1994_inactivation of anaerobic bacteria by various photo sensitized porphyrins or by hemin

8/8/2019 1994_Inactivation of Anaerobic Bacteria by Various Photo Sensitized Porphyrins or by Hemin

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CURRENTMICROBIOLOOYVol. 29 (1994), pp. 125-131 Cu r r e n tM i c r o b i o l o g yA n I n t e r n a t i o n a l J o u r n a l9 Springer-Verlag New York Inc. 1994

I na c t iv a t io n o f A na e r o b i c B a c te r ia by V a r i o us P ho to s e ns i t i z e dP o r phy ri ns o r by H e m i nY e s h a y a h u N i t za n , 1 H a n n a h M . W e x l e r,3,4 Syd ney M . F ineg old 2,4,51Health Science Research Center, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel2Medical Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, Los Angeles, CA, USA3Research Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, Los Angeles, CA, USA4Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA5Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA, USA

Ab s t r a c t . T h e p h o t o d y n a m i c e f fe c ts o f d e u t e r o p o r p h y r i n ( D P ) , h e m a t o p o r p h y r i n d e r iv a ti v e ( H P D ) ,h e m a t o p o r p h y r i n ( H P ) , o r p r o t o p o r p h y r i n ( P P ) o n a v a r ie t y o f a n a e r o b i c m i c r o o rg a n i s m s w e r ee xa m ine d in t h i s s tudy . The m a jo r i t y o f the spe c ie s , a m on g the 350 s t ra in s t e s t e d , w e r e i nh ib i t e d byc onc e n t r a t i ons o f < 2 .5 ~ g /m l o f l igh t - a c t iva t e d D P . S pe c i e s f ound to be r e s i s t a n t t o t h i s t r e a tm e n ti n c l u d e d Bilophila wadsworthia, Fusobacterium mortiferum, Fusobacterium varium, a n d Bacteroidesgracilis. The se spe c i e s w e r e i nh ib i t e d by c onc e n t r a t i ons o f > 60 p , g /m l o f D P . T he p o r phy r in -p r oduc in g spe c i e s, Porphyromonas a n d Prevotella spp, we re a l l inhib i te d by < 2 .5 txg/ml D P andl igh t . C om pa r ing the pho todyna m ic a c t iv i t y o f t he po r phy r in s u se d on Porphyromonas s t ra insr e su l t e d i n t he f o l low ing pa t t e r n : D P > H P D > H P > P P . Porphyromonas spp. , Gram-posi t ivec oc c i , a nd m a ny G r a m - pos i t i ve r ods ( e xc lud ing c lo s t r i d i a ) w e r e i na c t iva t e d by he m in ( a m e ta l -c on ta in ing po r phy r in ) a t 10 - 20 p .g /m l . H e m in inh ib i to r y a c t ion w a s no t a f f e c t e d by l igh t . B ind inga nd in se r t i on o f D P in to ba c t e r i a ( bo th i na c t iva t e d a nd non - ina c t iva t e d s tr a in s by D P a nd l i gh t )w e r e m o n i t o r e d b y t h e c h a r a c t er i s ti c f l u o r e s ce n c e b a n d o f b o u n d D P a t 6 2 2 n m . Porphyromonasspp . bound D P t i gh t ly , w he r e a s on ly l ow b ind ing w a s s e e n w i th B. wadsworthia a n d o t h e rD P - r e s i s t a n t spe c i e s . H igh b ind ing o f D P to B. wadsworthia c a n b e a c h i ev e d by p r e t r e a t m e n t o f t h eba c t e r i a w i th im ipe ne m o r c e f ox i t in , 13 -1ac ta m a ge n t s kn ow n to i n t e r f e r e w i th t he i n t e g r i ty o f t hece l l wa l l . I f ce ll wa l l in tegr i ty i s d is turbed (e .g . , by the se a gents) , ina c t iva t ion of B. wadsworthia byD P c a n o c c u r.

M e t a l - f r e e p o rp h y r i n s h a v e b e e n u s e d a s a n t i c a n c e rthe r a pe u t i c a ge n t s i n c l i n i c a l m e d ic ine . W he n a c t i -va te d by l ight , they ac t se lec t ive ly aga ins t so l id tum orsw i th m in im a l da m a ge to t he hos t [ 5 , 12 ]. R e s e a r c h int h e f ie l d o f p h o t o d y n a m i c th e r a p y h a s r e s u l te d i n t h ed e v e l o p m e n t o f a p h o t o d y n a m i c m o d a l i t y a g a i n s tm ic r oo r ga n i sm s . P o r phy r in - induc e d pho tody na m ic ef -fec t leads to e f f ic ient an t imic rob ia l k i ll ing upon i l lumi-na t ion o f G r a m - pos i t i ve ba c t e r i a , m yc op la sm a a ndyeas ts , but no t G ram -neg a t ive ce i l s [2 , 3, 10 , 13, 22].G r a m - ne ga t ive ba c t e r i a w e r e f oun d r e c e n t ly [ 26 ] t obe inh ib i t e d by pho tose ns i t iz a t i on o f po r phy r in s on lyi n t h e p r e s e n c e o f p o ly m y x i n n o n a p e p t i d e ( P M N P ) .P o lym yx in non a pe p t ide i s a non - tox ic a ge n t t ha td i so r ga n iz e s t he ba c t e r i a l m e m br a ne s t r uc tu r e [ 11 ,29 ] . Th i s d i so r ga n iz a t ion o f t he m e m br a ne s t r uc tu r ea l low s the po r phy r in t o b ind to t he c y top la sm icCorrespondence to: Y. Nitzan

