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Microb Ecol (1994) 27:57-63

MICROBIAL

ECOLOGY

1994 Springer-Verlag Ne w York Inc.

Effect of Therm al Oxidative Acidic Osmo tic or Nutritional

Stresses on Subsequent Culturability of scher ich ia cob in Seawater

P.M. Munro, R.L. C16ment, G.N. Flatau, M.J. Gauthier

Inst i tut National de la Sant6 et de la Recherche M6dicale, Unit6 303, C.E .R.B .O.M ., I Avenu e

Jean Lorrain, 06300 Nice, France

Received: 22 July 1993; Revised: 29 September 1993

Abstract. Survival o f stressed

Escherichia coli

with or w ithout the

rpoS

gene

was assessed after 2 and 6 days in sterile seawater. Cells were submitted to

thermal (48C), acidic (pH 5.1), oxidative (H202 lrnM), nutritional (C, N, P

starvation), or osm otic (NaCI 0.5M) stresses for periods ranging f rom 0 to 4 h.

We found a stress-mediated cross protection against seawater relative to con-

trols. Viability was higher when cells were acid, oxidatively, nutritionally or

osm otically stressed. Su rvival increased in cells stressed at 37C as compar ed

with 20C. With the exception of osmotic stress, we found that this stress-

induced cross protect ion was

rpoS

dependent.

Introduction

To su rvive in the marine environ men t, enteric bacteria have devel ope d a strategy in

which they shift into various states [for review see 18], undergoing dramatic

physiological and morphological changes [5, 15]. Nevertheless, i t has been shown

that the fate of these bacteria in seawater can d epend on the history o f the cells prior

to their arrival in this nutrient-poor environm ent [4]. For examp le,

Escherichia coli

and other enteric bacteria grown in a high-salt medium survive better in seawater

[3, 16]. The sam e kind o f survival enhancem ent has been described with stationary

phase cells [6], suggesting a poss ible adaptation to starvation via the express ion of

specific genes, also called survival genes [11, 20], related to nutrient-deprived

environm ents [ 13]. This resistance to starvation cou ld be under the control of stress

proteins [13].

This paper describes stresses-mediated resistance to seawater in

E. coli

and the

influence o f the duration o f stresses on cell culturability. W e su ggest that this cross

protection against seawater, achieved by a thermal, oxidative, acidic, osmotic, or

nutritional stress may b e in some cases under the control of

rpoS

a regulatory g ene

of central importance for early stationary phase-indu ced gene s in

E. coli

[7].

Correspondence to: P.M. Munro .

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58 P . M . M u nr o e t a l .

a t e r i a ls a n d e t h o d s

B a c t e r i a l S t r a i n s a n d G r o w t h C o n d i t i o n s

Escherichia coli M C 4 1 0 0 ( F - A arg-lac) U169araD139 rpsL150ptsF25 lbB5301 rbsR deoC relA1)

a n d R H 9 0 , a

rpoS

m u t a n t o f t h i s s t r a i n ( M C 4 1 0 0

rpoS359::Tn10)

[ 12 ] , w e r e u se d . S to c ks w e r e

m a i n t a i n e d a t - 7 0 C i n tr y p t ic s o y b r o t h (D i f c o L a b o r a to r i e s, D e t r o i t , M i c h . , U S A ) s u p p l e m e n t e d

w i th g lyc e r o l ( 10 v /v ) . F o r e xpe r im e n t s , c e l l s w e r e g r ow n a t 37 C in m in im a l M O P S m e d ium [ 17 ]

( pH 7 . 4 ) su pp le m e n te d w i th g luc ose ( 2 g / l ) a nd phosp ha te ( N a 2H P O 4 , 2 m M ) .

S t r e s s C o n d i t i o n s

B e f o r e t r a ns f e r t o s e a w a te r , c e l l s w e r e subm i t t e d t o one o f f i ve d i f f e r e n t s t r e s s c ond i t i ons : h igh

temp era ture (48C -+ 0 .2C) , h ig h osm ola r i ty (NaC1, 0 .5 M) , ac id i ty (pH 5 .1 -+ 0 .4) , h ig h pe roxide

c o n c e n t ra t i o n ( H 2 0 2 , 1 n ~ ) , a n d n u t r i e n t s t a rv a t io n ( M O P S m e d i u m w i t h o u t c a rb o n , n i t r o g e n , a n d

phosph o r us sou r c e s ) .

