Antibiotic susceptibility pattern of Neisseria gonorrhoeae strains isolated from five cities in India during

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1 RESEARCH ARTICLE Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 DOI 0.099/jmm Antibiotic susceptibility pattern of Neisseria gonorrhoeae strains isolated from five cities in India during Sangeeta V. Kulkarni, Manju Bala, 2 Syeeda A. Muqeeth, 3 G. Sasikala, 4 Amit P. Nirmalkar, R. Thorat, 5 H. Kambli, 5 Jyoti Sawant, Arun Risbud, Raman R. Gangakhedkar and Sheela V. Godbole, * Abstract Purpose. Emergence of multidrug resistance in Neisseria gonorrhoeae, an STI of public health significance is the biggest challenge to gonorrhoea control. Monitoring for antimicrobial resistance is essential for the early detection of emergent drug resistance patterns. Methodology. One hundred and twenty four N. gonorrhoeae strains were isolated between September 203-August 206 [82-New Delhi, 3-Pune, 3-Mumbai, 20-Secunderabad and 6-Hyderabad] to determine antimicrobial susceptibility and to compare the CLSI disc diffusion method with Etest for these strains. The results of the two methods were compared by using kappa statistics. Results. Ninety eight percent [CI: ] of isolates were resistant to ciprofloxacin, 52 % [CI: ] to penicillin, 56 % [CI: ] to tetracycline and 5 % [CI:.2 8.8] to azithromycin. All the strains were susceptible to spectinomycin, ceftriaxone and cefixime except for two strains which showed decreased susceptibility to ceftriaxone and cefixime. Kappa scores for penicillin, azithromycin, ciprofloxacin, ceftriaxone and cefixime showed that the CLSI method had high agreement with Etest while tetracycline had substantial agreement. Conclusion. Our data suggest that the disc diffusion method which is both cost effective and more feasible, can effectively be used routinely for monitoring antibiotic susceptibility in N. gonorrhoeae, in limited resource countries like India. We demonstrate the emergence of decreased susceptibility to ceftriaxone and cefixime and threshold levels of resistance to azithromycin in India. This underscores the importance of maintaining continued surveillance for antibiotic resistance in N. gonorrhoeae and a potential requirement for strategic change in guidelines in the not so distant future. INTRODUCTION Gonorrhoea caused by Neisseria gonorrhoeae (NG) a bacterial sexually transmitted infection (STI) remains a major global public health concern, primarily because of the development of resistant organisms. It is important not only for its propensity to cause long term reproductive tract morbidity in men and women but also for increasing the risk of human immunodeficiency virus acquisition []. The precise global burden of N. gonorrhoeae is difficult to establish because of the lack of diagnostic capability and reporting systems in many parts of the world. Recent WHO estimates of incidence suggest that approximately 78 million new cases of gonorrhea occur globally each year [2]. The Centers for Disease Control and Prevention (CDC) reported over cases in 204, making it the second most common notifiable infection in the United States [3]. Regular monitoring of antimicrobial susceptibility of N. gonorrhoeae is expected to be a standard practice. However it is not adequately implemented worldwide for various reasons which include invasive specimen collection procedures, the fastidious nature of the organism, a need for specialised culture media and trained personnel [4]. Moreover, as a consequence of emphasis on syndromic management of STIs and the introduction of nucleic acid amplification tests (NAATs); isolation of N. gonorrhoeae for susceptibility testing has reduced, especially in India. Received 2 October 207; Accepted 4 December 207 Author affiliations: ICMR-National AIDS Research Institute, Pune, Maharashtra, India; 2 Apex Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India; 3 Gandhi Medical College and Hospital, Secunderabad, India; 4 Osmania Medical College, Hyderabad, India; 5 The Humsafar Trust, Mumbai, India. *Correspondence: Sheela V. Godbole, sgodbole@nariindia.org Keywords: antibiotic resistance; India; N. gonorrhoeae. Abbreviations: AMR, antimicrobial resistance; CI, confidence interval; CLSI, Clinical and Laboratory Standard Institute; GC agar, Gonococcal agar; MDR, multidrug resistance; NARI, National AIDS Research Institute; NAATS, nucleic acid amplification tests; NG, Neisseria gonorrhoeae; PPNG, Penicillinase producing Neisseria gonorrhoeae; STI, sexually transmitted infection; TRNG, tetracycline resistant Neisseria gonorrhoeae ã 208 The Authors IP: On: Thu, 2 Dec :06:24

