Effective killing of Borrelia burgdorferi in vitro with novel herbal compounds

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds © he Au ho s, 2018. Published e sion Ka onen, Ka i; Gilbe , Leona Ka onen, K., & Gilbe , L. (2018). E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds. Gene al Medicine Open, 2(6), 1-4. h ps://doi.o g/10.15761/GMO.1000153 2018 Resea ch A icle Gene al Medicine Open ISSN: 2515-4737 Gen Med Open, 2018 doi: 10.15761/GMO.1000153 Volume 2(6): 1-4 E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds Ka i Ka onen* and Leona Gilbe Depa men o Biological and En i onmen al Science and Nanoscience Cen e , PO Box 35, FI-40014, Uni e si y o Jy askyla, Finland Abs ac In oduc ion: The ick-bo ne disease Lyme Bo eliosis is caused by Bo elia bac e ia. The disease can pe sis e en a e ea men wi h an ibio ics, which is why o he me hods o ea men a e needed. He bal compounds and phy ochemicals ha e been ecen ly examined in ela ion o e adica ing Bo elia bac e ia in i o. Objec i e: The possible an imic obial e ec o wo no el compounds, Biocidin Liquid and LSF B oad-Spec um Liposomal o mulas, was examined in he hopes o disco e ing an al e na i e me hod o e adica ion o Bo elia bac e ia. Me hods: The minimum inhibi o y concen a ions (MICs) and minimum bac e ial dea hs (MBDs), as well as, ime-kill e ec o each compound we e u ilized in he s udy. Resul s: The Liquid o mula e ec i ely killed he spi oche es wi h 1:10 dilu ion, while he MIC o he Liposomal o mula was 1:25. Mo eo e , he MIC o bo h compounds wi h Round Bodies was 1:50 and o bio ilms 1:10. Though long- e m e ec (MBD) was seen only wi h 1:5 dilu ions o bo h o mulas. Addi ionally, he killing e ec o each compound was obse ed al eady a 10 min pos - ea men . Conclusion: The s udy conduc ed he e p o ides new insigh in o he an imic obial e ec o he bal compounds. Fu he mo e, s udies such as hese a e equi ed in o de o disco e possible al e na i es o an ibio ics in he ba le agains Bo elia in ec ions. *Co espondence o: Ka i Ka onen, M.Sc, Depa men o Biological and En i onmen al Science and Nanoscience Cen e , PO Box 35, FI-40014, Uni e si y o Jy askyla, Finland, E-mail: [email p o ec ed] Key wo ds: Bo elia bu gdo e i, pleomo phic o m, an imic obial, pe sis e Recei ed: Decembe 18, 2018; Accep ed: Decembe 28, 2018; Published: Decembe 31, 2018 Abb e ia ions: B. bu gdo e i: Bo elia bu gdo e i; MIC: minimum inhibi o y concen a ion; MBD: minimum bac e ial dea h; RB: Round Body. In oduc ion The spi oche e bac e ia Bo elia bu gdo e i, ound in Ixodes icks, is he causa i e agen o Lyme Bo eliosis [1]. Un a o able condi ions, such as changes in pH, nu i ional deple ion, e en an ibio ics can lead he pleomo phic bac e ia o e e sibly al e hei o m in o Round Bodies (RBs) o bio ilms [2,3]. Fu he mo e, he immune sys em eac s di e en ly o he pleomo phic o ms wi h mac ophages diges ing and p ocessing RBs di e en ly han spi oche es [4]. Hence, examina ions o B. bu gdo e i need o include hese a ious o ms. Bo elia in ec ed pa ien s epo signs and symp oms anging om skin in lamma ion, a h i is and neu ological o ca diac impai