Correlation of resistance to streptomycin with age of the bacterial culture

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CORRELATION OF RESISTANCE TO STREPTOMYCIN WITH AGE OF THE BACTERIAL CULTURE

A T h e sis P re se n te d to th e F a c u lt y o f th e G ra d u a te S c h o o l U n iv e rs ity

of

S o u th e rn C a lif o r n ia

In P a r tia l F u lfillm e n t o f th e R e q u ir e m e n ts f o r t h e D egree M a ste r o f S c ie n c e

by M arv in R e itm a n F eb ru ary

1950

UMI Number: EP55016

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.Oissertaifott PiiblisJ'tiftg

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Th is thesis, w ri tt en by ...........................l A R V I ^ L R E I T M .N ...................................

under the gui d an ce of h. i.a „ F acu lty C o m m i t t e e , and a p p r o v e d by all its me mb ers , has been pre se nt e d to an d a c c e p te d by the Co un c il on G ra d u at e S t u d y an d R ese arc h in p a r ti a l fu lfill­ ment of the requ irements f o r the de gre e of

MASTER OF SCIENCE ..............................................

D ean

Faculty Committee

Chairman ...

TABLE OP CONTENTS CHAPTER

I. II.

PACE

BACKGROUND AND STATEMENT OF PROBLEM.................. EXPERIM ENTS.......................... ....

-

4

A p p earan ce o f r e s i s t a n t v a r i a n t s i n a g ro w in g c u l t u r e o f a s e n s i t i v e s t r a i n ...............................

4

P ro ced u re

. . . . .

.

.

.......................... ....

1

.........................................................

R e s u l t s a n d d i s c u s s i o n .................................... .....

4

.

5

Growth, p a t t e r n o f a r e s i s t a n t s t r a i n i n o c u l a t e d 11 i n t o a s e n s i t i v e c u l t u r e ................................. P r o c e d u r e .......................................................................... D isc u ss io n

11

...............................................

15

E f fe c t o f re d u c in g th e p ro p o rtio n of r e s i s t a n t c e l l s on t h e g r o w t h p a t t e r n o f t h e r e s i s t a n t s t r a i n in a p re d o m in a n tly s e n s i t iv e c u ltu r e

16

P r o c e d u r e ..........................................................................

16

D i s c u s s i o n .....................................................................

18

T est to d e te rm in e th e p r o d u c tio n o f a d i f f u s i b l e i n h i b i t o r y s u b s ta n c e by th e .............................................................. s e n sitiv e s tr a in

III.

P ro ced u re

.............................................................................

D isc u ss io n

. . . * .....................................................

DISCUSSION AND SUMMARY

.

18 18 19

..............................................

21

D i s c u s s i o n ......................... S u m m a r y ....................................................................................

21 22

B I B L IO G R A P H Y .......................................................................................................

24

L IST OF TABLES TABLE

I.

A.

II.

B*

III.

C.

IV .

D.

V.

E.

PAGE

Num ber o f B a c t e r i a R e s i s t a n t t o V a r i e d C o n c e n t r a t i o n s o f S t r e p t o m y c i n i n S a m p le s t a k e n fro m a B r o th G u ltu r e o v e r a F i f t y H o u r P e r i o d ( U s i n g S m o o th S t r a i n ) .....................

7

Humber o f S e n s i t i v e O r g a n is m s t o one R e s i s t a n t O r g a n is m ( P e r M l . ) a t S p e c i f i e d I n t e r v a l s ( U s i n g S m o o th S t r a i n ) ............................

7

Num ber o f B a c t e r i a R e s i s t a n t t o V a r i e d C o n c e n t r a t i o n s o f S t r e p t o m y c i n i n S a m p le s Taken fro m a B r o th C u ltu r e o v e r a F i f t y H o u r P e r i o d ( U s i n g S m o o th S t r a i n ) . . . . . Number o f S e n s i t i v e O r g a n is m s t o o n e R e s i s t a n t O rg a n ism ( P e r M l . ) a t S p e c i f i e d I n t e r v a l s ( U s i n g S m ooth S t r a i n ) ............................ Number o f B a c t e r i a R e s i s t a n t t o V a r i e d C o n c e n t r a t i o n s o f S t r e p t o m y c i n i n S a m p le s Taken fro m a B r o th C u ltu r e o v e r a F i f t y H our P e r i o d (U s in g Rough S t r a i n ) . . . . . . Number o f S e n s i t i v e O r g a n is m s t o o n e R e s i s t a n t O r g a n is m ( P e r M l . ) a t S p e c i f i e d I n t e r v a l s ( U s i n g R ough S t r a i n ) ................................. Number o f B a c t e r i a R e s i s t a n t t o V a r i e d C o n c e n t r a t i o n s o f S t r e p t o m y c i n i n S a m p le s Taken fro m a B r o th C u ltu r e o v e r a F i f t y H o u r P e r i o d ( U s i n g R ough S t r a i n ) . . . . . .

8

8

9

9

10

Number o f S e n s i t i v e O r g a n is m s t o o n e R e s i s t a n t O r g a n is m ( P e r N l . ) a t S p e c i f i e d I n t e r v a l s ( U s i n g R ough S t r a i n ) ...............................

10

The E f f e c t o f I n c r e a s i n g t h e Num ber o f R e s i s t a n t C e l l s I n i t i a l l y P r e s e n t on t h e G ro w th P a t t e r n o f R e s i s t a n t C e l l s i n a P re d o m in a n tly S e n s itiv e C u ltu re o f th e P aren t S tra in . . . . . ...............................................

13

Num ber o f S e n s i t i v e C e l l s t o C e l l (U sin g Rough S t r a i n )

13

One R e s i s t a n t .........................................

iv TABLE

V I.

F. V II.

V III.

PAGE

The E f f e c t o f I n c r e a s i n g t h e Num ber o f R e s i s t a n t C e l l s I n i t i a l l y P r e s e n t on t h e G r o w th P a t t e r n o f R e s i s t a n t C e l l s i n a P re d o m in a n tly S e n s itiv e C u ltu re o f th e P aren t S tra in ..................... . . . . . . . . . . Num ber o f S e n s i t i v e C e l l s t o C e l l (U s in g Rough S t r a i n )

.14

One R e s i s t a n t ..........................

14

R e s i s t a n t O r g a n is m s P e r M l. B r o t h C u l t u r e a s D e t e r m i n e d b y P l a t i n g s on S t r e p t o m y c i n A g a r , C o n t a i n i n g 1 . 1 U n i t s S t r e p t o m y c i n P e r M l. a t S p e c ifie d I n te r v a ls . . . . . . . . . . .

17

R e s i s t a n t O r g a n is m s P e r M l. B r o t h C u l t u r e a s D e te rm in e d by P l a t i n g s on S t r e p to m y c i n A g a r, C o n t a i n i n g 1 . 1 U n i t s S t r e p t o m y c i n P e r M l. a t S p e c ifie d I n te r v a ls

17

L IS T OP FIGURES

FIGURE 1.

S te p w ise M u ta tio n to R e s is ta n c e

2.

D e v elo p m en t o f R e s i s t a n t S t r a i n From a R e s is ta n t V a ria n t in P resen ce o f S tre p to m y c in . . . . . . . . ............................................