2 Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 In the absence of a vaccine, the control of gonorrhoea is achieved through effective antimicrobial therapy to treat the infection, reduce transmission and to prevent complications. Many countries including India, advocate syndromic approach for the management of STIs. N. gonorrhoeae has developed resistance to sulphonamides, penicillin, tetracycline, macrolides and fluoroquinolones in many parts of world [5, 6]. According to the WHO, the use of an antimicrobial in empiric treatment is discontinued when the rates of therapeutic failures and/or antimicrobial resistance reach a level of 5 % [7]. Over the past few years, gonococcal resistance to third-generation cephalosporins recommended as first choice of treatment has emerged rapidly in different parts of world. Consequently, the CDC currently recommends dual therapy with ceftriaxone plus azithromycin as treatment for uncomplicated gonorrhea [8]. Though there are no reports of treatment failure with cephalosporins from India, there are reports of treatment failure due to cephalosporins from Japan, Hong Kong, Norway, United Kingdom, France, Norway, Austria and South Africa [6, 9 ]. In the European, WHO Western Pacific and WHO Southeast Asian regions resistance or decreased susceptibility to ceftriaxone/cefixime was found to be 5 % whereas in Canada, United States and Africa it was found to be 5 % [6]. Furthermore, elevated minimum inhibitory concentrations (MICs) to cephalosporins have been reported from Japan, Australia, China, Hong Kong, USA, various parts of Europe and India [2, 3]. While there are no universally accepted guidelines for testing antimicrobial susceptibility of N. gonorrhoeae by disc diffusion method, different techniques are practiced, like the Clinical and Laboratory Standards Institute (CLSI) method, the Australian Gonococcal Surveillance Program method and the British Society for Antimicrobial Chemotherapy method. The recommended procedure for antimicrobial susceptibility testing of gonococci is a determination of the MIC by agar dilution or Etest. There have been sparse reports of comparison of these different methods of antimicrobial susceptibility [4 6]. Emergence of multidrug resistance (MDR) in N. gonorrhoeae can pose challenges in the control of gonorrhoea. Periodic monitoring of antimicrobial susceptibility of N. gonorrhoeae is essential for the early detection of the emergence of drug resistance for current use of treatment. Therefore, our study was conducted to determine antimicrobial susceptibility of N. gonorrhoeae isolates from five cities in India and to compare the disc diffusion method with Etest for seven antibiotics. METHODS Sample collection Specimens were obtained from men with symptoms of urethritis and women with symptoms of cervical discharge attending sexually transmitted disease clinics between September 203-August 206 from different cities (New Delhi, Pune, Mumbai, Hyderabad and Secunderabad) in India. Isolation and identification of N. gonorrhoeae isolates The specimens were inoculated directly either onto the Modified Thayer Martin medium or on chocolate agar and saponin lysed blood agar with vancomycin, colistin, nystatin, and trimethoprim supplement. The culture plates were incubated in a dessicator (candle jar) or plastic jar at 36 C with 5 % CO 2 and 45 % moisture for h. The swabs from Hyderabad and Mumbai were collected in Stuart transport medium and transported to NARI for culture. The colonies suspected to be N. gonorrhoeae were presumptively identified by Gram stain, oxidase and confirmed using superoxal tests, rapid carbohydrate utilisation test and Phadebact monoclonal GC kit (MKL Diagnostics). N. gonorrhoeae isolates were tested for b-lactamase production by the chromogenic cephalosporin method using nitrocefin discs (Becton, Dickinson and Company). All the N. gonorrhoeae isolates were stored at 70 C in nutrient broth containing 20 % glycerol at NARI [7]. Antimicrobial susceptibility testing Antimicrobial susceptibility testing for strains isolated from Delhi and Osmania Medical College, Hyderabad were performed at STD Reference laboratory at Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi. The antimicrobial susceptibility testing for strains isolated from Mumbai, Gandhi Medical College (Secunderabad) and Pune were performed at National AIDS Research Institute, Pune. The same methods were used in both settings. Inoculum preparation A homogenous suspension of 0.5 McFarland turbidity was prepared from 8 to 24 h old culture growth on GC agar base (Himedia laboratories) with % BBL Isovitalex Enrichment (Becton Dickinson). For both the methods, inoculum was prepared by emulsifying 2 3 colonies in 2.5 ml sterile saline (0.85 %). This suspension was used within 5 mins. All the plates for antimicrobial susceptibility testing were incubated at 36 C for 8 24 h [7]. CLSI disc diffusion method Antibiotic susceptibility was performed on GC agar base (Himedia laboratories) with % BBL Isovitalex enrichment (Becton Dickinson) with the following discs (Hi media laboratories, India and Oxoid, UK): penicillin (0IU), tetracycline (30 µg), ciprofloxacin (5 µg), ceftriaxone (30 µg), cefixime (5 µg), spectinomycin (00 µg) and azithromycin (0 µg). The results were interpreted by measuring the inhibition zone diameters and categorised as susceptible (S), intermediate (I) and resistant (R) using CLSI guidelines [8]. Etest method The Etest method was used as the reference method. The Minimum Inhibitory Concentration of penicillin, tetracycline, ciprofloxacin, ceftriaxone, cefixime, spectinomycin and azithromycin were determined by Etest method (Bio- Merieux) according to manufacturer s instructions on GC IP: On: Thu, 2 Dec :06:24

3 Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 agar base (Hi media laboratories, India and Difco Laboratories, USA) with % BBL Isovitalex enrichment (Becton Dickinson). The zone size was interpreted as susceptible, intermediate and resistant according to the CLSI guidelines [8]. Quality control strains World Health Organisation (WHO) reference strains A, K, L and G, received under the WHO Gonococcal Antimicrobial Surveillance Programme from South East Asia Region Reference Laboratory (New Delhi, India), N. gonorrhoeae ATCC and eight sensitive previously isolated strains (sensitive to penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone) preserved at NARI, were included as controls. Breakpoints for resistance were in accordance with the CLSI [8]. Statistical analysis Data were entered into Microsoft Excel and 00 % quality check was performed by a second person. Data analysis was performed using SPSS 5.0 (SPSS Inc). The susceptibility profile using the CLSI disc diffusion method was compared with the susceptibility profile obtained by the reference method i.e. the Etest. Discrepancies were categorised into three categories for each antibiotic, as minor (susceptible or resistant strain misinterpreted as intermediate), major (a susceptible strain misinterpreted as a resistant strain) and very major (a resistant strain misinterpreted as susceptible). Kappa statistics were used to determine the agreement between two methods. Kappa values of were considered as showing almost perfect agreement, Substantial agreement, Moderate agreement, Fair agreement, Slight agreement, <0 Less than chance agreement) [9]. RESULTS In all 24 N. gonorrhoeae strains were studied [82-Delhi (North), 3-Pune (West), 3-Mumbai (West), 20-Secunderabad (South East) and 6-Hyderabad (South East)]. Of these, 22 (98 %) were isolated from male patients. The men had a mean age of 29 years, while the two female patients were 33 and 36 years old. Majority were married (57 %), 68 (55 %) were educated up to higher secondary level (XII yrs. of education), 53 (43 %) were graduates while 3 (2 %) were illiterate. Ninety eight (79 %) patients were employed either in the government or private sector, 8 (6.5 %) were self employed, 6 (2.9 %) were students and 2 (.6 %) were housewives. The participants had a history of sexual exposure to persons other than spouse (46 %), female sex workers (3 %) and men having sex with men (23 %). Past history of STIs was reported by 06 (85 %) and 95 % did not