Research Antimicrobial susceptibility of Neisseria gonorrhoeae in Barcelona during a five-year period, 2013 to 2017 Paula Salmerón¹ , Belén Viñado¹ , Rachid El Ouazzani¹ , Marta Hernández¹ , María Jesús Barbera2,3 , Mireia Alberny⁴ , Mireia Jané⁵ , Nieves Larrosa1,6,7 , Tomás Pumarola1,6,7 , Yannick Hoyos-Mallecot1,6 , Judit Serra-Pladevall1,6,7 1. Microbiology Department, Vall d’Hebron Hospital Universitari, Barcelona, Spain 2. Drassanes-Vall d’Hebron Sexually Transmitted Infections Unit, Department of Infectious Diseases, Vall d’Hebron Hospital Universitari, Barcelona, Spain 3. Universitat de Barcelona, Barcelona, Spain 4. Medical Management of Primary Care Services, Institut Català de la Salut (ICS), Barcelona, Spain 5. Agència de Salut Pública de Catalunya, Generalitat de Catalunya, Barcelona, Spain 6. Microbiology group, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Barcelona, Spain 7. Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain Correspondence: Yannick Hoyos (yhoyos@vhebron.net) Citation style for this article: Salmerón Paula , Viñado Belén , El Ouazzani Rachid , Hernández Marta , Barbera María Jesús , Alberny Mireia , Jané Mireia , Larrosa Nieves , Pumarola Tomás , Hoyos-Mallecot Yannick , Serra-Pladevall Judit . Antimicrobial susceptibility of Neisseria gonorrhoeae in Barcelona during a five-year period, 2013 to 2017. Euro Surveill. 2020;25(42):pii=1900576. https://doi.org/10.2807/1560-7917.ES.2020.25.42.1900576 Article submitted on 16 Sep 2019 / accepted on 05 Mar 2020 / published on 22 Oct 2020 Introduction: Increasing rates of antimicrobial resist- Introduction ance in Neisseria gonorrhoeae cause problems for Gonorrhoea is the second most reported bacterial treating gonorrhoea. Aim: This observational study sexually transmitted infection (STI), after chlamydia. aimed to describe isolates from all patients found Untreated gonorrhoea can lead to pelvic inflamma- infected with N. gonorrhoeae, in Barcelona, Spain, tory disease and infertility in women and epididymitis between 2013 and 2017, and with available anti- and orchitis in men. In the European Union/European microbial susceptibility data. Methods: Minimum Economic Area countries, a total of 89,239 confirmed inhibitory concentrations (MICs) of penicillin (PEN), cases of gonorrhoea were reported in 2017 with an cefixime (CFM), ceftriaxone (CRO), azithromycin overall rate of 22.2 cases per 100,000 population [1]. (AZM), ciprofloxacin (CIP), spectinomycin (SPT), This represented a 17% increase over the previous fosfomycin (FOF) and gentamicin (GEN) were deter- year, which was particularly striking in certain groups, mined by E-test. Susceptibility was assessed using such as men who have sex with men (MSM), who rep- clinical breakpoints from the European Committee resented almost half of the cases in 2017, and the on Antimicrobial Susceptibility Testing. Time trends 25–34-year-old population [1]. for PEN, CFM, AZM and CIP were investigated using logistic regression. Results: Of 1,979 patients with In Spain, 8,722 cases of gonococcal infection were infection (2,036 isolates), 1,888 (95.4%) were men. reported in 2017 (rate: 18.74 per 100,000 inhabitants), Patient median age was 32 years. The proportions of with a very wide range of infection incidence among the isolates resistant to extended-spectrum cephalospor- different regions of the country, from 2.44 and 48.50 ins were low, with 0.3% (5/1,982) resistant to CRO and cases per 100,000 inhabitants. The highest rates were 4.9% (98/1,985) to CFM. AZM resistance prevalence registered in Catalonia (48.50), Balearic Islands (41.79) was 2.7% (52/1,981), including 16 isolates detected and Madrid (28.48) [2]. Of the 3,622 cases reported in in 2016 and 2017, with high-level resistance. For CIP, Catalonia in 2017, men accounted for 82% of the diag- 51.3% (1,018/1,986) of isolates were resistant, and noses. Data from 1,136 patients (31%) could be col- for PEN, 20.1% (399/1,985). All isolates were suscep- lected: 44% were MSM followed by heterosexual men tible to SPT. MIC50 and MIC90 values of GEN were 4 (22%) and women (20%) [3]. and 6 mg/L and of FOF 12 and 24 mg/L, respectively. Between 2013 and 2017, PEN and CFM resistance rates Neisseria gonorrhoeae, the bacterial species respon- each decreased from 28.1% (92/327) to 12.2% (70/572) sible for gonorrhoea, has developed resistance to the and from 8.3% (27/327) to 4.4% (25/572) (p ≤ 0.0073). different families of antibiotics used in the past, chal- In contrast, AZM resistance prevalence appeared to lenging future treatment. Currently, extended-spec- increase from 1.5% in 2014 (5/340) to 3.0% (17/572) trum cephalosporins (ESC) are the last-line treatment in 2017. No trend was identified for CIP. Conclusion: option for N. gonorrhoeae infection. Unfortunately, Antimicrobial susceptibility surveillance is important strains