(Received for publication, December 27, 1943)

Transcription

1 Published Online: 1 April, 1944 Supp Inf: Dwnladed frm jem.rupress.rg n Octber 16, 2018 PARA-AMINOBENZOIC ACID PRODUCTION BY STAPHYLOCOCCI Bx WESLEY W. SPINK, M.D., LEMUEL D. WRIGHT, PH.D.~ JEAN J. VIVINO, AND HELEN R. SKEGGS (Frm the Divisin f Internal Medicine, University f Minnesta Hspitals and Medical Schl, Minneaplis, and the Medical-Research Divisin, Sharp and Dhrne, Inc., Glenlden, Pennsylvania) (Received fr publicatin, December 27, 1943) It is nw recgnized that several species f bacteria may develp resistance t the antibacterial actin f the sulfnamides. Strauss, Dingle, and Finland (1) have shwn that pathgenic strains f staphylccci may becme resistant in vitr by expsing cultures t increasing cncentratins f the drugs. Vivin and Spink (2) cnfirmed these bservatins, and als pinted ut that the develpment f sulfnamide-resistant staphylccci may take place in the human bdy. Up t the present time, we have islated 28 strains f cagulase-psitive staphylccci frm 24 patients, which shwed varying degrees f sulfnamideresistance. A descriptin f these investigatins and their clinical significance will be presented elsewhere (3). The mechanism whereby bacteria becme adapted t grw in the presence f the sulfnamides has been the subject f numerus investigatins. The bservatins f Wds (4) first suggested that para-aminbenzic acid (PABA) was synthesized by certain species f rganisms, and that PABA, an essential bacterial metablite, cmpeted with the chemically related sulfnamides fr a psitin in a bacterial enzyme system. MacLed (5) bserved that cultures f a sulfnamide-resistant strain f Type I pneumcccus yielded a filtrate which inhibited the antibacterial actin f sulfapyridine fr B. cli t a greater degree than a filtrate prepared frm the parent sulfnamide-sensitive strain. Mirick (6) utilized a suspensin f a sil bacillus, which culd be specifically adapted t xidize PABA, and fund that this suspensin rapidly destryed the diaztizable sulfnamide-inhibiting substance present in the filtrate prepared frm a sulfnamide-resistant strain f pneumcccus. Mre recently Mirick (7) has presented a methd fr quantitating small amunts f PABA with the aid f specific adapted enzymes f this same sil bacillus. Landy and his assciates (8) have investigated with a micrbilgical assay tw sulfnamideresistant strains f staphylcccus supplied by us, and reprted that the sulfnamide inhibitr elabrated by these strains was PABA. Up t the present time, there has nt appeared any cnfirmatry evidence in supprt f Landy's cnclusin that the antisulfnamide actin f staphylccci is related t the synthesis f PABA by the bacterial cells. The purpse f this reprt is t present the results f bservatins shwing that the resistance 331