m e m b r a n e o f G r a m - n e g a t i v e b ac t e ri a . T h e p r e r e q u i -s i te for photosens i t iza t ion of a mic robia l ce l l i s theb i n d in g o f t h e p o r p h y r i n t o t h e c y t o p la s m i c m e m -br a ne . Th i s b ind ing i s s t r ong ly pH de p e nd e n t [ 6 ]. Th eim m e d ia t e i nh ib i t i on o f c e l l g r ow th i s a c c om pa n ie dby a lt e r a t i ons in p r o t e in , D N A , a nd R N A syn the s i s a sw e l l as pe r tu r b e d c e l l w a l l a nd c e l l m e m b r a ne syn the -sis [16 , 19, 20].F u r t h e r m o r e , h e m i n , a n i r o n - c o n t a in i n g p o r p h y -r in syn the s i z e d by m os t ba c t e r i a l spe c i e s, ha s show na n a n t iba c t e r i a l e f f e c t on Staphylococcus aureus a n do the r G r a m - pos i t i ve spe c i e s [ 15 , 21 ]. Th i s a n t iba c t e -r i al a c t i v it y is i nd e pe nde n t o f i l l um ina t ion . I t w a s a l sos h o w n t h a t a c o m b i n a t i o n o f d e u t e r o p o r p h y r i n a n dh e m i n h a s a s t r o n g e r e f fe c t t h a n t h a t o f t h e s e p a r a t ec ons t i t ue n t s a n d i s e qua l ly e ff i c ie n t w i th o r w i tho u ti l lumin a t ion [15].A lm os t no in f o r m a t ion is a vai l ab l e on the pho to -se ns i t iz a t i on o f a na e r ob ic ba c t e r i a by po r phy r in s a nd


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1 2 6 CURRENTMICROBIOLOGYVo1.29 (1994)l igh t . On the o ther hand , hemin i s an impor tan ts u p p l e m e n t f o r a n a e r o b i c b a c t e r i a l g r o w t h [ 27 ]. S i n c ep h o t o s e n s i t i z a t i o n o f b a c t e r i a l c e l ls is i n d e p e n d e n t o ft h e a n t i b i o t i c r e si s t a n c e s p e c t r u m o f t h e t r e a t e dp a t h o g e n , t h e a n t i b a c t e r i a l a c t i o n o f p o r p h y ri n s m a yu l t i m a t e l y h a v e t h e r a p e u t i c p o t e n t i a l.I n t h e p r e s e n t w o r k , w e s t u d i e d t h e p h o t o d y -n a m i c e f f e c t o f p o r p h y r i n s o n b o t h G r a m - p o s i t i v e a n dG r a m - n e g a t i v e a n a e r o b i c b a c t e r i a . T h e c o n n e c t i o nb e t w e e n p o r p h y r i n b i n d i n g a n d k il li ng w a s e v a l u a t e d .In add i t ion , the an t imicrob ia l e f fec t o f hemin ( in thed a r k ) , a s w e l l a s t h e e f f e c t s o f t h e c o m b i n a t i o n o fd e u t e r o p o r p h y r i n a n d h e m i n i n t h e d a r k , w a s i n ve s ti -g a t e d .M a t e r i a l s a n d M e t h o d sB a c t er i a l cu l t ures . Al l 350 st r a ins o f anaer ob ic bac t e r i a t es t ed we r ei s o l a t ed in t he C l in i ca l An aer ob ic B ac te r io logy L abor a to r y a t t heV e t e r a n s A d m i n i s tr a t i o n M e d i c a l C e n t e r , W e s t L o s A n g e l e s ( W a d -s wor th ) and we r e iden t i f i ed accor d ing to s t andar d metho ds [ 8 , 27 ].R e f e r e n c e s t ra i n s o f B . fragilis ( AT C C 25285) and B. thetaiotaomi-cron ( AT C C 29741) wer e us ed as con t r o l s i n t he an t imicr ob ia lsuscept ibi l i ty tes ts . These s t rains we re tes ted in at leas t 12 dif ferentexper imen t s and gave cons i s t en t ly t he s ame r es u l ts ( M I C s o f < 2 .5I xg/ml and 5 ixg /ml f o r DT , r es pec t ive ly ; M I C f o r hem in by bo ths t r a ins was > 60 txg/ml) . T he A T C C s t r a ins wer e ob ta ined f r om theAm er ican T ype C u l tu r e C o l l ec t ion ( R ockv i l l e , M ar y land) . S t r a inswer e s ubcu l tu r ed on B r uce l l a b lood agar bef o r e t es t i ng , andco lon ies wer e s us p ended in B r uce l l a b r o th ( B B L M icr ob io logySys tems, Becton Dickinson & Co. , Cockeysvi l le , Maryland) toob ta in 0 . 5 M cFa r l and un i t s o f tu r b id i ty .A nt i m i ero b i a l s us cept i b i l i t y t e s t s a nd pho t o s ens i t i za t i o n pro ce -d u r e . M inimal i nh ib i to r y concen t r a t ions ( M I C s ) o f t he var iousp o r p h y ri n s w e r e d e t e r m i n e d b y t h e N C C L S - a p p r o v e d , W a d s w o r tha g a r p l a te d i l u t io n t e c h n i q u e o u t li n e d i n N C C L S d o c u m e n t M l l - A 2[ 18] and the W ads wor th A naer o b ic B ac te r io logy M an ua l [ 27] .B r ie f ly , B r uce l l a agar ( B B L M icr ob io logy Syst ems) p l a t es con ta in -ing s e r i a l twof o ld d i lu t ions o f por phyr ins wer e inocu la t ed in ananaer ob ic chamb er ( An aer obe Sys tems , San J os e , C a l i f o r n i a ) wi th105 C F U p er s po t wi th a S t eer s r ep l i ca to r . A f t e r 48 h o f i ncuba t ionin anaer ob ic cond i t i ons a t 37~ M I C s wer e r ead as t he lowes tconcen t r a t ion o f t he por phyr in per mi t t i ng no g r owth . I n o r der t omain ta in pho tos ens i t i za t ion , t he p l a t es wer e p l aced wi th the i rinocu la t ed s u r f ace upw ar ds and i l l umina ted f o r 2 h in an anaer ob icchamber , wi th two 60- W tungs t en l amps p l aced 30 cm above thep la t es . P l a tes t es t ed in t he dar k wer e t r ans f e r r ed imm edia t e ly a f te rinocu la t ion in to a dar k con ta iner and incuba ted f o r 48 h as above .For t es t i ng the p r o t ec t ion o f s u lf hydr yl r eagen t s on he min ac t ion ,2-m erca ptoe than ol o r L-cys teine ( f inal concen trat ions , 10 -1 to 10 -5M ) wer e a dded to t he bac t e r i a p r io r t o hemin t r ea tmen t . B r uce l l aagar p l a t es us ed we r e a l l s upp lemen ted w i th 1 jxg /ml v i t amin K1 .Dur ing t es t s wi th the B. wadswor th ia s t rains , the med ium wass upp lem ented wi th 1% pyr uv ic ac id ( S igma, S t . L ou i s , M is s our i ). B .gracilis s t r a ins wer e t es t ed on B r uce l l a agar s upp lemented wi thv i t amin K1 and 0 . 3% f o r ma te - f uma r a t e ( f ina l concen t r a t ion) .P r e p a r a t i o n o f p o r p h y r i n s o l u t i o n s . Por phyr in s tock s o lu tions wer epr epa r ed by d i ss o lv ing 5 mg o f t he por phyr in ( powder ) i n 0 .2 ml 1 NNa OH ; 1 . 8 ml o f 0. 85% NaC 1 s o lu t ion was then adde d , and themix tu r e was vor t exed . S tock s o lu t ions wer e kep t i n t he dar k a t

