JAC Advance Access originally published online on January 13, 2005
Journal of Antimicrobial Chemotherapy 2005 55(2):178-181; doi:10.1093/jac/dkh524
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JAC vol.55 no.2 © The British Society for Antimicrobial Chemotherapy 2005; all rights reserved
Molecular evaluation of antibiotic susceptibility of Tropheryma whipplei in axenic medium
Unité des Rickettsies, CNRS UMR 6020, IFR48, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
* Corresponding author. Tel: +33-4-91-38-55-17; Fax: +33-4-91-83-03-90; Email: didier.raoult{at}medecine.univ-mrs.fr
Received 10 June 2004; returned 11 September 2004; revised 17 September 2004; accepted 6 November 2004
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Abstract |
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Objectives and methods:
Whipple's disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. Tropheryma whipplei is a slow-growing facultative intracellular bacterium that remains poorly understood. In vitro antibiotic susceptibility testing has previously been assessed in cells using a real-time quantitative PCR assay. In this study, we have evaluated the antibiotic susceptibility of three strains of T. whipplei grown in axenic medium using the same assay.
Results:
The active compounds in axenic medium were doxycycline, macrolide compounds, penicillin G, streptomycin, rifampicin, chloramphenicol, thiamphenicol, teicoplanin, vancomycin, amoxicillin, gentamicin, aztreonam, levofloxacin and ceftriaxone, with MICs in the range 0.06–1 mg/L. Cefalothin was less active, with MICs in the range 2–4 mg/L. We found that co-trimoxazole was active with MICs in the range 0.5–1 mg/L, and sulfamethoxazole alone was active with MICs in the range 0.5–1 mg/L. MICs of trimethoprim varied from 64–128 mg/L.
Conclusions:
Co-trimoxazole was effective in vitro, but this activity was due to sulfamethoxazole alone. These results were in accordance with the fact that T. whipplei does not contain the encoding gene for dihydrofolate reductase, the target for trimethoprim.
Keywords: Whipple's disease , antibiotics , real-time quantitative PCR , MICs
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Introduction |
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Whipple's disease is a systemic infection with a broad spectrum of clinical manifestations, caused by the bacterium Tropheryma whipplei.1 Among the various clinical manifestations of the disease, there are at least three different entities: endocarditis, neurological and classical Whipple disease with fever, weight loss, diarrhoea, polyarthritis, skin hyper-pigmentation and adenopathy.2 Currently, the recommended treatment for Whipple's disease consists of a combination of streptomycin (1 g) and benzylpenicillin (penicillin G; 1.2 million units) over a period of 14 days, followed by oral co-trimoxazole (trimethoprim–sulfamethoxazole; 160 mg/800 mg twice daily) for 1 year.3 Nevertheless, relapses have been reported after antibiotic withdrawal, even in situations where the regimen has been followed for months.4–6 This could be due either to a lack of bactericidal activity of antibiotics against the intracellular bacteria,7 or to an inadequate regimen schedule.6
T. whipplei is a facultative, intracellular, rod-shaped, Gram-positive, filamentous and aerobic bacterium, sized 0.5–2 µm.1,8 According to phylogenetic analysis, T. whipplei is classified as a member of the Actinomycetes, placed between the genus Cellulomonas and a rare group of Actinomycetes with group B peptidoglycan.8 The agent of Whipple's disease, T. whipplei, has been recently isolated using a cell culture model and characterized in a patient with endocarditis.1 This was confirmed by the cerebrospinal fluid culture from two patients.9 Culture of this bacterium is a very painstaking process, but can be achieved using several intracellular models, including a murine fibroblast cell line (MRC5) or human embryonic lung fibroblast cells.1 Currently in the literature only five strains have been isolated.1,9–11 Recently, independent studies by Bentley et al.9 and by Raoult et al.12 have reported the complete genome sequence of two T. whipplei strains. Analysis of the genomic sequence has revealed specific deficiencies in the predicted metabolism of T. whipplei. This information was successfully used to design a comprehensive axenic medium for the culture of the organism.13
