Enhanced Control of Listeria Monocytogenes by in Situ-produced Pediocin During Dry Fermented Sausage Production

8/6/2019 Enhanced Control of Listeria Monocytogenes by in Situ-produced Pediocin During Dry Fermented Sausage Production

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V o l . 5 8 , N o . 3PPLIED AND ENV IRONMENT AL M I C R O B I O L O G Y , M a r . 1 9 9 2 , p . 8 8 4 - 8 9 00 0 9 9 - 2 2 4 0 / 9 2 / 0 3 0 8 8 4 - 0 7 $ 0 2 . 0 0 / 0C o p y r i g h t 1 9 9 2 , A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y

E n h a n c e d C o n t r o l o f L i s t e r i a monocytogenes b y I n S i t u - P r o d u c e dP e d i o c i n d u r i n g Dry F e r m e n t e d S a u s a g e P r o d u c t i o n tP . M. FOEGEDING,* A . B . THOMAS, D . H . PILKINGTON, AND T . R . KLAENHAMMER

D e p a r t m e n t o f Food S c i e n c e , N o r t h C ar o l i n a S t a t e U n i v e r s i t y , R a l e i g h , N o r t h C a r o l i n a 2 7 6 9 5 - 7 6 2 4R e c e i v e d 9 A u g u s t 1 9 9 1 / A c c e p t e d 2 0 December 1 9 9 1

To d e t e r m i n e w h e t h e r p e d i o c i n i s p r o d u c e d a n d h a s e f f e c t i v e a n t i l i s t e r i a l a c t i v i t y d u r i n g f o o d f e r m e n t a t i o n ,s i x sausage f e r m e n t a t i o n t r i a l s were c o n d u c t e d w i t h a n t i b i o t i c - r e s i s t a n t , p e d i o c i n - p r o d u c i n g ( B a c + ) P e d i o c o c -cus a c i d i l a c t i c i P A C 1 . 0 ( S t r r R i F ) a n d an i s o g e n i c p e d i o c i n - n e g a t i v e ( B a c - ) d e r i v a t i v e u s e d as a c o n t r o l . Meatwas i n o c u l a t e d ( c a . 1 0 5 C F U / g ) w i t h a c o m p o s i t e o f f i v e L i s t e r i a m o n o c y t o g e n e s s t r a i n s , e a c h e l e c t r o t r a n s f o r m e dw i t h pGK12 ( C m r E m r ) . P . a c i d i l a c t i c i a n d L . m o n o c y t o g e n e s p o p u l a t i o n s were s e l e c t i v e l y e n u m e r a t e d b yp l a t i n g on m e d i a w i t h a n t i b i o t i c s . T h i s s t u d y i n d i c a t e d t h a t t h e d r y sausage f e r m e n t a t i o n process can r e d u c eL . m o n o c y t o g e n e s p o p u l a t i o n s . E f f e c t i v e i n a c t i v a t i o n o f L . m o n o c y t o g e n e s was o b s e r v e d when t h e pH a t t h e e n do f t h e f e r m e n t a t i o n p o r t i o n o f t h e process was < 4 . 9 . P e d i o c i n was r e s p o n s i b l e f o r p a r t o f t h e a n t i l i s t e r i a la c t i v i t y d u r i n g t h e f e r m e n t a t i o n i n e a c h o f t h e s i x t r i a l s . F u r t h e r m o r e , i n h i b i t i o n o f L . m o n o c y t o g e n e s d u r i n gd r y i n g was e n h a n c e d i n t h e presence o f p e d i o c i n i n t h e t h r e e t r i a l s i n w h i c h L . m o n o c y t o g e n e s c o u l d b e d e t e c t e dt h r o u g h o u t t h e d r y i n g process. T h u s , p e d i o c i n p r o d u c t i o n c o n t r i b u t e d t o a n i n c r e a s e i n s a f e t y d u r i n g b o t h t h ef e r m e n t a t i o n a n d d r y i n g p o r t i o n s o f sausage m a n u f a c t u r i n g .

F o o d - b o r n e l i s t e r i o s i s o u t b r e a k s h a v e b e e n a s s o c i a t e dw i t h t h e c o n s u m p t i o n o f c o l e s l a w ( 3 0 ) , m i l k ( 8 ) , a n d c h e es ep r o d u c t s ( 2 ) . Two cases o f l i s t e r i o s i s h a v e b e e n l i n k e d t oc o n s u m p t i o n o f p o ul t ry p r o d u c t s ( 3 , 1 9 ) . A l t h o u g h a m a j o ro u t b r e a k o f l i s t e r i o s i s r e l a t e d t o meat c o n s u m p t i o n h a s n o tb e e n i d e n t i f i e d , t h e a s s o ci a t i o n o f L i s t e r i a monocytogenesw i t h m e a t , i n c l u d i n g c u r e d a n d f e r m e n t e d p r o d u c t s , i s w e l ld o c u m e n t e d ( 6 , 7 , 2 5 , 2 6 , 3 2 ) . R e c e n t s t u d i e s h a v e shownt h a t L . monocytogenes may s u r v i v e t h e c o m b i n a t i o n o f l o wp H , l o w water a c t i v i t y , s o d i u m c h l o r i d e , a nd s o d i u m n i t r i t ea s s o c i a t e d w i t h t h e f e r m e n t a t i o n a n d d r y i n g o f meat ( 4 , 9 , 1 6 ,1 7 ) .I n h i b i t i o n o f L . monocytogenes b y b a c t e r i o c i n s p r o d u c e db y P e d i o c o c c u s a c i d i l a c t i c i may p r o v i d e a d d e d c o n t r o la g a i n s t t h i s p a t h o g e n i n f e r m e n t e d meat p r o d u c t s . P . a c i d i -l a c t i c i P A C 1 . 0 p r o d u c e s a b a c t e r i o c i n d e s i g n a t e d p e d i o c i nP A - 1 ( 1 1 ) a n d h a s i n h i b i t o r y a c t i v i t y d i r e c t e d a g a i n s t L.monocytogenes ( 1 2 , 2 8 ) . A l t h o u g h p e d i o c i n s ar e known t oi n h i b i t L . monocytogenes ( 5 , 1 2 , 1 4 , 2 8 , 3 3 ) , no one h a sc l e a r l y i n d i c a t e d t h a t b a c t e r i o c i n p r o d u c t i o n i n f o o d pro-v i d e s a d d e d s a f e t y a b o v e a n d b e y o n d t h a t p r o v i d e d b y o t h e rf e r m e n t a t i o n e n d p r o d u c t s . One p r o b l e m i s t h e l ac k o f amethod f o r e n u m e r a t i n g m i x e d p o p u l a t i o n s , p a r t i c u l a r l ywhen l o w t a r g e t p o p u l a t i o n s ar e e n c o u n t e r e d a n d e n r i c h -ment p r o t o c o l s ar e r e q u i r e d . A s e c o n d p r o b l e m i s t h e n e e df o r a proper c o n t r o l f e r m e n t a t i o n w h e r e a l l v a r i a b l e s e x c e p tt h e p o s s i b l e b a c t e r i o c i n p r o d u c t i o n a n d a c t i v i t y ar e h e l d

c o n s t a n t . To a dd re s s t h e s e i s s u e s , we e m p l o y e d a s t r e p t o -m y c i n - a n d r i f a m p i n - r e s i s t a n t b a c t e r i o c i n - p r o d u c i n g P . a c i d -i l a c t i c i s t r a i n a n d i t s i s o g e n i c d e r i v a t i v e , w h i c h h a d b e e nc u r e d o f t h e p l a s m i d e n c o d i n g b a ct e ri o c i n p r o du c t i o n, t of e r m e n t meat i n o c u l a t e d w i t h L . monocytogenes. The L .monocytogenes c e l l s were g e n e t i c a l l y m a r k e d by e l e c t r o -t r a n s f o r m a t i o n w i t h p l a s m i d pGK12, w h i c h e n c o d e s r e s i s -t a n c e t o c h l o r a m p h e n i c o l a n d e r y t h r o m y c i n ( 2 0 ) . T h u s , b o t h