+4~ Deu te r opor p hyr in I X d ihydr och lo r ide was ob ta ined f r omPor phyr in P r oduc t s ( L ogan , Ver mont ) . Hematopor phyr in d ihydr o-ch lo r ide , p r o topor phyr in I X, and hemin ( bov ine type I ) wer epur chas ed f r om S igma. Hem atopor ph yr in der iva t ive ( HPD ) waspr epar ed by t r ea t ing hematopor phyr in wi th a mix tu r e o f g l ac i a lace t i c ac id and s u l f u r ic ac id in a r a t io o f 19 :1 ( vo l /vo l ) andpr oces s ed as des cr ibed p r ev ious ly [ 4 ].P o l y ca t i o n i c pept i des a nd a nt i b i o t i c s . Poly-L- lys ine (hydrobro-mide) was ob ta ined f r om S igma. Po lymyxin nonapep t ide ( PM NP)was p r epar ed f r om po lymyxin B s u l fa t e ob ta ined f r om S igma by apr oces s des cr ibed p r ev ious ly [ 30] wi th mino r mo di f ica t ions . Pur i tyof PM NP ba tches was examined on ce l lu los e- coa ted a luminum f o i lT L C p l a t e s . I m i p e n e m w a s o b t a i n e d f r o m M e r c k , S h a r p a n dDo hm e ( W es t Po in t , Penns y lvan ia ) , and cef ox i t i n was pur chas edf r om S igma.F l u o r e s c e n c e m e a s u r e m e n t s . F l u o r e s c e n c e s p e c t ra w e r e m e a s u r e don a d ig i ta l s pec t r o f luor om eter ( Per k in - E lme r L umines cence s pec-t r ome te r mo del L S-50 , in t e r f aced to a da t a s t a t i on 7500 comp uter ) .T he exc i t a t i on wave leng th was 405 nm, and the emis s ion s pec t r awer e r ecor ded f r om 550 nm to 700 nm. S t r a ins wer e g r own inB r uce l l a b r o th s upp lemented wi th v i t amin K1 f o r 48 h in ananaer ob ic chamb er a t 37~ C ul tu r es wer e was hed twice wi th0 .85% NaC 1 by cen t ri f uga t ion ( 12 ,000 g , 20 min) and r es us pendedin 0 .1 M phos ph a te s a l ine buf f e r ( pH 6 .5 ) . R e la t ive f luor es cence a tthe emis s ion band o f 622 nm was ca l cu la t ed and cor r ec t ed to as t andar d num ber o f bac t e r i a ( 109 ce l l s /ml ) .C e l l e n v e l o p e p r e p a r a t io n . C el l s wer e g r own f o r 72 h in B r uce l l ab r o th s upp lem ented wi th 1 ixg /ml v i t amin K1 and 1% pyr uv ic ac id .Gr ow th was har ves t ed by cen t r i f uga t ion ( 12, 000g , 35 min) , and thepe l l e t was r es us p ended in 0 . 05 M T r i s - HC l buf f e r ( pH = 7 . 5)con ta in ing 0 . 01 M M gC 12 and 5 ixg /ml DN as e . C e l l s wer e b r oken byth r ee pas sages th r ough a F r ench p r es s u r e ce l l ( SL M I ns t r um ent sInc. , Urbana, I l l inois ) at 12,000 p.s . i . Cel l lysate was centr i fuged( 1200 g , 10 min) t o r em ove unbr oke n ce ll s. T he s uper na tan t wasaga in cen t r i f uged ( 45 , 000g , 45 min) , and the pe l l e t ( con ta in ing ce l lenve lop e) was was hed on ce wi th 0 . 05 M T r is -HC 1 ( pH = 7 . 5) andus ed immedia t e ly .

