Azithromycin-resistant Neisseria gonorrhoeae isolates in Guangzhou, China (2009–2013): coevolution with decreased susceptibilities to ceftriaxone and genetic characteristics

The global spread of multidrug-resistant Neisseria gonorrhoeae (N. gonorrhoeae) is a growing public health threat. Worryingly, clinical treatment failures with the extended-spectrum cephalosporins (ESCs) [1], the last remaining options for empirical first-line monotherapy, have recently been reported. Accordingly, dual antimicrobial therapies have been introduced in several countries [2, 3], including China [4]. These consist of ceftriaxone (250–500 mg intramuscularly) or cefixime (400 mg orally, if ceftriaxone is not an option) together with azithromycin (AZM, 1–2 g orally) for treatment of uncomplicated gonorrhea. AZM was recommended for patients with gonorrhea or coinfection with Chlamydia trachomatis in China around the year 2000 [5], and since then has been broadly used because of its wide availability and ease of administration. In China, AZM-resistant (AZM-R) N. gonorrhoeae were first identified during 2001–2003 [6], and the first AZM-R isolates were identified in Guangzhou in 2009 [7]. Treatment failures with 2 g of AZM and high-level AZM resistant N. gonorrhoeae isolates with a minimum inhibitory concentration (MIC) ≥ 256 mg/L have been verified in several countries [8, 9]. Recently, a surveillance of antimicrobial resistance in gonococci from Canada resulted in two high-level AZM-R isolates (MIC ≥ 2048 mg/L), which may be due to mutation A2143G (N. gonorrhoeae numbering) in the four copies of the 23S rRNA gene [9].

Mutations at various genes such as 23S rRNA, mtrR and penA (encoding penicillin-binding protein 2, PBP2), have been identified associating with chromosomally mediated resistance to AZM [10, 11] and ESCs [12] in N. gonorrhoeae. Globally, AZM-R together decreased susceptibility to ESCs in N. gonorrhoeae has only rarely been reported [9, 13]. In this study, we investigated the prevalence of decreased susceptibility to ceftriaxone (CRO^D) in AZM-R N. gonorrhoeae isolates, and the molecular characteristics of AZM-R N. gonorrhoeae isolates from January 2009 to November 2013 in Guangzhou, China.

The present study combines antimicrobial susceptibility determinations with molecular-based analysis of AZM-R in N. gonorrhoeae isolated from 2009 to 2013 in Guangzhou, China. As observed in other countries worldwide [9, 13, 18], an upward shift in MIC by year and a high percentage of AZM-R were identified in this study, which was also in line with a previous report in two Chinese cities, Nanjing and Chongqing, between 2008 and 2009 [5]. The WHO recommends discontinuation of the empirical use of an antibiotic once 5 % of locally acquired gonococcal isolates are resistant [19]. Accordingly, AZM has not been recommended as a monotherapy for gonococcal urethritis or cervicitis in China, as well as in many other countries worldwide. To improve treatment efficacy and to delay the further selection of cephalosporin-resistant N. gonorrhoeae, most current guidelines now recommend ESCs and AZM in combination as the first-line treatment for gonorrhea [2‐4, 9]. However, more recently, AZM-R N. gonorrhoeae has been rarely and sporadically reported to also have reduced susceptibility to ESCs [9, 13], threatening the future efficacy of current therapeutic recommendations. Similarly, the present study showed that the prevalence of combined AZM-MLR/CRO^D isolates significantly increased as compared with that in all identified N. gonorrhoeae isolates.