* C o r r e s p o n d i n g a u t h o r .t P a p e r F S 9 1 - 2 8 of t h e Journal S e r i e s o f t h e D e p a r t m e n t o f FoodS c i e n c e , N o r t h C a r o l i n a S t a t e U n i v e r s i t y , R a l e i g h , NC 2 7 6 9 5 - 7 6 2 4 .

p e d i o c o c c a l a n d l i s t e r i a l p o p u l a t i o n s w e r e r e a d i l y e n u m e r -a t e d o n a n t i b i o t i c - c o n t a i n i n g m e d i a . T h i s s t u d y i n d i c a t e dt h a t m e a t f e r me nt a t i o n i n a n d o f i t s e l f was e f f e c t i v e i nc o n t r o l l i n g L . m o n o c y t o g e n e s , e s p e c i a l l y i f t h e pH a t t h e e n do f f e r m e n t a t i o n was l e s s t h a n 4 . 9 . When t h e pH was n o tl o w e r e d s u f f i c i e n t l y , L . m o n o c y t o g e n e s s u r v i v e d t h e f e r m e n -t a t i o n a n d b a c t e r i o c i n c o n t r i b u t e d t o i n a c t i v a t i o n o f L .m o n o c y t o g e n e s d u r i n g t h e d r y i n g p r o c e s s .M A T E R I A L S AND M E T H O D S

B a c t e r i a l c u l t u r e s a n d m e d i a . L . m o n o c y t o g e n e s c u l t u r e s( S c o t t A [ c l i n i c a l i s o l a t e , s e r o t y p e 4 b , o r i g i n a l l y o b t a i n e df r o m C . D o n n e l l y , U n i v e r s i t y o f V e r m o n t ] , F 5 0 6 9 [ r a w m i l ki s o l a t e , s e r o t y p e 4 b , o r i g i n a l l y o b t a i n e d f r o m C . D o n n e l l y ] ,A T CC 1 9 1 1 5 [ c l i n i c a l i s o l a t e , s e r o t y p e 4 b , f r o m t h e A m e r i -c a n T y p e C u l t u r e C o l l e c t i o n , R o c k v i l l e , M d . ] , a n d NCF-U2K3 a n d NCF-F1KK4 [ l i q u i d w h o l e e g g i s o l a t e s f r o m o u rl a b o r a t o r y ] ) w e r e 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 H I ) b r o t h( D i f c o , D e t r o i t , M i c h . ) a t 3 7 C . B a c t e r i o c i n - p r o d u c i n g( B a c + ) P . a c i d i l a c t i c i P A C 1 . 0 s t r a i n s ( o b t a i n e d f r o m D .H o o v e r , U n i v e r s i t y o f D e l a w a r e ) a n d b a c t e r i o c i n - n e g a t i v e( B a c - ) P . a c i d i l a c t i c i P A C 1 . 0 ( 1 1 ) s t r a i n s w e r e g r o w n i nMRS b r o t h ( D i f c o ) a t 3 7 C . C u l t u r e s w e r e s t o r e d a t - 2 0 C i nd o u b l e - s t r e n g t h b r o t h w i t h 20% g l y c e r i n ( F i s h e r S c i e n t i f i cC o . , P i t t s b u r g , P a . ) . B e f o r e e x p e r i m e n t a l u s e , c e l l s w e r es u b c u l t u r ed t w i ce ( 1 % i n o c u l u m f o r 2 4 h ) . S o l i d medium wasp r e p a r e d b y a d d i n g 1 . 5 % g r a n u l a t e d a g a r ( D i f c o ) t o t h eb r o t h . T h e o v e r l a y s w e r e p r e p a r e d w i t h 0 . 7 5 % a g a r . G r o w t ha n d s e l e c t i o n o f a n t i b i o t i c - r e s i s t a n t o r g a n i s m s u s e d , u n l e s so t h e r w i s e n o t e d , 1 0 , u g o f c h l o r a m p h e n i c o l p e r m l a n d 5 , u go f e r y t h r o m y c i n p e r m l i n BHI o r 5 0 0 , u g o f s t r e p t o m y c i n p e rm l a n d 5 0 , u g o f r if am pi n p e r m l i n MRS. In a l l m e a tf e r m e n t a t i o n t r i a l s , 5 , u g o f c h l o r a m p h e n i c o l p e r m l a n d 2 , u go f e r y t h r o m y c i n p e r m l i n BHI w e r e u s e d f o r t h e r e c o v e r y o fL . m o n o c y t o g e n e s . A s p e c i f i c r e s u s c i t a t i o n s t e p was n o tn e e d e d t o s e l e c t i v e l y r e c o v e r h e a t e d l i s t e r i a c e l l s ( d a t a n o ts h o w n ) . T h e a n t i b i o t i c s w e r e f r o m S i g m a C h e m i c a l C o . ( S t .L o u i s , M o . ) .P l a s m i d DNA e x t r a c t i o n a n d v i s u a l i z a t i o n . P l a s m i d pGK12