showing resistance to ESC have been reported to timely detect new phenotypes and trends. worldwide [4-6]. In 2017, the European Centre for www.eurosurveillance.org 1 Table 1 Characteristics of patients included in the study on antimicrobial susceptibility of Neisseria gonorrhoeae isolates, Barcelona, Spain, 2013−2017 (n = 1,979 patients) 2013 2014a 2015b 2016 2017 TOTALc Characteristics Number % Number % Number % Number % Number % Number % Numbers of patients and isolates d Isolates 329 100 340 100 339 100 447 100 581 100 2,036 100 Patients 321 100 326 100 328 100 423 100 581 100 1,979 100 Sex Men 303 94.4 306 93.9 320 97.6 400 94.6 559 96.2 1,888 95.4 Women 18 5.6 18 5.5 8 2.4 23 5.4 22 3.8 89 4.5 Unknown 0 0.0 2 0.6 0 0.0 0 0.0 0 0.0 2 0.1 Age in years < 20 NA NA 14 6.1 9 2.8 12 2.8 9 1.5 44 2.8 20–29 NA NA 73 31.7 118 36.8 153 36.2 236 40.6 580 37.3 30–39 NA NA 91 39.6 121 37.7 167 39.5 214 36.8 593 38.1 40–49 NA NA 39 17.0 52 16.2 68 16.1 84 14.5 243 15.6 ≥ 50 NA NA 13 5.7 21 6.5 23 5.4 38 6.5 95 6.1 32 32 32 32 Median (range) NA NA NA NA NA 31 (5–75) NA NA (14–74) (14–72) (4–75) (4–75) Clinical setting Drassanes STI unit 214 66.7 232 71.2 209 63.7 275 65.0 362 62.3 1,292 65.3 Primary healthcare units 90 28.0 76 23.3 100 30.5 118 27.9 189 32.5 573 29 Other HUVH departments 17 5.3 18 5.5 19 5.8 30 7.1 30 5.2 114 5.8 Specimen Urethral/balanoprepucial 261 79.3 245 72.1 274 80.8 365 81.7 453 78.0 1,598 78.5 Rectal 38 11.6 53 15.6 48 14.2 56 12.5 88 15.1 283 13.9 Endocervical/vaginal 16 4.9 11 3.2 10 2.9 21 4.7 11 1.9 69 3.4 Pharynx 10 3.0 11 3.2 7 2.1 5 1.1 29 5.0 62 3.0 Other e 4 1.2 20 5.9 0 0.0 1 0.2 0 0.0 24 1.2 HUVH: Vall d’Hebron University Hospital; NA: not available; STI: sexually transmitted infection. a Age data available from 230 patients (from April 2014 to December 2014). b Age data available from 321 patients. c Age data available from 1,555 patients. d Number of isolates can be greater than number of patients because over the study period several patients had more than one episode of gonorrhoea. e Other includes: synovial fluid, abdominal abscess, perianal exudates. The denominators for the percentages concerning patients’ characteristics are the total numbers of patients with information available on the characteristic in question, for the year or period specified in the column header. Denominators for specimen percentages are total number of isolates. Disease Control and Prevention (ECDC) reported a Nowadays, most of the treatment guidelines recom- resistance rate to cefixime of 1.9% within the European mend dual antimicrobial therapy (500 mg intramuscu- Union/European Economic Area countries and no lar ceftriaxone + 1 g or 2 g oral azithromycin) [9-12]. The isolates showing resistance to ceftriaxone [7]. The rationale for gonococcal combination therapy using resistance rate to azithromycin was 7.5%; and high- different antimicrobials with different mechanisms level resistance (minimum inhibitory concentration of action is to potentially mitigate the spread of anti- (MIC) ≥ 256 mg/L) was detected in seven isolates [7]. microbial resistance [13]. Combination therapy has been reported as possibly related to the decline in This pathogen has moreover been able to acquire or prevalence of N. gonorrhoeae isolates with decreased develop nearly all known mechanisms of antimicro- susceptibility to ceftriaxone (DSC), defined as a bial resistance: target modification, inactivation of the MIC > 0.032 mg/L [14,15]. antimicrobial by enzymatic means, decreased influx of antimicrobials, and increased efflux of antimicrobials The aim of this study is to describe antimicrobial sus- [8]. This situation highlights the importance of carry- ceptibility of N. gonorrhoeae in isolates collected ing out resistance monitoring programmes, in order to between 2013 and 2017 in Barcelona, Spain. update therapeutic guidelines and to timely detect the emergence of multidrug-resistant strains. 2 www.eurosurveillance.org Table 2 Antimicrobial susceptibility of Neisseria gonorrhoeae isolates collected in Barcelona, Spain, 2013−2017 (n = 2,036) MIC50 MIC90 MIC range Susceptibility categorya Antimicrobial Susceptible Intermediate Resistant in mg/L in mg/L in mg/L Number % Number % Number % PENb 0.25 12 0.002 – > 32 135 6.8 1,451 73.1 399 20.1 CROc 0.016 0.047 < 0.016 – 0.38 1,977 99.7 0 0.0 5 0.3 CFM b 0.016 0.094 < 0.016 – 0.38 1,887 95.1 0 0.0 98 4.9 AZMd 0.125 0.25 < 0.016 – > 256 1,929 97.4 0 0.0 52 2.6 CIPe 0.38 > 32 < 0.002 – > 32 965 48.6 3 0.2 1,018 51.3 SPT 8 12 1.5 – 64 2,036 100 0 0.0 0 0.0 GENf 4 6 0.25– 24 NA NA NA NA NA NA FOF g 12 24 0.064 – 128 NA NA NA NA NA NA AZM: azithromycin; CIP: ciprofloxacin; CFM: cefixime; CRO: ceftriaxone; ECOFF: epidemiological cut-off value; EUCAST: European Committee on Antimicrobial Susceptibility Testing; FOF: fosfomycin; GEN: gentamicin; MIC: minimum