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CURRENT M ICROBIOLOGYVo l. 27 1993), pp. 295-300

u r r e n t

M i c r o b i o l o g y

9 S p r i n g e r -V e r l a g N e w Y o rk I n c . 1 99 3

M e c h a n i sm o f Ba c t e ri ci d a l c t i o n o f P h e n e t h y l l c o h o l i n

scherichia coli

J . J . L u c c h i n i , ~ N . B o n n a v e i r o , ~ A . C r e m i e u x , ~ a n d F . L e G o f f i c 2

1Laboratory of Microbiology, Faculty of Pharmacy, Marseil le, France; and 2Laboratory of Bioorganic and o f Biotechnology,

Paris, France

A b s t r a c t . T h e m e c h a n i s m o f b a ct e r ic i d a l a c ti o n o f p h e n e t h y l a l c o h ol P E A ) i n E . c o l i w h i c h

w a s p r e v i o u s l y d e m o n s t r a t e d t o b e d e p e n d e n t o n p r o te i n s y n t h e s i s , h a s b e e n i n v e s t i g a te d .

M u t a n t s r e s is t a n t to P E A w e r e s e l e c t e d , b u t th e r e s i st a n c e o b s e r v e d w a s a s s o c i a t e d w i t h a

c h a n g e i n p e r m e a t i o n . P E A e f fe c t s o n D N A , R N A , a n d p r o te i n s y n t h e s i s w e r e s t u d i ed w i t h

b a c t e r i o s t a t i c a n d b a c t e r i c id a l c o n c e n t r a t i o n s . S i m i l a r r e s u l t s i n h ib i t io n o f D N A s y n t h e s i s a n d

d e c r e a s e i n R N A s y n t h e si s ) w e r e o b t a i n e d w i th l e th a l c o n c e n t r a t io n s o f P E A i n c e ll s p r e t r e a t e d

w i t h c h l o r a m p h e n i c o l , a n d w i t h b a c t e ri o s t a ti c c o n c e n t r a t i o n s o f P E A in u n p r e t r e a t e d c e ll s. T h e

P E A i n t ra c e l lu l a r a c c u m u l a t i o n r e a c h e d a m a x i m u m w i t h i n 4 m i n a n d w a s n o t i n h ib i te d b y

K C N o r b y 2 ,4 - d in i tr o p h e n o l. T h e p r e s e n c e o f p h e n y l a c e t a l d e h y d e w a s d e m o n s t r a t e d i n b o t h

s t a t i o n a r y a n d e x p o n e n t i a l g r o w t h p h a s e c e l l s e x p o s e d t o P E A b u t n o t i n c e l ls p r e t r e a t e d w i t h

c h l o r a m p h e n i c o l . T h e s e r e s u lt s s u g g e s t e d t h a t t h e b a c te r i c id a l m e c h a n i s m o f a c t i o n o f P E A

i n v o l v e s i t s c o n v e r s i o n i n t o t h e c o r r e s p o n d i n g a l d e h y d e .

I n a p r e v i o u s s t u d y [ 13 ], it w a s s h o w n t h a t t h e b a c t e -

r i c id a l a c t i v i t y o f b o t h p h e n e t h y l a l c o h o l P E A ) a n d

b e n z y l al c o h o l B A ) o n E s c h e r i c h i a c o l i a n d S t a p h y -

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

s y n t h e s is w a s i n h i b it e d b y c h l o r a m p h e n i c o l . P h e n o -

l ic c o m p o u n d s p h e n o l , th y m o l , c a r v a c ro l ) a n d 2-

p h e n o x y e t h a n o l , w h i c h d e m o n s t r a t e d l e t h al p r o p e r -

t ie s a n d c a u s e d m e m b r a n e d a m a g e s s i m i l ar t o th o s e

d u e t o P E A a n d B A , w e r e n o t a f f e c t e d i n t h e i r b a c t e -

r i ci d a l a c t i v i ty b y p r o t e i n s y n t h e s i s f a i lu r e . C o n s e -

q u e n t l y , c e l l m e m b r a n e d i s o r g a n i z a t i o n , w h i c h is

c o m m o n l y r e t a in e d a s th e m e c h a n i s m o f a c t io n o f

p h e n o l s a n d a l c o h o l s , c a n n o t b e c o n s i d e r e d a s re -

s p o n s i b l e f o r t h e c e ll d e a t h i n th e c a s e o f P E A a n d

B A .

