In Vitro Activity of AZD0914, a Novel DNA Gyrase Inhibitor, against Chlamydia trachomatis and Chlamydia pneumoniae

Abstract

The in vitro activities of AZD0914, levofloxacin, azithromycin, and doxycycline against 10 isolates each of Chlamydia trachomatis and Chlamydia pneumoniae were tested. For AZD0914, the MIC₉₀s for C. trachomatis and C. pneumoniae were 0.25 μg/ml (range, 0.06 to 0.5 μg/ml) and 1 μg/ml (range, 0.25 to 1 μg/ml), respectively, and the minimal bactericidal concentrations at which 90% of the isolates were killed (MBC₉₀s) were 0.5 μg/ml for C. trachomatis (range, 0.125 to 1 μg/ml) and 2 μg/ml for C. pneumoniae (range, 0.5 to 2 μg/ml).

TEXT

Chlamydia trachomatis infection is the most common sexually transmitted infection in the United States, causing more than 1.4 million cases of cervicitis and urethritis each year (1). Chlamydia pneumoniae is a frequent cause of community-acquired respiratory infections, including pneumonia and bronchitis, in adults and children (2). Quinolones have excellent activity against a wide range of bacteria, including Chlamydia spp. (3). Antimicrobial activity of quinolones is mediated through inhibition of bacterial DNA gyrase and topoisomerase IV activities, which then inhibit bacterial DNA synthesis (3). AZD0914 is a member of a new class of antibacterials which incorporates a novel spiropyrimidinetrione that also targets DNA gyrase and topoisomerase IV through a novel mode of inhibition (4). AZD0914 has potent in vitro antibacterial activity against fluoroquinolone-resistant and multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, and Neisseria gonorrhoeae (4, 5).

We compared the in vitro activity of AZD0914 to those of levofloxacin, azithromycin, and doxycycline against 10 isolates each of C. trachomatis and C. pneumoniae.

The isolates of C. trachomatis tested included seven standard isolates from the ATCC (Manassas, VA, USA) [D-UW-57Cx (VR-878), E-BOUR (VR-348B), F-IC-CAL3 (VR-346), H-UW-43Cx (VR-879), I-UW-12Ur (VR-880), J-UW-36Cx (VR-886), and L2-434 (VR-902B)] and four clinical isolates [N18 (cervical), N19 (cervical), and 7015 (infant eye)]. The isolates of C. pneumoniae tested included four standard isolates from the ATCC [TW 183 (VR-2282), AR 39 (53592), CM-1 (VR-1360), and T 2043 (VR1355)] and six isolates from patients with community-acquired pneumonia, including isolates from bronchoalveolar lavage specimens from patients with human immunodeficiency virus infection and pneumonia from the United States (BAY 1, BAY13, BAL 18, BAL 19, BAL 37, and BAL 62).

AZD0914 (AstraZeneca), azithromycin (Sigma-Aldrich, MO, USA), levofloxacin (Sigma-Aldrich, MO, USA), and doxycycline (Sigma-Aldrich, MO, USA) were supplied as powders and solubilized according to the manufacturers' instructions. Drug suspensions were made fresh each time the assay was run. Susceptibility testing of C. trachomatis and C. pneumoniae was performed with HEp-2 cells grown in 96-well microtiter plates (6). Each well was inoculated with 0.2 ml of the test strain diluted to yield 10⁴ inclusion-forming units per ml; the plates were centrifuged at 1,700 × g for 1 h and incubated at 35°C for 1 h. Wells were then aspirated and overlaid with medium containing 1 μg/ml of cycloheximide and serial 2-fold dilutions of the test drugs. After incubation at 35°C for 72 h, cultures were fixed and stained for inclusions with fluorescein-conjugated antibody to the chlamydial lipopolysaccharide genus-specific antigen (Pathfinder; Bio-Rad, Redmond, WA). The MIC was the lowest antimicrobial concentration at which no inclusions were seen. The minimal bactericidal concentration (MBC) was determined by aspirating the antibiotic-containing medium, washing the wells twice with phosphate-buffered saline, and adding antibiotic-free medium. The infected cells were frozen at −70°C, thawed, passed onto new cells, incubated for 72 h, and then fixed and stained as described above. The MBC was the lowest antimicrobial concentration that resulted in no inclusions after passage. All tests were run in duplicate.

The MICs and MBCs for C. trachomatis and C. pneumoniae are shown in Tables 1 and 2. For AZD0914, The MIC₉₀s for C. trachomatis and C. pneumoniae were 0.25 μg/ml (range, 0.06 to 0.5 μg/ml) and 1 μg/ml (range, 0.25 to 1 μg/ml), respectively, and the minimal bactericidal concentrations at which 90% of the isolates were killed (MBC₉₀s) were 0.5 μg/ml for C. trachomatis (range, 0.125 to 1 μg/ml) and 2 μg/ml for C. pneumoniae (range, 0.5 to 2 μg/ml).

TABLE 1.

Activities of AZD0914 and comparator antibacterials against 10 isolates of C. trachomatis

Drug	MIC (μg/ml)			MBC (μg/ml)
	Range	50%	90%	Range	90%
AZD0914	0.06–0.5	0.125	0.25	0.125–1	0.5
Levofloxacin	0.125–0.5	0.25	0.25	0.125–1	0.5
Doxycycline	0.03–0.25	0.06	0.125	0.03–0.25	0.125
Azithromycin	0.004–0.03	0.008	0.015	0.008–0.03	0.015

TABLE 2.

Activities of AZD0914 and comparator antibacterials against 10 isolates of C. pneumoniae

Drug	MIC (μg/ml)			MBC (μg/ml)
	Range	50%	90%	Range	90%
AZD0914	0.25–1	0.5	1	0.5–2	2
Levofloxacin	0.5	0.5	0.5	0.125–2	2
Doxycycline	0.06–0.125	0.125	0.125	0.25–0.5	0.5
Azithromycin	0.03–0.06	0.06	0.06	0.06–0.25	0.25

The in vitro activity of AZD0914 against C. trachomatis was comparable to those of levofloxacin and doxycycline and 16-fold less than that of azithromycin, based on MIC₉₀ values. The in vitro activity of AZD0914 against C. pneumoniae was comparable with that of levofloxacin, 4-fold less than that of doxycycline, and 16-fold less than that of azithromycin, based on MIC₉₀ values. However, in vitro activity may not necessarily predict microbiologic efficacy in vivo against C. pneumoniae. For example, clarithromycin is 10- to 100-fold more active than erythromycin but was not more effective in the eradication of C. pneumoniae (7).

The in vitro activity of AZD0914 is comparable to those of several antibiotics with proven clinical efficacy against chlamydial infections (8). The results presented here suggest that AZD0914 may be effective for the treatment of infections due to C. trachomatis and C. pneumoniae. Furthermore, the in vitro susceptibility testing protocol used for this study is the only one whose results have been shown to correlate with clinical outcome and microbiologic eradication for infections caused by C. pneumoniae (8, 9). Of special interest is the fact that AZD0914 has activity against both C. trachomatis and N. gonorrhoeae (including drug-resistant strains), which may allow treatment of infections with strains of both species with a single drug. The role of AZD0914 in the treatment of C. trachomatis and C. pneumoniae infections will ultimately depend on the results of clinical studies that assess microbiologic efficacy.