Antibiotic Susceptibility of Tropheryma whipplei in MRC5 Cells

T. whipplei strainsThree T. whipplei isolates obtained in our laboratory and cultured in MRC5 fibroblast cells (ATCC number CCL-171) were tested in our study: Twist strain, isolated from a heart valve (24) and passaged 50 times; Endo-5 (6), isolated from a heart valve and passaged 44 times; and Slow2 (6), isolated from a duodenal biopsy and passaged 33 times. These data are presented in Table 1.

T. whipplei-infected MRC5 cells were grown in 150-cm² culture flasks (Falcon; Beckman) incubated at 37°C in a 5% CO₂-enriched atmosphere. Minimum essential medium (Life Technologies/Gibco-BRL and Cergy Pontoise) supplemented with 10% fetal bovine serum and 2 mM l-glutamine (Gibco) was used as the incubation medium (15). Cell infection rates were monitored three times a week by examining cells scraped from the flasks and stained with Gimenez stain (13).

Antibiotic solutionsThe antibiotics used in the study were tested in serial twofold dilutions as follows: doxycycline (0.5 to 8 μg/ml; Pfizer, Neuilly, France), levofloxacin (2 to 8 μg/ml; Hoechst Marion Roussel, Romainville, France), ofloxacin (0.5 to 8 μg/ml; Diamant, Puteaux, France), ciprofloxacin (0.5 to 8 μg/ml; Bayer Pharma, Sebs, France), erythromycin (0.06 to 10 μg/ml; Abbot, Rungis, France), telithromycin (0.5 to 8 μg/ml; Hoechst Marion Roussel), thiamphenicol (0.06 to 32 μg/ml; Sanofi Winthrop, Gentilly, France), rifampin (0.5 to 8 μg/ml; Cassenne, Puteaux, France), trimethoprim-sulfamethoxazole (0.06 to 32 μg/ml; Roche, Paris, France), gentamicin (0.25 to 10 μg/ml; Dakota Pharm, Creteil, France), amoxicillin (0.25 to 8 μg/ml; SmithKline Beecham, Nanterr, France), streptomycin (0.25 to 8 μg/ml; Diamant), ceftriaxone (0.25 to 100 μg/ml; Roche), penicillin G (0.06 to 10 μg/ml; Diamant), vancomycin (1 to 100 μg/ml; Dakota Pharm), clarithromycin (1 to 2 μg/ml; SmithKline Beecham), teicoplanin (Marion Merrell Dow, Paris, France), imipenem (0.25 to 10 μg/ml; Dakota Pharm), aztreonam (1 to 100 μg/ml; Sanofi Winthrop), cephalotin (1 to 100 μg/ml; Panapharma, Luitré-Fougeres, France), chloramphenicol (1 to 2 μg/ml; Coger, Paris, France), colimycin (Roche), hydroxychloroquine (1 to 2 μg/ml; Sanofi Synthelabo, Paris, France), and NH₄Cl (1 to 2 μg/ml; Coger, Paris, France).

Stock solutions were prepared according to the manufacturers' instructions and stored at −80°C until used. Working solutions were prepared extemporaneously by diluting stock solutions in minimal essential medium supplemented with 2 mM glutamine and 5% fetal bovine serum.

Antibiotic susceptibility testing of T. whipplei isolatesWe previously described a real-time PCR assay used to determine antibiotic susceptibility (18). Briefly, when cells were heavily infected, usually after 3 weeks of incubation, the cell supernatant was discarded from the flask, and the infected MRC5 cells were detached by using sterile glass beads with 5 ml of fresh medium. Cells were lysed by sonication (three 30-s sonications in ice at 60 mV) and centrifuged (15 min at 20,000 × g) to discard cell debris, and the supernatant was diluted 1:1,000 in culture medium. This suspension was used to infect confluent MRC5 monolayers in 48-well microtiter plates (D. Dutcher, Brumath, France), which were incubated at 37°C in 5% CO₂. Infected cells in three different wells were harvested every 3 days from days 0 to 12 postinfection. The growth kinetics of T. whipplei in MRC5 cells was determined by determining the numbers of genome copies in each cell suspension by quantitative PCR.