S u r v i v a l T e s t s

S ur v iva l t e s t s w e r e pe r f o r m e d in 100 m l o f a u toc l a ve d se a w a te r ( 113 C , 20 m in ) ( S S W ) ( C a pe o f N ic e ,

F r a nc e ) he ld i n 250 - m l E r l e nm e y e r fl a sks ( f u r the r r e f e r re d t o a s m ic r oc osm s) . C e l l s w e r e g r ow n in 100

m l o f M O P S m e d ium a t 37C a n d ha r ve s t e d at e a r ly e xpone n t i a l pha se o f g r ow th ( m 6o = 0 . 3 ) by

c e n t r i f uga t ion ( 27 , 000 g , 6 m in , 20 C ) . The y w e r e t he n su spe n de d , a t t he de ns i ty o f t he o r ig ina l

c u l tu r e , i n 6 s a m p le s ( 120 m l ) o f ne w M O P S m e d ium a nd subm i t t e d t o s t re s s c ond i t i ons . S usp e ns ions

w e r e i nc uba t e d a t 37 C , e xc e p t f o r t he r m a l s t r e s s ( 48 C ) . F o r e a c h su spe ns ion , a 20 - m l s a m p le w a s

c o l l e c t e d a ft e r 0 m in , 30 m in , l h , 2h , 3h , a nd 4h o f s t r e s s e xposu r e a n d c e n t r i f uge d ( 27 , 000 g , 6 r a in ,

20 C ) . C e l l s w e r e w a she d 3 t im e s w i th 25 m l o f S S W , the l a s t pe ll e t su spe nd e d in 2 m l o f S S W , a nd

th i s fi na l su spe ns ion w a s u se d to i noc u la t e m ic r oc osm s a t a de ns i ty o f 1 . 0 x 1 6 c f u /m l ( - +0 .5 x l 6

c f u /m l ) . M ic r o c osm s w e r e t he n inc uba t e d i n t he da r k a t r oom t e m pe r a tu r e ( 22 - 25 C ) .

S e pa r a t e s e t s o f e xpe r im e n t s , w h ic h f o l l ow e d the ba s i c p r o toc o l ou t l i ne d a bove , w e r e u se d to

e xa m ine the i n f lue nc e o f t e m pe r a tu r e on s t r e s s - induc e d c r o s s p r o t e c t i on a nd the de pe nde nc y o f c r o s s

p r o t e c t i on on the rpoS ge ne . S uspe ns ions w e r e e xpose d to s t r e s s c ond i t i ons a t 20 C f o r 3h f o r

c om pa r i son w i th t he r e su l t s ob t a ine d w i th e xposu r e a t 37 C . The r o l e o f rpoS w a s e x a m i n e d i n

e xpe r im e n t s c om pa r ing su r v iva l o f M C 4 100 a nd R H 9 0 s t r a in s . C e l l s w e r e e xpose d to s t r e s s c ond i t i ons

f o r l h a t 37C .

C u l t u r a b l e - C e l l s C o u n t s

A l l m ic r oc o sm s w e r e s a m p le d im m e d ia t e ly a f t e r i noc u la t i on (TO ) a nd a f t e r 2 a nd 6 da ys o f i nc uba t ion

in s e a w a te r. The c o lony f o r m ing a b i l i t y o f c e l l s w a s de t e r m ine d in t r ip l i c a t e by the m e m br a n e f il t r a t ion

t e c hn ique ( M ic r o F i l t r a t i on S ys t e m s f i l t e r s , po r e s i z e , 0 . 45 I xm ; M F S , D ub l in , C a l i f . , U S A ) on

nu t r i e n t a ga r ( D i fc o ) . A n a pp r op r i a t e d i l u t ion o f t he s a m p le s w a s u se d to y i e ld a num be r o f c o lon ie s

r a ng in g f r om a bou t 10 to 400 pe r p l a t e. C o lon ie s w e r e c o un te d on th e f il t e r s suppo r t e d on a ga r p l a t es

a f t e r a 24 - h i nc ub a t ion a t 37 C . L oss o f c u l tu r a b i l i t y w a s e xp r e s se d a s ( c f u /m l a f t e r 2 a nd 6 da ys o f

inc uba t ion in s e a w a te r ) / ( c f u /m l a t TO ) x 100 ( c u l tu r a b l e c e l l s) .

S t a t i st i c a l A n a l y s i s

A l l da t a p r e se n t e d a r e m e a ns f r om tw o se pa r a t e e xpe r im e n t s . A na ly s i s o f va r i a nc e w a s pe r f o r m e d on

the l og - t r a ns f o r m e d da t a i n o r de r t o s t a b i l i z e t he va r i a nc e .