PAGE 6

20

CHAPTER I

BACKGROUND AND STATEMENT OF PROBLEM The m e c h a n i s m w h i c h g o v e r n s h e r e d i t a r y c h a r a c t e r i s t i c s i n b a c t e r i a u s u a l l y m a i n t a i n s a r e l a t i v e l y hom ogeneous p o p u ­ la tio n under c o n sta n t c u ltu r a l c o n d itio n s. th e

o ccu rren ce

o f m u t a n t s among t h e

c e lls

In s p ite

of th is ,

can e a s i l y be d e ­

t e c t e d u s in g m ed ia w h ic h f a v o r g ro w th o f t h e v a r i a n t s b u t n o t of th e

Mn o r m a l n c e l l s .

S c h n e id e r (1948,

U sin g t h i s

t e c h n i q u e Ryan a n d

1949) c l e a r l y d e m o n s tra te d th e e n v iro n m e n ta l

f a c to r s re s p o n s ib le f o r th e e sta b lis h m e n t o f a h i s t id i n e in d e p e n d e n t s t r a i n fro m a d e p e n d e n t p a r e n t . d e s c rib e d th e i s o l a t i o n cal c h a ra c te ristic s,

of s tr a in s

S im ila r r e p o r ts

sh o w in g o p p o s i t e b i o c h e m i ­

(Z am enhof, 1 9 4 6 ; E n g le s b e r g and S t a n i e r ,

1949) an d e x tre m e d i f f e r e n c e s

in re s is ta n c e

(M ille r and

B o h n h o ff, 1947; Y e g ia n a n d Budd, 194 8 ) from t h a t o f th e p a re n t• F rom t h e a b o v e w o r k a n d o t h e r s and F in la n d ,

1948) i t

can be c o n clu d ed t h a t m u ta tio n s o c cu r

r e g u l a r l y i n g ro w in g c u l t u r e s a n d t h e m u ta n ts m u l t i p l y In th e p re s e n c e d e te rm in e d by th e

in flu e n c e

to c u ltu re ag e.

is

o f e n v iro n m e n ta l c o n d itio n s .

p a sse s th ro u g h a c y c le

re la tin g

e x te n t to w h ic h th e

o f th e p a re n t s t r a i n

The b a c t e r i a l c u l t u r e b e s i d e s tio n s ,

(B rau n , 1946; P a in e

sh o w in g g e n e t i c v a r i a ­

of g ro ss,

n o n -g e n e tic changes

These changes a s s o c i a t e d w ith th e

2

p h y s i o l o g i c a l g ro w th p h a s e s h a v e b e e n show n f o r m e t a b o l i s m (Moony a n d W in s lo w ,

1 9 3 5 ), s iz e

1 9 3 7 ), e le c tr o p h o r e tic p o t e n t i a l d u c tio n

(B ay n e-Jo n es a n d R hees,

re sista n c e

( H u n t i n g t o n a n d W in s lo w , (M o y e r, 1 9 3 6 ) , a n d h e a t p r o ­ 19 29 ).

1944; P r a t t and D u fren o y ,

(Y o u m an s,

The i n h i b i t o r y a c t i o n

1 9 4 8 ),

p e n ic illin

1948, c r y s ta l v io le t

Rahn, 1 9 4 4 ), and o t h e r a g e n ts

(B ig g e r,

( H o f fm a n a n d

(S h e r m a n a n d A l b u s ,

1 923).

o f s t r e p t o m y c i n , on t h e o t h e r h a n d , h a s

b e e n sh o w n t o b e e q u a l l y e f f e c t i v e (H a m re , R ake a n d D o n o v i c k , It

in th e

o f th e c u l t u r e d u r in g th e g ro w th p h a s e s have b e e n

sh o w n f o r s u l p h a n i l a m i d e

c e lls

V a ria tio n s

on y o u n g a n d r e s t i n g

1 946).

is e v id e n t th a t v a r ia tio n s

in re s is ta n c e

occur

r e g u l a r l y due to r a r e b u t c o n s t a n t m u ta tio n s i n g ro w in g c u ltu r e s and c u ltu r e a g e .

The p u r p o s e o f t h e p r e s e n t e x p e r i ­

m e n t s w as t o d e t e r m i n e w h e t h e r a n y r e l a t i o n s h i p c o u l d b e sho w n t o e x i s t b e t w e e n t h e c u l t u r e a g e a n d t h e n u m b e r o f r e s i s t a n t m u ta n ts. N e isse ria c a ta r r h a lis t e s t o rg a n ism b e c a u se i t c h a in s

A .T .C . # 7 9 0 0 w as c h o s e n a s t h e

i s n o n - s p o r u l a t i n g , d o e s n o t form

o r c lu m p s a n d g ro w s w e l l a t pH 7 . 8 ,

stre p to m y c in i s b o th s ta b le and a c t iv e .

a v a lu e a t w h ic h

The o r g a n i s m e x i s t s

a s a d i p l o c o c c u s a n d c o l o n i e s w ere assu m ed t o r e s u l t fro m ^ i n d i v i d u a l *1 o r g a n i s m s s i n c e sta n t.

t h e m o rp h o lo g y r e m a in e d c o n ­

The s m o o t h s t r a i n d u r i n g i s o l a t i o n i n s a l i n e was

c o n v e rte d In to a ro u g h s t r a i n as

ju d g e d by i t s

c u ltu ra l

3

ap p earan ces.

The s m o o t h s t r a i n p r o d u c e d l i t t l e

b r o t h and gave r i s e The r o u g h v a r i e t y

tu rb id ity

to co nvex c o lo n ie s h a v in g an even e d g e .

p ro d u ced a t u r b id b r o t h and c o lo n ie s p o s ­

se sse d c re n a te d b o rd e rs . S tre p to m y c in c a lc iu m c h lo r id e

c o m p l e x w as c h o s e n a s

t h e a g e n t a g a i n s t w h i c h r e s i s t a n c e w as t e s t e d d u e t o sta b ility

of th is

b a c te rio lo g y .

in

a n t i b i o t i c and i t s

th e

im p o rta n c e i n m e d ic a l

CHAPTER I I

• EXPERIMENTS I . . APPEARANCE OP RESISTANT VARIANTS IN A GROWING CULTURE OP A SENSITIVE. STRAIN P ro ced u re.

A 1 0 m l# t u b e o f n u t r i e n t b r o t h w a s i n o c u ­

l a t e d w i t h a lo o p o f f r e s h l y t r a n s f e r r e d N e i s s e r i a c a t a r r h a l i s w as i n c u b a t e d f o r t w e n t y - f o u r h o u r s a t 3 7 ° c e n tig ra d e *

P o l l o w i n g i n c u b a t i o n 5 ml* w e r e t r a n s f e r r e d

2 5 0 m l* n u t r i e n t b r o t h , p r e v i o u s l y r a i s e d and fro m t h i s

c u ltu re

to

to 37° c e n tig ra d e ,

th e p l a t i n g s w ere m ade.

A t s p e c i f i e d p e r i o d s o f 0, 2 , 5 , 1 m l* s a m p l e s w e r e t r a n s f e r r e d t o

1 2 , 2 4 , a n d 50 h o u r s ,

a se rie s

of p la te s

t o w h ic h

was a d d e d 2 0 m l . o f S t r e p t o m y c i n A s s a y A g a r c o n t a i n i n g a p p r o ­ p ria te

c o n c e n tra tio n s

o f stre p to m y c in *

P or c o n tr o ls th e

t e c h n i q u e w as s i m i l a r w i t h s t r e p t o m y c i n b e i n g o m i t t e d f r o m th e a g a r .