men s [5]. T ea men wi h an ibio ics ea ly on he in ec ion can clea he pa hogen om he body, howe e , pos - ea men pe sis ence by he bac e ia as asymp oma ic o wi h a mul iplici y o symp oms can occu [5]. I has been sugges ed ha he di e en pleomo phic o ms o Bo elia a e in ol ed in he a oidance o he immune sys em, and hus, in he pe sis ence o he disease [6]. Cu en ea men o Lyme bo eliosis elies solely on an ibio ics [5]. Howe e , s udies on an ibio ic e iciency wi h di e en pleomo phic o ms o Bo elia ha e shown o demons a e a ying e ec i eness o an ibio ics in killing he bac e ia [6,7]. The e o e, new app oaches o be e ea men a e equi ed. Fo cen u ies he bal compounds ha e been used as emedies o a ious ailmen s. Recen ly, he an imic obial possibili ies o a a ie y o phy ochemicals and he bal ex ac s agains Bo elia ha e been s udied [8-10]. Hence, s udies on na u al composi es migh o e new possibili ies o emedies o Lyme Bo eliosis. He e, wo comme cially a ailable he bal compounds, Biocidin Liquid and LSF B oad-Spec um Liposomal o mulas, we e es ed o hei e iciency in elimina ing di e en pleomo phic o ms o B. bu gdo e i in i o. Ma e ials and me hods Bac e ial s ain, cul u ing condi ions and es compounds All expe imen s we e conduc ed wi h in ec ious, luo escen B. bu gdo e i s ain GCB726 wi h GFP, which was g aciously p o ided by Geo ges Chaconas, Uni e si y o Calga y, Canada [11]. Ba bou - S oenne -Kelly medium (BSK II) [12], wi hou gela in and supplemen ed wi h 6% hea inac i a ed abbi se um (Sigma-Ald ich, S . Louis, USA) was used in he cul u ing o cells a +37 ºC. Low-passage numbe cells (≤ passage 8) we e used in all o he expe imen s. The es ed compounds, Biocidin® Liquid o mula and Biocidin® LSF B oad-Spec um Liposomal o mula, we e acqui ed om Bio-Bo anical Resea ch Inc. (CA, USA). As nega i e con ols o g ow h 100 µg/ml o doxycycline (Hexal®, Ge many) and 0.02 % H2O2 (Sigma-Ald ich) we e used. Un ea ed cells we e a posi i e con ol o g ow h. Ka onen K (2018) E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds Volume 2(6): 2-4 Gen Med Open, 2018 doi: 10.15761/GMO.1000153 Minimum inhibi o y concen a ion and minimum bac e ial dea h expe imen s The minimum inhibi o y concen a ions (MICs) o Biocidin® Liquid and Liposomal o mulas we e de e mined by incuba ing 15 x 106 B. bu gdo e i spi oche es in 3 ml o cul u e media wi h di e en dilu ions (1:5, 1:10, 1:25 and 1:50) o he compounds o 96 h [13]. The samples we e measu ed o luo escence wi h a spec opho ome e (Pe kin Elme , 2030 Mul ilabel eade Vic o ™ x4) e e y 24 h. RBs we e induced as p e iously desc ibed [13]. B ie ly, 15 x 106 B. bu gdo e i spi oche es we e incuba ed in s e ile dis illed H2O a +37 °C o 2 h be o e placing he cells in o 3 ml o esh media and adding he same dilu ions o each Biocidin® compound as wi h he spi oche e samples. Fluo escence measu emen s we e done in he abo e-men ioned manne . The MICs o bo h compounds wi h bio ilms we e de e mined by coun ing a 10 µl a ea in a C-Chip DHC-N01 Disposable Haemocy ome e (Sys em Neubaue Imp o ed, Digi al Bio) a 0 h and 72 h pos - ea men . App oxima ely 9000 bio