Antimicrobial susceptibilities and molecular typing of neisseria gonorrhoeae isolates at a medical centre in Taiwan, 2001–2013 with an emphasis on high rate of azithromycin resistance among the isolates

International Journal of Antimicrobial Agents 51 (2018) 768–774

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International Journal of Antimicrobial Agents j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

Antimicrobial susceptibilities and molecular typing of neisseria gonorrhoeae isolates at a medical centre in Taiwan, 2001–2013 with an emphasis on high rate of azithromycin resistance among the isolates Yen-Hung Liu a,b, Yu-Tsung Huang c,d, Chun-Hsing Liao c,e, Po-Ren Hsueh d,f,* a

Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan Department of Internal Medicine, Cardinal Tien Hospital, Taipei, Taiwan c Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwan d Department of Laboratory Medicine, National Taiwan; University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan e Department of Medicine, Yang Ming University, Taipei, Taiwan f Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan b

A R T I C L E

I N F O

Article history: Received 18 October 2017 Accepted 27 January 2018 Editor: A. Tsakris Keywords: Neisseria gonorrhoeae Antimicrobial resistance Ceftriaxone Azithromycin Multilocus sequence typing

A B S T R A C T

A high prevalence of gonococcal resistance to various antimicrobials and Neisseria gonorrhoeae isolates exhibiting resistance to extended-spectrum cephalosporins have been reported in the past few decades. A total of 226 N. gonorrhoeae isolates obtained from the National Taiwan University Hospital from 2001 to 2013 were evaluated. The minimum inhibitory concentrations (MICs) of the isolates to antimicrobials were determined by the agar dilution method and interpreted using the 2017 clinical breakpoints or epidemiological cut-off values recommended by the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). The genetic relatedness of these isolates was determined by multilocus sequence typing. None of the isolates was resistant to ceftriaxone and cefotaxime, and the resistance rates to cefixime, spectinomycin, cefpodoxime, ciprofloxacin, and penicillin were 0.4%, 0.4%, 13.3%, 91.6%, and 87.6%, respectively. The rate of isolates resistant to azithromycin was 14.6% (EUCAST criteria), which is higher than in previous surveillance studies. A total of 57 sequence types (ST) were identified, and ST1901, ST7365, and ST1927 prevailed. Isolates of ST8143 emerged after 2011. ST1901 isolates had relatively higher MIC values for ceftriaxone and azithromycin than those of the other STs. In conclusion, ceftriaxone remains an effective drug of choice for gonorrhoeal management in Taiwan. High rates of azithromycin resistance among N. gonorrhoeae isolates were found. The circulating ST1901 strains with high MIC values for ceftriaxone and azithromycin and the emerging ST8143 strains were alarming. © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

1. Introduction In 2012, the World Health Organization (WHO) estimated that 357 million new infections were diagnosed, and 1 in 4 were sexuallytransmitted infections (STIs), including chlamydia (131 million), gonorrhoea (78 million), syphilis (5.6 million) and trichomoniasis (143 million) [1]. Among the 78 million estimated new Neisseria gonorrhoeae infections, the highest number was in the WHO Western Pacific Region, which had up to 35.2 million cases [2]. In Taiwan,

* Corresponding author. Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan. E-mail address: [email protected] (P.-R. Hsueh).

the number of N. gonorrhoeae infections has increased gradually, from 2291 reported cases in 2010 (9.79 cases per 100 000 population) to 4475 reported cases in 2016 (19.00 cases per 100 000 population) [3]. There is no gonococcal vaccine; therefore, gonorrhoea treatment depends on effective and affordable antimicrobials [4]. However, a high prevalence of gonococcal resistance to various antimicrobials, including penicillins, cephalosporins, tetracyclines, fluoroquinolones, and macrolides, has been recorded in the past few decades [5–7]. Even more critical, extensively drug-resistant gonococcal strains, called ‘superbugs’, which carry high-level resistance to ceftriaxone, have been isolated in Japan, Australia, France, and Spain [8–11]. In Taiwan, the surveillance program of the GonococciNational Isolate Collection for Epidemiology 2010 (G-NICE 2010) was implemented between January and December 2010 and revealed that 70.34%, 89.60%, 2.95%, 3.73%, and 1.24% of the isolates

https://doi.org/10.1016/j.ijantimicag.2018.01.024 0924-8579/© 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Y.-H. Liu et al. / International Journal of Antimicrobial Agents 51 (2018) 768–774

