Decreased susceptibility of Neisseria gonorrhoeae isolates from Switzerland to Cefixime and Ceftriaxone: antimicrobial susceptibility data from 1990 and 2000 to 2012

Background Neisseria gonorrhoeae can rapidly develop resistance to antimicrobial agents. Over the last years, decreased gonococcal susceptibility to third-generation cephalosporins, especially cefixime, emerged worldwide. Therefore, current international guidelines recommend dual therapy for gonorrhoea with ceftriaxone plus either azithromycin or doxycycline. Gonococcal susceptibility data in Switzerland are sparse. Methods We investigated the prevalence of antibiotic susceptibility of N. gonorrhoeae in specimens collected between 1990 and 2012 at the University of Zurich, Switzerland. Minimum inhibitory concentrations (MICs) for cefixime, ceftriaxone, ciprofloxacin, and penicillin were determined by Etests. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to define reduced susceptibility. Results A total of 320 isolates were tested. Between 1990 and 2006 all tested samples were susceptible to both cephalosporins. Subsequently, the prevalence of elevated MICs for cefixime increased to 10.4% (2007/2008), 11.5% (2009/2010), and 11.4% (2011/2012); and for ceftriaxone to 2.4% (2007/2008), 4.7% (2009/2010), and 0% (2011/2012), respectively. The prevalence of resistance to ciprofloxacin (72.7%) and penicillin (22.7%) was high in 2011/2012. Conclusions Decreasing susceptibility of N. gonorrhoeae to third-generation cephalosporins in Switzerland supports treatment recommendations with ceftriaxone plus azithromycin or doxycycline. Health-care providers need to be aware of possible treatment failures with cephalosporins. Continued surveillance of gonococcal antimicrobial resistance is essential.


Background
Infections with Neisseria gonorrhoeae may cause serious complications, including pelvic inflammatory disease, infertility, peritonitis, and chronic pain, and it enhances HIV transmission [1,2]. Effective treatment of gonorrhoea is mandatory to control disease transmission and prevent sequelae. However, N. gonorrhoeae rapidly developed antimicrobial resistance to all previously recommended first-line drugs, including penicillins, tetracyclines, and fluoroquinolones. Third-generation cephalosporins, such as cefixime and ceftriaxone, remain the only readily available active antimicrobial class. In recent years, however, gonococcal strains with reduced susceptibility to cefixime emerged and spread worldwide [3]. Clinical treatment failures with cefixime have been reported in Japan, several European countries, Canada, and South Africa [4][5][6][7][8][9][10]. In 2009, the first treatment failures to ceftriaxone were observed in Australia [11] and 2010 in Sweden [12]. Currently, the identification of extensively drug-resistant (XDR) N. gonorrhoeae strains with high-level ceftriaxone resistance in Japan [13], France [14], and Spain [15], are of major concern. In addition to high ceftriaxone minimum inhibitory concentrations (MICs), these XDR strains are typically multidrug-resistant, exhibiting additional resistance to fluoroquinolones as well as to older drugs. Consequently, most treatment guidelines now recommend combination therapy with the injectable agent ceftriaxone plus either azithromycin or doxycycline instead of oral monotherapy with cefixime [16,17].
In Switzerland, a strong rise in N. gonorrhoeae infections was observed during the past decade. Notifications have increased nearly threefold, with 521 reported cases in 2003, and 1569 cases in 2012, of which 77% occurred in men [18]. Reporting gonorrhoea to public health authorities has been mandatory in Switzerland since 1988. However, gonococcal susceptibility data in Switzerland are sparse [19]. Due to the current N. gonorrhoeae resistance problem, improving gonococcal antimicrobial resistance surveillance is of utmost importance and high on the agenda of the WHO's "Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae, 2012" [20]. Local antimicrobial surveillance is essential for evidence-based treatment recommendations. Given the lack of data, we investigated the distribution of antibiotic susceptibility of N. gonorrhoeae in specimens collected between 1990 and 2012.

Methods
All isolates were obtained in the northeastern part of Switzerland and the region of Zurich between January 1990 and December 2012. Specimens were cultured from symptomatic gonorrhoea patients with only one isolate taken per patient in case they were infected at multiple sites. Species confirmation and susceptibility testing was performed at the Institute of Medical Microbiology, University of Zurich. Specimens were cultured on selective media, i.e. until 2009 on Thayer Martin agar (Difco; Becton, Dickinson and Company, Basel, Switzerland), supplemented with IsoVitalex, and after 2009 on VCA3 agar (BioMérieux, Marcy-l'Etoile, France). The commercial biochemical gallery Api NH (BioMérieux) was used for the identification.
The following data was available for all isolates collected between 2005 and 2012: specimen source, date specimen obtained, sex, age, and specimen site. For all isolates collected before 2005 only date of specimen collection was available. The study was approved by the ethics committee of Zurich (ethics approval number: KEK-ZH-Nr. 2012-0510). were collected from men, 35 (16.5%) from women, and in 9 (4.2%) samples data on sex and age was missing. Median age for males was 35 years (range 17 to 86 years) and for females 31 years (range 16 to 66 years). The isolates were obtained from the urethra (60%), cervix (13%), other locations (4%), including anorectum, pharynx, joint and eye, or from unknown sites (24%).

