- The study evaluated the in vitro activity of azithromycin, ceftriaxone, doxycycline, and various fluoroquinolones against a mutant strain of Neisseria gonorrhoeae. It determined the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of each drug. - Pharmacokinetic parameters were used to model concentrations over time and calculate the percentage of time drug levels remained above the MPC and within the mutant selection window (MSW). Time-kill assays measured bacterial killing at various time points. - All fluoroquinolones showed bactericidal activity and maintained levels above the MPC, suggesting they could prevent mutant selection. Azithromy

1. Bacterial Strains
• A gyrA mutant of the N. gonorrhoeae strain ATCC 49226,
designated m-49226, was studied.
Antimicrobials
• Analytical-grade azithromycin, ceftriaxone, doxycycline,
ciprofloxacin, levofloxacin, and moxifloxacin were evaluated.
Medium
• Gonococcal broth supplemented with 1% IsoVitaleX was used
for preparation of bacterial inocula and in time-kill studies.
• Gonococcal agar supplemented with 1% IsoVitaleX was used for
bacterial quantification and in MIC and MPC determinations.
Susceptibility Testing
• MIC values were determined using 105 colony-forming units
(CFU) of m-49226. The Etest® and broth microdilution
methodologies were used.
MPC Determinations – Figure 24
• MPCs were determined by plating 1010 CFU of m-49226 on agar
containing azithromycin, ceftriaxone, doxycycline, ciprofloxacin,
levofloxacin, or moxifloxacin in stepwise increasing
concentrations relative to each antimicrobial’s MIC. The lowest
concentration that inhibited mutant bacterial growth was
designated the MPC.
Figure 2. MPC Methodology4
Pharmacodynamic Modeling
• Pharmacokinetic parameters attained by azithromycin,
ceftriaxone, doxycycline, ciprofloxacin, levofloxacin, and
moxifloxacin were used to determine the relationship between
antimicrobial concentrations and the MSW.
Table 1. Pharmacokinetic Parameters
Time-Kill Assays
• The antimicrobial activities of each agent were tested for 24
hours, with bacterial quantification performed using samples
obtained at 0, 4, 8, and 24 hours.
• All time-kill assays were performed using concentrations of
antimicrobials equal to the above ƒCmax values and an inoculum
of ~108 CFU/mL of m-49226.
• Figures 3-8 illustrate the relationship between concentrations of the tested antimicrobials and the MSW. %T>MPC represents the % of the dosage interval that
concentrations exceed the MPC. %TMSW represents the % of the dosage interval that concentrations fall within the MSW.
Figure 9. Time-Kill Assay Results
Comparative in vitro evaluation of the activity of azithromycin, ceftriaxone, doxycycline, and
fluoroquinolones against mutant Neisseria gonorrhoeae
Dang X. Nguyen, Katie S. Eastman, Katharine R. Freudenberger, Emily G. Prescott, Doctor of Pharmacy Class of 2016
George P. Allen, Pharm.D., Associate Professor and Interim Chair, Department of Pharmacy Practice
• Neisseria gonorrhoeae is an urgent threat because of emerging
antimicrobial resistance. Resistance to recommended treatments
(azithromycin, ceftriaxone, and doxycycline) is increasing.1
• Fluoroquinolones are no longer recommended by the Centers for
Disease Control for the treatment of gonoccocal infections due to
resistance concerns.1
• In vitro data suggests that newer fluoroquinolones such as
moxifloxacin have activity against antimicrobial-resistant N.
gonorrhoeae.2
• The minimum inhibitory concentration (MIC) is the lowest
antimicrobial concentration that inhibits visible bacterial growth,
and is the standard measure of antimicrobial susceptibility.
• To minimize selection of resistant microorganisms, a novel
susceptibility parameter, the mutant prevention concentration
(MPC), may be used. The MPC is the MIC of the most resistant
first-step mutant in a large bacterial inoculum.3
• Resistance is promoted when antimicrobial concentrations fall
between the MIC and the MPC. This range of concentrations is
called the mutant selection window (MSW).3
• Pharmacokinetic parameters of antimicrobials may be used to
determine the relationship between antimicrobial concentrations
and the MIC, MPC, and MSW.
• Figure 1 shows the MIC, MPC, and MSW. Antimicrobials that
attain concentrations above the MIC but below the MPC will
inhibit the growth of susceptible bacterial populations but will not
inhibit the growth of mutant bacteria. Antimicrobial concentrations
above the MPC will inhibit the growth of both mutant and non-
mutant bacteria. An antimicrobial whose concentrations fall
above the MPC is an ideal therapeutic choice.3
Figure 1. The Mutant Selection Window
• We studied azithromycin, ceftriaxone, doxycycline, ciprofloxacin,
levofloxacin, and moxifloxacin against a fluoroquinolone-resistant
gyrA mutant strain of N. gonorrhoeae, m-49226.
• We also studied the combination of azithromycin with ceftriaxone
and azithromycin with doxycycline against m-49226.