were resistant to penicillin, ciprofloxacin, cefpodoxime, cefixime, and ceftriaxone, respectively [12]. In response to the increasing rates of drug resistance among gonococcal isolates, dual antimicrobial therapy with ceftriaxone and azithromycin is globally well-accepted as an empirical first-line gonorrhoea treatment [13,14]. Unfortunately, the first case of treatment failure with this recommended therapy due to a ceftriaxone- and azithromycin-resistant gonococcal strain has been reported in the United Kingdom [15]. Facing an era of extensively drug-resistant gonococcal strains, control of gonorrhoea depends on qualityassured epidemiological surveillance of antimicrobial resistance as well as timely diagnostics and effective antimicrobial treatment [4]. In this study, we investigated the antimicrobial resistance and genetic relatedness of N. gonorrhoeae isolates collected from a medical centre in Northern Taiwan during a 13-year period. 2. Patients and methods 2.1. Patients and setting Patients with N. gonorrhoeae infection were identified through the review of microbiology laboratory records at the National Taiwan University Hospital (NTUH) from January 2001 to December 2013. The NTUH is a 2900-bed, university-affiliated hospital with all medical and surgical specialties in Northern Taiwan and provides both primary and tertiary medical care. The medical records of patients with N. gonorrhoeae infection were retrospectively reviewed, including demographic data and infection sites. In the NTUH, the first-line regimen for definite therapy for inpatients and outpatients with uncomplicated gonococcal infections of the cervix and urethra was ceftriaxone 250 mg intramuscularly in a single dose. 2.2. Bacterial isolates A total of 279 non-duplicate isolates of N. gonorrhoeae were recovered from 279 patients who were treated at the NTUH from 2001 to 2013. The isolates were identified as oxidase-positive, Gramnegative, kidney-shaped diplococci with slightly concave adjacent

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surfaces in smears and confirmed by Vitek 2 identification system (bioMérieux, France). Among these isolates, 35 isolates were not preserved. The remaining 244 isolates were stored in trypticase soy broth with 20% glycerol at −70 °C. For testing, these isolates were retrieved from stored broth, inoculated on chocolate agar, and incubated at 37 °C in a 5% CO2- enriched atmosphere [16]. Among the 244 isolates, 18 were not viable after retrieval from stored broth. Thus, 226 isolates were used for analysis in this study (Fig. 1). The number of study isolates in each year was as follows: 2001 (n = 15), 2002 (n = 8), 2003 (n = 11), 2004 (n = 19), 2005 (n = 18), 2006 (n = 9), 2007 (n = 11), 2008 (n = 17), 2009 (n = 9), 2010 (n = 28), 2011 (n = 31), 2012 (n = 30), and 2013 (n = 20). 2.3. Antimicrobial susceptibility testing Minimum inhibitory concentrations (MICs) of the isolates to 12 antimicrobial agents were determined by the agar dilution method using GC agar (Difco GC medium base; BBL Microbiology Systems, Cockeysville, MD) with a 1% defined growth supplement (IsoVitaleXTM; BBL Microbiology Systems) [18]. N. gonorrhoeae ATCC 49226 was used as the control strain. The following antimicrobial agents were tested and obtained from their manufacturers: penicillin, ceftriaxone, cefixime, cefotaxime, cefpodoxime, ciprofloxacin, spectinomycin, doxycycline, and azithromycin from Sigma (St. Louis, MO, USA), gemifloxacin from LG Chem Investments (Seoul, South Korea), nemonoxacin from TaiGen Biotechnology, Co. Ltd. (Taipei, Taiwan), and tigecycline from Pfizer Inc. (New York, NY, USA) (Table 1). The MIC interpretive breakpoints used to determine susceptibility, intermediate susceptibility, and resistance to the agents tested were in accordance with the 2017 guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) [17,18] (Table 1). For azithromycin, the susceptibilities of the isolates were also categorized as wild-type (WT) and non-WT (NWT) based on epidemiological cut-off values (ECVs) as defined by the CLSI [18] (Table 1). No MIC breakpoints were recommended by either CLSI or EUCAST for doxycycline, gemifloxacin, nemonoxacin, or tigecycline [17,18]. Among the eight agents with both CLSI- and

Fig. 1. Annual rates of resistance to three selected antimicrobial agents for 226 Neisseria gonorrhoeae isolates recovered from patients treated at the National Taiwan University Hospital from 2001–2013. For defining azithromycin susceptibility, the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing were applied [17].

