BIOCHEMICAL CHARACTERISTICS AND NUTRITIONAL REQUIREMENTS OF PARACOLON BACILLI By IRVING OLITZKY A THESIS S u b m itte d t o t h e S ch o o l o f G rad u ate S t u d i e s o f M ichigan S t a t e C ollege of A g r i c u l t u r e and A p plied S cien ce i n p a r t i a l f u l f i l l m e n t of the req u irem en ts f o r the degree of DOCTOR OF PHILOSOPHY D epartm ent o f B a c te r io lo g y 1950 ACKNOWLEDGMENT I w i s h t o t h a n k D r . W. L. M allm an n u n d e r whose c a p a b l e d i r e c t i o n t h i s w ork was d o n e . The A u t h o r it it it it it ?{• "ifr it it * it it it it it it it it it CONTENTS INTRODUCTION....................................................................................................... 1 GENERAL MATERIALS AND METHODS................................................................ 8 BIOCHEMICAL CHARACTERISTICS OF THE PARACOLON CULTURES.................................................................................................................. 11 E x p e r i m e n t a l M e t h o d s .............................................................13 E x p e r i m e n t a l R e s u l t s ....................................... . . • NUTRITIONAL REQUIREMENTS OF PARACOLON CULTURES ....................... G eneral E xperim ental 15 25 M e t h o d s ................................... 27 G r o s s N u t r i t i o n a l R e q u i r e m e n t s ................................. 28 S p e c i f i c N u t r i t i o n a l R e q u i r e m e n t s .................................35 DISCUSSION.................................................................................................................. 45 CONCLUSIONS................................................................................. •................................50 REFERENCES...................................................................................................................52 INTRODUCTION I n t e r e s t in th e slo w -la c to se ferm enting b a c t e r i a of in ­ t e s t i n a l o r i g i n ( p a r a c o l o n s ) h a s grown c o n s i d e r a b l y i n t h e la s t decade. The m o u n t i n g c o n c e r n by b a c t e r i o l o g i s t s i n t h i s f i e l d h a s b e e n p r o m p t e d by s e v e r a l r e a s o n s ; fo re m o s t of which i s the acc u m u latio n of evidence of th e p o s s ib l e p a th o g e n ic i ty of paracolon b a c te r ia . I n v e s tig a to r s a s s o c ia te d w ith d ia g ­ n o s tic b a c te rio lo g y fin d i t hig h ly d e s ira b le t o know more a b o u t t h e s e b a c t e r i a , even though th e p a t h o g e n i c i t y of p a r a ­ colon b a c i l l i i s s t i l l q u e s t i o n e d b y many b a c t e r i o l o g i s t s . In any c ase th e a p p a r e n t r e l a t i o n s h i p o f th e p a ra c o lo n b a c t ­ e ria to th e salm onellae, s h i g e l l a e , a n d p r o t e u s m akes i t i m p o r t a n t t o a c c u m u l a t e more i n f o r m a t i o n on t h e s u b j e c t . Along w ith t h e i n t e r e s t i n t h e p a r a c o l o n s , b e c a u s e o f t h e ir p o ssib le p a th o g e n ic ity , th o se w orkers concerned w ith b a c t e r i a l taxonom y f i n d t h e s e o r g a n i s m s a f i e l d f o r f r u i t f u l research. A ll s tu d ie s have c l e a r l y i n d ic a t e d th e r e l a t i o n ­ ship of th e paraco lon b a c t e r i a to th e n o n-p ath o genic c o lifo rm a n d t h e p a t h o g e n i c S a l m o n e 1 l a - S h i g e 1l a g r o u p . The p a r a c o l o n s a p p e a r t o be t y p e s which a r e t h e r e s u l t o f t h e e v o l u t i o n a r y tre n d of e n te r ic b a c t e r i a l species-from th e t y p ic a l co liform s to th e w e ll d e li n e a t e d salm o n e lla e and s h i g e l l a e . H isto rical. R eferen ces to the p a ra c o lo n organism s have appeared i n the l i t e r a t u r e b e fo re the b e g in n in g of t h i s century. The o r g a n i s m s h a v e b e e n d e s i g n a t e d b y v a r i o u s t e r m s 1 such a s : a ty p ic a l co lifo rm s, a b e rra n t colifo rm s, e tc . s lo w -la c to se ferm en tin g c o l l , An o r g a n ! s m i s o l a t e d f r o m a c a s e o f g a s t r o - e n t e r i t i s was d e s i g n a t e d . , a s a ,,p a r a c o l l b a c l l l u s ,, b y W i d a l a n d N o b e c o u r t ( 1 8 9 7 ) on t h e b a s i s t h a t t h e s t r a i n ferm ented la c to s e slow ly. K e n n e d y , Cummings, a n d Marrow (1932) i n a stu d y o f a t y p i c a l l a c t o s e f e r m e n t e r s , p l a c e d th e p a ra c o lo n group i n a p o s i t i o n i n t e r m e d i a t e betw een th e r a p i d la c to s e - f e r m e n t in g colon b a c i l l i and th e n o n - l a c to s e - f e r m e n t ­ in g p a r a ty p h o id l$roup. Organism s which fe rm e n te d l a c t o s e s l o w l y o r a t y p i c a l l y w e r e c o n s i d e r e d p a r a c o l o n b a c t e r i a by Sandiford (1935). T o p le y and W ilson (1937) sta te d th a t the e n t e r i c - g r a m - n e g a t i v e b a c t e r i a --which do n o t f e r m e n t l a c t o s e sh o u ld n o t be i n c l u d e d i n th e p a r a c o l o n g ro u p . Throughout the l i t e r a t u r e the paracolon b a c te r ia a re considered e it h e r a s a d i s t i n c t h e te ro g e n o u s group o r a s a t y p i c a l s t r a i n s of e i t h e r th e t r u e colon b a c i l l i C lassificatio n . o r the p araty p h o id s. An a t t e m p t t o make t h e p a r a c o l o n g r o u p more i n c l u s i v e was r e p o r t e d b y S t u a r t , W h e e l e r , R u s t i g i a n a n d Zimmermann ( 1 9 4 3 ) . b a c illi in to These a u t h o r s grouped th e p a ra c o lo n (1) p a ra c o lo n A e r o b a o t e r . (2) p a ra c o lo n E s c h e r i ­ c h i a . and ( 3 ) p a ra co lo n i n te r m e d ia te s . Borman i n c o l l a b o r ­ a t i o n w i t h S t u a r t . a n d W heeler (1944) p ro p o se d g e n e r i c s t a t u s f o r t h e p a r a c o l o n s a n d i n t h e s i x t h e d i t i o n o f B e r g e y ' s M a n ual o f D e te rm in a tiv e B a c t e r io l o g y (B ree d , Murray, and H itc h e n s , 194 8) t h e . a e r o g e n i c p a r a c o l o n s a r e l i s t e d u n d e r t h r e e g e n e r a i n Appendix I . T ribe E sc h erich e ae , P aracolobactrum a e ro g e n o id e s sp. nov. 2 P araco lo b actru m Interm edium sp .n o v . Paracolobactrum co lifo rm e sp.n o v . The d e s c r i p t i o n a s g i v e n i n A p p e n d i x I . T r i b e E s c h e r l c h e a e i s : Genua A. P a r a c o l o b a c t r u m B o r m a n . S t u a r t . W h e e l e r . S h o r t r o d s c h a r a c t e r i z e d by c o n s i s t a n t l y d e l a y e d f e r m e n ­ ta tio n of lac to se (o ccasio n ally n e g a tiv e ). Glucose i s f e r ­ mented w ith f o r m a t io n o f v i s i b l e g a s . C e r t a i n forms a t t a c k c a r b o h y d r a t e s c h a r a c t e r i s t i c a l l y a t 2 0 ° t o 3 0 °C b u t n o t a t 37°0. A n tig en ic r e l a t i o n s h i p s to o th e r genera i n the fam ily a r e common, e v e n w i t h r e s p e c t t o m a j o r a n t i g e n s . The t y p e s p e c i e s i s P a r a c o l o b a c t r u m a e r o g e n o i d e s Borman, S t u a r t , and W heeler. I. A c e ty lm e th y lc a rb in o l produced. 1. Paracolobactrum aero g en o id e s II. A cety lm eth y lcarb in o l n o t produced. A. C itr ic a c id u t i l i z e d a s a so le source of carbon. 2. Pa r a c o l o b a c t rum i n t e r m e d i u m B. C i tr i c a c id n o t u t i l i z e d a s a so le source of carbon. 3. P a r a c o l o b a c t r u m c o l l form e The g e n e r i c a n d s p e c i e s d e s c r i p t i o n s f o r t h e a n a e r o g e n i c p a r a e o l o n s w ere g i v e n by Borman e t . a l . (1944) a s f o l l o w s : A e r o b i c , n o n - s p o r o g e n i c , gram n e g a t i v e r o d s . G lucose i s ferm ented a n a e r o g e n i c a ll y . L a c t o s e may o r may n o t b e attack ed . S a l ic i n n o t ferm ented. T his genus i s r e s e r v e d f o r f o r m s , a p p a r e n t l y t r a n s i t i o n a l , w h i c h do n o t c o n f o r m t o t h e d e s c r i p t io n s of o th e r genera of th e E n te r o b a c te r la c e a . T h i s t y p e s p e c i e s i s P r o s h i g e l l a d l s o a r Andrews comb, n o v . I. In d o le produced A. L actose fe rm e n te d , u s u a l l y l a t e 1. P r o s h i g e l l a d i s p a r (A n d rew s) com b,n o v . B. l a c to s e n o t ferm ented 2. P r o s h i g e l l a a l k a l e s c e n s ( A n dre w s) comb . n o v . II. In d o le n o t produced A. L actose fe rm e n te d , u s u a l l y l a t e 3* P r o s h l g e l l s o n n e l ( L e v i n e ) comb^ nov. For th e convenience o f th e r e a d e r th e term s p a ra c o lo n and P a rac o lo b ac tru m w i l l be used i n te r c h a n g e a b l y i n t h i s r e ­ p o r t when r e f e r r i n g t o a e r o g e n l c t y p e s ; th e term s a n a e ro g e n ic p a r a c o l o n a n d P r o s h i g e l l a w i l l be u s e d i n t e r c h a n g e a b l y when r e f e r e n c e i s made t o t h o s e t y p e s w h i c h a r e a