8 8 4


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L . M O N OCYT O GE N ES I N SAUSAGE 8 8 5( 2 0 ) , u s e d i n t h e e l e c t r o p o r a t i o n e x p e r i m e n t s , was i s o l a t e df r o m E s c h e r i c h i a c o l i J M 1 1 0 ( 2 1 ) . P l a s m i d s f o r m o l e c u l a rw e i g h t m a r k e r s w e r e i s o l a t e d f r o m E . c o l i V 1 5 7 ( 2 2 ) . L a r g e -s c a l e e x t r a c t i o n o f p l a s m i d DNA was b y t h e a l k a l i p r o c e d u r eo f R o d r i g u e z a n d T a i t ( 2 9 ) . P l a s m i d DNA was p u r i f i e dt h r o u g h c e s i u m c h l o r i d e - e t h i d i u m b r o m i d e e q u i l i b r i u m d e n -s i t y g r a d i e n t s ( 2 3 ) . S m a l l - s c a l e e x t r a c t i o n o f p l a s m i d DNAwas a s d e t a i l e d b y A n d e r s o n a n d McKay ( 1 ) , e x c e p t t h a tl y s o z y m e w a s i n c r e a s e d t o 1 5 m g / m l w i t h 3 0 m i n o f i n c u b a -t i o n a t 3 7 C a n d p h e n o l was r e m o v e d b y t w o e x t r a c t i o n sw i t h 3 0 0 , u 1 o f c h l o r o f o r m - i s o a m y l a l c o h o l ( 2 4 : 1 ) .E l e c t r o p o r a t i o n . L . m o n o c y t o g e n e s was e l e c t r o p o r a t e d a sd e t a i l e d b y L u c h a n s k y e t a l . ( 2 1 ) w i t h 2 . 5 x P E B e l e c t r o p o -r a t i o n b u f f e r a t 6 , 2 5 0 V c m - ' a n d 2 5 , u F i n 0 . 2 - c m c u v e t t e s .T r a n s f o r m a n t s o f s t r a i n s S c o t t A , A TCC 1 9 1 1 5 , a n d F 5 0 6 9w e r e s e l e c t e d f i r s t o n BHI a g a r w i t h c h l o r a m p h e n i c o l ( 7 . 5u g / m l ) a n d t h e n p l a t e d o n BHI a g a r w i t h e r y t h r o m y c i n ( 3 . 0, u g / m l ) . O t h e r t r a n s f o r m a n t s w e r e s e l e c t e d o n BHI a g a r w i t he a c h a n t i b i o t i c a t 3 , u g / m l .P l a s m i d s t a b i l i t y s t u d i e s . S i x s u c c e s s i v e 1% t r a n s f e r s o f L .m o n o c y t o g e n e s c o n t a i n i n g pGK12 i n BHI b r o t h w e r e i n c u -b a t e d a t 3 7 C f o r 2 4 h . A t e a c h t r a n s f e r i n t e r v a l , s a m p l e sw e r e d i l u t e d a n d p l a t e d o n BHI a g a r ; 1 0 0 i s o l a t e d c o l o n i e sw e r e t r a n s f e r r e d t o BHI a g a r w i t h c h l o r a m p h e n i c o l . T h ep e r c e n t l o s s o f pGK12 d u r i n g g r o w t h i n BHI b r o t h wasd e t e r m i n e d f r o m t h e p e r c e n t a g e o f c o l o n i e s t h a t di d n o t g r o wo n t h e BHI a g a r w i t h c h l o r a m p h e n i c o l .A f i v e - s t r a i n c o m p o s i t e c o n s i s t i n g o f e q u a l a l i q u o t s o ft r a n s f o r m e d L . m o n o c y t o g e n e s c u l t u r e s w a s u s e d t o i n o c u -l a t e a u t o c l a v e d ( 1 2 1 C , 1 5 m i n ) g r o u n d p o r k . T h e i n o c u l a t e dp o r k w a s i n c u b a t e d f o r 2 4 h a t 3 7 C . T h e p o r k c u l t u r e w a sm i x e d ( c a . 5% i n o c u l u m ) i n t o a u t o c l a v e d p o r k , a n d t h i sc u l t u r e was a l s o i n c u b a t e d f o r 2 4 h a t 3 7 C . A f t e r e a c h 2 4 - hi n t e r v a l , p o r t i o n s o f t h e p o r k c u l t u r e s w e r e d i l u t e d i n p e p -t o n e - w a t e r a n d p l a t e d o n BHI a g a r . T h e p e r c e n t l o s s o fp l a s m i d s w a s d e t e r m i n e d a s d e t a i l e d a b o v e .B a c t e r i o c i n d e t e c t i o n a n d a s s a y s . P . a c i d i l a c t i c i s t r a i n sw e r e e x a m i n e d f o r b a c t e r i o c i n p r o d u c t i o n b y d i r e c t ( 3 4 ) a n dd e f e r r e d ( 1 8 ) m e t h o d s . A m o d i f i c a t i o n o f t h e c r i t i c a l d i l u t i o na s s a y ( 2 4 ) w a s u s e d t o d e t e r m i n e t h e b a c t e r i o c i n t i t e r . S e r i a lt w o f o l d d i l u t i o n s o f s t e r i l e c u l t u r e s u p e r n a t a n t w e r e s p o t t e d( 1 0 , u l ) o n t o i n d i c a t o r l a w n s o f L a c t o b a c i l l u s s p . s t r a i n LA51( a l o w - p H - a n d a c i d - r e s i s t a n t o r g a n i s m f r o m H . F l e m i n g ,U . S . D e p a r t m e n t o f A g r i c u l t u r e , R a l e i g h , N . C . ) o r L . m o n o -c y t o g e n e s s t r a i n s a n d i n c u b a t e d f o r 1 6 t o 1 8 h a t 3 7 C .I n d i c a t o r l a w n s w e r e p r e p a r e d b y a d d i n g 0 . 1 m l o f a no v e r n i g h t c u l t u r e ( 1 8 h , 3 7 C ) t o 4 m l o f o v e r l a y a g a r . T h et i t e r was d e f i n e d a s t h e r e c i p r o c a l o f t h e h i g h e s t d i l u t i o ne x h i b i t i n g c o m p l e t e i n h i b i t i o n o f t h e i n d i c a t o r l a w n a n d w a se x p r e s s e d i n a c t i v i t y u n i t s p e r m i l l i l i t e r . B a c t e r i o c i n a s s a y sw i t h s a m p l e s n e u t r a l i z e d t o p H 7 . 0 w e r e i n c l u d e d t o e l i m i -n a t e i n h i b i t i o n d u e t o a c i d o r l o w p H .M u t a n t i s o l a t i o n a n d s c r e e n i n g . S p o n t a n e o u s m u t a n t s o f P .a c i d i l a c t i c i P A C 1 . 0 w i t h r e s i s t a n c e t o s t r e p t o m y c i n ( 2 , 0 0 0, u g / m l ) a n d r i f a m p i n ( 2 0 0 , u g / m l ) w e r e i s o l a t e d b y s e q u e n t i a ls e l e c t i o n o n M R S a g a r c o n t a i n i n g i n c r e a s i n g a n t i b i o t i c c o n -c e n t r a t i o n s . B a c - d e r i v a t i v e s w e r e i s o l a t e d f r o m a n t i b i o t i c -r e s i s t a n t c e l l s b y r e p e a t e d c u l t u r i n g i n b r o t h c o n t a i n i n g 2 0 , u ga c r i f l a v i n p e r m l a t 4 7 C f o r 1 8 h . A f t e r t h r e e t r a n s f e r s ,a p p r o p r i a t e d i l u t i o n s w e r e p l a t e d o n MRS a g a r w i t h s t r e p -t o m y c i n a n d r i f a m p i n a n d i n c u b a t e d f o r 2 4 h a t 3 7 C .C o l o n i e s w e r e r e p l i c a p l a t e d t o t e s t f o r b a c t e r i o c i n p r o d u c -t i o n a g a i n s t L a c t o b a c i l l u s s p . s t r a i n L A 5 1 . S u s p e c t e d Bac-c o l o n i e s w e r e p i c k e d f r o m t h e o r i g i n a l p l a t e a n d g r o w n i nMRS a t 3 7 C f o r 2 4 h . L o s s o f b a ct e r i oc in p r o d u c t i o n wasc o n f i r m e d b y d i r e c t a n d d e f e r r e d a n t a g o n i s m a n d b y p l a s m i d

v i s u a l i z a t i o n . P l a s m i d s w e r e e x t r a c t e d b y t h e m o d i f i e ds m a l l - s c a l e e x t r a c t i o n m e t h o d d e t a i l e d a b o v e .P r e p a r a t i o n o f b a c t e r i a l i n o c u l a f o r s a u s a g e f e r m e n t a t i o n . Af i v e - s t r a i n c o m p o s i t e o f t h e t r a n s f o r m e d L . m o n o c y t o g e n e sc u l t u r e s was u s e d f o r t h e i n o c u l a t e d m e a t s t u d i e s . S t r a i n sw e r e g r o w n i n d i v i d u a l l y i n BHI a t 3 7 C f o r 1 8 h . E q u a lp o r t i o n s o f e a c h c u l t u r e w e r e m i x e d t o y i e l d c a . 1 0 5 CFU/gwhen a d d e d t o t h e m e a t .P . a c i d i l a c t i c i w e r e s u b c u l t u r e d t w i c e i n MRS b r o t hc o n t a i n i n g 4 . 5 % N a C l a t 3 7 C f o r 1 8 h , c o n c e n t r a t e d b yc e n t r i f u g a t i o n ( 4 , 0 0 0 x g , 1 5 m i n ) , a n d r e s u s p e n d e d i ns t e r i l e 0 . 1 % D i f c o p e p t o n e - w a t e r f o r a n i n o c u l u m o f c a . 1 0 8C F U / g when a d d e d t o t h e m e a t .S a u s a g e m a n u f a c t u r e . S i x i n d e p e n d e n t s a u s a g e t r i a l s w e r ec o n d u c t e d , e a c h c o n s i s t i n g o f t w o s i m u l t a n e o u s f e r m e n t a -t i o n s w i t h e i t h e r t h e S t r r R i f ' B a c + P . a c i d i l a c t i c i s t r a i n o r i t si s o g e n i c Bac- d e r i v a t i v e . S a u s a g e was made f r o m f r e s h p o r ks h o u l d e r s . T h e s h o u l d e r s w e r e d e b o n e d , a n d t h e s u b c u t a n e -o u s f a t l a y e r was r e m o v e d . T h e d e f a t t e d p o r k was vacuump a c k a g e d a n d s t o r e d a t - 2 0 C f o r


