Department of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine (Collaborating Center of WHO), Faculty of Medicine, University of Heraklion, 1393/71409 Crete, Greece
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Abstract |
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Introduction |
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Infections caused by C. burnetii pose a serious problem, because none of the antibiotic regimens (including quinolones) proposed for the treatment of acute Q fever is bactericidal in vitro, which may explain the treatment failures in chronic Q fever infections. The antibiotic of choice remains tetracycline, but the optimal duration of antibiotic therapy for chronic Q fever remains undetermined, and treatment for 1 year and for indefinite periods has been suggested.2 Antimicrobial resistance could be one of the factors contributing to the treatment failures in chronic forms of the disease. Therefore, it is very important to monitor the possible development of resistance during these long periods of treatment.
Studies with Escherichia coli and other Gram-negative bacteria have shown that fluoroquinolones exert their antibacterial activities by inhibiting the DNA gyrase, an essential bacterial topoisomerase II.3,4 However, access to the target site is also a major determinant of antibacterial activity, the outer membrane being the major permeability barrier in Gram-negative bacteria.5
Decreased levels of drug accumulation, associated with a change in the outer membrane proteins (OMPs), have been identified as a second mechanism of resistance to fluoroquinolones in Gram-negative bacteria.3,4 An energy-dependent efflux pump, in association with altered OMPs, appears to play a critical role in reducing intracellular drug concentrations by pumping the drug back across the cytoplasmic membrane and, in some cases, the outer membrane.6
Little is known about the mechanisms of resistance to fluoroquinolones in C. burnetii. Recently, Musso et al.7 and Spyridaki et al.8 reported that a high level of in vitro resistance to quinolones in C. burnetii is associated with two distinct nucleotide mutations in gyrA gene. However, nothing is known about other possible mechanisms of resistance to quinolones, namely the elimination of antibiotics through an active efflux system and/or decreased outer membrane permeability. These mechanisms could also account for the differential antibiotic susceptibilities among the quinolone-susceptible and in vitro quinolone-resistant C. burnetii strains.
In this study, the uptake and intracellular accumulation of quinolones by susceptible and in vitro resistant C. burnetii strains were examined using pefloxacin as a representative compound.
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Materials and methods |
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We examined the uptake and intracellular accumulation of pefloxacin in two in vitro quinolone-resistant C. burnetii strains, RCB2 and RCB4 (MIC 3264 mg/L), and the parental quinolone-susceptible strains SCB2 and SCB4 (MIC 14 mg/L).8
All organisms were propagated within Vero cell cultures, and the MICs of quinolones were determined by the shell vial technique.8 Vero cells infected 15 days previously were used in the experiments.
Antibiotic and chemicals
Pefloxacin was purchased from Rhone Poulenc (Paris, France) and chemicals from Sigma Chemical Co. (St Louis, MO, USA).
Purification of C. burnetii from infected Vero cells
C. burnetii was purified from infected Vero cells by three freezethaw cycles (170°C, 37°C) followed by differential centrifugation. The purified preparations of C. burnetii were identified by the Gimenez stain. Concentrations of C. burnetii (organisms/mL) were determined by the shell vial technique.8
Uptake and intracellular accumulation of pefloxacin in purified C. burnetii isolates
Pefloxacin uptake was assayed by the method of Chapman & Georgopapadakou5 with slight modifications. Equivalent concentrations of each C. burnetii strain (108 organisms/mL) were suspended in 15 mM phosphate-buffered saline (PBS) adjusted to pH 4.5 and 7.2. The bacteria were equilibrated for 10 min at 37°C in a shaking water-bath in the presence and absence of 150 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and incubated with different concentrations of pefloxacin. For timekill studies, pefloxacin was added to a final concentration of 10 mg/L. After the addition of pefloxacin, 500 µL samples were removed at different time intervals, chilled on ice, diluted by the addition of 2.5 mL of ice-cold PBS (pH 4.5 and 7.2) and centrifuged for 5 min at 3800g. The pellet was washed once with 2 mL of ice-cold PBS, resuspended in 1 mL of 0.1 M glycine hydrochloride (pH 3) and shaken at 25°C for 15 h. The samples were then centrifuged at 3800g for 15 min at room temperature. The fluorescence in the supernatant was measured with a spectrofluorometer (Perkin Elmer, Norwalk, CT, USA) at excitation and emission wavelengths of 278 and 442 nm, respectively. The intracellular accumulation of antibiotic in the samples was calculated by comparison with standard curves of pefloxacin in 0.1 M glycine hydrochloride (pH 3) and is given in mg/L of bacteria.
The statistical test that was used to analyse the difference between the intracellular concentrations in quinolone-susceptible and in vitro quinolone-resistant C. burnetii strains, at different pH, was the MannWhitney U-test.
