Effect of Prolonged Treatment with Azithromycin, Clarithromycin, or Levofloxacin on Chlamydia pneumoniae in a Continuous-Infection Model

Persistent infections with Chlamydia pneumoniae have been implicated in the development of chronic diseases, such as atherosclerosis and asthma. Although azithromycin, clarithromycin, and levofloxacin are frequently used for the treatment of respiratory C. pneumoniae infections, little is known about the dose and duration of therapy needed to treat a putative chronic C. pneumoniae infection. In this study, we investigated the effect of prolonged treatment with azithromycin, clarithromycin, or levofloxacin on the viability of C. pneumoniae and cytokine production in an in vitro model of continuous infection. We found that a 30-day treatment with azithromycin, clarithromycin, and levofloxacin at concentrations comparable to those achieved in the pulmonary epithelial lining fluid reduced but did not eliminate C. pneumoniae in continuously infected HEp-2 cells. All three antibiotics decreased levels of interleukin-6 (IL-6) and IL-8 in HEp-2 cells, but this effect appeared to be secondary to the antichlamydial activity, as the cytokine levels correlated with the concentrations of microorganisms. The levels of IL-1β, IL-4, IL-10, tumor necrosis factor alpha, and gamma interferon were too low to assess the effect of antibiotics. These data suggest that the dosage and duration of antibiotic therapy currently being used may not be sufficient to eradicate a putative chronic C. pneumoniae infection

Differential Expressions of Genes Encoding Membrane Proteins between Acute and Continuous Chlamydia Pneumoniae Infections

Chlamydia pneumoniae is associated with several chronic human diseases, including chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. During chronic disease, organisms are believed to exist in a persistent phase that is not well understood at the genetic level. Long-term in vitro continuous infections are spontaneously persistent and are less susceptible than in vitro acute infections to treatment with antibiotics, and are therefore particularly relevant as an in vitro model of in vivo chronic disease. Real-time reverse transcriptase-PCR (r-t RT-PCR) was used to quantitate transcript copy numbers of 13 genes in continuous and acute infections with C. pneumoniae. The set of genes studied encodes proteins with known or predicted functions in the cell membrane, the inclusion membrane, cell division, metabolism, and immunopathology. Significant upregulation was seen for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in continuous cultures. The genes omcB, pmp1, and porB, all of which encode membrane proteins, shared similar patterns of expression over both acute and continuous profiles. These results show that Chlamydia in the long-term continuous model of persistence have a unique transcription profile, adding to our knowledge of regulation of this important stage of chlamydial growth

In Vitro Activities of Rifamycin Derivatives ABI-1648 (Rifalazil, KRM-1648), ABI-1657, and ABI-1131 against Chlamydia trachomatis and Recent Clinical Isolates of Chlamydia pneumoniae

ABI-1648 (rifalazil) is a semisynthetic rifamycin with potent bactericidal activity against intracellular respiratory bacteria, including Mycobacterium tuberculosis, and a long half-life (∼60 h) and thus can be administered once weekly. We therefore tested the in vitro activities of ABI-1648, its derivatives ABI-1657 and ABI-1131, azithromycin, and levofloxacin against 10 strains of Chlamydia trachomatis and 10 recent clinical isolates of Chlamydia pneumoniae. The MICs at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed for ABI-1648, ABI-1657, and ABI-1131 were 0.0025 μg/ml for C. trachomatis and 0.00125 to 0.0025 μg/ml for C. pneumoniae. ABI-1648, ABI-1657, and ABI-1131 were 10- to 1,000-fold more active than azithromycin and levofloxacin

Emergence of Resistance to Rifampin and Rifalazil in Chlamydophila pneumoniae and Chlamydia trachomatis