ilms in 3 ml o esh media we e ea ed he p e iously men ioned dilu ions o Biocidin® compounds and incuba ed a +37 °C o 72 h a e which he samples we e coun ed. Cell clus e s o o e 10 cells we e ega ded as bio ilms. The minimum bac e ial dea h (MBD) we e de e mined as has been done p e iously wi h al e a ions [13]. Samples o 300 μl om he MIC expe imen s om each pleomo phic o m om he 72-h ime poin , espec i ely, we e eseeded in o 3 ml o esh media and incuba ed o 3 weeks a +37 °C. A weeks 1 and 3 he change in pH (colo change) o he media was used o indica e g ow h (yes/no) and mic oscopy (Leica CTR5500, Ge many) was u ilized a week 3 o con i m he obse a ions. Time-kill expe imen s The a e o cell dea h o B. bu gdo e i was analyzed by adding he MIC dilu ions o he compounds (Liquid o mula = 1:10; Liposomal o mula = 1:25) o 6 x 106 spi oche es in 3 ml o cul u e media and coun ing he cells using a C-Chip haemocy ome e a 10, 20, 30, 60- and 120-min pos - ea men . The means o h ee sepa a e expe imen s we e coun ed and Excel was used in p oducing he g aph. Resul s MIC and MBD expe imen s The MICs o Biocidin® Liquid and Liposomal o mulas wi h spi oche es, RBs and bio ilms o B. bu gdo e i we e de e mined (Figu e 1). The MICs o bo h compounds wi h each B. bu gdo e i o m in Figu e 1 a e highligh ed wi h a black s a . Fi s , he MIC o he Liquid o mula wi h spi oche es demons a ed 1:10 dilu ion (Figu e 1A), while 1:25 dilu ion was enough wi h he Liposomal o mula (Figu e 1B), as hese we e he lowes dilu ions a which no g ow h was obse ed. Second, he MICs o bo h o mulas wi h RBs was de e mined o be he 1:50 dilu ion (Figu e 1C and 1D), as he ela i e luo escence alues ollowed hose o he nega i e con ols. Las , he MICs o bo h o mulas wi h bio ilms we e deduced by coun ing he bio ilms in 10 μl o each sample. Bo h compounds demons a ed an MIC o 1:10 dilu ion when cul u ed wi h bio ilms (Figu e 1E). A 72 h ime poin o he MIC expe imen s, samples we e pu o esh media and incuba ed o h ee weeks wi h he pu pose o de e mining he MBDs o bo h compound wi h each B. bu gdo e i pleomo phic o m. Below able exempli ies he esul s om wo sepa a e expe imen s o each B. bu gdo e i o m (Table 1). Fi s ly, he Liquid o mula demons a ed no g ow h wi h he 1:5 dilu ion du ing he whole h ee- week ime pe iod in all o he bac e ial o ms. On he hi d week g ow h was obse ed in each pleomo phic o m wi h he 1:10, 1:25 and 1:50 dilu ions excep he 1:10 dilu ion in RBs. Mo eo e , bo h spi oche e and bio ilm samples had g ow h wi h he 1:25 and 1:50 dilu ions al eady a week one, while he RB samples showed none. Hence, he sho - e m dilu ions o he Liquid o mula o each pleomo phic o m co ela ed hose o he MIC esul s. Howe e , only he 1:5 dilu ion esul ed in no g ow h long- e m wi h all pleomo phic o ms. Secondly, he esul s o he Liposomal o mula ollowed hose o he Liquid o mula, al hough mos o he spi oche e samples ha e been examined only once and he hi d week o 1:10 dilu ion wi h RBs is comple ely missing because o inapplicable da a. Howe e , simila ly o he Liquid o mula, he Liposomal o mula demons a ed no g ow h in he 1:5 dilu ion