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In many regions the susceptibility of Neisseria gonorrhoeae isolates to antimicrobial agents is rarely tested. The Gonococcal Antimicrobial Surveillance Program (GASP) in Cuba was established as part of a larger regional GASP program to facilitate the collection and reporting of antimicrobial susceptibility data for N gonorrhoeae isolates.The goal was to retrospectively determine the antimicrobial susceptibility and molecular epidemiology of 91 isolates of N gonorrhoeae isolated from 11 centers in Cuba.Isolates of N gonorrhoeae were collected and presumptively identified from 11 Cuban provincial health centers. They were then forwarded to the National Laboratory of Pathogenic Neisseria Havana for confirmatory identification and were subsequently analyzed at the Center for GASP in Ottawa. Isolates were tested for susceptibility to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, and azithromycin by the agar dilution method. To establish baseline data for molecular epidemiologic profiles, the auxotype (A), serovar (S), plasmid content (P), and TetM type of the isolates were determined. Certain A/S/P classes were further analyzed by pulsed field gel electrophoresis (PFGE).High percentages of the 91 N gonorrhoeae isolates were resistant to penicillin (68%) and tetracycline (83.5%), with 56% being penicillinase-producing (PPNG) and 64% carrying plasmid-mediated tetracycline resistance (TRNG; 50% were PP/TRNG). An additional 14% of the isolates carried chromosomal resistance (CMRNG) to either tetracycline or penicillin or both antibiotics. All isolates were susceptible to spectinomycin, ceftriaxone, and ciprofloxacin. However, nine isolates were resistant to azithromycin (MIC, ≥1.0 μg/ml), and 43 other isolates displayed reduced susceptibility to this antibiotic (MIC, 0.25–0.5 μg/ml). Although a total of 21 different A/S classes were identified, most of the isolates (61) belonged to three A/S classes: NR/IA-6 (35 isolates), NR/IB-1 (15 isolates), and P/IA-6 (11 isolates). Thirty-two of 45 PP/TRNG were A/S class NR/IA-6, and nine of the P/IA-6 isolates were TRNG. By contrast, most of A/S class NR/IB-1 (8) were CMRNG. PFGE analysis following digestion with Nhe I or Spe I further clustered the isolates into separate groups.This study demonstrates high percentages of N gonorrhoeae isolates with penicillin and tetracycline resistance in Cuba. As has been noted in other studies in the Caribbean region and Latin America, resistance and reduced susceptibility to azithromycin are developing as emerging problems. Since penicillin and tetracycline continue to be widely used for the treatment of gonococcal infections in Cuba, this study indicates the importance of antimicrobial susceptibility surveillance so that effective antibiotics may be recommended for treatment of gonococcal infections.