n a e r o g e n i c . P athogenicity. T h e r e h a v e "been many o b s e r v a t i o n s r e l a t i n g t o tfce p a t h o g e n i c i t y o f some o f t h e o r g a n i s m s i n t h e p a r a c o l o n group. I t i s beyond th e scope o f t h i s p a p e r to re v ie w a l l t h e work on t h e s u b j e c t o f p a t h o g e n i c i t y , h o w e v e r , c e r t a i n I o f t h e more i m p o r t a n t r e p o r t s w i l l b e c i t e d . E d w a r d s (194-5) r e p o r t e d on a p a r a c o l o n c u l t u r e i s o l a t e d fr o m a n i n f a n t s u f f e r i n g f r o m a c u t e c o l i t i s . Previous to t h i s N e te r (1944) re c o v e re d p a ra c o lo n b a c i l l i f r o m 22 p e r c e n t o f h e a l t h y i n f a n t s exam ined. F inding p araco lo n b a c te r ia in the i n t e s t i n a l d isc h a rg e s of a p p a re n tly h e a lth y in d iv id u a ls h a s somewhat c o m p l i c a t e d t h e p i c t u r e o f p o s s i b l e p a t h o g e n i c i t y . C h r i s t e n s e n ( 1 9 4 7 ) h a s r e p o r t e d on t h e d i s t r i b u t i o n o f P a r a ­ colobactrum organism s i n i n d i v i d u a l s r e s i d i n g i n an a r e a i n which e n t e r i c i n f e c t i o n s a r e h i g h l y en d em ic . He f o u n d P a r a ­ colobactrum i n p a t i e n t s w ith g a s t r o e n t e r i t i s i n a r a t i o t h r e e t o one w i t h r e s p e c t t o n o r m a l i n d i v i d u a l s . of L arge numbers o f S a lm o n e lla an d Shige11a s p e c i e s were a l s o o b t a i n e d from i n ­ d i v i d u a l s sh o w in g no s i g n s o f e n t e r i c d i s o r d e r s . which were p l a c e d i n t h e " A r i z o n a ' 1 g r o u p w e r e r e p o r t e d on i n d e t a i l by E d w a r d s , W e s t , a n d B r u n e r ( 1 9 4 7 a ) . of t h i s grpup were f i r s t S alm o nella. C u ltures R epresentatives re p o r te d to belong i n th e genus The s t r a i n s i n t h e A r i z o n a g r o u p a r e d e f i n i t e l y p a t h o g e n i c f o r a n i m a l s a n d p r o b a b l y a l s o f o r man. M c N a ir S c o t t , C o r i e l l , D a v i s , a n d B o l t z e s ( 1 9 4 7 ) r e p o r t e d on a n o u t b r e a k o f g a s t r o - e n t e r i t i s a t a c h i l d r e n ' s camp. R e c t a l swabs r e v e a l e d P a r a c o l o b a c t r u m i n 28 p e r c e n t o f t h e 43 p a t i e n t s a n d 10 f o o d h an d lers te s te d . Edwards e t . a l . 4 ( 1 9 4 8 ) r e p o r t e d on a * s e r o l o g i c a l l y r e l a t e d group d e s i g n a t e d th e "B ethesda" group. T w e n t y - t w o o f t h e 32 c u l t u r e s s t u d i e d w e r e i s o l a t e d f r o m e i t h e r p a t i e n t s o r food h a n d l e r s i n sm all o u tb re a k s o f e n te ritis.. A naerogenic s t r a i n s have a l s o been i n c r i m i n a t e d a s i n ­ c ite r s of e n te ric d iso rd e rs. The now fam o u s t y p e 2 9 9 1 1 f i r s t d e s c r i b e d by S t u a r t a n d R u s t i g i a n ( 1 9 4 3 ) was a l s o I s o l a t e d by o t h e r w o r k e r s ( G a l t o n , H e ss , a n d C o l l i n s 1947) from a person s u f f e r in g w ith d ia r r h e a . An o u t b r e a k o f d i a r r h e a c a u s e d by f r i c a s s e e d c h i c k e n s e r v e d i n a n Army o f f i c e r ' s m e s s was r e p o r t e d b y P l a s s ( 1 9 4 7 ) . T h i r t e e n o f t h e 16 p a t i e n t s who r e q u i r e d h o s p i t a l i z a t i o n w e re t h e s o u r c e o f o r g a n i s m s r e s e m b lin g ty p e 29911. It c a n be s e e n f r o m t h e s e few c i t a t i o n s t h a t d i a g n o s t i c b a c t e r i o l o g i s t s w i l l b e more a n d more c o n c e r n e d w ith the i s o l a t i o n and i d e n t i f i c a t i o n of th e p araco lo n b a c i l l i . B iochem ical and A n tig e n ic R e l a t i o n s h i p s . the paracolon b a c te r ia The r e l a t i o n s h i p o f to th e o t h e r e n t e r i c organism s h as b e e n s t u d i e d e x t e n s i v e l y on t h e b a s i s o f b i o c h e m i c a l c h a r a c t ­ e r i s t i c s and t h e i r a n t i g e n i c s t r u c t u r e . The b i o c h e m i c a l r e ­ l a t i o n s h i p s w i l l be d i s c u s s e d i n d e t a i l l a t e r b u t i t should b e s t a t e d now t h a t t h e c l a s s i f i c a t i o n o f t h e p a r a c o l o n b a c t e r i a on a b i o c h e m i c a l b a s i s a l o n e l e a v e s much t o b e d e s i r e d b e c a u s e o f th e extrem e v a r i a b i l i t y and g r a d a t i o n s o f r e a c t i o n s . A n t i g e n i c a n a l y s i s o f p a r a c o l o n c u l t u r e s h a s p r o v e d more fru itfu l even th o u g h t h i s a p p ro a c h h a s been ex ten d ed only t o a r e l a t i v e l y s m a ll number o f p a r a c o l o n t y p e s . On t h e b a s i s o f a n t i g e n i c s t r u c t u r e t h e p a r a c o l o n s form a g rad ed s e r i e s betw een t h e t r u e c o li f o r m s and th e s a lm o n e lla e and betw een t h e s a l m o n e l l a e an d th e s h i g e l l a e . A close re ­ la ti o n s h i p to the genus P ro te u s i s a ls o a p p a re n t. A ntigenic r e l a t i o n s h i p s t o o t h e r b a c t e r i a w e r e r e p o r t e d by W h e e l e r , S t u a r t , R u s t i g i a n , a n d Borman (194-3); E d w a r d s (194-5); S t u a r t a n d Van S t r a t u m (194-5); S t u a r t , W h e e l e r , a n d McGann ( 1 9 4 6 ) ; Hinshaw a n d M cN eil ( 1 9 4 7 ) ; a n d E d w a r d s , W e s t , a n d B r u n e r (1947b). E x c e l l e n t work on t h e a n t i g e n i c c l a s s i f i c a t i o n o f t h e p a r a c o l o n s was r e p o r t e d b y E d w a r d s e t . a l . (1947a); G a l t o n , a n d McGann ( 1 9 4 8 ) ; a n d E dw ard s e t . a l. S tu art, (1948). G alton a n d S t u a r t ( 1 9 4 9 ) r e p o r t e d on t h e u s e o f a p o o l e d P . I n t e r ­ medium a n t i s e r u m f o r u s e i n d i a g n o s t i c e n t e r i c b a c t e r i o l o g y . Purpose of th e P re s e n t Study. Because o f t h e r e l a t i o n s h i p o f i t h e p a r a c o lo n b a c t e r i a t o o t h e r e n te r ic o rg a n is m s , b o t h from th e view p o in t o f b io c h e m ic a l c h a r a c t e r i s t i c s and a n t i g e n i c s t r u c t u r e , i t was t h o u g h t p e r t i n e n t t o i n v e s t i g a t e t h e m e t a b ­ o l i c a c t i v i t i e s o f t h e p a r a c o l o n s a s e x p r e s s e d by t h e i r n u ­ t r i t i o n a l req uirem ents. T his phase o f th e problem i s Im p o rta n t i f one c o n s i d e r s t h e e v i d e n c e - w h i c h l e a d s t o t h e g e n e r a l sta te m e n t t h a t an i n c r e a s e i n th e d e g re e of p a r a s i t i s m i s a c c o m p a n i e d by l o s s e s i n enzyme s y s t e m s o f c e r t a i n m e t a b o l i c processes. I n t h e e n t e r i c g r o u p one c a n s e e t h i s pheno menon ro u g h ly i n t h e t r a n s i t i o n from th e t r u e c o l i f o r m s , w hich a r e re la tiv e ly n o n -fastid io u s, t o t h e s h i g e l l a e , many s t r a i n s o f w h i c h r e q u i r e one o r more g r o w t h f a c t o r s ( P o r t e r 1946). The i n a b i l i t y of th e paracolon ty p es to ferm ent la c to s e r a p id ly o r ferm ent la c to s e a t a l l , is suggestive of the lo s s or i m p a i r m e n t o f c e r t a i n enzyme s y s t e m s . The a n a e r o g e n i c p a r a ­ c o lo n s a r e r e l a t e d t o th e P r o t e u s which p ro d u ce s m a l l volumes o f gas a n d t o t h e Shige11a t y p e s which a r e d e f i n i t e l y a n a e r o ­ genic. S a lm o n e lla ty p h o s a which e x h i b i t s t h e g r e a t e s t p a t h o ­ g e n i c i t y of th e Salm onella ty p e s i s a n a e ro g e n ic . I t must be k e p t i n mind t h a t t h e b i o c h e m i c a l c h a r a c t e r i s t i c s a n d n u ­ t r i t i o n a l req u irem en ts under d is c u s s io n a r e tho se o f the "w ild ty p e " s t r a i n s . T rain in g or a d a p ta tio n r e s u l t s in a co m p lex ity of r e a c t i o n s , th e p roblem o f w hich i s beyond th e scope o f t h i s p a p e r . Thus t h e p r im a r y p u r p o s e o f t h e i n v e s t i g a t i o n t o be r e p o r t e d was t o d e t e r m i n e t h e n u t r i t i o n a l r e q u i r e m e n t s o f some p a r a c o l o n b a c t e r i a a n d t o r e l a t e t h e f i n d i n g s t o t h e b io ch em ical c l a s s i f i c a t i o n of th ese organism s. I t was a l s o t h e p u r p o s e t o g a t h e r more i n f o r m a t i o n t o s u b s t a n t i a t e t h e ro le of the p a ra co lo n s a s t r a n s i t i o n a l ty p e s of th e e n t e r i c b acteria. 