Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library

Abstract

Although antibiotic treatment for Lyme disease is effective in the majority of cases, especially during the early phase of the disease, a minority of patients suffer from post-treatment Lyme disease syndrome (PTLDS). It is unclear what mechanisms drive this problem, and although slow or ineffective killing of Borrelia burgdorferi has been suggested as an explanation, there is a lack of evidence that viable organisms are present in PTLDS. Although not a clinical surrogate, insight may be gained by examining stationary-phase in vitro Borrelia burgdorferi persisters that survive treatment with the antibiotics doxycycline and amoxicillin. To identify drug candidates that can eliminate B. burgdorferi persisters more effectively, we screened an Food and Drug Administration (FDA)-approved drug library consisting of 1524 compounds against stationary-phase B. burgdorferi by using a newly developed high throughput SYBR Green I/propidium iodide (PI) assay. We identified 165 agents approved for use in other disease conditions that had more activity than doxycycline and amoxicillin against B. burgdorferi persisters. The top 27 drug candidates from the 165 hits were confirmed to have higher anti-persister activity than the current frontline antibiotics. Among the top 27 confirmed drug candidates from the 165 hits, daptomycin, clofazimine, carbomycin, sulfa drugs (e.g., sulfamethoxazole), and certain cephalosporins (e.g. cefoperazone) had the highest anti-persister activity. In addition, some drug candidates, such as daptomycin and clofazimine (which had the highest activity against non-growing persisters), had relatively poor activity or a high minimal inhibitory concentration (MIC) against growing B. burgdorferi. Our findings may have implications for the development of a more effective treatment for Lyme disease and for the relief of long-term symptoms that afflict some Lyme disease patients.

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

“…Daptomycin is the only approved membrane-acting antibiotic that disrupts the proton motive force. The B. burgdorferi MIC to daptomycin was fairly high, 12 to 25 g/ml (Table 1), in accordance with published data (43). Daptomycin was highly bactericidal against B. burgdorferi, but a remaining subpopulation of persisters survived (Fig.…”

Persister Development by Borrelia burgdorferi Populations In Vitro

“…Several papers have been published on the MIC for doxycycline. The concentrations of B. burgdorferi that were used in the assays were either log 5 or log 6, and the MIC ranged from 0.25 g/ml to 0.5 g/ml, based on the criteria of inhibiting growth over a 3-to 5-day period (16,23,35). The results from the doxycycline MIC assays suggested that at early log phase, 0.25 g/ml is a sufficient MIC (Table 1; see also Fig.…”

“…However, multiple studies with animals have shown that B. burgdorferi is not fully eradicated with antibiotic treatment (16,17,26,(50)(51)(52). The development of slow-growing or dormant persisters in the presence of doxycycline (23,33), a microbiostatic antibiotic, would indicate that reliance on immune control coincident with treatment would be necessary for efficacy of antimicrobial therapy. Given the multiple strategies that B. burgdorferi utilizes to evade the immune response (53), the survival of persisters after treatment with doxycycline is a reasonable possibility.…”

“…Doxycycline acts on the bacterial 30S ribosomal subunit (19), while ceftriaxone can generally be described as having beta-lactam activity (20). Although previous clinical studies showed that doxycycline can be as effective as third-generation cephalosporin antibiotics like ceftriaxone (21,22), recent in vitro work suggests that doxycycline may not be as effective as ceftriaxone against stationaryphase bacteria (23). Importantly, the role of immune responses to infection is integral to the treatment mode for microbiostatic an-tibiotics like doxycycline, such that results from in vitro studies should not be overinterpreted to the in vivo situation.…”

“…The groups of cells were treated with increasing concentrations of doxycycline (0, 0.1, 0.25, 0.5, 1.0, and 2.5 g/ml) for 5 days. Previous studies have used not less than 72 h of treatment for MIC determination (27,32), but based on the time-dependent mechanism of doxycycline, we extended it to 5 days, as has also been reported previously (23,33). The MIC was quantified as the lowest concentration necessary to inhibit growth over a 5-day period.…”