N o r e s u lt s c o n c e r n i n g t h e p e n e t r a t i o n o f P E A

a n d B A i n t o ce l ls , o r th e m e c h a n i s m o f a c ti o n o f

B A h a v e b e e n p u b l i s h e d . B u t t h e l i te r a t u r e c o n t a i n s

s o m e s t u d ie s o n t h e m e c h a n i s m o f a c t io n o f P E A a t

g r o w t h - in h i b it i n g c o n c e n t r a t i o n s . B e r r a h a n d K o -

n e t z k a [2 ] a n d P r e v o s t a n d M o s e s [1 6] o b t a i n e d d i s -

c r e p a n t r e s u lt s o n t h e e ff e c ts o f P E A o n D N A , R N A ,

a n d p r o t e i n s y n t h e s i s . T h e s t u d i e s o f T r e i c k a n d

K o n e t z k a [ 2 1] , L a r k a n d L a r k [1 2], a n d M a s k e r a n d

E b e r l e [ 15 ] d e a l t w i t h D N A r e p l i c a t io n a n d t h e i n h i-

b i t i o n o f D N A s y n t h e s i s i n i ti a ti o n . R e s i s t a n t m u -

t a n ts w e r e d e s c r i b e d b y W a d a a n d Y u r a [2 2]. T h e

m u t a t i o n s , n a m e d d n a P , w e r e l o c a t e d b e t w e e n i l e

a n d m e t E i n t h e E . c o l i c h r o m o s o m e a n d w e r e r e-

l a t e d t o D N A r e p l i c a t io n a n d c e ll d i v i s io n . N o c o n -

f i r m a t io n o f th e r o l e o f P E A i n D N A r e p l i c a t i o n h a s

b e e n p u b l i sh e d .

T h i s s t u d y is f o c u s e d o n t h e m e c h a n i s m o f ac -

t i o n o f P E A i n E . c o l i e s p e c i a l l y t h a t i m p l i c a t e d i n

t h e l e th a l e f f e c t. I t r e p o r t s t h e d e s c r i p t i o n o f m u t a n t s

r e s i s t a n t t o P E A ; t h e e f f e c t s o f in h i b i t o r y a n d l e t h a l

c o n c e n t ra t io n s o f P E A o n D N A , R N A , a n d p r o te i n

s y n t h e s i s ; a n d t h e e v a l u a t i o n o f i ts a c c u m u l a t i o n i n

t h e ce ll . F u r t h e r m o r e , t h e h y p o t h e s i s o f a n e n z y -

m a t i c c o n v e r s i o n o f P E A i n to t h e c o r r e s p o n d i n g a l-

d e h y d e w a s i n v e s ti g a t ed .

M a t e r ia l s a n d M e t h o d s

Bacteria l stra ins cul ture me dia and growth condition s. E coli

K12 CR34 F-, thr-, leu-, thi- , thy-, lacy ), coded E. coli CR34,

was from our culture collection. Brain-h eart infusion BH I,

Difco) and the corresponding BH I Agar prepared by addit ion of

15 g agar/L) were used as culture media. Cells were obtained by

inoculation 1 ml) of an overnight culture in BH I flasks 100 ml),

placed at 30~ and shaken unti l the bacterial density reached

2 • 108 -+ 1 x 108 CFU /ml optical density = 0.2 ; 623 nm).