The b r o t h c u l t u r e was r e t u r n e d t o

a f t e r e a c h sa m p lin g and th e p l a t e s

th e in c u b a to r

w ere i n c u b a t e d a t 3 7 °

c e n tig ra d e f o r f iv e days b e fo re b e in g re a d . S tr e p to m y c in d i l u t i o n s w ere p r e p a r e d i n d i s t i l l e d w a te r p r e v io u s ly c o o le d and r e tu r n e d to

th e r e f r i g e r a t o r

a f t e r e a c h u s e ; t h e f i n a l d i l u t i o n was m ade i n 50 m l . o f m e lte d a g a r , h e l d a t 4 8 ° c e n t i g r a d e , by a d d i t i o n o f 1 m l. o f d ilu te d stre p to m y c in .

5

R e s u l ts an d B is c u ss I o n ,

It

is

seen in ta b le s I and I I

t h a t t h e num ber o f r e s i s t a n t o rg a n is m s t o rise s

to

.6 a n d .8 u n i t s

a maximum a t t h e t w e l f t h h o u r a n d t h e n f a l l s

s h a rp ly by th e tw e n ty - f o u r th h o u r.

C e lls r e s i s t a n t to 1 and

1 .2 u n i t s a p p e a r e d s p o r a d i c a l l y b u t s h o w e d l i t t l e m u ltip lic a tio n ,

o ff

e v id e n c e

of

t h e i r num ber re m a in in g c o n s t a n t f o r th e e n t i r e

p e rio d . T a b l e s I I I a n d IV p r e s e n t a s i m i l a r p a t t e r n ap p earan ce o r r e s i s t a n t c e l ls

f o r th e

in a s e n s i t i v e ro u g h c u l t u r e .

T h e r e w e r e a l a r g e r n u m b e r o f c e l l s r e s i s t a n t t o lo w c o n c e n ­ t r a t i o n s , b u t in th e h ig h e r re g io n s l i t t l e , In b o th s t r a i n s se n sitiv e

c e lls

to

i f a n y , g ro w th .

t a b l e A, B , C, B, t h e p r o p o r t i o n o f

re sista n t

c e lls

d u rin g th e e a r l y h o u rs b u t r is e s

\ } Xa lo w e st (re s is ta n t) s h a rp ly a f t e r th e f i f t h

h o u r. It

is

a p p a re n t th a t th e

on n o rm a l m u l t i p l i c a t i o n g ro w th i s

r e s i s t a n t c e l l s do n o t c a r r y

in th e b ro th c u ltu re f o r t h e i r

slo w o r a b s e n t .

As m u t a n t s f r o m a n o r m a l s t r a i n

th e y e v id e n tl y la c k e d th e a b i l i t y

to m u ltip ly ra p id ly in

t h e i r new a n d u n s u i t e d e n v i r o n m e n t . w as r a p i d l y

c o n s u m in g t h e n u t r i e n t s

t h e m ed ia l e s s th e se

c e lls

a n d g e n e r a l l y m ak in g

f a v o r a b l e f o r t h e slo w g ro w in g v a r i a n t s ,

soon fo u n d c o n d itio n s u n s u i t a b l e f o r g ro w th and

w ere t h e n e i t h e r i n h i b i t e d i.e .

As t h e n o r m a l s t r a i n

or e lim in a te d .

The r a r e m u t a n t s ,

t h e m o s t r e s i s t a n t c e l l s , w e r e e v e n m o re a l i e n t o t h e

6

m ed ia t h a n t h e l e s s e r r e s i s t a n t c e l l s

and th e r e f o r e

fa ile d

to

grow . A s p e c u l a t i v e d i a g r a m b a s e d o n a sc h e m e p r o p o s e d by R eed

(1 9 3 3 ) f o r th e R f-^ 3 c h a n g e s ,

is

s k e t c h e d b e lo w a s a

p o s s ib le e x p la n a tio n f o r changes o f s e n s i t iv e

c e lls

to r e s i s ­

ta n t b a c te ria . se n sitiv e

d iv is io n

se n sitiv e

in te rm e d ia te re sista n c e d iv is io n

in te rm e d ia te

oo h ig h re sista n c e

d iv is io n

h ig h re sista n c e

le th a l

F ig u re 1. C irc le s

S te p w ise M u ta tio n t o R e s is ta n c e

r e p r e s e n t g e n e s w h i c h a r e d i v i d e d am ong

d a u g h te r c e l l s d u rin g f i s s i o n . a s s o c i a t e d w ith an in c r e a s e a lte ra tio n g ro w th ,

a s s o c i a t e d w ith m eta b o lism ,

The m o s t d r a s t i c

c o m p le te ly u n a b le t o s u r v iv e

o r change i s

in r e s i s t a n c e and a p ro b a b le

in c h a r a c te r is tic s

e tc .

Each gene lo s s

change r e s u l t s

in a c e ll

i n t h e c u l t u r e m e d ia

th e stre p to m y c in dep en d ent c e l l s ) .

(p erh ap s

7 TABLE I NUMBER OP BACTERIA RESISTANT TO VARIED CONCENTRATIONS OP STREPTOMYCIN IN SAMPLES TAKEN PROM A BROTH CULTURE OVER A FIFTY-HOUR PERIOD (USING SMOOTH STRAIN)

S tre p to m y c in C o n c e n tra tio n in u n its /m l.

H ours 12

24

50

38

68

3

4

0

18

29

1

2

0

0

1

4

0

1

0

0

1

0

0

1

0

2

.6

10

4

.8

3

1. 1 .2 0 (C o n ­ tro l )

5.

4 * 5x 104 3 * 5x 104 2 . 6 x l 0 5 1 . 5 x l 0 7 5 * 5 x l Q 7 2*5x10®

TABLE A NUMBER OP SENSITIVE ! ORGANISMS TO ONE RESISTANT ORGANISM (PER ML. ) AT SPECIFIED INTERVALS (USING SMOOTH STRAIN) Age i n H ours

#

.6

S tre p to m y c in C o n c e n tra tio n .8

1.

0

.0 5 x 1 0 ®

. 02x 10®

• 0 5 x l0 ® «

2

.0 9 x 1 0 ®

.0 4 x 1 0 ® *

. 04x10®-:.-

5

.7x10®

. 01x 10®

..3 x 1 0 ®

12

200x 10®

.5 x 1 0 ®

4 .x l0 ®

24

200x 10®

60x10®

60x10®-::-

50

600x105

100x 106

200x 10® a b sen c e o f g ro w th o f r e s i s t a n t c e l l s , t h e r e f o r e p r o p o r tio n of Afc3-v e ) c e l l s w as a t l e a s t t h a t sh o w n (re s is ta n t)

8 TABLE I I NUMBER OP BACTERIA RESISTANT TO VARIED CONCENTRATIONS OP STREPTOMYCIN IN SAMPLES TAKEN PROM A BROTH CULTURE OVER A FIFTY-HOUR PERIOD (USING- SMOOTH STR A IN )

S tre p to m y c in C o n c e n tra tio n in u n its /m l.

0

2

5

12

24

50

.6

5

2

42

52

1

3

.8

1

1

1

5

1

2

1.

0

0

1

1

0

1

1 .2

0

0

•0

0

0

0

0 (C o n ­ tro l )

h o u rs

4 . 6 x l 0 5 1 . 7 x l 0 5 1 . 8x 10^

5 .7 x l0 6

6x l 07 2 . 8x 108

TABIE B NUMBER OP SENSITIVE i ORGANISMS TO ONE RESISTANT ORGANISM (PER ML. ) AT SPECIFIED INTERVALS (USING SMOOTH STRAIN) Age I n h o u rs

.6

S tre p to m y c in C o n c e n tra tio n .8

1.