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Table 1 Antibiotic susceptibility of 226 Neisseria gonorrhoeae isolates recovered from patients treated at the National Taiwan University Hospital from 2001–2013a. Agent

Penicillin CLSI EUCAST Ceftriaxone CLSI EUCAST Cefixime CLSI EUCAST Cefotaxime CLSI EUCAST Cefpodoxime CLSI EUCAST Ciprofloxacin CLSI EUCAST Spectinomycin CLSI EUCAST Azithromycin CLSI EUCAST

MIC (mg/L) Range

MIC50

MIC90

0.25– > 128

4

>128

0.015–0.25

0.015–0.5

0.03–0.5

0.03–4

0.008–128

16–128

0.03– > 32

0.06

0.06

0.12

0.25

16

32

0.5

Clinical breakpoints and/or ECVsb

No. (%) of isolates with indicated susceptibility category

S

I

R

S

I

R

≤0.06 ≤0.06

0.12–1 0.12–1

≥2 >1

0 (0) 0 (0)

28 (12.4) 28 (12.4)

198 (87.6) 198 (87.6)

≤0.25 ≤0.125

-

>0.125

226 (100) 224 (99.1)

-

2 (0.9)

≤0.25 ≤0.125

-

>0.125

225 (99.6) 216 (95.6)

-

10 (4.4)

≤0.5 ≤0.125

-

>0.125

226 (100) 151 (66.8)

-

75 (33.2)

≤0.5 -

-

-

196 (86.7) -

-

-

≤0.06 ≤0.03

0.12–0.5 0.06

≥1 >0.06

15 (6.6) 15 (6.6)

4 (1.8) 0 (0)

207 (91.6) 211 (93.4)

≤32 ≤64

64 -

≥128 >64

225 (99.6) 225 (99.6)

0 (0) -

1 (0.4) 1 (0.4)

≤1 (WT) ≤0.25

0.5

≥2 (NWT) >0.5

223 (98.7; WT) 94 (41.6)

99 (43.8)

3 (1.3; NWT) 33 (14.6)

0.12

0.12

0.25

1

32

32

1

ECV, epidemiological cut-off values; MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant; WT, wild-type; NWT, non-wild-type; CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing. a No MIC breakpoints are recommended by either CLSI or EUCAST [17,18] for gemifloxacin, nemonoxacin, doxycycline, and tigecycline. The MIC range (mg/L), MIC50 (mg/ L), and MIC90 (mg/L) were 0.25–32, 4, and 32, respectively, for doxycycline; 0.008–16, 2, and 8, respectively, for gemifloxacin; 0.03–16, 2, and 4, respectively, for nemonoxacin; and 0.03–1, 0.5 and 0.5, respectively, for tigecycline. b MIC breakpoints for susceptible, intermediate, and resistant isolates were in accordance with the 2017 CLSI M100 S27 [18] and the 2017 EUCAST breakpoint tables for the interpretation of MICs and zone diameters, version 7.1 [17].

EUCAST-recommended MIC interpretive criteria (clinical breakpoints or ECVs), identical criteria were found only for penicillin (benzylpenicillin) [17,18].

tients infected with HIV, and 45 (19.9%) were recovered from nonHIV-infected patients. The remaining 157 (69.5%) isolates were obtained from patients with unknown HIV-infection status.

2.4. Multilocus sequence typing (MLST)

3.2. Antimicrobial susceptibilities of the isolates

Genetic relatedness of the isolates was determined using MLST as previously described [19]. In the N. gonorrhoeae MLST scheme, internal fragments of the following 7 housekeeping genes, approximately 430–500 bp in length, were amplified and sequenced: abcZ (putative ABC transporter), adk (adenylate kinase), aroE (shikimate dehydrogenase), fumC (fumarate hydratase), gdh (glucose-6phosphate dehydrogenase), pdhC (pyruvate dehydrogenase subunit), and pgm (phosphoglucomutase). The sequences were submitted to the MLST website (https://pubmlst.org/neisseria/) to assign alleles and sequence types (allelic profiles).