7 GENERAL MATERIALS AND METHODS Source o f G u l t u r e a . The c u l t u r e s u s e d i n t h e e x p e r i m e n t s , t o b e d e s c r i b e d , w e r e o b t a i n e d f r o m b o t h a n i m a l a n d human sources. M i s s Neu o f t h e D e p a r t m e n t o f B a c t e r i o l o g y a n d P u b l i c H e a l t h v e r y g r a c i o u s l y s u p p l i e d c u l t u r e s w h i c h on p r e s u m p ti v e media a p p e a r e d t o be p a r a c o l o n t y p e s . These o rg an ism s were i s o l a t e d from th e i n t e r n a l o rg a n s and f e c a l m a t e r i a l o f p i g s which were u sed i n a s tu d y o f sw ine e n t e r i t i s . The human s t r a i n s w e r e s u p p l i e d f r o m t h e d i a g n o s t i c l a b o r a t o r y o f th e M ichigan D epartm ent of H e a l t h a t L an sin g t h r o u g h t h e c o u r t e s y o f D r . H. E . Gope. The human s t r a i n s w e r e i s o l a t e d f ro m f e c a l s p e c i m e n s w h i c h were s u b m i t t e d f o r d i a g n o s i s o r fo r ro u tin e exam inations. sta te In s titu tio n s , The s u b j e c t s i n c l u d e d i n m a t e s o f h o sp ital o b ste tric p a tie n ts, c a r r i e r s u s p e c t s , n u r s e s a n d new b o m i n f a n t s . w ere n u m b e r e d a n d a s s i g n e d a l e t t e r typ h oid C ultures "p" f o r c u l t u r e s f r o m p i g s a n d "Hu f o r c u l t u r e s f r o m h u m a n s . A l l c u l t u r e s w e r e t e s t e d f o r p u r i t y by r e p e a t e d d i l u t i o n p latin g s. A p p r o x i m a t e l y 20 p e r c e n t o f t h e c u l t u r e s w e r e f o u n d t o be P r o t e u s on t h e b a s i s o f u r e a s e p r o d u c t i o n (C h ris te n s e n 1946). S e v e r a l o f t h e c u l t u r e s were c o n t a m i n a t e d w ith th e t r u e c o lifo rm s . I t was t h o u g h t i m p o r t a n t t o i n c l u d e p a r a c o l o n t y p e s w h i c h h a d a l r e a d y b e e n i n v e s t i g a t e d i n o r d e r t o h a v e some m easure o f th e r e l i a b i l i t y of th e t e s t s used to d e te rm in e the biochem ical c h a r a c t e r i s t i c s of the c u ltu r e s . W ith t h i s i n m i n d , Dr. P . R. E d w a r d s was c o n t a c t e d a t t h e A g r i c u l t u r a l Experim ent S t a t i o n , L e x i n g t o n , K e n t u c k y a n d he k i n d l y s u p p l i e d f o u r r e p r e s e n t a t i v e s t r a i n s of t h e A riz o n a g ro u p . These were d esig n ated w ith the l e t t e r ing a n tig e n ic d e s c rip tio n A iAok t- "A" a n d c o r r e s p o n d t o t h e f o l l o w ­ s u p p l i e d by D r . E d w a r d s . 1 ,4 :1 ,2 ,5 7 :1 ,7 ,8 5 :1 3 ,1 4 1 ,2 :1 ,2 ,5 (CDA 144) (CDA X 184 ) (G a l 188) (A rizona, D .G .l) Commander L. A. B a r n e s o f t h e N a v a l M e d i c a l R e s e a r c h I n s t i t u t e a t B e t h e s d a , M a r y l a n d g r a c i o u s l y s u p p l i e d two s t r a i n s e a c h o f p a r a c o l o n t y p e 3 2 0 1 1 , p a r a c o l o n t y p e 100- 5- 1 3 , and p a r a c o lo n ty p e 29911. T h e s e w ere d e s i g n a t e d a s Nj^N^; N2 , N5 ; a n d N^, N j r e s p e c t i v e l y . P a r a c o l o n t y p e 1 0 0 - 5 - 1 3 was i s o l a t e d f r o m s t o o l s a m p l e s o f p a t i e n t s i n w a r d s o f a U .S . n a v a l h o s p i t a l f o l l o w i n g t h e o c c u r r e n c e o f many c a s e s o f d i a r r h e a (B arnes and Cherry 1946). A t o t a l o f 28 c u l t u r e s w e r e o b t a i n e d w i t h b i o c h e m i c a l r e a c t i o n s t h a t would p l a c e them i n t h e P . I n t e r m e d i u m g r o u p . C u l t u r e s were k e p t i n s t o c k a s s t a b c u l t u r e s i n semi­ s o l i d a g a r ( m o t i l i t y t e s t medium, D i f c o ) a n d r e f r i g e r a t e d . F r e s h s t o c k c u l t u r e s w e re made m o n t h l y . B e f o r e a n y e x p e r i m e n t s w e r e p e r f o r m e d t h e c u l t u r e s w e re t r a n s f e r r e d t i m e s on n u t r i e n t a g a r s l a n t s . serially three A l l I n o c u l a t i o n s w e re d o n e f r o m a c t i v e l y g r o w i n g 24 h o u r c u l t u r e s . B iochem ical C h a r a c t e r i z a t i o n . In o rd e r to se p a ra te the c u l t u r e s i n t o b i o c h e m i c a l t y p e s many o f t h e commonly u s e d 9 b i o c h e m i c a l t e s t s w e re e m p l o y e d . These I n c l u d e d t h e d e t e r ­ m i n a t i o n o f t h e IMVIC r e a c t i o n s , a c t i o n on c a r b o h y d r a t e s , urease production, t a r t r a t e u t i l i z a t i o n , m o t i l i t y , hydrogen s u l f i d e p r o d u c t i o n , etc. The t e s t B w ere r e p e a t e d a t p e r i o d o f two y e a r s . d e te rm in a tio n of g e la tin liq u efactio n , l e a s t th r e e tim es over a No c h a n g e s w e r e n o t e d i n t h e b i o c h e m i c a l r e a c t i o n s except f o r changes i n th e tim e r e q u i r e d to produce certain reactio n s. When t h e r e s u l t s w e r e t a b u l a t e d t h e c u l t u r e s w e re p u t i n t o f o u r g r o u p s : P aracolobactrum a e ro g e n o id e s P a rac o lo b ac tru m interm edium Paracolobactrum c o llfo rm e anaerogenic p araco lo n s N u t r i t i o n a l R equirem ents. The n u t r i t i o n a l r e q u i r e m e n t s ' a s e x p r e s s e d by n o r m a l g r o w t h i n p u r i f i e d m e d i a w e r e d e t e r m i n e d by s t a r t i n g w i t h w h a t was c o n s i d e r e d a c o m p l e t e medium a n d elim in a tin g f i r s t g en eral c o n s titu e n ts and then s p e c if ic co n stitu en ts. The c o m p l e t e medium c o n s i s t e d o f a m i x t u r e o f in o r g a n ic s a l t s , w ater s o lu b le v i ta m in s , and a source of a m in o a c i d s i n t h e f o r m o f c a s e i n h y d r o l y s a t e s . ion of a l l the vitam ins or a l l t h e amino a c i d s , a s a screening t e s t to f a c i l i t a t e e tc ., served t h e m ore d e t a i l e d d e t e r ­ m inations of s p e c i f i c n u t r i t i v e re q u ire m e n ts. 10 The e l i m i n a t ­ BIOCHEMICAL CHARACTERISTICS OF THE PARACOLON CULTURES The i d e n t i f i c a t i o n o f p a r a c o l o n b a c i l l i by t h e b i o c h e m i c a l t e s t s u s u a l l y employed i s f a r from b e in g a n e x a c t p r o c e d u r e . Those c h a r a c t e r i s t i c s w h i c h p l a c e t h e p a r a c o l o n s a s i n t e r ­ m e d i a t e s i n t h e e n t e r i c g r o u p a r e t h e same c h a r a c t e r i s t i c s which c o m p l i c a t e t h e e s t a b l i s h m e n t o f a p o s i t i v e d i a g n o s t i c sc h em e . On p r e s u m p t i v e d i f f e r e n t i a l m ed ia i t is d iffic u lt to d i f f e r e n t i a t e t h e p a r a c o l o n b a c i l l i from o rg an ism s of t h e S alm onella, S h i s e l l a . or P ro te u s g enera. I n many c a s e s t h e u s e o f more e x t e n s i v e b i o c h e m i c a l t e s t s h a s f a i l e d c l e a r l y to i d e n t i f y a c u lt u r e a s a p a ra c o lo n . T his i s p a r t i c u l a r l y t r u e o f p a r a c o l o n s t r a i n s which f e r m e n t l a c t o s e v e r y s lo w ly or n o t a t a l l . The b a s i c p r o b l e m o f f i n d i n g some c h a r a c t e r i s t i c o r group of c h a r a c t e r i s t i c s which would c l e a r l y i d e n t i f y a n o rg a n is m a s a p a r a c o l o n h a s b e e n t h e s u b j e c t o f much e x p e r i m e n t a l w o r k , A m u l t i p l e c a r b o h y d r a t e medium was p r o p o s e d b y C h i l t o n a n d Fulton (1946), T h e 'm e d iu m c o n t a i n e d a d o n i t o l , a e s c u l l n , s a l i c i n , a n d s u c r o s e w i t h two i n d i c a t o r s ; a n d f e r r i c ammonium c i t r a t e . brom -eresol-purple The t e s t f o r i n d o l e p r o d u c t i o n was a l s o p e r f o r m e d on t h e medium u s i n g K o v a c ’ s r e a g e n t . The a u t h o r s t e s t e d o v e r 250 c u l t u r e s - o f p a r a c o l o n s a n d f o u n d t h a t 44 p e r c e n t o f t h e c u l t u r e s f e r m e n te d a t l e a s t one o f t h e s u g a r s i n 24 h o u r s . T h i r t y - f i v e p e r c e n t were i n d o l e p o s i t i v e and fe r m e n ta tio n n e g a t iv e . T h i s l e f t 21 p e r c e n t o f t h e c u l t u r e s which were n e g a t i v e i n b o t h t e s t s . T esting fo r l a c t o s e f e r m e n t a t i o n i n 10 p e r c e n t l a c t o s e a g a r s l a n t s t h e y f o u n d t h a t 22 o f t h e 55 c u l t u r e s ( n e g a t i v e on b o t h t e s t s ) f e rm e n te d t h e c a r b o h y d r a t e i n 24 h o u r s . t h a t P r o t e u s a n d Shi s e l l a The a u t h o r s s t a t e d c u l t u r e s may b e p o s i t i v e on t h e m u l t i p l e c a r b o h y d r a t e medium d e s i g n a t e d a s AASS b r o t h . A lo n g t h e same l i n e s S c h a u b ( 1 9 4 8 ) paracolon b a c i l l i . s t u d i e d 56 s t r a i n s o f U s i n g AASS b r o t h , 33 p e r c e n t o f t h e c u l ­ t u r e s gave a p o s i t i v e r e a c t i o n . Twenty-one p e r c e n t o f t h e c u l t u r e s f e r m e n t e d l a c t o s e i n 24 h o u r s on t h e 1 0 p e r c e n t lacto se ag ar. How ev er, t h e a u t h o r s t a t e d t h a t 20 s t r a i n s o f n o n - l a c t o s e - f e r m e n t i n g gram n e g a t i v e b a c i l l i g a v e a n a c i d r e a c t i o n on t h i s medium. Sc h a u b t h e n p r o p o s e d a scheme f o r d i f f e r e n t i a t i n g paracolon b a c i l l i from S a l m o n e l l a . S h i s e l l a . a n d o t h e r gram n e g a t i v e b a c i l l i w h i c h w