We have recently developed a new genomic assay for the determination of antibiotic susceptibilities of T. whipplei in fibroblast cells using real-time PCR. We found that fluoroquinolones were not active and that this was associated with mutations in the DNA gyrase gene.14 In a second report. we found that macrolide compounds, aminoglycosides, penicillin, rifampicin, chloramphenicol and co-trimoxazole compounds were effective against T. whipplei grown in cells. Cephalosporin compounds, colimycin and aztreonam were less effective. We found heterogeneity in susceptibility to imipenem and glycopeptide compounds. Teicoplanin was active, whereas the three strains tested had MICs of 10 mg/L for vancomycin. Finally, because Coxiella burnetii and T. whipplei share similar features, especially their intracellular lifestyle in acidic vacuoles, we demonstrated that the combination of doxycycline and hydroxychloroquine, the current antibiotic regimen for chronic Q fever, was also bactericidal against T. whipplei.7
The aim of the present study was to evaluate the antibiotic susceptibility of the three strains in axenic medium and to compare the results with intracellular activity. We believe that in vivo discrepancies reported in the treatment of Whipple's disease may be partly explained by a difference in susceptibility of the bacteria extracellularly and intracellularly. This has previously been shown for other facultative intracellular bacteria such as Bartonella and Brucella.15
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Materials and methods |
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Organisms
Three T. whipplei isolates obtained in our laboratory and cultured in fibroblast cells were tested in this study.7 Twist strain was isolated from a cardiac valve sample, Endo-5 from a blood sample and Slow2 was grown from a duodenal biopsy.
Culture
T. whipplei isolates were cultured either in MRC5 cells or in axenic medium, as previously described.7,13
Evaluation of kinetics of growth in cells and in axenic medium
When heavy infection was detected (100% of infected cells), cell supernatant was discarded, and infected MRC5 cells were detached using sterile glass beads with 5 mL of fresh medium. Cells were lysed by sonication (30 s three times on ice, at 60 mV), and the resultant bacterial inoculum was diluted to 1:100 in culture medium and used to infect confluent MRC5 monolayers in 24-well microtitre plates. Conversely, bacteria in axenic culture were centrifuged at high speed for 10 min. The resulting bacterial inoculum was diluted to 1:100 in culture medium, cultured in 24-well plates and incubated at 37°C, in 5% CO2. One hundred microlitres of each infected well was collected in duplicate, either from axenic culture or by scraping infected MRC5 cells every 3 days for 12 days and stored at –80°C before being used for quantitative PCR assay.
Antibiotic solutions
Antibiotics used in this study were in serial two-fold dilutions as follows: doxycycline 0.5–4 mg/L (Pfizer, Neuilly, France), levofloxacin 0.06–8 mg/L (Hoescht Marion Roussel, Romainville, France), ofloxacin 0.5–8 mg/L (Diamant, Puteaux, France), ciprofloxacin 0.5–8 mg/L (Bayer Pharma, Sebs, France), erythromycin 0.06–4 mg/L (Abbot, Rungis, France), telithromycin 0.06–4 mg/L (Hoescht Marion Roussel, Romainville, France), thiamphenicol 0.06–8 mg/L (Sanofi Winthrop, Gentilly, France), rifampicin 0.25–4 mg/L (Cassenne, Puteaux, France), trimethoprim–sulfamethoxazole 0.06–128 mg/L (Roche, Paris, France), trimethoprim 0.06–128 mg/L (Roche, Paris, France), sulfamethoxazole 0.06–16 mg/L (Roche, Paris, France), gentamicin 0.06–4 mg/L (Dakota Pharm, Creteil, France), amoxicillin 0.06–8 mg/L (SmithKline Beecham, Nanterr, France), streptomycin 0.06–8 mg/L (Diamant, Puteaux, France), ceftriaxone 0.06–128 mg/L (Roche, Paris, France), penicillin G 0.06–10 mg/L (Diamant, Paris, France), vancomycin 0.06–10 mg/L (Dakota Pharm, Creteil, France), clarithromycin 0.5–2 mg/L (SmithKline Beecham, Nanterre, France), teicoplanin (Marion Merrell Dow, France), imipenem 0.25–10 mg/L (Dakota Pharm, Creteil, France), aztreonam 1-128 mg/L (Sanofi Winthrop, Gentilly, France), cefalothin 0.06–10 mg/L (Panapharma, Luitré-Fougeres, France) and chloramphenicol 1-2 mg/L (Coger, Paris, France).
