an in vitro study of antiseptics and antibiotics used in en~1

8/6/2019 An in Vitro Study of Antiseptics and Antibiotics Used in en~1

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Journal of Dental Research

DOI: 10.1177/002203455603500424011956; 35; 656J DENT RES

Olivier Pita Fajarda, Louis I. Grossman and Joan McShaneAn in Vitro Study of Antiseptics and Antibiotics Used in Endodontics

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AN IN VITRO STUDY OF ANTISEPTICS AND ANTIBIOTICS USED INENDODONTICSOLIVIER PITA FAJARDA, LOUIS I . GROSSMAN, AND JOAN McSHANE

From t h e F a c u l t y o f D e n t i s t r y , U n i v e r s i t y o f U r u g u a y a n d t h e S c h o o l o f D e n t i s t r y , U n i v e r s i t yo f P e n n s y l v a n i a , P h i la de l p h i a , P a .

THE o b j e c t o f t h i s s t u d y i s t o a s c e r t a i n t h e a c t i o n , i n v i t r o , o f s o m e a n t i s e p t i c sa n d a n t i b i o t i c s w h i c h a r e u s e d f o r t h e d e s t r u c t i o n o f m i c r o o r g a n i s m s i n

i n f e c t e d r o o t c a n a l s .T h e l a b o r a t o r y p r o c e d u r e s a r e d i v i d e d i n t o t h r e e p a r t s : ( 1 ) p r e l i m i n a r yt e s t s t o d e t e r m i n e t h e e f f i c a c y o f t h e a g e n t s i n g e n e r a l a n d t o e s t a b l i s h w h e t h e rt h e y a r e a d a p t e d t o c l i n i c a l p r a c t i c e ; ( 2 ) a q u a l i t a t i v e s t u d y b y t h e d i s c m e t h o d ;a n d ( 3 ) a q u a n t i t a t i v e s t u d y b y t h e s e r i a l d i l u t i o n m e t h o d .

I n o u r l a b o r a t o r y , t h e f l o r a o f s e v e r a l i n f e c t e d r o o t c a n a l s w e r e f o u n d t o b eo f a m i x e d t y p e when s e v e r a l t e e t h w e r e c o m p a r e d , b u t i n m o s t c a s e s t h e r o o tc a n a l s c o n t a i n e d o n l y o n e t y p e o f o r g a n i s m . T h e a l p h a s t r e p t o c o c c u s w a s f o u n dm o s t f r e q u e n t l y . I n s o m e c a s e s 2 t y p e s o f o r g a n i s m s w e r e f o u n d , b u t 1 t y p ee x c e e d e d t h e o t h e r i n n u m b e r s . M i x e d f l o r a w e r e n e a r l y a l w a y s o b s e r v e d i ne x p o s e d r o o t c a n a l s , o r i n t h e c a s e o f c o n t a m i n a t i o n d u r i n g t h e c o u r s e o fe n d o d o n t i c t r e a t m e n t . A n u m b e r o f p u l p l e s s t e e t h w i t h p e r i a p i c a l a r e a s w e r ef o u n d t o b e s t e r i l e .

W i t h t h e a d v e n t o f a n t i b i o t i c s , i t h a s b e c o m e n e c e s s a r y t o s t u d y t h e b a c -t e r i a l f l o r a , a s m o s t a n t i b i o t i c s a r e s e l e c t i v e i n t h e i r a c t i o n .I n o u r e x p e r i m e n t s , 4 t y p e s o f m i c r o o r g a n i s m s , S t r . f e c a l i s , S t a p h . a l b u s ,C . a l b i c o n s , a n d E . c o l i , w e r e u s e d b e c a u s e t h e y a r e m o s t c o m m o n l y f o u n d i n

i n f e c t e d r o o t c a n a l s .A f t e r s o m e p r e l i m i n a r y t e s t s , A n t i s t i n e , A s t e r o l , s o d i u m c a p r y l a t e , d e c y lammonium s u l f a t e , f r a d i c i n , n e o m y c i n , P e r a z i l , r i m o c i d i n , T e r r a m y c i n , a n dt h i o l u t i n w e r e s e l e c t e d f o r t h e t e s t s . O f t h e s e , A n t i s t i n e , A s t e r o l , s o d i u mc a p r y l a t e , d e c y l ammonium s u l f a t e , a n d P e r a z i l a r e n o t a n t i b i o t i c s .J a w e t z , G u n n i s o n , a n d S p e c k , ' who s t u d i e d t h e a n t a g o n i s m c a u s e d b y

