a School of Pharmacy and Department of Family Medicine, School of Medicine, Campus Box 7595, University of North Carolina at Chapel Hill, NC 27599-7595; b Viral and Rickettsial Branch, Centers for Disease Control and Prevention, Atlanta, GA; c Department of Pharmacy, University of North Carolina Hospitals; d Abbott Laboratories, Abbott Park, IL, USA
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Abstract |
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Introduction |
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In vitro studies assessing the activity of various antimicrobial agents for several of the Rickettsia and Bartonella spp. are limited and diverse in approach.69 The antimicrobial action of clarithromycin may be underestimated when susceptibility testing is used, if the activity of its metabolite is not considered, or if animal models are used in which the metabolite is not produced.10 For these reasons, this study investigated the in vitro susceptibility of Rickettsia akari, R. conorii, R. prowazekii, R. rickettsii, B. elizabethae, B. henselae and B. quintana to different concentrations of both clarithromycin and 14-hydroxyclarithromycin (the primary active metabolite of clarithromycin in humans),11,12 and tetracycline, in Vero cell culture chamber slides. An immunofluorescent antibody (IFA) technique was used to evaluate the inhibition of Bartonella spp. and Rickettsia spp. proliferation.
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Materials and methods |
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Coded antibiotics (14-hydroxy-clarithromycin, clarithromycin and tetracycline HCl; Abbott Laboratories, North Chicago, IL, USA) and a non-antibiotic placebo (d-lactose; Sigma Chemical Co., St Louis, MO, USA) were prepared and tested with each species. The study was blind for the investigators. Tetracycline was the coded, positive antibiotic control of inhibition in each trial. Antibiotic solutions, prepared in 1 mL aliquots, at a stock concentration of 32 mg/L, were flash-frozen and stored at 70°C.
A coded control using medium without antibiotic (i.e. lactose) was used to provide reference numbers of foci, and a non-coded negative control (without antibiotics) was also included. Immediately before testing, the frozen drug samples were thawed, and dilutions of the drugs were made using minimal essential medium (MEM) supplemented with 2 mM l-glutamine, 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (HEPES) buffer and 2% fetal bovine serum (FBS).
Bacterial strains and cell culture preparation
B. henselae (Houston-1 isolate, ATCC 49882), B. elizabethae F9251 (ATCC 49927), R. akari (ATCC VR-612), R. conorii (ATCC VR-613), R. prowazekii (ATCC VR-233) and R. rickettsii (ATCC VR-149) cultures were obtained from the American Type Culture Collection, Rockville, MD, USA. B. quintana (OK 90-268), originally a human isolate, was a CDC laboratory strain.
Bartonella spp. were plated on brainheart infusion agar supplemented with 5% rabbit blood (BBL; Becton Dickinson and Co., Cockeysville, MD, USA). When adequate growth had been obtained, the colonies were harvested, with brainheart infusion broth, from the surface of the blood agar plates, and washed twice in sterile phosphate-buffered saline (PBS), pH 7.2, 0.01 M (Gibco BRL/Life Technologies, Inc., Gaithersburg, MD, USA). Colonies were then added to sterile 2 mL Sarstedt freezer vials (Sarstedt, Inc., Newton, NC, USA), flash-frozen and stored at 70°C.
Rickettsia spp. were grown in Vero cells and passed through a filter, pore size 1.2 µm (Sigma Chemical Co., St Louis, MO, USA). On the basis of preliminary estimations, dilutions of rickettsiae were cultured in MEM supplemented with 2 mM l-glutamine, 10 mM HEPES buffer and 2% FBS; in order to produce 510 rickettsial focus forming units for each microscopic field examined.
Before testing, the Bartonella spp. inocula were passed through a 22-G hypodermic syringe, then filtered through a 1.2 µm filter to minimize possible artifacts owing to the presence of aggregates of organisms with host cell fragments. Following filtration, the organisms were diluted in MEM and 2% FBS, to yield an inoculum equivalent to 2030 foci in each microscope field of the Vero cell monolayer, 2 h post-inoculation.
