Services on Demand
Print version ISSN 0365-0596
On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.77 no.6 Rio de Janeiro Nov./Dec. 2002
CLINICAL, LABORATORY AND THERAPEUTIC INVESTIGATION
Activity of five antimicrobial agents in vitro against Neisseria gonorrhoeae*
Walter Belda JúniorI; Luiz Fernando de Góes SiqueiraII; Marcelo Menta S. NicoIII; Luiz Jorge FagundesIV
Professor at the Dermatology Service, "Faculdade de Medicina da Universidade
de São Paulo". Department supervised by Professor Evandro Rivitti
IIAssistant Professor at the Epidemiology Department, "Faculdade de Saúde Pública da Universidade de São Paulo"
IIIAssistant M.D. at the Clinic of Dermatology, "Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo"
IVAssistant Professor, "Faculdade de Saúde Pública da Universidade de São Paulo"
The use of antimicrobial drugs in the treatment of gonorrhea began in 1930 with
the use of sulfonamides. Through the years, other drugs such as penicillin,
spectinomycin, tetracycline among others, came into use. Although highly efficient
at first, with the passing of time these drugs began to present untoward therapeutic
results, because of the appearance of cases with chromosomic and plasmidial
resistance. Because of this, in order to establish a program to combat and control
a determined bacterial morbidity, it was necessary to carry out a program of
epidemiologic surveillance evaluating the sensitivity behavior of the etiologic
agents against the various therapeutic agents.
OBJECTIVES AND METHODS: The objective of this work was to evaluate the susceptibility of strains of Neisseria gonorrhoeae to the five drugs which are mostly used for the treatment of gonorrhea in Brazil (pencillin; cefoxitine; tetracycline; thiamphenicol and spectinomycine), by Minimum Inhibitory Concentration.
RESULTS AND CONCLUSION: We concluded that drugs such as cefoxitine, thiamphenicol and spectinomycine are still excellent drugs for the treatment of gonorrhea. Although penicillin continues to be effective, its use requires greater care, due to the emergence of resistant strains, and tetracycline must be absolutely avoided for the treatment of gonorrhea.
Key words: gonorrhea; neisseria gonorrhoeae; drug resistance; microbial sensitivity tests.
Gonorrhea represents one of humanity's oldest morbid states. Urethral secretions of venereal origin, supposedly gonococcicides, were already described by the Chinese during the empire of Huang Ti in 2637 BC. The utilization of antimicrobial agents in its therapeutics began in the 1930s with sulfonamides.1 However, in spite of being highly effective, resistance to these drugs grew quickly,2,3 since resistance mechanisms were favored by selective potency following the heavy use of these drugs. With the emergence of penicillin, the therapeutics of gonorrhea took new directions. Although in 1943 this drug proved effective, already by the end of the decade of the 1950's several reports pointed to a reduction in the sensitivity of gonococcus to penicillin.6-10 Starting from the beginning of the 80's, reports relating to the resistance to spectinomycin11,12and to cephalosporins13,14,15 also began to appear. From the above it is clear that a good strategy to combat and control certain bacterial morbidity should anticipate a program of epidemiologic vigilance, monitoring the sensitivity reactions of the etiological agents, as well as other procedures. From the epidemiological point of view it is known today that cultural issues in certain segments of the population are of capital importance in the phenomenon of selective potency16,17,18 of the various etiological agents. Among these cultural aspects that stand out are the indiscriminate use of antibiotics that, according to the World Health Organization are linked to social pressures arising from the stigma that accompanies sexually transmissible diseases.19 Self-medication, lay diagnosis,16,20,21 and attendance at drugstore counters, had occurred in 90% of the first consultations for sexually transmissible diseases.20,22,23 Finally, further complicating the cultural aspects in the continuation of the phenomenon of selective potency, there exists with equal importance the inopportune action of doctors, fomenting the excessive use of antimicrobial drugs, whether at private clinics, or under official recommendations in health programs.19
Even before the development of other phenotypic techniques, the behavior of sensitivity was, among other parameters, used to characterize the strains of Neisseria gonorrhoeae, his behavior was correlated to other serious diseases, as well as to the various geographical areas.24 However, the emergence of new techniques facilitated this work. At the present time the best tool for programs of epidemiologic surveillance, playing the part of an epidemiological indicator of sensitivity behavior to antimicrobial drugs, is the current and more precise methodology known as Minimum Inhibitory Concentration (MIC).25 The purpose of this present work is to evaluate the current sensitivity, through MIC to the five main drugs used in the treatment of gonorrhea in Brazil (penicillin; cefoxitin; tetracycline; thiamphenicol and spectinomycin).