3/7

8 8 6 F O E G E D I N G ET A L .F S 069

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F I G . 1 . A g a r o s e g e l e l e c t r o p h o r e s i s o f p l a s m i d DNA i s o l a t e df r o m p a r e n t a l a nd p G K 1 2 - t r a n s f o r m e d L . monocytogenes F 5 0 6 9 .L a n e s : A , E . c o l i V157 r e f e r e n c e p l a s m i d s ( s i z e s i n k i l o b a s e si n d i c a t e d b y a r r o w s ) ; B , pGK12 p l a s m i d DNA i s o l a t e d f r o m E . c o l iJ M 1 1 0 ; P a n d T , p a r e n t a l a n d t r a n s f o r m e d L . monocytogenesc u l t u r e s , r e s p e c t i v e l y .

a n d e r y t h r o m y c i n . P . a c i d i l a c t i c i was e n u m e r a t e d on M R Spour p l a t e s c o n t a i n i n g s t r e p t o m y c i n a n d r i f a m p i n a f t e r 7 2 ho f i n c u b a t i o n a t 3 7 C .The w e i g h t s o f f i v e sausages were r e c o r d e d b e f o r e a n dp e r i o d i c a l l y d u r i n g f e r m e n t a t i o n a n d d r y i n g a n d u s e d t oc a l c u l a t e t h e p e r c e n t w e i g h t l o s s d u r i n g f e r m e n t a t i o n a n dd r y i n g .The sausage pH a n d t i t r a t a b l e a c i d i t y , e x p r e s s e d as t h ep e r c e n t l a c t i c a c i d , were d e t e r m i n e d as d e t a i l e d b y S e b r a n e k( 3 1 ) . pH was m o n i t o r e d b y u s i n g a c o m b i n a t i o n e l e c t r o d e( F i s h e r S c i e n t i f i c C o . , P i t t s b u r g , P a . ) a n d an Accumet 9 1 0p H meter ( F i s h e r ) . T i t r a t i o n s were t o an e n d p o i n t p H o f 8 . 3 .

R ESUL TSE l e c t r o p o r a t i o n o f L . m o n o c y t o g e n e s . P l a s m i d p r o f i l e s o fp a r e n t a l a n d e l e c t r o t r a n s f o r m e d L . monocytogenes F 5 0 6 9

ar e shown i n F i g . 1 . A l l f i v e t r a n s f o r m e d s t r a i n s c o n t a i n e dt h e 4 . 4 - k b p l a s m i d ( d a t a n o t s h o w n ) . The a c q u i s i t i o n o fp l a s m i d pGK12 was c o n f i r m e d p h e n o t y p i c a l l y b y r e s i s t a n c et o 1 0 p g e a c h o f c h l o r a m p h e n i c o l a n d e r y t h r o m y c i n per m l .The g r o w t h r a t e s i n BHI o f t h e t r a n s f o r m a n t s were 0 . 7 t o 1 . 2t i m es t h o s e o f t h e p a r e n t c u l t u r e s .P l a s m i d s t a b i l i t y . The s t a b i l i t y o f pGK12 i n e a c h o f t h e f i v eL . monocytogenes grown i n BHI was 9 9 t o 1 0 0 % a f t e r s i xs u c c e s s i v e t r a n s f e r s w i t h o u t s e l e c t i v e pressure t o r e t a i n t h e

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FIG. 2 . Agarose ge l electrop h oresis of p l a s m i d DNAfrom P.acidilactici PAC1.0 Strr Rif Back and Strr R i f ' Bac- derivativestrains. Lanes: ref, E. coli V157 reference p la smids; +, pa re n t a lstrain PAC1.0 ( S t r r Rif Suc' B a c ' ) ; -,PAC1.0 derivative curedof the 9.4-kb p l a s m i d ( S t r r R i f ' Suc' B a c - ) . The 34.5- and 9.4-kbp l a s m i d s areindicated to the right.

p l as m i d . Plasmid stability in autoclaved ground pork withthe five strains asa composite culture ranged from 74 to 99%after twosuccessive transfers.

P. acidilactici mutant isolation and s c r e e n i n g . A tr r R i f 'chromosomal mutant of P. acidilacticiPAC1.0wasselectedto p e r m i t m o n i t o r i n g the starter culture p op u la tion d u r i n gthe fermentation process. The P. acidilactici PAC1.0 ( S t r rRif B a c ' ) strain wascompared with the p a renta l P. acidi-lactici PAC1.0 strains for growth characteristics and acidp r o d uc t i o n . The growth and acid p r o d u c t i o n rates of bothcultures werevirtua lly identical (data not shown), indicatingthat chromosomal marking of thePAC1.0 strain did not alterthese properties, which arei m p o r t a n t to sausage fermenta-tion.The Bac- control starter culture was isolated by cur i n g

the antibiotic-resistant Bac' culture of the 9.4-kb p l a s m i dthat encodes p e d i o c in p r o d u ct i o n. The p l a s m i d - c u r e d ( B a c - )derivative was m i s s i n g the 9.4-kb p l a s m i d (Fig. 2 ) . Theinability to produce bacteriocin wasverified by direct anddeferred antagonism assays against Lactobacillus Sp. strainLA51 (data not shown) and L. monocytogenes ( T ab l e 1 ) .

Pediocin antagonism against L. monocytogenes. Deferredand direct antagonism of P. acidilactici against parental andtransformed L. monocytogenes strains wasmeasured ( T ab l e1 ) . The parental strains and transformed L. monocytogenesderivatives were simi larly sensitive to p edi oci n. Therefore,the presence of pGK12 in L. monocytogenes had no sub-stantial effect onit s sensitivity to pediocin. Unusual d i f f e r -encesin the s e n s i t i v i t y or resistance to p e d i o c i n were not

T A B L E 1 . A n t a g o n i s m o f L . m o n o c y t o g e n e s s t r a i n s b e a r i n g pGK12 b y P . a c i d i l a c t i c i aD e f e r r e d a n t ag o n i s m ( zo n e s i z e i n mm) D i r e c t a n t a g o n i s m ( a c t i v i t y u n i t s / m l )L . m o n o c y t o g e n e ss t r a i n B a c + ( P A C 1 . 0 S t r r R i f ' B a c + S t r r R i f ' B a c - B a c + ( P A C 1 . 0 S t r ' R i f r B a c + S t r r R i f r Bac-p a r e n t ) p a r e n t )

S c o t t A 1 . 0 1 . 0 0 8 0 0 1 , 6 0 0 0A T CC 1 9 1 1 5 4 . 0 1 . 0 0 4 0 0 1 , 6 0 0 0F 5 0 6 9 5 . 0 2 . 0 0 8 0 0 1 , 6 0 0 0NCF-U2K3 5 . 0 2 . 0 0 8 0 0 1 , 6 0 0 0NCF-F1KK4 6 . 0 2 . 0 0 8 0 0 1 , 6 0 0 0a P h e n o t y p e s g i v e n i n c o l u m n s u b h e a d i n g s a r e t h o s e o f t h e P . a c i d i l a c t i c i s t r a i n s u s e d f o r a n t a g o n i s m a s s a y s .