inhibitory concentration; NA: not applicable; PEN: penicillin; SPT: spectinomycin. a According to EUCAST (2019) clinical breakpoints. b Antimicrobial susceptibility testing for PEN and CFM was performed on 1,985 isolates. c Antimicrobial susceptibility testing for CRO was performed on 1,982 isolates. d Antimicrobial susceptibility testing for AZM was performed on 1,981 isolates. e Antimicrobial susceptibility testing for CIP was performed on 1,986 isolates. f GEN was determined in the period from 2013 to 2016. g FOF was determined in 2015 and 2016. Methods azithromycin, spectinomycin, gentamicin, and fosfo- mycin were determined by means of the E-test method Study population (bioMérieux, France), as described by the Clinical and The study population consisted of all patients who Laboratory Standard Institute (CLSI) [16]. Interpretation were diagnosed with a N. gonorrhoeae infection and was performed using clinical breakpoints from the with isolates for which antimicrobial susceptibility European Committee on Antimicrobial Susceptibility data were available. When patients tested positive for Testing (EUCAST) [17], except for gentamicin and fos- gonorrhoea on multiple sites and more than one culture fomycin, since cut-off points for these antibiotics are was obtained, only the genital culture was included in not established by EUCAST (nor by CLSI). N. gonor- the study. rhoeae ATCC 49226 was used as a reference strain for antimicrobial susceptibility testing. Patients were attended in three different clinical set- tings: Drassanes-Vall d’Hebron Sexually Transmitted Descriptive and statistical analysis Infections unit, different medical departments of Vall Descriptive analyses of the study population were d’Hebron University Hospital and primary healthcare performed. Statistical analyses were conducted using units in Barcelona, Spain. Together these settings cover Stata (StataCorp, College Station, Texas, US). Trends 1,200,000 of the 1,620,343 inhabitants of Barcelona of penicillin, cefixime, azithromycin and ciprofloxacin (74.1%). resistance over the study period were calculated using logistic regression analyses. Culture Urethral, rectal, vaginal, endocervical, and/or phar- Determinants for resistance for penicillin (MIC > 1 mg/L), yngeal samples were cultured on selective Thayer– cefixime (MIC > 0.125 mg/L), azithromycin (epidemio- Martin medium and incubated at 35–37 °C in a 5% logical cut-off (ECOFF) > 1 mg/L) and ciprofloxacin CO2 atmosphere for 24–48 hours. Probable N. gonor- (MIC > 0.06 mg/L) were identified using logistic regres- rhoeae strains were identified by oxidase reaction sion analyses. Univariable and multivariable analyses (oxidase-positive) and mass spectrometry (MALDI- were performed. Differences with p < 0.05 were consid- TOF, Vitek MS system, Biomérieux, Marcy-I´Étoile, ered statistically significant. As there were very few France). N. gonorrhoeae strains were subcultured in strains that reached the 0.125 mg/L threshold of ceftri- order to obtain fresh colonies for antimicrobial suscep- axone resistance, we did not determine associations tibility testing (AST) and frozen at −80 °C in trypticase between potential factors for ceftriaxone resistance soy broth with 20% glycerol. and resistance to this antibiotic. Antimicrobial susceptibility AST was performed on fresh colonies (<24 hours). The MICs of penicillin, ceftriaxone, cefixime, ciprofloxacin, www.eurosurveillance.org 3 Figure Proportion of Neisseria gonorrhoeae isolates with different minimum inhibitory concentrations (mg/L) for (A) ceftriaxone (n = 1,982 isolates), (B) cefixime (n = 1,985 isolates) and (C) azithromycin (n = 1,981 isolates), by year, Barcelona, Spain, 2013−2017 A. Ceftriaxone B. Cefixime 100 100 90 90 80 80 Proportion of isolates (%) Proportion of isolates (%) 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 2013 2014 2015 2016 2017 2013 2014 2015 2016 2017 Year Year C. Azithromycin 100 90 Minimum inhibitory concentration (mg/L) 80 Proportion of isolates (%) >1 0.125 70 0.5–1 0.094 60 0.38 0.047–0.064 50 0.25 0.023–0.032 40 0.19 ≤0.016 30 20 10 0 2013 2014 2015 2016 2017 Year EUCAST: European Committee on Antimicrobial Susceptibility Testing; MIC: minimum inhibitory concentration. According to EUCAST, the MIC breakpoints for resistance to ceftriaxone and cefixime are identical and are MIC > 0.125 mg/L. The epidemiological cut-off for azithromycin is > 1mg/L. 4 www.eurosurveillance.org Ethical statement For azithromycin, most of isolates (54.6%; 1,082/1,981) As this was a retrospective study, no ethical approval showed MIC between 0.094 and 0.19 mg/L. Annual was needed. All data related to patients were coded to MIC50 and MIC90 values remain the same during the maintain confidentiality. study period, 0.125 and 0.25 mg/L, respectively. Nevertheless, the proportion of resistant isolates, Results