Results
The results of antimicrobial susceptibilities are summarized in Table 1

Discussion
In Switzerland, N. gonorrhoeae strains with decreased susceptibility to third-generation cephalosporins are emerging. We observed increasing rates of cefixime resistant strains, with a prevalence of 11% between 2007 and 2012. Moreover, ceftriaxone MICs are rising. The proportion of isolates with elevated ceftriaxone MIC was 2.2% between 2007 and 2012.
Our findings are in line with the worldwide documented decrease in cephalosporin susceptibility of N. gonorrhoeae. In the European Gonococcal antimicrobial susceptibility surveillance project (Euro-GASP), 9% of isolates displayed decreased susceptibility to cefixime (MIC ≥0.25 mg/L) in 2010. Cefixime resistance rates were >5% in 11 of 21 European countries, and >15% in 5 countries, including Austria, Denmark, Slovenia, Spain and Cyprus [22]. In the United States the percentage of cefixime resistant isolates (MIC ≥0.25 mg/L) increased from 0.1% in 2006 to 1.4% in 2011 [23]. The rise was most pronounced in the West with resistance rates of 17% in Hawaii and 6.4% in San Diego, California, in 2011 [17].
These are in vitro data and clinical cephalosporin resistance has not yet been defined due to the fact that so far there have been too few documented treatment failures in order to establish a clear relationship between MIC and clinical treatment failure. In a recently published Canadian cohort study the rate of clinical treatment failure associated with isolates of N. gonorrhoeae with a cefixime MIC of ≥0.12 mg/L was 25.0% compared to 1.9% of isolates with a cefixime MIC of <0.12 mg/L [9].
In 2008, we isolated one strain with a MIC for ceftriaxone of 0.25 mg/L. No decreased susceptibility to ceftriaxone (MIC ≥0.25 mg/L) was detected from 2004 to 2010 among Euro-GASP isolates. However, in correspondence to our data, ceftriaxone MICs increased from 2009 onwards [22]. In the United States the percentage of isolates with elevated ceftriaxone MICs (≥0.125 mg/L) rose slightly from 0% in 2006 to 0.4% in 2011 [17]. As displayed for cefixime, no  threshold for resistance to ceftriaxone in N. gonorrhoeae has been defined yet. Increasing MICs for cephalosporins, however, may precede the emergence of resistance.
We found high ciprofloxacin resistance rates of N. gonorrhoeae, with 72.7% in 2012. Our data demonstrates that during the 1990s and early 2000s, when ciprofloxacin was first choice for treatment, fluoroquinolone-resistant isolates emerged, and rapidly increased after 2000, correlating with the observation in other countries around the world [24][25][26]. By 2007, in the United States, and even earlier in other regions, including Europe and Australia, fluoroquinolones were no longer recommended for the treatment of gonococcal infections [27,28]. In Europe, ciprofloxacin resistance was 53% (MIC ≥1 mg/L) in 2010 [22], and 13.3% in the United States in 2011 (≥1 mg/L) [23].
The World Health Organization (WHO) recommends discontinuation of empirical use of an antibiotic once 5% of locally acquired gonococcal isolates are resistant [20]. The high rates of strains with decreased cefixime susceptibility and first isolates with elevated MICs for ceftriaxone shown by our data, support the recommendation of dual therapy for gonococcal infections with ceftriaxone plus either azithromycin or doxycycline. Combination regimens have been adopted as strategies for the treatment of other bacterial infections in the context of multidrug resistance development. The oral cephalosporin, cefixime, should no longer be used as first-line treatment.
In light of identified strains with decreased ceftriaxone susceptibility, health-care providers need to be vigilant for treatment failures, even in patients treated with recommended antibiotic regimens. In persons treated with alternative regimens, such as cefixime, test of cure is recommended. Continued local surveillance of gonococcal antimicrobial susceptibility is important. Due to the widespread use of nucleic acid amplification testing, culture for N. gonorrhoeae with antimicrobial susceptibility testing is not routinely done in clinical practice.
To the best of our knowledge, this is the first report on cephalosporin susceptibility testing of N. gonorrhoeae in Switzerland. Longitudinal data on antimicrobial resistance in gonococci over a period of more than two decades was assessed. Nevertheless, the study has several limitations. The level of coverage was relatively low, attributed in a large part to the low number of gonococcal cultures taken routinely in clinical practice. The samples were not systematically collected within a sentinella system, which may result in selection of specimens with a higher resistance rate. Not all strains were tested for cephalosporins, which particularly concerns isolates obtained before 2006. However, the subset of 20-30% of all isolates stored, none showed decreased susceptibility for cefixime and ceftriaxone. It is therefore unlikely that we missed a relevant resistance signal. Finally, the isolates were not tested for azithromycin.

Conclusions
We found a high prevalence of N. gonorrhoeae with decreased susceptibility to cefixime, first strains with elevated MICs for ceftriaxone, and high resistance rates to ciprofloxacin. Treatment of gonorrhea with a combination regimen with ceftriaxone plus azithromycin or doxycycline is supported. Our data contributes to the global antimicrobial resistance surveillance of N. gonorrhoeae, and emphasizes the importance of further gonococcal antimicrobial resistance testing.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions HK, RZ and RW designed the study. SL and JM provided gonococcal isolates. MDM and RZ performed the laboratory analyses. All authors analysed and interpreted the data. HK and PH prepared the paper. All authors read and approved the final manuscript.