wi h each o he pleomo phic o m. Fu he mo e, bo h spi oche es and bio ilms indica ed g ow h a week one al eady in he 1:25 and 1:50 dilu ions, while RBs exp essed no g ow h a week one in any o he dilu ions. The i s week MBD esul s o he Liposomal o mula wi h RBs and bio ilms co esponded o he MIC esul s. Howe e , when cul u ed wi h spi oche es he 1:25 dilu ion al eady indica ed g ow h a week one. Thus, con adic ing he MIC o he Liposomal o mula de e mined ea lie . Time-kill expe imen s The e ec i eness o he Biocidin® compounds on B. bu gdo e i spi oche es was de e mined in ime-kill expe imen s (Figu e 2). B. bu gdo e i spi oche es we e ea ed wi h he MICs o he compounds: 1:10 dilu ion o he Liquid o mula; 1:25 o he Liposomal o mula, and he cells we e coun ed a 10, 20, 30, 60- and 120-min pos - ea men . Bo h compounds induced cell dea h o B. bu gdo e i, wi h he Liquid o mula being sligh ly mo e e ec i e. Discussion Lyme bo eliosis is cu en ly he mos common ick-bo ne disease in he No he n hemisphe e (S anek e al. 2012) [14]. An ibio ics a e usually e ec i e agains Bo elia sp., howe e , pe sis ing o ms ha e been unco e ed capable o esis ing ce ain an ibio ics [6,15]. The e o e, in es iga ing no el possibili ies o elimina ing he pe sis ing bac e ia om he body is c ucial. He e wo die a y supplemen compounds om Bio-Bo anical Resea ch Inc. we e examined o hei abili y o e adica e he di e en pleomo phic o ms o B. bu gdo e i in i o. In ecen yea s, he e ec s o se e al phy ochemicals on Bo elia ha e been s udied and ound o be e ec i e agains he di e en pleomo phic o ms o he bac e ia [8-10,16-18]. Fo ins ance, S e ia whole lea ex ac has been ound o educe he size and numbe o Bo elia bio ilms [10]. Addi ionally, g ape ui seed ex ac has been demons a ed o wo k agains Bo elia spi oche es and RBs [16]. Recen ly hough, o egano oil was ound o be highly ac i e agains pe sis en (7day g ow h) o ms o Bo elia e en a low concen a ions [17]. Simila ly, o p e ious s udies, bo h o he compounds used he e diminished he g ow h o all B. bu gdo e i o ms as was demons a ed by he MIC and MBD expe imen s. The ac i e ing edien s in bo h compounds a y om be y ex ac s o essen ial oils om plan s. In e es ingly, bo h o he compounds used he e also ha e o egano oil, as well as, g ape ui seed ex ac as ac i e agen s in hem. Addi ionally, he numbe o spi oche es we e d as ically educed a e only 2 h pos - ea men wi h he Biocidin® compounds. This sugges s ha hese compounds could o e a possible ea men o Lyme bo eliosis, i no solely on hei own bu possibly when used Ka onen K (2018) E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds Volume 2(6): 3-4 Gen Med Open, 2018 doi: 10.15761/GMO.1000153 Spi oche es Round Bodies Bio ilms Week 1 Week 3 Week 1 Week 3 Week 1 Week 3 Posi i e con ol Yes Yes Yes Yes Yes Yes Doxycycline No No No No No No H2O2No No No No No No Liquid o mula 1:5 No No No No No No 1:10 No Yes No No No Yes 1:25 Yes Yes No Yes Yes Yes 1:50 Yes Yes No Yes Yes Yes Liposomal o mula 1:5 No* No* No No No No 1:10 No Yes* No --- No Yes 1:25 Yes* Yes* No Yes Yes Yes 1:50 Yes* Yes* No Yes Yes Yes Table 1. Minimum