Rickettsiae grow only intracellularly, and the antibiotic susceptibilities of these bacteria have been assessed by either plaque, dye uptake, or immunofluorescence assays, which are time-consuming. We used a quantitative PCR (with the LightCycler instrument) to assess the levels of inhibition of Rickettisa felis, R. conorii, and R. typhi DNA synthesis in the presence of various antibiotics. We established the kinetics of rickettsial DNA during growth and showed that R. conorii grows more quickly than R. typhi in cell culture, with maximum replication occurring after 5 and 7 days, respectively. The MICs of the antibiotics tested for R. conorii and R. typhi by the quantitative PCR assay were similar to those previously obtained by plaque and dye uptake assays. We found that R. felis is susceptible to doxycycline, rifampin, thiamphenicol, and fluoroquinolones but not to gentamicin, erythromycin, amoxicillin, or trimethoprim-sulfamethoxazole. The resistance of this new species to erythromycin is consistent with its current taxonomic position within the spotted fever group. We believe that quantitative PCR could be used in the future to simplify and shorten antibiotic susceptibility assays of other rickettsiae and other strict intracellular pathogens.Rickettsiae are strict intracellular bacteria belonging to the alpha group of the class Proteobacteria (15). Rickettsioses are zoonoses that have geographical distributions similar to those of their vectors. The genus comprises the spotted fever group rickettsiae and the typhus group rickettsiae, which includes Rickettsia prowazekii, the agent of epidemic typhus, and R. typhi, the agent of murine typhus (16). Since they are obligate intracellular bacteria, in vitro studies of their susceptibilities to antibiotics necessitate the use of cell culture systems. Using the plaque assay and the dye uptake assay (18), we have now described the antibiotic susceptibilities of most of the rickettsiae with the exception of R. felis, which, until recently, was not available to us. The two assays depend on the induction of cytopathic effects and plaque formation in cell cultures by the rickettsiae, but some rickettsiae do not normally cause cytopathic effects in primary cultures (18). Recently, Ives et al.(5-7) described a new assay that uses immunofluorescence staining, which avoids the problem of a lack of cytopathic effects. A lack of cytopathic effects occurs with R. felis, the agent of flea-borne spotted fever, which we have now isolated and cultured in a Xenopus laevis cell line (XTC-2 cells) (14) and in Vero cells. To determine the antibiotic susceptibility of this species we developed an original antibiotic assay using PCR with the LightCycler instrument. It is a new, real-time PCR technique which has been used to diagnose several bacterial infections (3) but which has not been used to determine antibiotic susceptibilities. The technique combines PCR amplification and detection of products in a single optically clear glass capillary tube, which enables rapid temperature transi...

In order to find new molecules for antiviral drug design, we screened 102 ethyl acetate extracts from New-Caledonian flora for antiviral activity against the dengue 2 virus RNA-dependant RNA polymerase (DV-NS5 RdRp). The leaf extract of Dacrydium balansae, which strongly inhibited the DV-NS5, was submitted to bioguided fractionation. Four biflavonoids ( 1- 4), three sterols ( 5- 7), and two stilbene derivatives ( 8- 9) were identified and evaluated for their antiviral potential on the DV-NS5 RdRp. Biflavonoids appeared to be potent inhibitors of DV-NS5 RdRp with IC (50)s between 0.26 and 3.12 µM. Inhibitory activity evaluations against the RNA polymerase from other Flaviviridae viruses allowed us to conclude that these compounds are specific inhibitors of the DV RNA polymerase. The strongest inhibitions were observed with hinokiflavone ( 4), but podocarpusflavone A ( 2) is the strongest noncytotoxic inhibitor of the DV-NS5 and it also displayed polymerase inhibitory activity in a DV replicon. A preliminary structure-activity relationship study (SARs) revealed the necessity of the biflavonoid skeleton, the influence of number and position of methoxylations, and the importance of a free rotation of the linkage between the two apigenin monomers of the biflavonoids. To the best of our knowledge, podocarpusflavone A ( 2) is the strongest noncytotoxic non-nucleotide molecule exhibiting a specific inhibitory activity against the RNA polymerase domain of DV-NS5 and thus is promising for chemotherapy development against dengue fever.