report history of antibiotic treatment exposure in the preceding 3 months. Antibiotic susceptibility The results of the antimicrobial susceptibility testing of all 24 isolates of Neisseria gonorrhoeae by CLSI disc diffusion technique are summarised in Table. Antimicrobial resistance profile by Etest method is shown in Table 2. All the strains were susceptible to spectinomycin, ceftriaxone and cefixime except for two strains which showed decreased susceptibility to ceftriaxone and cefixime (MIC=0.25 µg ml ). Ninety five percent [CI: ] of the strains were susceptible to azithromycin (MICs=< µg ml ) while 5 % [CI:.2 8.8] of the strains showed resistance (MIC= 8 µg ml ). All except two strains were resistant to ciprofloxacin. One of them showed intermediate susceptibility (MICs=0.5 µg ml ) and the other was susceptible (MIC=0.06 µg ml ) to ciprofloxacin. Of the resistant strains, 7 % [CI: ] were observed to have high level resistance to ciprofloxacin (MIC 4 µg ml ). Of the total isolates, 5 % were resistant to penicillin (MIC=-32 µg ml ) and 49 % showed intermediate susceptibility to penicillin (MIC= µg ml ). Forty six percent of the strains were resistant to tetracycline (MIC=2 32 µg ml ), 50 % [CI: ] strains showed intermediate susceptibility (MIC= µg ml ) while 4 % [CI: ] were susceptible to tetracycline (MIC= µg ml ). Penicillinase producing N. gonorrhoeae (PPNG) was observed in 37.8 % [CI: ] of the isolates and tetracycline resistant N. gonorrhoeae (TRNG) in 24. % [CI: ] of the isolates. Table. Antibiotic susceptibility pattern by CLSI disc diffusion technique for Neisseria gonorrhoeae isolates Antibiotic Class of antibiotic Susceptibility Profile (N=24) Resistant N (%) Intermediate/Decreased susceptibility N (%) Susceptible N (%) Penicillin Penicillins 64 (52 %) 60 (48 %) 0 Tetracycline Broad-spectrum 69 (56 %) 5 (4 %) 4 (3 %) Ciprofloxacin Fluoroquinolones 22 (98.4 %) (0.8 %) (0.8 %) Spectinomycin Aminoglycoside (00 %) Ceftriaxone Extended spectrum cephalosporins 0 2 (.6 %) 22 (98.4 %) Cefixime Extended spectrum cephalosporins 0 2 (.6 %) 22 (98.4 %) Azithromycin Macrolides 6 (4.8 %) 0 8 (95.2 %) IP: On: Thu, 2 Dec :06:24

4 Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 Table 2. Antibiotic susceptibility pattern and MIC range by Etest method for Neisseria gonorrhoeae isolates MIC values (µg ml ) Penicillin Tetracycline Ciprofloxacin Spectinomycin Ceftriaxone Cefixime Azithromycin R I S R I S R I S R I S R I S R I S R I S Total Two isolates which were interpreted as resistant by disc diffusion showed intermediate susceptibility by MIC values to penicillin (Tables and 2). Of the 4 isolates which were interpreted as resistant by disc diffusion, showed intermediate susceptibility and three were susceptible to tetracycline by MIC values. MDR (i.e. resistance to penicillin, tetracycline and ciprofloxacin) was noted in 35.5 % [CI: ] of all isolates. Different phenotypes of N. gonorrhoeae isolates are depicted in Table 3. There were no major discrepancies identified between CLSI and Etest methods. Table 4 shows a comparison of discrepancies of CLSI disc diffusion method and Etest as well as Kappa score for seven different antibiotics. Statistically, Kappa scores reveal that CLSI method was in almost perfect agreement with Etest for penicillin, azithromycin, ciprofloxacin, ceftriaxone, cefixime while tetracycline had substantial agreement with Etest. Kappa value for spectinomycin could not be calculated because there was only one category i.e. susceptible. DISCUSSION Our study shows a significant 35.5 % [CI: ] MDR (ciprofloxacin, penicillin and tetracycline) among N. gonorrhoeae strains in India and also highlights that levels of resistance to azithromycin are reaching the WHO designated cut-off in some regions of India. However, we also show that N. gonorrhoeae continue to remain largely susceptible to cephalosporins (ceftrixone and cefixime). The MDR strains isolated from our study were from New Delhi [59 %, CI: ], Secunderabad [27.3 %, CI: ] and Hyderabad [3.6 %, CI: ]. Previous studies from India have highlighted MDR resistance in New Delhi and Pune (20 40 %) [20, 2]. A large proportion of the strains in our study were from the New Delhi region followed by Hyderabad-Secunderabad. While the current study depicts emergence of MDR resistance