MATERIAL AND METHODS
This study was accomplished using strains of Neisseria gonorrhoeae obtained from patients presenting acute but not complex gonorrhea, of both sexes, being treated at the Sexually Transmissible Diseases Service of the Dermatology Division of the Hospital das Clínicas, Faculty of Medicine, University of São Paulo and Sexually Transmissible Diseases Service of the Centro de Saúde Escola Geraldo de Paula Souza, Faculty of Public Health, University of São Paulo.
In the period from July 1998 to February of 2000, 65 strains of Neisseria gonorrhoeae were isolated.
The strains were isolated in modified Thayer-Martin medium 26 and later identified by direct bacterioscopy with Gram coloration, by reaction of the enzyme cytochrome-oxidase and by reaction of acidification of sugars.27-31 The strains were also submitted to chromogenic cephalosporin test for isolation of the betalactamase enzyme.32-36
In the studied population 15 strains were isolated of Neisseria gonorrhoeae producing penicillins (NGPP).
All strains were submitted to the susceptibility test using MIC technique with dilution in Agar.37-40 In the sensitivity test, the five drugs that are the most used in the treatment of gonorrhea were utilized: penicillin, cefoxitin, tetracycline, thiamphenicol and spectinomycin. The drugs tested were prepared according to appropriate techniques41,42 and in such a way as to obtain the following final concentrations:
0.25; 0.5; 1; 2; 4 and 8mg/ml
Cefoxitin: 0.125; 0.25; 0.5; 1; 2; 4 and 8mg/ml
Tetracycline: 0.125; 0.25; 0.5; 1; 2; 4; 8 and 16mg/ml
Thiamphenicol: 0.125; 0.25; 0.5; 1; 2; 4; 8 and 16mg/ml
Spectinomycin: 7.5; 10; 12.5; 15; 17.5; 20; 32; 64 and 128mg/ml
The inoculations were prepared starting with a subculture of each Neisseria gonorrhoeae, with a variation of from 18 to 24 hours of growth in medium under appropriate conditions.26,31,43 The MIC was read from the lowest concentration required for total inhibition of bacterial growth. Interpretation of the results, summarized in table 1, was made according to norms of the National Committee for Clinical Laboratory Standards - NCCLS.38
The results of the sensitivity of the 50 strains of Neisseria gonorrhoeae not producing penicillinase are presented in table 2, and, likewise, the results of the 15 strains of NGPPs, in table 3. The comparative result is expressed in table 4, and the distribution among the 65 strains, according to the tested drugs, in table 5.
In relation to cefoxitin, the strains not producing penicillinase presented their MIC more frequently in 0.125mg/ml, while NGPPs demonstrated a greater tolerance to this drug, presenting a 0.5mg/ml MIC more frequently, followed by 2mg/ml. Table 5 shows homogeneity in the behavior of sensitivity of the NGPP strains, which present a smaller variation than those not producing penicillinase, however with higher levels of tolerance to cefoxitin than those not producing penicillinase. The phenomenon observed was the deviation in the mean and median values. Among the strains not producing penicillinase the mean was 0.515mg/ml, and median 0.125mg/ml, passing the NGPPs to present a mean of 1.084mg/ml and median 0.5mg/ml. The same occurs with MIC50, that passes from 0.0919mg/ml among those not producing penicillinase to 0.5833mg/ml among NGPPs. And MIC90 repeats the phenomenon, passing from 0.9545mg/ml to 1.7mg/ml among those not producing penicillinase and NGPPs, respectively. As can be seen, the process of toleration among the strains of Neisseria gonorrhoeae is disturbing.