ere n o t t r u e c o l i f o r m s . T h i s scheme was b a s e d on w h e t h e r t h e c u l t u r e s w e r e i n h i b i t e d on S . S . a g a r ( D i f c o ) o r on a s i m p l i f i e d d e s o x y c h o l a t e - c i t r a t e - t h i o s u l f a t e medium. Sc h a u b a l s o t e s t e d h e r c u l t u r e s i n u r e a medium o f C h r i s t e n s e n ( 1 9 4 6 ) . the Tw enty-nine p e r c e n t o f t h e c u l t u r e s w ere u r e a s e p o s i t i v e . The o n l y c h a r a c t e r i s t i c s w h i c h h a v e d e f i n i t e v a l u e f o r g r o u p i n g o f t h e p a r a c o l o n s a r e t h e IMVIC r e a c t i o n s . On t h i s b a s i s a l l p a r a c o l o n c u l t u r e s c a n be a r r a n g e d i n t o t h r e e g e n e r a a s p r o p o s e d by Borman, S t u a r t , a n d W h e e l e r ( 1 9 4 4 ) . In the i n v e s t i g a t i o n b e in g r e p o r te d a l l th e u su a l b i o ­ c h e m i c a l t e s t s were a p p l i e d t o t h e p a r a c o l o n c u l t u r e s a l o n g w i t h some o f t h e d i f f e r e n t i a l t e s t s c i t e d a b o v e E x p e r i m e n t a l M e th o d s A f t e r p u r i f y i n g t h e c u l t u r e s t h e y w e re s u b j e c t e d t o t h e biochem ical t e s t s . A l l i n o c u l a t i o n s w e r e made f r o m 2 4 h o u r n u t r i e n t a g a r s l a n t c u l t u r e s which had p r e v i o u s l y been t r a n s ­ fe r r e d d a ily a t l e a s t th re e tim es. IMVTQ R e a c t i o n . I n d o l p r o d u c t i o n was d e t e r m i n e d w i t h b o t h K o v a c ' s a n d P r i n g s h e i m ’ s r e a g e n t s u s i n g 48 h o u r c u l t u r e s i n peptone b r o t h . The m e t h y l r e d t e s t a n d t h e t e s t f o r p r o d u c t ­ i o n o f a c e t y l - m e t h y l - c a r b i n o l ( V . P . ) w e r e made on c u l t u r e s growing i n M .R .-V .P. b r o t h ( D i f c o ) . The i n d i c a t o r f o r t h e V . P . t e s t was a l p h a n a p t h o l a n d p o t a s s i u m h y d r o x i d e . C itrate u t i l i z a t i o n was d e t e r m i n e d on K o s e r ' s c i t r a t e b r o t h a n d Sim m o n 's c i t r a t e a g a r . Hydrogen S u l f i d e a n d U re a s e P r o d u c t i o n . The p r o d u c t i o n o f h y d r o g e n s u l f i d e was d e t e r m i n e d by t h e u s e o f T . S . I . a g a r (B altim ore B io lo g ic a l l a b o r a to r y ) , peptone b r o th w ith le a d a c e t a t e p a p e r s t r i p s , a n d SIM medium ( D i f c o ) . The l a t t e r medium was a l s o u s e d f o r d e t e c t i n g i n d o l e p r o d u c t i o n a n d m o tility . U r e a s e p r o d u c t i o n was d e t e c t e d by t h e m e t h o d s o f S t u a r t , Van S t r a t u m , a n d R u s t i g i a n and C h r is te n s e n (1946) (1941) (u re a b r o t h , D ifco ) (ureaagar, D ifco). T a r t r a t e u t i l i z a t i o n was t e s t e d on p h e n o l r e d t a r t r a t e agar (D ifco). F erm entation of C a rb o h y d ra tes. 13 The c a r b o h y d r a t e s a n d r e l a t e d s u b s t a n c e s u s e d f o r f e r m e n t a t i o n s t u d i e s were a s f o l l o w s : glucose, l a c t o s e , m alto se, D -m annitol, L -a ra b in o s e , t r e h a l o s e , D -g alacto se, ad o n ito l, D- su c ro se , D -xylose, sa lic in , d u lcito l, rhamnose, D - s o r b i t o l , and i n o s i t o l . were d i s s o l v e d i n p u r p l e b r o t h b a s e of 0 .5 or 1.0 p e r c e n t. raffin o se, The c a r b o h y d r a t e s (D ifco) in c o n c e n tr a tio n s S terilizatio n of the carbohydrate m ed ia was c a r r i e d o u t i n a n a u t o c l a v e f o r 10 m i n u t e s a t 1 2 1° C . The 10 m i n u t e p e r i o d was a d e q u a t e f o r s t e r i l i z a t i o n a s t h e t u b e s w e re p l a c e d i n t e s t t u b e r a c k s . The r e l a t i v e l y short h e a t in g p e rio d d id n o t h y d ro ly ze th e c a rb o h y d ra te s to any ap p reciab le e x te n t. The i n o c u l a t e d c a r b o h y d r a t e b r o t h s w e r e o b s e r v e d d a i l y and a l l n e g a t i v e t u b e s were k e p t f o r a t l e a s t 30 d a y s . la c to s e Ferm entation. Since th e d e m o n stra tio n of l a c to s e a c t i v i t y i s of g r e a t im portance i n th e c h a r a c t e r i z a t i o n of paracolon b a c i l l i , more e m p h a s i s was p l a c e d on t h e r e a c t i o n of the c u ltu re s in la c to se c o n t a i n i n g m ed ia t h a n i n t h e m e d i a c o n ta in in g th e O ther c a rb o h y d ra te s . Along w i t h i n o c u l a t i n g t h e c u l t u r e s i n t o 1 p e r c e n t l a c t o s e b r o t h t h e c u l t u r e s were a l s o i n o c u l a t e d i n t o 5 p e r c e n t l a c t o s e b r o t h a n d on 10 p e r cent la c to s e ag ar s la n ts (pu rple a g ar base, D ifco). H ershey and B ro n fe n b r e n n e r ( 1 9 3 6 ) , w orking w i t h slowla c to s e - ferm enting b a c i l l i , showed t h a t t h e i r c u l t u r e s g rew more r a p i d l y i n m ed ia c o n t a i n i n g h i g h e r c o n c e n t r a t i o n s o f lacto se. Darby a n d Mallm ann ( 1 9 3 9 ) d e m o n s t r a t e d t h a t B a c t o - tr y p t o s e produced l a r g e r b a c t e r i a l p o p u la tio n s than B actop e p t o n e when b o t h w e re c o m p a r e d i n g r o w t h c u r v e e x p e r i m e n t s . 14 A c c o r d in g ly , a lo n g w ith t h e o t h e r media c o n t a i n i n g l a c t o s e , a medium was u s e d w h i c h c o n t a i n e d t h e same i n g r e d i e n t s a s p u r p l e b r o t h b a s e ( D i f c o ) e x c e p t t h a t B a c t o - t r y p t o s e was u s e d i n a c o n c e n tr a tio n of 2 p e r c e n t i n s t e a d of p r o te o s e peptone N o . 3 . The c o n c e n t r a t i o n o f l a c t o s e i n t h i s medium was 1 p e r cent. A l l t h e m ed ia c o n t a i n i n g l a c t o s e w e r e s e e d e d a t t h e same t i m e a n d i n c u b a t e d a t 3 7° C . O b s e r v a t i o n s w e r e made d a i l y a n d p a r t i c u l a r a t t e n t i o n given to th e f i r s t breakdown. signs of carbohydrate A l l t u b e s s h o w in g n o r e a c t i o n w e r e k e p t f o r a t l e a s t 30 d a y s a n d s t o p p e r e d t o p r e v e n t e v a p o r a t i o n . G e l a t i n e L i q u e f a c t i o n . I n h i b i t i o n on S . S . A g a r , a n d R e a c t i o n oh AASS B r o t h . The a b i l i t y o f the c u l t u r e s to l i q u i f y g e l­ a t i n e was s t u d i e d b o t h i n g e l a t i n e s t a b t u b e s a n d by s t r e a k ­ i n g t h e c u l t u r e s on n u t r i e n t a g a r p l a t e s c o n t a i n i n g 0 . 4 p e r cent g e la tin e B acteria, ( M an ual o f M e t h o d s f o r P u r e C u l t u r e S t u d y o f 1947). A l l c u l t u r e s w e r e a l s o i n o c u l a t e d i n t o AASS b r o t h a n d d ilu te s u s p e n s i o n s o f t h e c u l t u r e s w e r e s t r e a k e d on S . S . a g a r p lates. E xperim ental R e su lts Ba se d on IMVIG r e a c t i o n s a n d w h e t h e r o r n o t g a s was f o r m e d d u r i n g t h e f e r m e n t a t i o n o f g l u c o s e , t h e c u l t u r e s w ere d iv id e d in to fo u r groups. The a e r o g e n i c s t r a i n s ( a c i d a n d g a s i n g l u c o s e b r o t h ) w ere p l a c e d i n t h r e e s p e c i e s - P . f o r m e , P. i n te r m e d i u m , an d P. a e r o g e n o i d e s . 15 c o ll- T ho se c u l t u r e s which p ro d u ced a t b e s t o n ly a b u b b le o f g a s i n g l u c o s e b r o t h were c o n s i d e r e d a n a e r o g e n i c p a r a c o l o n s . The c o m p l e t e b i o ­ chem ical r e a c t i o n s o f a l l the c u l t u r e s a r e p r e s e n te d i n T ables 1-4. Grouping of P a r a c o lo n C u l tu r e s W ith in th e S ioecles. Any arrangem ent of p a ra c o lo n c u lt u r e s i n t o groups w ith in the s p e c i e s i n w h i c h t h e y w e r e p l a c e d a s a r e s u l t o f t h e IMVIG r e a c t i o n m u st b e p u r e l y a r b i t r a r y . Thus, v a r i o u s a u t h o r s h a v e a r r a n g e d t h e i r c u l t u r e s i n t o g r o u p s on t h e b a s i s o f a c h a r a c t e r i s t i c o r group of c h a r a c t e r i s t i c s which t h e y c o n s i d e r im portant. For exam ple, s u c r o s e p o s i t i v e s t r a i n s c o u ld be g rouped t o g e t h e r a s d i s t i n c t from s u c r o s e n e g a t i v e s t r a i n s . As was s t a t e d p r e v i o u s l y t h e p u r p o s e o f t h i s i n v e s t i g a t i o n was t o com pare n u t r i t i o n a l r e q u i r e m e n t s w i t h b i o c h e m i c a l r e ­ actio n s. On t h i s b a s i s t h e o n l y a c c e p t a b l e g r o u p i n g i s t h a t o f c o m b i n i n g a l l c u l t u r e s sh o w in g t h e same b i o c h e m i c a l r e ­ a c t i o n s i n t o t h e same g r o u p . A l t h o u g h t h i s w o u ld n e c e s s a r i l y make f o r a l a r g e n u m b e r o f g r o u p s , a n d f o r some g r o u p s w h i c h a r e o n l y r e p r e s e n t e d by one c u l t u r e , i t i s above m entioned r e a s o n . ju stifie d fo r the I n T a b l e s 1 - 4 t h e