For antibiotic susceptibility testing, dilutions of antibiotics (prepared as described above) were added at serial twofold dilutions to the culture media of individual tissue culture plate wells after 48 h of incubation, in duplicate. Antibiotic-free wells served as growth controls, whereas uninfected MRC5 cell wells served as negative controls. During the antibiotic challenge experiment, cell cultures from wells were harvested every 3 days over a 12-day period, and cell suspensions were frozen at −80°C until DNA was extracted for quantitative PCR assays. The lack of toxicity of antibiotics to MRC5 cells was determined by examination of cell monolayers under an inverted microscope (Zeiss Axiovert 25; Carl Zeiss) at the time cell cultures were harvested. The MIC was defined as the lowest antibiotic concentration at which complete inhibition of bacterial growth was determined by measurement of DNA copies by quantitative PCR assay. The number of DNA copies present after 12 days was compared to the number of DNA copies at day 0 of the experiment. Experiments were carried out on three separate occasions, each time in duplicate, to determine consistency of results.

Controls used for antibiotic activityEscherichia coli ATCC 8739 and S. aureus CIP ATCC 49976 were obtained from the Pasteur Institute (Institut Pasteur, Marnes La Coquette, France) and used as antibiotic test controls (18). Antibiotic activities were determined by using Mueller-Hinton agar (bioMérieux) incubated at 37°C for 18 h. The activities of the various dilutions of the antibiotics described above were determined after 15 days of incubation at 37°C.

Bactericidal activity of antibioticsThe bactericidal activity or postantibiotic effect of doxycycline in combination with hydroxychloroquine was determined by subculturing 100 μl of harvested cell suspensions, collected on day 12 of the experiment, onto fresh cell cultures containing antibiotic-free culture medium. After 7 and 10 days of culture, sample cells were harvested as described above for real-time PCR and Gimenez staining. The bactericidal effect or the postantibiotic effect was defined as the lack of regrowth of bacteria after 10 days of subculture.

Quantitative PCR assay by using a LightCyclerT. whipplei DNA was extracted from 100-μl aliquots of infected MRC5 cells by using the MagNA Pure LC DNA Isolation Kit III (Roche Applied Science, Penzberg, Germany) as described by the manufacturer. The extracted nucleic acid was resuspended in a final volume of 100 μl and stored at −20°C until used in the quantitative PCR assay.

PCR was performed by using a LightCycler instrument (Roche Biochemicals, Mannheim, Germany) with 20-μl volume glass capillaries. The PCR mixture (20 μl) contained 2 μl of extracted DNA, 13.2 μl of H₂O, 1.6 μl of MgCl₂ (25 mmol), 1 μl (i.e., 10 pmol) of the forward primer ITS (5′-CCGAGGCTTATCGCAGATTG-3′), 1 μl (i.e., 10 pmol) of the reverse primer ITS (5′-GGTGACTTAACCTTTTTGGAG-3′) (7), and 2 μl of DNA-Master Hybridization Probes (Roche Diagnostics) containing Taq DNA polymerase, reaction buffer, a deoxynucleoside triphosphate mixture, and 3 mmol of MgCl₂ (10-fold concentrated). The PCR was performed with an initial denaturation at 95°C for 8 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 5 s, and extension at 72°C. A calibration curve for DNA quantification was established by amplifying 10-fold serial dilutions of DNA extracted from the primary T. whipplei inoculum used to infect MRC5 cells.

The specificity of the PCR products was verified by sequencing as previously described (18).