Prototrophy was checked on C m edium [5] at 30~

Add ress reprint requests to: Dr. A. Crem ieux, Laboratoire de M icrobiologie, Facult6 de Pharmacie, 13385 Marseille Cedex 5, France.

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96

CUSRENX MICROBIOLOGY Vol. 27 (1993)

Chemicals. Reagent sources were: phenethyl alcohol (PEA),

phenylacetaldehyde, benzaldehyde, and N-methyl-N'-nitro-N-

nitrosoguanidine (NTG), from Sigma Chemical Co. ; ethyl meth-

anesulfonate (EMS), from Aldrich Chemical Co.; potassium cya-

nide, from Prolabo; dinitrophenol (DNP), trichloroacetic acid,

sodium borohydride, and chloramphenicol, from Merck; [6-

3H]thymidine (22 Ci/mmole) , [5-3H]uridine (26 Ci/mmole), L-[2,3-

3H]aspartic acid (32 Ci/mmole), and [1-14C]phenethyl alcohol (24

mCi/mmole) from C EA (Commissariat ~ l'Energie Atomique, Gif-

sur-Yvette, France). [1J4C]Phenylacetaldehyde was prepared

from [1-14C]PEA according to the method o f Marshall and Flyn n

[14].

Mutagenesis experiments. Mutagenesis was performed on cells

grown to the mid and late logarithmic stage with NTG (125 ~g/

ml) and EMS (233 tzg/ml) respectively, according to the method

of Carlton and Brown [4]. Mutagenized bacteria were first grown

to stationary phase, then harvested and dispersed in fresh medium

placed at 30~ When the exponential phase of growth was

reached , resistant mutants were s elected by plating, on BHI Agar

sup pleme nted with 0.3 and 0.5 (v/v) PEA (25 and 42 mM

respectively), conve nient dilutions of the cultures to obtain ap-

proximately 100 CFU per plate. Plates were incubated at 30~

and 42~ until colonies were about 0.5 mm in diameter .

Selected colonies were coded by a capital letter (A to J,

related to each of the ten successive experiments performed),

the referen ce of the mutagenic agent NTG (a) or EMS (b) and a

number. Fo r each selected colony, prototrophy was checked and

resistance to PEA was evaluated by g rowth inhibition and lethal

effect experiments. For more accuracy, MIC and MBC determi-

nations were followed by IC and BC calculations as described

previously [13].

Evaluation of DNA, RNA, and protein synthesis. Labeled precur-

sors (1 /~Ci/mI) were added to the BHI cultures (exponential

phase of growth, 2 x 108 _+ 1 x 108 CFU/ ml) per for med at 30~

in a shaking water bath. After 40 rain, PEA (30, 50, 75, 100

raM) was added, an d incorpora tion of the labeled precursors was

monitored in the samples (0.5 ml) that were removed every 20

rain. Cells were harvested by centrifugation (5000 g, 5 rain),

washed two times with a S6rensen phos phate buffer (pH 7.4), and

lyse d by NaOH 1 M (15 rain, 78~ Macrom olecu les precip itated

by a cold 10 (m/v) trichl oroace tic acid solution (30 rain, 0~

were transferred onto a prewetted filter (Whatman GF/C) and

washed four times with 5 ml of 5 cold trichloroacetic acid and

four times with 2 ml of ethanol. Filters were dried (1 h, 80~

under reduced pressure) and counted (Beckman LS 1701) in 5

ml scintillation fluid (Amersham). Results were e xpress ed in cpm

reported to 1 ml of the bacterial culture. Furthermore, experi-

ments were performed on cells pretreated with chloramphenicol

(20 ~g/ml) 20 rain before PEA addition (75 raM).

Accumulation experiments. The accumulation of [1-t4C]PEA (1

Ci/mole) was stud ied at 25~ acordin g to the filtration metho d of

Lacroix et al. [10]. The PEA concentration tested was 2.6 mM

(1/10 inhibitory concentration) for kinetics determination.

