Clinical Trails of Essential Oils Again Tick Bourne Diseases
Antibiotics (Basel). 2018 Dec; seven(4): 89.
Identification of Essential Oils with Potent Activity against Stationary Phase Borrelia burgdorferi
Judith Miklossy
2International Alzheimer Research Heart, Prevention Alzheimer International Foundation, Martigny-Croix CP 16 1921, Switzerland; hc.niweulb@yssolkimhtiduj
Received 2018 Aug 28; Accepted 2018 Oct 10.
Abstract
Lyme disease is the most common vector borne-disease in the Usa (US). While the majority of the Lyme affliction patients can be cured with 2–4 weeks antibody treatment, about 10–xx% of patients proceed to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as i possibility. It has recently been shown that B. burgdorferi develops dormant persisters in stationary phase cultures that are not killed by the current Lyme antibiotics, and in that location is involvement in identifying novel drug candidates that more finer kill such forms. We previously identified some highly active essential oils with first-class action against biofilm and stationary phase B. burgdorferi. Here, we screened another 35 essential oils and institute ten essential oils (Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cuminum cyminum L. seeds, Cymbopogon martini var. motia Bruno grass, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, Amyris balsamifera L. wood, Thymus vulgaris L. leaves, Litsea cubeba (Lour.) Pers. fruits, Eucalyptus citriodora Hook. leaves) and the agile component of cinnamon bark cinnamaldehyde (CA) at a low concentration of 0.1% take stiff activeness against stationary phase B. burgdorferi. At a lower concentration of 0.05%, essential oils of Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cymbopogon martini var. motia Bruno grass and CA however exhibited strong activity against the stationary phase B. burgdorferi. CA as well showed potent activity confronting replicating B. burgdorferi, with a MIC of 0.02% (or 0.2 μg/mL). In subculture studies, the top five essential oil hits Allium sativum Fifty. bulbs, Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, and Litsea cubeba (Lour.) Pers. fruits completely eradicated all B. burgdorferi stationary stage cells at 0.1%, while Cymbopogon martini var. motia Bruno grass, Eucalyptus citriodora Claw. leaves, Amyris balsamifera L. wood, Cuminum cyminum Fifty. seeds, and Thymus vulgaris L. leaves failed to do then as shown by visible spirochetal growth after 21-day subculture. At concentration of 0.05%, but Allium sativum L. bulbs essential oil and CA sterilized the B. burgdorferi stationary phase culture, as shown by no regrowth during subculture, while Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers and Litsea cubeba (Lour.) Pers. fruits essential oils all had visible growth during subculture. Future studies are needed to determine if these highly active essential oils could eradicate persistent B. burgdorferi infection in vivo.
Keywords: Borrelia burgdorferi, persisters, biofilm, antimicrobial activity, essential oils
1. Introduction
Lyme disease, which is caused by the spirochetal organism Borrelia burgdorferi, is the nigh mutual vector borne-disease in the United States (United states) with most 300,000 cases a year [ane]. While the majority of the Lyme disease patients tin can be cured with the standard 2–4 weeks antibiotic monotherapy with doxycycline or amoxicillin or cefuroxime [2], about 36% of patients go on to suffer from persisting symptoms of fatigue, joint, or musculoskeletal pain, and neuropsychiatric symptoms, even half dozen months afterward taking the standard antibiotic therapy [3]. These latter patients suffer from a poorly understood condition, called mail service-handling Lyme disease (PTLDS) syndrome. While the cause for PTLDS is unclear and is likely multifactorial, the following factors may be involved: autoimmunity [four], host response to expressionless droppings of Borrelia organism [five], tissue impairment caused during the infection, and persistent infection. There take been various anecdotal reports demonstrating persistence of the organism despite standard antibiotic treatment [half dozen,7,8]. For example, civilisation of B. burgdorferi leaner from patients despite treatment has been reported as exceptional case reports [9]. In addition, in animal studies with mice, dogs and monkeys, it has been shown that the electric current Lyme antibiotic handling with doxycycline, cefuroxime, or ceftriaxone is unable to completely eradicate the Borrelia organism, as detected by xenodiagnosis and PCR [6,7,eight,10], but feasible organism cannot exist cultured in the conventional sense equally in other persistent bacterial infections, like tuberculosis afterwards handling [eleven,12].
