|Year : 2015 | Volume
| Issue : 1 | Page : 39-42
Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone
T Bharara1, P Bhalla1, D Rawat1, VK Garg2, K Sardana2, A Chakravarti1
1 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
2 Department of Dermatology and Sexually Transmitted Diseases, Maulana Azad Medical College, New Delhi, India
|Date of Submission||03-Sep-2013|
|Date of Acceptance||21-Mar-2014|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, Maulana Azad Medical College, New Delhi
Source of Support: None, Conflict of Interest: None
Context: Gonorrhoea is one of the most common sexually transmitted infections (STI) in developing countries and is a global health problem. Aims: To analyze the trend of antimicrobial susceptibility of Neisseria gonorrhoeae isolates over the years, in a tertiary care hospital of North India. Settings and Design: The study population comprised males with urethritis and females with endocervicitis attending STI clinic of our hospital. Materials and Methods: In our STI laboratory, all gonococcal isolates are subjected to antimicrobial susceptibility testing by disc diffusion method as per CLSI guidelines. β-lactamase production is determined by chromogenic cephalosporin test. Minimum Inhibitory Concentration (MIC) for ceftriaxone is determined by E-test. Statistical Analysis Used: Data were expressed as percentages. The differences in percentages were tested for statistical significance by using χ2 test and P values were determined. Results: The percentage of penicillinase producing N. gonorrhoeae (PPNG) increased from 8% in 1995-96 to 20% in 2004-05 and 88% in 2011-2013. Quinolone-resistant N. gonorrhoeae (QRNG) showed a significant increase from 12% in 1995-96 to 98.3% in 2004-05, while 84% isolates were found to be QRNG by 2011-2013. In January 2013 we detected our first gonococcal isolate with decreased susceptibility to third-generation cephalosporins; Ceftriaxone, Cefixime and Cefpodoxime (MIC for ceftriaxone = 0.19 μg/ml). Conclusions: The results of our study highlighted an alarming increase in the percentage of PPNG and QRNG strains over the years. Emergence of N. gonorrhoeae isolates with decreased susceptibility to third-generation cephalosporins is a cause of concern and thus emphasises the importance of antimicrobial susceptibility testing.
Keywords: Antimicrobial susceptibility, gonococcal antimicrobial surveillance program, Neisseria gonorrhoeae, penicillinase producing N. gonorrhoeae, quinolone-resistant N. gonorrhoeae
|How to cite this article:|
Bharara T, Bhalla P, Rawat D, Garg V K, Sardana K, Chakravarti A. Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone. Indian J Med Microbiol 2015;33:39-42
|How to cite this URL:|
Bharara T, Bhalla P, Rawat D, Garg V K, Sardana K, Chakravarti A. Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jul 10];33:39-42. Available from: http://www.ijmm.org/text.asp?2015/33/1/39/148374
| ~ Introduction|| |
According to World Health Organization (WHO) estimates, gonorrhoea is one of the most prevalent bacterial sexually transmitted infections (STI).  It is a major public health concern globally causing substantial morbidity and economic burden.  Over the last decade, Neisseria More Details gonorrhoeae has developed resistance against antimicrobial agents such as penicillin, tetracycline and quinolones in several countries including India. 
During 2009-12, the percentage of penicillinase- producing N. gonorrhoeae (PPNG), isolates from WHO South East Asian Region (SEAR) varied from 0% (Pune, Nagpur) to 88.9% (Bhutan), while the rates of resistance to tetracycline and resistance to ciprofloxacin varied from 9.8%-100% and 57%-100%, respectively.  The percentage of ciprofloxacin-resistant strains have varied in India not only in different parts of the country but also in the same city.  Further, resistance to spectinomycin and azithromycin has been observed very rarely in India, Bangladesh, Bhutan and Thailand. ,
Among WHO SEAR, decreased susceptibility to extended spectrum cephalosporins (ESCs) has been noted in India, Bhutan, Indonesia, Myanmar and Thailand, whereas resistance to ESCs has not yet been reported from these regions. 
The first reported treatment failure to cefixime occurred in Japan in 2002, followed by similar reports from other countries. , The emergence of resistance to oral cephalosporins like cefixime and more recent reports of H041 and F89 Ceftriaxone-resistant isolates from Japan (2009) and France (2010) is of grave concern. ,
In this article, we report the trend of antimicrobial resistance of N. gonorrhoeae isolates from 1995 to 2013 in our institute and the emergence of decreased susceptibility towards ceftriaxone.
| ~ Materials and Methods|| |
This study was carried out in the Department of Microbiology in conjunction with Department of Dermatology and STD of a tertiary care hospital of North India. The study population comprised males with urethritis, females with endocervicitis attending STI clinic of the hospital. All patients were included in the study after taking informed consent.