Detection of PEA and phenylacetaldehyde in bacterial cells by

thin layer chromatography. Bacteria (exponential growth phase,

2 x 108 + 1 • 108 CFU/ ml) wer e mixed with [1-14C]PEA (75

mM; 0.1 Ci), then incubated at 30~ Complementary experiments

were done with cells in stationary growth phase at 21~ and with

54

o

1

~ a

B

i 1 5 t i m e

i n m i n

Fig. 1. Effect of phenet hyl alcohol on viability of CR34 E coli

(A) and A27a mutant (B). PEA concentrations (raM): a, 25;

b, 50; c, 75; d, 100; e, 125; f, 150; g, 175; and h, 200. Ea ch po int

represen ts the mean of three indepen dent determinations _+

confidence limit (P = 0.95).

100 mM (0.1 Ci) of PEA. These experimental conditions were

previously shown to produce the standard lethal effect. After 5

and 30 rain of exposure, samples (0.5 ml, in duplicate) were

removed, treated or not treated for 5 min with sodium borohy-

dride (20 mg/ml), and the cells were then lysed by NaOH (1 M)

for 5 rain at 78~

Thin-layer chromatograph y was performed on 20 x 20 cm

silica gel 60 plastic-coated sheets , layer th ickness 0.2 ram, with

F 254 fluorescent indicator (Merck) by use as solvent system:

benz ene- ethy l acetate-a cetic acid (90 : 5 : 5) and a dev elopmen t

time of 1 h. Chromatograms were developed at 20~ over a dis-

tance of 15 cm in a p resaturated glass chambe r lined with filter-

paper. A Berthold LB 2723 counter was used to detect the radio-

active compounds.

R e s u l t s

I s o l a ti o n o f P E A r e s is t an t m u t a n t s . O n l y 4 o f t h e 1 0

a s s a y s p e r f or m e d w i t h N T G a n d n o n e o f t h e 1 0

a s s a y s p e r f o rm e d w i t h E M S a l l o w e d i s o l a t i o n o f

m u t a n t s . O f a b o u t 1 03 c o l o n i e s s c r e e n e d a f te r m u t a -

g e n e s i s , 3 7 w e r e s e l e c t e d o n p l a te s , i n c l u d in g P E A .

N o n e o f th e m u t a n t s w a s f o u n d t o b e t h e r m o s e n s i -

t i v e, b u t a u x o t r o p h y w a s o b s e r v e d f o r a ll o f t h e m .

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

g r o u p s a c c o r d i n g t o t h e ir a b i li t y t o g r o w i n t h e p r e s -

e n c e o f 0 .3 o r 0 .5 P E A a n d a c c o r d i n g t o t h e

m o r p h o l o g y o f c e ll s a n d c o l o n ie s . P E A r e s i st a n c e

w a s t h e n e v a l u a t e d i n g r o w t h i n h i b it i o n s t u d ie s w i t h

t h e m e t h o d s p r e v i o u s l y d e s c r i b e d [ 1 3 ]. T h e f ir s t

t h r e e g r o u p s f a i le d t o d e m o n s t r a t e a n y r e s i s t a n c e

t o P E A . T h e f o u r th g ro u p , c o m p o s e d o f 5 m u t a n t s

( A 2 7 a , A 3 1 a , A 3 3 a , A 3 7 a , D 6 6 a ) , e x h i b i t e d a r e a l

r e s i s t a n c e t o P E A , a s d e m o n s t r a t e d b y t e s t i n g b a c t e -

r i o s t a t i c ( M I C = 5 0 m M ; 4 7 . 8 --< I C m M ----- 5 3 . 1 ) a s

w e l l a s b a c t e r i c i d a l a c t i v i t y M B C = 2 0 0 m M ;

1 7 7 .5 --< B C m M - - 1 9 1 .4 ) . C o r r e s p o n d i n g v a l u e s f o r

t h e E coli C R 3 4 p a r e n t a l s t r a in w e r e M I C = 3 0

( I C = 2 7 . 4 ) a n d M B C = 1 0 0 ( B C = 9 5 . 9 ) .