R e s u l t sP h o t o i n a c t i v a t i o n s t u d ie s w e r e p e r f o r m e d w i t h G r a m -p o s i t i v e an d G r a m - n e g a t i v e s t r a in s o f a n a e r o b i c b a c -t e r i a (3 5 0 s t r ai n s ). T h r e e h u n d r e d a n d s i x t e e n s tr a i n sw e r e s c r e e n e d f o r t h e i r s u s c e p t i b i l i t y to d e u t e r o p o r -p h y r i n a n d l ig h t . T a b l e 1 l is ts th e c u m u l a t i v e p e r c e n t -a g e o f s t r a in s i n h i b i t e d b y p h o t o s e n s i t i z e d D P f o re a c h g r o u p o f m i c r o o r g a n i s m s . A m o n g t h e G r a m -n e g a t i v e a n a e r o b i c b a c t e r i a , a l l s t r a in s o f P o r p h y r o m o -n a s s p p . a n d P r e v o t e l l a s p p . w e r e i n h i b i t e d b y p h o t o -s e n s i t i z e d D P ( M I C s o f < 2 .5 j x g / m l ) . O f t h e s t ra i n so f B . f r a g i l i s , 9 0 . 4 % h a d a n M I C o f < 2 .5 ~ g / m l , a n do n l y o n e s t r a in ( o f 4 2 ) w a s n o t i n h i b i t e d b y c o n c e n t r a -t i o n s o f a b o v e 6 0 ~ g / m l D P a n d l i g h t. O n t h e o t h e rh a n d , a l l B . g r a c il is , B . w a d s w o r t h i a , F . m o r t i f e r u m ,a n d F . v a r i u m s t r ai n s w e r e n o t i n h i b i t e d b y p h o t o s e n -s i t iz a t i o n , e v e n w i t h D P a t c o n c e n t r a t i o n s o f > 6 0j x g / m l . B e t w e e n 9 2 . 3 % a n d 9 4 . 7 % o f t h e G r a m -p o s i t i v e a n a e r o b i c c o c c i a n d r o d s ( e x c e p t fo r C l o s -


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Y. N i tzan e t a l . : Inac t iva t ion o f Anaero b ic Bac te r iaTab le 1 . Cum ula t ive inh ib i t ion o f anaerob e spec ies by pho toac t iva ted d eu te ropo rphyr in

127

No. of < 2.5s t ra insMicroorgan ism s tes ted No . (%)

Stra ins inh ib i ted a t (p ,g /m l) by DP and l igh t: S t ra ins no tinh ib i ted by_< 5 < 10 < 20 > 60 ixg/ml DPNo. (%) No . (%) No . (%) No . (%)

G r a m - n e g a t i v e a n a e r o b e s:Bacteroidesfragilis 42 38 (90.4) 38 (90.4) 40 (95.2) 41 (97.6) 1 (2.4)Bacteroides thetaiotaomicronanddistasonis 28 11 (39.3) 21 (75.1) 23 (82.2) 26 (92.9) 2 (7.1)Bacteroidesgracilis 11 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 11 (100.0)O t h e r Bacteroides spp. 33 19 (57.6) 27 (81.8) 31 (94.0) 32 (97.0) 1 (3.0)Bilophila wadsworthia 33 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 33 (100.0)Fusobacterium mortiferuma n d variurn 11 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 11 (100.0)O t h e r Fusobacterium spp. 22 15 (68.2) 15 (68.2) 15 (68.2) 16 (72.7) 6 (27.3)Porphyromonas spp. 35 35 (100.0) 35 (100.0) 35 (100.0) 35 (100.0) 0 (0.0)Prevotella spp. 32 32 (100.0) 32 (100.0) 32 (100.0) 32 (100.0) 0 (0.0)Gram -pos i t ive anaerobes :Clostridium spp. 24 18 (75.0) 21 (87.5) 21 (87.5) 22 (91.7) 2 (8.3)Gr am (+ ) cocci 26 24 (92.3) 24 (92.3) 26 (100.0) 26 (100.0) 0 (0.0)Gra m (+ ) rods 19 18 (94.7) 18 (94.7) 18 (94.7) 18 (94.7) 1 (5.3)

Tab le 2 . Cum ula t ive inh ib i t ion o f anaerob es by hem inStra ins inh ib i ted a t v~g /m lb y h e m i n

No. of < 10s t ra insMicroorgan ism s tes ted No . (%)

Stra ins no ti n h i b i t ed b y< 20 < 40 > 60 p~g/ml heroinNo. (%) No. (%) No. (%)

Gram -nega t ive anaerobes :Bacteroidesfragilis 42 1 (2.4)O t h e r Bacteroides spp. 72 1 (1.4)Bilophila wadsworthia 33 0 (0.0)Fusobacterium spp. 33 5 (15.2)Porphyromonas spp. 35 26 (74.3)Prevotella spp. 32 5 (15.6)Gram -po s i t ive anaerobes :Clostridium spp. 24 2 (8.3)Gra m (+ ) cocci 26 19 (73.1)Gra m (+ ) rods 19 9 (47 .4 )

1 (2.4) 2 (4.8) 40 (95.2)7 (9.7) 7 (9.7) 65 (90.3)0 (0.0) 0 (0.0) 33 (100.0)7 (21.2) 7 (21.2) 26 (78.8)33 (94.3) 33 (94.3) 2 (5.7)11 (34.4) 16 (50.0) 16 (50.0)5 (20.8) 10 (41.6) 14 (58.4)20 (76.9) 24 (92.3) 2 (7.7)14 (73.7) 16 (84.2) 3 (15.8)

tridium) had an MIC of < 2.5 txg/ml DP. Only 75% ofClostridium spp. were inhibited by DP (MIC < 2.5ixg/ml), but the percentage of all Gram-positiveanaerobes that were not inhibited by more than 60Ixg/ml DP (in the light) was very low. All the strainstha t are photosensitized in the light by 2.5 ixg/ml DPwere able to grow even in the presence of 60 wg/mlDP in the dark.