A P P L . E N V I R O N . M I C R O B I O L .


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F I G . 3 . S a u s a g e f e r m e n t a t i o n i n 7 7 - m m - d i a m e t e r c a s i n g s , t r i a l 1 . S y m b o l s : 0a n d * , P . a c i d i l a c t i c i PAC 1 . 0 S t r r R i f ' ; Aa n d A, L .monocytogenes ( p G K 1 2 ) f i v e - s t r a i n c o m p o s i t e ; O a n d * , p H ; 0a n d * , t i t r a t a b l e a c i d i t y ( T . A . ) ; A , * , a n d * , B a c + f e r m e n t a t i o n ; A , [ 1 , a n d0 , Bac- f e r m e n t a t i o n . Arrows i n d i c a t e p o pu l at i o ns o f


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F I G . 4 . S a u s a g e f e r m e n t a t i o n i n 7 7 - m m - d i a m e t e r c a s i n g s , t r i a l 2 . S y m b o l s a r e a s d e f i n e d i n t h e l e g e n d t o F i g . 3 .D e t e c t a b l e L . monocytogenes r e m a i n e d a f t e r t h e 4 8 - hf e r m e n t a t i o n p e r i o d f o r a l l t r i a l s . The i n h i b i t o r y e f f e c t o fb a c t e r i o c i n c o u l d n o t b e e v a l u a t e d d u r i n g d r y i n g f o r t h r e et r i a l s i n w h i c h t h e pH was g e n e r a l l y 4 . 8 a t t h e e n d o f t h ef e r m e n t a t i o n p e r i o d ; L . monocytogenes p o p u l a t i o n s were


6/7

L . M O N OCYT O GE N ES IN SAUSAGE 8 8 9T A B L E 2 . S u m m a r y o f p i l o t p l a n t f e r m e n t a t i o n s

Summary o f e f f e c t c o fL . m o n o c y t o g e n e s p o p u l a t i o n a u s i n g B a c + o r B a c -C a s i n g B a c pH a t e n d o f s t r a i n d u r i n g :d i a m e t e r p h e n o t y p e End o f f e r m e n t a t i o n End o f d r y i n g f e r m e n t a t i o n( m m ) I n i t i a l l o gC F U / g L o g CFU/g d e c r e a s e b L o g CFU/g d e c r e a s e F e r m e n t a t i o n D y i n g

9 9 1 B a c + 5 . 0 0 ( 0 . 1 1 ) 2 . 8 0 ( 0 . 0 2 ) 2 . 2 0 5 . 0 0 4 . 8B a C - 5 . 0 1 ( 0 . 0 9 ) 3 . 0 0 ( 0 . 0 6 ) 2 . 0 1 5 . 0 1 4 . 9 ( + ) NA9 9 2 B a c + 4 . 9 6 ( 0 . 0 3 ) 3 . 0 4 ( 0 . 0 3 ) 1 . 9 2 4 . 9 6 4 . 8B a C - 4 . 4 8 ( 0 . 0 1 ) 3 . 9 8 ( 0 . 0 1 ) 0 . 5 0 4 . 4 8 4 . 8 + NA7 7 1 B a c ' 5 . 0 0 ( 0 . 0 7 ) 1 . 9 0 ( 0 . 0 1 ) 3 . 1 0 5 . 0 0 4 . 8B a c - 5 . 0 4 ( 0 . 0 2 ) 2 . 9 4 ( 0 . 0 1 ) 2 . 1 0 5 . 0 4 4 . 8 + NA7 7 2 B a c k 5 . 1 1 ( 0 . 0 3 ) 2 . 9 1 ( 0 . 0 4 ) 2 . 2 0 5 . 0 1 4 . 9Bac- 5 . 0 0 ( 0 . 0 6 ) 3 . 9 6 ( 0 . 0 2 ) 1 . 0 4 2 . 3 6 ( 0 . 0 2 ) 2 . 6 4 4 . 9 + +5 2 1 B a c + 4 . 8 5 ( 0 . 0 2 ) 3 . 5 2 ( 0 . 0 5 ) 1 . 3 3 2 . 1 2 ( 0 . 0 2 ) 2 . 7 3 5 . 2B a c - 3 . 8 4 ( 0 . 0 2 ) 2 . 9 5 ( 0 . 0 4 ) 0 . 8 9 1 . 9 3 ( 0 . 0 2 ) 1 . 9 1 5 . 1 ( + ) ( + )5 2 2 B a c + 4 . 9 1 ( 0 . 0 1 ) 3 . 2 9 ( 0 . 1 0 ) 1 . 6 2 0 . 7 4 ( 0 . 0 5 ) 4 . 1 7 5 . 0B a c - 4 . 7 8 ( 0 . 0 2 ) 4 . 7 6 ( 0 . 0 3 ) 0 . 0 2 2 . 6 6 ( 0 . 0 7 ) 2 . 1 2 5 . 0 + +a V a l u e s r e p o r t e d a r e m e a n s o f t r i p l i c a t e d e t e r m i n a t i o n s . V a l u e s w i t h i n p a r e n t h e s e s r e p r e s e n t s t a n d a r d e r r o r s ." D e c r e a s e s i n L . m o n o c y t o g e n e s p o p u l a t i o n s w e r e d e t e r m i n e d b y s u b t r a c t i n g t h e l o g p o p u l a t i o n a t t h e e n d o f e i t h e r f e r me n t at i o n o r d r y i n g f r o m t h e l o g i n i t i a lp o p u l a t i o n . T h u s , t h e s e d e c r e a s e s d o n o t r e f l e c t t h e o v e r a l l p o p u l a t i o n d i f f e r e n c e s d u r i n g t h e p r o c e s s .c + , a d v a n t a g e ( > 1 l o g u n i t d i f f e r e n c e ) ; ( + ) , q u e s t i o n a b l e a d v a n t a g e ( d i f f e r e n c e o f < 1 l o g u n i t ) ; NA , n o t a p p l i c a b l e ( i n c o n c l u s i v e d a t a ) .