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J.J. Lucchini et al.: Mechanism of Bactericidal Action of PEA 29 7

E

o

v

~ 1 6

~ 8

~ 4

E

o

a

e t i m L - - ~

~ o ~ 0 ~ o 0 ~ o m i

E

o

~

e e m

i i m m e i n ~

2 0 t 6 0 1 0 0 ~ n

m e i n

Fig. 2. Effect of phenethyl alco-

hol on thymidine A), uridine

B), and L-aspartic acid C) incor-

poration in growing cells of

CR34 E c o l l a: control; b, c, d,

e: assays performed in presence

of PEA mM) 30 MIC), 50, 75

MBC), 100 respectively. PEA

was added after 40 min arrow).

Each point represents the mean

of three inde pendent determina-

tions -+ conf iden ce limit P =

0.95).

Time killing curves obtained with E coli CR34

and the A27a mutant are shown in Fig. 1 data not

reported fo r the other mutants). The general features

of these curves were identical for the parental strain

and all mutants. Whatever the PEA concentration,

the 5 log reduction o f the number of viable cells was

reached within 5 min and was not strongly increased

by a prolonged time of exposure.

The BC/IC ratios for the E coli CR34 parental

strain and mutants A27a, A31a, A33a, A37a, and

D66a were 3.90, 3.90, 3.90, 3.79, 3.60, and 3.70,

respectively. This observation suggested that the

resistance found was not linked to a biochemical

mechanism of action and was probably owing to

a change in membrane permeation to PEA. This

hypothesis was verified in results reported further.

All mutants d isplayed a similar profile in the analysis

of the cell fatty acid conte nt, which differed mark-

edly from that of the parental strain data not

shown). This observation suggested that all the mu-

tants were identical, in agreement with the IC and

BC values.

PE A ef fec ts on D N A R N A and prote in synthes is .

The kinetics of incorporation o f labeled precursors

in the presence of increasing concentrations of PEA

are presented in Fig. 2. In CR34 cells, a concentra-

tion of 30 mM PEA inhibitory for growth) was suffi-

cient to cause an almost immediate cessation of

RNA and protein synthesis. At this concentration,

DNA synthesis was lowered but not stopped. A con-

centration of 50 mM blocked DNA synthesis immedi-

ately. Higher and bactericidal concentratio ns 75,

100 mM) of PEA caused a dec rease in the incorpora-

tion rate of precursors involved in RNA and protein

synthesis as well as in DNA synthesis. In CR34

cells pret reat ed with chloramphen icol, PEA 75 mM)

v

E

o

a

b o~

c

2 0 ~ 6 0 1 0 0 t i m e L ~

i n m i n

r

b

d

c

9 - , , b ,

2 0 ~ 6 0 i 0 0 t i m e

i n m i n

Fig. 3. Effect of phenethyl alcohol 75 mM) on thymidine A) and

uridine B) incor porat ion in CR34

E c o l i

cells pretreated with

chloramphenicol, a: control; b: chloramphen icol 20/~g/ml) added

at 40 min double arrow); c: PEA 75 mM) add ed in cont rol cells

at 40 min; d: PEA 75 mM) adde d in cells treate d with chloram -

phenicol at 60 rain. E ach point rep resent s the mean of three

inde pend ent deter minat ions -+ confi dence limit P = 0.95).

stopped RNA synthesis and reduced DNA synthe-

sis, as was ob served with 30 mM PEA in untre ated

cells Fig. 3).

Similar results were ob tained with the five mu-

tants not shown) when comparing data obtained

with the corresponding MIC and MBC of PEA.