according to the EUCAST breakpoint (ECOFF > 1mg/L) Between 2013 and 2017, 2,054 strains were isolated seemed to increase slightly from 1.5% (5/340) in from 1,979 patients. Eighteen strains were excluded 2014 to 3.0% (17/572) in 2017 (p = 0.5295). A total of because, in 16 patients, N. gonorrhoeae was recovered 16 strains showed high-level azithromycin-resistance from two different body sites, and in one patient, (MIC ≥ 256mg/L): seven in 2016 and nine in 2017. gonococcus was recovered from three different sites. These 16 strains accounted for almost a third (16/52) In the end, susceptibility testing was performed on of all azithromycin resistant strains. No high-level 2,036 N. gonorrhoeae strains isolated from 1,979 azithromycin-resistant gonococcal isolates were found patients, because 47 patients presented two different from 2013 to 2015. episodes and five patients presented three episodes during the study period. However, the proportion of isolates showing both DSC and azithromycin resistance appeared to decrease Of the 1,979 patients included, 1,888 (95.4%) were from 1.5% (5/329) in 2013 to 0.3% (2/581) in 2017. from men. 1,292 (65.3%) were tended in the Drassanes- Vall d’Hebron Sexually Transmitted Infections Unit. The prevalence of resistance to ciprofloxacin and peni- Furthermore, 573 patients (29.0%) presented in pri- cillin was 51.3% and 20.1%, respectively (Table 2). For mary healthcare units and 114 (5.8%) in other medical penicillin, the resistance rate presented a statistically departments of Vall d’Hebron University Hospital. For significant decline over the study years (p = 0.0000), 1,555 patients with data on age available, the median from 28.1% (92/327) in 2013 to 12.2% (70/572) in age was 32 years, and 1,173 (75.4%) were between 20 2017. In addition, 14.7% (300/2,036) of isolates were and 40 years old. A total of 1,598 (78.5%) of the 2,036 penicillase-producing N. gonorrhoeae (PPNG). For isolates studied were from urethral samples and 1,888 ciprofloxacin, the resistance rate fluctuated between (95.4%) patients were men (Table 1). 48.8 and 56.8% during the study period, with no clear trend. All isolates were susceptible to spectinomycin Antimicrobial susceptibility data during the study (Table 2). The MIC50 and MIC90 values for gentamicin period are shown in Table 2. were 4 and 6 mg/L, while for fosfomycin these were 12 and 24 mg/L, respectively (Table 2). The distribution of MIC values by year for ceftriaxone, cefixime and azithromycin is shown in the Figure (panels Table 3 summarises the determinants of resistance to A, B and C respectively). Most of the isolates (69.6%; the different antibiotics tested during the study. 1,380/1,982) showed a ceftriaxone MIC ≤ 0.016 mg/L. The percentage of ceftriaxone resistant isolates Antimicrobial resistance rates were compared between (defined as MIC > 0.125 mg/L) appeared to decrease isolates from primary healthcare units and the from 0.6% (2/324) in 2013 and 2014 to 0.2% (1/408) in Drassanes-Vall d’Hebron STI unit. Resistance rates of all 2016 (p = 0.4637). No isolates resistant to ceftriaxone antibiotics were higher in primary healthcare units than were found either in 2015 or 2017. The average preva- in Drassanes-Vall d‘Hebron STI unit, being statistically lence of decreased susceptibility to ceftriaxone (DSC; significant for penicillin (odds ratio (OR): 1.53; 95% con- 0.032 < MIC < 0.125 mg/L) throughout the study period fidence interval (CI): 1.20–1.95; p = 0.0006), cefixime was 15.8% (314/1,982), seemingly declining from 2013 (OR: 1.91; 95%CI: 1.22–3.01; p = 0.0050), azithromycin to 2015 (from 24.1% (78/324) to 7.4% (25/339)) but in (OR: 1.89; 95%CI: 1.07–3.31; p = 0.0272) and ciprofloxa- 2016 and 2017 the proportions of isolates with DSC cin (OR: 1.63; 95%CI: 1.33–1.99; p = 0.0000) (Table 3). returned to higher levels, 19.1% (78/408) and 14.3% (82/572), respectively. Discussion This study describes antimicrobial surveillance data The average percentage of isolates with cefixime from 2,036 N. gonorrhoeae isolates in Barcelona during resistance (MIC > 0.125 mg/L) at 4.9% was higher than a 5-year period. To our knowledge, this is the first report the average percentage with ceftriaxone resistance at in Spain describing antimicrobial resistance of such 0.3% (Table 2). Despite this, cefixime resistance rate a considerable number of N. gonorrhoeae isolates. decreased from 8.3% (27/327) in 2013 to 4.4% (25/572) Other studies have been published previously [18,19], in 2017 (p = 0.0073). For both ESC, the proportion of but the amount of strains included was much lower. isolates with MIC ≤ 0.016 mg/L appeared to increase Our findings show that the rate of ceftriaxone resist- from 2013 to 2015, from 35.2% (115/327) to 78.5% ance remains low and stable. These results agree with (266/339) for cefixime and from 61.1% (198/324) to data