Previous studies investigating AZM-R and CRO^D have focused on the mutations of three genes: 23S rRNA, mtrR and penA [9‐12]. Mutations at various positions within the central loop of domain V of the 23S rRNA gene are thought to lead to resistance by reducing the ability of the 23S rRNA protein to bind the antibiotics such as AZM [10]. Previous reports have shown that the mutation C2611T in any of the four alleles of the 23S rRNA can result in a lower level of resistance to AZM [10]. In addition, the mutation A2059G (Escherichia coli numbering) in at least three of the four alleles can confer AZM-HLR, and AZM-sensitive isolates containing this single allele mutation can quickly develop high-level resistance in the presence of erythromycin [10]. In the current study, only seven AZM-R isolates contained mutation C2611T, whereas the remaining large number of AZM-R isolates contained four wild-type alleles. These findings contrast, however, with other reports from Canada [9], the United States [11, 20] and the United Kingdom [10], which most of the AZM-MLR isolates contained this mutation was more than 80 %. Notably, all the above-mentioned seven AZM-R isolates that contained the mutation C2611T showed reduced susceptibility to ceftriaxone. The mutation A2059G, which is linked to AZM-HLR, was not detected in the present study. In the mtrR gene, specific mutations in the promoter or coding region can lead to decrease the MtrCDE efflux pump repression and subsequently increase export of the antimicrobials [20]. Loss of the MtrCDE efflux pump can significantly increase susceptibility to AZM, ESCs, penicillin, ciprofloxacin, and solithromyc in N. gonorrhoeae isolates [21]. In this study, a single nucleotide deletion in the mtrR promoter showed over 85 % in AZMR-R isolates, but not significantly difference between combined AZMR-R/CRO^S isolates and combined AZMR-R/CRO^D isolates, in line with a previous report that such a deletion may not implicate in CRO^D [12]. H105Y, the most prevalent single mutation, was not significantly difference in AZM-LLR isolates as compared to AZM-MLR isolates. Interestingly, the combined mutation in mtrR was only observed in AZM-MLR isolates, but not in in AZM-LLR isolates. Mutation in penA has been associated with decreased susceptibility or resistance to a number of beta-lactam agents, especially to ESCs [22]. The mutations G545S, I312M, and V316T in mosaic alleles of PBP2 were early suggested as important for the increased ESCs resistance [23]. However, a recent study revealed that these three mutations had only a little to effect on resistance, and their capacity to increase resistance to ESCs was dependent on the presence of other mutations in the mosaic alleles [24]. Mutation patterns XIII, XVII and XVIII have been reported to only present in CRO^D isolates in China [12] and Australia [22]. These three patterns contained G542S, P551S and/or P551L mutations, which have been associated with increased MIC of CRO [25]. However, their effects on the ceftriaxone MIC have not yet been proven by site-directed penA mutations in isogenic strain backgrounds [26] and the present study showed that these three patterns were present in both CRO^S and CRO^D isolates (data not shown). The most prevalent mutation pattern in AZMR-R isolates from Canada was XXXIV, which was showed to AZM-R together with reduced susceptibility to ESCs [9]. By comparison, this study revealed the most prevalent mutation patterns (V, XVIII and XXI) were the same in AZMR-LLR, AZMR-MLR, and combined AZMR-R/CRO^D isolates, respectively. Overall, the results of our study showed that the mutations of these three genes had the considerable diversity, and that either one of these three genes alone or combined mutation patterns of these three genes cannot account for AZM-MLR or combined AZM-R/CRO^D in our N. gonorrhoeae isolates.

NG-MAST analysis and the resulting development of a phylogenetic tree in this study also indicated a high degree of genetic diversity of N. gonorrhoeae in Guangzhou from 2009 to 2013. This observation may be due to the highly limited epidemiologic information of this study because our data do not represent the whole country, or the flow of large numbers of temporary population in Guangzhou, which lead to great opportunity for transport of new STs [27]. Also, AZM is extensively used in China [5] and has long elimination half-life. Thus we postulate that the diversity of STs developed, in part, in response to antibiotic pressure. Some STs that were observed in the current study (ST3356, ST1866, ST1766, and ST4313) had been noted in a previous report of emerging AZM-R N. gonorrhoeae in Nanjing and Chongqing, China [5], but these did not include STs (ST470, ST649, ST3158, ST1704, ST359, and ST696) that were reported previously in other countries and have been shown to account for most of the resistance or high-level resistance to AZM [9, 18, 28‐30].

AZM-R N. gonorrhoeae isolates are increasing and, in combination with CRO^D, have now been detected in Guangzhou. The combined AZM-R/CRO^D in N. gonorrhoeae is a relatively new phenomenon, and the mechanism for the emergence of these AZM-R/CRO^D isolates remains unknown and needs further studied. Therefore, antimicrobial susceptibility/resistance and the molecular epidemiology of AZM-R and/or CRO^D deserve continuous surveillance, which will be critical in providing valuable information for current and future therapeutic options in China.