Abstract
Background
Antimicrobial-resistant Neisseria gonorrhoeae is a major public health threat. Current CDC treatment guidelines for uncomplicated gonorrhoea recommend only ceftriaxone plus either azithromycin or doxycycline. Additional treatment options are needed.
Methods
We used antibiotic gradient synergy testing (the Etest) to evaluate antimicrobial combinations that included a third-generation cephalosporin (cefixime or ceftriaxone) plus azithromycin, doxycycline, gentamicin, rifampicin or fosfomycin. We tested each combination against 28 clinical N. gonorrhoeae isolates and four control strains of varying susceptibility profiles, and compared the results with those obtained using combination antimicrobial testing using agar dilution. We calculated the fractional inhibitory concentration index (FICI) for each combination to determine synergy, the results being interpreted as follows: FICI ≤ 0.5 = synergy; FICI > 4.0 = antagonism; and FICI > 0.5–4 = indifference.
Results
The combinations of a third-generation cephalosporin plus azithromycin, doxycycline, rifampicin, gentamicin or fosfomycin produced FICIs of indifference. The Etest and agar dilution methods produced comparable results.
Conclusions
Combinations of ceftriaxone plus rifampicin, gentamicin or fosfomycin may warrant further clinical investigation as treatments for gonorrhoea. Using the Etest for synergy testing is a viable method that has practical advantages over agar dilution.
Keywords: antimicrobial resistance, sexually transmitted diseases, novel therapies
Introduction
Gonorrhoea is the second most common reportable infection in the USA. Efforts to control gonorrhoea in the USA met with tremendous success between the 1970s and 1990s,1 but antimicrobial-resistant gonorrhoea has recently emerged as an urgent public health threat.2 Over the course of the last eight decades, Neisseria gonorrhoeae has developed and retained resistance to sulphonamides, penicillin, tetracyclines and quinolones,3 and the treatment of gonococcal infections currently relies on a single therapeutic antimicrobial class, the cephalosporins, of which the first-line therapy, ceftriaxone, is only available as an injectable drug.4 In the USA and the UK, the recommended treatment for N. gonorrhoeae includes the co-administration of a second agent, namely doxycycline or azithromycin, in order to increase treatment efficacy and prevent the emergence and transmission of antimicrobial resistance.
Some studies have observed in vitro synergy between third-generation cephalosporins and azithromycin,5–7 but others have not.8,9 Clinical data support the use of combination therapy with azithromycin over combination therapy with doxycycline,10–12 but whether or not these clinical outcomes correlate with in vitro data remains unknown. As the cefixime and ceftriaxone MICs continue to rise,13 and azithromycin resistance emerges,14–17 new combination therapies will be needed. One step in developing new combinations includes in vitro testing for synergy or antagonism. Although the Etest offers practical advantages over agar dilution and the chequerboard method, it has not been widely used for the synergy testing of N. gonorrhoeae.
In this study we sought to: (i) resolve the conflicting data regarding whether the combination of cefixime or ceftriaxone with azithromycin is synergistic against N. gonorrhoeae; (ii) determine whether the clinical data suggesting that a combination of cefixime and doxycycline is inferior to one of cefixime and azithromycin could be explained through in vitro studies; (iii) explore older antimicrobials in novel combinations for possible future treatment options; and (iv) compare synergy testing results obtained through the Etest method and agar dilution.
Materials and methods
We used antibiotic gradient synergy testing to evaluate antimicrobial combinations, which included a third-generation cephalosporin, either cefixime or ceftriaxone, plus a second antimicrobial agent. We tested all 10 combinations against 28 clinical N. gonorrhoeae isolates and four control strains with varying susceptibility profiles. Antimicrobial agents tested in combination with cephalosporins include azithromycin, doxycycline, gentamicin, rifampicin and fosfomycin. We elected to test azithromycin and doxycycline because both agents are part of currently recommended regimens, existing in vitro data are conflicting on azithromycin, and the clinical outcomes of combination therapy with doxycycline are poorer than those of combination therapy with azithromycin. We chose the other three antimicrobials as potential candidates for combination therapy because they met the following criteria: (i) they are not β-lactams and consequently have a different mechanism of action from that of cephalosporins; (ii) there is some evidence that each agent is synergistic with β-lactams in treating other bacterial infections;18–23 (iii) the drugs are not routinely used for gonorrhoea; and (iv) the drugs could be administered as part of a single-dose regimen.
