JAC Advance Access originally published online on June 30, 2008
Journal of Antimicrobial Chemotherapy 2008 62(4):845-847; doi:10.1093/jac/dkn282
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research letters |
Lovastatin, but not pravastatin, limits in vitro infection due to Coxiella burnetii
URMITE, CNRS-IRD UMR 6236, Faculté de Médecine et de Pharmacie, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
* Corresponding author. Tel: +33-491-32-43-75; Fax: +33-491-38-77-72; E-mail: jm.rolain{at}medecine.univ-mrs.fr
Keywords: minimum inhibitory concentration , inhibitory effect , Q fever
Data from clinical, animal and in vitro studies suggest that statins could have a beneficial effect in sepsis.1 Although little data exist about the effect of statins on strict intracellular bacteria such as Coxiella burnetii,2,3 we know that the genome of these bacteria contains the steroid biosynthesis pathway, and bacterial multiplication is achieved in a vacuole rich in cholesterol.3 Inhibition of cholesterol with different compounds could lead to limitation or inhibition of the bacterial growth.2,3 Moreover, Q fever, the disease induced by C. burnetii, remains difficult to treat, especially the chronic form. To the best of our knowledge, only the in vitro effect of lovastatin on the growth of C. burnetii has been studied previously.2,3 In the present study, we evaluated the growth inhibitory effect of two statins, lovastatin and pravastatin, on C. burnetii Nine Mile strain. Bacteria were grown in L929 cells either with or without pre-incubation for 2 days with statins at non-cytotoxic concentrations prior to infection. After bacterial infection, the media containing lovastatin or pravastatin were changed every 2 days. The two control groups not treated with statins consisted of infected cells alone or infected cells treated with doxycycline 4 mg/L. To evaluate bacterial growth in different conditions, we used a quantitative real-time PCR with a Taqman* probe targeting the Com1 gene. We compared bacterial growth in the control group (days 0–6) with that in the statin and doxycycline groups. When cells were treated with lovastatin at 0.4 mg/L, bacterial growth was reduced by 43% compared with untreated controls (P = 0.064), whereas pravastatin at 4 mg/L induced a 32% increase in the bacterial growth compared with untreated controls (P = 0.043) (Figure 1). Doxycycline at 4 mg/L induced bacterial inhibition >100% (P = 0.034). We also used an indirect immunofluorescence assay with double staining (C. burnetii and LAMP1)3 at day 7 post-infection to look at intravacuolar bacteria. More than 120 micro-photographs (Leica DM 2500, objective 100x oil, Nikon DS1-QM 1 Mp camera) were taken of each culture, and the mean size of the vacuoles containing fluorescent bacteria and the mean number of vacuoles per microscopic field (6400 µm2) were recorded using ImageJ software. Statistical analyses were performed using Epi Info 6.0 software (CDC, Atlanta, GA, USA). Figure 1 shows that lovastatin added prior to infection at 0.4 mg/L significantly reduced both the rate of infection [vacuoles significantly smaller than controls (P < 0.0001)] and the bacterial fluorescence/field. However, these effects were not found when lovastatin was added to cells after infection (P = 0.685) (Figure 1). Although pravastatin at 4 mg/L significantly induced smaller vacuoles (P = 0.01), the mean number of vacuoles was similar to that of the controls (Figure 1). This effect was not due to a decrease in the bacterial internalization at day 0 by cells pre-incubated with statins compared with the control group, as the mean number of internalized bacteria in the two groups was not statistically different (P > 0.05).
|
Our data are consistent with those of Howe and Heinzen,2,3 who reported C. burnetii growth inhibition with lovastatin at the same concentration. They also described morphologically that vacuoles were smaller when infected cells were treated with lovastatin compared with untreated cells. However, they did not use a quantitative method to prove this effect. Our method, in which we coupled imaging and software analysis, allows quantitative determination of differences in vacuole size and number.
Interestingly, pravastatin did not have any apparent effect in reducing C. burnetii infection. Pravastatin is hydrophilic and seems to be less effective than other statins in different models.4 This could be due to different pharmacokinetic properties compared with other statins4 and may explain the lack of activity of pravastatin in our C. burnetii model of infection. In our study, lovastatin was effective at a concentration achievable in the sera of patients treated with this drug. This drug does not seem to interfere with bacterial entry in cells, as internalization by cells was not different from controls, as also reported for Salmonella Typhimurium.5 We hypothesize that lovastatin indirectly reduces C. burnetii growth by modifying cholesterol-rich vacuoles. However, a minimal direct effect on bacteria could not eventually be eliminated, as the genome of C. burnetii contains some genes involved in the steroid biosynthesis pathway, especially that encoding the HMG CoA reductase, available at the KEGG web site (http://www.genome.jp/dbget-bin/get_pathway?org_name=cbu&mapno=00100). In vitro studies with statins in association with doxycycline could be performed in the future to look for a possible synergistic inhibitory effect on C. burnetii. Because the inhibitory effect of statins was seen only with pre-incubated cells, we believe that lovastatin may be effective in prophylaxis. These findings need to be confirmed using an animal model6 and/or epidemiological case–control study, especially in patients with chronic Q fever.
 |
Funding |
|---|
 Top Funding Transparency declarationsSupplementary dataReferences |
|---|
No specific funding for this work.
|
Transparency declarations |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
A colour version of Figure 1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
We thank Paul Newton for reviewing the manuscript prior to submission and Guy Vestris for technical assistance.
|
References |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
1 Terblanche M, Almog Y, Rosenson RS, et al. Statins: panacea for sepsis? Lancet Infect Dis (2006) 6:242–8.[CrossRef][Web of Science][Medline]
2 Howe D, Heinzen RA. Replication of Coxiella burnetii is inhibited in CHO K-1 cells treated with inhibitors of cholesterol metabolism. Ann N Y Acad Sci (2005) 1063:123–9.[CrossRef][Web of Science][Medline]
3 Howe D, Heinzen RA. Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism. Cell Microbiol (2006) 8:496–507.[CrossRef][Web of Science][Medline]
4 Corsini A, Bellosta S, Baetta R, et al. New insights into the pharmacodynamic and pharmacokinetic properties of statins. Pharmacol Ther (1999) 84:413–28.[CrossRef][Web of Science][Medline]
5
Catron DM, Lange Y, Borensztajn J, et al. Salmonella enterica serovar Typhimurium requires nonsterol precursors of the cholesterol biosynthetic pathway for intracellular proliferation. Infect Immun (2004) 72:1036–42.
6 Leone M, Honstettre A, Lepidi H, et al. Effect of sex on Coxiella burnetii infection: protective role of 17β-estradiol. J Infect Dis (2004) 189:339–45.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Parquet, S. Briolant, M. Torrentino-Madamet, M. Henry, L. Almeras, R. Amalvict, E. Baret, T. Fusai, C. Rogier, and B. Pradines Atorvastatin Is a Promising Partner for Antimalarial Drugs in Treatment of Plasmodium falciparum Malaria Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2248 - 2252. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||