from the southeastern part of India, the paucity of samples from western India (Pune and Mumbai) could be one of the explanations why MDR was not documented in this region. It is likely that extensive use of antibiotics without performing Table 3. Classification of 24 Neisseria gonorrhoeae isolates by phenotypes Phenotype Definition of phenotype Isolates n % PPNG TRNG PPNG/TRNG Chromosomally mediated resistance to penicillin Chromosomally mediated resistance to tetracycline Chromosomally mediated resistance to penicillin and tetracycline Sensitivity b-lactamase positive; Tetracycline MIC<6 µg ml b-lactamase negative; Tetracycline MIC6 µg ml b-lactamase positive; Tetracycline MIC6 µg ml Non-PPNG; MIC(penicillin)2 µg ml ; MIC(tetracycline)<2 µg ml Non-PPNG; MIC(penicillin)<2 µg ml ; MIC(tetracycline)2 µg ml and <6 µg ml Non-PPNG; MIC(penicillin)2 µg ml ; MIC(tetracycline)2 µg ml and <6 µg ml MIC<2 µg ml for penicillin and tetracycline 5 4. PPNG, Penicillinase producing Neisseria gonorrhoeae; TRNG, Tetracycline resistant Neisseria gonorrhoeae; MIC, minimum inhibitory concentration. IP: On: Thu, 2 Dec :06:24

5 Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 Table 4. Comparison of discrepancies between CLSI and Etest method for 24 Neisseria gonorrhoeae strains Antibiotics No. of discrepancies Kappa value Minor Major Very major Penicillin G % Tetracycline % Ciprofloxacin % Spectinomycin Ceftrixone % Cefixime % Azithromycin % susceptibility testing has generated not only an escalation of treatment failures, but also a positive selection pressure for MDR [9, 0]. Syndrome based treatment programs would need to factor in these data moving forward. Studies from United States and Europe have also reported high prevalence of MDR strains (20 55 %) while studies from African and South East Asian Countries have reported low prevalence ( 0 %) probably reflecting antibiotic access in these regions [22 26]. Resistance to ciprofloxacin was found to be 98.4 % [CI: ] in our study. The prevalence of ciprofloxacinresistant gonococci increased considerably from 67.3 % in 2003 to 99 % in 204 in India [27, 28]. Studies from South East Asian countries and the WHO Western Pacific Regions report high levels of ciprofloxacin resistance (70 00 %) [29, 30] while resistance to ciprofloxacin (5 60 %) is lower in United States, African and European countries [3, 3, 32]. However, less than 2 % resistance to ciprofloxacin was observed in the WHO Western Pacific region countries (Fiji and New Caledonia) [6]. Although levels (5 %) [CI: %] of azithromycin resistance were relatively low in our study they are approaching the 5 % cut-off accepted as a public health marker. These resistant strains have only been reported from Delhi both in our study and in the past [33]. However our recent data serve as an alert for the Indian subcontinent and there is a need to monitor clinical resistance to azithromycin based treatment in India. Azithromycin is a frequently used antibiotic in India for many ubiquitous medical conditions by medical practioners particularly due to its ease of administration and cost. Globally United States, Canada, Europe and Japan have reported high levels of azithromycin resistance (20 40 %) [34 36]. In order that India does not join these countries due care and attention to emerging azithromycin resistance needs to be given along with physician education. We report 00 % susceptibility to spectinomycin; however resistant strains have been reported from India (New Delhi and Kolkata) during the period [29] and reports from South East Asian Countries and WHO Western Pacific regions range between 5 0 % [37]. The emergence of resistance in adjoining regions of South East Asia should be taken seriously. Spectinomycin is not routinely used in India and this may explain the retention of efficacy of this antimicrobial. As expected, resistance to penicillin was found to be 5 % [CI: ] which was much higher than United States, Europe and African countries (0 40 %) [30, 35, 38 40]. The prevalence of penicillin resistance in India increased from 8 % in 983 (Mumbai) to 2 % (New Delhi) during and 48 % by 202 [33, 4]. Data from Pune showed an increase in penicillin resistance from 3 % in 996 to 00 % in 2007 [42]. However, since then, there has been a documented decline 48.4 % in 202 [2] similar to our study. The prevalence of PPNG 37.8 % (28.2 %-New Delhi and 9.6 %-Secunderabad) in this study was lower (47.9 %) than reports from Delhi during [33]. Studies from South East Asian countries and WHO Western Pacific Regions have reported 3 70 % PPNG strains [29] while Europe, Africa and USA have reported low prevalence (0. 