Twenty-five-year changing pattern of gonococcal antimicrobial susceptibility in Shanghai: surveillance and its impact on treatment guidelines

Background Antimicrobial resistance of Neisseria gonorrhoeae is a serious health problem in China. Gonococcal antimicrobial susceptibility has been monitored in Shanghai since 1988. In this study, we examined the changing pattern of gonococcal antimicrobial susceptibility based on data from N. gonorrhoeae isolates collected over the past 25 years. Methods Approximately 100–200 isolates each year (1988–2013) were tested for their susceptibility to penicillin (PEN), tetracycline (TET), ciprofloxacin (CIP), ceftriaxone (CRO) and spectinomycin (SPT), using the agar dilution method. Plasmid-mediated N. gonorrhoeae antimicrobial resistance, comprising penicillinase-producing N. gonorrhoeae (presumed PPNG) and high-level tetracycline resistance N. gonorrhoeae (presumed TRNG), were also determined. Breakpoints for susceptibilities followed those described by the Clinical and Laboratory Standard Institute and the European Committee on Antimicrobial Susceptibility Testing. Results A high proportion of isolates were resistant to PEN, TET and CIP, ranging from less than 20% at the beginning of the survey, increasing in the late 1990s and reaching over 90% in recent years. The proportion of isolates exhibiting plasmid-mediated resistance exceeded 38% for presumed PPNG and 20% for presumed TRNG in recent years. The proportion of CRO nonsusceptible isolates (MIC ≥ 0.125 mg/L) ranged from 7% to 13% in most of the study years. Almost all isolates were susceptible to SPT. The SPT MIC90 was 16–32 mg/L for 2008–2013. The proportion of CRO nonsusceptible-associated multiple-drug-resistant (MDR) isolates was over 5% in most of the study years. Conclusions N. gonorrhoeae isolates in Shanghai were resistant to PEN, TET and CIP. Furthermore, CRO nonsusceptible and MDR isolates were prevalent. N. gonorrhoeae isolates were also found to be susceptible to SPT. It is recommended that the CRO dose be increased from currently recommended 250 mg to 500 mg and that SPT be an alternative in treating urogenital gonorrhea. Our findings highlight the importance of both regional and national surveillance programs for the prompt modification of treatment guidelines, vital in responding to the changing pattern of gonococcal antimicrobial susceptibility. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0731-9) contains supplementary material, which is available to authorized users.


Background
Neisseria gonorrhoeae, a Gram-negative bacterium, is responsible for the sexually transmitted disease gonorrhea. In 2008, over 106 million new cases of N. gonorrhoeae were reported worldwide [1]. In China, infectious disease surveillance reports more than 100,000 new gonorrhea cases each year. In 2012, there were 95,263 gonorrhea cases reported in China with an incidence rate of 7.07 per 100,000 population. Gonorrhea ranked sixth in the most frequently reported infectious diseases in China [2]. There are currently no vaccines available for N. gonorrhoeae [3]. Antimicrobial treatment is essential to control this infectious disease. However, antimicrobial resistance (AMR) in N. gonorrhoeae is emerging as a significant public health problem worldwide [4,5]. In most areas of the world, N. gonorrhoeae has developed resistance to sulfonamides, penicillins, tetracyclines, macrolides and fluoroquinolones [6]. Decreased susceptibility, sporadic resistant isolates and clinical treatment failure to oral or parental thirdgeneration cephalosporins have also been observed [7][8][9][10]. Moreover, extensive/multiple-drug-resistant (MDR) N. gonorrhoeae isolates are commonly observed in various areas of the world [8,10].
In China, the burden of gonococcal penicillin (PEN) and tetracycline (TET) resistance reached high levels [11][12][13]. In response, fluoroquinolones were introduced as a treatment option. Since 1995, orally administered ciprofloxacin (CIP) has been recommended for the treatment of gonorrhea in China. However, as a result, the proportion of CIP resistance drastically increased from 15% in 1996 to nearly 100% in the mid-2000s in China [11][12][13]. The use of CIP for the treatment of gonorrhea has since been terminated in China [14,15]. Since 2007, ceftriaxone (CRO) has been recommended as the first line drug for the treatment of gonorrhea in China [14]. However, in Shanghai CRO has been used since 2000, and the presence and spread of gonococcal isolates with nonsusceptibility to CRO (minimal inhibitory concentration (MIC) = 0.125-0.25 mg/L) was reported in 11.9% of isolates in 2005 and 19.7% of isolates in this city in 2008 [11,12].
In this study, we used the data of the Shanghai Gonococcal Antimicrobial Susceptibility Surveillance Program from N. gonorrhoeae isolates collected over the past 25 years to analyze trends in AMR development in Shanghai. The AMR data were aligned with the timeline of treatment guidelines for gonorrhea in China. We found that N. gonorrhoeae isolates in Shanghai were resistant to PEN, TET and CIP, which were historically used for the treatment of gonorrhea in this region. Plasmid-mediated N. gonorrhoeae antimicrobial resistance has been prevalent in the past decades. High proportions of N. gonorrhoeae isolates are nonsusceptible to the currently recommended treatment regime of CRO (MIC ≥ 0.125). In Shanghai, MDR associated with the CRO nonsusceptible phenotype was prevalent in N. gonorrhoeae isolates. Our report indicates the usefulness of regional surveillance data in the modification of treatment guidelines and provides insights that will be important in future strategic planning for the treatment of gonorrhea.

