A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library

Abstract

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10–20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even when pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

Ceftriaxone Pulse Dosing Fails to Eradicate Biofilm-Like Microcolony B. burgdorferi Persisters Which Are Sterilized by Daptomycin/ Doxycycline/Cefuroxime without Pulse Dosing

“…As the round of pulse dosing treatment with ceftriaxone increased, the number of spirochetal forms and round body forms decreased in the log phase culture ( Figures 1A–D ). We noted after the first round of ceftriaxone treatment the number of planktonic spirochetes decreased dramatically while the number of round body forms increased slightly presumably due to conversion of spirochetal form to round body forms (Feng et al, 2016a) in both the 5 day old late log phase and the 10 day old stationary phase cultures. Two and three rounds of treatment could kill both spirochetal form and round body forms, and the fourth round of treatment eliminated nearly all planktonic B. burgdorferi cells but still could not eliminate the aggregated microcolony form in the 10 day stationary phase culture ( Figure 1H ).…”

“…Our previous studies suggest that drug combination treatment is more effective than single drug treatment and could eradicate the B. burgdorferi microcolony forms in vitro (Feng et al, 2015a, 2016a,b) and that daptomycin showed best activity against stationary phase and round body form of B. burgdorferi (Feng et al, 2014a, 2016a). The daptomycin/doxycycline combined with cefoperazone or cefuroxime is the most effective drug combination which could eradicate even the most resistant aggregated microcolony form of B. burgdorferi enriched in stationary phase cultures (Feng et al, 2015a, 2016b).…”

“…We found that daptomycin, which is the most effective antibiotic against B. burgdorferi persisters (Feng et al, 2014a, 2016a), fared poorly in pulse dosing experiments either singly ( Figure 2A ) or in drug combinations ( Figure 3A ), as it had even less activity than the ceftriaxone and doxycycline in the pulse dosing treatment ( Figure 2A ). Despite its excellent activity against non-growing B. burgdorferi persisters (Feng et al, 2014a), daptomycin is known to have limited activity against growing spirochetal forms as shown by a relatively high MIC (12.5–25 μg/ml) compared to ceftriaxone and doxycycline (Feng et al, 2014a).…”

“…These atypical forms can be considered part of the heterogeneous persisters (Zhang, 2014) as they have high tolerance to doxycycline and amoxicillin compared to growing spirochetal forms (Feng et al, 2014a, 2015a). Therefore, stationary phase cultures (7–15 day old) which are enriched in persisters have been used as a surrogate persister model for high-throughput drug screens against persister populations (Feng et al, 2014a, 2015a,b,d) and have been shown to have overlapping persister populations with amoxicillin tolerant round body persisters (Feng et al, 2016a). …”

Activity of Sulfa Drugs and Their Combinations against Stationary Phase B. burgdorferi In Vitro

“…These atypical forms have been shown to have increased tolerance to doxycycline and amoxicillin when compared to the growing spirochetal forms [13,14,15,16]. In addition, that the active hits from the round body persister screens [18] overlap with those from the screens on stationary phase cells [13] indicates the stationary phase culture or cells can be used as a relevant persister model. Therefore, stationary phase cultures (seven to 15 days old) enriched in persisters have been used as a model for high-throughput drug screens against persisters [13,14,19].…”