2 332 PARA-AMINOBENZOIC ACID PRODUCTION BY STAPHYLOCOCCI f staphylccci is clsely related quantitatively t the prductin f PABA by the bacteria. Methds and Materials Thrughut the present study Gladstne's (9) water clear synthetic medium has been utilized in which the presence f sulfnamide inhibitrs has been reduced t a minimum. The preparatin f this medium is described elsewhere (3). The standard test used fr detecting sulfnamide-resistant staphylccci was as fllws: Strains t be tested were grwn fr several generatins in the synthetic medium t insure against cntaminatin by any sulfnamide-inhibiting materials. 0.1 cc. f a 10-3 dilutin f a 24 hur culture in synthetic medium was added t a series f tubes cnraining 10 c. f the medium with varying cncentratins f sdium sulfathiazle. Under these cnditins the ttal number f bacteria added varied between 40,000 and 180,000. Sdium sulfathiazle was selected because repeated bservatins with the described standard test had shwn this cmpund t inhibit grwth f the staphylccci t a greater degree than either sulfanilamide, r the sdium salts f sulfapyridine, sulfadiazine, and sulfamerazine. After 48 hurs f incubatin at 37 C., bacterial grwth was ascertained and expressed in terms f turbidity with the use f the Evelyn phtelectric clrimeter. Ten su]fnamide-resistant and 5 nn-resistant strains f staphylccci, all cagulase-psitive and islated frm patients, were used in an attempt t btain a diaztizable substance prduced by the bacteria. 10 cc. f the synthetic medium was seeded with 0.1 cc. f a 10.3 dilutin f a 24 hur culture, and then incubated at 37 C. fr 96 hurs. Supernatants f the cultures were prepared by adding trichlracetic acid t a final cncentratin f 1.5 per cent and then centrifuging the cntents. The clrimetric methd f Brattn and Marshall (10) was used t determine the amunt f diaztizable material present in the supernatants, utilizing a standard PABA preparatin as a cntrl) The intensity f the clr was measured with the Evelyn phtelectric clrlmeter. A culture f an aerbic sil bacillus capable f being adapted t xidize PABA, thus preventing the develpment f a diaz clr, was btained thrugh the curtesy f Dr. Gerge S. Mirick f the Hspital f The Rckefeller Institute fr Medical Research. At the suggestin f Dr. Mirick, the bacillus was grwn fr apprximately 24 hurs in cc. f veal infusin brth, ph 7.8, in each f tw flasks. One flask cntained nly the inculated medium, and the secnd had 20 rag. per 100 cc. f pure PABA added. When maximum grwth was btained the bacterial cells were washed with phsphate buffer, ph 7.8. The washed cells, cncentrated in abut 10 cc. f phsphate buffer, were shaken fr 30 minutes with the previusly described supernatants, a sample f which had given a diaz clr reactin. The cells were similarly expsed t a slutin cntaining a knwn quantity f pure PABA. Immediately thereafter, the diaz test was perfrmed with the treated supernatants. The same prcedure was carried ut with cells nt grwn in the presence f PABA. T test the antisulfnamide actin f sulfnamide-resistant and sulfnamide- 1 Pure PABA was kindly supplied by Dr. D. F. Rbertsn f Merck & C., Inc.

3 w. w. spr~, L. D. wr~g~rr, j. j. VIVmG, H. X~. SKEGGS 333 sensitive strains f staphylccci, filtrates were prepared frm the representative strains by passing 72 hur cultures in synthetic medium thrugh a Seitz filter. The amunt f diaztizable substance in each filtrate was quantitated by the diaz reactin. The inhibitry effect f the filtrates upn the bacteristatic actin f sdium sulfathiazle was determined by adding t a series f tubes 1 cc. f the filtrate and 9 cc. f the synthetic medium cntaining the previusly described standard inculum f a sulfnamide-sensitive strain f staphylcccus. Varying cncentratins f sdium sulfathiazle were then added t each f the tubes. A cntrl series f tubes cntained varying amunts f pure PABA instead f the filtrates. The amunt f PABA used was that amunt in slutin which gave apprximately the same diaz reactin as the bacterial filtrates. The cntents f the tubes were incubated fr 48 hurs at 37 C. Attempts were made by tw f us t identify the diaztizable substance prduced by the staphylccci as being PABA by emplying the micrbilgical assay described by Lewis (11). Repeated failures were ascribed t a chemical cntaminatin f the cntrl medium, pssibly by PABA r ther grwth-stimulating substances fr the test rganism, LactbadUus arabinsus Mre desirable results were btained in a secnd labratry by utilizatin f the assay methd f Landy and Dicken (12). Material was prepared with the synthetic medium at Minneaplis and sent t Glenlden fr assay by the ther tw authrs f the present paper. In the first series f bservatins, filtrates prepared by Berkefeld filtratin were assayed. 5 cc. f each sample was autclaved at 15 punds pressure fr 15 minutes with 5 cc. f 0.2 N NaOH, and then neutralized with 0.2 N HC1. Acetbacter subxydans, as mdified by the additin f purines and pyrimidines (13), was emplyed. The medium and three sulfnamide-resistant strains (605, 611, and 620) were assayed at levels f 0.02 t 0.10 cc. f the riginal filtrate. The standard curve embraced a range f t 0.5 micrgram f PABA. Incubatin was carried ut fr 48 hurs at 30 C. Turbidity f the cultures, measured with the aid f the Klett-Summersn phtelectric clrimeter, was taken as an index f grwth. Tw ther series f assays were made accrding t the methd f Lewis (11). 1 cc. f each sample was autdaved with 10 cc. f a 1 N NaOH at 13½ punds pressure fr 30 minutes, and then neutralized with i N HC1. The sulfnamide-resistant cultures were assayed at levels f t cc. f the riginal sample, while the sulfnamide-sensitive strains were assayed at levels f t 0.05 cc. The standard curve cvered a range frm t micrgram f PABA. The basal medium was prepared accrding t the directins f Lewis. A 24 hur culture f Lactbacillus arabinsus 17-5, grwn in a medium free f PABA, was used as the inculum. Titratin f the acid prduced during 72 hurs' incubatin at 30 C. was used as a measure f the respnse f the rganism t PABA. RESULTS Diaztizable Substance Prduced by Staphylccci and Its Relatinship t Sulfnamide Resistance.--The results f the inhibitry effect f sdium sulfathiazle upn the grwth f 15 strains f staphylccci are presented in Table I. It is t be nted that the grwth f 5 strains was cmpletely inhibited by a cncentratin f less than 1 mg. per 100 c. f sdium sulfathiazle, whereas the inhibitin f 10 resistant strains was nly accmplished with a cncentra-

