Results of the growth assays showed a dose-dependent inhibition of C. burnetii
growth in response to increasing pentamidine concentrations, with a significant reduction (73.3% less than negative controls; P
< 0.05) in Coxiella
genome numbers at 1 μM (ca.. 0.6 μg/mL) pentamidine (). Increasing the dosage to 10 μM pentamidine did not significantly increase growth inhibition above that observed at the 1 μM MIC (). MIC results for pentamidine are comparable with the MIC obtained in vitro for doxycycline (ca. 1 μg/mL) and the C. burnetii
Nine Mile phase I strain (RSA493) [11
Fig. 1 Effect of varying concentrations of pentamidine on Coxiella burnetii growth. Coxiella genomes were quantified using quantitative polymerase chain reaction (qPCR)  after 96 h of growth in infected Vero cells in the presence of 0–10 μM (more ...)
A previous study examining maximal plasma concentrations of pentamidine during therapy for Trypanosoma gambiense
infections showed a range of 0.42 μM to 13.42 μM [12
]. These results suggest that concentrations above the in vitro MIC determined in this study could be attained in vivo during therapy.
To ensure that growth inhibition did not simply reflect a reduction in available host cells for C. burnetii to infect, Vero cells were cultured for 96 h in the presence of the same pentamidine concentrations as shown in . DNA was then extracted as above and genome quantities were determined by qPCR using a primer set specific for the Vero cell cytochrome oxidase gene. Results of the qPCR showed that regardless of pentamidine dosage, average cycle threshold (CT) values for the Vero cell cytochrome oxidase gene were not significantly different from untreated controls or from each other (). In addition, overt signs of cytotoxicity (e.g. altered morphology, loss of adherence, etc.) were not observed in Vero cells grown in the presence of pentamidine when cultures were examined by phase-contrast microscopy (data not shown). Live/dead staining using Trypan blue was also conducted and cytotoxicity was measured with a Cytotox96 Kit (Promega Corp., Madison, WI). However, no significant differences were found in the number of viable Vero cells that excluded Trypan blue per unit area or in levels of lactate dehydrogenase released to the culture supernatant (Cytotox96 assays) from Vero cells cultured for 96 h in the presence of 0–10 μM pentamidine (data not shown). Taken together, the results demonstrate that pentamidine does not affect host cell numbers at the concentrations employed in this study and suggest that pentamidine directly affects C. burnetii rather than the host cell in which the bacterium replicates ( and ).
Fig. 2 Effect of pentamidine on Vero host cell numbers during 96 h of growth. Quantitative polymerase chain reaction (qPCR) was used to compare Vero cell genome numbers using a primer set specific to its cytochrome oxidase (GenBank accession no. AY972804). Values (more ...)
To address the hypothesis that pentamidine targets and inhibits splicing by Coxiella
's group I intron RNAs, 35
S-GTP incorporation into spliced intron RNAs was assayed in vitro in the presence of 0, 25 or 50 mM pentamidine. Results of the experiment showed that 25 mM and 50 mM pentamidine significantly (P
< 0.05) reduced the splicing of Cbu.L1917 RNA by ca. 27% and 57%, respectively, compared with controls without the drug (). Cbu.L1951 was comparatively more resistant to splice inhibition and required 50 mM pentamidine to significantly (P
< 0.05) reduce its splicing activity by ca. 19% (). The disparity in Cbu.L1917 and Cbu.L1951 sensitivities to pentamidine may reflect differences in intrinsic splicing rates, where Cbu.L1917 is much more active in vitro possibly owing to the larger size of Cbu.L1951 (719 bases vs. 287 bases, respectively) and partial reliance on potential maturase function of its encoded homing endonuclease. Previous work has shown that group I introns display differential sensitivity to pentamidine [13
], possibly owing to specific RNA site interactions that subsequently affect folding and splicing rates [14
]. Regardless, since both introns must splice and re-ligate the shared precursor RNA to form a mature 23S rRNA, inhibition of either intron would impair growth by deceasing the translational efficiency of Coxiell
a. Thus, although the introns display differential sensitivity to inhibition by pentamidine, they constitute two potential targets for the drug in every 23S rRNA precursor of C. burnetii
. It was also observed that considerably greater quantities of pentamidine were needed to inhibit intron RNA splicing activities in vitro (25–50 mM) in contrast to inhibition of Coxiella
growth within Vero cells (>1.0 μM). These observations may be due to the tendency of pentamidine to accumulate in cellular lysosomes [15
], a characteristic that would, over time, increase the effective concentration of the drug in Coxiella
's phagolysosomal niche.
Fig. 3 Effect of pentamidine on RNA splicing by Coxiella burnetii group I intron RNAs in vitro. Values represent mean 35S-guanosine-5'-triphosphate (35S-GTP) incorporation into spliced RNAs [counts per min (CPMs) as a percent of the untreated control ± (more ...)
Whilst several studies have documented the utility of pentamidine for treatment of fungal (P. carinii
and Candida albicans
) and protozoal (Leishmania
) infections, this is the first report describing the antibacterial activity of the drug in vitro. In addition, we have identified two potential targets for the drug's antibacterial activity. From these data, we predict that any molecule that blocks the activity of Coxiella
's group I introns (e.g. other RNA splice inhibitors) constitutes a potential therapeutic agent for Q fever. Further, since both L1917 and L1951 group I introns are highly conserved elements that disrupt the single 23S rRNA gene of all eight genotypes of C. burnetii
], we predict that pentamidine would be an effective antimicrobial against multiple Coxiella
strains. Although the efficacy of pentamidine against C
in vivo remains to be determined, intron RNA splicing and formation of a mature 23S rRNA is a process that would also undoubtedly be required for replication in vivo. Thus, the drug has potential as an antimicrobial agent against C. burnetii
, especially during chronic Q fever where available therapeutic options are limited.