Recently, it has been demonstrated that B. burgdorferi can course various dormant non-growing persisters in stationary phase cultures that are tolerant or not killed by the current antibiotics that are used to treat Lyme disease [13,14,15,sixteen]. Thus, while the electric current Lyme antibiotics are practiced at killing the growing B. burgdorferi they have poor action confronting the non-growing persisters enriched in stationary phase culture [14,16,17]. Therefore, there is involvement to identify drugs that are more agile against the B. burgdorferi persisters than the electric current Lyme antibiotics. We used the stationary phase culture of B. burgdorferi as a persister model and performed loftier throughput screens and identified a range of drug candidates such as daptomycin, clofazimine, sulfa drugs, daunomycin, etc., which take strong activity confronting B. burgdorferi persisters. These persister active drugs act differently from the current Lyme antibiotics, as they seem to preferentially target the membrane. We found that the variant persister forms such as round bodies, microcolonies, and biofilms with increasing degree of persistence in vitro, cannot be killed past the current Lyme antibiotics or even persister drugs like daptomycin alone, and that they can only exist killed by a combination of drugs that kill persisters and drugs that kill the growing forms [fourteen]. These observations provide a possible explanation in support of persistent infection despite antibiotic treatment in vivo.
Although daptomycin has good anti-persister activity, it is expensive and is an intravenous drug and difficult to administer and prefer in clinical setting and it has limited penetration through blood encephalon barrier (BBB). Thus, there is interest to identify alternative drug candidates with high anti-persister action. Nosotros recently screened a panel of 34 essential oils and found the top three candidates oregano oil and its active component carvacrol, cinnamon bark, and clove bud equally having even better anti-persister action than daptomycin at forty μM [18]. To place more essential oils with strong activity against B. burgdorferi persisters, in this study, we screened an boosted 35 different essential oils and institute 10 essential oils (garlic, allspice, cumin, palmarosa, myrrh, hydacheim, amyris, thyme white, Litsea cubeba, lemon eucalyptus) and the active component of cinnamon bark cinnamaldehyde as having strong action in the stationary phase B. burgdorferi persister model.
ii. Materials and Methods
two.1. Organism and Civilisation Conditions
A depression passaged strain B. burgdorferi B31 5A19 was kindly provided by Dr. Monica Embers [15]. Firstly, we prepared the B. burgdorferi B31 culture in BSK-H medium (HiMedia Laboratories, Mumbai, Bharat), supplemented with 6% rabbit serum (Sigma-Aldrich, St. Louis, MO, United states) without antibiotics. Subsequently incubation for vii days in microaerophilic incubator (33 °C, v% COii), the B. burgdorferi culture went into stationary stage (~x7 spirochetes/mL), followed by evaluating potential anti-persister activity of essential oils in a 96-well plate (see below).
2.two. Essential Oils and Drugs
We purchased a console of essential oils (Plant Guru, Plainfield, NJ, Usa) and cinnamaldehyde (CA) (Sigma-Aldrich, St. Louis, MO, USA). The essential oils from Establish Guru company are tested past third party laboratory using GC/MS, and the GC/MS written report tin can be found on their website [19]. Dimethyl sulfoxide (DMSO)-soluble essential oils were prepared at 10% (v/v) in DMSO as stock solution, which was and so added with 7-solar day old stationary phase cultures at ration of ane:l to achieve 0.2% of essential oils in the mixture. The 0.2% essential oils were farther diluted to the stationary phase culture to become the desired concentration for evaluating anti-borrelia activity. DMSO-insoluble essential oils were directly added to B. burgdorferi cultures, then vortexed to form aqueous break, followed by firsthand transfer of essential oil aqueous suspension in serial dilutions to desired concentrations and then added to B. burgdorferi cultures. Doxycycline (Dox), cefuroxime (CefU), (Sigma-Aldrich, St. Louis, MO, United states), and daptomycin (Dap) (AK Scientific, Wedlock Urban center, CA, USA) were prepared at a concentration of 5 mg/mL in suitable solvents [20,21], then filter-sterilized past 0.2 μm filter and stored at −xx °C as stock solutions.
ii.three. Microscopy
Treated B. burgdorferi cell suspensions were checked with BZ-X710 All-in-One fluorescence microscope (KEYENCE, Itasca, IL, U.s.). The bacterial viability was evaluated by SYBR Dark-green I/PI assay, which was performed by computing the ratio of light-green/red fluorescence subsequently dying to make up one's mind the ratio of live and dead cells, as described previously [xvi,22]. The residual cell viability reading was obtained by analyzing 3 representative images of the same bacterial prison cell suspension taken by fluorescence microscopy. To quantitatively determine the bacterial viability from microscope images, software of BZ-Ten Analyzer and Image Pro-Plus were employed to evaluate fluorescence intensity, as we described previously [14].
ii.4. Evaluation of Essential Oils for Their Activities Against B. Burgdorferi Stationary Phase Cultures
To evaluate the possible activity of the essential oils confronting stationary phase B. burgdorferi, ten% DMSO-soluble essential oils or aqueous suspension of DMSO-insoluble essential oils were added to 100 µL of the seven-day old stationary phase B. burgdorferi culture in 96-well plate to obtain the desired concentrations. In the primary screen, each essential oil was assayed with last concentrations of 0.2% and 0.1% (v/v) in 96-well plates. Drugs of daptomycin, doxycycline, and cefuroxime were used every bit control with final concentration of xl μM. The active hits were checked further with lower concentrations of 0.ane% and 0.05%; all of the tests mentioned above were run in triplicate. All of the plates were sealed and incubated at 33 °C without shaking for seven days, and v% COtwo were maintained in the incubator.