4 334 PARA-AMINOBENZOIC ACID PRODUCTION BY STAPHYLOCOCCI tin f mg. r mre f the sulfnamide per 100 cc. f culture medium. The amunt f diaztizable substance fund in the supernatants f the cultures f the 15 strains was als cmpared with the resistance f the rganisms t the actin f sdium sulfathiazle. An aqueus slutin f pure PABA was used as a cntrl in quantitating the diaz reactin. Supernatants f the cultures were used instead f filtrates free f rganisms since the end results were the same with bth preparatins, and supernatants were btained with greater ease than TABLE I Diaztlnable Substance in Supernatants f Nn-Resistant and Sulfnamide-Resistant Strains f Staphylccci Grwn fr 96 Hurs at 37~C. Minimal cncentratin f Strain N. Diaztizable substance sdium sulfathiazle cmpletely inhibiting grwth Nn-reslaant strain, micrgrams f PABA per 100 c. mg. per 100. Less than 10 Less than 1 " " 10 " " 10 " " 10 " " 10 Resistant strains were the filtrates. As shwn in Table I, the sulfnamide-sensitive strains prduced less than 10 micrgrams per 100 cc. f diaztizable material, expressed in terms f PABA. On the ther hand, the sulfnamide-resistant strains prduced frm five t slightly ver ten times as much f the substance. It shuld be emphasized that the clr reactin cannt be measured accurately with the Brattn-Marshall technique when the cncentratin is belw 10 micrgrams per 100 cc. Nevertheless, the results shw a marked difference in the amunt f diaztizable substances prduced by these tw grups f staphylccci. Effect f a Sil Bacillus (Mirick), Specifically Adapted t Oxidize PABA, upn

5 W. W. SPINK, L. D. WRIGHT, J. J. VIVINO, It. R. SKEGGS 335 the Diaztizable Substance.--After the supernatants f the 15 cultures f staphylccci had each been expsed t the sil bacillus, accrding t the methd previusly described, n diaz reactin culd be develped. Cntrl samples f supernatants similarly treated with the parent strain f Mirick's sil bacillus, which had nt been adapted t xidize PABA, prduced the typical diaz clr. This is further evidence that the diaztizable material frmed by staphylccci is PABA. Effect f Bacteria-Free Filtrates f Nn-Resistant and Sulfnamide-Resistant Strains f Staphylccci upn the Antistaphylcccic Actin f Sdium Sulfathiazle.--Filtrates prepared frm bth sulfnamide-sensitive and sulfnamideresistant strains f staphylccci inhibited the bacteristatic actin f sdium sulfathiazle fr strains whse grwth was readily inhibited by the drug. TABLE II A ntisulfnamide Effect f Filtrates f Nn-Resistant and Resistant Strains f Staphylccci, and f P A B A, upn A nticta phylcccic Actin f Sdium Sul fathlazle Test material 0.01 Nn-resistant strain Nn-resistant strain 14 plus filtrate f nn-resistant strain Nn-resistant strain 14 plus filtrate f resistant strain Nn-resistant strain 14 plus 0.27 nag. per 100 cc. PABA... Cncentratin f sdium sulfathiazle, rag. per 100 c. 0.1 = minimum grwth. -- maximum grwth The results f a representative experiment are shwn in Table II. The grwth f strain 14 was inhibited cmpletely by 1 rag. per 100 cc. f sdium sulfathiazle, while the grwth f strain 605 required a cncentratin f 250 mg. f the sulfnamide. It is f interest t bserve that the filtrate f strain 14 inhibited the antistaphylccdc actin f the sulfathiazle fr this same strain. Reference t Table I shws that this strain prduced relatively small amunts f the diaztizable material. The filtrate f the sulfnamide-resistant strain 605 prduced a much mre marked inhibitin f the sulfnamide activity fr strain 14. In quantitating the diaz reactin f the filtrate f strain 605, it was calculated that the diaztizable substance represented apprximately 0.27 mg. per 100 cc. f PABA. This cncentratin f PABA acted t inhibit bacteristasis by sulfathiazle nearly as markedly as did the filtrate f strain 605. Micrbilgical Assay f Staphylcccic Filtrates fr PABA (Methd f Landy and Dicken).--In the first series f assays, the filtrates f tw sulfnamidesensitive strains f staphylccci and three strains resistant t sulfnamide

