We have established an in vitro model of long-term continuous Chlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.Chlamydia pneumoniae is a frequent cause of communityacquired pneumonia and bronchitis in adults and children. Like other chlamydial species, it can cause prolonged or chronic infections which may persist for months or years (11). These persistent infections have been implicated in the development of a number of chronic diseases, including chronic obstructive pulmonary disease, atherosclerosis, and asthma (20). However, whether persistent C. pneumoniae is a cause, a triggering cofactor, or an innocent bystander remains controversial.Persistent chlamydial infections can be established in vitro using several methods, including treatment with cytokines (2, 4, 16, 18) or antibiotics (5, 6) or by deprivation of certain nutrients (13). In all cases they have been described as having morphologically abnormal reticulate bodies (RBs), which suggests that they are somehow altered during their otherwise normal development.In the present study we describe the ultrastructural findings determined using an in vitro model of long-term continuous C. pneumoniae infection in HEp-2 cells, a respiratory epithelial cell line (14,17).Briefly, confluent HEp-2 cells were inoculated once with C. pneumoniae isolate TW-183 (ATCC VR2282) or CM-1 (ATCC VR1360) to achieve 100% infection. After 3 to 5 days, when lysis of most of the infected host cells was seen, the culture medium was replaced with fresh medium but without added cycloheximide. After 1 week of further incubation, growth of colonies of new host cells was observed. Since then, continuous C. pneumoniae cultures have been maintained for over 4 years by reseeding the infected host cells into new flasks to prevent overgrowth. No new cells or chlamydiae were added. C. pneumoniae remained viable and cultivable in this model.Two days prior to sampling, the continuously infected cells were seeded onto six-well plates. Cell monolayers were trypsinized, and infected cells were collected into 1.5-ml centrifuge tubes and fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. Samples were prepared for transmission electron microscopy by standard procedures (9). Samples were postfixed in osmium tetroxide, followed by uranyl acetate. The cells were then dehydrated in increasing concentrations of ethanol (50, 70, and 90%) and acetone (90 and 100%) and subsequently embedded in Spurr's epoxy resin. Ultrathin sections (50 to 100 nm in thickness) were prepared and collected onto 200-mesh copper grids, contrasted with 1% uranyl acetate and Reynolds lead citrate before being examined, and photographed using a JEOL 1200EX transmission electron microscope.Three types of chlamydial inclusions were observed by transmission el...

The in vitro activities of ABT 773, telithromycin (HMR 3647), azithromycin, clarithromycin, erythromycin, and levofloxacin were tested against 20 strains of Chlamydia pneumoniae. The MIC at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by ABT 773 were 0.015 g/ml (range, 0.008 to 0.015 g/ml). ABT 773 was the most active antibiotic tested in this study.The ketolides are a new class of macrolide antibiotics with a keto group replacing the L-cladinose moiety in position 3 and an alkyl-aryl extension at positions 11 and 12 of the lactone ring. The ketolides are acid stable and have activity against a broad range of respiratory pathogens, including multiresistant pneumococci, Haemophilus influenzae, Legionella species, and Mycoplasma pneumoniae (1,2,3,5,8). Chlamydia pneumoniae is an important cause of community-acquired respiratory infection in adults and children worldwide. Clinically, these infections cannot be readily differentiated from those caused by other "atypical" pathogens, such as M. pneumoniae. Data on the activity of ketolides against C. pneumoniae are limited; only the activity of telithromycin (HMR 3647) and HMR 3004 (RU 64004) has been evaluated so far (5, 10; F. Haider, F. Eb, and J. Orfila, Abstr. 35th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F165, 1995). Therefore, we compared the in vitro activity of ABT 773 (a new ketolide antibiotic), telithromycin, azithromycin, clarithromycin, erythromycin, and levofloxacin against 20 isolates of C. pneumoniae.Isolates of C. pneumoniae tested included two reference strains, TW 183 (ATCC VR-2282) and AR39 (ATCC 53592), an isolate from a child with pneumonia from Japan, J21 (ATCC 1435), W 6805, an isolate from an adult with pneumonia from Wisconsin, and 16 isolates from a large United States multicenter treatment study of adults with communityacquired pneumonia. ABT 773, telithromycin, azithromycin, erythromycin, clarithromycin, and levofloxacin were provided as powders and solubilized according to the instructions of the manufacturers. Susceptibility testing of C. pneumoniae was performed with cycloheximide-treated HEp-2 cells grown in 96-well microtiter plates (7). Each well was inoculated with 0.1 ml of the test strain diluted to yield 10 3 to 10 4 inclusion-forming units per ml; the plates were centrifuged at 1,700 ϫ g for 1 h and incubated at 35°C for 1 h. Wells were then aspirated and overlaid with medium containing 1 g of cycloheximide per ml and serial twofold dilutions of the test drug. 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; Kallestad Diagnostics, Chaska, Minn.). The MIC was the lowest antibiotic concentration at which no inclusions were seen.The minimal bactericidal concentration (MBC) was determined by aspirating the antibiotic-containing medium, washing wells twice with phosphate-buffered saline, and adding an...