The susceptibility results of the N. gonorrhoeae isolates to 12 antimicrobial agents interpreted by both CLSI and EUCAST criteria are summarized in Table 1 [17,18]. Among these isolates, 87.6% (n = 198) were resistant to penicillin, and >90% were resistant to ciprofloxacin. Only one isolate (0.4%) was resistant to spectinomycin. Among the third-generation cephalosporins tested, the rates of isolates resistant to ceftriaxone, cefixime, cefotaxime and cefpodoxime were 0%, 0.4% (n = 1), 0 % and 13.3% (n = 30), respectively, based on the CLSI criteria [18], and the rates of isolates resistant to ceftriaxone, cefixime, and cefotaxime were 0.9% (n = 2), 4.4% (n = 10), and 33.2% (n = 75), respectively, according to the EUCAST criteria [17]. Figures S1A and S1B illustrate the MIC distributions and accumulative percentages for each indicated azithromycin and ceftriaxone MIC against the 226 N. gonorrhoeae isolates. Among the 226 isolates, 94 (41.6%) exhibited an azithromycin MIC of ≤0.25 mg/L, 99 (43.8%) had an MIC of 0.5 mg/L, 30 (13.3%) had an MIC of 1 mg/L, 2 (0.9%) had an MIC of 2 mg/L, and one (0.4%) had an azithromycin MIC of 32 mg/L. Based on the clinical breakpoints designated for azithromycin by the EUCAST, 33 isolates (14.6%) were resistant (MICs of azithromycin of >0.5 mg/L). However, only 1.3% (n = 3) of the isolates tested were categorized as NWT (MICs of azithromycin of ≥2 mg/L) by CLSI criteria. The two isolates that were defined as resistant to ceftriaxone based on the EUCAST criteria both had an MIC of 0.25 mg/L for ceftriaxone, 0.12 mg/L for cefixime, ≥0.25 mg/L for cefotaxime, 32 mg/L for ciprofloxacin, 32 mg/L for spectinomycin, and 0.5 mg/L for azithromycin. The MIC90 values for gemifloxacin, nemonoxacin, and doxycycline were 8 mg/L, 4 mg/L, and 32 mg/L,

3. Results 3.1. Characteristics of isolates Among the 226 isolates of N. gonorrhoeae collected from 226 patients who were treated at the NTUH from 2001 to 2013, 109 (48%) were isolated from 2010 to 2013 (Fig. 1). Among these patients, 186 (82.3%) were male and the median age of these 226 patients was 31.2 years. Among these isolates, 172 (76.1%) were obtained from male urethral discharge, 36 (15.9%) from vaginal discharge, and 18 (8.0%) from other sites (seven from skin pus, four from eye discharge, three from blood, and one each from a surgical wound, gastric juice, synovial fluid, and a Bartholin abscess). Among these patients, serological and virological results for human immunodeficiency virus (HIV) infection were available only in 69 patients (30.5%). Twenty-four (10.6%) isolates were recovered from pa-

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respectively. For tigecycline, the MIC50 and MIC90 values were both 0.5 mg/L. Annual rates of isolate resistance to tested antimicrobial agents with recommended MIC breakpoints by either CLSI or EUCAST were steady during the study period, except cefpodoxime, ciprofloxacin and azithromycin. For penicillin, the resistant rate was >80% in each year. For ceftriaxone, cefixime, cefotaxime, and spectinomycin, no resistance was found during the study period, except 3.2% for cefixime in 2011 and 5.3% for spectinomycin in 2004. Fig. 1 shows the annual rates of isolates resistant to cefpodoxime (CLSI criteria), ciprofloxacin (CLSI criteria) and azithromycin (EUCAST criteria) from 2001 to 2013. The lowest rates (0%) of isolate resistance to cefpodoxime were found in 2001 and 2009, and the highest rate of resistance (33.3%) was noted in 2006. Isolates with high rates (>80%) of resistance to azithromycin were found from 2001 to 2003 and in 2006. 3.3. Isolates stratified by HIV infection status Isolates obtained from HIV-infected patients (n = 24) had lower resistance rates to penicillin (66.7%) and ciprofloxacin (66.7%) than those from HIV-non-infected patients (n = 45) (93.3% and 95.6%, respectively). 3.4. MLST of the isolates Among the 226 isolates, a total of 57 different sequence types (STs) were identified (Table 2). The five main STs included ST1901 (n = 32, 14.2%), ST7365 (n = 21, 9.3%), ST1927 (n = 18, 8.0%), ST1600 (n = 16, 7.0%), and ST7371 (n = 11, 4.9%). ST1901 was the most prevalent ST before 2007 (Fig. 2). After 2007, no predominant STs were noted, but the proportions of ST1927 and ST7365 isolates increased from 1.1% and 5.5% (prior to 2007) to 12.6% and 11.9% (after 2007), respectively. ST8143 isolates were first identified in 2012. 3.5. Antimicrobial susceptibilities of isolates within the prevalent STs Fig. 3 demonstrates the distribution of the azithromycin and ceftriaxone MICs among the three main STs. Twelve isolates (37.5%) of ST1901 (n = 32) and seven (33.3%) of ST7365 (n = 21) had MICs of ≥0.12 mg/L for ceftriaxone, whereas only four (22.2%) ST1927