common c h a r a c t e r ­ i s t i c s of th e f o u r p a r a c o lo n ty p e s (P . a e r o g e n o i d e s . P. i n t e r ­ medium, P. c o l l f o r m e , a n d a n a e r o g e n i c p a r a c o l o n s ) a r e l i s t e d and t h e c u l t u r e s a r e a rra n g e d i n t o groups a c c o rd in g t o t h e i r r e a c t i o n s i n th e b i o c h e m i c a l t e s t s which were v a r i a b l e w i t h i n the species. G e l a t i n e l i q u i f a c t i o n was n o t i n c l u d e d i n T a b l e s 1 - 4 a s t h e r e was m ark e d v a r i a t i o n s i n t h e r e s u l t s o f t h e two m e t h o d s o f d e t e r m i n i n g t h i s p r o p e r t y . T h is w i l l be T able 1 B io ch em ical c h a r a c t e r i s t i c s of P. c o lif o r m e c u l t u r e s u sed i n n u t r i t i o n experim ents Common c h a r a c t e r i s t i c s : A l l c u l t u r e s were i n d o l e and m eth y l r e d p o s i t i v e , V.P. n e g a t i v e ; u t i l i z e d t a r t r a t e a n d d i d n o t u t i l i z e c i t r a t e ; d i d n o t produce u re a se o r hydrogen s u l f i d e ; ferm ented g lu c o s e , m a l t o s e ,m a n n it o l , a r a b i n o s e , t r e h a l o s e , and g a l a c t o s e ; d i d n o t fe rm e n t r a f f i n o s e , a d o n i t o l , and in o sito l. R eactio n Group Number o f C u ltu res I n Grou p 1 (1) Q> W O o -P to oo u o5 q oo pp Cc Oo + 4 - rH O -P •rH hO rHh 2 C3 •H oq •H rH h cti 0) m K o H hi S B 2 4 C 2 4 D 2 4 — E 2 4 - F 1 4 — — — — 4 G 1 4 - - - - - H 1 4 I 1 + + - J 1 + + - K 1 - - L 1 - - 4 - 4 17 - 4 4 4 4 — — - 4 4 4 — 4 - - - - 4 4 - - - - - 4 _ _ + - - — + - 4 4 4 - - -h 'S co § p 5 - o a A 4 H CO Pp co co xj f*! Q) 4 - 4 4 Table 2 B iochem ical c h a r a c t e r i s t i c s o f p. interm ed!um c u l t u r e s used in n u t r i t i o n experim ents Common c h a r a c t e r i s t i c s : A l l c u l t u r e s w ere m o t i l e ; m e t h y l r e d p o s i t i v e ; V.P. n e g a t i v e ; u t i l i z e d t a r t r a t e an d c i t r a t e ; ferm ented g lu c o s e , l a c t o s e , m a n n ito l, m a l t o s e , s o r b i t o l , a r a b i n o s e , rham nose, t r e h a l o s e , and g a l a c t o s e ; d i d n o t f e r ­ ment a d o n l t o l o r i n o s i t o l . Raffinose & rH O -P •H O H Salicin CM 1 m 0) H O Xylose CO Sucrose Group Number o f C ultures I n Group Urease R eaction A 6 - + 4 - 4 4 - - B 5 + - - - 4 - - - C 3 + ♦ - 4 4 - 4 - D 3 4 - - - 4 4 - - E 2 + - - - 4 - 4 - F 2 ♦ - - 4 4 - 4 - G 2 + 4 - - 4 - 4 - H 2 + - - - 4 4 4 - I 2 - - 4 - 4 4 - - J 1 4 4 4 - 4 4 4 - K 1 4 - - - - - 4 - L 1 - 4 4 4 4 - 4 4 18 T able 3 B i o c h e m ic a l c h a r a c t e r i s t i c s o f P. a e r o g e n o i d e s c u l t u r e s used in n u t r i t i o n experim ents. Common c h a r a c t e r i s t i c s : A l l c u l t u r e s w e re V . P . n e g a t i v e a n d i n d o l e p o s i t i v e ; u t i l i z e d c i t r a t e , were m o t i l e ; f e r m e n te d g lu c o s e , m a n n ito l, m a lto se , x y lo s e , a r a b i n o s e , rhamnose, g a la c to s e and t r e h a l o s e ; d i d n o t ferm ent i n o s i t o l ; ' , R eactio n ft 0) u G ro up Number o f C u ltu res I n Group r—1 b> ft ft CD 0) P cd P O •H ft ccj isi ft H P P ft ctf p EH P ft P O P o ft 0) w n) CD P p ft CD to O w ft a •H rH O -P p —1 P P o o o p CO •H rH Cj CO P o cti ftl CO CM CD CO O 0) w o G •H i H O P •H P O rH O P •H ft ft tals * Norm al g r o w t h a s d e f i n e d i n t e x t . ** C o m p o s i t i o n o f medium same a s shown i n T a b l e 6 e x c e p t t h a t a d e n i n e , g u a n in e , a n d u r a c i l were o m i t t e d . 34 v itam in s, c ase in h y d ro ly sa te , h ydro ly sate. o r "both v i t a m i n s a n d c a s e i n F o u r c u l t u r e s i n t h i s g r o u p (Al t A2 , A^, H93) c o u l d be m a i n t a i n e d i n a medium c o n s i s t i n g o f i n o r g a n i c s a l t s , g lu c o se , and th e a d d i t i o n of e i t h e r th e v ita m in m ixture or casein hydro ly sate. C u l t u r e , A4 r e q u i r e d t h e v i t a m i n m i x t u r e w h e t h e r o r n o t c a s e i n h y d r o l y s a t e was i n c l u d e d i n t h e medium. From T a b l e 9 i t c a n b e s e e n t h a t t h e r e a r e no a p p a r e n t v itam in re q u ire m e n ts i n th e p. a e ro g e n o ld e s group. O utside o f the e ig h t n o n -e x a c tin g c u l t u r e s the rem ainder of th e c u l t u r e s i n t h a t g r o u p r e q u i r e a n a m in o s o u r c e - o f n i t r o g e n a s s u p p l i e d by c a s e i n h y d r o l y s a t e . S p e c i f i c N u t r i t i o n a l R equirem ents V itam in R eq u irem en ts. The d a t a o b t a i n e d i n t h e s t u d y o f t h e g r o s s n u t r i t i o n a l r e q u i r e m e n t s and i n o t h e r p r e l i m i n a r y ex­ p e r i m e n t s i n d i c a t e d t h a t o n l y two o f t h e e x a c t i n g s t r a i n s e x h ib ite d d e f i n i t e vitam in req u irem en ts. revealed th a t r ib o f la v in , S e v e ra l experim ents th ia m in , b i o t i n , and f o l i c a c i d w ere n o t e s s e n t i a l f o r n o r m a l g r o w t h o f t h e two c u l t u r e s P. i n t e r m e d i u m A4 a n d P . o o l i f o r m e H100. N i c o t i n i c a c i d was d e f i n i t e l y r e q u i r e d by t h e s e c u l t u r e s a s shown i n T a b l e 10 i n which i s r e c o r d e d t h e d a t a o b t a i n e d i n a t y p i c a l e x p e r im e n t concerning v itam in req u ire m e n ts. The d a t a i n T a b l e 10 w e r e o b t a i n e d u s i n g a b a s a l medium c o n t a i n i n g i n o r g a n i c s a l t s , g lu cose, a c id hydrolyzed c a s e in , p y rid o x in e , P.A .B ., p a n to ­ t h e n a t e , a n d n i c o t i n i c a c i d i n t h e same c o n c e n t r a t i o n s a s u s e d in a l l previous experim ents. V a r i a t i o n s on t h e b a s a l medium T a b l e 10 The e f f e c t o f t h e o m i s s i o n o f v i t a m i n s on t h e a c i d p r o d u c t i o n of th re e paracolon c u ltu r e s . Ml. o f .01N NaOH u s e d t o t i t r a t e a c i d produced** C ulture Hours o f i n c u b a t i o n H86 H100 A4 24 72 24 72 24 72 12.7 11.7 14.0 10 .7 1 2 .2 13.7 3.2 4.0 3.5 3.2 12.7 1 2 .2 Pyridoxine 1 2 .2 10.7 1 3 .0 11.7 12.7 10.7 Pantothenate 1 3 .0 1 1 .2 13.5 10.7 13.7 11.7 P.A .B . 13.7 1 1 .2 1 3 .2 1 1 .2 1 3 .2 11.7 2.3 2 .2 3.7 3 .2 12.7 10.7 B a s a l medium* m i n u s : None N ic o tin ic acid A ll * B a s a l medium c o n s i s t e d o f s a l t s , g l u c o s e , a c i d h y d r o l y z e d c a s e i n , n i c o t i n i c a c i d , p y r i d o x i n e , p a n t o t h e n a t e , a n d P.A .B . ** Amount o f NaOH shown a b o v e i s t o t a l u s e d i n t i t r a t i n g m in u s am ount n e e d e d t o t i t r a t e a n u n i n o c u l a t e d t u b e o f medium. 36 in c lu d e d the om ission of s i n g l e v ita m in s and th e om ission o f a l l the vitam in s. F o u r t u b e s o f e a c h medium w e r e p r e p a r e d f o r e a c h c u l t u r e a n d i n o c u l a t e d w i t h a d r o p o f a w a sh e d c e l l s u s p e n s i o n made f ro m a 24 h o u r n u t r i e n t a g a r s l a n t c u l t u r e . The i n o c u l u m was a p p r o x i m a t e l y 5 x 1 0^ c e l l s p e r 10 m l . medium. of The a c i d p r o d u c e d a f t e r 24 a n d 72 h o u r s i n c u b a t i o n a t 37°G was t i t r a t e d i n d u p l i c a t e t u b e s w i t h . 0 1 N NaOH. P . i n t e r m e d i u m H86 was i n c l u d e d i n t h i s e x p e r i m e n t i n o r d e r t o o b t a i n i n f o r m a t i o n on t h e s t i m u l a t o r y e f f e c t i f a n y on a c u l t u r e which d i d n o t r e q u i r e t h e v i t a m i n m ix t u r e s u p p le m e n t. I t can be s e e n f r o m t h e d a t a t h a t o n l y t h e o m i s s i o n o f n i c o t i n i c a c i d h a d a n y e f f e c t on t h e r e s p o n s e o f c u l t u r e A^ a n d H100. C u l t u r e H86 was n o t s t i m u l a t e d by t h e v i t a m i n s n o r i n h i b i t e d b y ..the l a c k o f a n y o r a l l t h e v i t a m i n s . H100 a n d C ultures c o u l d b e m a i n t a i n e d b y 24 h o u r s e r i a l t r a n s f e r s i n a medium c o n s i s t i n g o f s a l t s , g lu c o s e , and n i c o t i n i c a c id i n c o n c e n t r a t i o n s a s low a s .0 2 m i c r o g r a m s p e r m l. o f medium. An a m in o n i t r o g e n s o u r c e , t h o u g h s t i m u l a t o r y , was n o t r e qui r e d . U s i n g c u l t u r e H100, a n e x p e r i m e n t was d e s i g n e d t o o b t a i n i n f o r m a t i o n a s t o t h e optimum c o n c e n t r a t i o n o f n i c o t i n i c a c i d . U s i n g a s a l t s a n d g l u c o s e b a s a l medium, two s e t s o f t u b e s were p r e p a r e d c o n t a i n i n g i n c r e a s i n g amounts of n i c o t i n i c a c i d . The t u b e s o f media w e r e i n o c u l a t e d a s d e s c r i b e d i n t h e p r e v ­ i o u s e x p e r i m e n t a n d t i t r a t e d i n t r i p l i c a t e a f t e r 24 a n d 72 hours in c u b a tio n a t 37°0. The r e s p o n s e o f c u l t u r e H100 t o i n c r e a s i n g a m o u n t s o f n i c o t i n i c -a c i d i s shown i n F i g . 