosphorylase de thimidine sous forme de sang de cheval legerement lyse (de 2 a 10 % ) n'elimine pas *The terms co-trimoxazole and
trimethoprim-sulfamethoxazole are synonymous. tChief, department of microbiology, The Wellesley Hospital, Toronto; assistant professor, department of medical microbiology, University of Toronto Reprint requests to: Dr. W-D. Leers, The Wellesley Hospital, 160 Wellesley St. E,
Toronto, Ont. M4Y U3
tous les inhibiteurs dans ces epreuves, surtout si on utilise des geloses "indefinies", et on n'en obtient qu'une pousse
mediocre de H. influenzae. Si on ajoute plus de 2 % on obtient des plaques foncees, qui rendent difficile I'interpretation de la sensibilite. La gelose de Fildes pour "tester" H. influenzae peut-etre faite avec du sang de mouton ou de cheval, si on utilise une gelose "definie" convenable. On conseille d'employer la gelose de Wellcotest ou la DST (Oxoid) avec le milieu de Fildes pour H. influenzae ou avec du sang pour S. pyogenes, en vue de I'etude de la sensibilite in vitro au co-trimoxazole. L'addition de phosphorylase de thimidine, sous forme de sang de cheval lyse a 2 %, ne gene pas la lecture. Mais on ne obtient une pousse mediocre de H. Influenzae. Ce qui est important, c'est I'inoculation adequate des plaques. La croissance sur les plaques doit §tre legere, dense, mais jamais confluente. Une pousse trop energique peut rendre certaines souches "faussement resistantes" meme si on a utilise des milieux definis. Notre experience indique que nombre de souches de H. influenzae et de S. pyogenes considerees auparavant comme resistantes, peuvent avoir ete "faussement resistantes", en raison de l'emploi de milieux "indefinis ". Considerant nos resultats, nous estimons qu'on peut traiter au co-trimoxazole les infections respiratoires, a moins que des epreuves bacteriologiques ne prouvent que ce traitement est contreindique. En effet, dans des conditions adequates, H. influenzae et S. pyogenes sont generalement sensibles in vitro.
It is well known that in vitro sensitivity
testing for sulfonamides
presents problems that are due to inhibitors found in the various media. l>* But this is also true of other antibiotics, for example
gentamicin, where the magnesium content and the agar base may influence the minimal inhibitory concentration.3'4 The inhibitory action of some media towards sulfonamides It has can be inactivated by addition of lysed horse blood.1 been shown that this inhibitory action is not due to p-aminobenzoic acid, which competes with the sulfonamides, but to thymidine phosphorylase.5 Thymidine phosphorylase acts on thymidine, which can be used by the bacteria to counteract the action of sulfonamides. Thymidine is present in a variety of bacteriologic media. The action of thymidine phosphorylase and its influence on in vitro sensitivity testing of co-trimoxazole has been studied thoroughly.6"8 Bushby showed conclusively that the presence of thymidine in bacteriologic media may give erroneous results when bacteria Addition of are tested for the action of co-trimoxazole.6'7'9 to lysed horse blood, which contains thymidine phosphorylase, bacteriologic media would render many resistant strains sensi¬ tive in vitro. Bushby7 emphasized the importance of "defined" media with a low thymidine content and the addition of lysed horse blood, prepared by gentle freezing and thawing as described by Harper and Cawston.1 CMA JOURNAL/JUNE 14, 1975/VOL. 112 59S
For some microorganisms that need highly enriched media the use of defined media for sensitivity testing may present problems. Hemophilus influenzae, for example, grows only on enriched media with essential factors. Sensitivity testing of these bacteria on enriched media for antimicrobial agents other than sulfonamides presents usually no problems.10 But these enrich¬ ed media may contain inhibitory substances such as thymidine, which may give erroneous results when testing H. influenzae and Streptococcus pyogenes for co-trimoxazole sensitivity. Because of apparent in vitro resistance of H. influenzae to co-trimoxazole, some investigators caution against using cotrimoxazole in patients with bacterial respiratory infections until the etiologie microorganism has been identified and tested for its sensitivity. The in vitro resistance to co-trimoxazole of H. the influenzae as described by those authors was up to 6,5% ofthan strains tested.11"13 Amies found in addition that less 20% of strains of S. pyogenes were sensitive to co-trimoxazole in 13 vitro.