Transport of [1-14C]PEA into bacteria. The kinetics

of PEA accumulation are shown in Fig. 4. For E.

coli CR34, the accumula tion reached a maxim um at

25~ 4 min after add ition of [1-14C]PEA. With 2.6

mM external con centration , the maximal intracellu-

lar level was 12 pmol [1-~4C]PEA/108 bac ter ia. The

intracellular PEA conce ntration was abo ut 1.5-fold

higher than the extracellular concentration, These

values were calculated on the assumption of an E.

coli cell volume of about 1.76 • 10 -~2 ml [6]. For

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9 8

CURRENT MICROBIOLOGY VOI 27 1 9 9 3 )

4 8 6 33

time

iz min

F i g . 4. A c c u m u l a t i o n o f P E A b y C R 3 4 E coli

a ) a n d t h e r e s i s t a n t A 2 7 a m u t a n t b ) .

[ 1 -1 4 C ]P E A 2 . 6 m M ) w a s a d d e d t o t h e c e ll s u s -

p e n s i o n 1 08 C F U / m l ) a t 2 5 ~ E a c h p o i n t r e p -

r e s e n t s t h e m e a n o f th r e e i n d e p e n d e n t d e t e r m i -

n a t i o n s + c o n f i d e n c e l im i t P = 0 . 9 5 ).

T a b l e 1 . P E A a c c u m u l a t i o n in C R 3 4 E coil a n d i n a 2 7 a

m u t a n t

A c c u m u l a t i o n a p m o l /1 0 8 b a c t e r i a )

C R 3 4 A 2 7 a

I n h i b i t o r b 1 r a i n 3 2 r a i n 1 r a i n 3 2 r a i n

N o n e 8 . 7 -+ 0 . 7 1 1 . 5 _ + 0 . 4 0 . 2 + 1 . 0 9 . 2 - + 0 . 6

K C N 1 0 m M ) 7 . 2 • 0 . 3 9 . 1 + 0 . 3 1 . 5 + 1 . 2 8 . 3 -+ 0 . 9

D N P 5 m M ) 8 . 2 -+ 0 . 4 1 0 . 4 _ + 0 . 5 2 . 5 • 1. 1 8 . 9 • 0 . 6

M e a n v a l u e o f t h r e e d e t e r m i n a t i o n s _+ t h e c o n f i d e n c e l i m i t

P = 0 . 9 5 ) .

b I n h i b i t o r a d d e d 5 m i n b e f o r e P E A 2 . 6 r aM ) .

mutant A27a, accumulation of [1-14C]PEA reached

an identical maximum, bu t only after 32 min. At this

time, the intracellular PEA concentration was also

about 1.5-fold higher than the extracellular one.

Neither electron transport inhibitor KCN) nor

agents that uncou ple oxidative phosphorylation and

electron transport 2,4-dinitrophenol) were able to

inhibit [1-14C]PEA accumulation, or to change the

transport kinetics. Identical results were obtained

in bacter ia treated successively Table 1) or simulta-

neously by the inhibitor and PEA. These results

suggested that PEA accumulation by

E coli

is not

energy dependent.

Format ion o f phenylacetaldehyde in E coi l cells

On

thin-layer chromatogr ams, spots o f PEA and phenyl-

acetaldehyde were located at Rf 0.26 and 0.76 re-

spectively, as described in the literature for the sol-

vent system used [3].

Assays were performed with both stationary and

exponential growth phase ceils Fig. 5) and gave

similar results. In cells treated by PEA for 5 and 30

min, three radioacti ve com pou nds Rf 0.02, 0.25,

and 0.76) were detected. The first spot was located

close to the starting point. The th ird spot Rf 0.76),

corresponding to phenylacetaldehyde, was more

consi stent in cells exposed for 30 min to PEA Fig.

5-A,B). This spot disappeared in samples treated

with sodium borohydride, which stabilizes the Schiff

base formed from proteins and aldehydes, leading

the first spot Rf 0.02) to increase st rongly. In cells

exposed 30 min to PEA but pret reat ed 10 min be-

fore) with chloramphenicol, only two spots were

detected: Rf 0.02 and 0.26 Fig. 5-D).