The MICs of heroin (Fe-bound porphyrin) for316 strains were also tested. Cumulative percentagesof inhibited strains in each group of anaerobes arelisted in Table 2. Porphyromonas spp. and the Gram-positive anaerobes (cocci and rods excluding Clos-

tridium spp.) were inhibited by hemin. For most of thePorphyromonas strains (74.3%), the MICs were about10 Ixg/ml. No st rain was inh ibited by 5 ixg/ml hemin(hemin as a growth additive is normally added at 5ixg/ml). Porphyromonas spp. are the only Gram-negative anaerobic species shown to be significantlyinhibited by heroin. Anaerobic Gram-positive cocciwere also inhibited by hemin. Gram-positive rodswere less inhibited by heroin (47.4% were inhibited at10 ixg/ml hemin, and 84.2% were inhibited at 40ixg/ml). Thiolic agents, such as mercaptoethanol orcysteine (> 10 -2 M), can reverse the inhibition byhemin.


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1 2 8 CURRENTMICROBIOLOGYVol. 29 (1994)T ab le 3 . C um ula t ive inh ib i t i on of Porphyromonas s t r a ins by d i f f e r en t pho toac t iva t ed por phyr ins

S t r a ins inh ib i t ed a t ( ~g /ml ) by por phyr in and l i gh t

No . o f


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Y. Ni t zan e t a l .: I nac t iva t ion o f An aer ob ic B ac te r i a 12 91 2 0 [ ~ [1 0 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., , = ,

50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5040 . . . . . . . . . . . . . . . . . . . . . . . . .

= .: 200 5 6 9 1 1 2 0 2 3 2 5 3 0 3 2 1 3 7 1 2 1 3 1 6 2 4 2 9 3 2

STRAIN NUMBERFig . 2. R e la t ive f luor es cence o f DP ( 20 I ~g/ml) emis s ion a t 622 nm( exc i t a t ion a t 405 nm) wi th var ious P o r p h y r o m o n a s an d B i l o p h i l as tr a ins . I n t ens i t i es wer e co r r ec t ed to t he s ame n umb er o f ce l ls (109ce l l s /ml ) .

by the synerg is tic ef f ec t o f the an t ib io t ic an d ph o toac -t i v a ted D P co u l d t h e Bilophila cel l s be k i l l ed by smal lam o u n t s o f D P ( 1 0 C g /m l ) . T h e p o l y ca t i o n ic p ep -t ides , po lymyxin non ape p t id e and po ly L- lys ine , whichcan ex p o s e G r am - n eg a t i v e ae r o b i c b ac t e r ia l en v e -lopes , f a i led to expose the envelopes in B. wadswor-thia strains.

Di s cu s s i o nW e r ep o r t h e r e an ex t en si v e s t u d y o f p h o t o s en s i t iz a -t i o n o f an ae r o b es b y p o r p h y ri n s . A l t h o u g h t h e an t im i -crob ia l e f f ec t s o f i ron- f r ee porphyr ins in the i r exci teds t a t e h av e a l r ead y b ee n e s t ab l i s h ed an d r ev i ew edrecen t ly [15, 25], the k now ledge o f the ef f ec t s o f theseco m p o u n d s o n an ae r o b i c b ac t e r i a h a s r em a i n ed v e r yl imi ted . Only four s t r a ins o f Bacteroides fragilis, Clos-tridium perfringens, Peptococcus magnus, an d Pepto-streptococcus anaerobius [ 32 ] w e r e t e s t ed an d f o u n d t ob e i n ac t i v a t ed b y H P D an d l i gh t. I n t h e p r e s e n t s t ud y ,we employed 350 s t r a ins inc lud ing mos t c l in ica l lys i gn i fi can t an ae r o b i c s p ec i e s an d G r am - n eg a t i v e r o d sas wel l as Gram-pos i t ive rods and cocci . Th is inves t i -g a t i o n r ev ea l ed t h a t t h e p h o t o d y n am i c e f f ec t o fporphyr ins occur s in a l a rge var ie ty o f anaerob icb ac t e r i a . M o s t o f t h e an ae r o b i c s p ec i e s , b o t h G r amnegat ive an d G ram pos i t ive , a r e inact iva ted by ligh t-ac t i v a t ed d eu t e r o p o r p h y r i n ( D P ) a t co n cen t r a t i o n s o f< 2.5 txg /ml . Th e la t te r co ncen t r a t ion i s a t l eas t four -t o f iv e f o ld l o w er t h an t h e co n cen t r a t i o n s n ee d ed f o raerob ic bac ter ia [22 , 26]. On th e o the r hand , B .wadsworthia, F. mort i ferum, F. varium, a n d B. gracilisw er e n o t i n ac t i v a ted ev en a t co n cen t r a t i o n s o f > 6 0i ~/ m l D P . F r o m t h e o v e r a ll p h o t o i n ac t iv a t i o n p a t t e r nb y D P o f an ae r o b i c b ac t e r i a , i t s eem s t h a t t h eD P - i n ac t i v a t ed s p ec i e s a r e a l s o G r am - n eg a t i v e b ac t e -r i a . F u r t h e r m o r e , t h e s e D P - p h o t o i n ac t i v a t ed , G r am -

25 0

u ., 20 00Z

15 0O

p".* 10 0" 5 1 1

O

B - 1 3 B - 2 4

we ENV IM CEF NP P I . WC ENV IM CEF NP PLFig . 3. DP r e l a t ive emis s ion f luor es cence a t 622 nm ( exc i t a t i on a t405 nm) wi th two B i l o p h i l a w a d s w o r t h i a s t r a ins g r own in d i f f e r en tcond i t ions : ( A) I n t ac t who le ce l l s ( WC ) , g r own nor m al ly , s e r ved ascontrols ; (B) B i l o p h i l a ce l l enve lopes ( E NV) ; ( C ) B i l o p h i l a cel lsg r o w n w i th 1 0 0 ~ g / m l i m i p e n e m ( I M ) ; ( D ) B i l o p h i l a ce l l s g r ownwith 100 p~g/ml cefoxi t in (CE F); (E) B i l o p h i l a ce l l s g r own w i th 250ixg /ml po lymyxin non apep t ide ( NP) ; ( F ) B i l o p h i l a ce l l s g r own wi th250 ~g/ml polylys ine (PL) .