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Survival o f Stressed

Escherichia coli

in Seawater 9

t O

0

m

03

r j

o

. .Q

o

, 0 1

,001

,0001

,00001

,000001

T h e r m a l

0 3 0 6 0 12 0 1 8 0

1 0

,01

,001

4 0

O x i d a t i v e

,1

o i

,oo1 ~

o o o 1

z z ~

, o o o o l ~ N . N . N .~

0 30 60 120 180

O s m o t i c

~ ~

~I ~ ~ ~

0 30 ~ 120 180 240

2 4 0

A c i d i c

,1

,01

o o

o o o

0 30 60 120 180 240

S t a r v a t i o n

~

~ / / ;~

1 ~ ' A ;., ~: ~ / / / . / / /

r . . /~ 7 / , i i l ~ l t

l V tA / f l ; 1 // ,., 1~ : 7 /

,o o o, N . N . ~ ' ~ : > + '..~ . N

0 3 0 6 0 1 2 0 1 0 0 2 4 0

D u r a t io n o f s t r e s s ( m i n )

Fig. 1. Percen tage of culturable cells o f stressed E . c o l i M C 4100 after a 6-day incubation in

seawa ter. Cells were su bm itted to different stresses for different periods of t ime see M aterials and

M ethods) at 37C prior to their incubat ion in seawater . B a r s represent standard deviations of two

different experiments.

R e s u l t s

I n f l u e n c e

of Previous Stress on Resistance to Seawa ter

A f t e r 2 d a y s o f i n c u b a t i o n i n s e a w a t e r , n o s i g n i f ic a n t e f f e c t o f s tr e s s o n r e s i s t a n c e

o f E . c o l i t o s e a w a t e r w a s d e t e c t e d d a ta n o t s h o w n ) . H o w e v e r , d a ta o b t a in e d a f te r

6 d a y s o f in c u b a t i o n F i g . 1 ) s h o w e d t h a t a p r e v i o u s e x p o s u r e t o a s t re s s a l l o w e d a

b e t t e r r e s i s t a n c e t o s e a w a t e r .

W i t h t h e e x c e p t i o n o f a c i d i c s t r e ss , v i a b i l i ty i n c r e a s e d w i t h d u r a t i on o f e x p o s u r e

w i t h i n t h e t e s te d l im i t s ) u p t o a m a x i m u m l e v e l , w h i c h d i d n o t c h a n g e w i t h fu r t h er

e x p o s u r e . T h e d u r a t io n o f e x p o s u r e y i e l d i n g m a x i m u m v i a b il it y i n s e a w a t e r va r ie d

w i t h t h e s tr e s s fa c to r : 6 0 m i n f o r o s m o t i c a n d t h e r m a l s t r e s se s , 1 2 0 m i n f o r

s t a r v a t i o n s tr e s s , a n d 1 8 0 r a i n f o r o x i d a t i v e s t r e s s . F o r a c i d i c s t r e s s t h is l e v e l

i n c r e a s e d u p t o 1 2 0 m i n t h e n d e c r e a s e d .

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60 P.M. Mu nro et al.

tg~

0

_

;g

,000

,0000

,00000

T p o x ca r o s m

t r e s s e s

Fig. 2. Percentag e of culturable cells of stressed

E. coli MC 4100 after a 6-day incubation in

seawater. Cells we re previously stressed for 3 h at

20C T = no stress, pH = acidic stress,

ox = oxidative stress, car = starvation stress,

osm = osmotic stress). For ease of comparison,

data from the 37C experiment under the same

conditions Fig. l) have been included here.

Bars

represent standard deviations of tw o d ifferent

experiments.

T h e r e l a t i v e e f f e c t o f th e s e s t r e ss e s o n t h e r e s i s t a n c e o f c e l ls t o s e a w a t e r f o l l o w e d

t h e s e q u e n c e : o s m o t i c > s t a r v a t io n > o x i d a t i v e > a c id i c > t h e r m a l . O s m o t i c

s t re s s a l s o p r o v i d e d t h e b e s t p r o t e c t i v e e f f e c t w i t h i n t h e s h o r t e s t t i m e p e r i o d .

W h e n t h e s e s t r e s s e s w e r e a p p l i e d a t 20 C f o r 3 h , a s i g n i f i c a n t l e v e l o f p r o t e c t i o n

w a s o b s e r v e d o n l y i n t h e c a s e o f o s m o t i c P < 0 . 0 1 ) a n d s t a r v a t i o n s t r e s s e s

P < 0 . 0 5 ) F i g . 2 ) , t h u s c o n f i r m i n g t h e ir g r e a t e r e f f e c t . A t 2 0 C n o s i g n i f ic a n t

d i f f e r e n c e s i n v i a b i l i t y w e r e f o u n d f o r c e l l s r e c e i v i n g e i t h e r a c i d i c o r o x i d a t i v e

s t r e s se s .