from the ECDC [7]. From 2013 to 2015 the number 82.3% (279/339) for ceftriaxone (Figure). of isolates with MIC ≤ 0.016 mg/L of ceftriaxone and cefixime seemed to increase progressively. However, in 2016 and 2017 we observed an important decrease in www.eurosurveillance.org 5 Table 3 Determinants, according to logistic regression analysis, of resistance to penicillin (MIC > 1 mg/L), cefixime (MIC > 0.125 mg/L), azithromycin (ECOFF > 1 mg/L) and ciprofloxacin (MIC > 0.06 mg/L) in Neisseria gonorrhoeae isolates from primary healthcare units and Drassanes-Vall d’Hebron sexually transmitted infection unit, Barcelona, Spain, 2013– 2017 (n = 2,036) Number Univariable Multivariable Number Univariable Multivariable Total Total of Variable isolates % isolates % OR resistant OR (95%CI) p value OR (95%CI) p value of resistant OR (95%CI) p value p value tested tested (95%CI) isolates isolates Antibiotic Penicillin Cefimixime Year 2013 92 327 28.1 1 (ref) 1 (ref) 27 327 8.3 1 (ref) 1 (ref) 0.93 0.97 0.63 0.67 2014 88 339 26.0 18 339 5.3 (0.66–1.33) (0.68–1.38) (0.33–1.20) (0.35–1.29) 0.73 0.70 0.19 0.20 2015 73 339 21.5 0.0000 0.0000 5 339 1.5 0.0064 0.0073 (0.51–1.05) (0.49–1.01) (0.07–0.50) (0.07–0.52) 0.61 0.59 0.60 0.61 2016 76 408 18.6 23 408 5.6 (0.42–0.86) (0.42–0.85) (0.32–1.12) (0.32–1.16) 0.34 0.33 0.43 0.43 2017 70 572 12.2 25 572 4.4 (0.24–0.49) (0.23–0.47) (0.23–0.80) (0.23–0.80) Total 399 1,985 NA NA NA NA NA 98 1,985 NA NA NA NA NA Sex Male 388 1,895 20.5 1 (ref) 1 (ref) 88 1,896 4.6 1 (ref) 1 (ref) 0.55 0.1786 0.49 0.1067 3.36 0.0074 2.72 0.0303 Female 11 88 12.5 10 87 11.5 (0.23–1.31) (0.20–1.17) (1.38–8.16) (1.10–6.72) Total 399 1,983a NA NA NA NA NA 98 1,983a NA NA NA NA NA Clinical setting DVHSTI 242 1,303 18.6 1 (ref) 1 (ref) 46 1,303 3.5 1 (ref) 1 (ref) (n = 1,340b) 0.0041 0.0006 0.0064 0.0050 1.41 1.53 1.86 1.91 PH (n = 579b) 138 566 24.4 36 565 6.4 (1.12–1.79) (1.20–1.95) (1.19–2.92) (1.22–3.01) Total 380c 1,869 c NA NA NA NA NA 82c 1,868c NA NA NA NA NA Antibiotic Azithromycin Ciprofloxacin Year 2013 11 322 3.4 1 (ref) 1 (ref) 162 325 49.8 1 (ref) 1 (ref) 0.42 0.45 1.01 1.06 2014 5 340 1.5 167 340 49.1 (0.15–1.23) (0.15–1.30) (0.74–1.39) (0.77–1.45) 0.60 0.60 1.45 1.44 2015 7 339 2.1 192 338 56.8 (0.23–1.57) (0.23–1.57) (1.06–1.99) (1.05–1.98) 0.5295 0.5939 0.0771 0.0846 0.81 0.81 1.11 1.11 2016 12 408 2.9 213 407 52.3 (0.34–1.89) (0.35–1.90) (0.82–1.50) (0.82–1.50) 1.002 0.87 0.85 0.98 2017 17 572 3 284 576 49.3 (0.758– (0.40–1.88) (0.39–1.84) (0.74–1.30) 1.325) Total 52 1,981 NA NA NA NA NA 1,018 1,986 51.3 NA NA NA NA Sex Male 50 1,891 2.6 1 (ref) 1 (ref) 965 1,896 50.9 1 (ref) 1 (ref) 1.57 0.5410 1.45 0.6184 0.98 0.9312 0.99 0.9832 Female 2 88 2.2 53 88 60.2 (0.37–6.65) (0.34–6.22) (0.55–1.73) (0.56–1.77) Total 52 1,979a NA NA NA NA 1,018 1,984a NA NA NA NA NA Clinical setting DVHSTI 28 1,302 2.2 1 (ref) 1 612 1,303 47.0 1 1 (ref) (n = 1,340b) 0.0207 0.0272 0.0000 0.0000 1.94 1.89 1.63 1.63 PH (n = 579b) 23 563 4.1 332 567 58.6 (1.11–3.39) (1.07–3.31) (1.34–1.99) (1.33–1.99) Total 51c 1,865c NA NA NA NA NA 944c 1,870c NA NA NA NA NA CI: confidence interval; DVHSTI: Drassanes-Vall d’Hebron sexually transmitted infection unit; ECOFF: epidemiological cut-off value; MIC: minimum inhibitory concentration; NA: not applicable; OR: odds ratio; PH: primary healthcare unit. a There are two patients for whom sex is not known. b Number of isolates received by the setting. c Isolates from Vall d’Hebron University Hospital are not included in the statistical analyses relating to clinical setting. 6 www.eurosurveillance.org the percentage of strains with MIC ≤ 0.016 mg/L and an countries such as the United Kingdom and Australia increase in strains with higher MICs, which concur with in 2018 is concerning [24]. These strains show a high a report that isolates with DSC have been emerging in level of azithromycin resistance and they are also Europe in recent years [15]. resistant to ceftriaxone, resulting in resistance to the first line dual therapy for gonorrhoea (ceftriaxone In our work, we were able to observe a decrease in the intramuscularly and azithromycin orally) recommended resistance rate of both ESC throughout the study. The by European, Australian and World Health Organization reason that this decrease is statistically significant guidelines. The appearance and dissemination of these only for cefixime may be due to the small number of type of strains compromises the successful treatment ceftriaxone resistant strains. of this infection. This highlights the need to keep the capability to culture N. gonorrhoeae, in order to moni- Since dual therapy (ceftriaxone 500 mg + azithromy- tor antibiotic susceptibility and rapidly detect the cin 1 g) was implemented in Barcelona in 2012, resist- emergence of XDR strains. ance to ceftriaxone has started to decrease: 2.8% in 2012 (data not