In relation to tetracycline, as was observed in this work, there was a variation between the strains not producing penicillinase that was not found among the NGPPs. In spite of the fact that of the values of MIC90 for the two similar populations, the mean (0.985mg/ml) distribution frequency (0.5mg/ml) and MIC50(0.4583mg/ml) observed among those not producing penicillinase, compared with the statistics relative to NGPPs, were practically double.
As for the sensitivity behavior to thiamphenicol, the two populations studied (producers and non producers of penicillinase) presented a homogeneous variation. The mean (1.625mg/ml) and median, with bimodal values (0.5 and 1mg/ml), observed in the strains not producing penicillinase, represent values of 2.384mg/ml and 4mg/ml, respectively, among the NGPP strains. The values of MIC50 for thiamphenicol are a repetition of this behavior, 0.7333mg/ml among those not producing penicillinase and 1.7mg/ml among NGPPs; however, in relation to MIC90 for thiamphenicol, the observed alterations are negligible.
In relation to spectinomycin, the variation observed between the NGPP strains was greater than that observed among the strains not producing penicillinase. Although the average and MIC90had increased, MIC50 was less among the NGPPs. Although reports exist of chromosomal resistance to spectinomycin among the strains of Neisseria gonorrhoeae, occurrence of this phenomenon was not observed in the present work.
Finally, in relation to penicillin, for the NGPP strains all the values were above the sensitivity limits.
The detection of strains other than NGPPs that were presented with MIC and that fell in the intermediate classification of sensitivity between cefoxitin and thiamphenicol, and of NGPP strains with intermediate MIC of sensitivity between thiamphenicol and spectinomycin, is a warning signal. It reinforces the need for the use of more rigorous criteria in the prescription of these drugs that, in spite of still being highly effective in the treatment of gonorrhea, already present indications in the laboratory that suggest they may also be heading toward a loss of effectiveness in the treatment of the disease, with the possibility of allowing the emergence of chromosomal or even plasmidial resistance to these drugs.
In conclusion, drugs such as cefoxitin, thiamphenicol and spectinomycin as yet continue to constitute excellent drugs for the treatment of gonorrhea. Penicillin, although still effective, requires greater care in its use due to the appearance of resistant strains. In relation to tetracycline, its use in the treatment of gonorrhea in Brazil at this time presents risks and its prescription clearly should be contraindicated.
In view of the results obtained, it can be concluded that most of the tested drugs present satisfactory results in the treatment of gonorrhea.
1. Cookkinis AJ, Mcelligot GLM. Sulphanilamide in gonorrhea. Lancet 1938;2:355-62. [ Links ]
2. Carpenter CM, Ackerman H,Winchester ME. Correlation of in vitro sulfonamide resistance of the gonococcus with results of sulfonamide therapy.Am J Public Health 1944;34:250-54. [ Links ]
3. Goodale WT, Gould RG, Schwalb L. Laboratory identification of sulfonamide-resistant gonococci infections. JAMA 1943;123:547-9. [ Links ]
4. Lankford CE. The in vitro tolerance of the gonococcus for penicillin.Am J Syph Gonor Vener Dis 1945;29:56-63. [ Links ]
