IN VITRO ACTION OF SULFONAMIDES ON CLOSTRIDIA

SULFONAMIDE INHIBITORS1

G. B. REED, J. H. ORR AND R. W. REEDQueen's University, Kingston, Canada

Received for publication March 27, 1944

Local sulfonamide therapy has been shown to be markedly successful in gasgangrene wounds in experimental animals and man (see Reed and Orr, 1941,1942; Bliss, Long and Smith, 1942). The only evidence of in vitro bacterio-static action of sulfonamides against the gas gangrene clostridia is an earlyreport by Spray (1937). Spray found the growth of Clostridium tetani, C.novyi and C. septicum to be inhibited by sulfanilamide and disulfonamide inlow concentrations but C. welchii, C. tertium, C. sporogenes and C. histolyticumto be inhibited only by high concentrations of disulfonamide. This work mightbe criticised on the ground that the medium used was likely to be rich in sulfon-amide-inhibitor substances and that the inoculum used was very large. Onthe other hand Broh-Kahn (1939) and Fox (1940) raise some doubt as to thebacteriostatic action of sulfonamides under anaerobic conditions and Warren,Street and Stokinger (1939) question Spray's results on these grounds. Cliftonand Loewinger (1943), however, have shown that anaerobic growth of Esch-erichia coli is inhibited by sulfanilamide.

Spray's results are in agreement with the animal results insofar as theyindicate bacteriostatic action against certain species of Clostridium. Reed andOrr (1941 and 1942) have shown that in experimental gas gangrene in guineapigs six different sulfonamides give much more conspicuous therapeutic effectsin the case of C. welchii infections than in the case of infections by other speciesof Clostridium. Of the four principal gas gangrene species C. welchii was mosteffectively controlled by all the sulfonamides examined, C. sordellii least effec-tively controlled, while C. septicum and C. novyi fell in an intermediate posi-tion. In contrast, Spray found that sulfonamides he tested in vitro producedless bacteriostatic action on C. welchii than other species of Clostridium.

1. IN VITRO BACTERIOSTATIC ACTION IN PEPTONE MEDIA

Sulfanilamide, sulfapyridine, sulfaguanidine, sulfadiazine or sulfathiazole wasadded to peptone-thioglycollate broth (Reed and Orr, 1941), to make drugconcentrations of 1 to 200 mg per cent. These combinations were tubed in8-ml amounts in deep tubes and autoclaved. The species to be tested weregrown in this medium, without drug, for several culture generations. Twenty-four-hour cultures were filtered through cotton to remove clumps, dilutedserially in saline and the minimum inoculujm to initiate growth determined.In the test series, ten times the average minimum inoculum to initiate growth

I Part of an investigation aided financially by the Canadian National Research Council.233

234 G. B. REED, J. H. ORR AND R. W. REED

in the drug-free medium was introduced near the bottom of tubes of drug-freebroth and of the several drug doncentrations.Table 1 represents the lowest concentrations of some five sulfonamides to

inhibit the growth of the seven most important gas gangrene species of Clostrid-ium. It is apparent from the table that all five sulfonamides tested in pep-

TABLE 1Milligrams per cent in five sulfonamides to inhibit growth of seven species of ClostridiaInoculum ten times number of organisms required for same media without drug

SMALLEST CONCENTRATION SULFONAMIDES TO INHIBIT GROWTH

Sulfanilamide | Sulfapyridine Sulfaguanadine Sulfadiazine Sulfathiazole

mg % mg % mg % mg % mg %

C. welchiiI day................. 50 50 25 25 102 days ................ 100 100 100 100 1005 days................ 200 200 200 200 200

C. sporogenes1 day................. 50 50 25 25 102 days................ 100 100 100 100 1005 days................ 200 200 200 200 200

