Journal of Experimental Biology and Agricultural Sciences, February - 2016, Volume - 4 ( 1 )

1 " Journal oi bxpennienlal Biology and Agricultural Sciences

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INHIBITION OF QUORUM SENSING IN Chromokacterium violaceum CV026 B Y VIOLACEIN PRODUCED B Y Pseudomonas aeruginosa

Any r itnani . L W I r u i n /\yunmgivas ana H U S I I M U

- ^ r r * * ? - . ' a n u a r y 02.2<J16;Rev!SKm-Jamiary 27.2016. Accepted - February 20,2016 .'waiiacup i mirar — reoruary z u . zuro

K E Y W B S

A a f c - Q S

Pseudomonas aeruginosa

Extract

Chromobaciervtoiaceum C V 0 2 6

V i o l a o e w

M P F H t d C T

Q u o r u m sensing ( Q S ) regulates various act iv it ies o f bacteria such as b io f i lm forming, v irulence b»e*nfy s w a r m i n g , and pigment production. B a c t e r i u m Chromobacterhtm violaceum produced vio lacent has also been regulated by quorum sensing system. T h e a i m o f this study w a s to evaluate the Q S inhibit ion act iv i ty buvioiaceum produced by the extract o f Pseudomonas aeruginosa isolated from root o f Velnvna zizammdes P. aeruginosa cultured a n K i n g ' s B agar, then was extracted us ing ethyl acetate a s solvent. T h e extract w a s used to test a n t i - Q S properties on C. violaceum C V 0 2 6 at different concentration v i z 0.0 m g / m L (control ) , 2.5 m g / m L , 3.0 m g / m L and 3.5 m g / m L . V i o l a c e i n content o f culture w a s measured by a spectrophotometer at a wavelength o f 585 n m T h e extract A 2 .5 , 3.0. and 3.5 m g / m L concentration had a significant effect on die reduction o f v io lace in production by 3 1 . 6 % , 3 5 8 % a n d 7 0 3 % , respectively. W h i l e us ing a n extract at die same concentration l eve l there w a s no negative effect on the number o f C. violaceum C V 0 2 6 ce l ls found. T h e results o f study suggest d ia l among the various tested concentrations. 3.5 m g / m L extract inhibits Q S in C. violaceum C V Q 2 6 v i a v i o lace in production. T h u s , the extract o f f aeruginosa has a very high potential to develop a n t i - Q S .

' v c,pondasg a s & o r

E-mail : aoy&riaa@upi edu (Any Fitnam)

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1 I n t r o d u c t i o n

Q u o r u m sensing ( Q S ) i s a ce l l to ce l l communicat ion mechanism that a l lows bacteria to control gone expression in order to respond to the ce l l densitv C e l l densitv c a n stimulate synthesis o f s m a l l molecules (autoinduccrs) a n d Q S regulates m a m activities such as bioiummescence. btof i lm formation, v irulence factor, and s w a r m i n g (Defiant et a L . 2 0 1 3 ) A n t i - Q S is a phenomenon o f ce l l - ce l l communicat ion related to molecules as intermediate Molecules as a n a n t i - Q S agent cou ld rcolace an inducer molecule w h i c h is responsible for the induction o f protein expression to produce a vdureot agent. These a n t i - Q S molecules c a n be used as a medicine.

C violaceum is Gram-neaat ive bacter ia found m nature and A l t causes inlectioiis in human a n d animals . T h i s bacterium c a n cause septicemia and abscess in lungs and in the l iver (Petr i l o et a l . 1984V Q S i n C . violaceum retaliates v io lace in oiement production, antibiotic, hvdroeen cvanide and some enzymes. C. violaceum has many genes invo lved i n v io lace in production such as v i o l ) . v i o C , v i o A genes w h i c h i s arranged i n a n operon svstem mediated bv N - A c v l Homosenne Lactone ( A H L ) (August et al . 2000V O S olavs an important role m determining v irulence factors and it has lead scientists to find a new target in developing treatment for disease caused by bacterial m f r r t k u n ( V a s a n et a L , 2013V A c t m o b a d e n a . F v n u c u t e s . Cvanobactcna . Bacteroidetes and Protcobactena produce a n t i - Q S enzymes ( K a l i a . 2 0 1 3 ) . Noucoenaie A H L s as intermadiates o f A H L biosyntbetic pathway, a d i cyc l i c peptides, produced by bacteria as a n b - Q S or Q u o r u m Ouenchine ( O O ) molecules ( B a u e r & Robinson. 2 0 0 2 ) .

