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RESULTS ON TAXONOMY AND PHYSIOLOGICAL STATE OF BACTERIA DERIVED FROM
LASER-INDUCED SINGLE CELL ANALYSIS
B. Lindner, U. Seydel
To cite this version:
B. Lindner, U. Seydel. RESULTS ON TAXONOMY AND PHYSIOLOGICAL STATE OF BACTE-
RIA DERIVED FROM LASER-INDUCED SINGLE CELL ANALYSIS. Journal de Physique Collo-
ques, 1984, 45 (C2), pp.C2-565-C2-568. �10.1051/jphyscol:19842129�. �jpa-00223798�
JOURNAL DE PHYSIQUE
Colloque C2, supplkment au n"2, Tome 45, f k v r i e r 1984 page C2-565
RESULTS ON TAXONOMY AND PHYSIOLOGICAL STATE OF BACTERIA DERIVED FROM LASER-INDUCED SINGLE CELL ANALYSIS
B. L i n d n e r and U . Seydel
Research I n s t i t u t e BorsteZ, F.R.G.
Resume
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Dans c e t t e p u b l i c a t i o n on d 6 c r i t l ' a p p l i c a t i o n de l a m i c r o a n a l y s e p a r sonde l a s e r e t s p e c t r o g r a p h i e de masse ?i l ' e t u d e de c e l l u l e s b a c t e - r i e n n e s i s o l e e s . C e t t e methode donne des i n f o r m a t i o n s p r e c i e u s e s a u s s i b i e n s u r l e u r p h y s i o l o g i e que s u r l e u r c l assement taxonomique.A b s t r a c t
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I n t h i s paper t h e a p p l i c a t i o n o f l a s e r m i c r o p r o b e mass a n a l y s i s t o s i n g l e b a c t e r i a l : e l l s i s d e s c r i b e d . I t i s shown t h a t t h i s methodology y i e l d s i n f o r m a t i o n on t h e p h y s i o l o g i c a l s t a t e o f a b a c t e r i u m as w e l l as oni t s taxonomical p o s i t i o n .
I n t r o d u c t i o n : The l a s e r n i c r o p r o b e nass a n a l y s i s as s u p p l i e d w i t h t h e LAMMA-500 i*~GKt?iiakes p o s s i b l e t h e mass s p e c t r o m e t r i c a n a l y s i s o f s i n g l e b a c t e r i a l c e l l s because o f i t s h i g h l a t e r a l r e s o l u t i o n t o g e t h e r w i t h i t s low d e t e c t i o n l i m i t s f o r atomic as w e l l as m o l e c u l a r i o n s . The i n t r a c e l l u l a r c o n t e n t s o f c e r t a i n c a t i o n s c o r - r e l a t e s w i t h t h e p h y s i o l o g i c a l s t a t e o f t h e i n d i v i d u a l c e l l /I/. The measurement o f t h e s e q u a n t i t i e s f r o m a l a r g e r number o f s i n g l e c e l l s a l l o w s t h e c a l c u l a t i o n o f t h e i r d i s t r i b u t i o n s w i t h i n a p o p u l a t i o n . From t h e c h a r a c t e r o f t h e s e h i s t o g r a m s more d e t a i l e d i n f o r m a t i o n on t h e p h y s i o l o g i c a l s t a t e and i t s a1 t e r a t i o n s by e n v i r o n m e n t a l i n f l u e n c e s (e.g. c h e n o t h e r a p e u t i c s ) rnay be o b t a i n e d t h a n f r o m t h e i n e v i t a b l y
averaged d a t a f r o m t h e e s t a b l i s h e d mi c r o b i 01 o g i c a l g r o s s methods.