Bacterial isolates
We selected a panel of thirty-two N. gonorrhoeae strains based on their varying cefixime MICs as determined by the Etest (≤0.047 mg/L, n = 11; 0.064–0.094 mg/L, n = 10; ≥0.125 mg/L, n = 11). The panel included 28 unique clinical isolates collected from patients attending the Public Health Seattle-King County Sexually Transmitted Diseases Clinic between June 2007 and November 2012, and four N. gonorrhoeae quality control strains.24–26
Antimicrobial susceptibility testing, and synergy testing and interpretation
The MICs of each single antimicrobial agent were determined by Etest and by agar dilution prior to testing the antimicrobial agents in combination using the manufacturer's (bioMérieux, Inc., Durham, NC, USA) instructions for the Etest and standard CLSI25 recommendations for agar dilution. Synergy testing by Etest (cross or 90° angle formation method) and agar dilution was performed in duplicate, with the MIC of each antimicrobial in the combination read and interpreted using previously described methods.8,27,28 For each antimicrobial combination, we calculated the fractional inhibitory concentration index (FICI)5,28,29 by computing the ratio of the MIC of the combination divided by the MIC of the antimicrobial alone for each agent, and then adding those two ratios together (Equation A). The FICI data were interpreted using the following criteria: synergy, FICI ≤0.5; indifference, FICI > 0.5–4.0; and antagonism, FICI > 4.0.
 |
Discrepant MIC results and those combinations with FICI ≤ 1 were confirmed by performing an additional duplicate synergy test.
Statistical analysis
Results were stratified by the Etest cefixime MIC groups of ≤0.047 mg/L, 0.064–0.094 mg/L and ≥0.125 mg/L. We chose these groupings to explore differences between isolates with elevated MICs of cefixime, those with intermediate MICs and those with very low MICs. We calculated medians, and report both the median and range for each antimicrobial by cefixime group alone (MICA, MICB), and in combination [MICA(with B), MICB(with A)]. In order to compare the Etest and agar dilution, we converted the Etest MIC into the equivalent agar 2-fold dilution.30 We then used a Wilcoxon rank sum test to analyse the FICI differences. We assumed an alpha of 0.05.
Results
Gonococcal isolates
The Etest MICs for the 32 tested isolates of the seven tested antibiotics and a standard antimicrobial panel are shown in Table 1. By CDC standards,4 all but one of the isolates was susceptible to azithromycin (MIC < 2 mg/L). Although there are no CLSI breakpoints for doxycycline for N. gonorrhoeae, CLSI defines tetracycline resistance as ≥2 mg/L.25 Using this criterion, more than half of the isolates were considered to be resistant to doxycycline. There are currently no MIC breakpoints for the susceptibility of N. gonorrhoeae to gentamicin, fosfomycin and rifampicin. Fosfomycin MICs for other clinically important bacteria are in general high, with the breakpoints for the Enterobacteriaceae being susceptible ≤64 mg/L, intermediate 128 mg/L and resistant ≥256 mg/L.25
Table 1.