2.5. Essential Oil and Drug Susceptibility Testing
The alive and dead cells after vii-24-hour interval treatment with essential oils or antibiotics were evaluated using the SYBR Green I/PI assay combined with fluorescence microscopy, as described [16,22]. Briefly, the ratio of alive and dead cells was reflected past the ratio of green/red fluorescence, which was calculated through the regression equation and regression curve with to the lowest degree-foursquare fitting analysis.
To determine the Minimum inhibitory concentration (MIC) of cinnamaldehyde on growth of B. burgdorferi, the standard microdilution method was used and the growth inhibition was assessed past microscopy. ten% cinnamaldehyde DMSO stock was added to B. burgdorferi cultures (1 × tenfour spirochetes/mL) to get an initial suspension with 0.v% of cinnamaldehyde, and so a series of suspension was prepared past two-fold dilutions, with cinnamaldehyde concentrations ranging from 0.v% (=5 µg/mL) to 0.004% (=0.04 µg/mL). All of the experiments were carried out in triplicate. The B. burgdorferi cultures afterward treatment in 96-well microplate were incubated at 33 °C for vii days. Jail cell proliferation was assessed by the SYBR Green I/PI analysis combined with BZ-X710 All-in-Ane fluorescence microscope.
2.6. Subculture Studies to Assess Viability of Essential Oil-Treated B. Burgdorferi Organisms
Essential oils or control drugs were added into ane mL of seven-twenty-four hour period old B. burgdorferi stationary phase culture in 1.five mL Eppendorf tubes, incubated for seven days at 33 °C without shaking. Adjacent, cells were centrifuged and jail cell pellets were washed with fresh BSK-H medium (1 mL) followed past resuspension in 500 μL of the same medium without antibiotics. Then, 50 μL of cell suspension was inoculated into 1 mL of fresh BSK-H medium, incubated at 33 °C for 20 days for subculture. Cell growth was assessed using SYBR Green I/PI assay and fluorescence microscopy, as described in a higher place.
three. Results
three.1. Evaluating Activity of Essential Oils Against Stationary Phase B. Burgdorferi
In this written report, nosotros explored activity of another console of 35 new essential oils together with command drugs against a seven-mean solar day old B. burgdorferi stationary phase civilisation in 96-well plates incubated for seven days. Our previous study discovered that cinnamon bark essential oil showed very stiff activeness confronting B. burgdorferi culture at stationary phase fifty-fifty at 0.05% concentration [18]. To identify the active components of cinnamon bark essential oil, we besides added cinnamaldehyde (CA), the major ingredient of cinnamon bark, in this screen. Table 1 outlines the activeness of the 35 essential oils and CA against B. burgdorferi culture at stationary phase. Although the Litsea cubeba essential oil showed too strong autofluorescence to determine its activity at 0.2% concentration, all the other essential oil candidates, except parsley seed, showed significantly stronger activity (p < 0.05) than the doxycycline control (Table 1) at 0.2% concentration with SYBR Green I/PI assay. Among them, sixteen essential oils and CA at 0.2% concentration were found to accept strong activity against B. burgdorferi culture at stationary phase as compared to the control antibiotics doxycycline, cefuroxime, and daptomycin (Table one). Every bit previously described [23], we calculated the ratio of residual live cells and dead cells of microscope images using Epitome Pro-Plus software, which could eliminate the autofluorescence of the background. Using fluorescence microscopy, nosotros confirmed that, at 0.two% concentration, the xvi essential oils and CA could eradicate all live cells with simply dead and aggregated cells left every bit shown in Table ane and Effigy 1. At concentration of 0.1%, 10 essential oils (garlic, allspice, cumin, palmarosa, myrrh, hydacheim, amyris, thyme white, Litsea cubeba, lemon eucalyptus), and CA still exhibited significant activity (p < 0.05) over the current clinically used doxycycline (Tabular array 1; Effigy 2). Amid them, the about active essential oils were garlic, allspice, cumin, palmarosa, myrrh, and hydacheim considering of their remarkable activity even at 0.ane%, equally shown by totally cerise (dead) cells with SYBR Green I/PI assay and fluorescence microscope tests (Figure 1). CA as well showed very strong activity at 0.1% concentration. Although the plate reader information showed carrot seed and deep muscle essential oils had a pregnant activity (p < 0.05) compared with the doxycycline command, the microscope result did not confirm information technology due to loftier residual viability (60% and 68%, p > 0.05) (Table one). For the other six essential oils (cornmint, fennel sweetness, ho wood, birch, petitgrain, and caput ease), which showed strong action at 0.ii% concentration, we did non observe them to have higher activity than the doxycycline control at 0.1% concentration (Table 1, Figure 2). In addition, although essential oils of birch and Litsea cubeba take autofluorescence, which showed false high rest viability and interfered with the SYBR Green I/PI plate reader assay, they both exhibited strong activity against the stationary stage B. burgdorferi, as confirmed by SYBR Green I/PI fluorescence microscopy.
