Comparative In Vitro Susceptibilities and Bactericidal Activities of Investigational Fluoroquinolone ABT-492 and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas

Abstract

We determined in vitro susceptibilities for ABT-492 and other antimicrobials against Mycoplasma pneumoniae, Mycoplasma fermentans, Mycoplasma hominis, and Ureaplasma species. ABT-492 MICs were ≤1 μg/ml, and the agent was bactericidal against selected isolates of M. pneumoniae and M. hominis. ABT-492 has potential for treatment of infections due to these microorganisms.

ABT-492 (Abbott Laboratories, Abbott Park, Ill.) is an investigational fluoroquinolone with activity against gram-negative and gram-positive bacteria that cause respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarralis (3, 10, 11). This drug is known to be up to 64 times more active than other fluoroquinolones currently available for use against S. pneumoniae (3). This study was undertaken to evaluate the in vitro activity of ABT-492 against a large number of clinical isolates of mycoplasma and ureaplasma species known to cause disease in humans. ABT-492 was studied in comparison to other fluoroquinolones, macrolides, lincosamides, and doxycycline. Its bactericidal activities were assessed against these organisms by determining MBCs and by measuring the dynamics of bacterial killing with time-kill assays for selected isolates.

(This work was presented at the 103rd General Meeting of the American Society for Microbiology, Washington, D.C., 18 to 22 May 2003.)

The Mycoplasma pneumoniae isolates included 101 strains collected from the respiratory tracts of individuals with proven respiratory disease. Mycoplasma fermentans isolates (n = 13) were either clinical strains or were derived from the mycoplasma collection at the National Institutes of Health. Mycoplasma hominis isolates (n = 10) were derived from clinical specimens of the urogenital tract or wound cultures. Ureaplasma isolates (n = 22), which included both U. urealyticum and U. parvum, were derived from cultures of the urogenital tracts of adults or lower respiratory tracts of neonates.

The following antimicrobial agents were tested: ABT-492, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, clarithromycin, azithromycin, erythromycin, doxycycline, and clindamycin. Clindamycin was tested only against M. hominis and M. fermentans. Clarithromycin, azithromycin, and erythromycin were tested only against M. pneumoniae and Ureaplasma spp. Antimicrobial powders were dissolved according to the manufacturers' recommendations. ABT-492 was dissolved in 1 N NaOH and then diluted to the desired concentration in sterile deionized water. Stock solutions of each drug were prepared fresh on the day each assay was performed.

A broth microdilution method (9) was performed to determine MICs. A subgroup of randomly chosen isolates consisting of 13 M. pneumoniae, 3 M. hominis, 2 M. fermentans, and 2 Ureaplasma isolates was tested to determine MBCs of ABT-492 in comparison to those of other fluoroquinolones. Doxycycline, known to be bacteriostatic against mycoplasmas and ureaplasmas, was also tested for comparative purposes. MBC testing was performed directly from microtiter plates used to determine MICs as described previously (9). Bactericidal activity was identified when the MBC was no more than 2 dilutions (fourfold) greater than the MIC. For the time-kill assays, ABT-492 powder was dissolved and added to each of four tubes of SP 4 broth at concentrations equivalent to one, two, four, and eight times the MICs that had been determined for three isolates of M. pneumoniae and one isolate of M. hominis by procedures and quality control measures described previously (9). Bactericidal effect in this assay was defined as a reduction of ≥3 log₁₀ CFU (99.9%) from the original inoculum.

Summaries of the MICs of ABT-492 the other antimicrobial agents used in this study are shown in Table 1. All organisms were inhibited by ABT-492 at concentrations of 1 μg/ml or less.

TABLE 1.

Summary of broth microdilution MICs of ABT-492 and other antimicrobial agents for human mycoplasmas and ureaplasmas

Species (n) and drug	MIC (μg/ml)a
	Range	50%	90%
M. pneumoniae (101)
ABT-492	0.063-0.5	0.25	0.5
Ciprofloxacin	0.5-4.0	2.0	4.0
Gatifloxacin	0.016-0.25	0.125	0.125
Levofloxacin	0.5-2.0	0.5	1.0
Moxifloxacin	0.032-0.25	0.063	0.125
Azithromycin	≤0.001	≤0.001	≤0.001
Clarithromycin	≤0.001	≤0.001	≤0.001
Erythromycin	≤0.001-0.016	≤0.001	0.004
Doxycycline	0.063-1.0	0.25	0.5
M. fermentans (13)
ABT-492	≤0.001-0.016	0.008	0.016
Ciprofloxacin	0.016-0.125	0.063	0.125
Gatifloxacin	0.002-0.016	0.008	0.016
Levofloxacin	0.016-1.0	0.063	0.125
Moxifloxacin	0.004-0.016	0.016	0.031
Clindamycin	0.004-0.063	0.016	0.063
Doxycycline	0.031-1.0	0.125	0.5
M. hominis (10)
ABT-492	0.008-0.016	0.016	0.016
Ciprofloxacin	0.25-0.5	0.5	0.5
Gatifloxacin	0.031-0.063	0.063	0.063
Levofloxacin	0.125-0.5	0.25	0.5
Moxifloxacin	0.016-0.063	0.031	0.063
Doxycycline	0.008-64	8.0	32.0
Clindamycin	0.016-0.063	0.031	0.063
Ureaplasma spp. (22)
ABT-492	≤0.008-1.0	0.063	0.25
Ciprofloxacin	1.0-16.0	4.0	8.0
Gatifloxacin	0.25-1.0	0.5	1.0
Levofloxacin	0.063-1.0	1.0	2.0
Moxifloxacin	0.063-1.0	0.25	0.5
Doxycycline	0.016-0.25	0.125	0.125
Azithromycin	0.5-8.0	2.0	4.0
Clarithromcycin	0.016-1.0	0.125	0.25
Erythromycin	0.125-4.0	1.0	2.0