Antimicrobial susceptibility profiles of clinical isolates and reference strains of N. gonorrhoeae determined by Etesta
| Strain | Source | Year | MIC (mg/L) |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PEN | TET | DOX | SPT | CIP | CTX | CFM | CRO | AZM | GEN | RIF | FOF | |||
| 1 | urethra | 2011 | >32b | 48 | 24 | 8 | 4 | 0.19 | 0.047 | 0.064 | 0.25 | 8 | 32 | 24 |
| 2 | urethra | 2011 | 1 | 4 | 2 | 8 | 16 | 0.125 | 0.016 | 0.032 | 16 | 6 | 0.125 | 16 |
| 3 | rectum | 2012 | 1.5 | 4 | 3 | 8 | 16 | 0.38 | 0.032 | 0.047 | 1 | 6 | 0.25 | 16 |
| 4 | urethra | 2011 | 2 | 3 | 3 | 8 | 16 | 0.25 | 0.047 | 0.047 | 0.25 | 6 | 0.25 | 24 |
| 5 | urethra | 2012 | 1 | 3 | 2 | 8 | 16 | 0.19 | 0.016 | 0.032 | 0.25 | 8 | 0.094 | 12 |
| 6 | urethra | 2011 | 2 | 2 | 2 | 8 | 16 | 0.19 | 0.016 | 0.032 | 0.38 | 12 | 0.25 | 16 |
| 7 | controlc | — | 1 | 1 | 1 | 12 | 0.004 | 0.064 | 0.016 | 0.016 | 0.38 | 6 | 0.19 | 16 |
| 8 | pharynx | 2012 | 1.5 | 3 | 2 | 12 | 16 | 0.25 | 0.032 | 0.047 | 0.38 | 8 | 0.25 | 16 |
| 9 | pharynx | 2011 | 2 | 3 | 2 | 8 | 16 | 0.25 | 0.016 | 0.032 | 1.5 | 12 | 0.125 | 24 |
| 10 | controld | 1991 | >32b | 2 | 1 | >1024 | 0.015 | 0.094 | 0.016 | 0.032 | 0.25 | 4 | 0.25 | 24 |
| 11 | pharynx | 2008 | 1.5 | 2 | 2 | 8 | 16 | 0.25 | 0.032 | 0.047 | 0.38 | 8 | 0.125 | 12 |
| 12 | rectum | 2011 | 1.5 | 3 | 3 | 8 | 16 | 0.38 | 0.094 | 0.064 | 0.38 | 8 | 0.125 | 24 |
| 13 | pharynx | 2012 | 2 | 4 | 3 | 12 | 16 | 0.75 | 0.094 | 0.064 | 0.38 | 8 | 0.25 | 12 |
| 14 | urethra | 2010 | 1.5 | 4 | 3 | 8 | 16 | 0.5 | 0.094 | 0.064 | 0.38 | 8 | 0.25 | 12 |
| 15 | pharynx | 2012 | 1 | 3 | 1.5 | 8 | 16 | 0.25 | 0.094 | 0.064 | 0.25 | 6 | 0.125 | 16 |
| 16 | urethra | 2011 | 1.5 | 4 | 3 | 8 | 16 | 0.38 | 0.094 | 0.094 | 0.5 | 6 | 0.25 | 24 |
| 17 | urethra | 2010 | 1.5 | 3 | 3 | 8 | 16 | 0.38 | 0.094 | 0.064 | 0.5 | 8 | 0.125 | 16 |
| 18 | urethra | 2012 | 1.5 | 4 | 2 | 8 | 16 | 0.25 | 0.094 | 0.064 | 1 | 8 | 0.25 | 16 |
| 19 | urethra | 2012 | 2 | 3 | 2 | 8 | 16 | 0.5 | 0.094 | 0.064 | 0.5 | 6 | 0.125 | 8 |
| 20 | rectum | 2011 | 1 | 3 | 1.5 | 6 | 16 | 0.25 | 0.064 | 0.064 | 0.5 | 6 | 0.19 | 12 |
| 21 | cervix | 2012 | 1 | 4 | 2 | 8 | 16 | 0.19 | 0.064 | 0.064 | 0.38 | 3 | 0.125 | 16 |
| 22 | controle | 2003 | 3 | 3 | 2 | 12 | >32 | 1 | 0.38 | 0.125 | 0.25 | 4 | 0.38 | 16 |
| 23 | pharynx | 2008 | 1.5 | 3 | 3 | 8 | 16 | 0.75 | 0.19 | 0.094 | 0.5 | 8 | 0.094 | 24 |