C. novyi1 day................. 2 2 2 2 22 days................ 10 10 2 2 25 days................ 10 10 10 10 10

C. histolyticum1 day................. 2 2 2 1 12 days................ 6 6 3 3 35 days................. 10 10 10 10 10

C. carnis1 day................. 3 3 1 1 12 days................. 6 6 3 3 35 days................ 10 10 10 10 10

C. septicum1 day................. 1 1 1 1 12 days................ 3 3 2 2 25 days................ 5 5 3 3 3

C. sordellii1 day................. 1 1 1 1 12 days................ 1 1 1 1 15 days................ 3 3 2 2 2

tone-thioglycollate broth are highly bacteriostatic for C. sordellii, C. septicum,C. carnis, a little less bacteriostatic for C. novyi and C. histolyticum and onlyslightly bacteriostatic for C. sporogenes and C. welchii. It is recognized, asdiscussed in the next section, that the peptone in the medium will tend to in-hibit the action of the sulfonamides but all tests were in the same peptoneconcentration so that the results are comparable.

Quqantitative determinations of the number of viable organisms in cultures

IN VITRO ACTION OF SULFONAMIDES ON CLOSTRIDIA ,

containing increasing concentration of sulfathiazole lead to somewhat differenteonclusions. Though the gross cultures indicate that C. welchii continues tomultiply in relatively high concentrations of sulfonamides; graph (fig. 1) ofthe numbers of C. welchii after 24 hours in cultures containing increasing con-centrations of sulfathiazole indicates that even in very low concentrationsthere is an appreciable retardation in multiplication and that the retardationincreases with increasing concentration.

8E7\

6

24 -0

10 50 100MS. per cent

FIG. 1. Graph showing amount of growth, in 24 hours, of C. welchii in peptone-thiogly-collate broth containing increasing concentrations of sulfanilamide. Ordinates representlog of the number of organisms per ml after 24 hours' growth, abscissa concentration ofsuifathiazole in mg per cent.

2. IN VITRO BACTERIOSTATIC ACTION IN INHIBITOR-FREE MEDIUM

Substances which inhibit the in vitro bacteriostatic action of sulfonamidesare known to be widely distributed. Lockwood (1938) found the inhibitor inpeptone. McLeod (1940) demonstrated an inhibitor in various animal tissues,especially after autolysis. Stamp (1939), Fleming (1940), Green (1940),Blanchard (1941) and others found inhibitors in cultures or extracts of yeastand several species of bacteria. McLeod (1940) found that drug-fast strainsof pneumococci produced unusually large amounts of inhibitor substance.Woods (1940) demonstrated that para-amino-benzoic acid was the inhibitor inpeptone and a variety of other substances. The more recent work of Blisand Long (1941), Lamanna and Shapiro (1942), Kahn and Harris (1943), Landyand Streightoff (1943) indicates that other substances may be concerned insulfonamide inhibition but para-amino-benzoic acid appears to be the principalinhibitor in tissue and microorganism extracts.

Since the inhibitor substances are relatively small molecules it seemed prob-able that they should be removed by dialysis. Proteose peptone was dialyzedfor 3 to 4 days in cellulose sacs against running water. The residue, evaporatedto dryness and used in place of undialized peptone, in the previously mentioned

235

G. B. REED, J. H. ORR AND R. W. REED

medium, supported luxuriant growth of all species of Clostridium tested. How-ever, a slightly larger inoculum was necessary than in the case of the undializedpeptone medium. This restriction was partially but not completely removedby the addition of cysteine, tryptofane and certain growth factors. The dializedpeptone medium had a very low content of inhibitor substances as tested bythe Escherichia coli procedure discussed in the next section.The results from testing the bacteriostatic action of sulfanilamide for C.

welchii in this dialyzed peptone in contrast with the undialyzed peptone mediumis summarized in table 2. It is apparent that the sulfanilamide is effective inappreciably smaller concentrations in the inhibitor-free medium. It is equallyevident that even in this medium much more sulfanilamide is necessary toinhibit the growth of C. welchii than in the case of many other species ofClostridium.