bac e ial dea h o B. bu gdo e i spi oche es, Round Bodies and bio ilms ea ed wi h Biocidin® compounds Dilu ions o 1:5, 1:10, 1:25 and 1:50 we e used and g ow h was obse ed (yes/no) o h ee weeks. Cells wi hou a ea men we e a posi i e con ol o g ow h, while 100 μg/ml o doxycycline and 0.02% H2O2 we e used as nega i e con ol o g ow h. Resul s a e mainly om wo sepa a e assays. Dashed lines indica e incomple e da a, while he as e isk indica es only 1 epea *only 1 epea --- da a incomple e Figu e 1. Minimum inhibi o y concen a ions o Biocidin® compounds wi h B. bu gdo e i a a ious ime poin s 15 x 106 spi oche es (A and B) o Round Bodies (C and D) in 3 ml o cul u e media we e mixed wi h ei he Biocidin® Liquid (A and C) o Liposomal o mula (B and D) in a ious dilu ions (1:5, 1:10, 1:25 and 1:50) o 96 h. Panel E illus a es 9000 B. bu gdo e i bio ilms in 3 ml o cul u e media wi h he same dilu ions as abo e o Liquid o Liposomal o mula o 72 h. Cells wi hou a ea men we e a posi i e con ol o g ow h, while 100 μg/ml o doxycycline and 0.02 % H2O2 we e used as nega i e con ol o g ow h. Values o spi oche es and Round Bodies a e means om wo sepa a e expe imen s. The alues o bio ilms wi h Liquid o mula a e om jus one expe imen , while hose o he Liposomal o mula a e he means o h ee expe imen s. The black s a s indica e he minimum inhibi o y concen a ions (MICs) o each compound o he di e en pleomo phic o ms. Ka onen K (2018) E ec i e killing o Bo elia bu gdo e i in i o wi h no el he bal compounds Volume 2(6): 4-4 Gen Med Open, 2018 doi: 10.15761/GMO.1000153 syne gis ically wi h an ibio ics as has been done be o e wi h o he phy ochemicals [19]. Na u ally, s udies in ol ing an ibio ics, as well as, RBs and bio ilms should be conduc ed in o de o ealize he ull po en ial o hese compounds. S ill, he p omising esul s gained he e o hese wo compounds indica e new possibili ies o comba B. bu gdo e i in ec ions. Au ho ship and Con ibu o ship All au ho s mee he c i e ia o au ho ship. Acknowledgemen s We’d like o acknowledge he Schwa z ounda ion o hei con inuous suppo o ou esea ch endea o s. Funding This wo k was suppo ed by Bio-Bo anical esea ch Inc. Howe e , he au ho s de ised he s udy design, collec ed and analyzed he da a, and p epa ed he manusc ip wi hou inpu om he company. The decision o publish was he au ho s. Con lic s o in e es The au ho s ha e no con lic s o in e es . Re e ences 1. Bu gdo e W, Ba bou AG, Hayes SF, Benach JL, G unwald E (1982) Lyme disease-a ick-bo ne spi oche osis? Science 216: 1317-1319. [C oss e ] 2. Me iläinen L, He anen A, Schwa zbach A, Gilbe L (2015) Mo phological and biochemical ea u es o Bo elia bu gdo e i pleomo phic o ms. Mic obiology 161: 516-527. [C oss e ] 3. Mu gia R, Cinco M (2004) Induc ion o cys ic o ms by di e en s ess condi ions in Bo elia bu gdo e i. APMIS 112: 57-62. [C oss e ] 4. Me iläinen L, B ande H, He anen A, Schwa zbach A, Gilbe L (2016) Pleomo phic o ms o Bo elia bu gdo e i induce dis inc immune esponses. Mic ob.In ec [C oss e ] 5. S anek G, Wo mse GP, G ay J, S le F (2012) Lyme bo eliosis. The Lance 379: 461- 473. 