shown) to 0.0% in 2017. This could Although the resistance rate to ciprofloxacin observed be explained by the change in treatment regimen in this study is similar to that of neighbouring countries whereby, until 2011, gonorrhoea was treated with cef- [25], it is higher than that reported in eastern European triaxone 250 mg. On the other hand, the resistance rate countries such as Ukraine [26]. of azithromycin has increased slightly from 1.5% in 2014 to 3.0% in 2017 and high-level resistant isolates The breakpoints of gentamicin are not yet established (MIC ≥ 256 mg/L) were first detected in 2016 and 2017. by EUCAST. In this study it appears that N. gonor- An increase in azithromycin rate has been documented rhoeae does not have high MIC values for gentamicin in several European countries [15]. (MIC50 = 4mg/L and MIC90 = 6mg/L). These values are similar to those reported previously and suggest that Although the European and Spanish guidelines still rec- gentamicin can be a low-cost and efficacious alterna- ommend dual therapy [12,20], the increase in azithro- tive therapy [27]. In addition, other studies support the mycin resistance detected worldwide, not only in N. in vitro activity of gentamycin, used in combination with gonorrhoeae but also in other STIs such as Mycoplasma cefixime or ertapenem, to control the spread of multi- genitalium [21], brings into question the advisability drug-resistant (MDR) and XDR N. gonorrhoeae strains of this therapeutic strategy. In fact, since 2019, the [28]. British Association for Sexual Health and HIV (BASHH) has recommended monotherapy with ceftriaxone 1 g Similar to gentamicin, no clinical breakpoints for fosfo- [22]. However, some studies support that the selec- mycin exist. The review conducted by Tesh et al. sug- tion/induction of azithromycin resistance of N. gonor- gests that fosfomycin can be an alternative treatment rhoeae by the use of the current dual therapy is limited for gonococcal infection [29]. In this review, isolates [23], and it may be associated with the general use of with a MIC < 16 mg/L were considered as susceptible, azithromycin for respiratory infections or the treatment while those with MICs between 32 and 64 mg/L were of non-gonococcal urethritis [13]. moderately susceptible. These MICs are similar to those found by our study, but it is necessary to estab- The overall azithromycin resistance rate in our study lish susceptibility breakpoints in order to understand was 2.6% based on the EUCAST breakpoint (ECOFF is how to appropriately dose fosfomycin to treat N. gonor- 1 mg/L). This percentage is lower than that reported rhoeae infections [29]. by other Spanish groups such as Cobo et al. [18], who showed a resistance percentage of 13.8% in Almería. It is surprising that the resistance rates of almost all On the other hand, Fuertes de Vega et al. reported a antimicrobials tested were higher in isolates from resistance rate of 5.2% in Barcelona [19], which is more primary healthcare than in those from the STI unit. similar to our results. We must bear in mind that the One reason that might explain this is that 70% of the cut-off point used by these authors is that of prior Drassanes-Vall d’Hebron STI Unit patients are MSM. EUCAST report versions (0.5 mg/L). If we analyse the Although we do not have access to this information, we azithromycin resistance rate of our study according to hypothesise by deduction that the majority of patients this cut-off, the percentage of resistance strains would who attend primary healthcare units are men who have be 4.1%. sex with women (MSW) and if so, this might suggest that there are different N. gonorrhoeae populations The difference in the percentage of azithromycin resist- circulating in the two populations. ance between different regions of Spain could be due to divergence in treatment patterns or the circulation One limitation of this study is that antimicrobial sus- of different genotypes with greater ability to develop ceptibility tests of N. gonorrhoeae were performed antimicrobial resistance. mainly in symptomatic patients, since they are the ones from whom a sample is collected for culture. On other hand, the occurrence of extensively drug- Asymptomatic patients are diagnosed by nucleic resistant (XDR) strains of N. gonorrhoeae reported in acid amplification tests (NAAT). Another limitation www.eurosurveillance.org 7 Catalonia. Epidemiological surveillance of sexually transmitted is that when N. gonorrhoeae was isolated in multiple infections in Catalonia. Annual report 2017] Badalona: sites in one patient, antimicrobial susceptibility CEEISCAT; 2018. was only performed on genital samples. This could 4. Cámara J, Serra J, Ayats J, Bastida T, Carnicer-Pont D, Andreu A, et al. Molecular characterization of two high-level ceftriaxone- lead to the loss of strains with higher MIC values in resistant Neisseria gonorrhoeae isolates detected in Catalonia, pharyngeal carriers and consequently to underestimate Spain. J Antimicrob Chemother. 