Medic ina l plants have some endophytic bacteria w h i c h produces secondary metabolites w h i c h w o r k as antimicrobial a v e r t s (Strobe! . 2003V Nowadays , endoohvtcs are a source for nove l natural products i n modern medicine. industry a n d agriculture ( Y u et a l . , 2010) . Most novel natural products posscsing antraucrobiai act iv it ies have bees isolated from endophvtes.

Endophyt ic microbes have been isolated from various plant tissues such as roots, leaves a n d stems F i t n a n i et at. ( 2 0 1 3 ) have isolated and characterized 17 isolates from the root o f Vettveria zizamoides a n L u r i a B e t t a n i agar media. A m o n g these 5 isolates have been identified w i t h polyketide synthase gene by polymerase c h a m reaction ana lys i s . B a s e d o n I6S rRNA analvs is . one o f them is Pseudomonas aeruginosa ( F i t n a w et a L . 2013V ? aeruginosa is G r a m negative, aerobic and rod shaped bacteria belonging to the fami ly Pseudomonadaceae. Further , A l l u et a L ( 2 0 1 4 ) isolated and characterized endophytic / ' aeruginosa from red that pepper. Ant imi c rob ia l properties o f the isolated hacterial strain were characterized against fungal pathogen Colletotrichum. B a s e d o n the study, b ioc idal properties o f the P. aeruginosa were established and it w a s also suggested that isolated bacterium could erow in an art i f ic ial medium. T h e a i m o f this study w a s to evaluate the O S inhibit ion activit ies in term o f v io laceum

production inhibit ion by the extract o f P. aeruginosa isolated from root o f V. zaanundes

2 M a t e r i a l s a n d \f eth<»d*-

2 1 Preparation o f bacterial culture

C violaceum C V Q 2 6 w a s obtained from R e s e a r c h Centre Microb ia l D ivers i ty . Bogor Agr i cu l tura l Univers i ty . Indonesia, w h i l e the P. aeruginosa w a s isolated from the root o f V. zizanioides. Isolated P. aeruginosa w a s maintained cm the K i n g s B agar (Raptan 2 . 0 % ; K H j P O . 0 . 1 5 % : M g S O . 0 . 1 5 % glycerol ( 8 5 % ) 1 .5% ( v / v ) and 2 . 0 % Bac to agar ( D i f c o . Spark . U S A ) at 37°C. T h e culture was rejuvenated alter every t w o weeks W h i l e £ violaceum C V 0 2 6 w a s cultured i n L i r a Ber tau i ( L B ) agar a n d menbated at 2 7 * C Cu l ture w a s rejuvenated for the interval o f every 4 days. Before the treatment, r u b u r r w a s inoculated to L B broth a n d incubated at 2 7 " C fix 18-24 h in water bath a i d shaker

2.2 E x t r a c t i o n o f Endophyt ic P. aeruginosa

E x t r a c t i o n o f P. aeruginosa w a s carried out bv the method described by N i y a z A h a m e d ( 2 0 1 2 ) w i t h some modif ication B r i e f l y . P. aeruginosa w e r e cultured i n 1 0 m L of K i n g ' s B a n a (Peptoo 2 0 % . K H a P Q . 0 . 1 5 % M a S O , 0 1 5 % ) : g lycerol ( 8 5 0 % ) 1 5 % ( v / v ) ; B a c t o agar 2 0 % ( D i f c o . S o a k . U S A ) broth w i t h 3 7 " C i n temperature a n d 120 m i ' d m a o f s h a k i n g for 24 hours. T h e overnight culture w a s then inoculated into 9 0 m L o f the same m r r h u m a n d condition. After their stationary phase, cultures were moved into centrifuge tube and ccntnfuged at 10000 rev / ram fix 10 minutes. Supernal-uits were then moved into separating f lask and added by ethyl acetate ( M e r c k , N e w Y o r k . U S A ) ( 1 : 1 v / v ) . Separating tube w a s hand-shaken constantly for about 15 minutes and left for 20 m i n u t e T h e n the "pp**r ^niAm^ foyer that formed i n the contained organic matter w a s taken. E x t r a c t was concentrated by v a c u u m evaporator w i t h 5 0 " C i n temperature