D ep en d en ce o f S tr e s s - In d u ced Pr o t ec t i o n t o r p o S = k a t F )

W i t h t h e e x c e p t i o n o f o s m o t i c a l l y s t r e s s e d c e ll s , a s i g n i f ic a n t l y h i g h e r l e v e l o f

r e s i s t a n c e t o s e a w a t e r w a s o b s e r v e d w i t h s t r e s s e d r p o S c e l l s P < 0 . 0 5 f o r

o x i d a t i v e a n d a c i d i c s t r e s s a n d P < 0 . 0 1 f o r t h e r m a l a n d n u t r i t i o n a l s t r e s s e s )

r e l a t iv e t o t h e m u t a n t F i g . 3 ) . T h u s , c r o s s p r o t e c t i o n t o s e a w a t e r t h r o u g h o x i d a -

t i v e , a c i d i c , t h e r m a l , a n d n u t r i ti o n a l s t r e s s e s a p p e a r s t o b e

r p o S

d e p e n d e n t ,

w h e r e a s s u c h p r o t e c t i o n t h r o u g h o s m o t i c s t r e s s d o e s n o t .

i s cu s s i on

T h i s s t u d y d e m o n s t r a t e s a t h e r m a l , o x i d a t i v e , a c i d i c , o s m o t i c , a n d n u t r i t i o n a l

s t r e s s -i n d u c e d c r o s s p r o t e c t i o n a g a i n s t s e a w a t e r F i g . 1 ) . W h i l e f o r E. col i , p r o -

l o n g e d p e r i o d s i n s e a w a t e r r e p r e s e n t b o t h a n o s m o t i c a n d a n u t ri t io n a l s t re s s , w e

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Survival of Stressed Escherichia coli in Seawater 61

10

o

,01

,001

,0001

T tern p ox car osm

Stresses

Fig. 3. Percentage of culturable cells of stressed.

E. coli MC 4100 rpoS+) and RH 90 rpoS-) after

a 6-day incubation in seawater. Cells were

previously stressed for 1 h as described in

Materials and Methods (T = no stress,

tern = thermal stress, pH = acidic stress,

ox = oxidative stress, car = starvation stress,

osm = osmotic stress). Bars represent standard

deviations of two different experiments.

conc lude tha t the r e i s a mul t ip le - s t r es s - induced c ross pro tec t ion of E. co l i to

osmot ic and nu t r i t iona l cha l lenge .

This e f f ec t has b een ve r i f i ed for a l l the t e s ted s t r e s s f ac tor s , bu t i s mo re or l e s s

impor tan t depending on the s t r e s s . Osmot ica l ly s t r e s sed ce l l s and nu t r ien t - s ta rved

ce l l s show h igh er surv iva l r a tes in seaw ate r (F ig . 1 ). O ur da ta sugges t tha t th is i s

a l so depende nt on the t emp era ture a t whic h the ce l l s a r e s tr e s sed . P ro tec t ion aga ins t

seaw ate r is no t ach ieve d a f te r a 3 -h ac id ic or ox ida t ive s t r e s s a t 20C, whe reas ce l l s

s t r e s sed a t 37C in o the rwise the same condi t ions a r e approximate ly 10 t imes and

3 , 000 t i m e s , r e s pe c t i ve l y , m o r e r e s i st a n t t o s e a wa t e r t ha n t hos e t ha t a re uns t r e ss e d

(F ig . 2 ) . A s ign i f ican t l eve l o f p ro tec t io n wi th s t r e ss a t 20C i s ach ieved only wi th

osmotic and nutr i t ional s t ress (Fig. 2) .

P r e v i ous s t ud i e s f r om t h is l a bo r a t o r y ha ve a dd r e s s e d t he i n f lue nc e o f g r owt h

c ond i t ions on s u r v i va l o f E . co i l in seaw ate r [4] . Escher ich ia co l i ce l l s submi t ted to

an osm ot ic s t r e s s (growth in a h igh sa l t medi um ) or to a nu t r i t iona l s t r e s s ( s ta t ionary

pha s e c e l ls ) ha ve be e n s h own t o m a i n t a i n c u l t u r a b i li t y a f t e r p r o l ong e d pe r i ods i n

seaw ate r [3 , 6 ] .