2012;67(8):1858-60. https:// doi.org/10.1093/jac/dks162 PMID: 22566592 antimicrobial resistance. Moreover, due to the 5. de Curraize C, Kumanski S, Micaëlo M, Fournet N, La Ruche G, retrospective nature of the study, there is a lack of Meunier F, et al. Ceftriaxone-resistant Neisseria gonorrhoeae isolates (2010 to 2014) in France characterized by using demographic and epidemiological information. whole-genome sequencing. Antimicrob Agents Chemother. 2016;60(11):6962-4. https://doi.org/10.1128/AAC.01568-16 PMID: 27600036 Although our results are only representative of 6. Lahra MM, Ryder N, Whiley DM. A new multidrug-resistant Barcelona, the patients included in the current study strain of Neisseria gonorrhoeae in Australia. N Engl J Med. represent 62.1% of those reported in Catalonia [30]. 2014;371(19):1850-1. https://doi.org/10.1056/NEJMc1408109 PMID: 25372111 7. European Centre for Disease Prevention and Control (ECDC). In conclusion, we analysed the susceptibility of 2,036 Gonococcal antimicrobial susceptibility surveillance in Europe isolates in Barcelona from 2013 to 2017. We were able – Results summary 2017. Stockholm: ECDC; 2019. 8. Unemo M. Current and future antimicrobial treatment of to observe that susceptibility to ceftriaxone remains gonorrhoea - the rapidly evolving Neisseria gonorrhoeae high and resistance has decreased since dual ther- continues to challenge. BMC Infect Dis. 2015;15(1):364. https://doi.org/10.1186/s12879-015-1029-2 PMID: 26293005 apy was implemented in 2012. However, azithromycin 9. Workowski KA, Bolan GA, Centers for Disease Control resistance increased during the study period and high- and Prevention. Sexually transmitted diseases treatment level azithromycin resistant strains were isolated for guidelines, 2015. MMWR Recomm Rep. 2015;64(RR-03):1-137. PMID: 26042815 the first time in 2016. 10. Public Health Agency of Canada (PHAC). Canadian Guidelines Our study highlights the need to monitor antibiotic sus- on Sexually Transmitted Infections. Ottawa: PHAC; 2013. ceptibility and to perform molecular typing studies at a Gonococcal Infections Chapter. national level, which would allow identifying temporal 11. Australian Sexual Health Alliance (ASHA). Australian STI Management Guidelines for Use in Primary Care. Sydney: and geographical changes, to detect the emergence ASHA. [Accessed 12 Jun 2019]. Available from: http://www. and dissemination of new strains, and to maintain sti.guidelines.org.au/sexually-transmissible-infections/ gonorrhoea#management therapeutic guidelines updated. 12. Bignell C, Unemo M, Radcliffe K, Jensen JS, Babayan K, Barton S, et al. , European STI Guidelines Editorial Board. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int J STD AIDS. 2013;24(2):85-92. Acknowledgements https://doi.org/10.1177/0956462412472837 PMID: 24400344 13. Unemo M, Workowski K. Dual antimicrobial therapy for We would like to thank all the Primary Health Centers includ- gonorrhoea: what is the role of azithromycin? Lancet Infect ed in the study, Drassanes-Vall d’Hebron STI Unit and labo- Dis. 2018;18(5):486-8. https://doi.org/10.1016/S1473- ratory of Vall d’Hebron University Hospital. 3099(18)30162-2 PMID: 29523498 14. Hofstraat SH, Götz HM, van Dam AP, van der Sande MA, van Benthem BH. Trends and determinants of antimicrobial susceptibility of Neisseria gonorrhoeae in the Netherlands, Conflict of interest 2007 to 2015. Euro Surveill. 2018;23(36). https://doi. org/10.2807/1560-7917.ES.2018.23.36.1700565 PMID: None declared. 30205870 15. Day MJ, Spiteri G, Jacobsson S, Woodford N, Amato-Gauci AJ, Cole MJ, et al. , Euro-GASP network. Stably high azithromycin resistance and decreasing ceftriaxone susceptibility in Authors’ contributions Neisseria gonorrhoeae in 25 European countries, 2016. BMC Infect Dis. 2018;18(1):609. https://doi.org/10.1186/s12879- Salmerón P, Pumarola T, Larrosa N, Hoyos Y and Serra J con- 018-3528-4 PMID: 30509194 tributed to the design and carrying out of the study. 16. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 29th ed. CLSI supplement M100. Wayne, PA: CLSI; 2019. Viñado B, El Ouazzani R and Hernández M were responsible 17. The European Committee on Antimicrobial Susceptibility for the susceptibility testing of the strains. Testing (EUCAST). Breakpoints tables for interpretation of MICs and zone diameters. Version 9.0. Växjö: EUCAST; 2019. Jané M, Alberny M and Barbera MJ were responsible for the 18. Cobo F, Cabezas-Fernández MT, Cabeza-Barrera MI. review of the data. Antimicrobial susceptibility and typing of Neisseria gonorrhoeae strains from Southern Spain, 2012-2014. Enferm Infecc Microbiol Clin. 2016;34(1):3-7. https://doi.org/10.1016/j. eimc.2015.01.017 PMID: 25735717 References 19. Fuertes de Vega I, Baliu-Piqué C, Bosch Mestres J, Vergara Gómez A, Vallés X, Alsina Gibert M. Risk factors 1. European Centre for Disease Prevention and Control (ECDC). for antimicrobial-resistant Neisseria gonorrhoeae and Gonorrhoea. Annual epidemiological report for 2017. characteristics of patients infected with gonorrhea. Enferm Stockholm: ECDC; 2019. Infecc Microbiol Clin. 2018;36(3):165-8. https://doi. 2. Unidad de vigilancia del VIH y conductas de riesgo. Vigilancia org/10.1016/j.eimc.2016.11.012 PMID: 28094065 epidemiológica de las infecciones de transmisión sexual, 2017. 