For each Bartonella sp., following careful aspiration of the Vero cell supernatant medium (so as to not disrupt the confluent cell monolayers), each well of the chamber slides was inoculated with 0.25 mL of the diluted Bartonella- containing medium prepared as above. The slides were incubated at 35°C in a 5% CO2-enriched environment for 2 h, allowing time for the Bartonella spp. to establish attachment and entry to the host cells.13 At the end of the initial incubation period, the inocula were replaced by 0.25 mL of the antibiotic dilutions and re-incubated in 5% CO2 for 5 days. Fifteen different concentrations (0.00195, 0.00391, 0.00782, 0.0156, 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mg/L) of each antibiotic were tested 10 times with each species.
For Rickettsia spp., antibiotic-free Vero cells (0.25 mL of a 1.5 x 105 solution) were seeded in the wells of 16-well chamber microscope slides (Lab-Tek; NUNC Inc., Naperville, IL, USA) and incubated at 37°C until confluent at 3 days. Slides were incubated at either 35°C for R. prowazekii or 32°C for the remaining species in a 5% CO2-enriched atmosphere for 3 h, thus permitting sufficient time for rickettsiae to penetrate into the cytoplasm of the Vero cells. Inocula were removed carefully by aspiration and the monolayers were washed twice with MEM to remove extracellular rickettsiae that were partially attached to Vero cells or free in the medium. Infected monolayers were then replenished with medium containing the 15 different antibiotic concentrations and re-incubated in 5% CO2 for 5 days. Two 16-well slides were prepared for each agent, including controls, and each concentration of the antibiotics was tested 10 times with each species. Chamber slides were monitored daily for contamination.
Immunofluorescent assay testing
When the non-coded, non-antibiotic control-infected cells showed well-formed fluorescent foci, media were removed from all infected and antibiotic-treated monolayers. The cell culture monolayers were fixed immediately with methanol. To determine the inhibition of Rickettsia and Bartonella spp. proliferation, foci from all coded specimens were stained by an indirect immunofluorescent antibody method. Human anti-Rickettsia or Bartonella spp. antibody, derived from high-titre rabbit antisera to -irradiated whole rickettsiae or bartonella cells, was used as a primary antibody. Goat anti-human IgG fluorescein-labelled antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, USA) served as a secondary conjugate antibody. Based upon an approved CDC Animal Care and Use Committee protocol cited previously,14 the rabbit antibody was raised in rabbits after purifying the bacteria on a 66% diatrizoate meglumine and 10% diatrizoate sodium 10% (Renografin-76, Bristol-Myers Squibb Diagnostics, Princeton, NJ, USA) isopycnic gradient to help free them from Vero host cell debris and facilitate purification. Fluorescein antibody staining produced distinct foci, without background staining and with immune reagent specificity.
The actual titres of primary antisera or fluoresceinconjugated goat anti-human IgG were determined by titration of the reagents to endpoint titres with known positive samples, then using a dilution approximately two-fold less than the endpoint as a working dilution. Primary and secondary antibodies were incubated for 30 min each, followed by three 5 min washes with PBS and a single quick dip in distilled water before air-drying.
A Zeiss Axioskope epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY, USA) equipped with a 40x objective lens was used to count the immunofluorescent foci in each well. The findings were compared with controls. The number of immunofluorescent foci (i.e. clusters of multiple cells of rickettsiae, in one or more adjacent cells in the cell culture or individual bacterial cells of bartonella) in 50 random fields was determined for each well. Foci (either multiple or individual cells) in each of the four wells belonging to each antibiotic dilution were counted, and the mean for fluorescent foci per 50 fields was determined. The extent of antibiotic-induced foci inhibition was recorded for each dilution of antibiotic in comparison with the antibiotic-negative control. The 10 readings of each drug dilution were averaged, and the minimum antibiotic concentration that completely inhibited growth after 5 days of incubation was designated as the MIC. Statistical significance was assessed using Student's t test at the 95% confidence limit.