The urethral/endocervical samples were inoculated immediately after collection onto Modified Thayer Martin medium (MTM) and the inoculated plates were transported to the laboratory in a candle jar for further incubation in 3-7% CO 2 (candle jar) at 36°C ± 1°C for 48 hours. The colonies suspected to be N. gonorrhoeae were presumptively identified by Gram stain, oxidase test and superoxol test. Confirmation of identity was based on rapid carbohydrate utilisation test (RCUT). Gonococcal isolates were stored at −70°C in glycerol broths. 
Antimicrobial susceptibility testing
All isolates were examined for susceptibility to penicillin (10 IU), ciprofloxacin (5 μg), tetracycline (30 μg), ceftriaxone (30 μg), cefixime (5 μg), azithromycin (15 μg) and spectinomycin (100 μg) by disk diffusion method as per CLSI guidelines.  β-lactamase production is determined by chromogenic cephalosporin test. In addition minimum inhibitory concentration (MIC) for ceftriaxone was determined by E-test method [Figure 1].  N. gonorrhoeae ATCC 49226 strain and WHO reference strains A, C, E, G, J, K-Q were used as controls for disc diffusion and MIC testing.
|Figure 1: Minimum inhibitory concentration (MIC) of ceftriaxone by E test|
Click here to view
Criteria used for defining penicillinase-producing N. gonorrhoeae (PPNG), quinolone-resistant N. gonorrhoeae (QRNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were as follows:
- PPNG- N. gonorrhoeae isolates that are positive in the chromogenic cephalosporin test
- QRNG: N. gonorrhoeae isolates that are resistant to quinolones by disk diffusion testing as per CLSI 2011
- TRNG: N. gonorrhoeae isolates showing inhibition zone diameters of ≤19 mm as per CLSI 2011.
This centre has been participating in Gonococcal Antimicrobial Surveillance Program (GASP) organised by WHO-SEAR regional reference laboratory.
Data were expressed as percentages. The differences in percentages were statistically compared, tested for significance by using χ2 test and P values were determined.
| ~ Results|| |
The number of gonococcal isolates studied were 50 (48 males and 2 females) in 1995-96, 60 (52 males, 4 females and 4 sexual contacts) in 2004-05, and 25 (25 males) during 2011-2013. , Although N. gonorrhoeae isolated from sexual contacts had the same antimicrobial susceptibility profile they are included in analysis of results. A significant increase in PPNG has been observed over the years. The percentage of PPNG increased from 8% in 1995-96 to 20% in 2004-05 (χ2 = 2.268, P = 0.1321) and 88% in 2011-2013 (χ2 = 31.226, P < 0.0001). , QRNG showed a significant increase from 12% in 1995-96 to 98.3% in 2004-05 (χ2 = 80.557, P < 0.0001), while 84% isolates were found to be QRNG by 2011-2013 (χ2 = 4.215, P = 0.0401). Percentage of TRNG was found to be 28% in 1995-96, 18.3% in 2004-05 (χ2 = 0.953, P = 0.329) and 12% in 2011-2013 (χ2 = 0.157, P = 0.6918). Although the percentage TRNG has decreased over the years, chromosomally mediated resistance to tetracycline has increased. , All strains of N. gonorrhoeae isolated over the years from our institute were found to be sensitive to both azithromycin and spectinomycin. In January 2013, we detected our first gonococcal isolate with decreased susceptibility to third-generation cephalosporins; Ceftriaxone, Cefixime and Cefpodoxime by disk diffusion method, which was further confirmed by MIC testing (MIC for ceftriaxone = 0.19 μg/ml) using WHO K as control strain. The MIC90 of ceftriaxone for N. gonorrhoeae isolates has also increased from 0.016 μg/ml in 2004-05 to 0.023 μg/ml in 2011-13  [Figure 2].
| ~ Discussion|| |
The number of cases of gonorrhoea and hence gonococcal isolates has declined in our hospital over the years. It may be either due to actual decreasing incidence of gonorrhoea over the years or due to the fact that gradually fewer patients are reporting to the clinic because of easy availability of antimicrobials as per syndromic management of STIs in peripheral and private health set-ups.
Surveillance of antimicrobial resistance is required to monitor trends in established types of resistance and promptly identify new types of resistance. Data from this centre have highlighted an alarming increase in the percentage of PPNG and QRNG strains over the years. , Other studies from Nagpur (0%), Hyderabad (41.9%) and Kolkata (52.6%) have reported a lower percentage of PPNG, findings similar to our study were reported from another WHO SEAR country, Thailand in 2013.  Bhutan and Thailand have also reported a significant increase in QRNG strains over the years similar to our study. 
In our study the percentage of TRNG has decreased over the years with 12% TRNG during 2011-13. Ray et al., reported a higher percentage of TRNG strains from Hyderabad (45.6%) and Nagpur (33.8%) while, a similar percentage was reported from Delhi (7.4-10%). 