| 24 | pharynx | 2009 | 2 | 3 | 3 | 8 | 16 | 0.75 | 0.19 | 0.125 | 0.5 | 8 | 0.19 | 24 |
| 25 | controlf | — | 8 | 4 | 3 | 12 | 16 | 1 | 0.38 | 0.125 | 0.5 | 8 | 0.25 | 16 |
| 26 | urethra | 2012 | 2 | 3 | 2 | 8 | 16 | 0.5 | 0.125 | 0.064 | 0.38 | 8 | 0.064 | 16 |
| 27 | urethra | 2010 | 3 | 6 | 2 | 12 | >32 | 0.5 | 0.125 | 0.064 | 0.38 | 12 | 0.38 | 12 |
| 28 | pharynx | 2007 | 1.5 | 2 | 1 | 8 | 32 | 0.38 | 0.25 | 0.064 | 0.25 | 6 | 0.064 | 24 |
| 29 | urethra | 2012 | 2 | 3 | 3 | 8 | 16 | 0.75 | 0.125 | 0.064 | 0.5 | 6 | 0.125 | 24 |
| 30 | rectum | 2012 | 2 | 2 | 1.5 | 8 | 16 | 0.75 | 0.125 | 0.125 | 0.25 | 6 | 0.125 | 12 |
| 31 | pharynx | 2012 | 1.5 | 3 | 1.5 | 8 | 16 | 0.5 | 0.125 | 0.125 | 0.5 | 12 | 0.125 | 24 |
| 32 | rectum | 2008 | 2 | 3 | 2 | 8 | 32 | 0.5 | 0.19 | 0.064 | 0.38 | 8 | 0.125 | 16 |
PEN, penicillin; TET, tetracycline; DOX, doxycycline; SPT, spectinomycin; CIP, ciprofloxacin; CTX, cefotaxime; CFM, cefixime; CRO, ceftriaxone; AZM, azithromycin; GEN, gentamicin; RIF, rifampicin; FOF, fosfomycin.
aMICs were determined by the Etest (except the MIC of ciprofloxacin, which was determined by agar dilution).
bAll clinical isolates and reference strains were β-lactamase-negative by nitrocefin except strains 1 and 10, which were β-lactamase-positive, penicillinase-producing N. gonorrhoeae (PPNG).
cCLSI-recommended quality control strain for gonococcal susceptibility testing, ATCC 49226.
dWHO PPNG, spectinomycin-resistant reference strain, WHO O.
eWHO cephalosporin-non-susceptible, penA mosaic-positive reference strain, WHO K.
fCDC cephalosporin-non-susceptible reference strain, SPL-4.
Current therapies: cephalosporins plus azithromycin or doxycycline
The Etest results from combining cefixime or ceftriaxone with azithromycin or doxycycline are summarized in Table 2. The average FICI for both cephalosporins in combination with azithromycin was 1.26. No synergy or antagonism was seen with any of the combinations. Notably, in the presence of cefixime or ceftriaxone, the isolate with the highest azithromycin MIC (16 mg/L) had an azithromycin MIC of 3 mg/L, equivalent to a reduction of two doubling dilutions. However, a similar fold reduction in MIC was not seen with the other isolates where, on average, the azithromycin MIC was reduced by one-half to two-thirds in combination with a cephalosporin. The combinations of a third-generation cephalosporin and doxycycline produced an average FICI of 1.29, indicating indifference.
Table 2.