Collection of N. gonorrhoeae isolates
The Shanghai Gonococcal Antimicrobial Susceptibility Surveillance Program was established in 1988 and has been led by the Shanghai Skin Disease Hospital (SSDH), Shanghai, China. The number of gonorrhea cases reported by the SSDH was approximately 14-16% of the reported cases from the entire Shanghai city over the years [16]. The number of gonococcal isolates tested for antimicrobial susceptibility accounted for about 10% of the SSDH reported cases or 2.1% of reported cases in Shanghai in 2012. All of the surveyed N. gonorrhoeae isolates were from the Sexually Transmitted Infection Clinic of the SSDH.
Each year in the SSDH, approximately 100-200 N. gonorrhoeae isolates were collected from individual patients for antimicrobial susceptibility testing. The first 10-20 N. gonorrhoeae isolates each month were collected from patients with culture positive gonorrhea [12]. During the period from 1988 to 2000, N. gonorrhoeae isolates were collected from both male and female patients, and during the period from 2001 to 2013, N. gonorrhoeae isolates were only collected from male patients. N. gonorrhoeae was isolated and cultured as previously described [12].
Gonococcal isolates were collected as part of standard patient care, and ethics approval was not required for their use.

Determination of the minimal inhibitory concentrations (MIC)
The antimicrobial susceptibility of N. gonorrhoeae isolates was determined using the agar dilution method [17] at the Laboratory Diagnostic Centre of the SSDH. WHO reference strains A, B, C, D and E were used in susceptibility tests [12]. Five antimicrobial agents were tested against N.  China (1988China ( -2002 or purchased from Sigma-Aldrich (Distributor: the Shanghai ANPEL Scientific Instrument Co. Ltd., Shanghai, China). The MIC testing was performed on GC agar base supplemented with 1% IsoVitalex as previously described [12]. Each MIC determination was performed in duplicate. Gonococcal isolates with inconsistent CRO MICs between the duplicates were re-tested.
Determination of plasmid-mediated resistance in N. gonorrhoeae isolates Beta-lactamase producing N. gonorrhoeae (PPNG) isolates were determined using the chromogenic cephalosporin test before 2003 and the nitrocefin test in 2003-2013 (Oxoid; distributed by GuangZhou LOSO Science Ltd.) [12]. All N. gonorrhoeae isolates were tested for β-lactamase production. Plasmid-mediated tetracycline-resistant N. gonorrhoeae (TRNG) isolates were defined as those having a TET MIC of ≥ 16.0 mg/L. PP/TRNG isolates were defined as those isolates that were β-lactamase positive and had a TET MIC of ≥ 16 mg/L. Therefore, the characterizations for presumed PPNG and TRNG were solely based on the detection of β-lactamase and the TET MIC, respectively.

Criteria for resistant phenotypes of N. gonorrhoeae
Breakpoints for the susceptibilities to PEN, TET, CIP and SPT were those described by the Clinical Laboratory Standards Institute (CLSI) [17]. The classifications of resistance phenotypes included PEN resistance (MIC ≥ 2.0 mg/L), TET resistance (MIC ≥ 2.0 mg/L), CIP resistance (MIC ≥ 1.0 mg/L) and SPT resistance (MIC ≥ 128.0 mg/L). Chromosomally-mediated resistant N. gonorrhoeae (CMRNG) isolates exhibited a PEN MIC of ≥ 2.0 mg/L, a TET MIC of ≥ 2.0 mg/L and were not classified as PPNG or TRNG. For CRO susceptibility, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint was followed (nonsusceptible, MIC ≥ 0.125 mg/L) [18]. In this study, the susceptibilities to CRO in N. gonorrhoeae were further categorized as susceptible (MIC ≤ 0.03 mg/L), reduced susceptibility (MIC = 0.06) and nonsusceptible (MIC ≥ 0.125 mg/L) [11]. The MDR phenotypes of N. gonorrhoeae were defined as various combinations of the above resistant phenotypes (Table 1).