A u r e o m y c i n , T e r r a m y c i n , o r c h l o r a m p h e n i c o l o n t h e o n e h a n d , w i t h p e n i c i l l i no n t h e o t h e r , s h o w e d t h a t i t o c c u r s o n l y i f a n y o f t h e f o r m e r i s g i v e n e i t h e r w i t h .o r b e f o r e t h e l a t t e r , b u t n o t i f p e n i c i l l i n i s a d m i n i s t e r e d f i r s t . T h i s s e e m s t os h o w t h a t i t i s n o t a m u t u a l a n t a g o n i s m o r i n c o m p a t i b i l i t y o f t h e d r u g s , b u tr a t h e r a n i n t e r f e r e n c e b y c h l o r a m p h e n i c o l , T e r r a m y c i n , o r A u r e o m y c i n w i t h t h ea c t i v i t y o f p e n i c i l l i n . I n t e r f e r e n c e o f e f f e c t i s o b s e r v e d n o t o n l y among a n t i -b i o t i c s b u t a l s o w h e n an a n t i b i o t i c i s u s e d w i t h some o t h e r a g e n t , s u c h a s p e n i -c i l l i n w i t h b o r i c a c i d , o r w i t h s u l f a d i a z i n e u n d e r c e r t a i n c o n d i t i o n s .

R e c e i v e d f o r p u b l i c a t i o n A p r i l 2 8 , 1 9 5 4 . Revised F e b . 1 0 , 1 9 5 6 .6 5 6

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V o l u m e 3 5N u m b e r 4 ANTISEPTICS AND ANTIBIOTICS USED I N ENDODONTICS

On t h e o t h e r h a n d , s e v e r a l s t u d i e s h a v e d e m o n s t r a t e d a s y n e r g i s t i c e f f e c ta m o n g c e r t a i n a n t i b i o t i c s s u c h a s b e t w e e n p e n i c i l l i n a n d s t r e p t o m y c i n , a n dp e n i c i l l i n a n d b a c i t r a c i n , a n d a l s o b e t w e e n p e n i c i l l i n a n d s o m e s u l f o n a m i d e s .

MATERIALSA g e n t s . - A n t i s t i n e i s a s y n t h e t i c a n t i h i s t a m i n i c ( 2 p h e n y l - b e n z y l a m i n o -

m e t h y l - i m i d a z o l i n e h y d r o c h l o r i d e ) w i t h r e l a t i v e l y l o w t o x i c i t y . A s t e r o l i s t h ed i h y d r o c h l o r i d e o f 2 m e t h y l a m i n e - 6 - b e n z o t h i a z o l e c o n s i d e r e d t o h a v e a h i g hd e g r e e o f a c t i v i t y a g a i n s t f u n g i , i n c l u d i n g C . a l b i c a n s . S o d i u m c a p r y l a t e , t h es o d i u m s a l t o f c a p r y l i c a c i d , i s r e l a t i v e l y n o n t o x i c . F r a d i c i n i s a n a n t i b i o t i cd e r i v e d f r o m S t r e p t o m y c e s f r a d i a e . N e o n m y c i n i s a n a n t i b i o t i c h a v i n g a c y c l o -e t h a n o l t y p e s t r u c t u r e . P e r a z i l i s a n a n t i h i s t a m i n i c w h i c h h a s a p i p e r a z i n en u c l e u s a s a b a s e i n s t e a d o f t h e e t h y l e n e g r o u p . R i m o c i d i n i s a n a n t i b i o t i cd e r i v e d f r o m S t r e p t o m y c e s r i m o s u s . T e r r a m y c i n i s a n a n t i b i o t i c d e r i v e d f r o mS t r e p t o m y c e s r i m o s u s . T h i o l u t i n i s a n a n t i b i o t i c d e r i v e d f r o m S t r e p t o m y c e sa l b u s .

METHODM e t h o d A . - C u l t u r e s w e r e p r e p a r e d f r o m a 2 4 - h o u r g r o w t h o f o r g a n i s m s