• We used MSW testing and time kill assays to compare the
activity of various antimicrobials against N. gonorrhoeae.
• The MIC/MPC ratio was determined for each antimicrobial, and
the MSW was then calculated for each agent.
• Extracellular pharmacokinetic parameters obtained by
therapeutic doses of all antimicrobials were studied to calculate
the time within the MSW for each agent.
• A time-kill methodology was used to assess bacterial killing of all
antimicrobials, as well as antimicrobial combinations.
Objectives
Corresponding Author
George P. Allen, Pharm.D.
University of New England
College of Pharmacy
716 Stevens Avenue
Portland, ME 04103-2670
gallen3@une.edu
Background Methods Results
References
Conclusions
1. Bolan GA, Sparling PF, Wasserheit JN. The emerging threat of untreatable gonococcal infection. N Engl J Med 2012;366:485-7.
2. Allen GP, Hankins CD. Evaluation of the mutant selection window for fluoroquinolones against Neisseria gonorrhoeae. J Antimicrob Chemother 2009;64:359-63.
3. Drlica K. The mutant selection window and antimicrobial resistance. J Antimicrob Chemother 2003;52:11-17.
4. Blondeau JM. New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Vet Dermatol 2009;20:383-96.
• All of the tested fluoroquinolones showed bactericidal activity and are predicted to prevent mutant selection in N. gonorrhoeae.
• Azithromycin, ceftriaxone, and doxycyline are not predicted to prevent mutant selection.
• Although fluoroquinolones are not currently recommended for the treatment of gonococcal infections, they may represent alternatives in the future.
• Our results suggest a potential role for fluoroquinolones, particularly newer agents such as moxifloxacin, against N. gonorrhoeae.
Antimicrobial Dose ƒCmax (mg/L) T1/2 (hr)
azithromycin 2 g PO x1 0.28 68
ceftriaxone 250 mg IM x1 0.95 8
doxycycline 100 mg PO q12h 1.3 19.5
ciprofloxacin 500 mg PO q12h 2.08 4
levofloxacin 250 mg PO q24h 2.21 7
moxifloxacin 400 mg PO q24h 2.7 12
Disclosure
Authors of this presentation have the following to disclose concerning possible financial or personal relationships with commercial entities that may have a direct or indirect interest in the subject matter of this presentation:
All authors: nothing to disclose.
Time post-administration
Serumortissueantimicrobialconcentration
Concentrations within the MSW
will inhibit the growth of
susceptible bacterial populations
but will not inhibit the growth of
mutant bacteria.
Concentrations above the MPC
will inhibit the growth of both
mutant and non-mutant bacteria.
Concentrations that fall
below the MIC will not
inhibit mutant or non-
mutant bacteria.
Cmax
0 4 8 12 16 20 24
0.0
0.2
0.4
0.6
0.8
1.0
10
20
30
40
time (hours)
concentration(mg/L)
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
time (hours)
concentration(mg/L)
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
10
15
20
time (hours)
concentration(mg/L)
MIC
0.5
MPC
32
%T>MPC = 0%
%TMSW = 0%
MIC
0.03125
MPC
1
%T>MPC = 0%
%TMSW = 100%
MIC
1
MPC
16
%T>MPC = 0%
%TMSW = 30.8%
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0
2.5
3.0
time (hours)concentration(mg/L)
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0
2.5
time (hours)
concentration(mg/L)
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0
2.5
3.0
time (hours)
concentration(mg/L)
MIC
0.0625
MPC
0.25
%T>MPC = 100%
%TMSW = 0%
MIC
0.0625
MPC
0.25
%T>MPC = 91.7%
%TMSW = 8.3%
MIC
0.125
MPC
0.5
%T>MPC = 100%
%TMSW = 0%
0 4 8 12 16 20 24
0
1
2
3
4
5
6
7
8
9
10
À
À
À
À
q
q
q
q
¢
¢
¢
¢
˜
˜ ˜
˜
time (hours)
log10CFU/mL
• Figure 9 shows the results of time-kill assays for all antimicrobials. The dotted
line indicates the lower limit of detection of bacterial colonies (2 log10 CFU/mL).
• All fluoroquinolones achieved bactericidal activity (> 3 log10 CFU/mL kill).
• Ceftriaxone, alone and in combination with azithromycin, also achieved
bactericidal activity.
• Azithromycin and doxycycline, when used alone, did not achieve bactericidal
activity. The combination of azithromycin and doxycycline also failed to achieve
bactericidal activity.
Figure 3. Azithromycin Pharmacodynamics Figure 4. Ceftriaxone Pharmacodynamics Figure 5. Doxycycline Pharmacodynamics
Figure 6. Ciprofloxacin Pharmacodynamics Figure 7. Levofloxacin Pharmacodynamics Figure 8. Moxifloxacin Pharmacodynamics
growth control
doxycycline
azithromycin
ceftriaxone
azithromycin + doxycycline
ciprofloxacin
levofloxacin
moxifloxacin
azithromycin + ceftriaxone