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Results |
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Discussion |
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The Cmax/MIC ratio is determined from extracellular media supernatant concentrations. In traditional in vitro studies such as this, the assumption is that the antibiotic achieves the required inhibitory concentration at the site of infection. When treating intraphagocytic bacterial infections such as those caused by Bartonella and Rickettsia spp., an additional barrier in the form of phagocytic cell membrane must be breached to eradicate the infection. The same antibiotic may achieve different concentrations within different phagocytic cell types,2225 and the intracellular antibiotic concentration is only one of the many important variables contributing to the efficiency of bacterial killing. For example, the pKa of clarithromycin is 9.0,26 and with the decreased pH of the cellular environment, the ionized fraction (i.e. a pharmacologically inactive form) of clarithromycin would increase, to give higher than expected MIC values. However, clarithromycin is more acid stable than either erythromycin or azithromycin.21
Within the first 10 h of infection, R. rickettsii multiply and spread with apparent ease from host cell to host cell without immediate host cell lysis.27,28 Other species of rickettsiae grow and accumulate within the original infected cell, do not cross host cell membranes readily, and spread to other cells presumably as the architecture of the host cell disintegrates; therefore, the antibiotic penetration rate into the infected cell may be important for rapid containment of certain rickettsial infections. In this investigation, the rickettsiae were able to invade the host cells before antibiotic administration, thus providing discrimination between extracellular and intracellular antimicrobial activities.
Both erythromycin and doxycycline have been used to treat infections with Bartonella spp., and tetracycline or chloramphenicol has been used to treat infections with Rickettsia spp. These antibiotics penetrate eukaryotic membranes and concentrate within the cytoplasm of phagocytic cells.2931 These agents readily enter macrophages and leucocytes to achieve high intracellular concentrations; but different species of bartonella may behave differently within the endothelial cell or other cell types.
Effective intracellular, pericellular and lymph node antimicrobial concentrations are considered the sine qua non of successful treatment of cat scratch disease.7 The model demonstrated in this study is significantly different from an extracellular in vitro model, which investigated the effect of single antibiotic dilutions to render bartonella non-infectious.8 Our model for antimicrobial activity was based upon the ability of the antibiotic to inhibit bartonella growth in a cell culture environment. For bartonella infections, in vitro results will not adequately predict clinical benefit with most compounds, unless those agents achieve high intracellular concentrations and high concentrations in the lymph nodes. These results were also consistent with those previously obtained via plaque and dye uptake assays.6,7,9,32,33
Currently, there are no data for the intracellular antimicrobial concentrations in bartonella- and rickettsia-infected endothelial cells. Extracellular antimicrobial concentration may not be appropriate for intracellular pathogens such as bartonella or rickettsia, especially for some of the new macrolides that exhibit a large volume of distribution into cells and tissue. Studies to assess the antimicrobial activity of macrolide agents for Bartonella or Rickettsia spp. in both environments are needed.
Based upon the activity demonstrated in this in-vitro evaluation, clarithromycin, with its active metabolite, 14-hydroxy-clarithromycin, may have potential as an alternative to tetracycline and chloramphenicol in the treatment of infections caused by Rickettsia and Bartonella spp. In vitro antimicrobial data do not correlate directly with clinical efficacy, and animal models may not be an optimal environment to evaluate antimicrobial activity,34 particularly with respect to clarithromycin metabolism.9 Because clarithromycin and its 14-hydroxy derivative display different in vitro antibacterial activity, these results suggest that evaluation of both agents together may be more predictive of the potential of clarithromycin in the treatment of patients affected with either rickettsia- or bartonella-related diseases.
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Acknowledgments |
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Notes |
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References |
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Received 14 April 1999; returned 30 July 1999; revised 1 September 1999; accepted 26 October 1999