On closer investigation of these reports and the bacteriologic methods of testing described, it is clear that many of the media used were not suited for sensitivity testing with co-
trimoxazole.1 l'! 4»15
In order to clarify the controversy about the in vitro sensitivity of H. influenzae and S. pyogenes, we undertook a study: (1) to find a bacteriologic medium that would support growth of H. influenzae and S. pyogenes and that would not interfere with the in vitro sensitivity testing to co-trimoxazole; and (2) to determine if some of the previous studies of in vitro resistant strains of H. influenzae and S. pyogenes may have been, in fact, reported erroneously as "false-resistant" strains
because of inhibitors in the media.
Materials and methods H. influenzae and S. pyogenes All strains were isolated from clinical specimens in our diagnostic hospital laboratory. Sensitivity testing H. influenzae was tested against ampicillin as well as against co-trimoxazole. Strains were inoculated on round 100 x 15-mm plastic plates by emulsifying approximately 1 mm3 in 1 ml of trypticase soy broth. The sensitivity plates were then seeded using a cotton-tipped swab.16 The plates were incubated for
1.25 ug of trimethoprim Mg of ampicillin (BBL).
Sensitivity discs (a) Paper discs contained 23.75 ug of sulfamethoxazole and 60S CMA JOURNAL/JUNE 14, 1975/VOL. 112
10
Thymidine phosphorylase (TP) Oxalated horse blood was prepared by gentle freezing and thawing, as described by Harper and Cawston.1 It was then added to the media to provide a final concentration of 2 to 10%.
Statistical analysis No attempt was made to evaluate the difference in results because of the small number of microorganisms
statistically tested. Blood
Sheep or horse blood prepared by the Fildes digest method is referred to as "acid-lysed blood". The "gently" frozen and thawed horse blood in concentrations of 2 to 10%, which contains the thymidine phosphorylase, is referred to as "gently lysed blood" or "thymidine phosphorylase (TP)". Results and discussion
Hemophilus influenzae In our diagnostic hospital laboratory we use routinely Fildes medium with Columbia base agar (CAB) for isolation and sensitivity testing for H. influenzae. So far we have encountered only one strain that was resistant to ampicillin but many that
resistant to co-trimoxazole. Also, strains of S. pyogenes tested on horse blood agar with CAB were found to be resistant to co-trimoxazole in our routine diagnostic laboratory. The studies of Bushby6'7'9 and others8'18 convinced us that our results might not have been correct because of the Table I.Bacteriologic culture media were
Supplier or Abbreviation
Medium
manufacturer
CAB
DST
18 to 24 hours at 35°C in 5 to 10% CO*. As an alternative method for inoculation, selected colonies were suspended in 4 ml of Levinthal broth and incubated for 6 hours. A dilution of 1:100 of this suspension was then seeded with a swab as above. Although determination of minimal inhibitory concentration (MIC) would have contributed to this study, time did not allow for this procedure.