Stock solutions were prepared according to the manufacturers' instructions and stored at –80°C until use. Working solutions were prepared extemporaneously by dilution of stock solutions in axenic medium.
Antibiotic susceptibility testing of T. whipplei isolates in axenic medium
For antibiotic susceptibility testing, experiments were conducted in 24-well plates. Antibiotics were added at serial two-fold dilutions in culture media. Antibiotic-free wells served as growth controls. During antibiotic challenge, cultures were harvested every 3 days in duplicate for a total of 9 days, and frozen at –80°C until DNA extraction for quantitative PCR assays. The number of DNA copies after 9 days was compared with the number of DNA copies at day 0 of the experiment, as previously described.7 All experiments were carried out twice to confirm the results.
MICs
MICs were defined as the minimal antibiotic concentrations allowing complete inhibition of bacterial growth. This was determined by measurement of DNA copies by quantitative PCR assay, as compared with a growth control at the beginning of the experiment.
Controls used for antibiotic activity
Escherichia coli ATCC 8739 and Staphylococcus aureus C.I.P. ATCC 49976 were obtained from the Pasteur Institute (Institut Pasteur, Marnes La Coquette, France) and used as antibiotic test controls.14 Antibiotic activities were determined using Mueller–Hinton agar (bioMérieux) incubated at 37°C for 18 h. The activities of the various dilutions of the antibiotics used above were determined after 15 days of incubation at 37°C.
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Results |
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Specificity and sensitivity of PCR detection
The specificity of the PCR products was shown as a single sharp melting temperature obtained at 
87.5°C, as previously described.7
For each run, non-infected cells used as negative controls were always negative by PCR assay. Sensitivity was determined using a standard calibration curve obtained with 10-fold serial dilutions of T. whipplei. The standard curve was determined in each experiment to enable the results of all experiments to be correlated.
Growth kinetics of T. whipplei measured using real-time PCR
The results of quantification of DNA copies obtained after 9 days of culture, as well as doubling time for the three strains in axenic medium, are compared in Table 1 with results obtained in cells.
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Antibiotic susceptibility testing of T. whipplei strains
Growth kinetics allowed us to determine the best point for susceptibility testing: 9 days in axenic medium but 12 days in MRC5 cells.7 The antibiotic susceptibilities of T. whipplei obtained both in axenic medium and in cells from a previous study7 are presented in Table 2. The active compounds in axenic medium were doxycycline, macrolide compounds, penicillin G, streptomycin, rifampicin, chloramphenicol, thiamphenicol, teicoplanin, vancomycin, amoxicillin, gentamicin, aztreonam, levofloxacin, co-trimoxazole and ceftriaxone with MICs in the range 0.06–1 mg/L (Table 2). Trimethoprim alone was not active with MICs in the range 64–128 mg/L (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present study, we evaluated the antibiotic susceptibility of T. whipplei in axenic medium and compared the results with those previously obtained in cells using a real-time quantitative PCR assay, as described previously.7,14
The three strains displayed a homogeneous pattern of antibiotic susceptibilities in axenic medium (Table 2). Almost all the antibiotics were active, except fluoroquinolone compounds. This result was not surprising because resistance to fluoroquinolones is associated with mutations in the DNA gyrase gene.14 As compared with MICs found in cells (Table 2), the bacteria were globally more susceptible in axenic medium. We found that ß-lactams, including imipenem, were more active against extracellular bacteria (MICs in the range 0.06–1 mg/L) than against intracellular bacteria (MICs in the range 0.25–20 mg/L). Vancomycin was also more active in axenic medium (MICs in the range 1–2 mg/L versus 10 mg/L for intracellular bacteria). This result was more in accordance with the fact that use of vancomycin precludes the primary isolation and establishment of T. whipplei from clinical samples.10 Such discrepancies of activity could be due either to poor penetration into cells, or to inactivation of the drug in cells because of the acidic environment in which T. whipplei multiply.16 Aztreonam, the first monocyclic ß-lactam antibiotic, is effective against T. whipplei. In general, aztreonam has a high affinity for the protein-binding protein 3 (PBP-3) of aerobic Gram-negative bacteria. Conversely, it binds poorly to PBP sites of aerobic Gram-positive and anaerobic bacteria and consequently has relatively poor inhibitory effects against these bacteria.17 In the genome sequence of T. whipplei, at least three PBP encoding genes have been annotated, but affinity of these PBPs for aztreonam is unknown.