Sensitivity and specificityFigure 1 shows the melting curves obtained with standard concentrations of a previously quantified inoculum of T. whipplei (10⁸) and the specificity of the PCR product (a single sharp melting temperature obtained at ca. 87.5°C), as determined by 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. After resolution by agar electrophoresis, all T. whipplei PCR products showed a single band fragment. Sequences of this unique band were always 100% identical to the sequence of the ITS gene of T. whipplei (GenBank accession number AF248312).

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FIG. 1.

Melting curves obtained with standard concentrations of T. whipplei by PCR by measuring the amount of fluorescence (Df/dT) with a LightCycler. The specificities of the PCR products are shown by detection of a single peak at 88°C.

Growth kinetics of T. whipplei as determined by using real-time PCRFibroblast cells present a contact inhibition when monolayers are confluent, and thus the numbers of cells in each well were approximately the same (Fig. 2). The viability of cells was verified by dye staining. The growth of T. whipplei isolates was slow, with 2.3 ± 3.8 log increases in bacterial concentration over 12 days of incubation for Twist strain, 4.3 ± 4.65 log for Endo2 strain, and 1.8 ± 5.4 log for Slow2 strain. During this period, the doubling time of T. whipplei Twist was determined to be 35 h, whereas that of Endo2 was 32 h and that of Slow2 was of 48 h. Thereafter, bacterial growth plateaued.

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FIG. 2.

Growth kinetics of T. whipplei strains (Twist, Endo-5, and Slow2) cultured in MRC5 cells and determined by real-time quantitative PCR.

Antibiotic susceptibility testing of T. whipplei strainsTo be sure that the decrease in DNA copies when infected cells were treated by antibiotics was not due to a loss of viability, we have added in each PCR experiment controls, including the infected cells without antibiotics at day 0 and at day 12. Antibiotic susceptibilities against the three isolates by using real-time PCR were determined on day 12 because the three isolates reached their maximum growth after this time. This was confirmed by Gimenez staining. The three isolates of T. whipplei had a similar pattern of antibiotic susceptibility (Table 2). They were susceptible to doxycycline, macrolides, telithromycin, penicillin G, streptomycin, rifampin, teicoplanin, chloramphenicol, thiamphenicol, amoxicillin, and trimethoprim-sulfamethoxazole, with MICs ranging between 0.25 and 2 μg/ml. We found heterogeneity in the susceptibility to imipenem with the Twist strain being susceptible (MIC of 0.5 μg/ml), whereas Endo2 and Slow isolates were resistant (MIC of 10 μg/ml). To vancomycin the MICs were ≥10 μg/ml for the three strains. Antibiotics that were less active were cephalotin, colimycin, aztreonam, and the fluoroquinolones.

Antibiotic dilutions were found to be stable for 15 days and had the same activity against control strains over this time in the control experiments.

Bactericidal activity of combined therapyCultures of Twist strain treated with doxycycline (2 μg/ml) alone or doxycycline (2 μg/ml) and hydroxychloroquine (1 μg/ml) for 12 days, were reinoculated onto fresh confluent cells without antibiotics to evaluate the regrowth of the bacteria. The results of this experiment are shown in Fig. 3. The combination of doxycycline and hydroxychloroquine appeared to be bactericidal since no bacterial regrowth was detected by Gimenez staining or PCR assay, even after 10 days of subculture. When used alone, doxycycline was not bactericidal since regrowth of organisms occurred after 10 days of subculture as detected by PCR assay (+1.65 log of DNA copies) and also by Gimenez staining.

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FIG. 3.

Growth kinetics of T. whipplei Twist strain treated for 12 days with either doxycycline alone or doxycycline plus hydroxychloroquine. Antibiotics were removed after 12 days and samples were reinoculated in fresh confluent MRC5 cells. Regrowth of the bacteria was determined by quantitative PCR. Symbols: ♦, growth control; ▪, doxycycline (2 μg/ml); ▴, doxycycline (2 μg/ml) plus hydroxychloroquine (1 μg/ml).