Etest MICs (median and range) of indicated antibiotics alone and in combination
| MIC (mg/L), median (range) |
FICIa | Interpretation | ||||
|---|---|---|---|---|---|---|
| MICA | MICA(with B) | MICB | MICB(with A) | |||
| Cefixime and azithromycin | ||||||
| all isolates (n = 32) | 0.094 (0.016–0.38) | 0.064 (0.016–0.19) | 0.38 (0.25–16) | 0.19 (0.094–3) | 1.26 (0.908–1.5) | indifference |
| Ceftriaxone and azithromycin | ||||||
| all isolates (n = 32) | 0.064 (0.023–0.125) | 0.047 (0.016–0.094) | 0.38 (0.19–16) | 0.25 (0.125–2) | 1.25 (0.931–1.76) | indifference |
| Cefixime and doxycycline | ||||||
| all isolates (n = 32) | 0.094 (0.016–0.38) | 0.047 (0.016–0.25) | 1.75 (1–24) | 1 (0.5–16) | 1.33 (1–1.75) | indifference |
| Ceftriaxone and doxycycline | ||||||
| all isolates (n = 32) | 0.064 (0.023–0.125) | 0.047 (0.012–0.94) | 2 (1–24) | 1 (0.5–24) | 1.23 (0.833–1.5) | indifference |
MICA, cephalosporin (cefixime or ceftriaxone).
MICB, azithromycin or doxycycline.
MICA(with B), cefixime or ceftriaxone with azithromycin or doxycycline.
MICB(with A), azithromycin or doxycycline with cephalosporin.
aThe FICI data were interpreted using the following criteria: synergy, FICI ≤ 0.5; indifference, FICI > 0.5–4.0; and antagonism, FICI > 4.0.
Novel combinations
The novel combinations of cefixime or ceftriaxone plus gentamicin, rifampicin, or fosfomycin produced an FICI of indifference (Table 3). Notably, the mean FICI for the combination of ceftriaxone plus fosfomycin was 0.96 (SD 0.19), the lowest total FICI in this study. Similarly, for the subset of isolates with an elevated cefixime MIC (≥0.125 mg/L), cefixime plus fosfomycin produced an FICI of 0.83. In both groups, the relatively low FICI was primarily led by the isolates with elevated cefixime MICs.
Table 3.
Etest MICs (median and range) of the indicated antibiotics alone and in combination
| MIC (mg/L), median (range) |
FICIa | Interpretation | ||||
|---|---|---|---|---|---|---|
| MICA | MICA(with B) | MICB | MICB(with A) | |||
| Cefixime and gentamicin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.032 (0.016–0.047) | 0.016 (0.016–0.047) | 6 (4–12) | 3 (2–4) | 1.33 (0.864–1.67) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.094 (0.064–0.094) | 0.056 (0.032–0.064) | 7 (2–8) | 3.5 (1–6) | 1.18 (0.840–1.5) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.19 (0.125–0.38) | 0.094 (0.047–0.25) | 8 (2–12) | 4 (1–6) | 1.04 (0.679–1.42) | indifference |
| all isolates (n = 32) | 0.094 (0.016–0.38) | 0.047 (0.016–0.25) | 7 (2–12) | 3.5 (1–6) | 1.18 (0.679–1.67) | indifference |
| Ceftriaxone and gentamicin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.032 (0.023–0.064) | 0.032 (0.012–0.047) | 8 (4–16) | 4 (1.5–4) | 1.18 (0.75–2) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.064 (0.064–0.094) | 0.047 (0.032–0.064) | 8 (3–8) | 3.5 (1–4) | 1.07 (0.833–1.25) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.094 (0.064–0.125) | 0.064 (0.032–0.125) | 8 (4–12) | 3 (2–8) | 1.23 (0.762–1.5) | indifference |
| all isolates (n = 32) | 0.064 (0.023–0.125) | 0.047 (0.012–0.125) | 8 (3–16) | 4 (1–8) | 1.18 (0.75–2) | indifference |