0

.9 x 1 0 ®

.5x10®

.5 x 1 0 ® *

2

.9 x 1 0 ®

. 2x 10®

. 2x l o 6*-

5

. 4x10®

2x l 06

- 2x 10®*

2x 10®

6x 10®

12 24

1x 10® 600x10®

60x106

60x10® *

50 900x10® 100x 10® 300x10® •K- a b s e n c e o f g r o w t h o f r e s i s t a n t c e l l s , t h e r e f o r e p r o p o r t i o n of ) c e l l s w as a t l e a s t t h a t sh o w n (re s is ta n t)

9 TABLE I I I NUMBER OP BACTERIA RESISTANT TO VARIED CONCENTRATIONS OP STREPTOMYCIN IN SAMPLES TAKEN PROM A BROTH CULTURE OVER A FIFTY-HOUR PERIOD ( USING- ROUGH STR A IN )

S tre p to m y c in C o n c e n tra tio n in u n its /m l. •

0

2

5

h o u rs 12

24

50

.6

10 5

88

35 7

625

475

92

.8

8

6

5

5

4

6

1.

2

0

0

1

0

0

1 .2

1

0

0

1

0

0

0 ( C o n - 7 . 1x 106 6 . 9 x l 0 6 1 . 7 x l 0 7 tro l)

1 . 2x l 08 2 • I x l O 8 2 . 4 x l 0 8

TABLE C NUMBER OP SENSITIVE ORGANISMS TO iONE RESISTANT ORGANISM (PER M L .) AT SPECIFIED INTERVALS (USING ROUGH STRAIN)

Age i n h o u rs

&

.6

S tre p to m y c in C o n c e n tra tio n .8

1.

0

7 x l0 4

1x 10®

4x10®

2

8x l 04

1x 10 6

7x10® *

5

5 x l0 4

3 x l0 6

2x 10®*

12

20x l 04

20x 106

60x10®

24

4 0 x l0 4

50x106

100x 10®

50 40x10® 3 0 0 x l0 4 80x10® ab sen ce p f.g ro w th o f r e s i s t a n t c e l l s , th e r e f o r e p o r p o rtio n o f vg Q H S x t i v e ) c e I T s w a s a t l e a s t t h a t sh o w n (re s is ta n t)

10 TABLE IV NUMBER OP BACTERIA RESISTANT TO VARIED CONCENTRATIONS OP STREPTOMYCIN IN SAMPLES TAKEN PROM A BROTH CULTURE OVER A FIFTY-HOUR PERIOD (USING ROUGH STRAIN)

S tre p to m y c in C o n c e n tra tio n in u n its /m l.

H o u rs 0

2

5

12

24

50

.6

48

52

275

427

5

5

.8

5

1

2

3

1

2

1.

1

1

0

0

1

1

1 .2

0

0

0

0

1

1

0 (C o n - 5 . 8x 10 6 5 . 6 x l 0 6 7 . 8x l 06 tro l)

6 . 6x l 07 2 . 5 x 1 0 8 2 .5 x 1 0 8

TABLE D NUMBER OP SENSITIVE ORGANISMS TO ONE RESISTANT ORGANISM (PER M L.) AT SPECIFIED INTERVALS (USING ROUGH STRAIN)

Age i n ho u rs

•6

S tre p to m y c in C o n c e n tr a tio n .8

1.

0

1x 10®

1x 10 6

6x 10®

2

2x105

6x 10s

6x 10®

5

.3 x 1 0 ®

4x10®

8x 10®-::-

20x 10®

7 0 x l 0 6-::-

12 24

2x 10® 500x10®

200x 10®

200x 10®

50 500x10® 100x 10® 200x 10® * a b s e n c e 9E g ro w th , of* r e s i s t a n t c e l l s , t h e r e f o r e p o r p o r t i o n o f )56n s i t i v e ; c e l l s w as a t l e a s t t h a t s h o w n (re sx sta n t)

11

I n t h e p r e c e d i n g e x p e r im e n t t h e m u ta n ts w h ic h a p ­ p e a r e d d u r in g g ro w th o f th e p a r e n t c u l t u r e e s t a b l i s h e d as a s i g n i f i c a n t segm ent o f th e la tio n *

S im ila r in h ib ito r y e f f e c ts

w as o b s e r v e d b y Z a m e n h o f i n t e s t i n g m u ta n t g ro w th

(Z am enhof, 1 9 4 6 ) .

f a i l e d t o becom e t o t a l popu­

o f th e m u ta n t c u l t u r e fo r c itra te

The s e n s i t i v e

in d e p e n d e n t s t r a in had

b y a n e l i m i n a t i o n p r o c e s s b u t t h r o u g h a n u n kno w n m e c h a n i s m p re v e n te d th e s u r v iv a l o f th e v a r ia n t c e l l s its

s tra in

and r e t a i n e d

c h a ra c te ristic s*

The f o l l o w i n g e x p e r i m e n t w a s p l a n n e d t o d e t e r m i n e th e e f f e c t in itia lly c e lls

o f i n c r e a s i n g t h e num ber o f r e s i s t a n t c e l l s p r e s e n t on t h e g r o w th p a t t e r n

in a p re d o m in a n tly s e n s itiv e

num ber o f r e s i s t a n t tiv e

c e lls

in itia lly

c u l t u r e w as v e r y l o w , i t

of th e

c u ltu re *

a m o re d e f i n i t e

S in c e th e

p re s e n t in th e s e n s i ­

seem ed p r o b a b l e t h a t by

s t a r ti n g a c u ltu re w ith a la rg e r p ro p o rtio n c e lls ,

re s ista n t

of re sista n t

t r e n d c o u ld be o b se rv e d o r a d i f ­

f e r e n t p a t t e r n m ig h t r e s u l t .

T he n e x t e x p e r i m e n t w as s e t

up t o t e s t t h e s e p o s s i b i l i t i e s . II.

GROWTH PATTERN OP A RESISTANT STRAIN INOCULATED INTO A SENSITIVE CULTURE

E x p e rim e n ta l p ro c e d u re .

A r e s i s t a n t s t r a i n w as

o b t a i n e d f r o m a c o l o n y t h a t h a d g ro w n o n 1 .2 u n i t s

stre p to ­

m y c i n i n t h e p r e c e d i n g e x p e r i m e n t , a n d w as s t r u c k o n n u t r i ­ ent ag ar.

T h i s c u l t u r e w h e n e x a m i n e d w as f o u n d t o c o n s i s t

12

o f n o rm a l gram n e g a t i v e d i p l o c o c e i t h a t p r o d u c e d a n a l k a l i n e r e a c tio n in s e m i-s o lid d e x tro s e a g a r

(p h en o l re d in d ic a t o r )

t h a t w as i d e n t i c a l i n a r e a a n d i n t e n s i t y a s t h a t p r o d u c e d b y th e s e n s i t i v e

stra in ;

th e

c o lo r re a c tio n s

i n t h e tw o s t r a i n s

w e re s i m i l a r w hen e x a m in e d a t 1 , 2 , a n d 5 d a y i n t e r v a l s . One d r o p o f t h e r e s i s t a n t b r o t h c u l t u r e w as t r a n s ­ f e r r e d t o e a c h o f tw o f l a s k s b ro th .

c o n ta in i n g 250 m l. n u t r i e n t

To o ne o f t h e s e f l a s k s

i n o c u l a w as a d d e d 5 m l . o f t h e

c o n ta in in g th e r e s i s t a n t se n sitiv e

b ro th c u ltu r e .