Although rifamycins have excellent activity against Chlamydophila pneumoniae and Chlamydia trachomatis in vitro, concerns about the possible development of resistance during therapy have discouraged their use for treatment of chlamydial infections. Rifalazil, a new semisynthetic rifamycin with a long half-life, is the most active antimicrobial against C. pneumoniae and C. trachomatis in vitro, indicating its potential for treatment of acute and chronic C. pneumoniae and C. trachomatis infections. We investigated the effect of serial passage of two C. pneumoniae isolates and two serotypes of C. trachomatis in subinhibitory concentrations of rifalazil and rifampin on the development of phenotypic and genotypic resistance. C. trachomatis developed resistance to both antimicrobials within six passages, with higher level resistance to rifampin (128 to 256 μg/ml) and lower level resistance to rifalazil (0.5 to 1 μg/ml). C. pneumoniae TW-183 developed only low-level resistance to rifampin (0.25 μg/ml) and rifalazil (0.016 μg/ml) after 12 passages. C. pneumoniae CWL-029 failed to develop resistance to either drug. Two unique mutations emerged in the rpoB gene of rifampin (L456I) and rifalazil (D461E)-resistant C. pneumoniae TW-183. A single mutation (H471Y) was detected in both rifampin- and rifalazil-resistant C. trachomatis UW-3/Cx/D, and a unique mutation (V136F) was found in rifalazil-resistant BU-434/L(2). No mutations were detected in the entire rpoB gene of rifampin-resistant BU-434/L(2). This is the first description of antibiotic resistance-associated mutations in C. pneumoniae and of rifampin resistance in C. trachomatis not associated with mutations in the rpoB gene

Isolation and Antimicrobial Susceptibilities of Chlamydial Isolates from Western Barred Bandicoots

A range of species of Chlamydiales have previously been detected in a variety of Australian marsupials, including koalas and western barred bandicoots. Thirty-seven ocular, urogenital, or nasal swabs were obtained from 21 wild western barred bandicoots. Chlamydia culture and antibiotic susceptibility testing were performed for cycloheximide-treated HEp-2 cells in 96-well microtiter plates. Chlamydia spp. were isolated from 11 specimens from 9 (42.8%) bandicoots. All isolates were identified as Chlamydiales by conventional PCR with 16S and 23S rRNA gene primers specific to Chlamydiales and were confirmed to be Chlamydia pneumoniae by a C. pneumoniae-specific ompA-based real-time PCR assay and 16S rRNA and 23S rRNA gene signature sequence analyses. The MICs of azithromycin, doxycycline, ciprofloxacin, and enrofloxacin for 10 C. pneumoniae isolates from these bandicoots ranged from 0.015 to 1 μg/ml, 0.25 to 1 μg/ml, 0.25 to 2 μg/ml, and 0.25 to 0.5 μg/ml, respectively. The MICs at which 90% of isolates were inhibited and the minimal bactericidal concentrations were within the ranges reported previously for human isolates of C. pneumoniae

Genetic and Culture-Based Approaches for Detecting Macrolide Resistance in Chlamydia pneumoniae

Three clinical Chlamydia pneumoniae isolates for which the MIC of azithromycin increased after treatment were investigated for genetic evidence of macrolide resistance. Attempts to induce antibiotic resistance in vitro were made. No genetic mechanism was identified for the phenotypic change in these C. pneumoniae isolates. No macrolide resistance was obtained in vitro

Molecular Characterization of Chlamydophila Pneumoniae Isolates from Western Barred Bandicoots

Chlamydophila pneumoniae is an obligate intracellular respiratory pathogen that has been associated with pneumonia and chronic bronchitis, atherosclerosis, asthma and other chronic diseases in humans. However, C. pneumoniae is not restricted to humans, as originally thought, and can cause infections in several animal hosts. C. pneumoniae was isolated in cell culture from nine Western barred bandicoots (Perameles bougainville) from Australia. The sequences of five genomic regions were determined, including full-length sequences of the 16S rRNA and ompA genes and the ygeD-urk intergenic spacer, and partial sequences of the 23S rRNA and rpoB genes. Sequence analysis of the entire 16S rRNA and ompA genes from bandicoot isolates demonstrated that they were 98.2-98.3% similar to human isolates, 94.6-99.3% similar to the equine biovar and almost identical, with 99.5-99.9% similarity, to the koala biovar. Comparative genotyping of the variable domain 4 region of the ompA gene demonstrated that bandicoot isolates seemed to be identical to the animal genotype that has been recently identified in human carotid plaque specimens. Minor sequence polymorphism observed in ompA, 16S rRNA and rpoB genes of animal isolates, indicating genomic diversity within C. pneumoniae, may have important implications for diagnostic PCR assays leading to false negative results. Forty percent of selected published species-specific PCR assays were found to have sequence variability in primer and/or probe that might affect their performance in detecting bandicoot isolates of C. pneumoniae, or possibly other animal and human strains where minor sequence polymorphisms may be present. The data from this study support the previous observations that C. pneumoniae is not restricted to humans and may be widespread in an animal reservoir with a potential risk of transmission to humans