| Cefixime and rifampicin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.016 (0.016–0.047) | 0.016 (0.016–0.047) | 0.19 (0.064–32) | 0.064 (0.019–32) | 1.51 (1.08–2) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.094 (0.064–0.094) | 0.056 (0.023–0.094) | 0.125 (0.125–0.25) | 0.125 (0.064–0.125) | 1.44 (1.25–1.66) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.19 (0.125–0.38) | 0.094 (0.032–0.19) | 0.125 (0.064–0.38) | 0.094 (0.064–0.25) | 1.26 (1.08–1.75) | indifference |
| all isolates (n = 32) | 0.094 (0.016–0.38) | 0.047 (0.016–0.19) | 0.125 (0.064–32) | 0.094 (0.019–32) | 1.38 (1.08–2) | indifference |
| Ceftriaxone and rifampicin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.032 (0.023–0.064) | 0.032 (0.012–0.047) | 0.25 (0.094–32) | 0.125 (0.064–24) | 1.19 (1–2) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.064 (0.064–0.094) | 0.047 (0.023–0.064) | 0.128 (0.125–0.25) | 0.125 (0.064–0.19) | 1.49 (1.01–1.75) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.094 (0.064–0.125) | 0.047 (0.032–0.125) | 0.125 (0.064–0.25) | 0.094 (0.047–0.19) | 1.41 (1.02–2) | indifference |
| all isolates (n = 32) | 0.064 (0.023–0.125) | 0.0395 (0.012–0.125) | 0.158 (0.064–32) | 0.125 (0.047–24) | 1.44 (1–2) | indifference |
| Cefixime and fosfomycin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.016 (0.016–0.047) | 0.016 (0.016–0.047) | 16 (12–32) | 6 (0.75–12) | 1.22 (0.75–1.38) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.094 (0.064–0.094) | 0.047 (0.032–0.064) | 16 (8–32) | 8 (4–12) | 1.03 (0.833–1.25) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.19 (0.125–0.38) | 0.064 (0.047–0.19) | 24 (12–24) | 6 (4–12) | 0.845 (0.626–1.01) | indifference |
| all isolates (n = 32) | 0.094 (0.016–0.38) | 0.047 (0.016–0.19) | 16 (8–32) | 6 (0.75–12) | 1 (0.626–1.38) | indifference |
| Ceftriaxone and fosfomycin | ||||||
| cefixime MIC ≤ 0.047 mg/L (n = 11) | 0.032 (0.023–0.064) | 0.032 (0.012–0.047) | 16 (12–32) | 6 (3–12) | 1.01 (0.75–1.25) | indifference |
| cefixime MIC 0.064–0.094 mg/L (n = 10) | 0.064 (0.064–0.094) | 0.032 (0.023–0.064) | 16 (12–24) | 6 (4–12) | 0.992 (0.75–1.5) | indifference |
| cefixime MIC ≥ 0.125 mg/L (n = 11) | 0.094 (0.064–0.125) | 0.047 (0.032–0.064) | 24 (12–24) | 8 (4–8) | 0.845 (0.667–1.07) | indifference |
| all isolates (n = 32) | 0.064 (0.023–0.125) | 0.032 (0.012–0.064) | 16 (12–32) | 6 (3–12) | 0.977 (0.667–1.5) | indifference |
MICA, cephalosporin (cefixime or ceftriaxone).
MICB, second antimicrobial (gentamicin, rifampicin or fosfomycin).
MICA(with B), cefixime or ceftriaxone with azithromycin.
MICB(with A), second antimicrobial (gentamicin, rifampin or fosfomycin) with a cephalosporin.
aThe FICI data were interpreted using the following criteria: synergy, FICI ≤ 0.5; indifference, FICI > 0.5–4.0; and antagonism, FICI > 4.0.
Etest and agar dilution
The Etest and agar dilution tests produced significantly different mean FICIs, with agar dilution consistently resulting in higher FICI values (Table 4). However, these differences were generally relatively small in magnitude and did not lead to a different interpretation of FICI values.