( T h i s p r o c e s s w as d o n e i n d u p l i c a t e . ) At s p e c i f i e d i n t e r v a l s

1 m l. sa m p le s,

p re v io u s ly

d i l u t e d , w ere p l a t e d fro m e a c h f l a s k on n u t r i e n t a g a r and n u t r i e n t a g a r c o n t a i n i n g 1 .1 u n i t s The r e s u l t s

t h a t fo llo w a re re c o rd e d in o rg a n ism s/m l.

o f t h e 250 m l. b r o t h c u l t u r e s . th e r e s u l t s

s tre p to m y c in .

of p la tin g

The t a b l e s b e lo w r e p r e s e n t

t h e m ix e d an d r e s i s t a n t c u l t u r e

th e p re s e n c e and a b se n c e o f s tr e p to m y c in .

in

15 TABLE V

THE EFFECT OF INCREASING THE NUMBER OF RESISTANT CELLS INITIALLY PRESENT ON THE GROWTH PATTERN OF RESISTANT CELLS IN A PREDOMINANTLY SENSITIVE CULTURE OF THE PARENT STRAIN

ho u rs 24

48

.9

1400

2500

•

0

1700

2600

12

R e sista n t

n u trie n t agar

.1 6

o

.1 3 * H

stre p to m y c in

R e sista n t) S e n s itiv e ) 1 .9

.1 5

stre p to m y c in n u trie n t agar * a l l c o u n t s x 10^

290.

21*

14*

12.

32 0 0

3200.

P l a t i n g s w e r e m ade o n a g a r c o n t a i n i n g l * . l u n i t s s t r e p t o m y c i n a n d on a g a r i n t h e a b s e n c e S e n sitiv e

o f s tre p to m y c in *

c o u n ts r e p r e s e n t ' t o t a l num ber o f o rg an ism s

g r o w i n g on n u t r i e n t a g a r i n a b s e n c e o f s t r e p t o m y c i n ,

sin c e

no s i g n i f i c a n t d i f f e r e n c e w o u ld r e s u l t f r o m c o r r e c t i n g f o r num ber o f r e s i s t a n t c e l l s

p re se n t* TABLE E

NUMBER OF SENSITIVE CELLS TO ONE RESISTANT CELL (USING ROUGH STRAIN) ho u rs

0

12

24

48

S e n sitiv e org an i 1 .4 x 1 0 ® ^ /^ n e re sista n t o rg an ism

1*5x10^

2 .7 x 1 0 ®

2 .3 x 1 0 ®

14

TABLE V I

THE EFFECT OF INCREASING THE NUMBER OF RESISTANT CELLS INITIALLY PRESENT ON THE GROWTH PATTERN OF RESISTANT CELLS IN A PREDOMINANTLY SENSITIVE CULTURE OF THE PARENT STRAIN

h o u rs 0

12

24

48

stre p to m y c in

. 15#

12

1 600

28 00

n u trie n t agar

.1 7

13

1800

2900

.1 7

3

14

15

350

3400

2900

R e sista n t

R e s is ta n t and) se n s itiv e ) s tre p to m y c in n u trie n t ag ar # a l l c o u n t s x 10^

23.

TABLE F NUMBER OF SENSITIVE CELLS TO ONE RESISTANT CELL (USING ROUGH STRAIN)

h o u rs 0

12

24

48

1 .3 x l0 2

l.lx lO 2

2 .4 x 1 0 2

1•9x102

S e n sitiv e o rg a n ism s

one r e s i s t a n t o rg a n ism

15

D isc u ss io n .

The r e s u l t s

in d ic a te

t h a t th e

g ro w th o f

t h e r e s i s t a n t s t r a i n w as i n s h a r p c o n t r a s t w i t h t h a t o b ­ served fo r c u ltu re .

th e m u ta n ts i n i t i a l l y The r e s i s t a n t s t r a i n

to e s ta b lis h ra tio

its e lf

o c c u rrin g in a s e n s itiv e

is

seen

( T a b l e s V a n d V I)

and t o m a in ta in a p p ro x im a te ly th e

i n w h i c h i t w as i n o c u l a t e d

a n a la g o u s t o th e- r e s u l t s

(T a b le s E and F ) .

T h is i s

o b t a i n e d b y R yan an d S c h n e id e r

( 1 9 4 9 ) who f o u n d t h a t a m i x t u r e

of h is tid in e

d ep en d en t and

i n d e p e n d e n t o r g a n i s m s w h en g ro w n o n o p tim u m c o n c e n t r a t i o n s of h istid in e , tio n s .

re ta in e d th e ir i n i t i a l r a tio

f o r m any g e n e r a ­

T h e r e was a p p a r e n t l y a c h a n g e I n t h e g r o w t h c h a r a c ­

te ristic s

o f th e m u ta n t c e l l s

m y cin a g a r .

a f t e r h a v i n g g ro w n on s t r e p t o ­

A s s u m in g a c h a n g e t o h a v e o c c u r r e d ,

in g e x p la n a tio n is

th e

fo llo w ­

o ffered .

The m u t a n t c e l l d i f f e r s p h y s i o l o g i c a l l y , g ro w in g and u n a b le t o e s t a b l i s h i t s e l f b e f o r e

is

slo w

su c cu m b in g t o

t h e a d v e r s e c o n d i t i o n s p r o d u c e d i n t h e m ed ia b y t h e n o rm a l s tra in .

When t r a n s f e r r e d

g ro w th o f c o m p e tin g c e l l s

t o a m ed ia w h ic h p r e v e n t s th e th e m u ta n t i s

a b le to u n d erg o a

g r e a t d e a l o f u n d is tu rb e d m u l t i p l i c a t i o n and s im u lta n e o u s ly a s e le c tiv e p ro c e ss ta k e s p la c e . fin a lly

a p p e a r i n g on t h e s t r e p t o m y c i n a g a r r e p r e s e n t s a

se le c te d s tr a in c a p a b le

I n t h i s w ay t h e c o l o n y

o f now n o r m a l l y r e s i s t a n t i n d i v i d u a l s

of r a p id m u ltip lic a tio n even in th e absence of th e

drug and in th e p re s e n c e

of s e n sitiv e

c e lls .

It

was o b s e r v e d

16

t h a t w here th e

o r i g i n a l m u ta n ts fro m th e

w ere v e ry slo w t o m u l t i p l y fro m f o u r t o f i v e

se n sitiv e

on s t r e p t o m y c i n a g a r ,

days to produce d e f i n i t e

c u ltu re

re q u irin g

c o lo n ie s ,

r e s i s t a n t , s t r a i n p ro du ced e q u iv a le n t s iz e d c o lo n ie s to th r e e d a y s.

T h is , w o u ld s u p p o r t t h e

re q u ire d to s e le c t B ecause o f th e

out a s ta b le

in i­

s l o w a n d m o re

stra in .

l a r g e num ber o f r e s i s t a n t o rg a n is m s

in th e in o c u la th e c o n d itio n s w ere n o t a n a lo g o u s ,

i n one

c o n te n tio n th a t

t i a l m u l t i p l i c a t i o n o f th e m u ta n t i s n a t u r a l l y tim e i s

th e

o f th e o r i g i n a l e x p e rim e n t

s o t h e f o l l o w i n g e x p e r i m e n t w as p e r ­

fo rm e d i n w h ic h a s m a l l e r num ber o f r e s i s t a n t o r g a n is m s was i n i t i a l l y III.

p re s e n t th a n in th e p re c e d in g e x p e rim e n t.

