Results on Taxonomy and Physiological State of Bacteria Derived from Laser-induced Single Cell Analysis

I n t r o d u c t i o n : The l a s e r n i c rop robe nass a n a l y s i s as supp l i ed w i t h t h e LAMMA-500 i*~GKt?iiakes p o s s i b l e t h e mass spec t rome 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 oge the 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 mo lecu la r i ons . The i n t r a c e l l u l a r con ten ts 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 these q u a n t i t i e s f r om a l a r g e r number o f s i n g l e c e l l s a l l ows 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 popu la t i on . From t h e cha rac te r o f these h is tograms 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 env i ronmenta l i n f l u e n c e s (e.g. chenotherapeut ics) rnay be ob ta ined than f rom t h e i n e v i t a b l y averaged da ta f rom 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 ross methods. I n fo rma t i ons f rom t h e mo lecu la r ( f ragment) i o n s can a t p resent 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 ragmen ta t i on 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 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 mo lecu la 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 inu te d i f f e rences between d i f f e r e n t s t r a i n s even o f t h e sane spec ies 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 spec t ra o r c a l c u l a t i o n ' by hand' a l t hough p o s s i b l e /2/ r a t h e r inadequate. However, ex tens i ve s t a t i s t i c a l p ro 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 rug 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 ns t rumen t a h igh-energy UV-laser pu l se ( p u l s e d u r a t i o n 15ns) i s focused through t h e o b j e c t i v e of a l i g h t microscope on to t h e sample t o be analyzed and evapora tes 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 approx imate ly 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 mo lecu la r ( f ragment) i o n s a re 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 ) . For 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 va r i ous 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 harvested 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 t o avo id 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 water and brought onto Formvar coated Cu-gr idsand excess f l u i d was d ra ined 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 evapo ra t i on o f one s i n g l e c e l l a t a t ime . The i n f l u e n c e o f a drug ( 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-rat io was i n v e s t i ga ted w i t h E . c o l i grown i n m o d i f i e d An ton ' s medium /I/. For t h i s , t h e drug was added a t minimum i n h i b i t o r y concen t ra t i on (20 pmo l / l ) a t t h e beg inn ing o f t h e exponen t i a l growth. Un t rea ted c u l t u r e s were kep t as c o n t r o l s . A t d i f f e r e n t t imes d u r i n g growth 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 rom 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 shou ld be ment ioned 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 two 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 spec ies o f t h e sane genus o r between drugr 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 es tab l i shed . The d a t a p rocess ing i n v o l v e s t h e f o l l o w i n g s teps: 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 r e g i s t r a t i o n o f 90 spec t ra o f each s t r a i n conve rs ion 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 respec 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 spect ra , each o f which r e p r e s e n t i n g t h e average ove r 30 s i n g l e spec t ra 2. 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 desc r i bed i n / 3 / ) c a l c u l a t i o n o f we ight f a c t o r s f o r each mass, t hese 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 are determined 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 spec t ra 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 rom 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 cha rac te r i s t i c i t y resembles a s i g n a l t o no i se r a t i o . 3. C a l c u l a t i o n o f mathematical express ions 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 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 t h e Euc l i dean i n t e r p o i n t d is tance, we ighted by t h e c h a r a c t e r i s t i c i t i e s , can be taken as a numerical measure o f t h e d i s s i m i l a r i t y between two spec t ra computa t ion o f a d i s s i n i 1 a r i ty m a t r i x compr i s i ng t h e d i s s i m i l a r i t i e s between a l l spec t ra (when hav ing K spec t ra 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 nea res 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 2o 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 conven ient form. The procedure 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 ven i n /4/. Resu l t s and d i scuss ion : I n f i g . 1 t h e growth 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 iven. F ig .2 shows t h e cor respond ing d i s t r i b u t i o n s o f t h e 23Na,39K-ratio o f va r i ous samples t aken a t t h e t imes 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 va lues o f t h e Na,K-rat io due t o a l o n g e r a c t i o n o f t h e chemotherapeut ic. D i s t r i b u t i o n 4, however, i m p l i e s much more pronounced than t h e growth k i n e t i c s a recove ry o f t h e c e l l s . Furthermore, f r om t h e changes i n these h is tograms t h e beg i n n i n g o f t h e recove ry becomes ev iden 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 va lue f o r t h e Na,Kra t io over a l l c e l l s as i t i s obt a i n e d f r o m t h e gross methods can g i v e o n l y incomple te 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 mycobacter ia spec t ra 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 om t h e same s t r a i n a r e i n te rconnec ted . S t r a i n s be long ing t o d i f f e r e n t spec ies a r e we; 1 separated except those o f M.kansasi i and M.gast r i , which a re known t o be c l o s e l y r e 1 a ted i n biocheni ical terms.