In Vitro Antibacterial Activity of AZD0914 against Human Mycoplasmas and Ureaplasmas

Abstract

In this study, susceptibilities were determined for AZD0914, a spiropyrimidinetrione DNA gyrase inhibitor, azithromycin, doxycycline, and levofloxacin against Mycoplasma and Ureaplasma species. The activity of AZD0914 was comparable to that of levofloxacin and doxycycline against Mycoplasma genitalium and Mycoplasma pneumoniae. The AZD0914 MIC₉₀ against Mycoplasma hominis was 8-fold greater than that for levofloxacin. The AZD0914 MIC₉₀ against Ureaplasma species was 4-fold less than that for azithromycin and 8-fold less than that for levofloxacin and doxycycline.

TEXT

A medical need exists for new antibacterial agents, particularly compounds for the treatment of bacterial infections by multidrug-resistant pathogens and sexually transmitted infections. We previously reported on the in vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor (AstraZeneca Pharmaceuticals LP, Waltham, MA) with potent in vitro antibacterial activity against staphylococci (including methicillin- and vancomycin-resistant strains), streptococci, Neisseria gonorrhoeae, Legionella pneumophila, Clostridium difficile, Chlamydia trachomatis, and Chlamydia pneumoniae (1–3). In this study, the in vitro activity of AZD0914 and three comparators, levofloxacin, doxycycline, and azithromycin, was evaluated against five Mycoplasma and Ureaplasma species that are important human pathogens of the respiratory and urogenital tracts. The organisms tested included clinical isolates and reference strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma pneumoniae, Ureaplasma parvum, and Ureaplasma urealyticum. Three macrolide-resistant M. pneumoniae isolates were tested, two of which were previously published (4). The M. hominis and Ureaplasma strains included clinical isolates that contained tetM, which mediates tetracycline resistance. Ureaplasma strains containing mutations in 23S rRNA that confer macrolide resistance, and others that contained mutations in DNA gyrase and/or topoisomerase IV that confer resistance to fluoroquinolones, were also included (5, 6). A summary of the bacterial strains tested is provided in Table 1.

TABLE 1.

Summary of bacterial strains tested^a

Species (no. of isolates)	Dates isolated	Body site	Location/comments
M. genitalium (11)	1980–1988	Urethra (5), synovial fluid (1), throat (3), unknown (2)	7 clinical isolates from SC, Denmark, and Japan; 4 type strains (ATCC 33530, ATCC 49805, ATCC 49897, and ATCC 49899)
M. pneumoniae (21)	2009–2014	Bronchoalveolar lavage fluid (13), sputum (1), nasopharynx (1), throat (3), respiratory tract not specified (2), cerebrospinal fluid (1)	20 clinical isolates from AL and CO; 1 type strain (ATCC 29342-B7)
M. hominis (21)	1991–2013	Endometrium (15), cervix/vagina (3), rectum (1), wound (1), unknown (1)	20 clinical isolates from AL, AK, CA, CO, KY, OH, PA, TX, and Washington, DC; 1 type strain (ATCC 23114)
Ureaplasma spp. (21)	1997–2013	Pleural fluid (1), tracheal aspirate (2), placenta (1), rectum (1), tissue (1), urethra (4), vagina (6), unknown (5)	20 clinical isolates from AL, CA, MO, and OH; 1 type strain (ATCC 33175)

^aThe total number of bacterial strains for each species includes 1 ATCC type strain used for MIC quality control tests, each as specified by the CLSI (7).

MIC determination and interpretation were performed in accordance with the published guideline of the Clinical and Laboratory Standards Institute for testing human mycoplasmas (7) and included appropriate quality control strains. Mycoplasmacidal (minimal bactericidal concentration [MBC]) concentrations were determined and time-kill studies performed for AZD0914 and levofloxacin, as previously described (8). MBCs within two 2-fold dilutions of the MIC value were considered to be bactericidal. In the time-kill studies, a bactericidal effect was defined as a reduction of ≥3 log₁₀ CFU (99.9%) from the original inoculum.