Effect of 0.ii% essential oils on the viability of stationary phase B. burgdorferi. A vii-24-hour interval old B. burgdorferi stationary phase culture was treated with 0.two% (v/v) essential oils for vii days followed by staining with SYBR Green I/PI viability assay and fluorescence microscopy.
Event of 0.1% essential oils on the viability of stationary phase B. burgdorferi. A vii-twenty-four hour period former B. burgdorferi stationary phase civilisation was treated with 0.1% (five/v) essential oils for seven days followed by staining with SYBR Green I/PI viability assay and fluorescence microscopy.
Table 1
Effect of essential oils on a seven-solar day onetime stationary phase B. burgdorferi a.
| Essential Oils and Command Drugs | Plant and Extracted Office | Residual Viability (%) afterward 0.ii% EO or twoscore μM Antibiotic Handling | Rest Viability (%) later 0.i% EO Treatment | ||||
|---|---|---|---|---|---|---|---|
| Plate Reader b | Microscope c | p-Value e | Plate Reader b | Microscope c | p-Value e | ||
| Doxycycline | -- | 73 ± 4 | 66 ± 2 | i.000 | -- | -- | -- |
| Cefuroxime | -- | 58 ± three | 55 ± three | 0.0016 | -- | -- | -- |
| Daptomycin | -- | 28 ± 5 | 21 ± 1 | 0.0004 | -- | -- | -- |
| Cinnamaldehyde | -- | 26 ± five | 0 | 0.0002 | 54 ± two | 28 | 0.0016 |
| Garlic | Allium sativum L. (syn. Porrum sativum (L.) Rchb.), bulbs | 25 ± four | 0 | 0.0001 | 24 ± half dozen | 18 ± 2 | 0.0002 |
| Allspice | Pimenta officinalis Lindl., berries | 21 ± four | 0 | 0.0001 | 30 ± vi | 25 ± 3 | 0.0005 |
| Myrrh | Commiphora myrrha (T. Nees) Engl. (syn. Balsamea myrrha (T. Nees) Oken), resin | 32 ± 3 | 0 | 0.0001 | 35 ± 6 | 25 ± ii | 0.0009 |
| Hydacheim | Hedychium spicatum Thymus (syn. Gandasulium spicatum (Buch.-Ham. ex Sm.) Kuntze), flowers | 34 ± iv | 23 ± 2 | 0.0002 | 38 ± 7 | 26 ± i | 0.0017 |
| Litsea cubeba | Litsea cubeba (Lour.) Pers. (syn. Benzoin cubeba (Lour.) Hatus., Persea cubeba (Lour.) Spreng.), fruits | 98 ± four | ND d | -- | 77 ± 4 | 27 ± three | (0.00004) |
| Palmarosa | Cymbopogon martini var. motia Bruno, grass | 26 ± v | 0 | 0.0002 | 35 ± five | 29 ± 2 | 0.0004 |
| Lemon eucalyptus | Eucalyptus citriodora Hook. (syn. Corymbia citriodora (Claw.) Thousand.D. Hill & 50.A.S. Johnson), leaves | 35 ± 6 | 0 | 0.0006 | 39 ± 7 | 29 ± iv | 0.0015 |
| Amyris | Amyris balsamifera L. (syn. Elemifera balsamifera (L.) Kuntze), wood | 32 ± 3 | 4 ± 2 | 0.0001 | 38 ± five | 29 ± iii | 0.0006 |
| Cumin | Cuminum cyminum L., seeds | 31 ± 3 | 0 | 0.0001 | 31 ± 6 | 30 ± 1 | 0.0005 |
| Thyme white | Thymus vulgaris L. (syn. Origanum thymus (L.) Kuntze), leaves | 37 ± 2 | 26 ± ii | 0.0001 | 36 ± 1 | 30 ± ii | 0.0001 |
| Carrot seed | Daucus carota L., seeds | 38 ± 4 | 5 ± 3 | 0.0004 | 40 ± 3 | threescore ± two | 0.0003 (0.0705) |
| Head ease | Synergy blend | 41 ± iii | 25 ± 3 | 0.0003 | 74 ± 4 | 65 ± 1 | 0.8008 |
| Deep muscle | Synergy blend | 42 ± 4 | three ± two | 0.0004 | 56 ± four | 68 ± iv | 0.0060 (0.3911) |
| Birch | Betula lenta L., bark | 86 ± 5 | 22 ± 2 | (0.00001) | 91 ± 4 | 69 ± 2 | -- |
| Ho wood | Cinnamomum camphora (L.) J. Presl (syn. Cinnamomum camphora (50.) Nees & Eberm., Cinnamomum camphora (L.) Siebold), twigs and bawl | 36 ± 4 | 3 ± 2 | 0.0004 | 69 ± five | seventy ± 3 | 0.3078 |