The in vitro activities of iclaprim, a novel dihydrofolate reductase inhibitor, azithromycin, and levofloxacin were tested against 10 strains of Chlamydia trachomatis and 10 isolates of Chlamydia pneumoniae. For C. trachomatis and C. pneumoniae, the iclaprim MIC and minimal bactericidal concentration at which 90% of isolates were inhibited (MIC 90 and MBC 90 ) were 0.5 g/ml, compared to an azithromycin MIC 90 and MBC 90 of 0.125 g/ml and levofloxacin MIC 90 s and MBC 90 s of 1 g/ml for C. trachomatis and 0.5 g/ml for C. pneumoniae.Chlamydia trachomatis infection is the most common sexually transmitted infection in the United States, causing more than 3 million cases of cervicitis and urethritis every year. Chlamydia pneumoniae is a frequent cause of community-acquired respiratory infections, including pneumonia and bronchitis, in adults and children. Dihydrofolate reductase (DHFR) inhibitors, specifically trimethoprim, have been used for many years to treat infections due to a wide range of bacteria, usually in combination with sulfonamides. The antimicrobial activity against bacterial pathogens is mediated through inhibition of thymidylate synthesis and therefore nucleic acid synthesis. Since mammalian cells do not synthesize folic acid, human purine synthesis is not affected significantly. Even though chlamydiae synthesize folate, trimethoprim (TMP) or TMP-sulfamethoxazole cannot be used for the treatment of chlamydial disease, because trimethoprim is not active against chlamydiae (1).Iclaprim (formerly AR-100), a novel DHFR inhibitor, has potent activity against gram-positive and gram-negative bacteria, including F-2025, 2002). There are no data on the activity of iclaprim against Chlamydia spp.; however, trimethoprim has previously been reported to have no significant activity against C. trachomatis in vitro (1). Therefore, we compared the activity of iclaprim, a new DHFR inhibitor, with the activities of azithromycin and levofloxacin against C. trachomatis and C. pneumoniae. Antimicrobial agents were supplied as powders and solubilized according to manufacturers' instructions. Iclaprim (Arpida, Basel, Switzerland), azithromycin (Pfizer, New York, N.Y.), and levofloxacin (Ortho Pharmaceuticals, Raritan, N.J.) were used. In addition, TMP (Roche, N.J.) was tested against two isolates of C. pneumoniae (TW183 and CM-1). Susceptibility testing of C. pneumoniae was performed in cell culture by using HEp-2 cells grown in 96-well microtiter plates as previously described (3). Each experiment was set up in duplicate plates. Each well was inoculated with 0.1 ml of the test organism diluted to yield 10 3 to 10 4 inclusion-forming units per ml, centrifuged at 1,700 ϫ g for 1 h, and incubated at 35°C for 1 h. Wells were then aspirated and overlaid with 0.2 ml of medium containing 1 g of cycloheximide per ml and serial twofold dilutions of the test drug. After incubation at 35°C for 72 h, the cultures in one plate were fixed and stained for inclusions with fluorescein-conjugated antibody to the lipopolysaccharide genus ...