I n f o r m a t i o n s f r o m t h e m o l e c u l a r (fragment) i o n s can a t p r e s e n t be drawn o n l y v i a mass f i n g e r p r i n t s because - due t o y e t unknown f r a g m e n t a t i o n processes i n v o l v e d i n t h e l a s e r i o n i z a t i o n o f a complex b i o l o g i c a l m a t r i x
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i t i s n o t p o s s i b l e t o r e - l i a b l y i d e n t i f y s i n g l e m o l e c u l a r peaks. The f i n g e r p r i n t s comprise a l a r g e number o f nass peaks w i t h o f t e n m i n u t e d i f f e r e n c e s between d i f f e r e n t s t r a i n s - even o f t h e sane s p e c i e s-
making v i s u a b l e i n s p e c t a t i o n o f t h e s p e c t r a o r c a l c u l a t i o n ' b y hand'-
a l t h o u g h p o s s i b l e /2/ - r a t h e r inadequate. However, e x t e n s i v e s t a t i s t i c a l p r o - cedures a l l o w t h e d i f f e r e n t i a t i o n between e.g. d r u g s e n s i t i v e and t h e r e s p e c t i v e r e s i s t a n t s t r a i n f r o ~ i a l i m i t e d number o f s i n g l e c e l l nass f i n g e r p r i n t s .M a t e r i a l s and Methods: B r i e f l y , i n t h e LAMMA i n s t r u m e n t a h i g h - e n e r g y UV-laser p u l s e ( p u l s e d u r a t i o n 15ns) i s focused t h r o u g h t h e o b j e c t i v e of a l i g h t microscope o n t o t h e sample t o be analyzed and e v a p o r a t e s and p a r t l y i o n i z e s i t a t a l a t e r a l r e s o l u t i o n down t o a p p r o x i m a t e l y 1 pm, dependent on sample c o n d i t i o n s and on t h e l a s e r energy chosen. The produced atomic and m o l e c u l a r (fragment) i o n s a r e r e - g i s t e r e d by means o f a t i m e - o f - f l i g h t mass spectrometer a t a mass r e s o l u t i o n o f about 300 ( f o r t h e l e a d i s o t o p e s ) .
F o r s i n g l e c e l l a n a l y s i s t h e b a c t e r i a ( E . c o l i and v a r i o u s mycobacterium s t r a i n s , see below) were t r e a t e d as f o l l o w s : t h e c e l l s were h a r v e s t e d from t h e a p p r o p r i a t e growth medium and washed t w i c e q u i c k l y
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t o a v o i d i o n d i s l o c a t i o n-
i n d e s t i l l e d w a t e r and b r o u g h t o n t o Formvar coated C u - g r i d s a n d excess f l u i d was d r a i n e d o f f w i t h t i s s u e . T h i s way a wide spread d i s t r i b u t i o n o f t h e b a c t e r i a was achieved a l l o w i n g t h e l a s e r e v a p o r a t i o n o f one s i n g l e c e l l a t a t i m e .The i n f l u e n c e o f a d r u g ( n i t r o f u r a n d e r i v a t i v e HN32) on t h e Na,K-ratio was i n v e s t i - g a t e d w i t h E . c o l i grown i n m o d i f i e d A n t o n ' s medium /I/. For t h i s , t h e d r u g was added a t minimum i n h i b i t o r y c o n c e n t r a t i o n ( 2 0 p m o l / l ) a t t h e b e g i n n i n g o f t h e e x p o n e n t i a l growth. U n t r e a t e d c u l t u r e s were k e p t as c o n t r o l s . A t d i f f e r e n t t i m e s d u r i n g g r o w t h
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842129
C2-566 JOURNAL DE PHYSIQUE
b a c t e r i a l samples were prepared and up t o 200 c e l l s were analyzed f r o m each sample and t h e d i s t r i b u t i o n o f t h e 23Na,39K-ratio was determined. ( I t s h o u l d be mentioned t h a t t h i s r a t i o must n o t n e c e s s a r i l y r e v e a l t h e t r u e i n t r a c e l l u l a r r a t i o because o f d i f f e r e n t i o n i z a t i o n e f f i c i e n c i e s f o r t h e t w o c a t i o n s . )
F o r t h e d i f f e r e n t i a t i o n between v a r i o u s s p e c i e s o f t h e sane genus o r between d r u g - r e s i s t a n t s t r a i n s v i a mass f i n g e r p r i n t s an e x t e n s i v e computerized e v a l u a t i o n p r o - cedure was e s t a b l i s h e d .
