Antibiotic susceptibility of Tropheryma whipplei in MRC5 cells

Abstract

Whipple's disease is considered a rare chronic disease with a broad spectrum of clinical manifestations. Several antibiotics have been used for the treatment of this disease, and the current reference treatment was determined empirically on the basis of only a few clinical observations. Patients should be treated for months, and many relapse after antibiotic withdrawal. We report here the first extensive study on the susceptibilities of three reference strains of Tropheryma whipplei to antibiotic in cell culture by using a real-time PCR assay as previously described. We found that doxycycline, macrolides, ketolides, aminoglygosides, penicillin, rifampin, teicoplanin, chloramphenicol, and trimethoprim-sulfamethoxazole were active, with MICs ranging from 0.25 to 2 microg/ml. Vancomycin was somewhat active at an MIC of 10 microg/ml. We found heterogeneity in the susceptibility to imipenem, with one strain being susceptible and the two other strains being resistant. Cephalosporins, colimycine, aztreonam, and fluoroquinolones were not active. We also demonstrated that a combination of doxycycline and hydroxychloroquine was bactericidal. This combination has been shown to be active in the treatment of patients suffering from chronic infections with Coxiella burnetii, a bacterium that is also found intracellularly in acidic vacuoles. We believe, then, that this combination therapy should be further evaluated in clinical trials for the treatment of Whipple's disease.

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.

FIG. 2.

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

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).