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Table 2 Distribution of sequence types (STs) among the 226 isolates of Neisseria gonorrhoeae. STs with ≥5 isolates are listed in the table. Sequence type (ST)

No. (%) of isolates

1901 7365 1927 1600 7371 6714 1601 7363 7822 7360 8143 9903 6813 10241 Othersa

32 (14.2) 21 (9.3) 18 (8.0) 16 (7.0) 11 (4.9) 9 (4.0) 8 (3.5) 7 (3.1) 7 (3.1) 6 (2.7) 6 (2.7) 6 (2.7) 5 (2.2) 5 (2.2) 69 (30.5)

a Includes four each of ST1583, ST1902, ST7827, ST8126, three each of ST1587, ST1588, ST1928, ST8122, two each of ST1920, ST1963, ST8781, ST9304, ST9363, ST9900, and one each of ST1579, ST1580, ST1582, ST1584, ST1590, ST1596, ST1597, ST1598, ST1889, ST1895, ST1905, ST1921, ST6720, ST6960, ST7356, ST7367, ST7823, ST7828, ST8120, ST8123, ST8125, ST8156, ST8417, ST9362, ST10214, ST10314, ST10633, ST10899, and ST11179.

isolates (n = 18) exhibited MICs ≥0.12 mg/L for ceftriaxone. Nine (28.1%) ST1901 isolates had MICs of >0.5 mg/L for azithromycin, and neither of ST7365 and ST1927 had azithromycin MICs of >0.5 mg/L. 3.6. STs with high MIC values for cephalosporins The two isolates with a ceftriaxone MIC of 0.25 mg/L belonged to ST1901 and ST1588 and were collected in 2003 and 2013, respectively. The nine isolates exhibiting a cefixime MIC of >0.25 mg/L belonged to six different STs: ST1901 (n = 2), ST7363 (n = 2), ST9903 (n = 2), ST1587 (n = 1), ST7360 (n = 1), and ST9363 (n = 1). There were 21 isolates with a cefotaxime MIC of 0.5 mg/L and, among them, 13 different STs were found, with a predominance of ST1901 (n = 4, 19%). There were 30 isolates with a cefpodoxime MIC of >0.5 mg/L and, among them, 15 different STs were present, with a predominance of ST1901 (n = 5, 16.7%).

Fig. 2. Distributions of isolates belonging to the three most prevalent sequence types (ST1901, ST1927, and ST7365) and the emerging ST (ST8143) among the 226 Neisseria gonorrhoeae isolates recovered from patients treated at the National Taiwan University Hospital from 2001–2013.

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Fig. 3. Minimum inhibitory concentration (MIC) distributions of azithromycin (A) and ceftriaxone (B) for Neisseria gonorrhoeae ST1901 (n = 32), ST7365 (n = 21), and ST1927 (n = 18) isolates recovered from patients treated at the National Taiwan University Hospital from 2001–2013.

3.7. Antimicrobial susceptibilities of the STs collected from vaginal discharges Among the 36 isolates recovered from vaginal discharges, four (4/8, 50%) were ST1601 and five (5/9, 55.6%) were ST6714. Three (75%) of vaginal ST1601 and three (60%) of vaginal ST6714 isolates had a ceftriaxone MIC of 0.12 mg/L. Regarding azithromycin MIC values for vaginal isolates, one (25%) of vaginal ST1601 isolates and no vaginal ST6714 isolates exhibited MICs of >0.5 mg/L. 4. Discussion In the age of easy international travel and complex sexual networks, comprehensive approaches to address the problem of antimicrobial resistance in N. gonorrhoeae are important for stopping the emergence of untreatable gonorrhoea infections. Meanwhile, clinical practice guidelines should be updated by local, qualified and intensified surveillance as empirical antimicrobial regimens should not be administered if the antimicrobial resistance reaches a level of 5% [4]. Our study indicated that none of the isolates tested were resistant to ceftriaxone in vitro using the CLSI criteria; thus, ceftriaxone