The d a t a p r o c e s s i n g i n v o l v e s t h e f o l l o w i n g s t e p s :
1. Measurement and d a t a p r e p a r a t i o n f o r a m u l t i v a r i a t e a n a l y s i s
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r e g i s t r a t i o n o f 90 s p e c t r a o f each s t r a i n-
c o n v e r s i o n t o l i n e - s p e c t r a and n o r m a l i z a t i o n w i t h r e s p e c t t o t o t a l i n t e n s i t y t o c o r r e c t f o r v a r i a t i o n s i n sample s i z e and l a s e r i n t e n s i t y-
d a t a r e d u c t i o n f o r each s t r a i n t o 3 s p e c t r a , each o f which r e p r e s e n t i n g t h e average o v e r 30 s i n g l e s p e c t r a2. Checking f o r s t a t i s t i c a l s i g n i f i c a n t d i f f e r e n c e s ( t h i s a n a l y s i s f o l l o w s a procedure d e s c r i b e d i n / 3 / )
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c a l c u l a t i o n o f w e i g h t f a c t o r s f o r each mass, t h e s e s o - c a l l e d c h a r a c t e r i s t i c i-
t i e s a r e d e t e r m i n e d f o r each mass by comparing t h e d e v i a t i o n i n t h e peak i n - t e n s i t i e s w i t h i n s p e c t r a o f t h e same s t r a i n ( i n t r a - s t r a i n d e v i a t i o n ) w i t h t h a t d e r i v e d f r o m t h e d i f f e r e n t s t r a i n s ( i n t e r - s t r a i n d e v i a t i o n ) . The c h a r a c t e r - i s t i c i t y resembles a s i g n a l t o n o i s e r a t i o .
3. C a l c u l a t i o n o f mathematical e x p r e s s i o n s f o r d i s s i m i l a r i t i e s between two s p e c t r a
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each spectrum - reduced t o t h e n masses w i t h t h e h i g h e s t c h a r a c t e r i s t i c i t i e s-
i s i n t e r p r e t e d as a p o i n t i n a n-dim. room
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t h e E u c l i d e a n i n t e r p o i n t d i s t a n c e , w e i g h t e d by t h e c h a r a c t e r i s t i c i t i e s , can b e t a k e n as a numerical measure o f t h e d i s s i m i l a r i t y between two s p e c t r a-
c o m p u t a t i o n o f a d i s s i n i 1 a r i t y m a t r i x c o m p r i s i n g t h e d i s s i m i l a r i t i e s between a l l s p e c t r a (when h a v i n g K s p e c t r a t h e m a t r i x c o n s i s t s o f K ( K - 1 ) / 2 elements) 4. E v a l u a t i o n o f t h e d i s s i m i l a r i t y m a t r i x-
c a l c u l a t i o n o f t h e n e a r e s t neighbours o f each spectrum-
g r a p h i c a l r e p r e s e n t a t i o n i n a 2- o r 3-dim. p l o t t o show t h e i n f o r m a t i o n s t o r e d i n t h e m a t r i x i n a more c o n v e n i e n t form. The p r o c e d u r e i s based on a n o n l i n e a r mapping a l g o r i t h m g i v e n i n /4/.R e s u l t s and d i s c u s s i o n : I n f i g . 1 t h e g r o w t h k i n e t i c s o f t h e u n t r e a t e d c o n t r o l and o f H N 3 2 - t r e a t e d sample a r e g i v e n . F i g . 2 shows t h e c o r r e s p o n d i n g d i s t r i b u t i o n s o f t h e 23Na,39K-ratio o f v a r i o u s samples t a k e n a t t h e t i m e s marked i n t h e growth k i n e t i c s .
C l e a r l y , t h e d i s t r i b u t i o n s 1-3 show i n c r e a s i n g broadening and a s h i f t o f t h e median t o h i g h e r v a l u e s o f t h e Na,K-ratio due t o a l o n g e r a c t i o n o f t h e chemotherapeutic.
D i s t r i b u t i o n 4, however, i m p l i e s - much more pronounced t h a n t h e growth k i n e t i c s
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a r e c o v e r y o f t h e c e l l s . Furthermore, f r o m t h e changes i n t h e s e h i s t o g r a m s t h e be- g i n n i n g o f t h e r e c o v e r y becomes e v i d e n t e a r l i e r . The shapes o f t h e d i s t r i b u t i o n s i m p l y a l s o t h a t an averaged v a l u e f o r t h e N a , K - r a t i o o v e r a l l c e l l s as i t i s ob- t a i n e d f r o m t h e g r o s s methods can g i v e o n l y i n c o m p l e t e i n f o r m a t i o n .