TABLE 2Bacteriostatic action of sulfanilamide on C. welchii in dialyzed and non-dialyzed peptone

medium

DIALYZED PEPTONE NON-DIALYZED PEPTONE

DAYSINCUBATION Concentration sulfanilamide in mg per cent

10 25 50 100 150 200 10 25 50 100 150 200 250 300

days1 + + + - - - + + + + + - - -2 + + + + - - + + + + + + - -5 + + + + + -+ + + + + + + -

These in vitro results in the two types of media are, in general, in agreementwith Spray's (1937) findings and stand in conflict with the in vivo results,namely: C. welchii which is most effectively controlled in the animal body isleast inhibited in the test tube; C. sordellii which is least successfully controlledin the animal body is among the species most effectively inhibited in the testtube.The most likely explanation of this apparent contradiction appears to be

associated with sulfonamide-inhibiting substances produced by the organismsthemselves.

3. SULFONAMIDE INHIBITORS IN CULTURE FILTRATES

C. welchii and C. sordellii were grown in the dialyzed peptone medium for24 hours, centrifuged and filtered through sintered glass filters (Jena 5 on 3).The filtrates of, both cultures and sterile media were tested for sulfonamide in-hibitors by the E. coli method of Bliss and Long (1941). Serial dilutions ofthe filtrates were added to Bliss and Long's synthetic medium containing 10mg per cent of sulfanilamide, all brought to the same volume with the mediumand all inoculated with E. coli (107 ml of a twenty-four hour culture).

Results summarized in table 3 indicate that the filtrates of C. welchii upto a 1-32 dilution contain enough sulfanilamide-inhibiting substance to destroy

.236

IN VITRO ACTION OF SULFONAMIDES ON CLOSTRIDIA

the bacteriostatic effect of 10 mg per cent of sulfanilamide and 1-64 dilution

enough to reduce the bacteriostatic effect of 10 mg per cent of sulfanilamide.In contrast a 1-2 dilution of C. sordellii filtrate contains only enough inhibitorpartially to inhibit 10 mg per cent of the drug. Filtrates of the sterile mediumin which the clostridia were grown contain only a trace of inhibitor.A second experiment was carried out in the same manner as the first except

that the cultures of C. welchii and C. sordellii were incubated in the dialyzedpeptone medium for one-half to ten days, filtered, and the filtrates tested forsulfonamide inhibitors as in the first experiment.The results are summarized in fig. 2 (solid lines). The log of the concentra-

tion of sulfonamide inhibitor (the minimum concentration of filtrate whichpermits the growth of E. coli in the presence of 10 mg per cent of sulfanilamide)is plotted against the age of the culture from which the filtrates were collected.

TABLE 3Sulfonamide inhibitor activity of filtrates of 96-hour cultures of C. welchii and C. sordellii in

dialyzed peptone mediumTitrations were made by determining the concentration of extract which will inhibit the

growth of E. coli in the presence of 10 mg per cent of sulfanilamide.

DILUTION OF FILTRATE

1-2 1-4 1-8 1-16 1-32 1-64 1-128

Days incubation of E. coli

1 2 3 5 1 2 3 S 1 2 3 5 1 2 3 5 1 2 3 5 1 2 3 5 1 2 3 5

C. welchii ... + +++1+ ++1 ++1| - - - --|--|- - - -

C. sordellii.....---ASterile medium . .- - - + - - - - - - - - - - - - - - - - - - - - - -

+ indicates heavy growth, - indicates no growth.

The C. welchii filtrate curve rises sharply from a titre of 1 to 2 at the start to1-32 after 12 hours' incubation of the C. welchii culture and then slowly for thenext ten days to a titre of 1-320. This is in sharp contrast to the C. sordelliifiltrate which shows a titre of 1-2 at the start and shows no increase in in-hibitor for the first four days of incubation. Between four and six days' incuba-tion the titre rises sharply to 1-8 and at ten days reaches 1-9. The suddenincrease in the inhibitor titre at four days may indicate that a significantnumber of C. sordellii cells are by that time undergoing autolysis. (SeeMcLeod [19401 on the increase in inhibitor in animal tissue undergoing autol-ysis.)