6. Sha ma B, B own AV, Ma luck NE, Hu LT, Lewis K (2015) Bo elia bu gdo e i, he Causa i e Agen o Lyme Disease, Fo ms D ug-Tole an Pe sis e Cells. An imic ob. Agen s Chemo he 59: 4616-4624. 7. Sapi E, Kau N, Anyanwu S, Luecke D, Da a A e al. (2011) E alua ion o in- i o an ibio ic suscep ibili y o di e en mo phological o ms o Bo elia bu gdo e i. In ec D ug Resis 4: 97-113 [C oss e ] 8. Feng J, Wei ne M, Shi W, Zhang S, Sulli an D, e al. (2015) Iden i ica ion o addi ional an i-pe sis e ac i i y agains Bo elia bu gdo e i om an FDA d ug lib a y. An ibio ics 4: 397-410. [C oss e ] 9. Goc A, Niedzwiecki A, Ra h M (2015) In i o e alua ion o an ibac e ial ac i i y o phy ochemicals and mic onu ien s agains Bo elia bu gdo e i and Bo elia ga inii. J.Appl.Mic obiol 119: 1561-1572. [C oss e ] 10. Theophilus PAS, Vic o ia MJ, Soca as K, Filush KR, Gup a K, e al. (2015) E ec i eness o S e ia ebaudiana whole lea ex ac agains he a ious mo phological o ms o Bo elia bu gdo e i in i o. Eu opean Jou nal o Mic obiology and Immunology 5: 268-280. [C oss e ] 11. Mo ia y TJ, No man MU, Cola usso P, Bankhead T, Kubes P, e al. (2008) Real- ime high esolu ion 3D imaging o he lyme disease spi oche e adhe ing o and escaping om he ascula u e o a li ing hos . PLoS Pa hog 4: e1000090. [C oss e ] 12. Ba bou AG (1984) Isola ion and cul i a ion o Lyme disease spi oche es. Yale J.Biol. Med 57: 521-525. [C oss e ] 13. De e LL, Jo gensen JH, Ba bou AG (1992) In i o an imic obial suscep ibili y es ing o Bo elia bu gdo e i: a mic odilu ion MIC me hod and ime-kill s udies. J Clin Mic obiol 30: 2692-2697. 14. Hubalek Z (2009) Epidemiology o lyme bo eliosis. Cu P obl De ma ol 37: 31-50. [C oss e ] 15. Sapi E, Bas ian SL, Mpoy CM, Sco S, Ra elle A, e al. (2012) Cha ac e iza ion o bio ilm o ma ion by Bo elia bu gdo e i in i o. PloS one 7: e48277. [C oss e ] 16. B o son Ø, B o son S (2007) G ape ui seed ex ac is a powe ul in i o agen agains mo ile and cys ic o ms o Bo elia bu gdo e i sensu la o. In ec ion 35: 206- 208. [C oss e ] 17. Feng J, Zhang S, Shi W, Zubce ik N, Miklossy J, e al. (2017) Selec i e essen ial Oils om spice o culina y he bs ha e high ac i i y agains s a iona y Phase and Bio ilm Bo elia bu gdo e i. F on ie s in medicine 4: 169. [C oss e ] 18. Goc A, Niedzwiecki A, Ra h M (2017) Recip ocal coope a ion o phy ochemicals and mic onu ien s agains ypical and a ypical o ms o Bo elia spp. J.Appl.Mic obiol. [C oss e ] 19. Goc A, Niedzwiecki A, Ra h M (2016) Coope a ion o Doxycycline wi h Phy ochemicals and Mic onu ien s Agains Ac i e and Pe sis en Fo ms o Bo elia sp. In e na ional Jou nal o Biological Sciences 12: 1093. [C oss e ] Figu e 2. Ra e o cell dea h a e Biocidin® ea men s 6 x 106 Bo elia bu gdo e i spi oche es in 3 ml o media we e ea ed wi h 1:10 Biocidin® Liquid o mula o 1:25 Biocidin® Liposomal o mula o check he a e o cell dea h. Cells we e coun ed a 10, 20, 30, 60 and 120 min pos - ea men . Rep esen ed a e he mean alues o h ee sepa a e expe imen s. Bo h compounds d as ically educed B. bu gdo e i spi oche e coun a e 2 h pos - ea men Copy igh : ©2018 Ka onen K. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.