Correlation of in Vitro Sulfonamide Resistance of the Gonococcus with Results of Sulfonamide Therapy*T CHARLES M

Home , Sulfanilamide, Sulfathiazole, Sulfonamide (medicine)

Mar., 1944

Correlation of in vitro Sulfonamide Resistance of the Gonococcus with Results of Sulfonamide Therapy*t CHARLES M. CARPENTER, M.D., F.A.P.H.A., AND HELEN ACKERMAN University of Rochester, School of Medicine and Dentistry, Rochester, N. Ir.

AND MILLARD E. WINCHESTER, M.D., F.A.P.H.A., AND JANE WHITTLE Glynn County Board of Health, Brunswick, Ga.

PREVIOUS studies have demon- efficacy of sulfathiazole in patients with strated that in vitro the gonococcus gonococcal infection in New York City, acquires resistance to gradually in- and the susceptibility of the patient's creased concentrations of sulfanilamnide, strain of gonococcus to the drug in vitro. sulfapyridine, sulfathiazole, and sulfa- The present report records the results diazine.l1 It has been shown, further- of treatment with sulfathiazole on 105 more, that induced resistance of an patients with gonococcal infection and organism to one sulfonamide compound the in vitro behavior to sulfanilamide usually increases resistance to other and sulfathiazole of the strains of the derivatives of sulfanilamide.385 These gonococcus isolated from those patients. observations, together with an apparent This study was carried out at the decrease in the therapeutic efficacy of Glynn County Board of Health, sulfonamides in gonorrhea, suggested the Brunswick, Ga. investigation of a possible correlation between the laboratory and clinical MATERIALS AND METHODS findings. Information as to the preva- Patients with symptoms of uncom- lence of sulfonamide resistant strains of plicated urethritis of cervicitis reporting the gonococcus in a community follow- at the venereal disease clinic were ex- ing the widespread use of sulfonamides amined for evidence of gonococcal in- would in addition provide data of epi- fection. The film and cultural methods demiologic significance. Cohn and were used to verify the diagnosis of the Seijo 6 have recently reported a direct disease. Those from whom the gono- relationship between the therapeutic coccus was isolated were treated with a single course of therapy consisting of * Presented before the Epidemiology Section of 20 gm. of sulfathiazole administered in the American Public Health Association at the one of the two following manners: 3 Seventy-second Annual Meeting in New York, N. Y., October 14, 1943. gm. daily for 6 days and 2 gm. on the t This study was carried out in cobperation with 7th day or 4 gm. daily for 5 days. In the. Division of Venereal Diseases, U, S. Public Health Service, Washington, D. C. a few instances sulfapyrazine was sub- [2501 Vol. 34 SULFOlNAMIDE THERAPY 251 stituted for sulfathiazole. The patients Five ml. of medium in test tubes were returned to the clinic for examination inoculated with 0.1 ml. of a 24 hour once a week for at least 3 weeks, and Douglas's broth culture of the strain at each visit films and cultures were to be tested. A tube of blood broth again taken. A series of three negative without drug was also inoculated to bacteriologic examinations together with serve as a control. After 48 hours of the absence of clinical evidence of dis- incubation at 370 C. subcultures were ease comprised the criteria of cure. made to " chocolate " agar plates which Thus, patients responding to one course were similarly incubated, and the of therapy were considered " cured," presence or absence of growth was and those requiring additional treatment recorded. When growth occurred in any were designated " failures." The 105 tube containing drug, the strain was patients included in this study were designated " resistant." If no growth classified according to race and sex as was detected, the strain was considered follows: Negro, 79; white, 26. The "nonresistant." The minimal concen- Negroes comprised 54 males and 25 trations of 5 mg. per cent and 1 mg. females; the white patients, 13 males per cent of sulfanilamide and sulfa- and 13 females. Included in this group thiazole, respectively, were selected be- were only those patients with no history cause they were significantly higher of previous treatment with sulfonamide than the concentrations which were compounds. bactericidal for strains of the gono- The strain of gonococcus isolated coccus isolated from patients readily from each patient before treatment was cured with a single course of sulfona- tested in vitro for growth in blood-broth mide therapy. media containing sulfanilamide and sulfathiazole. As a rule, the tests were RESULTS carried out as soon as possible after iso- Of the 105 selected patients with lation, the strains examined for the most gonococcal infection from whom Neis- part being between the 5th and 10th seria gonorrhoeae was isolated prior to generations. In addition to the 105 sulfonamide therapy, 88, or 84 per cent, strains of the gonococcus recovered' were " cured " with one course of 20 from this group of patients, similar gm. of either sulfathiazole or sulfa- tests were made on 109 strains isolated pyrazine. The remaining 17, or 16 per in this same community. The observa- cent, failed to be cured with the same tion period of the study extended from amount of drug. May 1, 1942, to August 1, 1943. Seventy-seven strains of the gono- The medium was prepared in the coccus, 87.5 per cent, isolated from the following manner. Sulfanilamide to 88 patients designated as " cured ' yield final concentrations of 5, 10, 20, failed to grow in vitro in any of the and 40 mg. per cent was added to concentrations, 5, 10, 20, and 40 mg. Douglas's broth containing 0.05 per per cent sulfanilamide or in concentra- cent each of sodium phosphate and tions of 1, 2, 5, and 10 mg. per cent sodium nitrate. After sterilization in sulfathiazole. The remaining 11 strains, the autoclave at 121° C. for 10 min- 12.5 per cent, on the other hand, re- utes, 5 ml. of defibrinated lapine blood sisted the action of one or both drugs were added to each 100 ml. of the in vitro. medium. The sulfathiazole broth was Thirteen strains of the gonococcus, prepared in a similar manner except 76.5 per cent, recovered from 17 pa- that concentrations of 1, 2, 5, and 10 tients that failed to be cured likewise mg. per cent of the drug were employed. remained viable when exposed to the 252 AMERICAN JOURNAL OF PUBLIC HEALTH Mar., 1944