During our study we found one N. gonorrhoeae isolate with decreased susceptibility to all the third generation cephalosporins. In November 2013, Bala et al., reported 0.1%-84.2% N. gonorrhoeae isolates with decreased susceptibility to third generation cephalosporin in the WHO SEAR including India.  Treatment failure to cefixime has been reported from several countries like Japan, France, Canada, Austria and Norway. ,, Recently ceftriaxone resistant isolates have been identified in Japan (2009), France (2010) and Spain (2011). ,,
All the gonococcal isolates in our study were sensitive to azithromycin and spectinomycin. Bala et al., reported <5% gonococcal isolates resistant to azithromycin from WHO SEAR.  Spectinomycin-resistant strains have recently been reported from India (New Delhi and Kolkata) and Bhutan. , A small number of spectinomycin-resistant strains have been reported from China and Vietnam also.  Spectinomycin is not easily available in India and this may explain the retention of efficacy of this antimicrobial.
The very high rate of antimicrobial resistance among N. gonorrhoeae isolates may be an indicator of the overuse and misuse of antimicrobials due to over-the-counter availability, self-medication or prescription by unqualified practitioners.  Since the study was conducted in a tertiary care hospital with a large turnaround of OPD attendees including patients referred from various centres, there is a possibility that patients present to our centre after having already been treated elsewhere thus magnifying the problem of drug resistance. Also, as the number of isolates in the present study was small, a study with larger number of patients would be required to give a better evidence of the problem of drug resistant N. gonorrhoeae in the community.
Increase in N. gonorrhoeae isolates which are resistant to multiple antimicrobial agents is now a serious problem.  Emergence of N. gonorrhoeae isolates with decreased susceptibility/resistance to ceftriaxone, as a consequence of excess utilisation of oral third-generation cephalosporins for other infectious conditions, may pose a serious threat in the management of gonorrhoea in countries like India. Thus, continuous monitoring of antimicrobial susceptibility of all gonococcal strains circulating in a community should be performed to prevent treatment failures and further spread of resistant strains. At the same time, a search for new effective agents should be initiated as a solution to the problem. Progress towards a vaccine is urgently needed.
| ~ References|| |
Sethi S, Golparian D, Bala M, Dorji D, Ibrahim M, Jabeen K, et al
. Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae
isolates from India, Pakistan and Bhutan in 2007-2011. BMC Infect Dis 2013;13:35.
Bala M, Kakran M, Singh V, Sood S, Ramesh V, Members of WHO GASP SEAR Network. Monitoring antimicrobial resistance in Neisseria gonorrhoeae in selected countries of the WHO South-East Asia Region between 2009 and 2012: A retrospective analysis. Sex Transm Infect 2013;89:28-35.
Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae
in France: Novel penA
mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 2012;56:1273-80.
Yokoi S, Deguchi T, Ozawa T, Yasuda M, Ito S, Kubota Y, et al
. Threat to cefixime treatment for gonorrhoea. Emerg Infect Dis 2007;13:1275-7.
Ohnishi M, Golparian D, Shimuta K, Saika T, Hoshina S, Iwasaku K, et al
. Is Neisseria gonorrhoeae
initiating a future era of untreatable gonorrhea?: Detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob Agents Chemother 2011;55:3538-45.
Laboratory Diagnosis of Gonorrhoea. WHO Regional publication, South East Asia series No. 33. Geneva: World Health Organization; 1999. [Last accessed on 2013 Dec 15].
Clinical and Laboratory Standards Institute (CLSI) document M100-S21. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. 2011;31:92-6.
Bhalla P, Sethi K, Reddy BS, Mathur MD. Antimicrobial susceptibility and plasmid profile of Neisseria gonorrhoeae
in India (New Delhi). Sex Transm Infect 1998;74:210-2.
Khaki P, Bhalla P, Sharma P, Chawla R, Bhalla K. Epidemiological analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping. Indian J Med Microbiol 2007;25:225-9.
Ray K, Bala M, Kumari S, Narain JP. Antimicrobial resistance of Neisseria gonorrhoeae
in selected World Health Organization Southeast Asia Region countries: An overview. Sex Transm Dis 2005;32:178-84.
Unemo M, Golparian D, Syversen G, Vestrheim DF, Moi H. Two cases of verified clinical failures using internationally recommended first-line cefixime for gonorrhea treatment, Norway, 2010. Euro Surveill 2010;15.
Camara J, Serra J, Ayats, J, Bastida T, Carnicer-Pont, Andreu A, et al
. Molecular characterization of two high-level ceftriaxone-resistant Neisseria gonorrhoeae
isolates detected in Catalonia, Spain. J Antimicrob Chemother 2012;67:1858-60.
Bala M, Ray K, Salhan S. First case of spectinomycin resistant Neisseria gonorrhoeae
isolate in New Delhi, India Sex Transm Infect 2005;81:186-7.
Surveillance of antibiotic resistance in Neisseria gonorrhoeae
in the WHO Western Pacific Region, 2002. Commun Dis Intell Q Rep 2003;27:488-91.
Brhlikova P, Harper I, Jeffery R, Rawal N, Subedi M, Santhosh M. Trust and the regulation of pharmaceuticals: South Asia in a globalised world. Global Health 2011;7:10.
[Figure 1], [Figure 2]