High Activity of Selective Essential Oils against Stationary Phase Borrelia burgdorferi

Although the majority of patients with Lyme disease can be cured with the standard 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from post-treatment Lyme disease syndrome (PTLDS). While the cause for this is debated, one possibility is due to persisters not killed by the current Lyme antibiotics. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been carefully studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persisters. We found that many essential oils had varying degrees of activity against B. burgdorferi, with top 5 essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing more activity than the persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top 3 hits, oregano, cinnamon bark and clove bud, completely eradicated all viable cells without regrowth in subculture. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combinations in vitro and in vivo for improved treatment of persistent Lyme disease. IMPORTANCE There is a huge need for effective treatment of patients with Lyme disease who suffer from PTLDS. Recent in vitro and in vivo studies suggest that B. burgdorferi develops persisters that are not killed by the current Lyme antibiotics as a possible contributor to this condition. Although essential oils are used by patients with Lyme disease with variable improvement in symptoms, their anti-borrelia activity has not been carefully studied. Here we found that not all essential oils have adequate anti-borrelia activity and identified some highly potent essential oils (oregano, cinnamon bark, clove bud) that have even higher anti-persister and anti-biofilm activity than the persister drug daptomycin. Carvacrol was found to be the most active ingredient of oregano oil and have the potential to serve as a promising oral persister drug. Our findings may have implications for developing improved treatment of persisting Lyme disease.

, as well as persistent infection due to B. burgdorferi persisters that are not killed by the current antibiotics used to treat Lyme disease (8)(9)(10). 69 Various studies have found evidence of B. burgdorferi persistence in dogs (11), mice (8,9), 70 monkeys (10), as well as humans (12) after antibiotic treatment, however, viable organisms 71 are very difficult to be cultured from the host after antibiotic treatment.

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In log phase cultures (3-5 day old), B. burgdorferi is primarily in motile spirochetal form 74 which is highly susceptible to current Lyme antibiotics doxycycline and amoxicillin, 75 however, in stationary phase cultures (7-15 day old), increased numbers of atypical variant 76 forms such as round bodies and aggregated biofilm-like microcolonies develop (13,14). 77 These atypical forms have increased tolerance to doxycycline and amoxicillin when 78 compared to the growing spirochetal forms (13)(14)(15)(16). In addition, that the active hits from the 79 round body persister screens (17) overlap with those from the screens on stationary phase 80 cells (13) indicates the stationary phase culture contains overlapping persister population and 81 can be used as a relevant persister model for drug screens to identify agents with anti-82 persister activity. Using these models,we identified a range of drugs such as daptomycin, 83 clofazimine, anthracycline antibiotics, and sulfa drugs with high activity against stationary 84 phase cells enriched in persisters through screens of FDA-approved drug library and NCI 85 compound libraries (13,18

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Evaluation of essential oils for activity against stationary phase B. burgdorferi. We 100 evaluated a panel of 34 essential oils at four different concentrations (1%, 0.5%, 0.25% and 101 0.125%) for activity against a 7-day old B. burgdorferi stationary phase culture in the 96-well 102 plates with control drugs for 7 days. Consistent with our previous studies (13, 20), 103 daptomycin control was shown to have high activity against the B. burgdorferi stationary 104 phase culture, with a dose-dependent increase in killing activity resulting in a near total 105 clearance of B. burgdorferi cells at the 40 µM concentration (Figure 1). Five essential oils 106 (bandit, oregano, clove bud, geranium bourbon and cinnamon bark) at 1% concentration 107 showed more activity against the stationary phase B. burgdorferi culture than 40 µM 108 daptomycin with the plate reader SYBR green I/PI assay (Table 1). We found some essential 109 oils have autofluorescence which severely interfered with the SYBR Green I/PI plate reader 110 assay, but we were able to identify and resolve this issue present in some samples by 111 fluorescence microscopy. As we previously described (21), we directly calculated the green (live) cell ratio of microscope images using Image Pro-Plus software, which could eliminate 113 the background autofluorescence. Using SYBR Green I/PI assay and fluorescence 114 microscopy, we additionally found 18 essential oils that showed more or similar activity 115 against the stationary phase B. burgdorferi at 1% concentration compared to the 40 µM 116 daptomycin, which could eradicate all live cells as shown by red (dead) aggregated cells 117 (Table 1; Figure 1A). At 0.5% concentration, 7 essential oils (oregano, cinnamon bark, clove 118 bud, citronella, wintergreen, geranium bourbon, and patchouli dark) were found to have 119 higher or similar activity against the stationary phase B. burgdorferi than 40 µM daptomycin 120 by fluorescence microscope counting after SYBR Green I/PI assay (Table 1; Figure 1B).