20. GESIDA. Documento de consenso sobre diagnóstico y [HIV and risk behaviors surveillance unit. Epidemiological tratamiento de las infecciones de transmisión sexual en surveillance of sexually transmitted infections, 2017]. Madrid: adultos, niños y adolescents. 2017. [Consensus document Centro Nacional de Epidemiología, Instituto de Salud Carlos on diagnostic and treatment of adult and teenage/ III/Plan Nacional sobre el Sida, Dirección General de Salud adolescent sexually transmitted infections]. [Accessed 09 Pública, Calidad e Innovación; 2019. Jul 2019]. Available from: http://gesida-seimc.org/category/ 3. Centre d’Estudis Epidemiològics sobre les Infeccions de guias-clinicas/otras-guias-vigentes/ Transmissió Sexual i Sida de Catalunya (CEEISCAT). Vigilància 21. Barberá MJ, Fernández-Huerta M, Jensen JS, Caballero epidemiològica de les infeccions de transmissió sexual a E, Andreu A. Mycoplasma genitalium Macrolide and Catalunya. Informe anual 2017. [Center of Epidemiological Fluoroquinolone Resistance: Prevalence and Risk Factors Studies on Sexual Transmitted Infections and AIDS of Among a 2013-2014 Cohort of Patients in Barcelona, Spain. 8 www.eurosurveillance.org Sex Transm Dis. 2017;44(8):457-62. https://doi.org/10.1097/ OLQ.0000000000000631 PMID: 28703723 22. Fifer H, Saunders J, Soni S, Tariq Sadiq S, FitzGerald M. British Association for Sexual Health and HIV national guideline for the management of infection with Neisseria gonorrhoeae. 2019. [Accessed 04 Jul 2019]. Available from: https://www. bashh.org/guidelines 23. Clifton S, Town K, Furegato M, Cole M, Mohammed H, Woodhall SC, et al. Is previous azithromycin treatment associated with azithromycin resistance in Neisseria gonorrhoeae? A cross- sectional study using national surveillance data in England. Sex Transm Infect. 2018;94(6):421-6. https://doi.org/10.1136/ sextrans-2017-053461 PMID: 29511067 24. Jennison AV, Whiley D, Lahra MM, Graham RM, Cole MJ, Hughes G, et al. Genetic relatedness of ceftriaxone-resistant and high-level azithromycin resistant Neisseria gonorrhoeae cases, United Kingdom and Australia, February to April 2018. Euro Surveill. 2019;24(8):1900118. https://doi.org/10.2807/1560- 7917.ES.2019.24.8.1900118 PMID: 30808445 25. Harris SR, Cole MJ, Spiteri G, Sánchez-Busó L, Golparian D, Jacobsson S, et al. , Euro-GASP study group. Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey. Lancet Infect Dis. 2018;18(7):758-68. https://doi.org/10.1016/S1473- 3099(18)30225-1 PMID: 29776807 26. Boiko I, Golparian D, Krynytska I, Bezkorovaina H, Frankenberg A, Onuchyna M, et al. Antimicrobial susceptibility of Neisseria gonorrhoeae isolates and treatment of gonorrhoea patients in Ternopil and Dnipropetrovsk regions of Ukraine, 2013-2018. APMIS. 2019;127(7):503-9. https://doi.org/10.1111/apm.12948 PMID: 30903707 27. Mann LM, Kirkcaldy RD, Papp JR, Torrone EA. Susceptibility of Neisseria gonorrhoeae to Gentamicin- Gonococcal Isolate Surveillance Project, 2015-2016. Sex Transm Dis. 2018;45(2):96-8. https://doi.org/10.1097/ OLQ.0000000000000693 PMID: 29324629 28. Singh V, Bala M, Bhargava A, Kakran M, Bhatnagar R. In Vitro Synergy Testing of Gentamicin, an Old Drug Suggested as Future Treatment Option for Gonorrhoea, in Combination With Six Other Antimicrobials Against Multidrug-Resistant Neisseria gonorrhoeae Strains. Sex Transm Dis. 2018;45(2):127-31. https://doi.org/10.1097/OLQ.0000000000000708 PMID: 28876284 29. Tesh LD, Shaeer KM, Cho JC, Estrada SJ, Huang V, Bland CM, et al. Neisseria gonorrhoeae and fosfomycin: Past, present and future. Int J Antimicrob Agents. 2015;46(3):290-6. https://doi. org/10.1016/j.ijantimicag.2015.05.007 PMID: 26145201 30. Carmona G, Ruiz L, Fernández C, Jané M. Butlletí Epidemiològic de Catalunya. Març 2019. [Accessed 16 Jun 2019]. Available from: http://canalsalut.gencat.cat/web/.content/_Actualitat/ Butlletins/Promocio_proteccio_salut/bec_butlleti_ epidemiologic_de_catalunya/2019/bec-mar-2019.pdf License, supplementary material and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made. Any supplementary material referenced in the article can be found in the online version. This article is copyright of the authors or their affiliated in- stitutions, 2020. www.eurosurveillance.org 9