This study reports the antimicrobial susceptibility/resistance and molecular characteristics of N. gonorrhoeae isolates from India, Pakistan, and Bhutan in 2007–2011. A high prevalence of resistance was observed for ciprofloxacin (94%), penicillin G (68%), erythromycin (62%), and tetracycline (55%). These data are largely in accordance with previous surveys in the Southeast Asian region. Accordingly, in India, penicillin resistance has varied from 20% to 79%, tetracycline resistance from 0% to 97%, and ciprofloxacin resistance from 11% to 100% [34, 49]. In Sri Lanka, 97% and 8.2% resistance have been reported to penicillin and ciprofloxacin, respectively, and in Bangladesh resistance to ciprofloxacin, penicillin, and tetracycline was found to be 76%, 33% and 57%, respectively . Finally, in Pakistan 92%, 87%, and 78% resistance to ofloxacin, penicillin G, and tetracycline, respectively, has been noted .
Thus, in the present study none of the isolates was susceptible to ciprofloxacin, with 94% and 6% of the isolates being resistant and intermediately susceptible, respectively. gyrA mutations (S91F) were found universally, with many of the isolates containing additional mutations in the quinolone resistance determining regions of the gyrA and parC genes (Table 2), which confer a high level of resistance to fluoroquinolones (e.g., ciprofloxacin) [28, 30, 38]. The very high rate of fluoroquinolone resistance may be an indicator of the overuse and misuse of this class of antimicrobials in this region of the world, as caused by over-the-counter availability, unregulated and counterfeit medicines, self-medication or unqualified practitioners who prescribe a full range of treatments [49, 50]. Although fluoroquinolones are no longer recommended for first-line treatment of gonorrhoea in most parts of the world [4, 11, 51–54], most worryingly they are still being used excessively by, in particular, private practitioners and quacks in Southeast Asia.
Another very commonly prescribed antimicrobial in the Southeast Asian region is azithromycin, which is frequently used in syndromic management of STIs because of the convenience of single oral dose therapy for many infections and its efficacy against several STI pathogens, including N. gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Haemophilus ducreyi, Klebsiella granulomatis, and Treponema pallidum[53, 55]. Previously, only some single N. gonorrhoeae isolates with resistance to azithromycin have been reported in India and Bangladesh [36, 37]. However, in the present study only 77% of the isolates were susceptible to azithromycin and 23% showed resistance (7.7%) or intermediate susceptibility (15%), showing its unsuitability for use as an empirical first-line therapy for gonorrhoea in this region. Furthermore, resistance to azithromycin, including very high-level resistance, has been described in many countries globally [4, 11, 12, 28, 56–60]. Of the 15 isolates that showed resistance or intermediate susceptibility to azithromycin in the present study, 13 (87%) showed mutations in the mtrR promoter, which enhances the expression of the MtrCDE efflux pump, exporting azithromycin out from the gonococcal cells and thus confers increased azithromycin MICs in these isolates [11, 12, 28, 29].
Among the isolates examined in this study, no resistance was observed to ceftriaxone, cefixime, and spectinomycin. Accordingly, ceftriaxone, cefixime, and spectinomycin can be recommended as an empirical first-line therapy of gonorrhoea in this region, although judicious use of these antimicrobials (particularly spectinomycin) is imperative. Some single isolates with spectinomycin resistance and strains with “less susceptibility” to ceftriaxone have been reported in India and Bangladesh [32–34, 37]. However, few isolates from this region have been examined and mostly disc diffusion methods (with lower breakpoints for “less susceptibility” to ceftriaxone and resistance to spectinomycin compared with international MIC-determining resistance methods) for antimicrobial susceptibility testing have been used rather than internationally validated, quality assured MIC-determining methods. Nevertheless, our data, including MICs of up to 0.064 mg/L for ceftriaxone and cefixime, emphasise the importance of promptly implementing expanded antimicrobial resistance surveillance in India, Pakistan, and Bhutan. Corresponding to the absence of resistance to ESCs, no penA mosaic alleles or A501-altered PBP2 amino acid sequences were found in any of the isolates. These alterations of the penA gene are critical for resistance to cefixime and ceftriaxone [11–13, 16, 19].
Disquietingly, a majority of the gonococcal infections (as well as other STIs) in India, Pakistan, and Bhutan remain undiagnosed (using laboratory testing), or go unreported, or both. STIs are also still considered a taboo, and accordingly, people do not visit physicians with their ailment. In other cases the patients have been inadequately treated multiple times when they reach the hospitals, or the samples are inadequately transported to the laboratories, both of which diminish the likelihood of recovering gonococcus in culture. Moreover, most of the laboratories are not well equipped to provide adequate culture and characterisation facilities. Hence, there is a need to strengthen the existing system by providing more resources and training in this region for enhanced surveillance and detection of gonorrhoea and other STIs.
The N. gonorrhoeae population in India, Pakistan, and Bhutan was found to be highly diversified, with 49 NG-MAST STs identified among the 65 isolates examined (Additional file 1: Table S1). The high number of unique STs (n=39) may be a consequence of random sampling (only viable isolates examined) over several years, sub-optimal diagnostic procedures, incomplete epidemiological surveillance and ineffective contact tracing, local emergence of new STs, and import of strains from abroad. Nevertheless, some minor ST clusters were identified, indicating multiple sexual transmission chains. Furthermore, of the 18 isolates from Pakistan, one isolate was assigned ST368 that also was found in India, suggesting circulation of a few common lineages between the two countries, whereas 11 isolates were of new STs. One isolate from India also belonged to ST1407, which has been shown to be a internationally spread successful gonococcal clone that accounts for the majority of the decreased susceptibility and resistance to ESCs and treatment failures with cefixime worldwide [12, 17–19, 61, 62]. This clone has also shown its ability to develop high-level resistance to ceftriaxone . Surprisingly, the ST1407 isolate from India did not contain the penA mosaic allele XXXIV (instead it contained the penA allele XXXV)  that has been strongly associated with ST1407 [12, 17, 20, 61, 63]. Accordingly, despite showing high-level resistance to ciprofloxacin and intermediate susceptibility to azithromycin, the Indian ST1407 isolate displayed low MICs of ceftriaxone (0.008 mg/L) and cefixime (<0.016 mg/L). The seven isolates from Bhutan (belonging to ST6061, ST6062, ST6063, and ST6064) did not share STs with any of the isolates from India or Pakistan.
Because of the small number of isolates examined in the present study, the selection bias for these isolates cultured during several years and the high number of gonococcal infections remaining undiagnosed using laboratory testing in the included countries, the results of the present study need to be interpreted with caution. In future studies, additional isolates will hopefully be available as well as epidemiological data linked to the gonococcal isolates.