5. Mahoney JF, Ferguson C, Buchholtz M. The use of penicillin sodium in the treatment of sulfonamide-resistant gonorrhea in men:a preliminary report. Am J Syph Gonor Vener Dis 1943;27:525-28. [ Links ]
6. Curtis FR,Wilkinson AE. A comparison of the in vitro sensitivity of gonococci to penicillin with the results of treatment. Br J Vener Dis 1958;34:70-8. [ Links ]
7. Reyn A, Korner B, Bentzon MW. Effects of penicillin, streptomycin and tetracycline on Neisseria gonorrhoeae isolated in 1944 and in 1957. Br J Vener Dis 1958;34:227-39. [ Links ]
8. Reyn A. Sensitivity of Neisseria gonorrhoeae to antibiotics. Br J Vener Dis 1961;37:145-57. [ Links ]
9. Thayer JD, Field FW, Magnuson HJ. The sensitivity of gonococci to penicillin and its relationship to penicillin failure. Antibiotic chemother 1957;7:306-10. [ Links ]
10. Willcox RR. Treatment problems of gonorrhea. Bull WHO 1961;24:307-319. [ Links ]
11. Stolz E, Zwart HGF, Michel MF. Activity of eight antimicrobial agents in vitro against Neisseria gonorrhoeae. Br J Vener Dis 1975;51:257-64. [ Links ]
12. Thornsberry C, Jaffe H, Brown ST. Spectinomycin resistant Neisseria gonorrhoeae. JAMA 1977;237:2405-6. [ Links ]
13. Faruki H, Kohmescher RN,Mckinney WP. A community based outbreak of infection with penicillin-resistant Neisseria gonorrhoeae not producing penicillinase. N Engl J Med 1985;313:607-11. [ Links ]
14. Ison CA, Gedney J, Easmon CSF. Chromossomal resistance of gonococci to antibiotic. Genitourin Med 1987;63:239-3. [ Links ]
15. Zenilman JM, Nims LJ, Menegus MA. Spectinimycin resistant gonococcal infections in the United States 1985-1986. J Infect Dis 1987;156:1002-4. [ Links ]
16. Goh CL, Meija P, Sing EH. Chemoprofilaxis and gonococcal infections in prostitutes. Int J Epidem 1984;13:344-6. [ Links ]
17. Perine PL, Morton RS, Piot P. Epidemiology and treatment of penicillinase-producing Neisseria gonorrhoeae. Sex Transm Dis 1979;6:152-8. [ Links ]
18. Sparling PF. Treatment of gonorrhea: what effect will antibiotic resistance have in future? Sex Transm Dis 1979;6:120-5. [ Links ]
19. Organizacion Panamericana de la Salud (OPS). Neisseria gonorrhoeae. Resisência a multiples antibioticos. Bol Epidem 1985;6:7-10. [ Links ]
20. Bestane WJ. A gonorreia e outras uretrites na cidade de Santos. Rev Ass Méd Brás 1978;24:133-8. [ Links ]
21. Soibelman M, Amaral LR, Palmini AF. Indicações de medicamentos por balconistas de farmacias em Porto Alegre. Rev Ass Méd Brás 1986;32:79-83. [ Links ]
22. Bestane WJ, Meira AR, Meloni W. Tratamento da cistite em farmácias de São Paulo. Rev Ass Méd Brás 1980;26:185-6. [ Links ]
23. Bestane WJ, Meira AR, Krasucki MR. Alguns aspectos da prescrição de medicação para o tratamento da gonorréia em farmácias de Santos. Rev Ass Méd Brás 1980;26:2-3. [ Links ]
24. Wiesner PJ, Handsfield HH, Holmes KK. Low antibiotic resistance of gonococci causing disseminated gonococcal infection. N Engl J Med 1973;288:1221-4. [ Links ]
25. Knapp JS. Laboratory methods for the detection and phenotypic characterization of Neisseria gonorrhoeae strains resistant to antimicrobial agents. Sex Transm Dis 1988;15:225-31. [ Links ]
26. Rellog DS, Holmes KK, Hell GA. Laboratory dianosis of gonorrhea.Washington, DC. American Society for Microbiology,1976 (CUMITECH 4). [ Links ]
27. Carlson BL, Calnan MB, Goodman RE. Phadebact monoclonal GC-OMMI test for confirmation of Neisseria gonorrhoeae. J Clin Microbiol 1987;25:1982-6. [ Links ]
