Antimicrobial susceptibility of Neisseria gonorrhoeae in Greece: data for the years

Journal of Antimicrobial Chemotherapy (2006) 57, 775 779 doi:10.1093/jac/dkl040 Advance Access publication 21 February 2006 Antimicrobial susceptibility of Neisseria gonorrhoeae in Greece: data for the years 1994 2004 Maria Stathi 1, Alexandros Flemetakis 2, Vivi Miriagou 1, Helen Avgerinou 2, Kyriakos P. Kyriakis 1, Antonios N. Maniatis 3 and Eva Tzelepi 1 * 1 The National Reference Center for Neisseria gonorrhoeae, Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece; 2 Microbiology Laboratory of the Andreas Sygros Hospital for Skin and Venereal Diseases, Athens, Greece; 3 Department of Medical Microbiology, University of Thessaly, Larissa, Greece Introduction Received 29 November 2005; returned 4 January 2006; revised and accepted 27 January 2006 Objectives: Surveillance data concerning antimicrobial susceptibilities of Neisseria gonorrhoeae isolated in Greece during the 11 year period 1994 2004 are presented. Methods: Antimicrobial susceptibilities of all gonococcal isolates received by the Greek National Reference Center for N. gonorrhoeae during the study period were determined in terms of MICs using Etest. Trends in yearly isolation frequencies by susceptibility category were estimated for defining significant changes in overall susceptibility figures. Results: Cefotaxime and spectinomycin retained undiminished activity against all isolates throughout the study period. High rates of resistance and intermediate susceptibilities were noticed for penicillin, tetracycline and erythromycin, and even for norfloxacin and ciprofloxacin. A substantial portion (16.5%) of the gonococcal samples consisted of multiresistant strains exhibiting resistance to two or more agents of different antibiotic classes. Although annual rates of low-level chromosomal resistance decreased, high-level resistance owing to the presence of penicillin- and tetracycline-resistance plasmids increased. Fluoroquinolone resistance also showed a significant increasing trend after 1996, reaching a peak rate of 11.3% in 2004. Conclusion: Third-generation cephalosporins and spectinomycin should be considered as first-choice drugs for the empirical treatment of gonorrhoea in Greece. Keywords: N. gonorrhoeae, gonorrhoea, antibiotic resistance, fluoroquinolones Materials and methods The importance of monitoring antimicrobial resistance in Neisseria gonorrhoeae for disease management and control is well established. 1 3 The recent emergence of gonococci with reduced susceptibility to the currently recommended antibiotics for gonorrhoea therapy, including fluoroquinolones and some cephalosporins, reinforces the need for continuous surveillance of the susceptibility trends in N. gonorrhoeae. The Greek National Reference Center for N. gonorrhoeae (NRCNG) has been conducting continuous surveillance on microbiological characteristics of gonococcal infection since 1984, and in previous publications has reported antibiotic susceptibility data for N. gonorrhoeae strains isolated in Greece up to 1993. 4 In this article, we present data for the years 1994 2004. A total of 1027 gonococcal strains submitted to the Greek NRCNG between January 1994 and December 2004 were included in the study. The strains were non-repetitive (one per patient) and represented 71.8% of all 1431 gonorrhoea cases reported to the NRCNG during this 11 year period; the remainder of the reported cases escaped culture, relying for diagnosis on symptoms and Gram-staining. Of the strains, 908 (88.4%) were consecutively isolated from the cases seen in the outpatient clinic of the Andreas Sygros Hospital, Athens, which is the largest clinic in Greece for venereal diseases, receiving the main bulk of gonorrhoea cases throughout the country. The remaining 119 strains (11.6%) were sporadically isolated, most of them from occasional culturing of the cases seen in the