1. It j+ftri+R"- ilOO to ; in n lh o t inl-c ise:io •hf47r'-A L to E rE F K t t l-f-rrP pncm: £ E S ± t£ L is m f- eof ±H±hEE!± +r:!±!±Li ■; i t r H ± h h 2; ho nr t l t r a t l T i+ - ‘±L!± ‘. .r1-h1-i_ l.l.w pQ'Lprc i± l± rn± t lour t i t r a tion XtXLTD'U Rti+ff trqiilrl fci -HK[|.?w -MOK-HliW I I I mm l±Sti thbhEttli E EEtigfcS L11XI •r r f±h±!± f 'h t x i . j : ! : ! rt+H-t+R rogra m-irUr? H:EhSfi n ic o l n i - c - a c l rrrf. r r tttr r ri i ± t t ± t ± i ± i f f i H i i - r p e r - m l . —o med;.um rh tttL l i ± t t ± i f t T c a n be s e e n t h a t t h e p e a k i n a m o u n t o f a c i d p r o d u c t i o n i s r e a c h e d w i t h a c o n c e n t r a t i o n o f .0 2 m i c r o g r a m s o f n i c o t i n i c a c i d p e r m l. o f medium i n t h e 24 h o u r t i t r a t i o n a n d a c on­ c e n t r a t i o n o f .0 1 m i c r o g r a m o f n i c o t i n i c a c i d p e r m l . of medium i n t h e 72 h o u r t i t r a t i o n . A l o n g w i t h t h e two c u l t u r e s e x h i b i t i n g a d e f i n i t e v i t a m i n r e q u i r e m e n t , t h e r e were s e v e r a l c u l t u r e s t h a t c o u ld be main­ t a i n e d i n a medium c o n s i s t i n g o f s a l t s , acid . g l u c o s e , and n i c o t i n i c Tlie n i c o t i n i c a c i d c o u l d b e r e p l a c e d by t h e a m i n o n i t r o g e n s o u r c e s w hich f o r a l l p r a c t i c a l p u r p o s e s were f r e e of v itam ins. .For e x a m p l e , c u l t u r e s A^, A-j_, H46, H77, a n d H86 e x h i b i t e d t h e same g r o w t h r e s p o n s e on e i t h e r a medium composed of s a l t s , sa lts, g l u c o s e , a n d n i c o t i n i c a c i d o r one composed o f g lu c o s e , and d l - tr y p to p h a n e . N itro g e n Source R eq u irem en ts. T h o se a e r o g e n i c c u l t u r e s , w h i c h r e q u i r e d c a s e i n h y d r o l y s a t e f o r n o rm a l growth,.were s t u d i e d i n more d e t a i l w i t h r e s p e c t t o o t h e r s o u r c e s o f a m i n o n i t r o g e n , since i t seemed d e f i n i t e th a t an in o rg a n ic source of n itro g e n was u n s u i t a b l e f o r t h e n o r m a l g r o w t h o f t h e s e e x a c t i n g s t r a i n s . U s i n g a b a s a l medium o f t h e i n o r g a n i c s a l t s a n d g l u c o s e t h e f o l l o w i n g a m in o n i t r o g e n s o u r c e s w e re a d d e d t o s e p a r a t e b a t c h e s o f t h e b a s a l medium. 1) 2) Acid h y d r o l y z e d c a s e i n - v i t a m i n - f r e e casam ino a c i d s , (D ifco). ( C o n c e n t r a t i o n i n m edium -0.1 p e r c e n t . ) P e r o x i d e t r e a t e d p e p t o n e * - L y m a n . M o s e l e y , Wood,and Hale (1946) r e p o r t e d t h a t t h e t r e a t m e n t o f p e p to n e * K i n d l y s u p p l i e d by Mr. W.S. B o n i e c e o f t h e D e p a r t m e n t o f B a c t e r i o l o g y , M ichigan S t a t e C o lle g e . 39 3) 4) w ith hydrogen p e ro x id e r e s u l t e d i n a d e s t r u c t i o n of m ethionine, c y s tin e , try p to p h a n e, and ty r o s in e . ( C o n c e n t r a t i o n i n m edium -0.1 p e r c e n t . ) S i m u l a t e d c a s e i n h y d r o l y s a t e - composed o f 18 am ino a c i d s i n c o n c e n t r a t i o n s com parable t o t h o s e found in casein. W illia m s (1 9 4 2 ) , Block and B o l l i n g (1945). (See T ab le 1 1 .) d l - t r y p t o p h a n e ( u s e d i n c o n c e n t r a t i o n same a s i n th e sim ulated c a se in h y d r o ly s a te .) The m e d i a w e re i n o c u l a t e d i n a m a n n e r s i m i l a r t o t h a t d e s c r i b e d e a r l i e r a n d when p o s s i b l e t h e c u l t u r e s w ere c a r r i e d i n t h e m ed ia f o r a t l e a s t t h r e e t r a n s f e r s . The r e s u l t s o f t h i s e x ­ p e rim e n t a r e t a b u l a t e d i n Table 12. The d a t a f o r e n z y m a t i c c a s e i n h y d r o l y s a t e medium w e r e t h o s e o b t a i n e d i n t h e p r e l i m i ­ nary experim ents. I t can b e s e e n f r o m T a b l e 12 t h a t t h e o n l y a m i n o n i t r o g e n s o u r c e a d e q u a t e f o r a l l t h e c u l t u r e s t e s t e d was t h e m i x t u r e o f 18 a m in o a c i d s ( s i m u l a t e d c a s e i n h y d r o l y s a t e ) . The two c a s e i n h y d r o l y s a t e s ( a c i d and e n z y m a tic ) were a d e q u a te f o r a l l b u t on e c u l t u r e . The p e p t o n e w h i c h h a d b e e n t r e a t e d w i t h h y d r o g e n p e r o x i d e was a d e q u a t e f o r o n l y s e v e n o f t h e 33 c u ltu res te ste d . N i n e t e e n o f t h e c u l t u r e s c o u l d be m a i n ­ t a i n e d i n a medium c o n t a i n i n g p e r o x i d e t r e a t e d p e p t o n e b u t t h e g r o w t h i n e a c h t r a n s f e r was g r e a t l y d e l a y e d . The amino n i t r o g e n r e q u i r e m e n t o f t h r e e c u l t u r e s (H46, H48, a n d H57) was s a t i s f i e d w i t h J u s t one a m in o a c i d - t r y p t o p h a n e . Nine c u l t u r e s i n t h e P. In te rm e d iu m gro u p co u ld be m a i n t a i n e d i n a medium c o n t a i n i n g o n l y t r y p t o p h a n e a s a s o u r c e o f a m in o n i t r o g e n b u t h e r e a g a i n g r o w t h i n e a c h t r a n s f e r was d e l a y e d when c o m p a red t o t h e r e s p o n s e i n a more c o m p l e t e medium. In o r d e r t o d e te r m i n e which o f t h e amino a c i d s , d e f i c i e n t 40 T a b l e 11 C om position of t h e s im u la te d c a s e i n h y d r o l y s a t e . Grams p e r l i t e r o f medium Amino a c i d DL-tryptophane 0.0 4 0 L -cy stin e 0 .0 0 4 L -glutam ic a c id 0.218 D L -ly sin e (m onohydrochloride) 0.150 L -p ro lin e 0.0 8 0 D L-valine 0.1 5 8 D L -aspartic a c id 0.144 D L -leucine 0.194 L - a r g i n i n e (m onohydrochloride) 0.0 5 2 DL-alpha a l a n i n e 0.112 glycine L -h istid in e 0 .0 0 4 (m onohydrochloride) 0.030 D L -serine 0.1 1 6 L -h ydroxyproline 0.020 D L -threonine 0.0 7 8 L -ty ro sin e O.O65 D L -m ethionine 0.0 6 2 D L -phenylalanine 0.100 T a b l e 12 The g r o w t h o f p a r a c o l o n c u l t u r e s on a s a l t s - g l u c o s e medium w i t h v a r i e d s o u r c e s o f a m in o n i t r o g e n . * Amino N i t r o g e n Source P . co llfo rm e (3 c u l t u r e s ) P . Interm edium (2 0 c u l t u r e s ) P .aerogenoldes (LQ c u l t u r e s ) ♦ do) Enzym atic casein h y d ro ly sate ♦ ( 3)** Acid c a s e i n hy d ro ly sate + (' 2) + (19) + ( 9) i ( 1) ♦ d ( 1) ♦a( i) P eroxidetreated peptone ♦ ( 3) ♦ ( 3) ♦ a(i3) ♦ i) i - - ( 3) Sim ulated casein h ydrolysate ♦ ( 3) d l-try p to p h an e ♦ ( 2) - ( 1) ♦ (19) ♦a( l ) ( i) ( +d( 6) ( i) + ( 20) ♦ ( 10) ♦ ( 1) 1 ( 5) - ( 5) - ( 1) ♦d( 9) + ( 9) * ♦ I n d i c a t e s norm al grow th a s d e f in e d i n t e x t . +d i n d i ­ c a t e s d e l a y e d r e s p o n s e on one o r more t r a n s f e r s . + in d i­ c a t e s c u l t u r e c o u ld n o t be m a in ta in e d i n t r a n s f e r s . - i n d i c a t e s no g r o w t h f r o m f i r s t i n o c u l a t i o n . ** f i g u r e i n b r a c k e t shows n um ber o f c u l t u r e s e x h i b i t i n g i n d i c a t e d r e s p o n s e i n medium. 42 i n p e ro x id e t r e a t e d p e p to n e , a r e r e q u i r e d f o r norm al grow th o f some o f t h e c u l t u r e s , a s e r i e s o f e x p e r i m e n t s was c o n d u c t e d u s i n g a b a s a l medium composed o f s a l t s , t r e a t e d peptone, try p to p h a n e , glucose, peroxide- cystine, m e th i o n in e , a n d t r y o s i n e . By e l i m i n a t i n g one o r more o f t h e a m i n o a c i d s , n i n e v a r i ­ a t i o n s o f t h e b a s a l medium w e r e u s e d a n d s e e d e d a s p r e v i o u s l y d e sc rib e d in the v itam in ex p erim en ts. Acid p r o d u c t i o n , i n d u p l i c a t e t u b e s o f e a c h medium, f o r e a c h c u l t u r e was d e t e r ­ mined a f t e r 24 a n d 72 h o u r s i n c u b a t i o n . The r e s u l t s o f a t y p i c a l e x p e r i m e n t a r e shown i n T a b l e 13. The r e s u l t s c le a r ly i l l u s t r a t e a d e f i n i t e requirem ent f o r c y s tin e or m ethionine. As l o n g a s one o f t h e two a m i n o a c i d s was p r e ­ s e n t t h e medium was n u t r i t i o n a l l y a d e q u a t e . The e l i m i n a t i o n of try p to p h a n e o r t y r o s i n e s i n g l y o r i n c o m b in atio n s had no e f f e c t on t h e r e s p o n s e o f t h e c u l t u r e s t e s t e d . C u l t u r e s H55 a n d H66 would n o t grow a t a l l when t h e medium was d e f i c i e n t in c y s tin e and m ethionine. C u l t u r e H l l w o u l d grow i n t h e d e f i c i e n t medium, h o w e v e r , g r o w t h w a s d e l a y e d a n d t h e c u l t u r e c o u l d n o t b e m a i n t a i n e d by s e r i a l t r a n s f e r s i n t h e medium. S e v e r a l o f t h e a n a e r o g e n i c c u l t u r e s were se e d e d i n t o t h e medium c o n t a i n i n g t h e 18 amino a c i d s . I t was o b s e r v e d t h a t t h i s medium w o u l d s u p p o r t m o d e r a t e g r o w t h on c o n t i n u e d s u b c u l t u r e b u t a g a i n i t was o n l y by t h e a d d i t i o n o f y e a s t e x t r a c t t h a t l u x u r i a n t grow th co u ld be a c h i e v e d . 