g r o w n i n b r a i n h e a r t i n f u s i o n b r o t h f o r S t r . f e c a l i s , S . a l b u s , a n d E . c o l i , a n di n S a b o u r a u d ' s medium f o r C . a l b i c a n s . T h e a g e n t s t o b e t e s t e d f o r t h e i ra n t i b a c t e r i a l e f f e c t w e r e p r e p a r e d i n d i l u t i o n s o f 5 a n d 5 0 m g . p e r c u b i c c e n t i -m e t e r o f s o l v e n t . I n a d d i t i o n , d i l u t i o n s o f 1 0 0 m g . p e r c u b i c c e n t i m e t e r o f s o l v e n tw e r e m a d e i n t h o s e c a s e s w h e r e t h e c h e m i c a l a g e n t s w e r e t o b e u s e d a g a i n s tS t r . f e c a l i s o r C . a l b i c a n s b e c a u s e o f t h e g r e a t e r r e s i s t a n c e o f t h e s e m i c r o o r g a n -i s m s . I n t h o s e c a s e s w h e r e t h e a n t i b i o t i c o r c h e m i c a l a g e n t w a s n o t s o l u b l e i nw a t e r , i t w a s f i r s t d i s s o l v e d i n a l i t t l e p r o p y l e n e g l y c o l a n d t h e n i n w a t e r .T h e a m o u n t o f p r o p y l e n e g l y c o l u s e d w a s n e g l i g i b l e f r o m a n a n t i b a c t e r i a l s t a n d -p o i n t a s d e t e r m i n e d b y t e s t . G r a d u a t e d p i p e t t e s w e r e u s e d f o r t r a n s f e r r i n gs o l u t i o n s o f t h e c h e m i c a l s o r d i l u t i o n s o f t h e m i c r o o r g a n i s m s . T o e a c h t e s t t u b ew a s t r a n s f e r r e d 0 . 1 c . c . o f t h e c h e m i c a l s o l u t i o n a n d 0 . 1 c . c . o f t h e s u s p e n s i o no f m i c r o o r g a n i s m s . F r e s h p i p e t t e s w e r e u s e d f o r e a c h t r a n s f e r .

A s c o n t r o l s , 4 0 t u b e s o f c u l t u r e medium w e r e u s e d c o n t a i n i n g t h e s a m ea m o u n t o f c u l t u r e medium a n d t h e s a m e a m o u n t o f b a c t e r i a l s u s p e n s i o n , b u tw i t h o u t t h e a d d i t i o n o f c h e m i c a l s . T h e t u b e s w e r e i n c u b a t e d a t 3 7 0 C . e x c e p tt h e S a b o u r a u d ' s m e d i u m , w h i c h w a s i n c u b a t e d a t r o o m t e m p e r a t u r e . I n t h o s ec a s e s w h e r e . t h e r e w a s n o e v i d e n c e o f g r o w t h , s u b c u l t u r e s w e r e m a d e .

R e s u l t s : A l l c u l t u r e s w i t h S t r . f e c a l i s w e r e p o s i t i v e when t h e c h e m i c a l sw e r e u s e d i n a c o n c e n t r a t i o n o f 5 m g . p e r c u b i c c e n t i m e t e r . I n a 5 0 m g . p e rc u b i c c e n t i m e t e r c o n c e n t r a t i o n , A n t i s t i n e , d e c y l ammonium s u l f a t e , a n d T e r r a -m y c i n c u l t u r e s w e r e n e g a t i v e w h i l e a l l o t h e r s w e r e p o s i t i v e . A l l c u l t u r e s w e r ep o s i t i v e a g a i n s t C . a l b i c a n s e x c e p t t h i o l u t i n i n a c o n c e n t r a t i o n o f 1 0 0 m g . p e rc u b i c c e n t i m e t e r . N e o m y c i n , P e r a z i l , T e r r a m y c i n , a n d t h i o l u t i n i n b o t h 5 m g .a n d 5 0 m g . c o n c e n t r a t i o n s were e f f e c t i v e a g a i n s t S t a p h . a t b u s . A l l t h e r e s t s h o w e dg r o w t h . A g a i n s t E . c o l i , A n t i s t i n e , d e c y l ammonium s u l f a t e , n e o m y c i n , P e r a z i l ,

6 5 7



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658 FAJARDA, GROSSMAN, AND MC SHANE J . D R e s .T e r r a m y c i n , a n d t h i o l u t i n s h o w e d n o g r o w t h i n b o t h 5 m g . a n d 5 0 m g . p e r c u b i cc e n t i m e t e r c o n c e n t r a t i o n . C u l t u r e s w i t h A s t e r o l , s o d i u m c a p r y l a t e , f r a d i c i n , a n dr i m o c i d i n w e r e p o s i t i v e o n e x a m i n a t i o n .

