1. Efficacy of gentamicin in combination with other different antimicrobials: optimism to treat and combat superbug Neisseria gonorrhoeae Vikram Singh 1, 2, Aradhana Bhargava 1 and Ravi Bhatnagar 2 1 Apex Regional STD Teaching, Training & Research Centre, VMMC & Safdarjung Hospital, New Delhi, India. 2 SunRise University, Rajasthan, India.

2. Background  The emergence of resistance or treatment failure to third generation cephalosporins have left gonorrhoea untreatable.  With no promising alternatives to treatment of N. gonorrhoeae, it has now emerged as a “Superbug” with XDR strains.  This exemplifies the challenge of emerging to a situation reminiscent of the pre-antibiotic era.  The crisis of AMR to the last available antimicrobials has led to recommendations for dual antimicrobial therapies.

3.  With no new therapeutic drugs in the drug-delivery pipeline, dual antimicrobial therapy may be the only option in near future.  Gentamicin is an inexpensive and effective alternative treatment option for both developed & resource-poor settings.  CDC & NIH trial in 2013: gentamicin/ azithromycin combination 100% effective.

4. Objectives  To find out the prevalence of resistance to gentamicin.  To evaluate the synergistic, additive, indifferent or antagonistic effects of gentamicin in combination with six different antimicrobials.  To evaluate the currently recommended dual regimens.

5. Methodology Study period:  October 2012 to June 2015. Study Settings:  Apex Regional STD Teaching, Training & Research Centre, VMMC & Safdarjung Hospital, New Delhi, India. N. gonorrhoeae strains tested:  55 including 45 clinical isolates, 9 WHO reference strains and ATCC 49226.

6. Microscopic confirmation of N. gonorrhoeae

7. Isolation and identification of N. gonorrhoeae Specimens cultured on Chocolate agar GC medium base with saponin lysed blood and VCNT inhibitors. 35-36ºC, 5% CO 2, 18-24 hours incubation. Identification of N. gonorrhoeae isolates Typical colonies on culture agar media GNDC in microscopy Positive oxidase reaction Positive superoxol test Sugar utilization test - RCUT.

8. Identification tests of N. gonorrhoeae

9. Antimicrobial Susceptibility Testing Cefixime (IX) Ceftriaxone (TX) Azithromycin (AZ) MIC of N. gonorrhoeae strains determination by Etest method on GC agar base with 1% isovitalex Gentamicin (GM) Spectinomycin (SH) Moxifloxacin (MX) Ertapenem (ETP) Gentamycin susceptibility criteria MIC ≤4 µg/ml = Susceptible MIC 8-16 µg/ml = Less susceptible MIC ≥ 32 µg/ml = Resistant

10. Dual antimicrobial testing  Six combinations of gentamicin & two currently recommended tested by Etest fixed ratio method.  Synergistic, additive, indifferent or antagonistic effect were determined by the fractional inhibitory concentration Index (FICI) FICI = MIC AB /MIC A + MIC BA /MIC B Where : MIC A - MIC of drug A alone MIC B - MIC of drug B alone MIC AB - MIC of A in the presence of drug B MIC BA - MIC of B in the presence of drug A

11. Category interpretation criteria based on FICI values: Statically geometric mean of MICs & FICIs calculated for all isolates in each antimicrobial combination. Quality control for all in vitro procedures was performed using the WHO reference strains C, F-P and ATCC 49226.

12. A A A A B Etest fixed ratio method for Dual antimicrobial combination testing.

13. Results Out of 55 strains: Gentamicin susceptible=48, LS=7

14. Synergistic, additive, antagonistic effects of Gentamicin dual antimicrobial combinations Antibiotic combinations Tested Strains Synergistic (%) Additive (%) Indifference (%) Antagonistic (%) IX+GM557 (12.7)15(27.3)33 (60)0 GM+ETP5519 (34.5)23 (41.8)13 (23.7)0 TX+GM559 (16.4)10 (18.2)30 (54.5)6 (10.9) SC+GM555 (9.1)11 (20)35 (63.6)4 (7.3) AZ+GM555 (9.1)10 (18.2)35 (63.6)5 (9.1) GM+MX555 (9.1)14 (25.4)29 (52.8)7 (12.7) Total no. of combination evaluated 33050 (15.1)83 (25.2)175(53)22 (6.7)

15. GM+ETP  Geometric mean of gentamicin = 2.84 µg/ml 0.2 µg/ml (p value= <0.0001).  Geometric mean of Ertapenem= 0.01 µg/ml 0.006 µg/ml (p value= <0.0001).  Geometric mean of FICI for GM+ETP = 0.56 IX+GM  Geometric mean of Cefixime =0.02 µg/ml 0.01 µg/ml (p value = 0.03).  Gentamicin geometric mean =2.84 µg/ml 0.05 µg/ml  Geometric mean of FICI was 0.84 Recommendations Novel antimicrobial combinations for future treatment of MDR or XDR N. gonorrhoeae

16. In vitro synergistic effects in combination of Gentamicin with Ertapenem GM + ETP: highest level of in vitro synergistic effect in 19 (34.5%) strains and additive effect in 23 (41.8%) strains 4 µg/ml 0.008 µg/ml ETP: 0.002 µg/ml GM: 0.064 µg/ml

17. Currently recommended combinations for management of N. gonorrhoeae The geometric mean of FICI for TX+AZ = 1.28 (Indifference effect) The geometric mean of FICI for IX+AZ =0.98 (additive effect)

18. Summary and conclusions  Based on in vitro dual antimicrobial testing significant in vitro synergism without any antagonism was observed for: I.Gentamicin plus Ertapenem II.Cefixime plus Gentamicin  Above combinations are more effective than the combinations presently recommended for treatment (cephalosporin plus azithromycin).

19.  These combinations also warrant for an up-to date, comprehensive and randomized controlled treatment trials.  These dual therapies will be promising approach to reduce the resistance and overcome the fear of superbug crisis of N. gonorrhoeae.  These will help to change the treatment guidelines of gonorrhea.

20. Acknowledgment  Indian Council of Medical Research  Leemma Peter, Monika Kakran & Naveen Joshi

21. Thank You!