2.3 A n t i - Q S assay against C . violaceum C V 0 2 6

A n a l y s i s o f the a n t i - Q S assay o f P. aeruginosa extract w a s conducted as reported bv K n s h n a n et a l . ( 2 0 1 2 ) w o n modif ication. One colony o f C. violaceum C V 0 2 6 w a s inoculated in a 50 m L L B broth medium and incubated f a r 1 8 -2 4 b at 2TC w i t h 110 r e v / m m S t a t i o n s F o u r m L o f culture ( O D « , = l 2 ) m i x e d w i t h 2 0 m L L B (10 .0 a / L T n r t o n e . 5.0 a/L Y e a s t E x t r a c t . 5.0 g /L N a d ) ( D i f c o . S p a r k . U S A ) molted agar. N-hexanoyl -L-homoser ine- lactone ( C 6 - H S L ) ( S i g m a , S t L o u i s , U S A ) d isso lved i n D M S O 1 0 0 % , w a s also added to agar w i t h 1.2 pgftnL f inal concentration. T h e agar was m i x e d and poured into a Pe t r i d i sh a n d w e l l s were made tn the center o f the sol idi f ied agar plate. Three replicates were then made. A 4 0 p L o f P aeruginosa extract at 0 0, 2 3 , 3.0, 3.5 m g / m L concentration w a s put mto the w e l l , respectively T h e plates were kept • the uwubatcu Tor 18-24 h at B "(.' and checked for inhibition o f the v io lace in

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edssssm stasias in C/vomoboctenum Wrthraiw CVU26 bv V w i h e f wodnced bv Purado 105

T a b i c 1 Cul ture composit ion for each flask related v io lace in quantif ication

c r n m p m m o T o t a l \ a h u m

C V 0 2 6 ( O D M O O . I ) 1.1857 m L 1 x 10* C T T J / m l . n o t . « i m i . ' mi . i A . " * p i . O . i x f i t J a n .

D M S O 1 % 140 p L 0.07 % ( v / v )

Contro l - L B M e d i u m 1998 m L 2 n d . C V 0 2 6 ( O D w o O . l ) 1.860 m L 1 x 1 0 8 C F U / m L

D M S O 1 % 140 p L 0 . 0 7 % ( v / v j

L B x ! < ? ! — 2 ™ L C V 0 2 6 ( O D u o O . l ) 1.857 m L 1 x 1 0 * C F U / m L

• S S L ( I 2.4 j»L V . l l f l l — 1111

E x t r a c t 1 140 p L 0.0; 2.5; 3.0; 3.5 m g / m L

2 .4 V i o l a c e i n Quanti f i cat ion

apectrophoTometry as described by C h o o et a l . ( 2005 ) . T a b l e 1

from each flask w a s centrifuged at 13000 rev /nun for 5 m m T h e —-prr~"*—* - . v ~ • ' • r d f d . . . u . i - ~>»-« v /eshed 2 trmes usmg buffer phosphat O n e m L D M S O w a s m i x e d w i t h ihc pure* - j " * vu i t exeu vigorously fix 3 0 s u o u ! the v io lace in disso lved completely. T h e cultures were then centrifuged at 13000 rev /mm for 10 mm. T h e abscrbancc o f supernatant w a s r e a d using the spectrophotometer (585 ran) to measure o f v tolacein concentration

2 3 A n a l y s i s o f C violaceum C V 0 2 6 C e l l V i a H t y

plate Plates were incubated in 27°C for 2 4 hours. Zone o f inhibit ion around the disc was then observed (Sas idharan et a l . .

Reduct ion i n v io lace in productions i n C . violaceum C V 0 2 6 are concentration dependent and reduced w i t h the increasing P aeruginosa extract concentration. Highest concentration o f P aeruginosa extract ( 3 .5mg /ml ) resulting in lower production o f v io lace in as s h o w n i n F i g u r e 1 . P. aeruginosa extract cou ld have influenced the mechanism o f O S m C violaceum Through decreasing v io lace in production. T h e extract cou ld have also interfered autoinducer production or mterferred w i t h the expression o f the gene.