EFFECT OF REDUCING THE PROPORTION OF RESISTANT CELLS ON THE GROWTH PATTERN OF THE RESISTANT STRAIN IN A PREDOMINANTLY SENSITIVE CULTURE

E x p e rim e n ta l p ro c e d u re . tu re

of th e s e n s itiv e

F iv e m l. fro m a b r o t h c u l ­

s t r a i n w as t r a n s f e r r e d t o a f l a s k

c o n t a i n i n g 250 m l. n u t r i e n t b r o t h . l/lO O d ro p o f t h e

To o n e f l a s k w a s a d d e d

r e s is ta n t b ro th c u ltu re ,

1 / 5 0 0 o f a d r o p f r o m t h e sam e r e s i s t a n t P l a t i n g s w e r e m ad e o f 1 m l . a t sp e c ifie d

in te rv a ls,

n u trie n t ag ar.

to

th e

o th e r

c u ltu re .

s a m p le s fro m e a c h f l a s k

u s i n g s t r e p t o m y c i n 1 .1 u n i t s / m l .

17

E f f e c t of re d u c in g th e p ro p o rtio n on t h e g r o w t h p a t t e r n se n sitiv e

of re s is ta n t

c e lls

of re s is ta n t

c e lls

in a p re d o m in a n tly

c u ltu re . TABLE ¥11

RESISTANT ORGANISMS PER ML. BROTH CULTURE AS DETERMINED BY PLATINGS ON STREPTOMYCIN AGAR, CONTAINING 1 . 1 UNITS STREPTOMYCIN PER ML. AT SPECIFIED INTERVALS B ro th in o c u la te d w ith 1/100 d r o p re sista n t 5 m l. s e n s i t i v e 1 /5 0 0 d ro p re sista n t 5 /m l. s e n s i t i v e

Age i n h o u r s 24 . 12

0

48

1 . 8x 10^

2 .9 x l0 4

4•5x10^

6 .7 x l0 4

2 .4 x 1 0 1

2 . 2x l 04

3 .2 x l0 4

4 . x lO 4

TABLE V I I I RESISTANT ORGANISMS PER ML. BROTH CULTURE AS DETERMINED BY PLATINGS ON STREPTOMYCIN AGAR, CONTAINING 1 . 1 UNITS STREPTOMYCIN PER ML. AT SPECIFIED INTERVALS

B ro th in o c u la te d w ith l/lO O d ro p re sista n t 5 m l. s e n s i t i v e 1 /5 0 0 d ro p re sista n t 5 m l. s e n s i t i v e

0

Age i n h o u r s 12 24

48

1 .5 x 1 0 2

2 .4 x 1 0 4

4 .1 x l0 4

5x104

1 .9 X 1 0 1

2 . 0x 104

3x10 4

3 .1 x l0 4

18

D isc u ss io n .

T a b l e s V I I a n d ¥ 1 1 1 show t h e a b i l i t y

th e r e s i s t a n t o rg an ism s t o m u l t i p l y n o r m a lly ; c o n t r a s t t o th e g ro w th o f m u ta n ts

th is

in a s e n s itiv e

in sh arp

c u ltu re .

The f a c t t h a t e v e n w h e r e l e s s

th a n tw e n ty -fiv e r e s i s t a n t

o rg a n is m s w ere p r e s e n t i n t h e

in o c u la ,

th e r e s i s t a n t s t r a i n

e s ta b lis h e d i t s e l f as a s u b s ta n tia l p o rtio n of th e p o p u la tio n ,

( T a b l e s V, V I , V I I , V I I I ) ,

to ta l

su p p o rts th e a s ­

su m p tio n o f a change i n g ro w th c h a r a c t e r i s t i c s w i t h th e d e v elo p m e n t o f a r e s i s t a n t

of

a sso c ia te d

s tra in .

T h e r e w a s e v i d e n t l y some i n h i b i t o r y a c t i o n b y t h e g ro w in g c u l t u r e f o r th e r e s i s t a n t

c e lls

r e a c h a maximum a t

a b o u t th e tw e n ty - f o u r t h h o u r, h o w ever, th e

se n sitiv e

do n o t show s u b s t a n t i a l i n c r e a s e s b e y o n d t h i s f o l l o w i n g e x p e r i m e n t w as s e t u p t o o f a d i f f u s i b l e p ro d u ced d u rin g th e w h ic h m ig h t e x p la in th e IV .

age.

c e lls The

t e s t f o r th e p ro d u c tio n l a t e r ho u rs

o f g ro w th

l i m i t a t i o n o f g ro w th .

TEST TO DETERMINE THE PRODUCTION OP A DIFFUSIBDE INHIBITORY SUBSTANCE BY THE SENSITIVE STRAIN N e isse ria

th e s u rfa c e

c a ta rrh a lis

(se n sitiv e )

w as s t r e a k e d a l o n g

of n u trie n t a g ar in a P e tr i d is h .

The p l a t e

a f t e r b e i n g i n c u b a t e d f o r f o r t y - e i g h t h o u r s was o v e r l a i n w ith a th in

l a y e r o f a g a r p re v io u sly ^ - in o c u la te d w ith th e

re sista n t s tra in .

The p l a t e w as a g a i n i n c u b a t e d a n d o b ­

s e r v e d a f t e r tw o d a y s a n d o n e w e e k p e r i o d s f o r z o n e s o f *

a b o u t f i v e m in u te s e a r l i e r

.

19

in h ib itio n ,

ho z o n es o f i n h i b i t i o n w ere o b s e r v e d i n th e

o v e rla in a g a r. The o n l y c o n c l u s i o n t h a t s u b s ta n c e does n o t d i f f u s e

th a t

in liq u id

su b s ta n c e t h a t

is

th a t a

f r o m bhe o l d e r c u l t u r e w h i c h c a n

i n h i b i t th e r e s i s t a n t c e l l s su g g e sts

can b e draw n i s

un d er th e above c o n d itio n s .

c u ltu re

in h ib ito ry ,

it

It

is n o t a s p e c if ic to x ic

how ever no d e f i n i t e

sta te m e n t

c a n be m ade. D isc u ss io n .

The r e s u l t s

g ro w th c h a r a c t e r i s t i c s

o f th e m u ta n t c e l l s

c u ltu r e and th e r e s u l t i n g

se e m s f e a s i b l e ?

in r e l a t i o n

An i n t e r p r e t a t i o n

in

in th e p a re n t

re sista n t s tra in ;

re p o rte d s im ila r o b se rv a tio n s p en d en t m u ta n ts.

p o in t to a d if f e r e n c e

Zam enhof (1 9 4 6 )

to , c i t r a t e

on th e b a s i s

in d e ­

of m u ta tio n

-D uring m u l t i p l i c a t i o n b a c t e r i a p r o d u c e a

num ber o f a b n o rm a l c e l l s

o r m u ta n ts w h ic h a r e n o t a d e q u a te ly

p r e p a r e d f o r r a p i d g r o w t h a n d a r e u n a b l e t o g ro w I n a c r o w d e d c u ltu re . it

is

I f th e e n v iro n m e n t i s

a l t e r e d t o f a v o r th e m u ta n t

a b le to m u ltip ly , a lth o u g h slo w ly a t f i r s t ,

p ro cess

o f s e l e c t i o n am ong i t s

sta b le ,

r e s i s t a n t and in d ep e n d en t s t r a i n .

and by a

own p r o g e n y e s t a b l i s h a

e x p e rim e n t s tre p to m y c in se rv e d to

In th e p re s e n t

i n h i b i t th e

se n sitiv e

c e l l s a n d I n t h i s w ay p r e v e n t e d t h e e l i m i n a t i o n o f t h e m u ta n ts.