Venereal Hospital of Thessaloniki, North... *Corresponding author. Tel: +30-210-6478-810; Fax: +30-210-6423-498; E-mail: tzelepi@pasteur.gr... 775 Ó The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Stathi et al. Table 1. Annual antimicrobial susceptibility rates in Neisseria gonorrhoeae strains isolated in Greece during the period 1994 2004 Antimicrobial agent Susceptibility parameters 1994 (n = 68) 1995 (n = 67) 1996 (n = 55) 1997 (n = 65) 1998 (n = 113) 1999 (n = 98) 2000 (n = 76) 2001 (n = 140) 2002 (n = 99) 2003 (n = 104) 2004 (n = 142) Penicillin G % PPNG 7.4 7.5 5.5 9.2 13.3 16.3 13.2 11.4 13.1 12.5 9.9 % CMPR 5.9 11.9 7.3 12.3 8.0 2.0 6.6 3.6 1.0 1.9 6.3 % intermediate 58.8 52.2 60 60 54 61.2 60.5 49.3 45.5 55.8 73.2 % susceptible 27.9 28.3 27.3 18.5 24.7 20.4 19.7 35.7 40.4 29.8 10.6 MIC range (mg/l) MIC50 (mg/l) 0.25 0.19 0.25 0.5 0.19 0.19 0.25 0.125 0.125 0.094 0.19 MIC90 (mg/l) 3 3 1.5 3 16 32 8 6 12 24 2 Cefotaxime % susceptible 100 100 100 100 100 100 100 100 100 100 100 MIC range (mg/l) to 0.5 to 0.5 MIC50 (mg/l) 0.012 0.008 0.016 0.016 0.012 0.008 0.016 0.012 0.008 0.012 0.016 MIC90 (mg/l) 0.094 0.094 0.094 0.094 0.094 0.064 0.064 0.047 to 0.094 Spectinomycin %intermediate 1.5 % susceptible 100 100 100 98.5 100 100 100 100 100 100 100 MIC range (mg/l) 1 32 1 32 1 32 8 64 1 32 0.75 24 2 32 1 24 2 24 1 32 1 16 MIC50 (mg/l) 8 12 8 16 12 12 12 12 8 12 8 MIC90 (mg/l) 16 16 16 32 16 16 16 16 16 16 16 Tetracycline % TRNG 2.9 4.5 1.8 6.1 6.2 3.1 5.3 4.3 3.0 10.6 6.3 % CMTR (>1) 25.0 29.8 34.6 38.5 12.4 6.1 18.4 5.7 3.0 0 6.3 % intermediate 64.7 64.2 61.8 55.4 77.0 81.6 71.0 83.6 75.8 71.1 83.1 % susceptible 7.4 1.5 1.8 0 4.4 9.2 5.3 6.4 18.2 18.3 4.2 MIC range (mg/l) 32 0.064 48 0.064 64 0.19 128 0.047 48 to 32 MIC50 (mg/l) 0.5 0.75 1 1 0.5 0.38 0.5 0.38 0.25 0.25 0.38 MIC90 (mg/l) 3 3 2 3 2 1 2 1.5 0.75 6 1.5 0.047 to 64 to 64 Erythromycin % ERNG 0.7 % CMER 25.0 19.4 30.9 30.8 16.8 9.2 26.3 13.6 8.1 10.6 23.2 % intermediate 41.2 50.7 49.1 55.4 48.7 45.9 47.4 60.7 60.6 60.6 55.6 % susceptible 33.8 29.9 20 13.8 34.5 44.9 26.3 25.0 31.3 28.8 21.1 MIC range (mg/l) to 6 to 3 to 6 0.125 to 4 0.023 to 4 to 96 to 3 0.023 2 0.047 6 MIC 50 (mg/l) 0.5 0.5 0.75 0.75 0.5 0.38 0.75 0.75 0.5 0.75 0.75 MIC90 (mg/l) 2 2 2 2 2 1 1.5 2 1 1.5 1.5 Norfloxacin % resistant 1.5 2.7 2.0 5.3 6.4 9.1 9.6 11.3 %intermediate 3.6 9.2 15.0 5.1 1.4 1.0 % susceptible 100 100 96.4 89.3 82.3 92.9 94.7 92.2 89.9 90.4 88.7 MIC range (mg/l) to 0.094 to 96 to 1 to 1.5 to 12 to 48 776

Antimicrobial susceptibility of N. gonorrhoeae in Greece MIC 50 (mg/l) 0.023 0.023 0.023 0.023 0.023 0.023 MIC90 (mg/l) 0.047 0.064 0.064 0.38 0.5 0.064 0.094 0.094 0.5 0.094 6 Ciprofloxacin % resistant 1.5 1.0 5.3 3.6 6.1 9.6 11.3 % intermediate 3.6 3.1 12.4 5.1 3.6 3 % susceptible 100 100 96.4 95.4 87.6 93.9 94.7 92.8 89.9 90.4 88.7 to 0.75 to 0.25 to 0.023 MIC range (mg/l) to 0.023 MIC50 (mg/l) 0.004 0.004 0.004 0.006 0.006 0.006 0.004 0.004 0.006 0.004 0.004 MIC 90 (mg/l) 0.012 0.016 0.016 0.064 0.094 0.023 0.016 0.094 0.016 Nalidixic acid % QRNG 5.4 10.8 18.6 8.1 5.3 7.9 10.1 9.6 11.3 % non-qrng 100 100 94.6 89.2 81.4 91.9 94.7 92.1 89.9 90.4 88.7 PPNG, penicillinase-producing gonococci; TRNG, tetracycline-resistant gonococci harbouring Tet-M plasmids; ERNG, one highly erythromycin-resistant N. gonorrhoeae strain; QRNG, quinolone-resistant isolates grown on GC agar containing 64 mg/l nalidixic acid (MIC > 64 mg/l); CMPR, CMTR and CMER, isolates chromosomally resistant to penicillin, tetracycline and erythromycin, respectively; MIC50 and MIC90, the lowest MIC values inhibiting 50 and 90% of the isolates, respectively. Greece, and some from sporadic cases encountered in other non- STD-specialized hospitals located in Athens, Piraeus and Ioannina. This strain collection represented the total of the viable isolates obtained in all reporting hospitals without any kind of selection. Of the isolates, 1006 were from males and 21 from females. The limited number of strains from females probably reflects, at least in part, a preference among women to attend private medical practitioners, scarcely reporting gonorrhoea cases to the NRCNG. 