n eg a t i v e an ae r o b es d o n o t r eq u i r e m em b r an e d i s o rg a -n i zi n g ag en t s li k e po l y m y x in n o n a p ep t i d e ( P M N P ) . I tw as s h o w n p r ev i o u s l y t h a t t h e p h o t o i n ac t i v a t i o n o faerob ic bacter ia i s d i f f er en t [22 , 26] . Only Gram-p o s i t i v e ae r o b i c b ac t e r i a a r e p h o t o i n ac t i v a t ed d i -r ec t ly b y D P an d l ig h t [ 22 ]. G r am - n eg a t i v e ae r o b i cb a c t e r ia r e q u i re t h e p r e s e n c e o f P M N P f o r p h o t o s e n -s i t i z a t i o n an d i n ac t i v a t i o n . D P n eed s P M N P t o en -ab l e i t t o p en e t r a t e t h r o u g h t h e b ac t e r i a l b a r r i e r i no r d e r t o b i n d t o t h e i n n e r m em b r an e [ 2 6 ] an d t op r o d u ce t h e p h o t o d y n a m i c e f f ec t an d k i l li n g o f t h ece ll . A s i n d i ca t ed ab o v e , w i t h i n t h e G r am - n eg a t i v ean ae r o b i c s p ec i e s t h e r e a r e t w o g r o u p s : ( 1 ) t h o s ew h o s e o u t e r b a r r ie r p e r m i t s t h e p e n e t r a t i o n o f D P t ot h e i n n e r m em b r an e an d w h i ch a r e p h o t o i n ac t i v a t edan d ( 2 ) t h o s e w h o s e b a r r i e r ( p r o b a b l y t h e o u t e rm e m b r a n e ) i s n o t p e r m e a b l e t o D P a n d w h i c h a r et h e r e f o r e n o t p h o t o i n ac t i v a t ed . S u r p r i s i n g l y , t h eG r am - n eg a t i v e an a e r o b i c s p ec i e s t h a t a r e n o t i n h i b -i t ed b y p h o t o s en s i t i z ed D P a r e n o t a f f ec t ed ev en b yt h e p r e s e n c e o f P M N P o r p o l y l y s i n e a n d r e m a i nun inh ib i ted .

B y t h e f l u o r e s cen ce p a r am e t e r s em p l o y ed i n th i ss t ud y , w e co u l d d i f f e r en ti a t e t h e n a t u r e o f t h e en v i r o n -m en t i n w h i ch t h e D P m o l ecu l e r e s i d es i n t h em i c r o b i a l b a r r ie r s . M eas u r e m e n t s o f t h e f lu o r e s cen ces p ec t r u m o f D P ( ex c i ta t i o n a t 40 5 n m ) w er e u s ed t oi n t e r p r e t t h e d i f fe r en t b i n d in g ab i li t y o f D P t o Porphy-r o m o n a s s t r a ins and to Bilophila s t r a i n s . W e f o u n d aco r r e l a t io n b e t w e en t h e ex t en t o f D P b i n d i n g a n d t h eobserved pho todynamic ef f ec t (k i l l ing o f the ce l l s ) inthese two species . I t i s c lear tha t P o r p h y r o m o n a ss t ra i n s b i n d en o r m o u s q u an t i t i e s o f D P an d co n s e -


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1 3 0 CURRENT MICROBIOLOGYVol . 29 (1994)

quently are killed by the photodynamic process thatfollows upon illumination. On the other hand, Bilo-phi la strains bind relatively small amounts of DP andare not killed by the photosensitization reaction evenwhen high concentrations of DP are used. The bandat 622 nm is the typical emission of DP in a low-polarity medium [15]. The low-polarity phase in thebacterial cell that results in this band for DP is thecytoplasmic membrane, as was previously shown foraerobic Gram-positive [6] and for aerobic Gram-negative bacteria (in the presence of PMNP) [26].The possibility that the Bilophila cytoplasmic mem-brane is not capable of binding DP was ruled out byshowing that the envelope preparation of Bilophilastrains can bind DP at least as well as intact Porphy-romonas cells. Thus, disorganizing or disrupting thebacterial barriers and exposing the inner membraneswill allow DP to bind. However, agents that disruptthe outer membranes, such as PMNP or polylysine,did not increase DP binding to Bilophila. It may bethat the Bilophila and other Gram-negative anaer-obes have a more complicated outer membranestructure or that they cannot be disrupted by thepolycationic agents. A recent review [28] indicatedthat in anaerobic Gram-negative bacteria, PMNB orpolycationic agents do not increase permeability ofthe outer membrane. Removal of the outer barriersand exposure of the inner membrane was accom-plished by the antibiotics imipenem and cefoxitin.The presence of these antibiotics in the growthmedium causes the release of lipopolysaccharidefrom the bacteria [7] (70-80% of the lipopolysaccha-ride can be released in their presence). Only thesynergistic effect of imipenem or cefoxitin and DPallowed the porphyrin to bind to the bacterial cell (tothe inner membrane) and to kill the cell uponphotosensitization. Again, the important role of thebinding of the photosensitizer molecule to the innermembrane is indicated. Furthermore, there are por-phyrin molecules other t han DP (e.g., HPD, HP, andPP) that do not photoinactivate these bacteria to thesame extent as DP. It was shown in this study thatPorphyromonas spp. are less inactivated by HPD orHP and are still less inactivated by PP, indicating thatthe inactivation by porphyrins may also depend onthe chemical structure of each molecule, which deter-mines its ability to penetrate the bacterial barriersand to reach the inner membrane.

The mechanism by which the porphyrin moleculeexerts its photodynamic effect and the major targetsites of inactivation within the bacterial cell are underfurther investigation. From preliminary results itseems that as a consequence of the anaerobic condi-

tions that exist during the time period of the photody-namic process, the photochemical reaction takingplace is of the type I mechanism [14, 31]. Hydroxylfree radical (OH-) and probably other free radicalsare produced and as a result kill the cells. In aerobicbacteria singlet oxygen [102] and hydroxyl free radi-cals inhibited the bacteria [24[.