As summarized in Table 2, AZD0914 showed in vitro activity against M. genitalium comparable to that of levofloxacin and doxycycline. The MIC range for these three drugs was within four 2-fold dilutions (0.25 to 2 μg/ml). The AZD0914 MICs, which ranged from 0.5 to 1 μg/ml for M. genitalium, showed that it was less potent than azithromycin (MIC range, <0.001 μg/ml). The MIC₉₀ for AZD0914 against M. pneumoniae (1 μg/ml) was equivalent to that of levofloxacin and 2-fold higher than that for doxycycline (0.5 μg/ml). Most M. pneumoniae isolates had azithromycin MICs of <0.001 μg/ml, but 3 strains were chosen for testing because they had azithromycin MICs of 16 and 32 μg/ml and contained mutations in 23S rRNA. AZD0914 maintained in vitro potency against these 3 macrolide-resistant isolates comparable to that for those isolates that were fully macrolide susceptible. Among the species tested, AZD0914 had the lowest overall activity against M. hominis (MIC₅₀ and MIC₉₀, 4 μg/ml; maximum MIC, 8 μg/ml). The doxycycline MICs for M. hominis isolates without tetM ranged from 0.03 to 2 μg/ml, while the MICs for the 3 isolates with tetM were 4 to 16 μg/ml. The corresponding tetracycline MICs were 32 μg/ml for those isolates (data not shown). The AZD0914 MICs were not affected by the presence of tetM. The MIC₉₀ for AZD0914 (4 μg/ml) was 8-fold greater than that for levofloxacin (0.5 μg/ml) and was equivalent to that of azithromycin, a drug that is not usually very active against this species. The MIC₉₀ for AZD0914 against Ureaplasma spp. was 1 μg/ml. There was no difference in the AZD0914 MICs against levofloxacin-resistant ureaplasmas (MICs, 8 to 32 μg/ml) and levofloxacin-susceptible isolates (MICs, ≤4 μg/ml). Similarly, among 5 doxycycline-resistant Ureaplasma isolates (MIC range, 4 to 8 μg/ml), the MICs for AZD0914 were not affected (MIC range, 0.25 to 2 μg/ml). The AZD0914 MIC for the single macrolide-resistant isolate of U. urealyticum (azithromycin MIC, 32 μg/ml) was 2 μg/ml, which was only one 2-fold dilution higher than the AZD0914 MIC₉₀ for this drug. Overall, AZD0914 was 4-fold more potent than azithromycin (MIC₉₀, 1 versus 4 μg/ml) against Ureaplasma species.

TABLE 2.

MIC summary for AZD0914 and 3 comparators tested against human mycoplasmas and ureaplasmas

Organism (no. of isolates)	Antimicrobial agent	MIC data (μg/ml)
		Range	MIC50	MIC90
M. genitalium (11)	AZD0914	0.5–1	0.5	1
	Azithromycin	<0.001	<0.001	<0.001
	Doxycycline	0.25–1	0.25	1
	Levofloxacin	0.5–2	1	2
M. pneumoniae (21)	AZD0914	0.5–1	0.5	1
	Azithromycin	<0.001–32	<0.001	16
	Doxycycline	0.125–0.5	0.25	0.5
	Levofloxacin	0.5–1	1	1
M. hominis (21)	AZD0914	2–8	4	4
	Azithromycin	1–8	4	4
	Doxycycline	0.03–16	0.06	4
	Levofloxacin	0.125–1	0.5	0.5
Ureaplasma spp. (21)	AZD0914	0.125–2	0.5	1
	Azithromycin	1–32	2	4
	Doxycycline	0.016–8	0.125	8
	Levofloxacin	0.25–32	1	8

The MBCs for a clinical isolate of M. pneumoniae and a reference strain for M. genitalium (ATCC 33530) for AZD0914 and levofloxacin were 2 times the respective MIC values, indicating bactericidal activity. Both drugs were also bactericidal for M. hominis (strain ATCC 23114), with an AZD0914 MBC of 2 times the MIC, whereas the levofloxacin MBC was 4 times the MIC. Neither drug was bactericidal against a reference strain (U. urealyticum ATCC 33175) and a clinical isolate of U. urealyticum, with MBCs that were >4 times the MICs.