5 N. gonorrhoeae identification was performed with conventional procedures, including Gram-staining, oxidase reaction and carbohydrate utilization (Neisseria 4H, Amersham-BioRad-Diagnostics Pasteur). The MICs of penicillin, cefotaxime, spectinomycin, tetracycline, erythromycin, ciprofloxacin and norfloxacin were determined using Etest according to the instructions of the manufacturer (AB Biodisk, Sweden). Tests were performed on GC agar supplemented with Vitox (Oxoid). The plates were inoculated with a bacterial suspension of turbidity equal to 0.5 on the McFarland scale and incubated at 35 C in the appropriate conditions of CO 2 (5%) and humidity (70%). The N. gonorrhoeae strains WHO A to WHO D were used as quality control standards. For susceptibility categorization, the breakpoints proposed by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) were used, when available. 6 Generally accepted breakpoints were adopted for erythromycin (MIC 2 mg/l for resistance and 0.25 mg/l for susceptibility) and norfloxacin (MIC 1 mg/l for resistance and 0.25 mg/l for susceptibility). 4 For categorization of susceptibility results according to these breakpoints, MIC values lying between marginal 2-fold concentrations were rounded to the nearest higher concentration. Penicillinaseproducing (PPNG) strains were detected using a nitrocefin assay. The presence of Tet-M plasmids in highly tetracycline-resistant gonococci (TRNG) was confirmed by amplification of the tetm gene using PCR as described previously. 7 The characterization of strains as quinolone resistant (QRNG) was based upon growth on GC agar containing 64 mg/l nalidixic acid, indicative of gyra or gyra plus parc mutations. 8 Statistical analysis based on least squares (linear model) was carried out to define trends in yearly isolation frequencies by susceptibility category. Results and discussion During the 11 year period of the study, the NRCNG received a total of 1027 isolates, with annual numbers ranging between 55 in 1996 and 142 isolates in 2004. Increments in the total number of isolates collected each year did not reflect any significant change in the relative incidence of gonorrhoea cases, which maintained its low rate over the study period. 5 Results from antimicrobial susceptibility testing of gonococcal strains isolated annually are presented in Table 1. Cefotaxime and spectinomycin retained undiminished activity against all the isolates throughout the study period, with only one strain, isolated in 1997, presenting intermediate susceptibility to spectinomycin. Ciprofloxacin, followed by norfloxacin, was active against the majority of the isolates, but a significant trend (P < 0.001) to increasing incidence of fluoroquinolone-resistant gonococci was noticed. Penicillin, tetracycline and erythromycin exhibited poor in vitro activities, with MIC 50 values in the intermediate range and MIC 90 values steadily in the resistance range. High rates of both plasmid-mediated and low-level chromosomally-mediated resistance contributed to this figure, but the most pronounced 777

Stathi et al. effect resulted from the vast numbers of intermediately susceptible gonococci. Plasmid-mediated resistance was represented by 144 (14%) of the 1027 isolates, of which 90 (8.8%) were PPNG, 29 (2.8%) TRNG, and 25 (2.4%) were PP/TRNG carrying both penicillinand tetracycline-resistance plasmids. Moreover, 20 of the isolates that possessed resistance plasmids were simultaneously resistant to one or both of the fluoroquinolone agents tested (data not shown). In terms of annual rates, plasmid-mediated resistance ranged from 5.5.3% for penicillin and from 1.8 to 10.6% for tetracycline, with the highest isolation frequencies