TABLE 1 Correlation of Effect of Sulfonamides on Neisseria gonorrhoeae in Vitro with Sulfonamide Therapy in 105 Patients Resistance to Sulfonamides Clinical Results with Sulfonamide Therapy of Strains of Gonococcus - - -- Isolated before Treatment Cured * Uncured -- No. of No. of No. of Strain Strains Per cent Patients Per cent Patients Per cent " Nonresistant " 81 77 77 95 4 5 " Resistant " 24 23 1 1 46 13 54 Total 105 100 88 84 17 .16

* Criteria of cure consisted of three negative bacteriologic examinations at weekly intervals together with the absence of clinical evidence of gonorrhea. above concentrations of sulfanilamide *ing the first 6 months of the study were and sulfathiazole in vitro, while 4 " resistant," whereas observations made strains, 23.5 per cent, did not survive during the last 3 months showed that the action of the drugs. 59.3 per cent of all strains examined A correlation between the curative. were " resistant " (Table 2).

TABLE 2 Trend of Prevalence of in Vitro Sulfonamide Resistance of the Gonococcus in Br-unswick, Ga. Cultures Cultures "Nonresistant" "Resistant" Cultures to Sulfonamides to Sulfonamides "Resistant" Time Intervals Tested in Vitro in Vitro Cultures No. No. No. Per cent May, 1942-October, 1942 46 39 7 15.2 November, 1942-January, 1943 45 33 12 26.7 February, 1943-April, 1943 69 46 23 33.3 May, 1943-July, 1943 54 22 32 59.3 Total 214 140 74 34.-6 effect of the drugs in vivo and the re- DISCUSSION sistance of the specific organism in vitro Cohn and Seijo 6 working with 107 showed that of 81 patients infected strains of the gonococcus isolated from with " nonresistant " strains, 77, 95 per 101 patients in New York City have cent, were "cured," but that of 24 reported that 100 per cent of the pa- patients infected with "resistant" tients with " nonresistant " strains were strains, only 11, 46 per cent, were cured, but only 18 per cent of the pa- " cured " with an equivalent amount of tients with " resistant " strains re- the compound (Table 1). tovered. In our study, 95 per- cent of Of a total of 214 strains of the gono- the patients with " nonresistant " strains coccus isolated prior to treatment at responded favorably to sulfonamide Brunswick, Ga., during a 15 montb therapy as well as 46 per cent of the period from May 1, 1942, to August 1, patients with " resistant " strains. Thus 1943, and tested for sulfonamide re- in the group reported by Cohn and Seijo, sistance, 74, or 34.6 per cent, were only 3 of 17 patients with " resistant " " resistant " to sulfanilamide and sulfa- strains of the gonococcus were cured, thiazole in vitro. Of greater signifi- whereas in our group, 11 of 24 patients cance, however, is the finding that only with " resistant " strains recovered. A 15.2 per cent of these strains tested dur- valid comparison of the results obtained Vol.34 SULFONAMIDE THERAPY 253 in the two studies cannot be made be- of the gonococcus known to have been cause each patient of Cohn and Seijo re- exposed to sulfonamides. ceived during the first course of therapy Routine tests for the sulfonamide only a total of 14 gm. of drug. Our resistance of a strain of gonococcus in patients, on the other hand, were given vitro are as yet impractical because of a total of 20 gm. each. the time and expense involved. The The investigation of the resistance in present study points out, however, one vitro of recently isolated strains of the of the difficulties encountered in carry- gonococcus to concentrations of sul- ing out with sulfonamide therapy an fonamide compounds comparable to intensive program for the community those obtained in the blood of patients control of gonorrhea. after sulfonamide therapy has provided It is interesting to speculate on information of value in shedding