N. gonorrhoeae has become less susceptible to CRO
During the period from 1995 to 2013, 3153 N. gonorrhoeae isolates were examined for their susceptibility to CRO (Table 2). Overall, 71.90% isolates were susceptible to CRO (MIC ≤ 0.03 mg/L); 20.30% had a reduced susceptibility phenotype (MIC = 0.06 mg/L) and 7.80% exhibited a CRO nonsusceptible phenotype (MIC ≥ 0.125 mg/L). The highest CRO MIC value for the N. gonorrhoeae isolates was 0.25 mg/L, which accounted for 0.92% of the isolates tested (29/3153).
The proportions of CRO non-susceptible isolates were over or equal to 5% in all years since 1998, and were around 7%-13% in the last 3 years (2011 to 2013). The proportion of isolates showing reduced susceptibility to CRO ranged from 12% to 40% from 1998 to 2013. From 1998 to 2013, the MIC 50 values for CRO were between 0.015 mg/L and 0.06 mg/L, and the MIC 90 values were 0.06-0.125 mg/L ( Table 2).

Discussion
In China, antibiotic resistance of N. gonorrhoeae has become an increasing problem over the past few decades in the treatment of gonorrhea infection. Since the mid-1990s, a high proportion of N. gonorrhoeae isolates sampled in Shanghai were resistant to PEN, TET and CIP. Plasmid-mediated resistance has been highly prevalent since the early 2000s. The proportion of N. gonorrhoeae isolates exhibiting a CRO nonsusceptible phenotype (MIC ≥ 0.125 mg/L) has been greater than 5% since 1998. In 2013, 13.76% of isolates exhibited a CRO MIC of ≥ 0.125 mg/L, suggesting that the effectiveness of the currently recommended treatment regime for gonorrhea is diminishing. MDR phenotypes associated with PEN, TET and CIP resistance were prevalent. N. gonorrhoeae isolates exhibiting a CRO nonsusceptible phenotype associated MDR were also found to be prevalent in recent years. In Shanghai, N. gonorrhoeae isolates were found to be susceptible to SPT.
The CRO MIC of N. gonorrhoeae isolates has been extensively monitored. In the United States, the proportion of isolates with elevated CRO MICs (≥0.125 mg/L) ranged from 0.1% to 0.4% between 2006 and 2012 [19].
In the present study, we found that the proportion of N. gonorrhoeae isolates with CRO MICs ≥ 0.125 mg/L in Shanghai (1996-2013) greatly varied from year to year, ranging from 0% (1995) to 19.72% in 2008, and 13.76% in 2013. Our findings were consistent with those of a survey of CRO susceptibility in Nanjing, China [24,25]. The proportion of CRO less susceptible N. gonorrhoeae isolates (CRO MIC ≥ 0.06 mg/L) was 17.86-50.5% during the period from 1999 to 2006 in Nanjing [24]. Chen et al. reported that the proportion of isolates with a CRO MIC = 0.125 mg/L was 39.4% in 2007 and 11.3% in 2012 [25]. The proportion of CRO nonsusceptible isolates (MIC ≥ 0.125 mg/L) ranged from 22% to over 48% in Nanning in 2000-2012 [26], much higher than that detected in Shanghai. Regional differences in gonococcal AMR within a country have also been demonstrated in other countries. In Canada, isolates of N. gonorrhoeae from the province of Saskatchewan retained a high prevalence of susceptibility to antibiotics, including CIP and PEN and CRO. Unlike reports of reduced susceptibility to third-generation cephalosporins (CRO or cefixime) from other regions of Canada and the rest of the world, all isolates collected during 2003-2008 from Saskatchewan were fully susceptible to CRO and cefixime [27]. Approximately 7% of the isolates (n = 23) showed a CRO MIC of 0.03-0.06 mg/L, and 93% of the  [29]. Indeed, cefixime treatment failure was found in 3.09% of the investigated cases (9/291) in Toronto between 2010 and 2011 [9]. Taking all of these data into account, it is clear that regional monitoring of N. gonorrhoeae AMR is as important as national surveillance programs. Currently, the recommended therapy for gonorrhea is a single dose of oral or parental third-generation cephalosporin. The doses of CRO used for the treatment of uncomplicated gonorrhea vary among countries; typically 250-500 mg per dose is injected intramuscularly, but in Japan 1 g is administered intravascularly [14,23,[30][31][32][33][34]. A landmark study suggested that increasing the dose of CRO is associated with a decreased proportion of CRO nonsusceptible N. gonorrhoeae isolates in the UK [4]. However, this decrease may not be sustainable [21]. We observed that the proportion of CRO nonsusceptible isolates transiently increased from 5% in 2007 when CRO became a recommended drug in China, to over 19% in 2008, 11% in 2009 and 13% in 2013. In Shanghai, CRO at a single low dose (250 mg) has been used to treat uncomplicated gonorrhea since 2000, and the proportion of CRO nonsusceptible N. gonorrhoeae isolates remains fairly stable. Based on our findings and reports from others in China [24][25][26], CRO remains an effective antimicrobial for the treatment of uncomplicated gonorrhea. Since CRO nonsusceptible N. gonorrhoeae isolates are prevalent in China, a higher dose of CRO (500 mg) is recommended, at least in some areas such as Shanghai, Nanjing and Nanning [24][25][26].