r e q u i r e d f o r g r o w t h o f L . m o n o c y t o g e n e s ; t h i s may l i m i tm e t a b o l i c e n er g y g en er at i o n a n d h i n d e r p l a s m i d r e t e n t i o nu n d e r n o n s e l e c t i v e c o n d i t i o n s . In t h e s e s t u d i e s , t h e a u t o -c l a v e d g r o u n d p o r k d i d n o t c o n t a i n a d d e d c a r b o h y d r a t e .W i t h t h e a d d i t i o n o f g l u c o s e t o t h e s a u s a g e f o rm u l a ti o n,h i g h e r p l a s m i d s t a b i l i t y may h a v e o c c u r r e d . T h i s was n o te v a l u a t e d .A n t i b i o t i c - r e s i s t a n t m u t a n t s o f P . a c i d i l a c t i c i P A C 1 . 0w e r e s e l e c t e d t o p e r m i t e n u m e r a t i o n o f t h e s t a r t e r c u l t u r ed ur i ng s a u s a g e m a n u f a c t u r e . T h e P . a c i d i l a c t i c i P A C 1 . 0 S t r rR i f T B a c + s t r a i n g r e w s l i g h t l y s l o w e r i n BHI t h a n d i d t h ep a r e n t c u l t u r e , b u t a c i d p r o d u c t i o n a n d p e d i o c i n p r o d u c t i o nw e r e c o m p a r a b l e t o t h o s e o f t h e p a r e n t a l P . a c i d i l a c t i c i .T h e s e d a t a s u g g e s t e d t h a t t h i s m u t a n t w o u l d p e r f o r m a c c e p t -a b l y ( g r o w a n d p r o d u c e a c i d a n d b a c t e r i o c i n a t a d e q u a t er a t e s a n d i n a p p r o p r i a t e a m o u n t s ) i n t h e s a u s a g e f e r m e n t a -t i o n e x p e r i m e n t s .H a r r i s e t a l . ( 1 3 ) u s e d c h r o m o s o m a l l y e n c o d e d a n t i b i o t i cr e s i s t a n c e t o q u a n t i t i a t e p o p u l a t i o n s o f a m i x e d s t a r t e rc u l t u r e o f L a c t o c o c c u s l a c t i s a n d L e u c o n o s t o c m e s e n t e r o i -d e s d u r i n g a m o d e l s a u e r k r a u t f e r m e n t a t i o n . T h e a p p r o a c hu s e d b y H a r r i s a n d c o w o r k e r s was s i m i l a r t o t h a t u s e d t om o n i t o r P . a c i d i l a c t i c i i n t h i s s t u d y . T h e s e t w o m e t h o d s t og e n e t i c a l l y m a r k o r g a n i s m s o f i n t e r e s t , w i t h e i t h e r p l a s m i d -o r c h r o m o s o m e - e n c o d e d d e t e r m i n a n t s , p e r m i t s e l e c t i o n o fc u l t u r e s f r o m a m i x e d b a c k g r o u n d m i c r o f l o r a a n d p r o v i d em o d e l s t o m o n i t o r m i n o r i t y p o p u l a t i o n s o f i n t e r e s t i n o t h e rf o o d f e r m e n t a t i o n s y s t e m s o r o t h e r m i c r o b i o l o g i c a l l y c o m -p l e x s y s t e m s . In t h i s r e s e a r c h , Cmr a n d E mr e n c o d e d b yp l a s m i d d e t e r m i n a n t s p e r m i t t e d e n u m e r a t i o n o f L . m o n o c y -t o g e n e s when i t r e p r e s e n t e d a s l i t t l e a s 1 0 - 8 o f t h e p o p u l a -t i o n .P . a c i d i l a c t i c i P A C 1 . 0 h a d p r e v i o u s l y b e e n r e p o r t e d t oc o n t a i n two p l a s m i d s o f 3 4 . 5 a n d 9 . 4 k b , d e s i g n a t e d a spSRQ10 a n d p S R Q 1 1 , r e s p e c t i v e l y ( 1 0 ) . T h e p r e s e n c e o f t h e3 4 . 5 - k b p l a s m i d ( p S R Q 1 0 ) c o r r e l a t e s w i t h a s u c r o s e - f e r -m e n t i n g p h e n o t y p e ( S u c + ) , a n d t h e p r e s e n c e o f t h e 9 . 4 - k bp l a s m i d ( p S R Q 1 1 ) c o r r e l a t e s w i t h b a c t e r i o c i n p r o d u c t i o n( B a c + ) . T h e a b i l i t y t o c u r e b a c t e r i o c i n - p r o d u c i n g p l a s m i d si n P . a c i d i l a c t i c i , r e s u l t i n g i n a b a c t e r i o c i n - n e g a t i v e p h e n o -

t y p e ( B a c - ) , was d e m o n s t r a t e d b y G o n z a l e z a n d Kunka ( 1 1 )a n d H o o v e r e t a l . ( 1 4 ) . T h e Bac- c o n t r o l s t a r t e r c u l t u r e wasi s o l a t e d b y c u r i n g t h e a n t i b i o t i c - r e s i s t a n t B a c + c u l t u r e o f t h e9 . 4 - k b p l a s m i d ; t h e 3 4 . 5 - k b p l a s m i d a n d s u c r o s e f e r m e n t i n ga b i l i t y w e r e r e t a i n e d b y t h e c u l t u r e . T h i s was i m p o r t a n t ,s i n c e i t r e s u l t e d i n i d e n t i c a l g e n o m e s i n Bac+ a n d Bac-s t r a i n s e x c e p t f o r t h e 9 . 4 - k b p l a s m i d , p r o v i d i n g a c o n t r o ld i f f e r i n g f r o m t h e b a c t e r i o c i n - p r o d u c i n g t e s t c u l t u r e o n l y i ni t s i n a b i l i t y t o p r o d u c e b a c t e r i o c i n .To a c c o u n t f o r d i f f e r e n t c o n d i t i o n s t h a t may o c c u r d u r i n gf e r m e n t a t i o n a n d t h e i r p o s s i b l e e f f e c t s o n b a c t e r i o c i n p r o -d u c t i o n a n d i n h i b i t i o n , t h r e e s a u s a g e c a s i n g s i z e s w e r e u s e d .T h e c a s i n g d i a m e t e r a f f e c t s t h e r a t e o f h ea t p e n e t r a t i o n a n dc o n s e q u e n t l y t h e r a t e s o f m e t a b o l i s m , i n c l u d i n g a c i d p r o d u c -t i o n , m o i s t u r e l o s s , w a t e r a c t i v i t y r e d u c t i o n , a n d o t h e rf a c t o r s t h a t c o n t r i b u t e t o t e r m i n a t i n g t h e f e r m e n t a t i o n .Th e s u r v i v a l o f L . m o n o c y t o g e n e s d u r i n g f e r m e n t a t i o na n d d r y i n g o b s e r v e d i n t h i s s t u d y was c o n s i s t e n t w i t h d a t ar e p o r t e d b y o t h e r s ( 4 , 9 , 1 5 - 1 7 ) . T h i s s t u d y i s u n i q u e ,h o w e v e r , b e c a u s e i t c l e a r l y d e m o n s t r a t e s t h a t a n t a g o n i s mo f L . m o n o c y t o g e n e s may b e e n h a n c e d b y b a c t e r i o c i np r o d u c t i o n i n s i t u a n d b a c t e r i o c i n a c t i v i t y t h r o u g h o u t t h ef e r m e n t a t i o n p r o c e s s . B e r r y e t a l . ( 4 ) r e p o r t e d t h a t a b a c t e -r i o c i n - p r o d u c i n g s t a r t e r c u l t u r e e f f e c t i v e l y i n h i b i t e d L .m o n o c y t o g e n e s d u ri ng s a u s a g e f e r m e n t a t i o n . T h e i r s t u d ywas n o t d e s i g n e d t o d e t e c t t h e c o n t r i b u t i o n o f b a c t e r i o c i np e r s e a n d c o m p a r e d f e r m e n t a t i o n s w i t h a b a c t e r i o c i n -p r o d u c i n g P . a c i d i l a c t i c i s t r a i n w i t h t h o s e w i t h a n o n -b a c t e r i o c i n - p r o d u c i n g P e d i o c o c c u s p e n t o s a c e o u s s t r a i n .O u r s t u d y d i f f e r s b e c a u s e i t c l e a r l y s h o w s t h a t b a c t e r i o c i np r o d u c t i o n , i n c o m b i n a t i o n w i t h o t h e r f e r m e n t a t i o n e n dp r o d u c t s , i nc r ea s e s t h e i n h i b i t i o n o f L . m o n o c y t o g e n e sd u r i n g f e r m e n t a t i o n a n d d r y i n g .T h i s r es e ar ch i n d i c a t e s t h a t meat f e r m e n t a t i o n , m i n i m a lh e a t i n g a f t e r f e r m e n t a t i o n , a n d d r y i n g o f s a u s a g e r e d u c e d L .m o n o c y t o g e n e s p o p u l a t i o n s b y 5 l o g u n i t s o r m o r e , r e g a r d -l e s s o f w h e t h e r b a c t e r i o c i n p r o d u c t i o n o c c u r r e d . E f f e c t i v er e d u c t i o n i n L . m o n o c y t o g e n e s p o p u l a t i o n s c o r r e l a t e d w i t ha n a d e q u a t e pH d r o p ( b e l o w 4 . 9 ) d u r i n g t h e f e r m e n t a t i o np o r t i o n o f t h e p r o c e s s . T h e pH a f f e c t s t h e i n h i b i t o r y r o l e o f