T h e v iab i l i t y o f C . violaceum C V G 2 6 cel l w a s assessed by a total plate count ( T P C ) One hundred m L o f C violacem C V 0 2 6 culture w i f e P. aeruginosa extract w a s centrifuged at 13000 rev /mm for 10 n a n to remove remaining extract i s the culture. Later on the pellets were washed 2 times i n 100 m L P 0 4 2 and excess o f P. aeruginosa extract w a s discarded. T h e culture w a s added to 100 m L Mid le r H m t o n B r o t h ( M H B ) O n e m L o f di luted culture to factors o f 10 1 — 10"* and one m L o f culture from factors 10"6 — 10"* pour into M u l k r H m t o n A g a r ( M H A ) . T h e plates were incubated at 27°C for 18-24 h T h e total o f v iab le bacteria w a s then counted ( C h o o et a l . , 2 0 0 5 ) .

2 £ Ant ibacter ia l A s s a y

A s antsmcroUMU assav w a s anarvzea against C. viomcein ~ - ~- ..•i.»<^--..r.-. ...» jjffUJJ WWA--*! ! - f > - —1

culture w a s ser ia l ly di luted to factors o f 10 ' — 10"8 raid pourn into a n M H A m r d i n m T h e mixture w a s then left to be sohdffied and incubated at 27~C tor 18-24 hours T h e touu , iihh- bacteria! ce l l was i T y " counted. T o ensure * h i " !os* a paper disc dif fusion assay w a s also taken. 100 p L overnight culture o f C. violaceum C V 0 2 6 ( O D « » = 0 . 1 ) w a s spread on M H A Paper discs containing extracts w i th current concentrations (0 .0 , 2.5, 3.0, 3.5 m g / m L ) were loaded or to the

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C w H I < «urnl rVm«ml ' mj ml * Vmil ml I BMW) | DMto t r e a t m e n t

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r i a » c i Decreas ing v io iaceui production by P. aeruginosa

extract

T a b l e 2 shows the result o f quantif ication o f v io lace in production in the presence o f P. aeruginosa extract. V i o l a c e i n Dfouucoon b y cultures i n foe presence o i P. aeruginosa extract w a s s ^ n i S c s s f l v different flvrr. S i c cuiVcre •••-S*-*"-aeruginosa extract (control ) . Highest inhibit ion i n the v i o lace in production w a s reported at the highest concentration ( 3 3 m g / m L ) .

Jenmal o f Experimental Biology and Agricultural Sciences hap ."www jebas erg

106 i c t a l

T a M e 2 Decreas ing Tota l V io lace in production by Pseudomonas aeruginosa 1 x i r a c i using Spectrophotometer

3.n m g T i i t 3.5 my m l E + C V H : 6 - H S L 0 9 9 5 4 0 .17* 0 4 * 8 * 0 l c 0 132 4 0 0 1 9 " Control ( C V + C 6 - H S L ) 1.23 1 0 08* Control ( C V - C 6 - H S L ) 0 0285 i 0 0 0 2 e

Statist ical analys is us ing M a n n - W h i t n e y U test; Dif ferent superscripts shows a significant difference ( P < 0 0 5 ) , F E s t i a c t , C V ( hromobactermm violaceum: H S L : Homo Serme Lactone

Accord ing to K a l i a & Pirohit ( 2 0 1 1 ) v io lace in production TiTipht have reduced because o f the act iv i ty o f gene i n n i h a m g Q S signal w h i c h can either iranhit or rcauce v iotacein producnon. T h e y aiso reported that structure o f the s ignal w a s disrupted and the receptor sites w i t h antagonist signal analogues were blocked. Hcut icr ct a l . ( 2 0 0 6 ) suggested that P. aeruginosa has fv-t3-oxo-dodecrax>vi)-honioserine lactone ( 3 -oxo-C l i - H S i . i a n d h-buianoyi -bomosenne lactone ( C 4 - H S L ) . T h e y regulate the expression o f a lot o f genes in accordance w i t h the induction o f the transcription o f i o s l a n d rhli, a s autoinducers