Media The bacteriologic culture media and their abbreviated symbois used in the text are described in Table I. H. influenzae was grown on a variety of agar bases with Fildes digest:17 1 g of pepsin is dissolved in 150 ml of saline. Fifty millilitres of sheep blood or horse blood and 6 ml of pure concentrated HCI are added. The extract is incubated for 1 to 24 hours at 50°C and the pH adjusted to 7.4 with 20% NaOH. Then 0.5 ml of 0.25% chloroform is added and the mixture shaken vigorously. The extract is heated for 20 minutes at 56°C to remove the chloroform and added to agar for a final concentration of 5%. S. pyogenes was grown on 5% sheep or horse blood agar using different agar bases.17
(Difco). (b) Paper discs contained
WT MH Fildes
SB HB (oxal)
HB (def) LB TP
Thymidine phosphorylase prepared by gently freezing and thawing of horse blood
(1) (2) (3) (4) (5) (6) (7) (8) (9)
(9)
Baltimore Biological Laboratories, Baltimore, Maryland Oxoid Ltd., supplied by Medox Co., Ottawa Burroughs-Wellcome, Montreal Supplied by Canlab, Toronto
CowanandSteel17
Woodlyn Farms, Guelph, Ont. Connaught Laboratories Limited, Diagnostic Division, Willowdale, Ont. Trypticase soy broth with 5% Fildes enrichment Harper and Cawston1
inhibitory effect of some of our media on the action of co-trimoxazole on these bacteria. We therefore tested our Fildes medium, which was prepared with acid-lysed sheep blood and CAB. As recommended by Bushby9 we added 10% gently lysed horse blood prepared according to the method of Harper and Cawston1 by freezing and thawing, which contains the TP. It must be appreciated that H. influenzae grows aerobically only in the presence of the X factor (hemin) and the V factor, a coenzyme, nicotinamide adenine dinucleotide, which are suppli¬ ed by the addition of acid-hydrolysed sheep or horse blood. Therefore//. influenzae will not grow on nonenriched "defined" media such as Wellcotest agar (WT), Oxoid sensitivity agar (DST) or Mueller-Hinton sensitivity agar (MH). When the gently lysed blood (TP) is added to the various agar bases the H. influenzae does not grow, indicating that this additive does not contain the necessary growth factors. We also compared acid-lysed horse blood with acid-lysed sheep blood in the Fildes medium to see if the results of the sensitivity testing would produce any difference. Table 11 shows the result with 16 strains of H. influenzae. There is no significant difference between acid-lysed sheep and horse blood as enrichment for the Fildes medium. Also the addition of TP did not make any difference. We assumed that the acid-lysed horse blood did not contain TP because it might have been destroyed during the process of preparation. The gently lysed horse blood, containing TP, has to be lysed very carefully by freezing and thawing in order to preserve the enzyme.1 Howev¬ er, we did not study this question further. The Fildes medium with 5% acid-lysed blood and 10% gently lysed blood (TP) resulted in a very dark medium, which made Table II.Sensitivity of 16 strains of H. influenzae to co-trimoxazole Fildes medium with CAB and sheep or horse blood
on
CAB with acid-lysed horse blood + TP 10%
reading the sensitivities very difficult. We therefore had to use a lower concentration of TP. We again prepared Fildes medium with acid-lysed horse and sheep blood (Table III). The TP was prepared in the form of oxalated or defibrinated horse blood because we wanted to see if this would make any difference. The gently lysed horse blood was added to give a 2% concentration, which resulted in plates that could easily be read. The addition of 2% gently lysed horse blood (TP) resulted in a higher number of sensitive strains than when we tested without TP. Whether oxalated or defibrinated blood was used made no difference, nor whether acid-lysed horse or sheep blood was used as Fildes enrichment. From the foregoing it seems obvious that there were some inhibitory substances in the medium, either in the agar base or in the acid-lysed blood. We therefore decided to try different "defined" agar bases instead of the "undefined" CAB for preparing the Fildes agar. The term "defined" means that the medium has a very low thymidine content.6 The term "undefin¬ ed" means that the thymidine content is not known. The results of these experiments are shown in Table IV. Acid-lysed sheep blood was used throughout. Nineteen strains were tested but some of the WT plates without TP were contaminated so that only seven strains could be compared. The data confirm the results of the previous experiment, in which it
was shown that the addition of TP rendered some "resistant" strains sensitive to co-trimoxazole. When a "defined" agar base (DST) was used, however, all but one of the strains were found sensitive without the additional TP and all were sensitive when 2% TP was added. If Fildes agar was prepared with WT all strains were found sensitive, regardless of the addition of TP. The same experiment was performed using the Fildes agar prepared with acid-lysed horse blood instead of sheep blood (Table IV). The number of resistant strains with or without TP in CAB differs by only one. In regard to the DST agar base there is no difference with or without TP, and WT without TP produces one resistant strain. If these results are compared no significant difference is apparent whether acid-lysed sheep or horse blood is used as enrichment, for all agar bases tested. However, the "undefined" CAB with the Fildes extract contains inhibitors that seem to be only partially inactivated by the addition of gently lysed horse blood (TP). It seems, therefore, that the acid-lysed horse and sheep blood, which contain the X and V factors, do not contain inhibitors that interfere with the action of co-trimoxazole on H. influenzae in vitro. The inhibitors are in the "undefined" agar
14
Table IV.Sensitivity of 19 strains of H. influenzae on Fildes medium with acid-lysed sheep or horse blood and different agar bases and 2% TP
Table III.Sensitivity of 28 strains of H. influenzae on Fildes medium with CAB and 2 % TP made from oxalated or defibrinated blood Media Number of strains Resistant
Sensitive
Medium
CAB CAB with acid-lysed sheep blood 11
17
CAB + TP
CAB with acid-lysed sheep blood + TP (oxal.) 15
13
DST
13
DST 4- TP
18
WT
13
WT + TP
blood + TP
15 acid-lysed sheep CAB with acid-lysed horse blood 10 CAB with acid-lysed horse blood + TP (oxal.) 15 CAB with acid-lysed horse blood + TP (defib.)15
CAB with
(defib.)