M e t h o d B . I n o r d e r t o s t u d y t h e a c t i o n o f t h e c h e m i c a l o b j e c t i v e l y , a g a rp o u r - p l a t e s w e r e made c o n t a i n i n g 1 0 p e r c e n t o f s t e r i l e h o r s e b l o o d , a n d t h ea g a r w a s t h e n i n o c u l a t e d w i t h a s u s p e n s i o n o f o r g a n i s m s a g a i n s t w h i c h t h ec h e m i c a l s o l u t i o n w a s t o b e t e s t e d . D i s c s o f s t e r i l e f i l t e r p a p e r w h i c h h a d b e e ns u b m e r g e d i n a s o l u t i o n o f t h e c h e m i c a l f o r 2 4 h o u r s p r e v i o u s l y a n dt h e n d r i e d t h o r o u g h l y w e r e p l a c e d o n t h e s u r f a c e o f t h e a g a r i m m e d i a t e l y a f t e ri t s i n o c u l a t i o n w i t h t h e m i c r o o r g a n i s m s . T h e p l a t e s w e r e t h e n i n c u b a t e d f o r4 8 h o u r s a t 3 7 0 C . , e x c e p t S a b o u r a u d ' s m e d i u m , w h i c h w a s k e p t a t room t e m p e r a -t u r e . Z o n e s o f i n h i b i t i o n w e r e a p p a r e n t i n m o s t c a s e s a f t e r 2 4 h o u r s a n d o b -s e r v a t i o n s w e r e made b o t h a f t e r 2 4 a n d 4 8 h o u r s . I n a d d i t i o n t o t h e c h e m i c a l sp r e v i o u s l y t e s t e d , a e r o s p o r i n , e n d o m y c i n , h u m u l o n , l u p u l o n , h e x a h y d r o l u p u l o n ,a s p e r g i l l i c a c i d , a n d g l i o t o x i n w e r e a d d e d t o t h e t e s t s .

S i n c e o n e c a n n o t d e t e r m i n e m i c r o s c o p i c a l l y w h e t h e r a z o n e o f i n h i b i t i o n i st o t a l o r o n l y p a r t i a l , t h e z o n e s w e r e d i v i de d i n t o s m a l l a r e a s , w o r k i n g f r o m t h eb o r d e r o f t h e d i s c t o t h e p e r i p h e r y o f t h e z o n e o f i n h i b i t i o n . S a m p l e s w e r et a k e n f r o m e a c h o f t h e s e a r e a s , t r a n s f e r r e d t o c u l t u r e m e d i u m , a n d i n c u b a t e d .S m e a r s w e r e t h e n m a d e f o r m i c r o s c o p i c e x a m i n a t i o n .

R e s u l t s : I t w a s f o u n d t h a t i n a d d i t i o n t o p a r t i a l o r t o t a l i n h i b i t i o n , s o m ec h e m i c a l s p r o d u c e d t w o w e l l - d e f i n e d z o n e s o f i n h i b i t i o n , e . g . , n e o m y c i n a g a i n s tS t r . f e c a l d i s , g l i o t o x i n a g a i n s t E . c o l i , a e r o s p o r i n a g a i n s t E . c o l i , P e r a z i l a g a i n s ts t a p h y l o c o c c u s , e t c . I n s o m e c a s e s a t h i r d z o n e a p p e a r e d w h i c h may b e c o n -s i d e r e d a z o n e o f s t i m u l a t i o n . S o m e t i m e s t h i s z o n e was s i t u a t e d b e t w e e n t h ez o n e s o f i n h i b i t i o n , a t o t h e r t i m e s i t was o u t s i d e t h e z o n e s o f i n h i b i t i o n .

M e t h o d C . - A f t e r d e t e r m i n i n g t h e q u a l i t a t i v e e f f e c t o f t h e c h e m i c a l s , a ne f f o r t w a s made t o d e t e r m i n e t h e i r q u a n t i t a t i v e e f f e c t . O n l y t h o s e c h e m i c a la g e n t s w h i c h a p p e a r e d t o b e r e l a t i v e l y e f f e c t i v e w e r e i n c l u d e d i n t h e s e t e s t s .