28. Dillon JR, Carballo M, Pauze M. Evaluation of eight methods for identification of pathogenic Neisseria species:Neisseria-KWIK;RIM-W;gonobiotest;minitek; gonochek II;gonogen;phadebact monoclonal;GC-OMMI test and Syva microtrak test. J Clin Microbiol 1988;26:493-9. [ Links ]
29. Laughon BE, Ehret JM,Tanino TT. Fluorescent antibody for confirmation of Neisseria gonorrhoeae. J Clin Microbiol 1987;25:2388-91. [ Links ]
30. Shtibel R, Toma S. Neisseria gonorrhoeae: evaluation of same methods used for carbohydrate utilization. Can J Microbiol 1978;24:177-81. [ Links ]
31. Siqueira LFG. O laboratorio nas doenças sexualmente transmissíveis III. Bol Inform Union 1984;34:3-4. [ Links ]
32. Montgomery K, Raymond L, Drew WL. Chromogenic cephalosporin spot test to detect beta-lactamase in clinically significant bacteria. J Clin Microbiol 1979;9:205-7. [ Links ]
33. O´Callaghan CH, Morris A, Kirby SM. Novel method for detection of beta-lactamase by using a chromogenic cephalosporin substrate. Antimicrob Agents chemother 1972;1:283-8. [ Links ]
34. Sng EH, Yeo KL, Rajan VS. Comparison of methods for the detection of penicillinase-producing Neisseria gonorrhoeae. Br J Vener Dis 1980;56:311-3. [ Links ]
35. Sng EH, Yeo KL, Rajan VS. Simple method for detecting penicillinase-producing Neisseria gonorrhoeae and staphylococcus aureus. Br J Vener Dis 1981;57:141-2. [ Links ]
36. Waitkins SA, Anderson RD. Failure of the fluorescent antibody reaction to identify penicillinase producing gonococci. J Clin Pathol 1982;35:215-8. [ Links ]
37. National Commitee for Clinical Laboratory Standards (NCCLS). 1985. Methods for diluition antimicrobial susceptibility for bacteria that grow aerobically. Approved satndard. M7-A5. NCCLS, Villa Nova, P.A. [ Links ]
38. National Commitee for clinical Laboratory Standards (NCCLS). 1987. Performance standards antimicrobial susceptibility testing: second informational supplement.M-100,52. NCCLS. Villa Nova. P.A. [ Links ]
39. National Commitee for Clinical Laboratory Standards (NCCLS). 1988. Methods for diluition antimicrobial susceptibility tests for bacteria that grow aerobically. Tentative standard. M7-T2, 8. NCCLS. Villa Nova. P.A. [ Links ]
40. Wasington II JA. Susceptibility tests agar diluition. IN: Lennette EH, Ballows A, Hausler WJ.- Manual of clinical microbiology.4a.ed.Washington,DC,American Society for Microbiology, 1985, p.967-71. [ Links ]
41. Anhalt JP,Washington II JA. Preparation and storage of antimicrobial solutions. IN: Lennette EH,Ballows A,Hausler WJ.-Manual of clinical microbiology, 4a.ed., Washington, DC, American Society for Microbiology, 1985, p.1019-1020. [ Links ]
42. Finegold SM, Baron EJ. Metodos para evaluar la efectividad antimicrobiana.IN: Bailey & Scott - Diagnóstico microbiológico. 7ª. ed. Buenos Aires. Ed. Medica Panamericana, 1989,p.190-210. [ Links ]
43. Stiers E, Foltz EL, Graves B. A inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Atimicrob Agents chemother 1959;9:307-11. [ Links ]
Walter Belda Júnior
Av. Açocê, 162
Moema SP 04075-020
Phone/Fax: (11) 5051-1921 / 5051-5141
Received in November,
29th of 2001
Approved by the Consultive Council and accepted for publication in July, 31st of 2002
* Work conducted at the Service of Sexually Transmitted Diseases of the Dermatology Division, "Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo" and at the Service of Sexually Transmitted Diseases, "Centro de Saúde Escola Geraldo de Paula Souza da Faculdade de Saúde Pública da Universidade de São Paulo".