13 *Some plates contaminated
CMA JOURNAL/JUNE
14, 1975/VOL. 112 61S
base, in our case the CAB. However, it may be possible that 2% lysed horse blood (TP) may not be present in sufficient quantity to inactivate the inhibitors in the "undefined" CAB. Therefore, our next set of experiments included the addition of the gently lysed horse blood (TP) as 2, 4 or 6%. Four of 10 strains were sensitive and 6 were resistant. The 6% TP produced the same difficulty in reading the results that we found with the 10% preparation. The Fildes agar already contains 5% of acid-lysed blood, so that a very dark plate is produced if another 6 or 10% of gently lysed blood is added. The results of this experiment are interesting. The addition of TP, regardless of concentration, made no difference on the in vitro sensitivity of H. influenzae to co-trimoxazole. We then compared different defined agar bases without the addition of TP and included the MH in
our series (Table V). With WT or DST agar base all but one strain in DST agar are sensitive, thus confirming the previous results. A surprise is the result with the MH. One half of the strains are resistant, the same proportion as with the CAB. Three of the five strains
correspond.
experiment included only a small number of different media bases and compared the various agar bases with and without the additions of thymidine phosphorylase (Table VI). As expected, more than one half of the strains of H. influenzae were resistant to co-trimoxazole when tested on Fildes medium with CAB. This result changed somewhat when Since this last
strains, in our next experiment we used only 4% TPF with four
was added, but still about one third of the strains were found to be resistant in vitro. DST agar base showed two strains resistant without the addition of TPF. The MH showed one strain resistant regardless of the presence of TP. With WT all strains tested were sensitive, regardless of the addition of TP. Therefore the incorporation of WT into the Fildes medium gives results for testing H. influenzae for co-trimoxazole sensitivity that are unequivocal. Addition of gently lysed horse blood (TP) to Fildes media made reading of sensitivities difficult. It also resulted in scanty growth of H. influenzae. We think that the gently lysed blood contains inhibitors to the V factor, since the addition of a "staphylococcus streak" allowed heavy growth on the medium around the staphylococcus colonies. We also compared two methods of inoculation. One method was to use a freshly emulsified suspension of organisms from the agar plate. The other method was to use a 1:100 dilution of a
TP
Table VI.Sensitivity of 24 strains of H. influenzae on Fildes medium with different agar bases and 4 % TP, comparing two different
inoculating techniques
62S CMA JOURNAL/JUNE 14, 1975/VOL. 112
6-hour growth of actively multiplying organisms in Levinthal broth. The latter method produced a slightly less dense nonconfluent growth than the first method. The results are shown in Table VI. There are some minor, nonsignificant differences with the CAB and the DST without TP. All other results are the same.