| Petitgrain | Citrus aurantium L, trees and leaves | 38 ± 3 | 19 ± ii | 0.0002 | 71 ± 4 | 70 ± 3 | 0.4743 |
| Fennel sweet | Foeniculum vulgare Mill., seeds | twoscore ± 5 | 2 ± 1 | 0.0006 | 72 ± 3 | 75 ± four | 0.6235 |
| Cornmint | Mentha arvensis, leaf | 35 ± 5 | 0 | 0.0004 | 68 ± 4 | 85 ± one | 0.1359 |
| Citrus smash | Synergy alloy | 51 ± v | >70 | 0.0039 | 71 ± 5 | >70 | 0.5865 |
| Nutmeg | Myristica fragrans Houtt., seeds | 43 ± four | >70 | 0.0008 | 71 ± 4 | >70 | 0.6533 |
| Alive | Synergy blend | xl ± 4 | >70 | 0.0004 | 71 ± 3 | >70 | 0.5228 |
| New beginning | Synergy alloy | 48 ± 4 | >70 | 0.0013 | 75 ± 4 | >lxx | 0.5107 |
| Happy | Synergy blend | 47 ± four | >seventy | 0.0009 | 78 ± 2 | >70 | -- |
| Meditation | Synergy blend | 55 ± 4 | >70 | 0.0041 | 79 ± 4 | >70 | -- |
| Deep wood | Synergy blend | 61 ± 1 | >seventy | 0.0039 | 79 ± 3 | >70 | -- |
| Copaiba | Copaifera officinalis (Jacq.) L., balsm | 51 ± 2 | >70 | 0.0007 | 79 ± two | >70 | -- |
| Balsam fir | Abies balsamea (Fifty.) Mill. (syn. Peuce balsamea (L.) Rich.), needles | 57 ± five | >70 | 0.0124 | 80 ± 1 | >lxx | -- |
| Juniper Berry | Juniperus communis L., berries | 56 ± v | >70 | 0.0086 | 82 ± 3 | >70 | -- |
| Camphor | Cinnamomum camphora (50.) J. Presl (syn. Cinnamomum camphora (L.) Nees & Eberm., Cinnamomum camphora (L.) Siebold), forest | 58 ± 3 | >lxx | 0.0047 | 82 ± 3 | >70 | -- |
| Vetiver | Vetiveria zizanioides (Fifty.) Nash (syn. Phalaris zizanioides L.), root | 41 ± iii | >70 | 0.0003 | 82 ± 5 | >70 | -- |
| Fir needle | Abies sibirica Ledeb. (syn. Pinus sibirica (Ledeb.) Turcz.), needles | 60 ± iii | >lxx | 0.0109 | 83 ± 5 | >seventy | -- |
| Sleep tight | Synergy alloy | 57 ± five | >70 | 0.0130 | 85 ± half-dozen | >70 | -- |
| Turmeric | Curcuma longa L. (syn. Kua domestica (L.) Medik.), root | fifty ± 2 | >70 | 0.0007 | 93 ± iii | >seventy | -- |
| Elemi | Canarium luzonicum (Blume) A. Grayness (syn. Pimela luzonica Blume), resin | 58 ± 3 | >70 | 0.0059 | 95 ± two | >70 | -- |
| Parsley seed | Petroselinum sativum Hoffm., seeds | 64 ± five | >seventy | 0.0645 | 97 ± 3 | >seventy | -- |
The pinnacle 10 essential oils and CA (residual viability lower lx%) were chosen to evaluate their activities and explore their potential to eradicate B. burgdorferi cultures at stationary phase that harbor large numbers of persisters using lower essential oil concentrations (0.i% and 0.05%). We did the confirmation tests with one mL stationary phase B. burgdorferi in 1.5 mL Eppendorf tubes. At 0.i% concentration, the tube tests confirmed the active hits from the previous 96-well plate screen, although the action of all essential oils decreased slightly in the tube tests when compared to the 96-well plate tests (Tabular array ii, Effigy three). At a very low concentration of 0.05%, we noticed that garlic, allspice, palmarosa, and CA still exhibited potent activity against the stationary stage B. burgdorferi, canonical by few remainder greenish aggregated cells shown in Tabular array ii and Effigy 3. Meanwhile, we too found CA showed strong activity against replicating B. burgdorferi, with an MIC of 0.02% (equal to 0.2 μg/mL).