( a m i K m r l i DMSO :<ncml i DMSO-HSU - HSI I

T r e a t B K M

Figure 2 V i a b l e ce l l o f C . violaceum C V 0 2 6 i n the presence o f P. aeruginosa extract

T a W c 3 shows total population o f C violaceum ( log C F U / m L ) In culture w i t h extracts and coctrois. T o t a l C . violaceiax

C V 0 2 6 population d id not show anv signif icant difference — med ia w i th addition 0.0, 2 3 , 3.0 a n d 3 3 ™g'"* : B M I f t i c ' i i c s n K r i rcveares turn P. aeruvmoso exffact in metes !flW iti fha-nre the vtatue ecus and mat me extract o u mx ca=cr - i r -death.

T h e comparison o f total bacteria also c a n be observed from figure 2. T h e decreasing v io lace in w a s nor cbhowed by decreasing o f total v iable bacteria. M e a n w h i l e , antibacterial assay w h i c h carried by disc d i f fus ion iiiTlhp'd. showed a s i m i l a r result that P. aeruemosa had no inhibitory act iv i ty aea ins l t vioiaceum C V o u h . T h e resuh o f these assays m a c a w s that extract o f P aentgmnsa has potency as anft -QS m ( '

G C - M S had h a m i mtm i a r r f r evea f cd that the extracts n f P I K I W I I W K I h — B M H »»*LQS ctxmpamds Accocding la P r a t i w i C O O ) P. aengjnosa c x t r - c t contains 3-( 1 - pnenvt - 2.3dii ivdro - 1 H - isomdoi - 2 - y i ) p r o p a n - l - o i a n d lH4soiBdok-1 .3(2Fr ) -dithione B o t h compounds are indole derivat ives and s imi lar type o f indole der ivat ives had been isolated b o m Escherichia coli by L i & Y o u n g (2013) a n d established a n t i - Q S act ivity against C. vioiaceum Staoilarly. Romano et a l ( 2014 ) reported the mechanism of this compound i n inhibit ing quorum sensing by inhibit ing the act ivity o f vioA gene w h i c h is one o f m a n y genes contain*^ -a vioABCD operoo that is v e r y cruc ia l for v io taccm p r o d u c t n a i n C violaceum. S i m i l a r research showed that endbptty. fiingus p~ bohsc-* f o u r t h ? «••?«« - « ^ .

(Sylibum mananum) produces po lyhydroxy anthraqwnoncs as quorum sensing inhibitor (F igueroa et a L , 2014).

T a b l e 3 Ant imicrob ia l A s s a y Aga inst C. vioiaceum C V 0 2 6

l o r a l v iab le h a c t e r i a f l no ( F l m l i > H i 2.5 in j n i l 3.0 m u n i l 3.5 ms n i l

E 4 C V 0 2 6 4 C 6 - H S L 8.86 4 0 16* 8 8 1 4 0 4 1 * 8.65 4 0 3 2 *

Control ( C 6 - H S L ) 9 18 1 0 028* Control ( - C 6 - H S L ) 9 1 1 i 0 073*

Statist ical analys is by D u n c a n test; Dif ferent superscripts shows a signif icant difference ( P < 0 .0S ) ; E - E x t r a c t ; C V : dm i » < u a t vioiaceum: H S L : H o m o Serme Lactone

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Sflhdaaoa of Qaonm i c n a t a ChromobacUrmm vwlacnmm CV026 by Vmtacem produced by M l

O v e r a l l , this study shows that P. aeruginosa isolated from the soot o f V. rrrnmrufies produces secondary metabolites w h i c h has a n t i - Q S properties. Furthermore, P. aeruginosa extract cotdd inhibit v io lace in production i n C. violaceum culture without k i l l i n g the ce l l . Further research w i l l pur i fy the extract to cet a single compound and be analyzed as a n a n t i - Q S compound.

A c k n o w l e d g e m e n t s

T h e authors are grateful for the Indonesia Univers i ty o f Educat ion , Bandung , Indonesia for t h e n financial assistance for this research experiment.

C o n f l i c t of* Interest

Authors w o u l d hereby l ike to declare that there i s no conflict o f interests that could possibly arise.

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