The m u t a n t s ,

on t h e

bypass th e b a c t e r i o s t a t i c

o t h e r h a n d , w ere a b le t o

a c t i o n o f th e d ru g , p ro b a b ly by

t h e i r p o s s e s s i o n o f a n a l t e r n a t i v e m e ta b o lic p a th w a y , and

20

m u ltip lic a tio n ensued.

As t h e

r e s i s t a n t p o p u la tio n w ith in

th e c o lo n y i n c r e a s e d , n a t u r a l s e l e c t i o n e lim in a te d th e i n ­ fe rio r

c e l ls and as a r e s u l t

sta b le

and r e s i s t a n t

tra te s

th e d e v elo p m e n t o f a r e s i s t a n t

stra in .

th e s u rv iv o rs re p re s e n te d a The f o l l o w i n g f i g u r e

illu s ­

s t r a i n as e x p la in e d

above: S u g g e s t e d b y D r . E m i l e B o g a n o f O l i v e View S a n i t o r i u m . s e n sitiv e

c e ll

CD ]“•• o rig in a l r e s is ta n t v a rie n t

O O O O > o.

d ie s

Low r a t e o f i n c r e a s e P r o c e s s d o e s n o t go t o c o m p l e t i o n i n a d r u g - f r e e m ed ia F ig u re

2.

R a p id i n c r e a s e A s t a b l e and R e sista n t s tr a in em erges

D e v e l o p m e n t o f R e s i s t a n t S t r a i n P ro m a R e s i s t a n t V a ria n t in P re se n c e o f S tre p to m y c in

21

CHAPTER I I I DISCUSSION AND SUMMARY D isc u ss io n . a c q u isitio n a ta b rin e et a l,

I t H as p r e v i o u s l y b e e n s h o w n t h a t t h e

of re s is ta n c e to

s u lp h a n ila m id e ,

a c rifla v in e ,

( S e v a g a n d G -ots, 1 9 4 8 ) a n d s t r e p t o m y c i n

(O g in sk y

1 9 4 9 ) was a c c o m p a n i e d b y c h a n g e s i n some m e t a b o l i c

p ro cesses.

T hese chan g es i n d i c a t e d t h a t s y n t h e t i c

w ays n o t s u s c e p t i b l e re sista n t th e d ru g s.

to dru g in te r f e r e n c e ,

p ro v id e d th e

o r g a n i s m s w i t h s u r v i v a l v a l u e w hen e x p o s e d t o O g in sk y e t a l

m y cin I n h i b i t s

( 1 9 4 9 ) h a v e sh o w n t h a t s t r e p t o ­

an o x id a tiv e r e a c tio n ,

c e ta te -p y ru v a te

c o n d e n sa tio n .

p ro b a b ly th e

The a b i l i t y

a b o v e c o n d e n s a t i o n w as a b s e n t i n t h e in d ic a tin g

p a th ­

o x a la -

to e f f e c t th e

re sista n t s tra in

th a t th e a b i l i t y

t o g ro w i n t h e p r e s e n c e o f

s t r e p t o m y c i n d e p e n d s on t h e

d e v elo p m e n t o f a l t e r n a t i v e

r e a c tio n s p e r m ittin g th e

c e l l to d isp e n se w ith t h i s

con­

d e n sa tio n . S t r e p t o m y c i n w as f o u n d t o p r e c i p i t a t e b a c t e r i a l n u c le ic a c id s tio n

(D o n o v ick e t a l ,

o f enzym es o r g e n e t i c

a c t i o n i s n o t known. no m en a

(ch an g es

L u ria

1948) b u t w h e th e r an a l t e r a ­

fa c to rs (1 9 4 7 )

i s a consequence o f such s t a t e d t h a t tfT h e s e p h e ­

i n d u c e d b y m u c l e i c a c i d s ) p o i n t t o a m o re

a c c e s s ib le g e n e tic

sy s te m in b a c t e r i a

to e x i s t in h ig h e r o rg a n ism s,

sin c e

th a n h as b een p ro v ed

In th e

fo rm er th e

22 g e n e tic d e te rm in a n ts

can be r e a c h e d and a l t e r e d by s p e c i f i c

c o m p o n e n ts s u p p l i e d f r o m t h e th e re fo re

e x is ts th a t

o u t s i d e . tf

th e r o le

The p o s s i b i l i t y

o f s t r e p t o m y c i n w a s m o re

d i r e c t i n th e d e v elo p m e n t o f r e s i s t a n c e to i t

th an i s

a ttrib u te d

i n t h e p r e c e d i n g d i a g r a m , h o w e v e r t h e sc h e m e was

b a s e d on t h e o r i e s m o st g e n e r a l l y a c c e p t e d a t t h e p r e s e n t tim e . Sum m ary. c u ltu re s

E x p e r im e n ts w ere p e rf o r m e d i n w h ic h b r o t h

o f N e i s s e r i a c a t a r r h a l i s w ere t e s t e d a t s p e c i f i e d

i n te r v a l s f o r th e p re se n c e a n ts .

I t was f o u n d t h a t maximum n u m b e r s o f r e s i s t a n t

a p p ea re d a t th e

c e n tra tio n s

c e lls

t w e l f t h h o u r , b u t by th e t w e n ty - f o u r t h h o u r

t h e i r n u m b e rs h a d f a l l e n

th e

o f stre p to m y c in r e s i s t a n t v a r i ­

o ff sh a rp ly .

At th e h i g h e s t c o n ­

o f s t r e p t o m y c i n t h e r e was no d e f i n i t e

re s is ta n t c e lls

tre n d as

a p p e a re d s p o r a d ic a lly th ro u g h o u t th e

te s te d p e rio d . The r a t i o d u r in g th e f i r s t

of r e s is ta n t

to

se n sitiv e

c e l l s w as h i g h e r

tw e lv e h o u rs th a n d u rin g th e re m a in in g

p e rio d . E x p e rim e n ts to f o llo w

th e g ro w th o f r e s i s t a n t

w h ic h w ere i n o c u l a t e d i n t o a c u l t u r e I n d ic a te d th a t th e r a t i o

of re s is ta n t

of se n sitiv e to s e n s itiv e

re m a in e d a p p r o x im a te ly c o n s t a n t f o r th e p e rio d .

e n tire

c e lls c e lls

te s te d

An e x p l a n a t i o n b a s e d on c u r r e n t c o n c e p t s

a t i o n was o f f e r e d t o r e c o n c i l e

c e lls

of v a ri­

th e p o o r g ro w th o f th e

25

o r i g i n a l m u ta n t

w ith ,

th e

c o m p e titiv e g ro w th a b i l i t y

r e s i s t a n t s t r a i n t o w h ic h i t A

re s u lts.

u l t i m a t e l y gave r i s e .