S t a r va t i on - i nduc e d c r o s s p r o t e c t i on o f E. co l i aga ins t hea t o r H202 [8] and

os m ot i c c ha l l e nge [ 9 ] , a nd s t a r va t i on - i nduc e d c r o s s p r o t e c t i on o f E. co l i and

m a r i ne Vibr io spp . aga ins t l e tha l t empera tures [10] have been c lea r ly demon-

s t ra t ed . On t he o t he r ha nd , t he s a m e k i nd o f t he r m o t o l e r a nc e a nd r e s i s t a nc e a ga i n s t

hyd r oge n pe r ox i de c a n be o s m o t i c a l l y s t i m u l a t e d i n E. co i l [7].

Co mb ar ro e t a l. [ 2] have a l r eady in ves t iga ted the e f f ec t o f an ox ida t ive s t r e s s a t

23 - 25 C on f u r t he r s u r v i va l o f E. co l i in seawate r th rough a t r ans ien t incuba t ion

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62 P . M . M unr o e t a l .

f rom anaerob ic g rowth med ium ( in t es t i na l o r u r inary l i ke cond i t i ons ) t o aerob ic

f resh o r was t e water . Th ey foun d the percen t age o f cu l tu rab l e ce l l s a ft e r 7 days i n

seaw ater t o be t he sam e as i n t h is s t ud y ( abou t 0 .0001) fo r a 3 -h incubat ion t ime in

wa s t e water . H ow eve r , t he cond i t i ons used in bo th s tud i es canno t a l l ow any fu r ther

conclus ion .

The da t a p resen t ed he re supp or t t he observa t ions t ha t mu l t ip l e - s tr ess r es i s t ance i s

as e f f i c i en t fo r ce l l s i n cu l tu re as i n seaw ater su rv iva l .

T h e d e p e n d e n c e o n rpoS of c ross p ro tec t i on aga ins t seaw ater induce d b y thermal ,

oxidat ive, acid ic , and nut r i t ional s t resses (Fig . 3) i s suppor ted by the f indings that

Ka tF ( - -Rpo S) m utan t s fa i l t o deve lop thermores i s t ance , o sm ores i s t ance , o r r es i s-

t ance t o H202 and have r educed v i ab i l i t y under severa l s t a rva t ion cond i t i ons [12 ,

19] . Thus,

rp o S

which has been iden t i f i ed as a cen t ra l r egu la to r o f t he genera l

s t a rva t ion r esponse [12 , 14 ] , appear s a l so t o be impor t an t i n mul t i p l e - s t r ess - in -

duc ed r es is t ance t o seaw ater . I t has a l so been dem ons t ra t ed [12 , 14 ] t ha t severa l

rpoS-dependent

pro t e ins were n o t osmo t i ca l l y i nduced , t hus e xp la in ing the f ac t t ha t

osm ot i c c ross p ro t ec t i on aga ins t seaw ater is no t

rpoS-dependent.

I n s u m mar y , i n d ep en d en tl y o f li g h t an d p r ed a ti o n , l o n g k n o w n t o b e s o me o f t h e

ma in f ac to r s a f f ec ti ng the su rv iva l o f bac t e r i a i n seaw ater [1 ] , enhanc ed r es i s t ance

o f

E. coli

t o seawater can be ach i eved by a p rev ious ac id i c , t hermal , ox ida t ive ,

osm ot i c , o r nu t ri ti ona l s t r ess . W i th the exc ep t ion o f osm ot i c s t r ess , t hi s c ross

p ro t ec t i on i s p robab ly assoc i a t ed wi th rpoS a pu t a t i ve s igma fac to r o f cen t ra l

impor t ance fo r ear ly s t a t i onary -phase- induced genes [7 ] . I t has been assumed tha t

rpoS

i s a l so o f majo r impor t ance i n su rv iva l o f

E. coli

i n f eas t and f am ine [7]

cond i t i ons i n t he env i ronm ent , and w e sugg es t tha t i t i s espec i a l l y impor t an t du r ing

the s t r ess - inducing t r ans i t f rom the hum an in tes t ina l o r u r inary t r ac t t o seaw ater v i a

w as t ew a t e r .

S t u d i es a r e u n d e r w ay t o ch ec k f o r a p o s s i b l e

de novo

pro t e in syn thes i s depen-

denc e o f th i s mul t i p l e - s tr ess - induced c ross p ro t ec t i on aga ins t seaw ater .

A c k n o w l e d g m e n t s . W e t h a n k D r . R e g i n e H e n g g e - A r o n i s f o r k i n d l y p r o v i d i n g t he E . c o l i s t r a in s . W e

a r e g r at e f ul t o D r . J o h n D o l a n f o r h e lp f u l c o m m e n t s a n d r e v i e w s o f t h e m a n u s c r ip t . T h i s w o r k w a s

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