Similar results were obtained with mutant A27a

data not shown) except that phenylacetaldehyde

formation was delayed.

D i s c u s s i o n

Since a specific mechanism of action of PEA, one

dependent on an efficient protein synthesis, might

be suspected, our first aim was to obtain resistant

mutants. Such mutants have been described by

Wada and Yura [22]. Their level of resistance was

very low MIC of the mutants, 21-23 mM; MIC of

wild strain 20 mN). Un fortuna tely, the muta nts were

lost, and no further results were reported on the

mutation, named dna P, which was related to DNA

replication and cell division. We obtained five mu-

tants demonstrating a strong level of resistance to

PEA. Their behavior in antimicrobial activity test-

ing, as well as in other studies, suggested that the

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J . J . Lu c c h i n i e t a l .: M e c h a n i sm o f B a c t e ri c i da l A c t i o n o f PEA 2 9 9

A

r

D

. . . . . . . . 8 ; 2 1 ~ ~ 2 ~

4 [ c , ]

F i g . 5 . P h e n y l a c e t a l d e h y d e f o r m a t i o n i n C R 3 4 E coli c e l l s e x p o -

n e n t i a l g r o w t h p h a s e ) t r e a t e d w i th [ I J a C ] P E A 7 5 m M ) . C h r o m a -

t o g r a m s : A ) , ce l l s t r e a t e d f o r 5 m i n w i t h P E A ; B ) , ce l ls tr e a t e d

f o r 3 0 m i n w i t h P E A ; C ) , c e ll s t r e a t e d f o r 3 0 m i n w i t h P E A ,

t h e n w i t h s o d iu m b o r o h y d r i d e 2 0 m g / m l ) ; D ) , c e l ls p r e t r e a t e d

1 0 m i n w i t h c h l o r a m p h e n i c o l 2 0 tx g /m l ) b e f o r e a d d i ti o n o f P E A

f o r 3 0 m i n .

mutations were related to drug penetration. Conse-

quently they could not be retained for determination

of a specific mechanism o f action.

Several studies have dealt with the effects of

PEA on DNA, RNA, and protein synthesis. Most

of them were performe d with subinhibitory concen-

trations of PEA. Our results PEA concentration =

MIC) agree with those published by Prevost and

Moses [16] and late r by Masker and Eberle [15]:

RNA and protein syntheses were immediately

stopped, the DNA synthesis being only lowered.

The rupture of the DNA -membr ane association was

hypot hesize d by Ma sker and Eberle [15], but Brun-

ner and Treick [1] demonstrated that c oncentrations

over 50 mu were needed to provoke such effects.

Rosenk ranz et al. [17, 18] demonstrated that mRNAs

were not conc erned with PEA action. Other studies

reported the effects of PEA on the inhibition of

T a b l e 2 . A n t i b a c t e r i a l a c t i v i t y o f p h e n e t h y l a n d b e n z y l

a l c o h o l s a n d o f t h e c o r r e s p o n d i n g a l d e h y d e s o n E coli

G r o w t h

i n h i b i t i o n L e t h a l e f f e c t

M I C I C M B C B C

ra M ) mM ) mM ) mM )

PE A 3 0 2 7 .4 1 0 0 9 5 .9

Ph e n y l a c e t a l d e h y d e 1 1 .6 2 0 1 8 .6

B A 4 0 4 2 3 0 0 2 6 8

B e n z a c e t a l d e h y d e 1 0 8 . 9 6 0 4 7 .1

phage replication [9], on sporulation, and on spore

germination [19]. More recently, Tachibana and Yo-

nei [20] published on t he inhibition of excision repai r

in cells treated by PEA.

At lethal concentrat ions 75 mM for the parental

strain, 150 mM for the mutants), our results showed

the immediate decrease in precursors incorporated

during DNA, RNA, an d protein synthesis . This was

consistent with a damaged bacterial membrane and

with macromolecule leakage [7, 15].