is still the drug of choice and an effective empirical treatment for gonorrhoea. However, two (0.9%) isolates with an elevated ceftriaxone MIC (0.25 mg/L) were found in this study. N. gonorrhoeae strains with elevated ceftriaxone MICs have caused treatment failure in pharyngeal gonorrhoea in Japan [8], Australia [20], and Sweden [21]. Therefore, in addition to effective ceftriaxone treatment, the continuous monitoring of drug resistance and early identification of potential treatment failure, particularly for pharyngeal gonorrhoea, are necessary for the management of N. gonorrhoeae infections. Azithromycin has been extensively used for sexually transmitted diseases and respiratory tract infections [13,22]; this has impacted the susceptibility of N. gonorrhoeae due to subtherapeutic azithromycin concentrations and their consequent selection of resistance [23]. Not surprisingly, the emergence of high-level azithromycin resistance for N. gonorrhoeae, defined by an azithromycin MIC >256 mg/L, has been reported in many countries [6,24,25]. Surveillance of antibiotic resistance in N. gonorrhoeae in the WHO Western Pacific and South East Asian Regions showed very low (<1%) or no resistance in Cambodia, Vietnam, India and Australia, whereas 34% resistance was reported in Mongolia [26]. The mean azithromycin resistance rate in Europe in 2014 was 7.9%, ranging from 0% in Cyprus, Estonia and Iceland to 40% in Greece [27].

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In our study, the rate of isolates resistant to azithromycin was 14.6% (EUCAST criteria), which was higher than in previous surveillance studies, except the data from Mongolia, Greece, Ireland, and Portugal [26,27]. Due to the high resistance rate (>5%) of N. gonorrhoeae to azithromycin in Taiwan, the efficacy of the recommended dual therapy, ceftriaxone plus azithromycin, deserves further investigation and cautious evaluation in Northern Taiwan. ST1901 was the most prevalent ST among the N. gonorrhoeae isolates during the 13-year study period. Similarly, from 2010 to 2012, in the Kyoto and Osaka area, ST1901 had taken over as the most significantly prevalent ST (ST1901, 40.9%; ST7359, 19.2%; and ST7363, 17.1%), and it is currently responsible for most of the decreased susceptibility to extended-spectrum cephalosporins in Europe [5,28]. However, we noticed that no specific ST strain, including ST1901, has been predominant after 2007, whereas the proportion of ST1927 and ST7365 strains increased (from 1.1% to 12.6% and from 5.5% to 11.9%, respectively), and ST8143 (6 isolates) abruptly emerged after 2012. The newly emerging ST8143 should be monitored to determine if it will follow the pattern of ST1901 and become highly prevalent locally and globally. Earlier studies showed that cephalosporin-resistant N. gonorrhoeae may be clonal; however, there were no specific ST predominated isolates with high MIC levels to cephalosporin in our study, 2 STs with an MIC of 0.25 mg/L for ceftriaxone, 7 STs with an MIC of >0.25 mg/L for cefixime, 13 STs with an MIC of 0.5 mg/L for cefotaxime and 30 STs with an MIC of >0.5 mg/L for cefpodoxime. Lo et al. [29] reported that 11 ceftibuten-resistant N. gonorrhoeae isolates shared the same Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types, and Ameyama et al. [30] found that most mosaic PBP2 isolates with a cefixime MIC ≥0.125 mg/L from Japan were also strongly linked via pulse-field gel electrophoresis analysis. In contrast, a study from Sweden showed heterogeneity among their gonococcal isolates with reduced antibiotic susceptibility [31]. Peng et al. also demonstrated that isolates with ceftriaxone MICs ≥0.125 mg/L were not limited to particular genotypes [32]. The various numbers of collected N. gonorrhoeae strains and assorted discrimination methods for N. gonorrhoeae differentiation in these previous studies may account for the perplexing inconsistency. First, our study and the studies from Sweden and Peng et al. analysed more than 100 N. gonorrhoeae isolates (226, 331, and 128 isolates, respectively) [31,32], whereas the studies from Lo et al. and Ameyama et al. studied fewer isolates (44 and 77 isolates, respectively) [29,30]. Second, two discrimination methods, NG-MAST and full-length porB gene sequences, were applied in the reports from Sweden and Peng, whereas Lo et al. and Ameyama et al. use only one discrimination method in their studies [29–32]. We also found that ST1901 isolates generally exhibited higher MICs for both ceftriaxone (MIC ≥0.12 mg/L in 37.5% ST1901 isolates) and azithromycin (MIC >0.5 mg/L in 28.3% ST1901 isolates) than those of another common ST, ST1927 (ceftriaxone MIC ≥0.12 mg/L in 22.2% isolates, and no isolates with azithromycin MIC >0.5 mg/L). Allen et al. also detected similar results in Canada, indicating the clonal co-evolution (NG-MAST 3158) of azithromycin resistance and reduced susceptibility to ceftriaxone in Ontario since 2012; however, the mechanism for the emergence of this phenomenon is unknown [33]. More studies focusing on the exploration of the possible mechanisms and molecular epidemiology of these strains to avoid their global spread are important. This study had several limitations. First, there was selection bias because all the isolates were collected from symptomatic patients at one medical centre in Northern Taiwan and some of the isolates were recovered from patients who were referred from other hospitals due to the failure of gonorrhoea treatment. Nationwide data from clinics, regional hospitals and medical centres are needed to fully assemble epidemiological information on N. gonorrhoeae infection. Second, some patient data were missing, including HIV-