Interpretation of these results is complicated if the test cultures of E. coliare incubated for several days. When this is done E. coli, which fails to growin twenty-four hours, presumably due to bacteriostatic action of the sulfonamide(not inhibited by the filtrates of C. welchii or C. sordellii), grows out after fourto five days. Five-day readings of a set of E. coli test cultures is shown infig. 2 (broken lines) in contrast to twenty-four-hour readings of the same test

237

G. B. REED, J. H. ORR AND R. W. REED

cultures (fig. 2, solid lines). This probably means that the E. coli orgnmsintroduced as inoculum, after standing several days in the medium, produceenough sulfonamide inhibitor considerably to supplement that introduced withthe C. welchii and C. sordellii filtrates. However, even with this complication

wz - S dar ncubationGwelche 1 tet E.col,;VC.weIchigL COL

I filtr-te. 4/ -----_-o_--

0 i0.to'GC.sordeIlg,0. t f'itrd t e= 2. xtx C. welcha,filtratet ' /

if31 ~~~~24hour incub4tion;'f | ~~~~~~te-stE.coli

Z / C~~~~~~~..sordelhil filtnat4e0

0 8 2 - 4 6 8 loDays

FIG. 2. Graphs showing concentration of sulfonamide inhibitor in filtrates of cultures ofC. welchii and C. sordellii of various ages. Ordinates represent concentration of inhibitor,i.e., the dilution of filtrate which permits growth of E. coli in the presence of 10 mg per centof sulfonamide. Abscissa represent the age of the cultures from which filtrates wereprepared.

Lower curves (solid lines) 24 hours' incubation of the E. coli test organism. Uppercurves (broken lines) 5 days' incubation of the E. coli test organism.

it is still apparent (fig. 2, broken lines) that C. welchii is producing more in-hibitor than the C. sordellii.

4. SULFONAMIDE INHIBITORS IN EXTRACTS OF CLOSTRIDIUM

Stamp (1939), Green (1940) and others have shown that extracts as well asbroth filtrates of various species of bacteria contain sulfonamide inhibitors.

Extracts of C. welchii and C. sordellii were prepared according to Green's(1940) method as follows: the growth from seventy-two-hour anaerobic cultures

238

IN VITRO ACTION OF SULFONAMIDES ON CLOSTRIDIA

TABLE 4Sulfonamide inhibitor activity of six extract fractions of C. welchii and C. sordellii

Titrations were made by determining the concentration of extract which will permit thegrowth of E. coli in synthetic media containing 10 to 100 mg per cent of sulfanilamide.

2. 1st 48 hr NH4OHextraction

+ indicates heavy growth, i indicates slight growth, - indicates no growth.

on meat-infusion agar was suspended in distilled water (growth from twelvepetri dishes in 250 ml) and the suspensions of the two species brought to thesame opacity in a photoelectric nephelometer. After standing one hour atroom temperature in distilled water, the suspensions were centrifuged and the

239

G. B. REED, J. H. ORR AND R. W. REED

supernatant filtered through sintered glass (fraction 1). The organisms werethen resuspended in 60 ml N/25 NH40H and incubated at 37 C with continuousshaking, centrifuged, the supernatant adjusted to pH 7.2 and filtered (fraction2). The organisms were again suspended in 60 ml N/25 NH40H and treatedas the first ammonia extraction (fraction 3).

After removal of portions for testing, fractions 2 and 3 were combined,evaporated to half volume at 40 C, and precipitated at 10 C with nine volumesof alcohol. The alcohol-soluble portion was evaporated to dryness, redissolvedin 15 ml of water and filtered (fraction 4).The residue of organisms from the ammonia extraction was resuspended in

50 ml of water and allowed to autolyse for nine days at 37 C, centrifuged and

TABLE 5Sulfonamide inhibitor activity of two water extracts at 37 C with continuous shaking of

C. welchii and C. sordelliiTitrations were made by determining the concentration of extract which will inhibit the

growth of E. coli in the presence of 10 mg per cent of sulfanilamide.

C. WELCE[II EXTRACT DILUTION C. SORDELLII EXTRACT DILUTION

1-5 1-25 1-125 1-5 1-25 j 1-125

Days incubation E. coli

Fraeion:lsthrsate |2 3 15 1-2 3 5 112 3 5 1 213 5 1 2|3 5 1 213 5Fraction 1: 1st 3 hrs water

extract at 37 C withshaking..........++++++++++++-+++------- -+

Fraction 2: 2nd 3 hrs waterextract at 37 C withshaking.........