Streptococcus pyogenes We wanted to find out if TP would have
an
effect
on
the
sensitivity of S. pyogenes to co-trimoxazole when grown on sheep or horse blood agar. All 15 strains became resistant. Blood agar made from sheep or horse blood produced the same results, rendering all strains resistant. The addition of TP did not alter this pattern. This is in contrast to the same experiment with H. influenzae (Table III), in which one third of the strains
found sensitive and the addition of TP rendered at least of the resistant strains sensitive. The result is in contrast, also, to previous reports, in which it is postulated that the addition of 0.25 to 3% gently lysed horse blood would render almost any medium suitable for sensitivity testing with were some
trimethoprim.2 then
We tested 30 strains of S. pyogenes on horse blood agar with different agar bases. As already mentioned, Amies found more than 80% of S. pyogenes in vitro resistant to co-trimox¬ azole.13 He used a tryptose blood agar base with 5% sheep blood as sensitivity medium. Our results of testing the in vitro sensitivity of 30 strains of S. pyogenes to co-trimoxazole on different blood agar bases are Table V.Sensitivities of 10 strains of H. influenzae on Fildes medium with different agar bases
Medium
Number of strains Sensitive Resistant
CAB
5
DST
9
WT
10
MH
5
Table VII.Sensitivity of 30 strains of S. pyogenes on horse blood agar with different agar bases and 4 % TP, comparing two different
inoculating techniques
shown in Table VII. Columbia blood agar base rendered all but one strain resistant to co-trimoxazole, and 2 out of 30 were sensitive with the addition of TP. Wellcotest blood agar base showed all 30 strains sensitive and DST blood agar base rendered one strain resistant. The four resistant strains on MH showed in vitro sensitivity if TP was added. These results indicate that the undefined Columbia blood agar base is unreliable for testing S. pyogenes for co-trimoxazole sensitivity, regardless of the presence of TP. After incubation the WT turns light brown, which makes the reading of S. pyogenes hemolysis at times difficult. However, there was never doubt as to the in vitro sensitivity of the organism on this account. As with H. infiuenzae, we tested two different methods of inoculation. The first method is fast, using a freshly emulsified suspension. The second, time-consuming method is a 6-hour broth culture used in a dilution of 1:100. The latter method produces lighter growth than the first one. Some of the strains on CAB that were found to be resistant with the "fast inoculation method" were found to be sensitive with the 6-hour incubation method. The addition of TP did not change the pattern. On the other hand, the 6-hour incubation method rendered all strains sensitive when tested on the other three blood-agar base media. It may be concluded that a lighter inoculum with actively multiplying S. pyogenes produces a higher degree of in vitro sensitivity when testing for co-trimoxazole sensitivity. These results indicate that S. pyogenes is sensitive to co-trimoxazole in vitro if proper agar bases and a light inoculum are used for the preparation of the blood agar. The high incidence of resistant S. pyogenes as reported previously may indeed be due to the culture media used.1 . The technical assistance of Mrs. Kay Arthurs, chief technologist, and Mr. William Tennant, charge technologist, is gratefully acknowledged.
