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. 2015 Aug;59(8):4616-24.
doi: 10.1128/AAC.00864-15. Epub 2015 May 26.

Borrelia Burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells

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Borrelia Burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells

Bijaya Sharma et al. Antimicrob Agents Chemother. .
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Abstract

Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the United States. When treated early, the disease usually resolves, but when left untreated, it can result in symptoms such as arthritis and encephalopathy. Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In this study, we examined the ability of B. burgdorferi to form persisters. Killing growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from the exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters. Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture.

Figures

FIG 1
FIG 1
Killing of B. burgdorferi by antibiotics. (a) Time-dependent killing. Antibiotics were added to an exponentially growing culture; samples were taken over time, washed, diluted, and plated in semisolid BSK-II medium for CFU counts. The culture was treated with amoxicillin (Amox) (6 μg/ml) or ceftriaxone (Cef) (3 μg/ml) (n = 9). (b to d) Dose-dependent killing. A late exponential culture of B. burgdorferi culture was exposed to antibiotics for 5 days, and surviving cells were determined by CFU count. The culture was treated with amoxicillin (b), ceftriaxone (c), or doxycycline (Dox) (d) (n = 6). Error bars represent standard errors.
FIG 2
FIG 2
Growth-dependent persister formation in B. burgdorferi. Growth in BSK-II medium was determined by CFU count. Persister levels were determined by taking samples from the growing culture, exposing to antibiotic for 5 days, and counting CFU. (a) Amoxicillin (Amox) (6 μg/ml) (n = 6); (b) ceftriaxone (Cef) (3 μg/ml) (n = 6). Error bars represent standard errors.
FIG 3
FIG 3
Persister formation is not heritable. Colonies recovered from a persister experiment before and after antibiotic treatment were used to inoculate fresh BSK-II medium. The colonies were allowed to grow for 3 days and treated with the same antibiotic used in the original persister experiment for 5 days (Amox, 6 µg/ml; Cef, 3 µg/ml). Persister levels of the colonies recovered after antibiotic treatment (right two bars) were not significantly different than the colonies recovered before antibiotic treatment (left two bars) (n = 5). Error bars represent standard errors. Amox, amoxicillin; Cef, ceftriaxone.
FIG 4
FIG 4
Killing of B. burgdorferi with drug combinations. (a) Time-dependent killing of late exponential B. burgdorferi cultures exposed to the indicated antibiotics in combination. Amoxicillin (Amox) (6 μg/ml), ceftriaxone (Cef) (3 μg/ml), and doxycycline (Dox) (2.5 μg/ml) (n = 6). (b) Killing of late exponential B. burgdorferi exposed to gemifloxacin (Gemi) (1.5 μg/ml) and/or spectinomycin (Spec) (160 μg/ml) singly or in combination (n = 6). An aliquot was taken at indicated time points, washed, diluted, and plated on semisolid BSK-II medium for CFU counts. Error bars represent standard errors.
FIG 5
FIG 5
Killing of B. burgdorferi by daptomycin. Time-dependent killing of stationary-phase B. burgdorferi exposed to daptomycin (81 μg/ml) (n = 3). Error bars represent standard errors.
FIG 6
FIG 6
Killing of B. burgdorferi by mitomycin C (MMC). (a, c) Time-dependent killing of B. burgdorferi. Three independent cultures of B. burgdorferi either at late exponential phase (a) or stationary phase (c) of growth were treated with MMC, 0.8 μg/ml (4× MIC) or 1.6 μg/ml (8× MIC). (b) Dose-dependent killing of late exponential cultures of B. burgdorferi after 5-day exposure to increasing concentrations of MMC. An aliquot was taken at indicated time points, washed, diluted, and plated on semisolid BSK-II medium for CFU counts (n = 6). The x axis is the limit of detection. Asterisks represent eradication to the limit of detection.
FIG 7
FIG 7
Pulse dosing results in effective killing of B. burgdorferi persisters. Late exponential cultures of B. burgdorferi were treated with ceftriaxone (Cef) (3 μg/ml) (a) or amoxicillin (Amox) (6 μg/ml) (b) for 5 days. This represents the first round of killing. The cultures were then washed and allowed to recover in fresh BSK-II medium for 24 h. They were then treated again with amoxicillin (6 μg/ml) or ceftriaxone (3 μg/ml) for a further 5 days to give the second round of killing. This was repeated for a total of four rounds of killing with a 24-h period of growth in fresh medium between each round. Bars represent the averages of at least three independent cultures (n = 3 to 6), and error bars represent standard errors. The x axis is the limit of detection. Asterisks represent eradication to the limit of detection.

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