+ indicates heavy growth of E. coli, - indicates no growth of E. coli.

filtered (fraction 5). A part of fraction 5 was precipitated with alcohol andthe alcohol-soluble portion collected as fraction 4 (fraction 6).

In each instance the C. welchii and C. sordellii organisms were subjected tothe same treatment with the same volume of extracting fluids.

Sulfonamide-inhibitor activity of the six fractions of each filtrate was de-termined by the same E. coli procedure, used in previous experiments, exceptthat the E. coli synthetic medium was made up with 10 to 100 mg per cent ofsulfanilamide. Results are summarized in table 4. It is apparent that theprimary water extract (fraction 1) of C. welchii yielded a high concentrationof drug inhibitor while the corresponding C. sordellii fraction yielded a smallconcentration of drug inhibitor. The entire ammonia extracts and the alcohol-soluble portion of the ammonia extracts (fractions 2, 3 and 4) of C. welchiiyielded a considerable concentration of drug inhibitor. The correspondingextracts of C. sordellii yielded a scarcely measurable amount of drug inhibitor.The entire autolysate and the alcohol-soluble portion of these fractions (5 and6) of C. welchii yielded a small amount of drug inhibitor while the correspond-ing extracts of C. sordellii gave still lower amounts.

240

IN VITRO ACTION OF SULFONAMIDES ON CLOSTRIDIA

In another experiment, suspensions of C. welchii and C. sordellii containingthe same number of organisms were extracted with distilled water for threehours at 37 C with continuous shaking, centrifuged and filtered (fraction 1).The sedimented organisms were resuspended in water and similarly treated foranother three hours (fraction 2). This is similar to the first part of the preced-ing experiment except that the water extraction was for three rather than onehour and with continuous shaking.

Titrations made with E. coli as in the previous experiment, are summarizedin table 5. The long extraction with continuous shaking, it is apparent, resultsin recovery of a large amount of drug inhibitor from C. welchii and a smaller'amount of less active inhibitor from C. sordeUii. However, the contrast be-tween the amounts of inhibitor produced by the two species is less evident thanin the previous experiment where the water extraction was at room tempera-ture with occasional shaking.

These results with extracts are in agreement with the culture filtrates inshowing that C. welchii produces a large amount and C. sordellii a much smalleramount of sulfonamide inhibitor.

DISCUSSION

From the results presented in this paper it is apparent that if a sulfonamide-containing medium is inoculated with C. welchii, or other species which pro-duces a relatively large amount of inhibitor, the preformed inhibitor includedin the inoculum or the inhibitor produced by the organisms of the inoculumwill tend to neutralize the bacteriostatic effect of the sulfonamide. The smallerthe inoculum, the smaller the actual or potential inhibitor, and therefore thegreater opportunity for the bacteriostatic action to come into play.

Since some species of Clostridium produce much larger amounts of sulfon-amide-inhibitor than others it follows that those species which produce thelarger amounts, as C. welchii, will be less influenced by sulfonamides thanthose species, such as C. sordellii, which produce little inhibitor.

This difference in sulfonamide-inhibitor production satisfactorily explains thein vitro bacteriostatic action of sulfonamides for the several species of clostridia.This, however, still leaves unexplained the observed fact that species like C.welchii which produce large amounts of inhibitor and against which the sul-fonamides exert slight in vitro bacteriostatic action are effectively controlled inthe animal body by the same sulfonamides; or that species like C. sordelliiwhich produce little inhibitor and against which sulfonamides exert markedin vitro bacteriostatic action are much less effectively controlled in the animalbody by the same sulfonamides. It will be shown in a later paper that there isa marked difference in the behavior of sulfonamide-inhibitors in the test tubeand in living animal tissues.

SUMMARY

1. It has been shown that sulfonamides exert an in vitro bacteriostatic actionon all pathogenic species of Clostridium tested.