Effect of active essential oils on stationary phase B. burgdorferi. A ane mL B. burgdorferi stationary phase culture (seven-day old) was treated with 0.i% (A) or 0.05% (B) essential oils (labeled on the image) in 1.5 mL Eppendorf tubes for 7 days followed by staining with SYBR Greenish I/PI viability analysis and fluorescence microscopy.
Table 2
Comparison of pinnacle 10 essential oil activities against stationary stage B. burgdorferi with 0.1% and 0.05% (v/v) treatment and subculture a.
| Essential Oil Treatment | Residual Viability after 0.one% Essential Oil Treatment | Balance Viability afterwards 0.05% Essential Oil Treatment | ||
|---|---|---|---|---|
| Treatment b | Subculture c | Handling b | Subculture c | |
| Drug free control | 93% | + | 93% | + |
| Daptomycin+Doxycycline+Cefuroxime d | 18%d | − d | N/A | N/A |
| Garlic | thirty% | − | 33% | − |
| Allspice | 34% | − | 48% | + |
| Myrrh | 42% | − | 41% | + |
| Hydacheim | 44% | − | 61% | + |
| Litsea cubeba | 68% | − | 69% | + |
| Palmarosa | 39% | + | 67% | + |
| Lemon eucalyptus | 46% | + | 79% | + |
| Amyris | 48% | + | 71% | + |
| Cumin | 42% | + | lx% | + |
| Thyme white | forty% | + | 76% | + |
| Cinnamaldehyde | 34% | − | 56% | − |
3.2. Subculture Studies to Evaluate the Action of Essential Oils Against Stationary Phase B. burgdorferi
To validate the capability of the essential oils in eradicating B. burgdorferi cells at stationary phase, we performed subculture studies by incubating essential oils treated cells in fresh BSK medium after the removal of the drugs with washing, as previously described [14]. We picked the top 10 active essential oils (garlic, allspice, myrrh, hydacheim, Litsea cubeba, palmarosa, lemon eucalyptus, amyris, cumin, and thyme white) to further ostend whether they could eradicate the stationary phase B. burgdorferi cells at 0.ane% or 0.05% concentration past subculture experiments after the essential oil exposure (Table 2). At 0.1% concentration, we did not find any regrowth in samples of the peak v hits, including garlic, allspice, myrrh, hydacheim, and Litsea cubeba (Table 2, Figure 4A). Yet, palmarosa, lemon eucalyptus, amyris, cumin and thyme white could not eradicate B. burgdorferi cells at stationary phase every bit many spirochetes were still visible afterwards 21 days' subculture (Figure 4A). The subculture study likewise confirmed the strong activity of CA by showing no growth of spirochete after treatment with 0.1% CA. At concentration of 0.05%, we did non observe spirochetal regrowth in the garlic essential oil treated samples that were subcultured for 21 days (Effigy fourB), which indicates that garlic essential oil could completely impale all B. burgdorferi forms even at 0.05% concentration. On the other mitt, the other four agile essential oils (allspice, myrrh, hydacheim and Litsea cubeba) at a concentration of 0.05% could not sterilize the B. burgdorferi culture at stationary phase, since spirochetes were visible later on 21 days subculture (Figure 4B). Similar to the previous subculture outcome of cinnamon bark essential oil [eighteen], 0.05% cinnamaldehyde sterilized the B. burgdorferi stationary phase culture as shown by no regrowth afterwards 21 days subculture (Figure fourB), indicating that the agile component of cinnamon bark essential oil is attributable to cinnamaldehyde.
Subculture of B. burgdorferi after handling with essential oils. A B. burgdorferi stationary stage civilization (seven-solar day one-time) was treated with the indicated essential oils at 0.1% (A) or 0.05% (B) for seven days followed by washing and resuspension in fresh BSK-H medium and subculture for 21 days. The viability of the subculture was examined past SYBR Green I/PI stain and fluorescence microscopy.
