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In Vitro Activity of Garenoxacin against Chlamydia spp.

Chlamydophila (Chlamydia) pneumoniae is considered an important pathogen causing respiratory tract infections, such as pharyngitis, sinusitis, bronchitis and pneumonia. Chlamydia trachomatis causes human genitourinary tract infections and is associated with neonatal conjunctivitis and pneumonitis. Chlamydophila (Chlamydia) psittaci is related to human respiratory tract infections acquired from infected animals. Recently, quinolones have come into use for the treatment of chlamydial infectious diseases in addition to macrolides and tetracyclines. Garenoxacin (T-3811ME) is a des-fluoro (6) quinolone with a broad spectrum of in vitro activity against both Gram-positive and -negative bacteria, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae 1. In particular, garenoxacin shows efficient activity against pathogens associated with community-acquired respiratory tract infections, such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and also atypical pathogens, such as Chlamydia spp., Legionella spp. and Mycoplasma pneumoniae. It was reported that garenoxacin showed excellent activity against Chlamydia spp. in vitro using different cell lines of LLC-MK2 cells 2 and HEp-2 cells 3-5. Although the standardization of inspection methods of C. pneumoniae is the aim of organizations such as the CDC in multiple facilities 6, susceptibility testing methods for antimicrobials of Chlamydia spp. are not yet standardized and the cells used for the measurement vary by facility. In Japan, HeLa229 cells or HL cells have been used for antimicrobial susceptibility testing against Chlamydia spp 7,8. We hope that our study can help standardize susceptibility method testing of Chlamydia spp. In this study, we investigated the antichlamydial activity of garenoxacin using HL cells for C. pneumoniae and HeLa 229 cells for C. trachomatis and C. psittaci, (commonly used cell lines), in comparison with that of levofloxacin, gatifloxacin, clarithromycin and minocycline. For minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations, the following strains of C. pneumoniae were tested against two reference strains: TW-183 and AR39 (obtained from the Washington Research Foundation, Seattle, WA, USA), and eight clinical isolates: KKpn-1, KKpn-24, KKpn-27, TFC-62, YK-41, AC14, AC-51, AC-121. All clinical isolates of C. pneumoniae were isolated in Japan from 1989 to 2000. AC-14, AC-51 and AC-121 strains were from children and the other five strains were from adults. We tested three reference strains of C. trachomatis, D/UW3/Cx, E/UW-5/Cx, L2/434/Bu (obtained from the National Institute of Infectious Diseases, Tokyo, Japan) and one reference strain of C. psittaci, Budgerigar-1 (obtained from the National Institute of Infectious Diseases). The two cell lines used were obtained from the National Institute of Infectious Diseases: HL cells for C. pneumoniae and HeLa 229 cells for C. trachomatis and C. psittaci. Eagle’s minimum essential medium (EMEM; Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Tissue Culture Biologicals, Tulare, CA) was used for cell growth of C. pneumoniae and C. trachomatis, and EMEM (Nissui Pharmaceuticals Co., Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated FBS (JRH Biosciences, Lenexa, KS, USA) for C. psittaci. The antimicrobial agents tested were garenoxacin (synthesized in Toyama Chemical Co., Ltd., Tokyo, Japan), levofloxacin (LKT Laboratories, Inc., Saint Paul, MN, USA), gatifloxacin (extracted by Toyama Chemical Co., Ltd. from available tablets; Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan), clarithromycin (LKT Laboratories, Inc.) and minocycline (Wyeth K. K., Tokyo, Japan). Susceptibility testing of Chlamydia spp. was performed by modified standard methods of the Japan Society of Chemotherapy 7, 8. Each cell line was cultured in 24-well plates and cell monolayers were used for the test. Each well was inoculated with 1 x 103 to 104 inclusion-forming units (IFU) of each Chlamydia strain per well, and centrifuged at 900 x g for 1 h. The plates were then incubated for 1 h at 35°C for C. pneumoniae, at 37°C for C. trachomatis and C. psittaci. Inocula were aspirated and replaced with 1 mL of the culture medium with serial two-fold dilutions of the test REPRINT