of PPNG and TRNG strains in the years 1999 and 2003, respectively (Table 1). Despite fluctuations, the rates of plasmid-mediated resistance increased during the study period (P < 0.05 for PPNG and marginally significant for TRNG) (Figure 1). An additional portion of the gonococcal population studied, consisting of 180 (17.5%) of the total isolates, were chromosomally resistant to penicillin, tetracycline or erythromycin, with half of them (90 isolates) exhibiting cross-resistance to more than one of these agents (data not shown). Combined resistance to penicillin and tetracycline, exhibited by the so-called CMRNG isolates, accounted for 3.6% of the total gonococcal sample. In contrast to plasmid-mediated resistance, the frequency of isolates chromosomally resistant to either agent decreased (P < 0.01), probably reflecting a reduction in the presence of multidrug-resistant outbreak strains documented in Greece between the years 1991 and 1998 (Figure 1). 9 The rates of chromosomal resistance were greater for erythromycin than for tetracycline and much less pronounced for penicillin. The level of resistance to penicillin exhibited by PPNG strains ranged from 2 mg/l and that of plasmid-mediated resistance to tetracycline exhibited by TRNG strains from 6 to 128 mg/l. Notably, the lower levels of plasmid-mediated resistance to these agents overlapped with those conferred by low-level chromosomal resistance (MICs up mg/l for penicillin and up to 4 mg/l for tetracycline), underlining the necessity for confirmatory tests, such as nitrocefin assay and Tet-M PCR, for the reliable classification of gonococcal isolates in the PPNG and TRNG categories. This particularly refers to the characterization of plasmid-mediated resistance to tetracycline, which is generally considered as conferring MIC values 16 mg/l. 2 Although lower MICs of tetracycline are unusual for TRNG strains, 15 of the TRNG isolates in this study were inhibited by concentrations of 6 8 mg/l. TRNG strains inhibited by MIC values <16 mg/l have also been recently reported from Canada. 10 The most important observation in the present study is the establishment of QRNG strains in the Greek community. QRNG strains were only sporadically isolated in Greece before 1994 4 and were absent in the first 2 years of the study period. In the years 1997 and 1998, the annual MIC 90 values of fluoroquinolones were slightly increased, mainly due to the presence of isolates intermediately susceptible to these agents. Apart from this event, which has been attributed to clonal spread of an outbreak strain, 11 QRNG strains with high levels of fluoroquinolone resistance were isolated in progressively increasing frequencies after 1996, reaching a rate of 11.3% in the year 2004 (P < 0.001) (Table 1 and Figure 1). Similar findings have been reported recently from most other countries in Europe and worldwide. 12 Irrespective of the resistance type and pattern, 35.5% (364 strains) of all gonococci isolated during the study period were resistant and an additional 61% intermediately susceptible % of isolates 40 35 30 25 20 15 10 5 0 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Year to at least one of the antibiotics tested. Moreover, approximately half of the resistant strains (16.5% of all isolates) showed multiple-resistance patterns, including two or more different antibiotic classes (data not shown). In conclusion, antimicrobial susceptibility of N. gonorrhoeae isolated in Greece during the past decade was characterized by high rates of resistance and intermediate susceptibility to penicillin, tetracycline, erythromycin and fluoroquinolones. To our knowledge, none of these agents is currently used as first-line therapy for gonorrhoea, at least in the clinical settings reporting to the NRCNG. However, resistance profiles documented here cannot be easily interpreted in relation to antimicrobial policies, since national guidelines do not exist. Different prescribing policies may be followed depending on each hospital s directives and probably based on international guidelines combined with local clinical experience. Moreover, the unregulated use of various antibiotics, including penicillins, macrolides and fluorinated quinolones, that are available over the counter in Greece may have contributed to the establishment of resistance. 