4. Give-and-take
We recently institute that many essential oils have amend action against B. burgdorferi cells at stationary stage than the current clinically used antibiotics for treating Lyme disease [18]. Here, we screened another panel of 35 new essential oils using B. burgdorferi culture at stationary phase every bit a persister model [16]. Previously, we plant that 23 essential oils had strong activity at i% concentration, merely only 5 of them showed good activity at a lower concentration of 0.25% [xviii]. To identify the essential oils that accept activity against B. burgdorferi persisters at low concentrations, we performed the screen at 0.two% and 0.ane% concentrations in this study. Some essential oils, such every bit Litsea cubeba oil, showed high autofluorescence after SYBR Light-green I/PI stain, which significantly interfered with the SYBR Dark-green I/PI assay (Table 1, Effigy 1). Still, using lower concentration (0.i%) and fluorescence microscopy, we were able to verify the results from the SYBR Greenish I/PI analysis and eliminate the problem of autofluorescence with some essential oils. Some other limitation of the SYBR Green/PI assay is that not all cells turning red are dead, and farther subculture studies are needed to verify whether the PI- stained blood-red cells are indeed expressionless after drug exposure. In this study, we identified 18 essential oils (at 0.two% concentration) that are more than active than 40 μM daptomycin (a persister drug command that could eradicate B. burgdorferi stationary stage cells), from which ten essential oils stand up out as having a remarkable activity even at 0.one% concentration (Table 1). Amid them, garlic essential oil exhibited the best activity as shown by the lowest remainder viability of B. burgdorferi at 0.1%. In the subsequent comparison studies, the garlic essential oil highlighted itself as showing a sterilizing activeness even at a lower concentration of 0.05%, considering no Borrelia cells grew up in the subculture study (Table 2). Garlic as a common spice has been used throughout history as an antimicrobial, and a variety of garlic supplements have been commercialized equally tablets and capsules. The antibacterial activity of garlic was described by ancient Chinese, and in more recent times, by Louis Pasteur in 1858. Although allicin, an antibacterial compound from garlic, is shown to have antibacterial activity against multiple bacterial species [24,25], it has not been well studied on B. burgdorferi, specially the non-growing stationary phase organism, despite its anecdotal clinical use by some patients with Lyme disease (http://www.natural-homeremedies.com/blog/best-dwelling-remedies-for-lyme-affliction/; http://lymebook.com/blog/supplements/garlic-allimax-allimed-alli-c-allicin/). In this study, garlic essential oil was identified as the most stiff candidate having activeness confronting stationary phase B. burgdorferi, and its activity is equivalent to that of oregano and cinnamon bark essential oils, the two nigh agile essential oils against B. burgdorferi we identified in our previous written report [xviii].
Additionally, we found four other essential oils, allspice, myrrh, hydacheim, and Litsea cubeba that showed excellent activity against B. burgdorferi at the stationary phase, though the extracts or essential oils of these 4 plants were reported to possess antibacterial activeness on other leaner. Allspice is a commonly used flavoring agent in food processing and is known to have antibacterial activities on many organisms [26]. Myrrh as a traditional medicine has been used since ancient times. In mod times, myrrh is used as an antiseptic in topical and toothpaste [27]. Information technology has been shown that some sesquiterpene components of myrrh, including furanodien-six-one, methoxyfuranoguai-ix-en-eight-one possess in vitro bactericidal, and fungicidal activity confronting multiple pathogenic bacteria, including Eastward. coli, Due south. aureus, P. aeruginosa, and C. albicans [28], but these two most active compounds were non detected in our samples [xix] (Tabular array 3). Hydacheim essential oil is extracted from the flower of Hedychium spicatum plant which is commonly known as the ginger lily plant. The methanol extract of H. spicatum is reported to have antimicrobial activity against many leaner, including Shigella boydii, Eastward. coli, Due south. aureus, P. aeruginosa, and K. pneumoniae [29]. Litsea cubeba is also used in traditional Chinese medicine for a long time. Its essential oils from stalk, alabastrum, leaf, bloom, root, fruit parts are also reported to exhibit antibacterial activity on B. subtilis, E. coli, East. faecalis, S. aureus, P. aeruginosa, and Grand. albicans [xxx]. Based on these studies and application of allspice, myrrh, hydacheim, and Litsea cubeba, it would be of interest to develop effective regimens to fight against Lyme illness in the hereafter.
Table 3
The top 5 major compositions of the three about active essential oils.
