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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 .
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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.