Hemin, the iron-containing protoporphyrin, hasbeen shown to possess antibacterial activity that wasnot dependent on illumination. Porphyromonas spp.,Gram-positive cocci, and many Gram-positive rods(excluding clostridia) were found to be inactivated byhemin. This ph enome non was fou nd previously withGram-positive aerobic bacteria [9, 21] but not withGram-negative aerobic cells. In the present study, the'strains were shown to have an MIC to hemin of 10-20~g/ml. This concentration is only two- to fourfoldhigher than the amount of hemin recommended as agrowth additive for anaerobes (5 ~g/ml). Heroinaffects the cells not by photosensitization but ratherthrough an oxidation-reduction process in connectionwith peroxide [14]. With anaerobic bacteria as withaerobes [23], we found tha t thiol reagents can protectfrom hemin inactivation. Thus, the antibacterial ef-fect of hemin must be different from the antimicro-bial effects of the metal-free porphyrins, where thebacteria are not protected from killing by thiols. Itshould be noted that excess exogenous heroin (as asupplement) can lead to cell inactivation instead ofincreasing growth.

The antibacterial action of porphyrins on anaer-obes may ultimately have therapeutic potential. Theseresults suggest new possibilities for therapy of anaero-bic bacterial infections. This modality is indep endentof antibiotic resistance of the pathog en and can alsoact synergisticallywith antibiotics to which the patho-gen is resistant. Some of these porphyrins havealready been used for clinical treatments [17], and thesource of light can be achieved by optic fiber even inbody cavities. In addition, it seems from preliminaryresults on the fluorescence of bound porphyrins tobacteria th at porphyrins may be good probes to definesubgroups in species found to be a single genus, suchas Bilophila wadsw orthia [1].A C K N O W L E D G M E N T SThis work was supported in part by VA Merit Review MedicalResearch Funds and in part by Fujisawa Pharmaceuticals Co.(Deerfield, Illinois).L i t e r a t u r e C i t e d1. Baron EJ, Summanen P, Downes J, Roberts MC, Wexler H,Finegold SM (1989)Bilophila wadsworthia,gene. nov. and sp.


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2 . B er to l in i G , Dal l ' Acqua M , Vas s o le r M , Sa lva to B , J o r i G( 1984) B ac te r i a l and yeas t ce l l s as model s f o r s tudy ing hemato-por phyr in pho tos ens i t i za t ion . I n : Andr eon i A and C ubeddu R( eds ) Por phyr ins i n t umor pho to ther apy . New Yor k : P l enumPress, pp 177-184

3 . B er to l in i G , Vei l A , Gr os s a to A, J o r i G ( 1985) T he ph o tos ens i -t i z ing ac t iv ity o f hematopor ph yr in on m olecu les . J G en M icr o -biol 131:2217-2223

4 . D o u g h e r t y T J , K a u f m a n J E , G o l d f a r b A , W e i s h a u p t K R ,B oyle D, M i t t e lm an A ( 1978) Pho to r ad ia t ion ther ap y f o r t het r ea tme n t o f mal ignan t t umo r s . C ancer R es 38 :2628-2635

5 . D o u g h e r t y T J , B o y l e D G , W e i s h a u p t K R , H e n d e r s o n B A ,P o t e r W R , B e l l n i e r D A , W i t y k K E ( 1 9 8 3 ) P h o t o r a d i a t i o nther apy c l in i ca l and d r ug advances. I n : Kes s e l D an d D ough-er ty T J ( eds ) Por phyr in pho tos ens i t iza t ion . New Yor k : P l en umPress , pp 3-136 . E hr enber g B , M al ik Z , Ni t zan Y ( 1985) F luor es cen t s pec t r a lchanges o f hematopor phyr in der iva t ive upon b ind ing to l i p idvesicles. S . a u r e u s an d E . c o l i ce l l s . Pho tochem Pho tob io l41:429-4357. Evans ME, Pol lack M (1993) Effect of ant ibiot ic class andconcen t r a t ion on the r e l eas e o f l i popo lys acchar ide f r om E s c h -er ich ia co l i . J Infec t Dis 167:1336-13428 . H o l d e m a n L V , C a t o E P , M o o r e W E C ( 1 9 77 ) A n a e r o b elabora tory manua l , 4th ed. Blacksburg, Virginia: VirginiaPo ly techn ic I ns ti t u t e and S ta t e Univer s i ty

9 . J am es SM , T agg J R ( 1987) I nh ib i t ion o f mutans s t r ep tococc ib y t h e h e m a t i n f o r m e d o n b l o o d - c o nt a i n in g m e d i a b y p r o te o -ly t ic en t e r ococc i . FE M S M icr ob io l L e t t 44 :95-99

10 . Kv e l lo - S tens t r om AG , M oan J , Gr unbor g G, E k lund T ( 1980)Pho todynam ic inac t iva t ion o f yeas t ce l l s s ens i t i zed by hemato-por phyr in . Pho tochem Pho tob io132 :349- 352

11 . L am J , Hi ldebr and t J , Schu tze E , Wenze l AF ( 1986) M em-br ane d i s o r gan iz ing p r oper ty o f po lymyxin B nonapep t ide . JAnt imicr ob C hemother 18 :9 - 15

12 . L and E J ( 1984) Por phyr in pho to ther apy o f hum an cancer . I n tJ Ra diat Bio146:219-22313 . M al ik Z , G ozhans ky S , Ni t zan Y ( 1982) E f f ec t s o f pho toac t i -

va t ed haem atopor ph yr in der iva t ive on bac t e r i a and an t ib io t i cres is tance. Microbios Let t 21:103-112