6 336 PARA-AMINOBENZOIC ACID PRODUCTION BY STAPHYLOCOCCI activity were investigated. As a cntrl, the amunt f active material in the synthetic medium was determined. The results are given in Table III. Slight grwth stimulatin was fund t ccur in the uninculated synthetic medium. The 3 sulfnamide-resistant strains prduced apprximately 30 t 40 times the PABA prduced by the 2 sulfnamide-sensitive strains. Fr cmparisn, Table III als includes the cncentratin f sdium sulfathiazle required t inhibit cmpletely the grwth f each strain f staphylcccus. Cmparative Micrbilgical Assay f PABA in Cell-Free Filtrates and in the Medium with Staphylccci Present (Methd f Lewis).--Since the first series was carried ut with the filtrates f staphylcccic cultures, there existed the pssibility that a prtin f the PABA had been remved in the prcess f TABLE HI PABA Cntent f Staphylcccic Filtrates (Metkd f Landy and Dicken), As Cmpared wltk the Cncentratin f Sdium Sul fatklanle Required t Inhibit in Vitr Grwth f the Cultures Sample Medium... Strain 7*... " 14"... " 605~... " 611~... " 620~... * Nn-resistant t sulfnamide actin. Resistant t sulfnamide actin. PABA micrgrams per 100 c O Minimal cncentratin f sdium sulfathiazle cmpletely inhibiting grwth rag. per I00 ~. Less than 1 c~ gt filtering the cultures thrugh the Berkefeld candles. Therefre, a secnd series f assays was made in which the amunt f PABA was determined simultaneusly in cell-free filtrates and in the medium cntaining the fully grwn cells. These results are presented in Table IV. Bth preparatins yielded the same amunt f PABA. In the light f these findings, there appeared t be n advantage in assaying cell-free filtrates fr PABA, and in subsequent determinatins, the riginal medium with the fully grwn cells was utilized. It is t be nted that the values f PABA btained with the methd f Lewis were cnsiderably greater than thse btained with the methd f Landy and Dicken. In additin, the yield f PABA was much greater with the sulfnamide-resistant strains f staphylccci in cmparisn with the nn-resistant strains. Cmparisn f PABA Prductin by Staphylccci (Methd f Lewis) with the Cncentratin f Sdium Sulfathiazle Required t Inhibit the in Vitr Grwth

7 TABLE IV Cmparative Assay f PABA (Methd f L~.~) with Cell-Free Filtrates and with thoriginal Culture Medium Cntaining Staphylccci Minimal cncentratin f Strain and material assayed PABA sdium sul~athiazle cmpletely inhibiting grwth 7 --cells* filtrate* cells* filtrate*... 7c--~ells~... 7c--filtrate~... 14c---cells~ " ~ " ~... * Nn-resistant t sulfnamide actin. :~ Resistant t sulfnamide actin. micrgrams per 100 e rag. per 100 c. Less than 1 " " 1 " " 1 cc O TABLE V Cmparisn f PABA Prductin by Staphylccci (Methd f Lewis) with the Cncenlralin f Sdium Sulfathiazle Required t Inhibit in Vitr Grwth f the Cultures Minimal cncentratin f Strain PABA sdium sulfathiazle cmpletely inhibiting grwth 14" 104" 715" 716" 717" 724* 727* 14C~ 116~ 117~ 604~ 605~ 66~ 611~ 616~: 619~ 628~ 629~ mlcrgrams per 100 ~ rag. per 100 c. Less than * Nn-resistant t sulfnamide actin. Resistant t sulfnamide actin. 337