light whether " resistant " strains of the on the future course of sulfonamide gonococcus were distributed in man therapy in gonorrhea. Evidence has previous to the era of sulfonamide been accumulated to indicate that the therapy, or whether strains of the or- extensive use of sulfonamide compounds, ganism have acquired resistance as a chiefly sulfathiazole, for the treatment result of contact with the drug. Fur- of gonococcal infection within a period ther investigations including stidies of of 15 months has markedly increased strains isolated prior to the " sulfona- the prevalence of " resistant " strains mide era " may answer this question. in the community in which the study was carried out This finding may pos- SUMMARY sibly be attributed to the elimination of The in vitro response to sulfathiazole a high percentage of the " nonresistant" and sulfanilamide of 105 strains of strains by chemotherapy, as well as to Neisseria gonorrhoeae has been corre- the development of " resistant " strains lated with the clinical responses of the from exposure to the drugs in vivo. patients from whom the organisms were Inasmuch as the gonococcus acquires isolated. resistance to sulfonamides in vitro, un- Of the group of patients from whom doubtedly the organism becomes re- " nonresistant " strains were isolated, sistant to the drugs in vivo. 95 per cent were " cured " in 5 days The marked increase in the number with 20 gm. of sulfathiazole. On the of " resistant " strains isolated from un- other hand, only 46 per cent of the treated patients at the venereal disease group with " resistant" strains re- clinic-is striking. It is known that dur- sponded to an equivalent amount of ing the period of observation numerous sulfonamide therapy. patients in the community obtained An increase in the prevalence of sulfathiazole from local pharmacists pre- sulfonamide-resistant strains of Neis- sumably for the treatment of gonococcal seria gonorrhoeae from 15 to 59 per cent infection. Theretore, self-treatment in 15' months was observed in Glynn may have cured many patients infected County, Ga., from May, 1942, to with "nonresistant " strains of the August, 1943, following the extensive gonococcus. Thus a proportion of pa- use of sulfonamide drugs as a part of tients attending the clinic may have a special program for the control of comprised those who failed to cure gonorrhea. themselves. It must be pointed out, REFERENCES however, that every effort was made to 1. Boak, R. A., Charles, R. L., and Carpenter, exclude previously treated patients from C. M. Tolerance of the Gonococcus In Vitro for Increasing Concentrations of Sulfanilamide. Am. A. the study in order to eliminate strains Advancement Sc., Publ. No. 11, pp. 118-119. 254 AMERICAN JOURNAL OF PUBLIC HEALTH Mar., 1944

2. Westphal, L., Charles, R. L., and Carpenter, Sulfapyridine, Sulfathiazole, and Sulfamethylthiazole. C. M. The Development of Sulfapyridine-Fast Ann. Ixt. Med., 14:1001-1031 (Dec.), 1940. Strains of the Gonococcus. Ven. Dis. Inform., 21: 6. Cohn, A., and Seijo, I. Further Observations 183-186 (June), 1940. on the Correlation between Clinical and in Vitro 3. Lankford, C. E., Scott, V., and Cooke, W. R. Reactions of Gonococcus Strains to Sulfathiazole. Am. Studies of Sulfonamide Resistance of the Gonococcus. J. Syph., Gonor., & Ven. Dis., 27:301-308 (May), J. Bact., 45:201 (Feb.), 1943. 1943. 4. Kirby, W. M. M. Development of Sulfa- thiazole-Resistant Gonococci in Vitro. Proc. Soc. Exper. Biol. & Med., 52:175-176 (Mar.), 1943. 5. Lowell, F. C., Strauss, E., and Findland, M. NoTE: Sulfapyrazine was supplied by Observations on Susceptibility of Pneumococci to Mead Johnson and Company, Evansville, Ind.

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