t e s t f o r th e p re s e n c e of a d i f f u s i b l e

s u b s ta n c e from th e s e n s i t i v e

o f th e

in h ib ito ry

c u ltu re y ie ld e d n e g a tiv e

BIBLIOGRAPHY

24

A v e r y , 0 . T . , M acL eod, C. M ., a n d M c C a r t y , M ., “ S t u d i e s on th e C h e m ica l N a tu re o f th e S u b s ta n c e I n d u c in g T r a n s ­ f o r m a tio n o f th e Pneum onococcus T y p e s ," J o u r n a l o f E x p e r i m e n t a l M e d i c i n e , 7 9 : 1 3 7 - 1 5 8 , 1944* B a y n e - J o n e s , S . , a n d R h e e s , H. S . , “ B a c t e r i a l C a l o r i m e t r y * 2m R e la tio n s h ip o f H oat P ro d u c tio n t o P h ases o f th e C u ltu re C y c le , “ J o u r n a l o f B a c te r io lo g y , 1 7 s1 2 3-140, 1929. B i g g e r , J . W ., “ T r e a t m e n t o f S t a p h y l o c o c c u s I n f e c t i o n s w i t h P e n i c i l l i n by I n t e r m i t t e n t S t e r i l i z a t i o n , “ L ancet 2 :4 9 7 -5 0 0 , 1944. B r a u n , W ., “ D i s s o c i a t i o n i n B r u c e l l a A b o r t u s , ” J o u r n a l o f B a c t e r i o l o g y , 5 1 :3 2 7 - 3 4 9 , 1946 D o n o v i c k , R . , B a y a n , A* P . , C a n a l e s , P . , a n d P a n s y , P . , “ The I n f l u e n c e o f C e r t a i n S u b s t a n c e s o n t h e A c t i v i t y o f S tre p to m y c in . 3. D iffe re n tia l E ffe c ts of V a r i o u s E l e c t r o l y t e s o n t h e A c t i o n o f S t r e p t o m y c i n ,'1 J o u r n a l o f B a c t e r i o l o g y , 5 6 : 1 2 5 - 1 3 7 , 194*8. E n g l e s b e r g , E . , a n d S t a n i e r , R. Y . , “ The R e l a t i o n s h i p B e tw e e n G r o w th a n d M u t a t i o n i n P s e u d o m o n a s P l u o r e s c e n s , “ J o u r n a l o f B a c te r io lo g y , 5 8 :1 7 1 -1 8 0 , 1949. H am re, D . , R a k e , G . , a n d D o n o v i c k , R . , “B a c t e r i o d i d a l A c t i o n o f S tre p to m y c in ," F e d e ra tio n P r o c ., 5 :2 4 7 , 1946. H o f f m a n , C. E . , a n d R a h n , H . , “ B a c t e r i o c i d a l a n d B a c t e r i o ­ s t a t i c A c tio n o f C r y s ta l V i o l e t ," J o u rn a l of B a c te r io ­ lo g y , 4 8 :1 7 7 -1 8 6 , 1948. H u n t i n g t o n , E . , a n d W in s lo w , C. E . A . , “ C e l l S i z e a n d M e ta b o lic A c t i v i t y a t V a rio u s P h a se s o f th e B a c t e r i a l C u ltu re C y c le ," J o u r n a l o f B a c te r io lo g y , 3 3 :1 2 3 -1 4 4 , 1947. L u r ia , S . E ., "R e ce n t A dvances i n B a c t e r i a l G e n e tic s ," B a c t e r i a l R e v ie w s, 1 1: 1947. M i l l e r , C. P . , a n d B o h n h o f f , M ., " D e v e l o p m e n t o f S t r e p t o ­ m y cin R e s i s t a n t V a r i a n t s o f M e n in g o c o c c u s ," S c i e n c e , 1 0 5 :6 2 0 -6 2 1 , 1947. M ooney, G . , a n d W in s lo w , C. E . A . , “ The M e t a b o l i c A c t i v i t y o f V a r i o u s C o l o n G ro u p O r g a n is m s a t D i f f e r e n t P h a s e s o f th e C u ltu re C y c le ," J o u r n a l of B a c te r io lo g y , 3 0 :4 2 7 -4 4 0 , 1935. “

25

M o y e r, L . S . , ’’C h a n g e s I n t h e E l e c t r o k i n e t i c P o t e n t i a l o f B a c t e r i a a t V a rio u s P h a se s o f th e C u ltu r e C y c l e ,” J o u rn a l of B a c te r io lo g y , 3 2 :4 3 3 -4 6 4 , 1929, O g i n s k y , S m i t h , a n d U m b r e i t , ’’The A c t i o n o f S t r e p t o m y c i n . I. The N a t u r e o f t h e R e a c t i o n I n h i b i t e d , ” J o u r n a l o f B a c t e r io lo g y , 5 8 :7 4 7 -7 5 9 , 1949. P a i n e , I . P . , a n d F i n l a n d , M ., ’’B a c t e r i a S e n s i t i v e t o , R e s i s t a n t t o a n d D e p e n d e n t on S t r e p t o m y c i n , ” J o u r n a l o f B a c te r io lo g y , 5 6 :2 0 7 -2 1 8 , 1948. P r a t t , R . , a n d D u f r e n o y , J . , ’’C y t o c h e m i c a l I n t e r p r e t a t i o n o f t h e M e c h a n is m o f P e n i c i l l i n A c t i o n , ” B a c t e r l o l o g i c a R e v ie w s, 1 2 :7 9 0 1 0 3 , 1 9 4 8 . R e e d , G . , ”A H y p o t h e t i c a l V iew o f B a c t e r i a l V a r i a t i o n , ” J o u rn a l o f B a c te r io lo g y , 2 5 :5 8 0 -5 8 6 , 1933. R yan, P . J . , a n d S c h n e i d e r , L. K ., °The C o n s e q u e n c e s o f M u ta tio n D u rin g t h e B ro w th o f B io c h e m ic a l M u ta n ts o f E sc h e ric h ia c o l i . I. The P a t t e r n o f A d a p t i o n o f H i s t i d in e l e s s C u ltu re s ,* 1 J o u rn a l o f B a c te r io lo g y , 5 6 :6 9 9 -7 0 8 , 1948. R y a n , F . J . , a n d S c h n e i d e r , L . K . , f,The C o n s e q u e n c e s o f M u ta tio n D u rin g th e B ro w th o f B io c h e m ic a l M u ta n ts of E sc h e ric h ia c o ll. IV . The M e c h a n is m o f I n h i b i t i o n o f H i s t i d i n e I n d e p e n d e n t B a c t e r i a by H i s t i d i n e l e s s B a c t e r i a , 0 J o u r n a l of B a c te r io lo g y , 5 8 :2 0 1 -2 1 3 , 1949. S e v a g , M. G . , a n d D o t s , J . S . , T,E n z y m a t i c S t u d i e s o n t h e M e c h a n is m o f t h e R e s i s t a n c e o f P n e u m o c o c c u s t o D r u g s , 0 J o u r n a l o f B a c t e r i o l o g y , 5 6 :7 3 7 -7 4 8 , 1948. S h e r m a n , J . M ., a n d A l b u s , W. R . , ’’P h y s i o l o g i c a l Y o u t h i n B a c te ria ,* * J o u r n a l o f B a c t e r i o l o g y , 8 :1 2 7 - 1 3 9 , 1923. Y e g i a n , D . , a n d B u d d , V . , f,A V a r i a n t o f M y c o b a c t e r i u m R a n ae R e q u ir in g S tre p to m y c in f o r G ro w th ,0 J o u r n a l o f B a c t e r i o ­ lo g y , 5 5 :4 5 9 -4 6 1 , 1948. Y o u m a n s, A. S . , **The R e l a t i o n s h i p o f t h e Age o f t h e B a c t e r i a l C u l t u r e t o t h e D e l a y i n S u l f o n a m i d e B a c t e r i o s t a s i s , ** J o u r n a l o f B a c te r io lo g y , 5 5 :5 0 3 -5 1 5 , 1948. Z a m e n h o f , S . , l,S t u d i e s o n B a c t e r i a l M u t a b i l i t y ; The Time o f A .p p e a r a n c e o f t h e M u t a n t i n E s c h e r i c h i a c o l i , * * J o u r n a l o f B a c t e r i o l o g y , 5 1 :3 5 1 -3 6 1 , 1946.