14 . M al ik Z , Hana n ia J , Ni t zan Y ( 1990a) New t r ends in pho tob io l -ogy : bac t e r i a l e f f ec t s o f pho toac t iva t ed po r phyr in s - - an a l t e r na-t ive appr oach to an t imicr ob ia l d rugs. J Pho to chem Pho tob io l B5:281-29315 . M al ik Z , L adan H, Ni t zan Y, E hr enber g B ( 1990b) T hebac te r i a l ac t iv i ty o f a deu te r opor phyr in - hemin mix tu r e onGr am - pos i t i ve bac t e r i a . A micr ob io log ica l and s pec t r os cop ics tudy . J Pho toc hem Pho tob io l B 6 :419-430

16 . M al ik Z , L adan H, E hr enber g B , Ni t zan Y ( 1992) B ac te r i a land v i r a l pho todynamic inac t iva t ion . I n : Hender s on B W andDougher ty T J ( eds . ) Pho todynamic ther apy- - bas i c p r inc ip l es

and c l in i ca l app l i ca t ion . New Yor k : M ar ce l Dekker I nc . , pp97- 113

17 . M ar cus SL ( 1992) C l in ica l pho todynam ic ther apy : t he con t inu-ing evo lu t ion . I n : Hender s on B W and Dougher ty T J ( eds . )Pho todyna mic T her a py- - B as i c P r inc ip l es and C l in i ca l App l i ca -t i on . New Yor k : M ar ce l De kker I nc . , pp 217- 268

18 . N at iona l C om mi t t ee f o r C l in ica l L abor a to r y S tand ar ds ( 1990)M ethods f o r an t imicr ob ia l s us cep t ib i l i t y t es t i ng o f anaer ob icbac te r i a , 2nd ed . , Vo l 9 , Number 15 . Appr oved s t andar dsM l l - A 2 . V i l l a n o v a , P e n n s y l v a n i a : N a t i o n a l C o m m i t t e e f o rC l in i ca l L abor a to r y S tandar ds

19 . Ni r U, L adan H, M al ik Z , Ni t zan Y ( 1991) I n v ivo e f f ec t s o fp o r p h y ri n s o n b a c t e r i a l p la s m i d D N A . J P h o t o c h e m P h o t o b i o lB 11:295-306

20 . Ni t zan Y, Gozhans ky S , M al ik Z ( 1983) E f f ec t o f pho toac t i -va t ed her na topor phyr in der iva t ive on the v i ab i l it y o f S t a p h y l o -c o c c u s a u r e u s . C ur r M icr ob io l 8 :279-284

21 . N i t zan Y, L adan H, M al ik Z ( 1987a) Gr ow th inh ib i to r y e f f ec tof hemin on s taphylococci . Cu rr Micro biol 14:297-284

22 . Ni t zan Y, Sha inber g B , M al ik Z ( 1987b) T he pho todynamicef f ec t o f deu te r opor phyr in on Gr am- pos i t i ve bac t e r i a . C ur rMicrobiol 15:251-25823 . N i t zan Y, L adan H, Gozh ans ky S , M al ik Z ( 1987c) C har ac t e r -i za t ion o f hem in an t imicr ob ia l ac t ion on S t a p h y l o c o c c u s a u r e u s .FE M S M icr ob io l L e t t 48 :401-- 406

24 . Ni t zan Y, Sha inber g B , M al ik Z ( 1989) T he mechan i s m ofpho todynam ic inac tiva t ion o f S t a p h y l o c o c cu s a u r e u s b y d e u t e r o -por phyr in . C ur r M icr ob io l 19 :265- 269

25 . Ni t zan Y, M al ik Z , E hr enber g B ( 1991) Pho tos ens i t i za t ion o fmicr ob ia l ce l l s . I n : E R ik l i s ( ed ) Pho tob io logy and pho to -ther a py- - t he s c i ence and i ts app l ica t ions . New Yor k : P l en umPress, pp 815-820

26 . Ni t zan Y, Gu t t e r man M , M al ik Z , E hr en ber g B ( 1992)I nac t iva t ion o f Gr am- nega t ive bac t e r i a by pho tos ens i t i zedpor phyr ins . Pho tochem Pho tob io155 :89- 96

2 7 . S u m m a n e n P , B a r o n E J , C i tr o n D M , S t r o n g C A , W e x l e r H M ,F inego ld SM ( 1993) Wads wo r th anaer ob ic bac t e r io logy manua l ,5 th ed . B e lm ont , C a l i f o r n i a : S t a r Pub l i s h ing

28 . Vaa r a M ( 1992) Agen t s t ha t i nc r eas e the per me ab i l i t y o f t heou te r mem br ane . M icr ob io l R ev 56:395- 411

29 . Vaar a M , Vaar a T ( 1983a) Po lyca t ions as ou te r membr aned i s o r gan iz ing agen t s . An t imicr ob Agen t s C hemother 24 :114-12 2

30 . V aar a M , Vaar a T ( 1983b) Po lyca t ions sens i t ize en te r i c bac t e -r i a t o an tib io t ics . An t im icr ob Agen t s C he mo ther 24 :107-113

3 1 . V a n S t e v e n in c k J, T u s se n K , B o e g h e i m J P J , V a n D e r Z e e J ,D u b b e l m a n T M A R ( 19 8 6) P h o t o d y n a m i c g e n e r a t i o n o f h y -d r oxy l r ad ica ls by hema topor phyr in der iva t ive and l i ght . Pho to -chem Pho tob io144 :711- 716

3 2 . V e n e z i o F R , D i V i n c e n z o C , S h e r m a n R , R e i c h m a n M , O r i g i-t a n o T C , T h o m p s o n K , R e i c h m a n O H ( 1 9 8 5 ) B a c t e r i c i d a le f f ec t s o f pho to r ad ia t ion ther ap y wi th hem atopor phy r in der iva-t ive. J Infe ct Dis 151:166-169

1994_inactivation of anaerobic bacteria by various photo sensitized porphyrins or by hemin

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