We thank Miss Elizabeth Mmmo for the diligent preparation of the manuscript. References I. HARPER GJ, CAWSTON WC: The in vitro determination of the sulphonamide sensitivity of bacteria. I Pathol Bacteriol 57: 59, 1945 2. DARRELL JH, GARROD LP, WATERWORTH PM: Trimethoprim: laboratory and clinical studies. I C/in Pathol 21: 202. 1968 3. DUNCAN IB: Susceptibility of 1500 isolates of Pseudomonas aeruginosa to gentamicin, carbenicillin, colissin, and polymyxin B. Antio,icrob Agents Chemother 5: 9, 1974 4. GARROD LP, WATERWORTH PM: Effect of medium composition on the apparent sensitivity of Pseudomonas aeruginosa to gentamicin. I (liii Pathol 22: 534, 1969 5. BUSHBY SRM, HITCHINGS GH: Trimethoprim. a suiphonamide potentiator. Br I Chemother 33: 72, 1968 6. BUSHBY SRM: Sensitivity testing with trimethoprim-sulfamethoxazole. MedJAust(suppl) 1:10.1973 7. Idem: Trimethoprim-sulfamethoxazole: in vitro microbiological aspects. I Infect Dis 128 (suppl): 442, 1973 8. KOCH AE, BURCHALL ii: Reversal of the antimicrobial activity of trimethoprim by thymidine in commercially prepared media. Appi Microbial 22: 812, 1971 9. BUSHBY SRM: Combined antibacterial action in vitro of trime.hoprim and sulphonamides. The in vitro nature of synergy. Postgrod Med 1 45 (suppl): 10, 1969 10. WILLIAMS JD, ANDREWS J: Sensitivity of Haemophilus influenzae to antibiotics. Br Med J 1: 34, 1974 I. MAY JR. DAVIES J: Resistance of Haemophilus influcnzac to trimethoprim. Br MedJ 3: 376, 1972 12. BURNS MW, DEVITT L: Trimethoprim-sulfamethoxazole. Problems in treating lower respiratory tract infections. Med I Aust (suppl) 1: 62, 973 13. AMIES CR: Trimethoprim-sulfameshoxazole: in vitro sensitivity of 1000 clinical isolates. Can Med Assoc J 110: 336, 1974 14. BUSHBY SRM: Haemophilus influenzae apparently resistant to trimethoprim. Br Med 1 3: 50, 1973 15. BUSHBY SRM, BUSHBY MB: Haemophilus influenzae apparently resistant to trimethoprim. Presented at 8th Internat Congress Chemother, Athens, 1973 16. BAUER AW, KIRBY WMM, SHERRIS JC, et al: Antibiotic susceptibility testing by a standardized disk method. An, I C/in Pathol 45: 493, 1966 17. COWAN ST. STEEL KJ: Manual for the identification of Medical Bacteria. Cambridge U Pr, 1966 18. WATERWORTH PM, DAVIES J: Practical aspects of testing sensitivity to trimethoprim and sulfonamide. Postgrad Med 1 45 (suppl): 21, 1969
Synergy of trirnethoprim-sulfamethoxazole S.RM. BUSHBY, PH D
The basis for the synergy of the antibacterial drugs trimethoprim (TMP) and sulfamethoxazole (SMX) is well documented.1'2 Both drugs interfere with the biosynthesis of the folate coenzymes and thus ultimately affect the biosynthesis of proteins and nucleic acids. The sulfonamide acts as a competitor for para-aminobenzoic acid (PAB) in the formation of dihydropteroic acid by dihydropteroate synthetase.3 This acid is condensed with glutamic acid to form dihydrofolic acid, which is then reduced to tetrahydrofolic acid by dihydrofolate reductase. This reduction is essential in the formation of the coenzymes and it is at this step that TMP acts through binding with the dihydrofolate reductase.4 SMX and TMP therefore act in the same biochemical pathway and the enhancement of activity from their simultaneous administration is due to their actions being sequential. The blocking of the enzyme dihydropteroate synthetase by Reprint requests to: Dr. S.R.M. Bushby, Weilcome Research Laboratories, 3030 Cornwallis Rd., Research Triangle Park, NC 27709, USA
sulfonamides and of dihydrofolate reductase by TMP is competitive, and the amount of inhibitor needed to reduce the biosynthesis of dihydropteroic acid or tetrahydrofolic acid to levels below those essential for growth depends on the amount of substrate present. Because the sulfonamide acts before TMP in the biosynthesis, its role in the dual action is merely to reduce the amount of dihydrofolic acid against which TMP competes. If the competition between TMP and dihydrofolic acid were linear, then the effects of the dual action would be no more than additive, but because the competition by TMP increases relatively with decreases in dihydrofolic acid, the effects are synergic.5 Another factor that will also affect the dual action is that TMP affects not only the de novo synthesis of tetrahydrofolic acid but also its recycling. In the biosynthesis of thymine, at the stage at which uridylate is reduced to thymidylate, tetrahydrofolic acid is reoxidized to dihydrofolic acid, which is then returned to the tetrahydrofolate pooi by the action of dihydrofolate reductase; in the presence of TMP this recycling is inhibited and in conjunction with the partial cut-off CMA JOURNAL/JUNE 14, 1975/VOL. 112
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