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

Figures & data

Figure 1 (A) Growth curve of B. burgdorferi strain B31 in vitro. (B) Representative images of the log phase (3-day culture) and stationary phase of B. burgdorferi B31 strain (7-day culture), observed with fluorescent microscopy using the SYBR Green I/PI stain (×400 magnification). The arrows indicate multiple morphological forms of B. burgdorferi in stationary phase.

Figure 2 Susceptibility of log phase (3 days) and stationary-phase (7 days) B. burgdorferi to 50 µM drugs after a 5-day treatment. The percentages of residual live cells were determined using the SYBR Green I/PI assay.

Table 1 Activity of top 27 active hits with better activity than the current Lyme disease antibiotics against stationary-phase B. burgdorferi persisters ^a

Drugs (50 μM)	Residual viable cells^b	Residual viable cells^c	Ratio of green/red fluoresce
			Primary screening	Rescreening	Rescreening	P-value^d
Control	93%	94%	8.67	8.38	8.59	-
Amoxicillin^e	76%	76%	7.98	7.86	7.82	1.000000
Doxycycline^e	75%	67%	7.62	7.35	7.58	0.233596
Penicillin G^e	75%	68%	7.41	7.68	7.92	0.699416
Tetracycline^e	54%	50%	7.59	6.14	7.18	0.102366
Ceftriaxone^e	50%	44%	6.74	6.89	6.78	0.000182
Cefuroxime^e	49%	43%	6.59	6.84	6.67	0.000317
Clarithromycin^e	70%	65%	7.70	7.36	7.59	0.038775
Azithromycin^e	77%	80%	8.33	8.10	7.92	0.071492
Daptomycin	35%	28%	6.10	6.20	6.09	0.000008
Clofazimine	45%	32%	6.56	6.23	6.02	0.000599
Cefoperazone	37%	34%	6.54	6.32	6.23	0.000126
Carbomycin	41%	37%	6.37	6.81	6.32	0.001045
Vancomycin	48%	38%	6.65	6.58	6.37	0.000152
Cephalothin	49%	40%	6.74	6.49	6.55	0.000133
Cefotiam	42%	43%	6.41	7.55	6.21	0.000503
Cefmetazole	-	43%	6.80	7.38	6.00	0.045064
Cefepime	-	44%	6.67	7.16	6.45	0.006368
Amodiaquin	-	45%	6.79	6.44	6.85	0.000946
Streptomycin	-	45%	6.72	6.93	6.76	0.000175
Ticarcillin	-	46%	6.82	6.72	6.93	0.000163
Cefonicid	-	46%	6.86	7.54	6.07	0.067661
Piperacillin-tazobactam	47%	47%	7.18	6.47	6.98	0.009594
Cefdinir	-	48%	6.88	7.51	6.29	0.049107
Ceforanide	-	48%	6.89	7.49	6.33	0.043847
Cefmenoxime	-	48%	6.82	7.59	6.32	0.058674
Bismuth	-	48%	6.94	6.82	6.92	0.000082
Ceftizoxime	-	49%	6.94	6.83	7.03	0.000223
Ceftibuten	51%	49%	6.81	6.78	7.27	0.004888
Amphotericin B	-	50%	7.14	6.88	6.87	0.000783
Cefamandole	-	50%	6.71	7.73	6.52	0.076304
Quinine hydrobromide	-	50%	7.00	6.85	6.88	0.000124
Cyclacillin	51%	53%	6.81	6.88	7.64	0.045210
Colistin	50%	54%	7.15	7.26	7.23	0.000319
Sulfameter		54%	7.13	7.46	6.98	0.009635
Tigecycline	58%	51%	6.98	7.06	6.96	0.001557

a Stationary-phase B. burgdorferi (7-day old) cells were treated with drugs for 7 days. The line above clarithromycin refers to antibiotics used to treat Lyme disease.

b Residual viable B. burgdorferi was assayed by epifluorescence microscope counting.

c Residual viable B. burgdorferi was calculated according to the regression equation and ratio of Green/Red fluorescence obtained by SYBR Green I/PI assay.

d P-values of the standard t-test for the treated group versus a control group treated with amoxicillin, which is known to have poor activity against stationary-phase persisters.

e Currently recommended antibiotics for Lyme disease.⁵

Table 2 Comparison of the MIC values and anti-persister activity of selected antibiotics against B. burgdorferi

Antibiotics	MIC (μg/mL)	Cmax (μg/mL)^a	Activity against persisters
Doxycycline	≤0.25	3.6–4.6	−
Amoxicillin	≤0.25	1.5–13.8	−
Metronidazole	25	12.5–19.4	−
Daptomycin	12.5–25	57.8–93.9	++++
Clofazimine	6.25	0.47–0.7	+++
Carbomycin	≤0.25	0.625	+++
Cefoperazone	≤0.25	111–375	+++
Cefotiam	≤0.25	30–170	++
Vancomycin	0.2–0.4	19–23	+
Tazobactam	12.5	14.8–33.8	−
Sulfamethoxazole	≤0.25	46.3	−

a C_max values are derived from the literature.

Figure 3 Representative images of stationary-phase B. burgdorferi strain B31 treated with different antibiotics (50 µM) followed by staining in the SYBR Green I/PI assay (×400 magnification). (A) Drug-free control, (B) Doxycycline, (C) Amoxicillin, (D) Daptomycin, (E) Cefoperazone, (F) Clofazimine, (G) Carbomycin, (H) Cefotiam, and (I) Tetracycline.