T h e t e s t s w e r e c a r r i e d o u t b y t h e s e r i a l d i l u t i o n m e t h o d i n t h e f o l l o w i n gm a n n e r : S e v e n t u b e s w e r e e a c h f i l l e d w i t h 4 . 5 c . c . o f b r a i n h e a r t i n f us i o n b r o t h ;0 . 5 c . c . o f a 2 4 - h o u r c u l t u r e o f t h e s e l e c t e d o r g a n i s m s was a d d e d t o t h e f i r s tt u b e , w h i c h w a s s h a k e n t o d i s t r i b u t e t h e o r g a n i s m s e v e n l y ; 0 . 5 c . c . was t h e nr e m o v e d f r o m t h e f i r s t t u b e a n d t r a n s f e r r e d t o t h e s e c o n d t u b e , a n d t h i s p r o -c e d u r e w a s c o n t i n u e d u n t i l e a c h o f t h e t u b e s was i n o c u l a t e d . T h e n 0 . 5 o f t h i si n o c u l a t e d medium w a s t r a n s f e r r e d t o a t u b e c o n t a i n i n g m o l t e n a g a r , a n d aP e t r i p l a t e w a s p o u r e d . T h i s w a s r e p e a t e d i n t r i p l i c a t e f o r e a c h t u b e f o r at o t a l o f 2 1 p l a t e s . I n c u b a t i o n was c a r r i e d o u t a t 2 0 C . , 3 7 C . , a n d 4 5 0 C .f o r 4 8 h o u r s . I t was f o u n d t h a t 3 7 0 C . was t h e o p t i m u m t e m p e r a t u r e f o rg r o w t h , f o l l o w e d b y 2 0 0 C . a n d 4 5 0 C . , r e s p e c t i v e l y . L i t t l e d i f f e r e n c e w a sf o u n d b e t w e e n b r a i n h e a r t i n f u s i o n b r o t h a n d S a b o u r a u d ' s medium f o r g r o w -i n g C . a l b i c a i s . T h e n u m b e r o f c o l o n i e s w h i c h d e v e l o p e d u p o n t h e s e p l a t e s w a sc o u n t e d o v e r a F r o s h c o u n t e r . Where t h e c o l o n i e s w e r e n u m e r o u s , o n l y o n e -f i f t h o f e a c h d i s h was c o u n t e d a n d m u l t i p l i e d b y 5 . An a v e r a g e o f t h e 3 p l a t e swas t a k e n i n e a c h c a s e . I n some c a s e s , c o u n t i n g was i m p o s s i b l e o w i n g t o t h el a r g e n u m b e r o f c o l o n i e s .



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V o l u m e 3 5 ANTISEPTICS AND ANTIBIOTICS USED I N ENDODONTICS 659N u m b e r 4SUMMARY AND CONCLUSIONS

1 . A n u m b e r o f a n t i b i o t i c s a n d o t h e r c h e m i c a l a g e n t s w e r e t e s t e d a g a i n s t4 t y p e s o f m i c r o o r g a n i s m s c o m m o n l y f o u n d i n i n f e c t e d r o o t c a n a l s , S t r . f e c a l i s ,S t a p h . a t b u s , E . c o l i , a n d C . a t b i c a n s .2 . F r a d i c i n , t h i o l u t i n , s o d i u m c a p r y l a t e , a n d A s t e r o l w e r e e f f e c t i v e a g a i n s tC . a t b i c a n s , t h e m o s t e f f e c t i v e b e i n g f r a d i c i n .

3 . O f t h o s e a g e n t s t e s t e d , d e c y l ammonium s u l f a t e a n d n e o m y c i n w e r e m o s te f f e c t i v e a g a i n s t E . c o l i .4 . T e r r a m y c i n a n d P e r a z i l w e r e e f f e c t i v e a g a i n s t b o t h S t a p h . a l b u s a n dS t r . f e c a l i s .5 . T h e p o s s i b i l i t y o f c o m b i n i n g 2 o r m o r e a g e n t s i n o r d e r t o p r o d u c e

a w i d e r a n t i b a c t e r i a l s p e c t r u m t h a n e a c h h a s c o m m e n d s i t s e l f . S u c h f a c t o r s a sc o m p a t i b i l i t y , s t a b i l i t y , p o s s i b l e i r r i t a n t e f f e c t , e t c . , w o u l d n e e d t o b e t a k e n i n t oc o n s i d e r a t i o n .

6 . T h e m o r e c o m m o n l y u s e d a n t i b i o t i c s , s u c h a s p e n i c i l l i n , s t r e p t o m y c i n , o rb a c i t r a c i n , w e r e n o t i n c l u d e d i n t h e s e t e s t s a s t h e i r a n t i m i c r o b i a l e f f e c t h a s b e e ns t u d i e d b y o t h e r s .

REFERENCE1 . J a w e t z , E . , G u n n i s o n , J . B . , a n d S p e c k , R . S . : A n t i b i o t i c S y n e r g i s m a n d A n t a g o n i s m ,N e w E n g l a n d J . M e d . 2 4 5 : 9 6 6 , 1 9 5 1 .


an in vitro study of antiseptics and antibiotics used in en~1

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