4 Results of this study illustrate that third-generation cephalosporins and spectinomycin must be considered as first-choice drugs for the empirical treatment of gonorrhoea in Greece. Acknowledgements PMPR PMTR FQR CMR Linear (PMPR) Linear (PMTR) Linear (FQR) Linear (CMR) Figure 1. Trends of various types of gonococcal resistance during the period 1994 2004. PMPR, plasmid-mediated resistance to penicillin (PPNG); PMTR, plasmid-mediated resistance to tetracycline (TRNG); FQR, fluoroquinolone resistance (to ciprofloxacin or norfloxacin or both); CMR, chromosomal resistance to at least one of the antibiotics penicillin, tetracycline and erythromycin exhibited by non-ppng/non-trng/non-fqrng isolates. We thank our colleagues for their contributions to gonorrhoea surveillance in Greece: Efi Fragouli, MD (Microbiology Laboratory of the Andreas Sygros Hopsital, Athens); Agnes Kalogeropoulou, MD, and Maria Daniilidou, MD (Microbiology Laboratory of the Venereal Hospital of Thessaloniki); Stamatina Levidiotou, MD (Department of Microbiology, Medical School, University of Ioannina); Dimitra Gianneli, MD (Microbiology Laboratory of the Tzanion General Hospital, Piraeus); and Athena 778

Antimicrobial susceptibility of N. gonorrhoeae in Greece Argyropoulou, MD (Microbiology Laboratory of the Evangelismos General Hospital, Athens). Transparency declarations None to declare. References 1. Tapsal J. Current concepts in the management of gonorrhoea. Expert Opin Pharmacother 2002; 3: 147 57. 2. Ison CA, Martin IMC and the London Gonococcal Working Group. Susceptibility of gonococci isolated in London to therapeutic antibiotics: establishment of a London surveillance system. Sex Transm Infect 1999; 75: 107 11. 3. Lowndes CM, Fenton KA and the ESSTI (European Surveillance of STIs) Network. Surveillance systems for STIs in the European Union: facing a changing epidemiology. Sex Transm Infect 2004; 80: 264 71. 4. Tzelepi E, Avgerinou H, Kyriakis KP et al. Antimicrobial susceptibility and types of Neisseria gonorrhoeae in Greece data for the period 1990 to 1993. Sex Transm Dis 1997; 24: 378 85. 5. Kyriakis KP, Tzelepi E, Flemetakis A et al. Epidemiologic aspects of male gonococcal infection in Greece. Sex Transm Dis 1999; 29: 43 8. 6. NationalCommitteefor ClinicalLaboratoryStandards. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA, 2002. 7. Gascoyne-Binzi DM, Hawkey PM, Heritage J. The distribution of the Tet M determinant in tetracycline-resistant Neisseria gonorrhoeae. J Antimicrob Chemother 1994; 33: 1011 6. 8. Deguchi T, Yasuda M, Nakano M et al. Quinolone-resistant Neisseria gonorrhoeae: correlation of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV with antimicrobial susceptibility profiles. Antimicrob Agents Chemother 1996; 40: 1020 3. 9. Mavroidi A, Tzouvelekis LS, Kyriakis KP et al. Multidrug-resistant strains of Neisseria gonorrhoeae in Greece. Antimicrob Agents Chemother 2001; 45: 2651 4. 10. Greco V, Ng LK, Catana R et al. Molecular epidemiology of Neisseria gonorrhoeae isolates with plasmid-mediated tetracycline resistance in Canada: temporal and geographical trends (1986 1997). Microb Drug Resist 2003; 9: 353 60. 11. Mavroidi A, Tzouvelekis LS, Tassios PT et al. Characterization of Neisseria gonorrhoeae strains with decreased susceptibility to fluoroquinolones isolated in Greece during 1996 1999. J Clin Microbiol 2000; 38: 3489 91. 12. Dan M. The use of fluoroquinolones in gonorrhoea: the increasing problem of resistance. Expert Opin Pharmacother 2004; 5: 829 54. 779