| Essential Oil | Components | Content a |
|---|---|---|
| Garlic Allium sativum bulbs | Diallyl disulfide | 19% |
| (E)-1-Allyl-two-(prop-i-en-1-yl) disulfane | 15% | |
| Disulfide, methyl 2-propenyl | 6% | |
| ii-Vinyl-4H-1,3-dithiine | 6% | |
| (E)-methyl 1-propenyl sulfide | iv% | |
| Allspice Pimenta officinalis berries | Eugenol | 82% |
| β-Caryophyllene | half-dozen% | |
| Methyleugenol | 5% | |
| α-Humulene | 1% | |
| α-Selinene | 0.47% | |
| Myrrh Commiphora myrrha resin | Curzerene | 38% |
| Furanoeudesma-1,iii-diene | 24% | |
| β-Elemene | seven% | |
| Lindestrene | 7% | |
| E-Elemene | three% |
Although the active essential oils we identified have potent activity against stationary phase cells of B. burgdorferi in vitro, their activity in vivo is unknown at this time. In future studies, nosotros will use GC-Mass Spectrometry to identify the active ingredients of the active essential oils and confirm their activity against growing and non-growing B. burgdorferi. Once we place the active components of agile essential oils drug combination studies can be performed to enhance activeness against persister leaner. In addition, we will written report the mechanism of action of the active compounds in the near hereafter. The pharmacokinetic (PK) profile of the agile compounds in these agile essential oils will be assessed and their effective dosage and toxicity will exist determined in vivo. In our previous study, we institute that the cinnamon bark essential oil showed first-class action against stationary phase B. burgdorferi [eighteen], here we plant CA is an active component of cinnamon bawl essential oil. CA could eradicate the stationary phase B. burgdorferi at 0.05% concentration as no regrowth occurred in subculture (Table two). This indicates that CA possess similar activity against stationary phase B. burgdorferi as carvacrol, which is the one of the most agile compounds against non-growing B. burgdorferi nosotros identified from natural products [18]. Furthermore, CA is observed to be very active confronting growing B. burgdorferi cells with an MIC of 0.two µg/mL. The antibacterial action of CA was besides reported on some other bacteria. The mechanism of the antibacterial action of CA has been studied on unlike microorganisms, which suggests that its antibacterial activeness is mainly through interaction with the cell membrane [31]. CA as a common favoring agent in nutrient processing is also used equally food preservative to protect fauna feeds and human food from pathogenic leaner [31]. CA is considered as a safe compound for mammals, equally the median lethal dose LD50 of CA is 1850 ± 37 mg/kg past oral assistants in the acute toxicity study with oral administration rat model [32]. These findings suggest that CA could exist a skillful drug candidate for farther evaluation against B. burgdorferi in hereafter studies. We also want to point out that the safety of using essential oils and their components needs more thorough inquiry; for example, the intravenous toxicity of carvacrol is considerably college than oral toxicity [33]. Thus, appropriate creature studies are necessary to ostend the condom and activity of CA and other active essential oils in animal models earlier human studies.
This study used B. burgdorferi stationary phase cultures enriched in persisters equally a persister model for essential oil screens. The reason we used this model is that studies with tuberculosis persister drug pyrazinamide (PZA), which is more than active confronting stationary phase cells and persisters than against log phase growing cells and shortens the therapy [34,35], propose that drugs active against stationary phase cells/persisters will be more constructive at curing persistent infections than drugs active against growing cultures. This has been shown in the example of colistin as a persister drug for E. coli when being used together with quinolone or nitrofuran could more effectively eradicate urinary tract infection in mice [36]. Hereafter studies are needed to determine if the essential oils active confronting non-growing stationary phase B. burgdorferi cultures enriched in persisters are more effective in eradicating persistent B. burgdorferi infections in animal models than the current Lyme antibiotics which are mainly active confronting growing Borrelia.
5. Conclusions
In summary, nosotros identified some boosted essential oils that have stiff activity against stationary-phase cells of B. burgdorferi. The nearly active essential oils include garlic, allspice, myrrh, hydacheim, and Litsea cubeba. Among them, garlic oil could completely eradicate stationary stage B. burgdorferi with no regrowth at 0.05%, and the others could reach the aforementioned activity at 0.ane%. Additionally, cinnamaldehyde is identified to be an active ingredient of cinnamon bawl oil with very strong activity confronting B. burgdorferi stationary phase cells. Future studies will be carried out to identify the active components in the candidate essential oils, and to determine their in vitro activity alone or in combination with other active essential oils or antibiotics against B. burgdorferi sensu lato strains, including B. burgdorferi, B. garinii and B. afzelii, and assess their prophylactic and efficacy against B. burgdorferi in fauna models earlier human being trials.
Acknowledgments
We acknowledge the support by Global Lyme Brotherhood, LivLyme Foundation, NatCapLyme, and the Einstein-Sim Family Charitable Fund.
Writer Contributions
Conceptualization, Y.Z. and J.One thousand.; Methodology, J.F., W.S., K.M.T. and C.J.M.; Validation, J.F., W.S., G.Grand.T. and C.J.M.; Formal Analysis, J.F. and W.S.; Writing-Original Draft Preparation, J.F. and Y.Z.; Writing-Review & Editing, Y.Z., J.K.; Supervision, Y.Z.; Funding Acquisition, Y.Z.
Funding
This enquiry was funded in office by Global Lyme Brotherhood, LivLyme Foundation, NatCapLyme, and the Einstein-Sim Family Charitable Fund.
Conflicts of Interest
The authors declare no disharmonize of interest.
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