Detailed data on trends of MDR of N. gonorrhoeae in China are limited. However, with the consideration of the high resistant proportions of gonococcal isolates to PEN, TET, and CIP, non-susceptible CRO-associated MDR should not be ignored in China. We observed that over 5% of isolates were non-susceptible CRO-associated MDR in Shanghai in recent years.
One of the major purposes of antimicrobial susceptibility surveillance is to provide information that may indicate the need for modification of treatment guidelines. The World Health Organization recommended that when the prevalence of resistant gonococci to an antimicrobial agent rises to 5%, the antimicrobial should no longer be used [35]. In China, PEN had been the treatment of choice for gonorrhea until quinolones were introduced in 1993, PEN was removed from the treatment regime in 2000 [14,15]. Before 2000, the recommended dose of PEN continually increased because of a progressive decline in susceptibility. Penicillin was removed from the treatment regime 10 years after it was discontinued in 1985 in most regions of the world, simply because of the availability of replacing antibiotics. In 1993, oral fluoroquinolones (CIP and ofloxacin) were recommended for gonorrhea treatment, and at that time CIP resistance was already 15% in China [12,13]. Four years after (1997), CIP resistance reached over 80% (100% in 2000), while the drug remained in the treatment regime until 2007 [14].
Recently potential novel treatment strategies have been explored [6,7]. A strategy of combining older drugs was discussed [36][37][38]. However, there is no evidence that this combination of older drugs produces synergistic effects. Considering the problems of MDR, this strategy should be used with caution in China. A high prevalence of MDR to almost all currently available drugs in Shanghai limits options for combined therapy. New antibiotics are needed but are largely unavailable [38]. It is therefore worth considering and evaluating SPT as an alternative first line drug for the treatment of uncomplicated urogenital gonorrhea in China. However, SPT is not suitable for the treatment of pharyngeal gonorrhea even though the prevalence of resistance is low in most areas of the world [39]. The MICs of SPT need to be closely monitored, and any signs of increased SPT MIC warrant a thorough investigation and close attention by the policy makers.
The current study has a number of limitations. 1) All of the N. gonorrhoeae isolates tested in this study were collected from the Sexually Transmitted Infection Clinic of the SSDH, and only a small number of isolates were tested, accounting for 2.1% of the total reported cases in 2012 in Shanghai. 2) A lack of demographic and clinical data prevented any comparisons of N. gonorrhoeae antimicrobial resistance profiles with patient characteristics; 3) Molecular subtyping of the isolates was not performed. This would provide insight into whether there is clonal spread of resistant isolates and should be the focus of future studies. 4) The agar dilution MIC testing method has an intrinsic error rate of plus/minus one dilution. While using a breakpoint of 0.125 mg/L for CRO nonsusceptible isolates, some isolates may actually have MICs of 0.06 mg/L.

Conclusions
In summary, antimicrobial resistance of N. gonorrhoeae to PEN, TET and CIP is highly prevalent in Shanghai. A high proportion of N. gonorrhoeae isolates were found to be CRO nonsusceptible (MIC ≥ 0.125 mg/L). Plasmidmediated resistance and MDR are also prevalent in China. N. gonorrhoeae isolates are susceptible to SPT and this antibiotic needs to be further evaluated as a treatment option for urogenital gonococcal infections. Our findings suggest that the CRO dose should be increased to 500 mg in areas with a high prevalence of CRO nonsusceptible isolates. We propose that combination therapy using older antibiotics should be used with caution unless drug synergistic effects can be confirmed. Finally, both national and regional antimicrobial susceptibility surveillance programs are important to inform policy makers in a timely manner on the appropriate modifications of treatment guidelines.

Additional file
Additional file 1: Figure S1