8 338 PARA-AMINOBENZOIC ACID PRODUCTION BY STAPHYLOCOCCI f the Cultures.--The third series f assays was accmplished with 18 strains f staphylccci, 7 f which were cnsidered sensitive t the in ~itr actin f sdium sulfathiazle while the remaining 11 were resistant. Lewis' methd fr assaying PABA was utilized. In the preparatin f the samples, the cells were nt remved frm the culture medium. The cmparative results are shwn in Table V. It is bvius that the sulfnamide-sensitive strains prduced less YABA than the resistant strains. Hwever, the results btained with the resistant strains were incnstant. In several instances, the prductin f YABA culd nt be directly crrelated with the in vitr resistance f the rganisms t sdium sulfathiazle. As examples, strain 606 prduced nly 0.30 micrgram f PABA per cc., and yet a cncentratin f mg. f sdium sulfathiazle per 100 cc. was required t inhibit cmpletely the grwth f this rganism. Similar results were btained with strain 628. On the ther hand, strains 604, 611, and 616 prduced larger amunts f PABA and required nly the same amunt f the sulfnamide t inhibit their grwth. These incnsistencies will be discussed. It is f interest that the mst resistant strain (605) yielded the highest assay f PABA. DISCUSSION The freging data ffer further evidence that the develpment f sulfnamide-resistant strains f staphylccci is related quantitatively t the prductin f PABA by this species f bacteria. Nevertheless, this des nt rule ut the pssibility that anther mechanism r ther mechanisms may be invlved in the develpment f such resistance. The incnstant results which we have btained with tw different methds f assaying PABA merit cmment. It is believed that these discrepancies are assciated with several surces f errr which are inherent in any bilgical assay, particularly thse relating t the assay f PABA. As Mirick (7) has pinted ut, there may be substances clsely related chemically t PABA which may stimulate grwth f the test rganism. Furthermre, when substances f unknwn cmpsitin are supplied t a medium deficient fr prmting grwth, factrs unrelated t the ne f specific interest may activate grwth. Finally, rganisms may adapt themselves t grw in a deficient medium. These alteratins in bilgical activity may be s slight that they cannt be detected readily. The difficulties encuntered in assaying PABA in an unknwn mixture may be reslved by applying the technique described by Mirick (7) in which a sil bacillus may be specifically adapted t xidize PABA. Accrding t Mirick, the specific adaptive enzymes f this bacillus can be used t identify as little as 10 micrgrams f PABA. SUM3~ARY 1. Strains f staphylccci prduce diaztizable materials which can be cnverted t a dye, and the intensity f the clr reactin can be quantitated

9 W. W. SPINK, L. D. WRIGHT, J. J. VIVINO, H. R. SKEGGS 339 in the same manner as p-aminbenzic acid. The sulfnamide-resistant strains which we have studied prduce mre diaztizable substance than nn-resistant strains. 2. The develpment f a clr by the diaztizable substance can be inhibited by expsing the substance t a sil bacillus (Mirick) specifically adapted t xidize PABA. 3. The diaztizable substance prduced by staphylccci inhibits the antistaphylcccic actin f sdium sulfathiazle t apprximately the same degree as equivalent amunts f pure p-aminbenzic acid. 4. Tw micrbilgical methds fr assaying p-aminbenzic acid were emplyed fr quantitating the amunt f this material prduced by staphylccci. In general, the sulfnamide-resistant strains prduced mre p-aminbenzic acid than the sulfnamide-sensitive strains. The incnstant results btained with these bilgical assays are discussed. BIBLIOGRAPHY 1. Strauss, E., Dingle, J. H., and Finland, M., J. Immunl., 1941, 42, Vivin, J. J., and Spink, W. W., Prc. Sc. Exp. Bil. and Meg., 1942, 50, Spink, W. W., and Vivin, J. J., J. Clin. Inv., in press. 4. Wds, D. D., Brit. J. Exp. Path., 1940, 9.1, MacLed, C. M., J. Exp. Meg., 1940, 70., Mirick, G. S., J. Clin. Inv., 1941, 0.0, Mirick, G. S., J. Exp. Meg., 1943, 78, Landy, M., Larkum, N. W., Oswald, E. J., and Streightff, F., Science, 1943, 97, Gladstne, G. P., Brit. J. Exp. Path., 1937, 18, Brattn, A. C., and Marshall, E. K., Jr., J. Bil. Chem., 1939, 128, Lewis, J. C., J. Bil. Chem., 1942, 146, Landy, M., and Dicken, D. M., J. Bil. Chem., 1942, 146, Landy, M., and Streightff, F., Prc. Sc. Exp. Bil. and Meg., 1943, 80., 127.