Time-kill studies of M. pneumoniae (strain ATCC 29342-B7) tested against AZD0914 and levofloxacin at concentrations of 0.5 to 8 times the MIC demonstrated a bactericidal effect, with a >3-log₁₀ CFU (99.9%) reduction for AZD0914 after 48 h at 8 times the MIC, after 72 h at 4 times the MIC, and after 96 h at the MIC. Levofloxacin demonstrated a bactericidal effect at 4 and 8 times the MIC after 48 h, at 2 times the MIC after 96 h, and at the MIC after 120 h of incubation. Time-kill studies of M. genitalium (strain ATCC 33530) showed a bactericidal effect for AZD0914 after 24 h at 4 and 8 times the MIC and after 48 h for 2 times the MIC. A bactericidal effect was demonstrated for levofloxacin after 24 h of incubation at concentrations at and greater than the MIC.

Mycoplasma and Ureaplasma species can cause significant disease in respiratory and urogenital tracts (9). M. pneumoniae is an important cause of tracheobronchitis and community-acquired pneumonia in children and adults. Both M. genitalium and U. urealyticum can cause male urethritis, and M. genitalium also causes female cervicitis and pelvic inflammatory disease. Invasive infections of the bloodstream, central nervous system, and lungs sometimes occur in neonates due to M. hominis and Ureaplasma species. Invasive disease may also occur in adults in the setting of immunodeficiency. Treatment options for mycoplasmal and ureaplasmal infections are no longer clear-cut, since macrolide resistance is becoming very common in M. pneumoniae in Asia and is spreading gradually to Europe and North America (4, 10). The rates of tetracycline resistance may approach 50% in Ureaplasma spp. in some areas, and resistance to macrolides and fluoroquinolones has been well documented among the genital mycoplasmas (5, 6, 10). Patients who are immunosuppressed and those who have received numerous courses of antibiotics over time are at greater risk for having infections with drug-resistant organisms (10). For these reasons, new agents that are not affected by cross-resistance to other drug classes, such as macrolides, tetracyclines, and fluoroquinolones, are needed.

This study has demonstrated that AZD0914 has in vitro activity against M. genitalium, M. pneumoniae, U. urealyticum, and U. parvum that is generally comparable to that of levofloxacin, and its potency was unaffected by the presence of mutations conferring fluoroquinolone resistance, as has been shown with other microorganisms, including staphylococci and N. gonorrhoeae (3). Furthermore, resistance to macrolides and tetracyclines in Mycoplasma and Ureaplasma spp. appeared not to have any significant effect on the MICs of AZD0914. The AZD0914 MIC₉₀ was 4-fold less than that of azithromycin against Ureaplasma spp., making it the most active drug among all agents tested against these organisms. Similar to levofloxacin, AZD0914 showed bactericidal activity against M. genitalium, M. pneumoniae, and M. hominis, with MBCs of 1 to 2 times the MIC. It should be noted, however, that the AZD0914 MICs against M. hominis were several dilutions higher than those of levofloxacin, with the MIC₅₀ ranging from 2 to 8 μg/ml and an MIC₉₀ of 4 μg/ml. AZD0914 was not bactericidal against U. urealyticum, consistent with prior data for fluoroquinolones, such as garenoxacin, against these organisms (8). These data suggest that AZD0914 may be a potentially useful agent for further development as a possible treatment for infections caused by human mycoplasmas and ureaplasmas in the urogenital or respiratory tract.