Table 4.
Comparison of the Etest (rounded to equivalent 2-fold dilution MICs) and agar dilution synergy testing: FICIs for 10 antimicrobial combinations against 32 strains of N. gonorrhoeae
| Combination | FICI, median (range) |
P valuea | |
|---|---|---|---|
| Etest | agar | ||
| Cefixime and azithromycin | 1.25 (0.73–1.5) | 1.75 (1.5–2) | <0.001 |
| Ceftriaxone and azithromycin | 1.5 (0.73–2) | 2 (1.5–2) | <0.001 |
| Cefixime and doxycycline | 1.25 (0.98–2) | 2 (1.48–2) | <0.001 |
| Ceftriaxone and doxycycline | 1.5 (0.75–2) | 2 (1.5–2) | <0.001 |
| Cefixime and gentamicin | 1 (0.605–1.5) | 1.5 (1.25–2) | <0.001 |
| Ceftriaxone and gentamicin | 1.25 (0.73–2) | 1.5 (1.25–2) | 0.0001 |
| Cefixime and rifampicin | 1.49 (1–2) | 2 (1.48–2) | <0.001 |
| Ceftriaxone and rifampicin | 1.5 (0.96–2) | 2 (1.48–2) | 0.0002 |
| Cefixime and fosfomycin | 0.98 (0.73–1.5) | 1.5 (1.25–2) | <0.001 |
| Ceftriaxone and fosfomycin | 1 (0.49–1.5) | 1.5 (1.13–1.5) | <0.001 |
aP value derived from Wilcoxon rank sum.
Discussion
We found no synergy or antagonism with any of our 10 combination antimicrobial therapies for N. gonorrhoeae. Our findings support those of Pereira et al.,8 who reported a lack of synergy with the combination of a third-generation cephalosporin plus azithromycin, in contrast to other studies, which reported synergy with this combination.5,7,31 We did not find laboratory evidence to explain the clinical differences10–12 between the combination of a third-generation cephalosporin and doxycycline, and a third-generation cephalosporin and azithromycin. We did not find strong evidence of synergy among any of the novel combinations, although the combination of cefixime or ceftriaxone plus fosfomycin trended in that direction. Notably, although none of the combinations was synergistic, neither were they antagonistic, suggesting that they may have clinical utility. Finally, utilizing the Etest, our findings remained consistent when compared with the traditional method of agar dilution.
In vitro synergy studies of the combination of a third-generation cephalosporin plus azithromycin have reported disparate results, which may be due to differences in the isolates themselves (that is, Japanese5 versus British8), but are more likely a consequence of differences in the methods employed to assess synergy. Both published studies observing synergy or partial synergy5,7 have used the chequerboard method. In contrast, studies that have not observed synergy, like ours, have used agar dilution,8,9 the method we used to confirm our findings from the Etest. Etest studies have also employed different methods and produced disparate results.6,9 In a recently presented study, Wind et al.9 used the same methods as we present here and reported similar results. In contrast, Golparian et al.6 incubated the first Etest strip for 1 h at room temperature, and then replaced it with a second Etest strip and observed synergism. We chose not to use this method because we were concerned about the possibility of a delayed recovery of N. gonorrhoeae32–34 after pre-incubating the inoculated plates with the Etest strips for 1 h at room temperature without ambient CO2, a growth requirement for N. gonorrhoeae. The consistency of our results between agar dilution and the Etest suggests that the Etest is a reasonable, relatively simple alternative to agar dilution. Whether the Etest or agar dilution for N. gonorrhoeae synergy testing is better than chequerboard testing remains unknown.