2. The order of effectiveness is sulfathiazole, sulfadiazine, sulfapyridine,sulfanilamide.

241

G. B. REED, J. H. ORR AND R. W. REED

3. The species of Clostridium against which sulfonamides exert the greatestin vitro bacteriostatic action are those which, like C. sordellii, produce leastinhibitor; those against which the sulfonamides exert least bacteriostatic actionare those which, like C. welchii, produce most inhibitor.

4. This difference in sulfonamide-inhibitor production has been demonstratedin filtrates of broth cultures and in water and ammonia extracts of the or-ganisms.

REFERENCESBLANCHARD, K. C. 1941 The isolation of p-aminobenzoic acid from yeast. J. Biochem.,

140, 919-925Biss, E. A., AND LONG, P. H. 1941 Observations on the mode of action of sulfanilamide.

The antibacteriostatic effect of methionine. Bull. Johns Hopkins Hosp., 69, 14-38.Biss, E. A., LONG, P. H., AND SMITH, D. G. 1941 Chemotherapy of experimental gas

gangrene and tetanus infections in mice. War Med., 1, 799-810.BROH-KAHN, R. H. 1939 The bacteriostatic action of sulfanilamide under anaerobic con-

ditions. Science, 90, 543-544.CLIFTON, C. E., AND LOEWINGER, I. E. 1943 Sulfanilamide activity against E8cherichia

coli under anaerobic conditions. Proc. Soc. Exptl. Biol. Med., 52, 225-277.FLEMNG, A. 1940 Observations on the bacteriostatic action of M and B 693 and the

influence thereon of bacteria and peptone. J. Path. Bact., 50, 69-81.Fox, C. L., JR. 1940 Is sulfanilamide bacteriostatic under "anaerobic" conditions?

Science, 91, 477-478.GREEN, H. N. 1940 The action of sulfonamide. Brit. J. Exptl. Path., 21, 38-64.KAHN, H. I., AND HARRIS, J. S. 1943 Purines, amino acids, peptones and pancreas as

antagonists and potentiators of sulfonamide in E. coli. J. Pharm. Exptl. Therap., 77,1-16.

LAMANNA, C., AND SHAPIRO, I. M. 1942 Sulfanilamide bacteriostasis in the presence ofmercuric chloride and p-aminobenzoic acid. J. Bact. 45, 384-392.

LANDY, M., AND STREIGHTOFF, E. 1943 Effects of purines on sensitivity of the Acetobac-ter Suboxydans assay for p-aminobenzoic acid. Proc. Soc. Exptl. Biol. Med., 52,127-128.

LOCKWOOD, J. S. 1938 The effect of sulfanilamide in serum and blood on haemolytic strep-tococcus in vitro. J. Immunol., 35, 155-193.

McLEOD, C. M. 1940 Inhibition of bacteriostatic action of sulfanilamide drugs by sub-stances of animal and bacterial origin. J. Exptl. Med., 72, 217-232.

REED, G. B., AND ORR, J. H. 1941 Chemotherapy in experimental gas gangrene. Lancet,I, 376-379.

REED, G. B., AND ORR, J. H. 1941a Rapid identification of the gas gangrene anaerobes.War Med., 1, 493-510.

REED, G. B., AND ORR, J. H. 1942 Local chemotherapy of gas gangrene. War Med., 2,59-78.

SPRAY, R. S. 1937 Bacteriostatic action of prontosil soluble sulfanilamide and disulfan-ilamide on the sporulating anaerobes commonly causally associated with gaseous gan-grene. J. Lab. Clin. Med., 23, 609-615.

STAMP, T. C. 1939 Bacteriostatic action of sulphanilamide in vitro, influence of fractionsisolated from haemolytic streptococci. Lancet, II, 10-17.

WARREN, J., STREET, J. A., AND STOKINGER, H. E. 1939 Influence of sulfanilamide andrelated compounds upon oxidation reduction potentials of hemolytic streptococcus.Proc. Soc. Exptl. Biol. Med., 40, 208-212.

WOODS, D. D. 1940 The relation of p-aminobenzoic acid to mechanism of action of sulfan-ilamide. Brit. J. Exptl. Path., 21, 74-90.

242