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infection status, sexual orientation, travel history and possible sexual networks, and no isolates from the rectum and oropharynx were obtained. Consequently, the magnitude of antimicrobial resistance among specific core populations may be incorrectly estimated. In this study, HIV infection data were available for only approximately 30% of patients. Interpretation of the findings on lower resistance rates to penicillin and ciprofloxacin among isolates recovered from HIV-infected patients compared with those from HIVnon-infected patients should be prudential. Lastly, we applied only one method, MSLT, to type the N. gonorrhoeae isolates. Unfortunately, correctly characterizing all the strains and community epidemics by employing a single typing method is impossible, particularly considering that the highly recombinogenic feature of Neisseria spp., i.e., the exchange of DNA fragments by natural transformation, makes it challenging to investigate phylogeny [34]. Despite these limitations, this study outlined information regarding the antimicrobial resistance and molecular epidemiology of N. gonorrhoeae in Northern Taiwan. In conclusion, ceftriaxone is an effective drug of choice for gonorrhoeal management, but the efficacy of dual therapy, ceftriaxone plus azithromycin, should be closely monitored and carefully re-evaluated because of a high azithromycin resistance rate (14.6%) in N. gonorrhoeae as well as circulating ST1901 strains with high MICs for ceftriaxone and azithromycin. Regarding the global spread of antimicrobial-resistant N. gonorrhoeae, close monitoring of circulating ST1901, and emerging ST8143, strains as well as the susceptibility of gonococcal isolates cannot be emphasized enough for N. gonorrhoeae control. Acknowledgments Funding: None. Competing interests: None declared. Ethical approval: Not required. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2018.01.024. References [1] WHO. Sexually transmitted infections (STIs). 2016. Available from: http://www.who.int/mediacentre/factsheets/fs110/en/. [2] Unemo M, Bradshaw CS, Hocking JS, de Vries HJC, Francis SC, Mabey D, et al. Sexually transmitted infections: challenges ahead. Lancet Infect Dis 2017;17:e235–79. [3] WHO. Sexually transmitted infections (STIs). 2017. Available from: http://www.who.int/mediacentre/factsheets/fs110/en/. [4] Unemo M. Current and future antimicrobial treatment of gonorrhoea—the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infect Dis 2015;15:364. [5] Shimuta K, Unemo M, Nakayama S, Morita-Ishihara T, Dorin M, Kawahata T, et al. Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance. Antimicrob Agents Chemother 2013;57:5225–32. [6] Chisholm SA, Neal TJ, Alawattegama AB, Birley HD, Howe RA, Ison CA. Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales. J Antimicrob Chemother 2009;64:353–8. [7] Lahra MM, Enriquez RP, National Neisseria N. Australian Gonococcal Surveillance Programme annual report, 2015. Commun Dis Intell Q Rep 2017;41:E. [8] Ohnishi M, Golparian D, Shimuta K, Saika T, Hoshina S, Iwasaku K, et al. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob Agents Chemother 2011;55:3538–45. [9] Lahra MM, Ryder N, Whiley DM. A new multidrug-resistant strain of Neisseria gonorrhoeae in Australia. N Engl J Med 2014;371:1850–1. [10] Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 2012;56:1273–80.

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