Our findings failed to find laboratory evidence to explain the clinical treatment differential between combinations of cephalosporins with doxycycline and cephalosporins with azithromycin.10,11 In theory, combining a bacteriostatic drug (doxycycline) with a bactericidal one (β-lactam) may inhibit the growth required for the latter's mechanism of action.35 Our findings suggest that the clinical treatment failures witnessed with the combination of cefixime and doxycycline in the previous studies are likely to be a result of the relatively high proportion of N. gonorrhoeae strains in the USA1 and in Europe36 that are tetracycline resistant, or perhaps the poor activity of tetracyclines for pharyngeal gonorrhoea, and not of biochemical interactions.
Given the widespread concerns about antimicrobial-resistant gonorrhoea, treatment recommendations in the future are likely to continue to promote two-drug regimens. Our findings suggest several potential new candidate combinations. Gentamicin plus an extended-spectrum cephalosporin is one option, although gentamicin alone has been used to treat gonorrhoea in Malawi for many years.31,37 While a meta-analysis estimated the efficacy of single dose therapy to be only 91.5%,38 which does not exceed the thresholds defined by the WHO and CDC for effective therapies,3 a regimen that combined gentamicin with a cephalosporin would probably be more effective, even against organisms with somewhat elevated cephalosporin MICs. Gentamicin (240 mg intramuscularly) and 2 g of azithromycin was recently found to be highly effective in treating the largely azithromycin-susceptible N. gonorrhoeae found in the USA.39 Ceftriaxone plus gentamicin could be a combination regimen active against organisms with macrolide resistance, although this requires clinical evidence.
Rifampicin plus cefixime is another potential combination regimen that has the advantage of being orally administered. Rifampicin has been widely used in combination with other antimicrobials to treat infections such as tuberculosis40 and staphylococcal osteomyelitis associated with prosthetic material.41 Trials undertaken in the 1980s found that rifampicin plus erythromycin was highly effective in the treatment of gonorrhoea, with an efficacy of 98%.42,43 However, to our knowledge, there are no clinical data on the efficacy of single-dose cephalosporin and rifampicin combination therapy, and trials of such a regimen would need to carefully assess the treatment's impact on antimicrobial resistance.
Although fosfomycin is seldom used in the USA, fosfomycin plus cefixime or fosfomycin plus ceftriaxone were the final candidate combination regimens evaluated in our study, and were the only regimens for which we observed an FICI < 1.0. Fosfomycin is FDA-approved in the USA as an oral formulation (fosfomycin tromethamine) for the treatment of urinary tract infections.44,45 The drug is bactericidal and interrupts the cell wall synthesis of both Gram-positive and Gram-negative organisms at a step proximal to that of β-lactams.44,45 A 1969 study found that an older oral formulation of fosfomycin was ineffective in the treatment of gonococcal urethritis.46 However, an intramuscular formulation of the drug was used successfully in a clinical trial of gonorrhoea treatment,47 and the currently available oral version of the drug is more bioavailable than the formulation tested in the 1960s.44 As with the other regimens we tested in vitro, additional research is required before fosfomycin can be used in the treatment of N. gonorrhoeae.
In summary, we found no evidence of synergy or antagonism among 10 combinations of a third-generation cephalosporin plus azithromycin, doxycycline, rifampicin, gentamicin or fosfomycin. Combination therapy with extended-spectrum cephalosporins and rifampicin, gentamicin, or fosfomycin may be candidates for clinical trials as we prepare for an era of ever increasing antimicrobial-resistant N. gonorrhoeae.
Funding
This work was supported by the National Institutes of Health [Contract # HHSN272200800026C to (L. A. B.) and T32AI007140-36 to (L.A.B.)] and two institutional grants at the University of Washington: the Walt Stamm Fellow Support Award and the UW Housestaff Award (both to L. A. B.).
Transparency declarations
L. A. B. and M. R. G. have received research support from Hologic Gen-Probe. O. O. S. and M. R. G. have received research support from Melinta Therapeutics and Cempra Pharmaceuticals. K. K. H.: none to declare.
Acknowledgements
We thank Susan R. Swanzy and Carissa R. Kono for technical assistance.
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