^a50% and 90%, MIC₅₀ and MIC₉₀, respectively.

For M. pneumoniae, the ABT-492 MIC at which 90% of the isolates tested are inhibited (MIC₉₀) (0.5 μg/ml) was fourfold higher than those of gatifloxacin and moxifloxacin, twofold less than that of levofloxacin, and eightfold less than that of ciprofloxacin. Fluoroquinolones were less potent overall against M. pneumoniae than macrolides, with the maximum macrolide MIC equal to 0.016 μg/ml. ABT-492 MBCs for 7 of 13 isolates were less than or equal to four times the MICs, indicating bactericidal activity (Table 2). The MBCs of all of the other fluoroquinolones were less than or equal to four times the MIC for 10 to 12 of the 13 M. pneumoniae isolates tested. Conversely, doxycycline showed primarily a bacteriostatic effect with 11 of 13 MBCs being at least eight times higher than the corresponding MICs. Time-kill assay results for three M. pneumoniae isolates showed that ABT-492 killed 99.9% of all three isolates after 24 h when tested at eight times the MIC, after 48 h at concentrations equivalent to twice the MIC or greater, and after 96 h at the MIC.

TABLE 2.

Summary of MBCs for ABT-492 and other agents against M. pneumoniae

Drug	No. of isolates with MBCa:
	1× MIC	2× MIC	4× MIC	≥8× MIC
ABT-492	0	0	7	6
Ciprofloxacin	0	3	9	1
Levofloxacin	0	2	8	3
Moxifloxacinb	0	7	4	1
Gatifloxacin	2	6	5	0
Doxycycline	0	0	2	11

^an = 13.

^bn = 12.

The ABT-492 MIC₉₀ for M. fermentans (0.016 μg/ml) was equal to that of gatifloxacin, was twofold less than that of moxifloxacin, and was eightfold less than those of levofloxacin and ciprofloxacin. All fluoroquinolones tested except levofloxacin and ciprofloxacin were somewhat more potent than clindamycin and doxycycline against this mycoplasma. MBCs for two of two M. fermentans isolates were within 2 dilutions of the MICs, indicating bactericidal activity. Other fluoroquinolones also showed bactericidal activities for at least one of the two isolates tested, whereas doxycycline was bacteriostatic.

The M. hominis ABT-492 MIC₉₀ (0.016 μg/ml) was 4-fold less than the gatifloxacin and moxifloxacin MIC₉₀s and 32-fold less than the levofloxacin and ciprofloxacin MIC₉₀s, making it the most potent fluoroquinolone tested against this mycoplasma. ABT-492 was fourfold more active than clindamycin and had similar activity against five doxycycline-susceptible isolates and five doxycycline-resistant isolates that were tested. ABT-492 MBCs for two of three M. hominis isolates were within 2 dilutions of the MICs, indicating bactericidal activity similar to that of the other fluoroquinolones, whereas doxycyline was bacteriostatic. The time-kill assay showed a bactericidal effect for ABT-492 against M. hominis after 12 h of incubation at concentrations of four and eight times the MIC and after 48 h at concentrations of two or more times the MIC.

For Ureaplasma spp., the ABT-492 MIC₉₀ (0.25 μg/ml) was 2-fold less than the moxifloxacin MIC₉₀, 4-fold less than the gatifloxacin MIC₉₀, 8-fold less than the levofloxacin MIC₉₀, and 32-fold less than the ciprofloxacin MIC₉₀, making it the most potent fluoroquinolone tested against this organism. The activity of ABT-492 was comparable to those of clarithromycin and doxycycline. MBCs for both ureaplasma isolates were ≥8 dilutions higher than MICs, indicating bacteriostatic activity for ABT-492 against this organism. None of the other fluoroquinolones or doxycyline yielded MBCs that were within 4 dilutions of the corresponding MICs.

This study has quantitated the inhibitory activity of a new fluoroquinolone, ABT-492, against clinical isolates of human mycoplasmas and ureaplasmas and demonstrated bactericidal activity against a subgroup of these organisms. MIC and MBC data for ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin are consistent with previously published results for these drugs tested against human mycoplasmas and ureaplasmas (1-9). The results of this study support the future clinical development of ABT-492 as a potential treatment for diseases caused by these organisms.