V O L . 5 8 , 1 9 9 2


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8 9 0 F O E G E D I N G E T AL.o t h e r f e r m e n t a t i o n p r e s e r v a t i o n f a c t o r s , s u c h a s h e a t i n g ,a c i d , a n d d r y i n g . I n s i t u b a c t e r i o c i n p r o d u c t i o n e n h a n c e d t h ei n h i b i t i o n o f L . m o n o c y t o g e n e s d u r i n g b o t h f e r m e n t a t i o na n d d r y i n g . T h i s a d d e d i n h i b i t i o n w o u l d b e e s p e c i a l l y i m p o r -t a n t i f a s u f f i c i e n t l y l o w pH w e r e n o t a c h i e v e d d u r i n g t h ef e r m e n t a t i o n .Where a c i d p r o d u c t i o n may b e i n s u f f i c i e n t , b a c t e r i o c i np r o d u c t i o n w o u l d f a c i l i t a t e t h e r e d u c t i o n o f a n y r e m a i n i n g L .m o n o c y t o g e n e s . S t a r t e r c u l t u r e s , s u c h a s P . a c i d i l a c t i c iP A C 1 . 0 , t h a t p r o d u c e b a c t e r i o c i n s i n h i b i t o r y t o L . mono-c y t o g e n e s c o u l d b e u t i l i z e d i n m e a t f e r m e n t a t i o n s t o p r o v i d ea d d e d i n h i b i t i o n a g a i n s t t h i s p a t h o g e n . T h u s , u t i l i z a t i o n o fb a c t e r i o c i n - p r o d u c i n g s t a r t e r c u l t u r e s i s r e c o m m e n d e d t oh e l p c o n t r o l L . m o n o c y t o g e n e s i n m e a t f e r m e n t a t i o n s . A l s o ,u s i n g b a c t e r i o c i n - p r o d u c i n g c u l t u r e s i n o t h e r f o o d f e r m e n t a -t i o n s may p r o v e t o b e b e n e f i c i a l i n c o n t r o l l i n g t h i s o r o t h e rf o o d - b o r n e p a t h o g e n s . F o r a p p l i c a t i o n , i t s h o u l d b e r e c o g -n i z e d t h a t b a c t e r i o c i n - r e s i s t a n t s t r a i n s o f L . m o n o c y t o g e n e smay a r i s e d u e t o p r o l o n g e d o r r e p e a t e d e x p o s u r e t o b a c t e -r i o c i n s i n a l a b o r a t o r y o r f o o d p r o d u c t i o n s y s t e m .

AC KNOW L E DGM ENTST h i s r e s e a r c h w a s f u n d e d i n p a r t b y t h e N a t i o n a l L i v e s t o c k a n dM e a t B o a r d a n d b y t h e N o r t h C a r o l i n a A g r i c u l t u r a l R e s e a r c hS e r v i c e .

REFERENCES1 . A n d e r s o n , D . G . , a n d L . L . M c K a y . 1 9 8 3 . S i m p l e a n d r a p i dm e t h o d f o r i s o l a t i n g p l a s m i d DNA f r o m l a c t i c s t r e p t o c o c c i .A p p l . E n v i r o n . M i c r o b i o l . 4 6 : 5 4 9 - 5 5 2 .2 . A n o n y m o u s . 1 9 8 5 . L i s t e r i o s i s o u t b r e a k a s s o c i a t e d w i t h M e x i -c a n - s t y l e c h e e s e - C a l i f o r n i a . M o r b i d . M o r t a l . W e e k l y R e p .3 4 : 5 7 - 5 9 .3 . A n o n y m o u s . 1 9 8 9 . L i s t e r i o s i s a s s o c i a t e d w i t h c o n s u m p t i o n o ft u r k e y f r a n k s . M o r b i d . M o r t a l . W e e k l y R e p . 3 8 : 2 6 7 - 2 6 8 .4 . B e r r y , E . D . , M. B . L i e w e n , R . W. M a n d i g o , a n d R . W. H u t k i n s .1 9 9 0 . I n h i b i t i o n o f L i s t e r i a m o n o c y t o g e n e s b y b a c t e r i o c i n -p r o d u c i n g P e d i o c o c c u s d u r i n g t h e m a n u f a c t u r e o f f e r m e n t e ds e m i d r y s a u s a g e . J . F o o d P r o t . 5 3 : 1 9 4 - 1 9 7 .5 . B h u n i a , A . K . , M. C . J o h n s o n , a n d B . R a y . 1 9 8 8 . P u r i f i c a t i o n ,c h a r a c t e r i z a t i o n a n d a n t i m i c r o b i a l s p e c t r u m o f a b a c t e r i o c i np r o d u c e d b y P e d i o c o c c u s a c i d i l a c t i c i . J . A p p l . B a c t e r i o l . 6 5 :2 6 1 - 2 6 8 .6 . B r e u e r , V . J . , a n d 0 . P r a n d i . 1 9 8 8 . N a c h w e i s v o n L i s t e r i e n u n dd e r e n Vorkommen i n H a c k f l e i s c h u n d M e t t w u r s t e n i n O s t e r r e -i c h . A r c h i v . L e b e n s m i t t e l h y g . 3 9 : 2 5 - 5 6 .7 . F a r b e r , J . M . , T i t t i g e r , a n d L . G o u r . 1 9 8 8 . S u r v e i l l a n c e o fr a w - f e r m e n t e d ( d r y - c u r e d ) s a u s a g e s f o r t h e p r e s e n c e o f L i s t e r i as p p . C a n . I n s t . F o o d S c i . T e c h n o l . J . 2 1 : 4 3 0 - 4 3 4 .8 . F l e m i n g , D . W . , S . L . C o c h i , K . L . M a c D o n a l d , J . B r o n d u m ,

P . S . H a y e s , B . D . P l i k a y t i s , M. B . H o l m e s , A . A u d u r i e r , C . V .B r o o m e , a n d A . L . R e i n g o l d . 1 9 8 5 . P a s t e u r i z e d m i l k a s a v e h i c l eo f i n f e c t i o n i n a n o u t b r e a k o f l i s t e r i o s i s . N . E n g l . J . M e d .3 1 2 : 4 0 4 - 4 0 7 .9 . G l a s s , K . A . , a n d M. P . D o y l e . 1 9 8 9 . F a t e o f L i s t e r i a m o n o c y -t o g e n e s i n p r o c e s s e d m e a t p r o d u c t s d u r i n g r e f r i g e r a t e d s t o r a g e .A p p l . E n v i r o n . M i c r o b i o l . 5 5 : 1 5 6 5 - 1 5 6 9 .1 0 . G o n z a l e z , C . F . , a n d B . S . K u n k a . 1 9 8 3 . P l a s m i d t r a n s f e r i nP e d i o c o c c u s s p p . : i n t e r g e n e r i c a n d i n t r a g e n e r i c t r a n s f e r o fp I P 5 0 1 . A p p l . E n v i r o n . M i c r o b i o l . 5 1 : 1 0 5 - 1 0 9 .1 1 . G o n z a l e z , C . F . , a n d B . S . K u n k a . 1 9 8 7 . P l a s m i d - a s s o c i a t e db a c t e r i o c i n p r o d u c t i o n a n d s u c r o s e f e r m e n t a t i o n i n P e d i o c o c -c u s a c i d i l a c t i c i . A p p l . E n v i r o n . M i c r o b i o l . 5 3 : 2 5 3 4 - 2 5 3 8 .1 2 . H a r r i s , L . J . , M. A . D a e s c h e l , M. E . S t i l e s , a n d T . R . K l a e n h a m -m e r . 1 9 8 9 . A n t i m i c r o b i a l a c t i v i t y o f l a c t i c a c i d b a c t e r i a a g a i n s tL i s t e r i a m o n o c y t o g e n e s . J . F o o d P r o t . 5 2 : 3 8 4 - 3 8 7 .1 3 . H a r r i s , L . J . , H . P . F l e m i n g , a n d T . R . K l a e n h a m m e r . 1 9 9 0 . Us eo f L e u c o n o s t o c m e s e n t e r o i d e s a n d n i s i n - p r o d u c i n g L a c t o c o c -c u s l a c t i s s t r a i n s a s a p a i r e d s t a r t e r c u l t u r e s y s t e m f o rs a u e r k r a u t . F E MS M i c r o b i o l . R e v . 8 7 : P 1 0 0 .