43 T a b l e 13 The e f f e c t o f t h e o m i s s i o n o f a m i n o a c i d s on t h e a c i d p r o d u c ­ tio n of th re e paracolon c u ltu re s . Ml. o f . 01N NaOH u s e d t o t i t r a t e a c i d produced** C ulture H ll H66 H55 24 72 24 72 24 72 10.0 9 .8 9.0 9.0 9.5 8.5 T ryptophane 9 .5 9.5 8 .5 8.8 9 .5 7.5 C ystine 9.0 10.0 7 .5 8 .8 8.5 8 .5 Tyrosine 9 .5 10.0 7 .5 9.0 8.5 8 .0 10.0 1 1 .0 9 .0 9.0 7.0 8.0 C ystine and M ethionine 0 .5 9 .5 0 .0 0.0 0.0 0.0 T ryptophane and T yrosine 9 .5 8.5 9 .8 7.3 7.3 8.5 C ystine, T yrosine, and M ethionine 0.3 10.5 0.0 0.0 0.0 0.0 T ryptophane, T y ro sin e , and M ethionine 9 .8 9 .5 8 .0 7.0 8.0 8.5 A ll 0 .0 10.5 0 .3 0 .0 0.0 0 .0 Hours o f i n c u b a t i o n B a s a l medium* m i n u s : None M ethionine * B a s a l medium c o n s i s t e d o f s a l t s , g l u c o s e , p e r o x i d e t r e a t e d p e p to n e , t r y p to p h a n e , c y s t i n e , t y r o s i n e , and m e th io n in e . ** Amount o f NaOH shown a b o v e i s t o t a l u s e d i n t i t r a t i n g m in u s a m o u nt n e e d e d t o t i t r a t e a n u n i n o c u l a t e d t u b e o f medium. 44 DISCUSSION The d a t a o b t a i n e d f r o m t h e p r e c e d i n g e x p e r i m e n t s b r i n g s t o l i g h t some v e r y i n t e r e s t i n g r e l a t i o n s h i p s b e t w e e n t h e t a x ­ onomic g r o u p i n g o f p a r a c o l o n c u l t u r e s on t h e b a s e s o f b i o ­ c h e m i c a l c h a r a c t e r i s t i c s a n d t h e i r g r o u p i n g on t h e b a s i s o f n u t r i t i o n a l requirem ents. The c u l t u r e s s t u d i e d c o u l d be c o n v e n i e n t l y g r o u p e d i n t o one o f f o u r m a i n t y p e s on t h e b a s i s o f t h e i r IMVIC r e a c t i o n a n d w h e t h e r o r n o t t h e y f e r m e n t e d glucose w ith the p ro d u c tio n o f gas a lo n g w ith a c i d . fu rth e r attem p t to sep arate paracolon c u ltu r e s , se p a ra tio n i n t o groups w ith in th e s p e c ie s , purely a r b i t r a r y procedure. Any th at i s , can o n ly be a The a u t h o r h a s d i v i d e d t h e s p e c i e s i n t o g r o u p s o f c u l t u r e s e x h i b i t i n g t h e same b i o c h e m ­ ic a l ch aracteristics. As h a s b e e n s t a t e d p r e v i o u s l y , th is a p p r o a c h p r o d u c e d many g r o u p s w i t h i n t h e s p e c i e s a n d some g r o u p s w h i c h w e re o n l y r e p r e s e n t e d b y one c u l t u r e . hom ogeniety of th e s e b io c h e m ic a l groups i s The c a r r i e d o v e r .w ith resp e c t to the n u t r i t i o n a l requirem ents of the c u ltu r e s in the group. A s t u d y o f t h e d a t a o b t a i n e d f r o m t h e e x p e r i m e n t s on t h e g r o s s n u t r i t i o n a l r e q u i r e m e n t s r e v e a l s t h a t i n o n l y two i n s t a n c e s d i d c u l t u r e s i n t h e same b i o c h e m i c a l g r o u p e x h i b i t d i f f e r e n t g ross n u t r i t i o n a l req u irem en ts. To a l a r g e e x t e n t t h e h o m o g e n i e t y o f n u t r i t i o n a l r e q u i r e ­ ments i s grouping. s t i l l a p p a r e n t when r e l a t e d t o t h e l a r g e r t a x o n o m i c Thus i n t h e P . c o l l fo rm e s p e c i e s 85 p e r c e n t o f t h e c u l t u r e s t e s t e d h a d t h e same g r o s s n u t r i t i o n a l r e q u i r e m e n t In t h e P. Interm edium s p e c i e s t h e r e were t h r e e n u t r i t i o n a l g r o u p s r e p r e s e n t e d by 3 0 , 1 7 , a n d 50 p e r c e n t o f t h e c u l t u r e s tested . Two n u t r i t i o n a l g r o u p s i n t h e P . a e r o g e n o l d e s s p e c i e s a c c o u n t e d f o r 44 a n d 56 p e r c e n t o f t h e c u l t u r e s i n t h e species. The a n a e r o g e n i c c u l t u r e s w ere h o m ogenous i n t h a t no ne o f t h e c u l t u r e s c o u l d be m a i n t a i n e d i n t e s t medium No. 1 . and t h a t y e a s t e x t r a c t su p p lie d the n e c e s s a r y grow th f a c t o r s . A p e r s p e c t i v e o f th e r e l a t i o n s h i p betw een s p e c i e s g ro u p in g and g r o s s n u t r i t i o n a l r e q u i r e m e n t s can be o b t a i n e d from th e d a t a s u m m arize d i n T a b l e 1 4 . When t h e 34 n o n - e x a c t i n g c u l t u r e s ( n o r m a l g r o w t h on i n ­ o r g a n i c s a l t s and g l u c o s e ) were a n a l y z e d w ith r e s p e c t t o t h e i r r e a c t i o n s on t h e b i o c h e m i c a l t e s t s i t was f o u n d t h a t t h e o n l y common b i o c h e m i c a l c h a r a c t e r i s t i c o f t h e s e c u l t u r e s was t h a t they d id n o t produce hydrogen s u l f i d e . O t h e r common b i o ­ c h e m i c a l c h a r a c t e r i s t i c s w e re a l s o common t o a l l t h e a e r o g e n i c c u l t u r e s a n d t h u s h a d no a p p a r e n t s i g n i f i c a n c e . Hydrogen s u l f i d e p r o d u c t i o n was a v a r i a b l e c h a r a c t e r i s t i c o f t h e amino nitro g en req u irin g c u ltu re s . The two n i c o t i n i c a c i d - r e q u i r i n g c u l t u r e s w e r e i n d i f f e r e n t s p e c i e s , h o w e v e r , t h e y w e re a l i k e i n 17 o u t o f 22 of th e biochem ical r e a c t i o n s . I t was r a t h e r s u r p r i s i n g t h a t o n l y two c u l t u r e s p r o v e d t o h a v e d e f i n i t e v i t a m i n r e ­ quirem ents. The c u l t u r e s w h i c h h a d a n a p p a r e n t r e q u i r e m e n t f o r e i t h e r t h e v i t a m i n m i x t u r e o r a n a m in o n i t r o g e n s o u r c e a r e o f i n t e r e s t a s t h i s o b s e r v e d phe nomenon seems t o b e a case of a n i c o t i n i c a c id - tr y p to p h a n e r e l a t i o n s h i p . I t has b e e n shown by B e a d l e , M i t c h e l l , a n d Nyc ( 1 9 4 7 ) t h a t k y n u r e n i n e 46 T a b l e 14 A summary o f t h e r e l a t i o n s h i p b e t w e e n g r o u p i n g o f p a r a c o l o n b a c i l l i by b i o c h e m i c a l c h a r a c t e r i s t i c s and t h e i r g ro ss n u t r i t i o n a l requirem ents. Number o f c u l t u r e s r e q u i r i n g : Inorganic n itro g en P .co lifo rm e (20 c u l t u r e s ) Amino n itro g en A ccessory grow th facto rs 17 2 1*# P.interm edium (3 0 c u l t u r e s ) ^ 9 20* 1#* P.aerop;enoides T lS c u l t u r e s ) 8 10 anaerogenic paracolons (9 c u l t u r e s ) T otals 9 34 41 ^ *>cvf 11 * F o u r c u l t u r e s i n t h i s g r o u p c o u l d do w i t h o u t a m in o n i t r o g e n i f s u p p lie d w ith th e v ita m in supplem ent and v ic e v e r s a . ** N i c o t i n i c a c i d . Yeast e x t r a c t . 47 I s an i n t e r m e d i a t e i n th e fo rm a tio n o f n i c o t i n i c a c i d , p re c e d ­ ing th e oxypyridine c a rb o x y lic a c id s . S i n c e one o f t h e norm al p ro d u c ts of try p to p h a n e m etabolism i s k y n u r e n in e ,th e r e i s th e p o s s i b i l i t y t h a t the p a ra co lo n c u lt u r e s in q u e stio n r e q u i r e e i t h e r p r e f o r m e d n i c o t i n i c a c i d o r t h a t t h e y can s y n t h e s i z e n i c o t i n i c a c i d from k y n u re n in e i f they a r e supplied w ith try p to p h a n e (K oser, 1948), I t h a s b e e n shown t h a t a n a d e q u a t e s o u r c e o f a m i n o n i t r o g e n f o r t h e a e r o g e n i c p a r a c o l o n s v a r i e s f ro m j u s t one amino a c i d ( t r y p t o p h a n e ) f o r some c u l t u r e s t o a m i x t u r e o f a m in o a c i d s f o r t h e o t h e r c u l t u r e s . E x c e l l e n t growth i s a c h i e v e d i n a medium c o n t a i n i n g 18 a m i n o a c i d s ; i n some c a s e s t h i s medium s u p p o r t s g r o w t h b e t t e r t h a n d o e s a medium c o n t a i n ­ ing a hy d ro ly sate of casein . T his i s t o be e x p e c te d a s t h e h y d r o l y s i s o f c a s e i n by a c i d r e s u l t s i n some d e s t r u c t i o n o f try p to p h a n e and c y s t in e . H y d r o l y s i s o f p r o t e i n s by enzymes h a s a s i t s m ain d i s a d v a n t a g e t h e f a c t t h a t o f t e n t h e hy ­ d r o l y s i s i s n o t com plete and t h e amino a c i d s o f th e p r o t e i n m a t e r i a l a r e n o t a s r e a d i l y a v a i l a b l e a s i n a m ix tu re of am ino a c i d s . On t h e b a s i s o f n u t r i t i o n a l r e q u i r e m e n t s t h e p a r a c o l o n b a cilli s t u d i e d , w h i c h w e re c l a s s i f i e d a s P . i n t e r m e d i u m . P. a e r o g e n o i d e s . a n d a n a e r o g e n i c p a r a c o l o n s seem t o b e more c lo s e ly r e l a t e d to th e S alm onella, S h i g e l l a , and P ro teu s g e n e r a t h a n do t h o s e c u l t u r e s w h i c h w e r e c l a s s i f i e d a s P. c o ll form e. In t h i s connection i t i s I n t e r e s t i n g to note t h a t a l l evidence of p a th o g e n ic ity o f p a ra c o lo n c u lt u r e s 48 c o n c e rn s th o s e c u l t u r e s w hich c o u ld be c l a s s i f i e d a s P. I n te r m e d iu m . P. a e r o g e n o i d e s . an d a n a e r o g e n i c p a r a c o l o n s . C u l t u r e s i n t h e P . c o l l f o r m e s p e c i e s , f o r t h e m o st p a r t , c a n s y n t h e s i z e a m i n o a c i d s a n d g r o w t h f a c t o r s a n d t h u s would b e l e s s d e p e n d e n t on t h e i r h o s t f o r e s s e n t i a l n u t r i e n t s . The r e l a t i o n s h i p o f n u t r i t i o n a l r e q u i r e m e n t s o f b a c t e r i a t o t h e i r p a t h o g e n i c i t y i s a s u b j e c t on w h i c h much work h a s been done. The work r e p o r t e d h e r e o f f e r s a t l e a s t a s u g g e s tio n t h a t such r e l a t i o n s h i p s do e x i s t . 