Persister Development by Borrelia burgdorferi Populations In Vitro

Abstract

Doxycycline is an antibiotic commonly used to treat Lyme disease and other bacterial infections. The MIC and minimum bactericidal concentration (MBC) for Borrelia burgdorferi have been investigated by different groups but were experimentally established in this study as a function of input cell density. We demonstrated that B. burgdorferi treated in the stationary phase has a higher probability of regrowth following removal of antibiotic. In addition, we determined experimentally and mathematically that the spirochetes which persist posttreatment do not have a longer lag phase but exhibit a lower growth rate than untreated spirochetes. Finally, we found that treating the spirochetes by pulse-dosing did not eliminate growth or reduce the persister population in vitro. From these data, we propose that B. burgdorferi persister development is stochastic and driven by slowed growth.

FIG 1

Relationship between B. burgdorferi population density and MIC of doxycycline. The largest cell density with a population decrease from day 0 to day 5 is plotted against the lowest concentration of doxycycline required to cause the decrease. The relationship suggests a linear correlation, with an R² of 0.8721. For each data point, corresponding to input cell density (log 5 to log 8), 4 tubes are represented; error bars indicate standard errors of the means.

FIG 2

Number of motile B. burgdorferi spirochetes per concentration of doxycycline on day 5 posttreatment in the MBC assay. A growth inhibition assay was performed whereby cultures of differing initial concentrations and seed concentrations were treated with multiple concentrations of doxycycline. The number of motile spirochetes per culture was determined 5 days after the cessation of treatment. No growth was seen in any culture at this time point with treatments of 25 and 50 μg/ml. Error bars indicate standard errors of the means.

FIG 3

Pulse-dose assay. (A) Representative graph of the pulse-dose assay; (B) time to regrowth for each consecutive cycle of the assay. B. burgdorferi cultures (n = 34) were seeded initially at 7 × 10⁵ cells/ml, treated with 50 μg/ml of doxycycline, and then switched to doxycycline-free medium. Cultures were monitored for regrowth, and then those that regrew (n = 5) were treated again before they reached their initial concentration. The process was repeated 2 more times for those that regrew (n = 2). The time to regrowth after each treatment cycle is displayed. Error bars indicate SD. No significant difference was observed in the time to regrowth between cycles 2 and 3.