The three strains of T. whipplei were highly susceptible to macrolides, doxycycline, rifampicin, aminoglycosides and thiamphenicol.
We found that co-trimoxazole was similarly active in axenic medium and in cells,7 although it has been noted that the encoding sequence for dihydrofolate reductase (DHFR), the target enzyme for trimethoprim, was absent.18 Since the encoding sequence of dihydropteroate synthase (DHPS), the target enzyme for sulfamethoxazole, is present in the genome of T. whipplei, our hypothesis was that activity of co-trimoxazole was only due to sulfamethoxazole.7 We confirm our hypothesis since trimethoprim alone was not active against T. whipplei, whereas sulfamethoxazole alone can inhibit the growth of the bacteria. Among available complete genome sequences, several bacteria share similar features: absence of DHFR and presence of the other enzymes of the folate pathway.19 For example, the genome of Campylobacter jejuni does not contain the encoding sequence for DHFR but does contain the encoding sequence for DHPS.19 This pathogenic bacterium has been regarded as endogenously resistant to trimethoprim in vitro, but remains susceptible to a trimethoprim–sulfamethoxazole combination.
In vivo, co-trimoxazole is not always effective for the treatment of Whipple's disease and an 30% failure or relapse rate has been reported even after a 1 year course of treatment.2
The failures and relapses frequently reported could be due firstly to a lack of bactericidal activity of sulfamethoxazole at low pH where the bacteria lives.16
Indeed, we have previously demonstrated that only the association of doxycycline with hydroxychloroquine was able to kill the intracellular bacteria.7
Secondly, relapses could be due to the acquisition of resistance against sulfamethoxazole during the course of the treatment, especially by mutation in the DHPS encoding gene. There is one report of acquisition of resistance to co-trimoxazole after long and repeated treatment using this antibiotic.20
Finally, failure and relapses could be due to an inadequate concentration of antibiotic at the site of infection (cardiac valve or cerebrospinal fluid).
Because Whipple's disease is often a chronic infection, with T. whipplei living in acidic vacuoles of host cells, we suggest a therapeutic regimen including administration of doxycycline and hydroxychloroquine to eradicate the intracellular organisms. We believe that higher dosing of co-trimoxazole in association with doxycycline and hydroxychloroquine may improve the treatment of chronic Whipple's disease, especially for neurological forms of the disease. Moreover, this association may also prevent the acquisition of resistance to sulfamethoxazole. Clinical trials are needed to confirm our suggestion.
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Acknowledgements |
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We thank Pat Kelly and Esther Platt for reviewing the manuscript prior to submission.
This work was supported by the Fifth Framework programme of the European Union (QLG1-CT-2002–01049) and A. Boulos benefited by a grant from the Fondation pour la Recherche Médicale (FRM, France).
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