20 % PPNG) [3, 43, 44]. It may also be useful to continue to track trends in resistance to penicillin moving forward to watch for a possible reversal in the burden of resistant strains. Significantly, our study found two isolates with decreased susceptibility to ceftriaxone and cefixime, which currently form the backbone of syndromic management in India. Both of these strains were from New Delhi. Decreased susceptibility has also been previously reported from New Delhi, India ( %) [29, 33]. Cephalosporins form the mainstay for genital discharge therapy in India and again our data could be an important and early warning signal for emergence of resistance. Low level resistance to ceftriaxone and cefixime has been reported from Europe, United States and in Asia (0.3.4 %) while Belgium, Denmark, Greece, Norway, Hong Kong, Japan and Korea have already reported 5 % resistance to cephalosporins [3, 6, 32, 45, 46]. Our study showed significant agreement between CLSI and Etest method for antibiotic susceptibility testing of penicillin, ciprofloxacin, ceftriaxone, spectinomycin, azithromycin and cefixime, most of which are antibiotics of interest in long term. The need for increased surveillance of N. gonorrhoeae isolates requires an accurate and simple method to determine antimicrobial susceptibilities. Our data support the CLSI method of disc diffusion as an equally useful tool for AMR surveillance, especially in lower middle income countries due to ease of availability, lower cost, feasibility and accuracy. In spite of many efforts it was a challenge to obtain strains from western region (Pune Mumbai) as there has been an overall decrease in the presentation of symptomatic cases with frank urethral/cervical discharge in outpatient clinics. Thus, the New Delhi region contributed a majority of the strains in our study, followed by the southeastern region of India. It is likely that our study findings are driven by this unequal distribution and less reflective of the western region of India. IP: On: Thu, 2 Dec :06:24

6 Kulkarni et al., Journal of Medical Microbiology 208;67:22 28 In conclusion, while we document a high resistance to ciprofloxacin, penicillin and tetracycline, our study highlights that ceftriaxone and cefixime continue to be useful as an empirical first-line therapy for gonorrhea in India. Cefixime along with azithromycin is recommended by the national programme in syndromic management of gonorrhea. The emergence of resistance to azithromycin and documentation of decreased susceptibility to ceftriaxone and cefixime underscores the importance of maintaining a continued surveillance for antibiotic resistance in N. gonorrhoeae. CLSI test method would be equally useful method for susceptibility test and may be utilised to increase AMR surveillance for N. gonorrhoeae in lower-resource Indian laboratories. Funding information This study was a part of an extramural project funded by the Indian Council of Medical Research. Acknowledgements Authors thank Mr Vivek Anand, Ms Shruta Rawat, Dr Alpana Dange (Humsafar trust, Mumbai), Mr Bindumadhav Khire (Samapathik Trust, Pune) and Dr Shraddha Bapat (NARI) for providing support for collection of samples. Authors are grateful to Mrs Leelamma Peter, Mr Naveen Chandra Joshi and Dr Vikram Singh (Safdarjung hospital, New Delhi) for all the technical help. The authors also thank the Ex-Director of NARI for extending support for this study. We gratefully acknowledge the support of Indian Council of Medical Research, New Delhi for funding this study. Conflicts of interest The authors declare that there are no conflicts of interest. Ethical statement This study was approved by the Research Ethics committee of ICMR- National AIDS Research Institute, Pune (NARI/EC Protocol number: 202 7). References. Holmes KK, Sparling PF, Stamm WE, Piot P, Wasserheit JN et al. Sexually Transmitted Diseases, 4th ed. 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