I n f i g . 3 t h e 2-dim. n o n l i n e a r map o f t h e d i s s i m i l a r i t y m a t r i x r e p r e s e n t i n g t h e r e l a t i o n s h i p between 21 m y c o b a c t e r i a s p e c t r a i s shown. P o i n t s ( s p e c t r a ) o r i g i n a t i n g f r o m t h e same s t r a i n a r e i n t e r c o n n e c t e d . S t r a i n s b e l o n g i n g t o d i f f e r e n t s p e c i e s a r e we; 1 separated except t h o s e o f M.kansasi i and M . g a s t r i , which a r e known t o be c l o s e l y r e 1 a t e d i n biocheniical terms.
,f/
is 3b 3;
40
i s 50Time / h
Fig.1: Growth k i n e t i c s of E.Coli c e l l s t r e a t e d with t h e drug HN32 ( a n i t r o f u r a n
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d e r i v a t i v e ) a t minimal i n h i b i t o r y concentration and of t h e r e s p e c t i v e untreated c o n t r o l .Fig.2: D i s t r i b u t i o n s of t h e 23Na,39K-ratio within b a c t e r i a l c e l l populations
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t r e a t e d with HN32 a t minimal i n h i b i t o r y c o n c e n t r a t i o n . The histograms correspond t o t h e various sampling times marked i n f i g . 1 .JOURNAL DE PHYSIQUE
F i g . 3 : N o n l i n e a r , 2-dim. map o f 21 rnycobacterium s p e c t r a o f v a r i o u s species.
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STRESS:
.
8277 MAN; 3/3 93. ITERATIONM. AVlUM
M. KANSAS1 I
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F i g . 4 : N o n l i n e a r , 2-dim. map, showing t h e numerical c o r r e l a t i o n between - M . t u b e r c u l o s i s b a c t e r i a r e s i s t a n t t o d i f f e r e n t drugs.
STRESS, . 0 2 0 6 KAN: 3/4 62. ITERATION
CAPREOMYCIN R.
VlOMYClN R.
.
KANAMYCIN R.
CONTROL
The r e s u l t o f t h e numerical r e 1 a t i o n s h i p o f 4 M . t u b e r c u l o s i s s t r a i n s r e s i s t a n t t o d i f f e r e n t chemotherapeutics and t h e r e s p e c t i v e s e n s i t i v e s t r a i n ( c o n t r o l ) i s g i v e n i n f i g . 4 . The b a c t e r i a made r e s i s t a n t t o kanamycin and t o v i o m y c i n can n o t be d i f - f e r e n t i a t e d c l e a r l y ( p o s s i b l y due t o a c r o s s r e s i s t a n c e o f t h e b a c t e r i a ) .
F i g . 3 F i g . 4
These f i r s t r e s u l t s demonstrate t h a t t h e computerized f i n g e r p r i n t e v a l u a t i o n of t h e l a s e r d e s o r p t i o n mass s p e c t r a o f s i n g l e c e l l s may o f f e r u s e f u l i n f o r m a t i o n f o r m i c r o - b i 01 o g i c a l r e s e a r c h .
/1/ B. L i n d n e r and U. Seydel, J. Gen. M i c r o b i o l
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129, 51 (1983)/2/ U. Seydel and H.-J. Heinen, i n 'Recent Developments i n Mass Spectrometry i n B i o c h e m i s t r y and M e d i c i n e ' , Vol. 6, Eds: A. F r i g e r i o and M. McCamish, E l s e v i e r , Amsterdam (1980), p . 489
/3/ W. Eshuis, P.C. Kistemaker, H.L.C. Meuzelaar, i n ' A n a l y t i c a l P y r o l y s i s ' , Eds: C.E.R. Jones and C.C.A. Cramers, E l s e v i e r , Amsterdam (19771, p. 151 /4/ J.K. K r u s k a l , Psychometrika