In this work we confirm, by biochemical studies,

our previous results obtained from bacterial counts

showing that PEA lethal effect can be suppressed

when protein synthesis has been inhibited [13]. In

cells pretreated by chloramphenicol, DNA and RNA

syntheses were not inhibited by PEA, even when

the normal rate of their synthesis was not comp letely

restored. The protein involved in the mechanism of

bactericidal action of PEA could be a membrane

carrier needed for PEA accumulation, an enzyme

able to transform PEA into an active compound, or

any protein leading to cell death by an unknown

phenomenon.

Because of the common behavior of Gram-

negative and Gram-positive bacteria treated with

PEA [7], the first hypothesis appears improbable.

Results on PEA accumulation in E coli demon-

strated that the pheno menon was not energy depen-

dent. As suggested by Lang and Rye [11], PEA

would enter the cell through the lipidic bilayer of

the membrane or through transmembrane channels.

Since the need for protein synthesis was demon-

strated by PEA and BA, but not by oth er bactericidal

compounds phenoxyethanol and phenols), we have

hypothesized that PEA and BA might be converted

into their corresponding aldehyd e by an alcohol de-

hydrogenas e. The lethal properties o f phenylace tal-

dehyde and benzaldehyde were shown at concentra-

tions one-fifth those of PEA and BA Table 2). Thin-

8/11/2019 Bf 01575995


3 CURREN T MICROBIOLOGYVO1. 27 1993)

l a y e r c h r o m a t o g r a p h y s t u d i e s c o n f i r m e d t h i s h y -

p o t h e s i s f o r P E A , b y d e m o n s t r a t in g t h e p r e se n c e o f

a c o m p o u n d i d en t if ie d a s p h e n y l a c e t a l d e h y d e i n E .

coli c e l l s , b e c a u s e 1 ) t h e R f v a l u e o f t h e n e w s p o t

f o u n d w a s t h a t e x p e c t e d a n d 2 ) t h is s p o t w a s n o t

d e t e c t e d i n e x t r a c t s t r e a te d b y s o d i u m b o r o h y d r i d e ,

w h i c h s t a b i l i z e s t h e S c h i f f b a s e f o r m e d f r o m p r o -

t e i n s a n d a ld e h y d e s . B e s i d e s , i n c e l ls p r e t r ea t e d w i t h

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

C o n s e q u e n t l y , i t m a y b e p r o p o s e d t h at th e m e c h a -

n i s m o f b a c t e r ic i d a l a c t i o n o f P E A i n v o l v e s a n o n -

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

a l c o h o l d e h y d r o g e n a s e , f o l l o w e d b y t h e f o rm a t i o n

o f p h e n y l a c e t a l d e h y d e . I n t ra c e l lu l a r a n d n o n s p e c i f i c

d e n a t u r a t i o n o f p r o t e in s w o u l d p r o b a b l y o c c u r ,

c a u s i n g c e l l d e a t h .

E coli

m u t a n t s l a c k i n g a l c o h o l d e h y d r o g e n a s e

h a v e b e e n d e s c r i b e d i n th e li t e r a tu r e [ 8 , 2 3 ] . S t u d y -

i n g t h e l e th a l a c t i v it y o f P E A a n d B A o n s u c h s t r a i n s

w o u l d b e a c o n v e n i e n t w a y t o d e m o n s t r a te t h e r o le

o f a l c o h o l d e h y d r o g e n a s e i n t h e b a c te r i ci d a l m e c h a -

n i s m o f a c ti o n o f t h e s e a l c o h o l s. H o w e v e r , s in c e

a l c o h o l d e h y d r o g e n a s e i s e x p r e s s e d m o r e i n

E coli

c e l l s g r o w t n a n a e r o b i c a l l y [ 8 ] , t h e e f f e c t o f a e r o b i c

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

i n t e s t i n g t h e a n t i b a c te r i a l p r o p e r t i e s o f P E A a n d

B A s h o u l d b e d e t e r m i n e d .

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