49 CONCLUSIONS 1. The c l a s s i f i c a t i o n o f p a r a c o l o n b a c i l l i on t h e b a s i s o f r e a c t i o n s i n t h e b i o c h e m i c a l t e s t s u s u a l l y em p lo y ed h a s a s i t s main d i s a d v a n t a g e t h e e x t r e m e v a r i a b i l i t y o f r e a c t i o n s encountered. F o r t h e m o s t p a r t , p a r a c o l o n c u l t u r e s c a n be g r o u p e d on t h e b a s i s o f IMVIC r e a c t i o n a n d t h e f e r m e n t a t i o n of glucose. W ithin th e s p e c i e s g ro u p in g (P. c o l i f o r m e , P. I n t e r m e d i u m , a n d P . a e r o g e n o i d e s . ) can be f o u n d many t y p e s w h i c h d i f f e r i n r e a c t i o n on one o r more o f t h e b i o c h e m i c a l tests. 2. The d e l a y e d f e r m e n t a t i o n o f l a c t o s e , w h i c h i s c onsidered a very im p o rtan t c h a r a c t e r i s t i c of the p araco lo n b a cilli, is in i t s e l f a v ariab le c h a ra c te ris tic . lacto se f e r m e n t a t i o n c a n b e slow o r r a p i d a c c o r d i n g t o t h e medium e m p loy ed f o r t e s t i n g t h i s c h a r a c t e r i s t i c . The u s e o f a g a r s l a n t s c o n t a i n i n g l a c t o s e i n a c o n c e n t r a t i o n o f 10 p e r c e n t and a s u ita b le I n d i c a to r i s a v a lu a b le a id in the ra p id d e te c tio n of la c to s e ferm en tatio n . 3. The i n t e r m e d i a t e n a t u r e o f t h e p a r a c o l o n b a c i l l i was a p p a r e n t a l s o i n t h e i r n u t r i t i o n a l r e q u i r e m e n t s . Both e x a c t i n g an d n o n - e x a c t i n g s t r a i n s were e n c o u n t e r e d . 4. The P. c o l l ' f o r m e c u l t u r e s w ere more c l o & e l y r e l a t e d t o t h e E s c h e r i c h l a - A e r o b a c t o r g r o u p on t h e b a s i s o f n u t r i t ­ io n a l requirem ents. R e q u ire m e n ts o f t h e P. I n te r m e d iu m . P. a e r o g e n o i d e s . a n d a n a e r o g e n i c p a r a c o l o n s w e re s i m i l a r t o t h e 50 P r o t e u s . S a lm o n e lla . and S h i g e l l a g e n e ra . 5. Gro w th f a c t o r r e q u i r e m e n t s i n t h e a e r o g e n i c p a r a ­ colons a re l im i te d t o n i c o t i n i c a c i d . Amino a c i d r e q u i r e m e n t s can b e s a t i s f i e d i n some c u l t u r e s w i t h j u s t o ne a m ino a c i d try p top h an e. cu ltu res. C y s t i n e o r m e t h i o n i n e i s r e q u i r e d by s e v e r a l O t h e r c u l t u r e s r e q u i r e a m i x t u r e o f amino a c i d s a s s u p p l i e d by a s i m u l a t e d c a s e i n h y d r o l y s a t e ( 1 8 a m ino a c i d s ) o r by a c i d o r e n z y m a t i c h y d r o l y s a t e s o f c a s e i n . 6. A n a e r o g e n i c p a r a c o l o n s grow p o o r l y i n t h e s y n t h e t i c media e m p lo y e d . The a d d i t i o n o f y e a s t e x t r a c t r e s u l t e d i n l u x u r i a n t growth. 51 REFERENCES B a r n e s , L. A. a n d C h e r r y , L. B. 1946 A g r o u p o f p a r a c o l o n organism s h av in g a p p a r e n t p a th o g e n i c i t y . Am. J . P u b . H e a lth , 3 6 , 481-483. B e a d l e , G-. W., M i t c h e l l , H. K . , a n d Nyc, J . P . 1947 K ynurenine a s a n i n t e r m e d i a t e i n th e f o r m a tio n o f n i c o t i n i c a c i d f r o m t r y p t o p h a n e by N e u r o s p o r a . P r o c . N a t l . A c ad. S c i. U .S ., 22, 155-158. B l o c k , R. J . a n d B o l l i n g , D. 1945 The a m i n o a c i d c o m p o s i t ­ io n o f p r o t e i n s and fo o d s. C h a r l e s C. Thomas, S p r i n g ­ f i e l d , 111. Borman, E. K . , S t u a r t , C. A . , a n d W h e e l e r , K. M. 1944 Taxonomy o f t h e f a m i l y E n t e r o b a c t e r i a c e a e . J . B act., 48, 351-367. B r e e d , R. S . , M u r r a y , E. G. D . , a n d H i t c h e n s , A. P . B e r g e y 's manual o f d e t e r m i n a t i v e b a c t e r i o l o g y . W i l l i a m s a n d W i l k i n s C o . , B a l t i m o r e , Md. 1948 6 th ed. C h i l t o n , M. L. a n d F u l t o n , M. 1946 A p r e s u m p t i v e medium f o r d i f f e r e n t i a t i n g p a r a c o l o n f ro m S a l m o n e l l a c u l t u r e s . J. Lab . C l i n . M e d ., 2 1 , 8 2 4 - 8 2 7 . C h r i s t e n s e n , W. B. 1946 Urea d e c o m p o s i t i o n a s a means o f d i f f e r e n t i a t i n g P r o t e u s and p a r a c o l o n c u l t u r e s from each o t h e r a n d fro m S a l m o n e l l a a n d S h i g e l l a t y p e s . J . B act., 2 2 , 461-466. C h r i s t e n s e n , W. B, 1947 C o m p a r a t i v e d i s t r i b u t i o n a n d p o s s i b l e p a th o g e n ic ity of Paracolobactrum sp e c ie s in an a re a h ig h ly en d em ic f o r e n t e r i c i n f e c t i o n s . J . B a c t . , 22, 517-324. D a r b y , C. W. a n d M a llm a n n , W. L. 1939 S t u d i e s on m e d i a f o r co lifo rm organism s. J . Am. W a t e r WorkB A s s o c . , 2 1 , 6 8 9 - 706 . D ifco m anual. 1948 D i f c o L a b o r a t o r i e s I n c . , D e t r o i t . E d w a r d s , P . R. 1945 A p a r a c o l o n - l i k e b a c i l l u s i s o l a t e d fro m c o l i t i s i n an i n f a n t . J . B a c t . , 4g., 5 1 3 - 5 1 4 . 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F . , C o r i e l l , L. L . , D a v i s , H . , a n d B o l t j e s , B. H. A s e r i o l o g i c a l t y p e o f p a r a c o l o n a s a p r o b a b l e cause o f an epidem ic o f g a s t r o e n t e r i t i s . J. B act., 54 , 277-278. 53 N e t e r , E. 1945 P a r a c o l o n a n d P r o t e u s b a c i l l i i n t h e f e c e s of healthy in f a n t s . J . P e d i a t . , 26, 390-394. P e l c z a r , M. J . J r . , a n d P o r t e r , J . R. 1940 The u t i l i z a t i o n o f n i c o t i n i c a c i d a n d r e l a t e d p y r i d i n e compounds by t h e P r o te u s group o f o rg a n is m s . J . B a c t . , 2£» 4 2 9 - 4 3 5 . P e l c z a r , M. J . J r . , a n d P o r t e r , J . R. 1943 The n u t r i t i o n o f P r o t e u s Morgan! a m i n o a c i d a n d g r o w t h f a c t o r r e q u i r e ­ m ents. Arch. B iochem ., 2, 323-332. P e t e r s o n , W. H. a n d P e t e r s o n , b a c te r ia to v ita m in s and R e v s., £ , 49-109. M. S. 1945 R e l a t i o n of o t h e r growth f a c t o r s . B a c t. P l a s s , H. F. R. 1947 O u t b r e a k o f d i a r r h e a l d i s e a s e a s s o c ­ i a t e d w ith p a ra c o lo n . J . Lab. C l i n , M e d ., j52 , 8 8 6 - 8 8 8 . P o r t e r , J . R. 1946 B a c t e r i a l c h e m i s t r y a n d p h y s i o l o g y . J o h n W iley a n d S o n s , I n c . , N. Y. S a n d i f o r d , B. R. Path. B a ct., S c h a u b , I . G. b a cilli. 1935 The p a r a c o l o n g r o u p o f b a c t e r i a . 41, 7 7 -88. J. 1948 The c u l t u r a l d i f f e r e n t i a t i o n o f p a r a c o l o n B u l l . Jo h n s Hopkins H o s p . , 82, 36 7 -3 8 2 . S t u a r t , C. A . , G a l t o n , M. M . , a n d McGann, V. 1948 A n t i g e n i c r e l a t i o n s h i p s o f 765 P a r a c o l o b a c t r u m i n t e r m e d i u m c u l t u r e s . J . B a c t., ^ 6 , 411-417. S t u a r t , C. 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M ., R u s t i g i a n , R . , a n d Zimmerman, A. 1943 B i o c h e m i c a l a n d a n t i g e n i c r e l a t i o n s h i p s o f the paracolon b a c t e r i a . J . B a c t . , 42, 101-119. 54 T o p l e y , W. W. C . , a n d W i l s o n , G-. S. 1937 The p r i n c i p l e s o f b a c t e r i o l o g y and im m unity. W i l l i a m Wood a n d C o . , B altim ore. W h e e l e r , K. M . , S t u a r t , G. A . , R u s t i g i a n , R . , a n d Borman, E . K . 1943 S a l m o n e l l a a n t i g e n s o f c o l i f o r m b a c t e r i a . J. B a ct., 41, 59-66. W id a l a n d N o b e c o u r t 1897 S e r o r e a c t i o n d a n s une i n f e c t i o n a p a ra co lib ac illi. S e m a in e M e d i c a l e , No. 3 6 . C i t e d i n N e t e r , E. R. a n d C l a r k , P . 1944 O b s e r v a t i o n on t h e p o ssib le p a th o g e n ic ity of paracolon b a c i l l i a s i n c i t a n t s o f d i a r r h e a l d i s e a s e s i n i n f a n t s and c h i l d r e n . Am. J . D ig e s tiv e D i s e a s e s , 11, 356-360. W i l l i a m s , R. J . , a n d B e e r s t e c h e r , J r . , E. 1942 I n t r o d u c t i o n to biochem istry. 2nd e d . D. Van N o s t r a n d C o . , I n c . , N . Y . 55