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However, bandit thieves oil, while having good activity at 1%, had significantly less activity 122 at 0.5% and lower concentrations (Table 1). Among the effective hits, 5 essential oils 123 (oregano, cinnamon bark, clove bud, citronella, and wintergreen) still showed better activity 124 than 40 µM daptomycin at 0.25% concentration (Table 1; Figure 1C). Eventually, oregano, 125 cinnamon bark, and clove bud were identified as the most active essential oils because of 126 their remarkable activity even at the lowest concentration of 0.125%, which showed similar 127 or better activity than 40 µM daptomycin (Table 1; Figure 1D).

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To further compare the activity of these active essential oils and find whether they could 130 eradicate stationary phase B. burgdorferi at lower concentrations, we evaluated 6 essential 131 oils (oregano, cinnamon bark, clove bud, citronella, geranium bourbon, and wintergreen) at 132 even lower concentrations at 0.1% and 0.05%. We noticed that oregano could not wipe out    (Table 2), we used subculture to further confirm whether the top 6 active essential oils 155 (oregano, cinnamon bark and clove bud, citronella, geranium bourbon, and wintergreen) could eradicate the stationary phase B. burgdorferi cells at 0.1% or 0.05% concentration. At 157 0.1% concentration, the subculture results were consistent with the above drug exposure 158 results. We did not find any regrowth in samples of three top hits, oregano, cinnamon bark showed better activity than the cinnamon bark at 0.05% concentration (Table 2) Previous in vitro studies showed that certain essential oils have bacteriostatic and/or bactericidal activity against on multidrug resistant Gram-negative clinical isolates (23). In 179 this study, we tested 34 essential oils from different plants on non-growing stationary phase 180 B. burgdorferi as a model of persister drug screens. We were able to identify 23 essential oils 181 that are more active than 40 μM daptomycin at 1% concentration, 3 of which, i.e. oregano, 182 clove bud and cinnamon bark, highlighted themselves as having a remarkable activity even at 183 a very low concentration of 0.125% (Table 1). Among them oregano and cinnamon bark 184 essential oil had the best activity as shown by completely eradicating B. burgdorferi even at 185 0.05% concentration. In a previous study, oregano essential oil was found to have 186 antibacterial activity against Gram-positive and Gram-negative bacteria (22). Here, for the 187 first time, we identified oregano essential oil as having a highly potent activity against 188 stationary phase B. burgdorferi. We tested three major ingredients of oregano essential oil 189 (carvacrol, p-cymene and α-terpinene) on B. burgdorferi, and found carvacrol is the major 190 active component, which showed similar activity as the complete oregano essential oil 191 (Figures 2 and 3). In addition, we noted that oregano essential oil can dramatically reduce the 192 size of aggregated biofilm-like microcolonies compared to the antibiotic controls ( Figure 1).

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After treatment with 0.25% oregano essential oil, only some dispersed tiny red aggregated 194 cells were left in the culture ( Figure 1C). Interestingly, we observed that amount and size of We also noted that some essential oils such as oregano and cinnamon bark had relatively high 209 residual viability percentage (Table 2) at low concentration of 0.05% but their treated B. Meanwhile, we found that at a high concentration (above 1%) lemongrass or oregano 219 essential oil showed apparent high residual viability percentage by the SYBR Green I/PI plate 220 assay, compared with the microscopy counting data (Table 1, Figure 1A). This may be caused 221 by strong autofluorescence of these essential oils that severely interfere with the SYBR Green 222 I/PI assay. We studied the emission spectral of lemongrass essential oil using Synergy H1 223 multi-mode reader and found lemongrass essential oil emits the strongest autofluorescence.