Preparation and analysis of OMPs
The purified preparations of C. burnetii were washed once in PBS (pH 7.4) and sonicated in ice by ten 30 s bursts at an output of seven with 30 s pauses between each burst (VirTis sonifier, Gardiner, NY, USA). Sonic extracts were centrifuged at 7500g for 5 min and washed twice. After centrifugation the supernatant was centrifuged at 14 000g for 30 min. The membrane pellet was then incubated in 3% N-lauroyl sarcosyl for 45 min at room temperature. After centrifugation at 14 000g for 45 min, the pellet containing the OMPs was resuspended in 0.5 M TrisHCl (pH 6.8). The protein concentrations in the outer-membrane preparations were determined by the colorimetric protein microdetermination method (Sigma-Aldrich, Germany) and were adjusted to 1.5 mg/mL. Samples were stored at 20°C until use. OMP profiles were examined by SDS PAGE.9
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Results and discussion |
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The uptake of pefloxacin by C. burnetii was non-saturable in the range 1040 mg/L when it was measured up to 10 min after its addition. Uptake augmented linearly when extracellular pefloxacin concentrations increased from 10 to 40 mg/L. In the following experiments, pefloxacin was added to a final extracellular concentration of 10 mg/L. Pefloxacin uptake by the purified RCB2, RCB4, SCB2 and SCB4 C. burnetii strains was examined in the absence and presence of CCCP at pH 7.2 and 4.5 (Figure 1). Without CCCP and pH 7.2, the levels of uptake by the strains RCB2 and RCB4 were lower than those of the SCB2 and SCB4, indicating higher penetrability of pefloxacin into quinolone-susceptible C. burnetii strains (Figure 1
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The fact that both quinolone-susceptible and quinolone-resistant C. burnetii strains exhibit a reduced intracellular concentration of pefloxacin in an acidic environment indicates the existence of an additional contributing mechanism of resistance that has developed in the acidic pH of the phagolysosomes, where the microorganism multiplies.1
OMPs
To further investigate the differences of permeability between the in vitro quinolone-resistant and the parental quinolone-susceptible C. burnetii strains, the OMPs were analysed by SDSPAGE and compared (Figure 2). Differences between the quinolone-susceptible and the in vitro quinolone-resistant C. burnetii strains were not observed.9,10 Consequently, the different accumulation of pefloxacin by the RCB2 and RCB4 strains does not seem to be related to different OMPs.
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Notes |
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References |
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2 . Raoult, D. (1993). Treatment of Q fever. Antimicrobial Agents and Chemotherapy 37, 17336.[ISI][Medline]
3 . Cambau, E. & Gutmann, L. (1993). Mechanisms of resistance to quinolones. Drugs 45, 1523.[ISI][Medline]
4 . Wolfson, J. S. & Hooper, D. C. (1985). The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrobial Agents and Chemotherapy 28, 5816.[ISI][Medline]
5 . Chapman, J. S. & Georgopapadakou, N. H. (1988). Routes of quinolone permeation in Escherichia coli. Antimicrobial Agents and Chemotherapy 32, 43842.[ISI][Medline]
6 . Cohen, S. P., Hooper, D. C., Wolfson, J. S., Souza, K. S., McMurry, L. M. & Levy, S. B. (1988). Endogenous active efflux of norfloxacin in susceptible Escherichia coli. Antimicrobial Agents and Chemotherapy 32, 118791.[ISI][Medline]
7 . Musso, D., Drancourt, M., Osscini, S. & Raoult, D. (1996). Sequence of quinolone resistance-determining region of gyrA gene for clinical isolates and for an in vitro-selected quinolone-resistant strain of Coxiella burnetii. Antimicrobial Agents and Chemotherapy 40, 8703.[Abstract]
8 . Spyridaki, I., Psaroulaki, A., Aransay, A., Scoulica, E. & Tselentis, Y. (2000). Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP. Journal of Clinical Laboratory Analysis 14, 5963.[ISI][Medline]
9 . Wen, B.-H., Yu, S.-R., Yu, G.-Q., Li, Q.-J. & Zhang, X. (1991). Analysis of proteins and lipopolysaccharides from chinese isolates of Coxiella burnetii with monoclonal antibodies. Acta Virology 35, 53844.[ISI][Medline]
10 . Mallavia, L. P. & Samuel, J. E. (1989). Genetic diversity of Coxiella burnetii. In Intracellular Parasitism, (Moulder, J. W., Ed.), pp. 11726. CRC Press, Boca Raton, FL.
Received 23 July 2001; returned 19 September 2001; revised 6 November 2001; accepted 9 November 2001