1 4 . H o o v e r , D . G. , P . M. W al s h , K . M. K o l a e t i s , a n d M. M. D a l y .1 9 8 8 . A b a c t e r i o c i n p r o d u ce d b y P e d i o c o c c u s s p e c i e s a s s o c i -a t e d w i t h a 5 . 5 - m e g a d a l t o n p l a s m i d . J . F o o d P r o t . 5 1 : 2 9 - 3 1 .1 5 . J o h n s o n , J . L . , M. P . D o y l e , a n d R . G . C a s s e n s . 1 9 9 0 . L i s t e r i am o n o c y t o g e n e s a n d o t h e r s p p . i n m e a t a n d m e a t p r o d u c t s : ar e v i e w . J . F o o d P r o t . 5 3 : 8 1 - 9 1 .1 6 . J o h n s o n , J . L . , M. P . D o y l e , R . G . C a s s e n s , a n d J . L . S c h o w n i .1 9 8 8 . F a t e o f L i s t e r i a m o n o c y t o g e n e s i n t i s s u e s o f e x p e r i m e n -t a l l y i n f e c t e d c a t t l e a n d i n h a r d s a l a m i . A p pl . E nv i ro n . M i c r o -b i o l . 5 4 : 4 9 7 - 5 0 1 .1 7 . J u n t t i l a , J . , J . H i r n , P . H i l l , a n d E . N u r m i . 1 9 8 9 . E f f e c t o fd i f f e r e n t l e v e l s o f n i t r i t e a n d n i t r a t e o n t h e s u r v i v a l o f L i s t e r i am o n o c y t o g e n e s d u r i n g t h e m a n u f a c t u r e o f f e r m e n t e d s a u s a g e .J . F o o d P r o t . 5 2 : 1 5 8 - 1 6 1 .1 8 . K ek es s y, D . A . , a n d J . D . P i g u e t . 1 9 7 0 . New m e t h o d f o rd e t e c t i n g b a c t e r i o c i n p r o d u c t i o n . A p p l . M ic ro b i o l . 2 0 : 2 82 - 2 8 3 .1 9 . K e r r , K . G . , S . F . D e a l l e r , a n d R . W. L a c e y . 1 9 8 8 . M a t e r n o - f e t a ll i s t e r i o s i s f r o m c o o k - c h i l l e d a n d r e f r i g e r a t e d f o o d . L a n c e t i i :1 1 3 3 .2 0 . K o k , J . , J . M. B . M. Va n D e r V o s s e n , a n d G . V e n e m a . 1 9 8 4 .C o n s t r u c t i o n o f p l a s m i d c l o n i n g v e c t o r s f o r l a c t i c s t r e p t o c o c c iw h i c h a l s o r e p l i c a t e i n B a c i l l u s s u b t i l u s a n d E s c h e r i c h i a c o l i .A p pl . E nv i r o n . M i c r o b i o l . 4 8 : 7 2 6 - 7 3 1 .2 1 . L u c h a n s k y , J . B . , P . M. M u r i a n a, a n d T . R . K l a e n h a m m e r .1 9 8 9 . A pp l i ca t i o n o f e l e c t r o p o r a t i o n f o r t r a n s f e r o f p l a s m i dDNA t o L a c t o b a c i l l u s , L a c t o c o c c u s , L e u s o n o s t o c , L i s t e r i a ,P e d i o c o c c u s , B a c i l l u s , S t a p h y l o c o c cu s , E nt e ro c o cc u s , a n dP r o p i o n i b a c t e r i u m . M o l . M ic ro b i ol . 2 : 6 3 7- 6 46 .2 2 . M a c r i n a , F . L . , D . J . K o p e c k o , K . R . J o ne s , D . J . A y e r s , a n dS . M . C o w e n . 1 9 8 2 . 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C o n t a m i n a t i o n de s v i a n d e se t d e s p r o d u i t s d e c h ar c ut e ri e p a r e n H a u t e - V i e n n e . S c i . A l i -m e n t . 5 : 1 7 5 - 1 8 0 .2 6 . N i c o l a s , J . A . , a n d N . V i d u a d . 1 9 8 7 . C o n t r i b u t i o n a l ' e t u d e d e sL i s t e r i a p r e s e n t e s d a n s l e s d e n r e e s d ' o r i g i n e a n i m a l e d e s t i n e e sa l a c o n s o m m a t i o n h u m a i n e . R e c . M e d . V e t . 1 6 3 : 2 8 3 - 2 8 5 .2 7 . N i e l s e n , J . W . , J . S . D i c k s o n , a n d J . D . C r o u s e . 1 9 9 0 . Use o f ab a c t e r i o c i n p r o d u c e d b y P e d i o c o c c u s a c i d i l a c t i c i t o i n h i b i tL i s t e r i a m o n o c y t o g e n e s a s s o c i a t e d w i t h f r e s h m e a t . A p p l . E n -v i r o n . M i c r o b i o l . 5 6 : 2 1 4 2 - 2 1 4 5 .2 8 . P u c c i , M. J . , E . R . V e d a m u t h u , B . S . K u n k a , a n d P . A .V a n d e n b e r g h . 1 9 8 8 . I n h i b i t i o n o f L i s t e r i a m o n o c y t o g e n e s b yu s i ng b a ct er i oc in P A - 1 p r o d u c e d b y P e d i o c o c c u s a c i d i l a c t i c iP A C 1 . 0 . A p pl . E nv i ro n . M i c r o b i o l . 5 4 : 2 3 4 9 - 2 3 5 3 .2 9 . R o d r i g u e s , R . L . , a n d R . C . T a i t . 1 9 8 3 . R e c o m b i n a n t DNAt e c h n i q u e s : a n i n t r o d u c t i o n . A d d i s o n - W e s l e y P u b l i s h i n g C o . ,R e a d i n g , M a s s .3 0 . S c h l e c h , W. F . , P . M. L a V i g n e , R . A . B o r t o l u s s i , A . C . A l l e n ,E . V . H a l do ne , A . J . W o r t , A . W. H i g h t o w e r , S . E . J o h n s o n ,S . H . K i n g , E . S . N i c h o l l s , a n d C . V . B r o o m e . 1 9 8 3 . E p i d e m i cl i s t e r i o s i s - e v i d e n c e f o r t r a n s m i s s i o n b y f o o d . N . E n g l . J . M e d .3 0 8 : 2 0 3 - 2 0 6 .3 1 . S e b r a n e k , J . 1 9 7 8 . 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