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The peak fluorescence of lemongrass essential oil is at 520 nm that overlaps with the green 225 fluorescence of SYBR Green I dye (peak is at 535 nm). The strong autofluorescence caused 226 the abnormal residual viability percentage (above 100% in Table 1) using SYBR Green I/PI 227 plate assay. We also found oregano essential oil emits autofluorescence at 535 nm, which 228 pushed the green/red fluorescence ratio higher than their true values (Table 1). However, we 229 were able to solve this problem by using fluorescence microscopy as a more reliable measure 230 to confirm the results of SYBR Green I/PI plate reader assay (13, 21). In a previous study, it has been found that volatile oil from Cistus creticus showed growth 241 inhibiting activity against B. burgdorferi in vitro (24)  Although we found several essential oils (oregano, cinnamon bark, clove bud) that have 250 excellent sterilizing activity against B. burgdorferi stationary phase cells in vitro (Table 1)

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Identification of active components or active component combinations from essential oils 256 may help to eliminate the quality difference of natural products. However, we were able to 257 identify carvacrol as the most active ingredient in oregano essential oil, and its 258 pharmacokinetics has been studied as a feed addition in pigs (25) and topical oil in cattle 259 (26). In the rat model, the calculated LD50 of carvacrol is 471.2 mg/kg (27). We noticed that 260 the 0.05% of carvacrol used here, which is equivalent to 0.48 μg/mL or 3.2 µM and 261 completely eradicated B. burgdorferi stationary phase cells in subculture (Figure 3), is lower 262 than the peak plasma concentration (3.65 μg/mL) in the swine study (25). These findings 263 favor the application of carvacrol in future treatment studies. Importantly, carvacrol seems to 264 be more active than daptomycin, the most active persister drugs against B. burgdorferi (13,265 14). In this study, 0.1% carvacrol (6.4 µM) showed much higher activity (2% residual 266 viability) than 5 µM daptomycin (45% residual viability) (Table 1 and 2). In addition, 0.05% 267 carvacrol (3.2 µM) could eradicate B. burgdorferi stationary phase cells with no regrowth in 268 subculture, but 10 µg/mL daptomycin (6.2 µM), by contrast, could not completely kill B. In summary, we found that many essential oils had varying degrees of activity against 281 stationary phase B. burgdorferi. The most active essential oils are oregano, cinnamon bark, 282 and clove bud, which seem to have even higher activity than the persister drug daptomycin. A 283 particularly interesting observation is that these highly active essential oils had remarkable  Microscopy. The B. burgdorferi cultures were examined using BZ-X710 All-in-One 314 fluorescence microscope (KEYENCE, Inc.). The SYBR Green I/PI viability assay was 315 performed to assess the bacterial viability using the ratio of green/red fluorescence to 316 determine the live:dead cell ratio, respectively, as described previously (13,34). This residual 317 cell viability reading was confirmed by analyzing three representative images of the bacterial 318 culture using epifluorescence microscopy. BZ-X Analyzer and Image Pro-Plus software were 319 used to quantitatively determine the fluorescence intensity. concentrations. In the primary essential oil screen, each essential oil was assayed in four 326 concentrations, 1%, 0.5%, 0.25% and 0.125% (v/v) in 96-well plate. The active hits were 327 further confirmed with lower 0.1% and 0.05% concentration; all tests were run in triplicate.

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All the plates were incubated at 33°C and 5% CO 2 without shaking for 7 days when the 329 residual viable cells remaining were measured using the SYBR Green I/PI viability assay and 330 epifluorescence microscopy as described (13,34).

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Antibiotic susceptibility testing. To qualitatively determine the effect of essential oils in a 333 high-throughput manner, 10 μl of each essential oil from the pre-diluted stock was added to 334 7-day old stationary phase B. burgdorferi culture in the 96-well plate. Plates were sealed and 335 placed in 33°C incubator for 7 days when the SYBR Green I/ PI viability assay was used to 336 assess the live and dead cells as described (13) carvacrol suspension was two-fold diluted from 0.5% (4.88 µg/mL) to 0.008% (0.08 µg/mL).

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All experiments were run in triplicate. B. burgdorferi culture was incubated in 96-well 352 microplate at 33 °C for 7 days. Cell proliferation was assessed using the SYBR Green I/PI 353 assay and BZ-X710 All-in-One fluorescence microscope (KEYENCE, Inc.).

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Subculture studies to assess viability of the of essential oil-treated B. burgdorferi 356 organisms. A 7-day old B. burgdorferi stationary phase culture (500 μl) was treated with 357 essential oils or control drugs for 7 days in 1.5 ml Eppendorf tubes as described previously 358 (14). After incubation at 33 °C for 7 days without shaking, the cells were collected by 359 centrifugation and rinsed with 1 ml fresh BSK-H medium followed by resuspension in 500 μl 360 fresh BSK-H medium without antibiotics. Then 50 μl of cell suspension was transferred to 1 361 ml fresh BSK-H medium for subculture at 33 °C for 20 days. Cell proliferation was assessed 362 using SYBR Green I/PI assay and epifluorescence microscopy as described above.