Influence of prior growth conditions on low nutrient response of Escherichia coli

in seawater
MICHELJ. GAUTHIER,'
PATRICKM. MUNRO,AND VIOLETTE
A. BREITTMAYER
Institut national de la santk et de la recherche rne'dicale, Unite' 303, 1 avenue Jean Lorrain, F06300 Nice, France
Received April 12, 1988

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 05/10/13

For personal use only.

Accepted November 24, 1988

V. A. 1989. Influence of prior growth conditions on low nutrient
GAUTHIER,M. J., MUNRO,.'F M., and BREITTMAYER,
response of Escherichia coli in seawater. Can. J. Microbiol. 35: 379-383.
By use of experimental microcosms, it was demonstrated that the survival of Escherichia coli in nutrient-free seawater
depended on the age of cells and on some physicochemical conditions during their prior growth. Cells grown in a bacteriological medium, with an acid or an alkaline pH, at high temperature (44"C), or in the absence of oxygen were more sensitive
to exposure to seawater of low nutrient content. In contrast, some complex media allowed production of cells adapting more
rapidly to seawater. Cells grown in urine were far more sensitive than those grown in all bacteriological media tested. The
sensitivity of all cells was highest when they were harvested during the early exponential phase of growth.
Key words: Escherichia coli, seawater, growth, survival.

GAUTHIER,M. J., MUNRO,P. M., et BREITTMAYER,

V. A. 1989. Influence of prior growth conditions on low nutrient
response of Escherichia coli in seawater. Can. J. Microbiol. 35 : 379-383.
En utilisant des microcosmes expkrimentaux, il a CtC dCmontrC que la survie d'Escherichia coli dans de l'eau de mer
exempte de matibres nutritives dCpendait de l'lge des cellules et de certaines conditions physicochimiques prkvalant lors de
la croissance antkrieure des bactCries. Les cellules cultivCes dans un milieu bactCriologique, a pH acide ou basique, B une
tempkrature Clevte (44"C), ou en absence d'oxygbne Ctaient plus sensibles B l'eau de mer contenant peu de matibres nutritive~.Par contre, certains milieux de culture complexes permettaient une production de populations bactkriennes qui s'adaptaient plus rapidement l'eau de mer. Les bactCries cultivCes dans de l'urine Ctaient beaucoup plus sensibles que les bactCries
recueillies de n'importe lequel des milieux bacttriologiques utilisCs. Peu importe les conditions de croissance, la sensibilitC
bactkrienne Ctait plus grande lorsque les cellules Ctaient rCcoltCes au dCbut de la phase exponentielle de croissance.
Mots clds : Escherichia coli, eau de mer, croissance, survie.
[Traduit par la revue]

Introduction
There have been many studies on the decay of Escherichia
coli and other indicators of faecal pollution in marine environments, both in situ (Faust et al. 1975; Vasconcelos and Swartz
1976; Chamberlin and Mitchell 1978; Dawe and Penrose
1978; Hood and Ness 1982; Anderson et al. 1983; Lessard
and Sieburth 1983; Rhodes et al. 1983) and in laboratory
experiments (Anderson et al. 1979; Xu et al. 1982; Guthrie
and Scovill 1984; Roszak et al. 1984).
In vitro analysis of starvation survival in seawater generally
involves the use of microcosms, i.e., samples of seawater
inoculated with cells previously grown in a bacteriological
medium and which are washed exhaustively to eliminate any
traces of extraneous organic and inorganic matter. Recently
published data showed that the ability of E. coli to survive in
seawater depends on the composition of the medium used for
its previous growth, and especially on the presence of salts in
this medium (Gauthier et al. 1987; Munro et al. 1987). The
aim of this work was to estimate the influence of several
physicochemical parameters prevailing during the previous
growth of cells such as composition, pH, and NaCl content of
the medium, incubation temperature, and absence of oxygen,
on the starvation survival of E. coli in seawater microcosms.
Materials and methods
Bacterial strain
All tests were performed with Escherichia coli 12, a nontypable
enterotoxigenic E. coli that produces labile enterotoxin (strain LT+)
and was isolated from human stools in Bangladesh. This strain was
'Author to whom correspondence should be addressed.
Printed in Canada 1 Imprim6 au Canada

kindly provided by Dr. J. M. Scheftel, FacultC de MCdecine, Strasbourg, France.

Growth conditions
Stock cultures were maintained in liquid nitrogen. The survival of
strain LTf in seawater was analysed with cells grown at 37C in 6
different liquid bacteriological media: (i) nutrient broth (NB, Difco
Laboratories, Detroit, MI); (ii) tlyptic soy broth (TSB, Difco);
(iii) Mueller-Hinton broth (MHB, Bio-MCrieux, Marcy I'Etoile,
France); (iv) colonization factor antigen broth (CFAB, Evans et al.
1977); (v) mineral medium M 9 supplemented with glucose (2 g/L,
Maniatis et al. 1982); and (vi) m-FC broth (m-FCB) (Difco). It was
also analysed with cells grown on urine, freshly emitted by a healthy
male individual and filter sterilized (membrane pore size 0.22 pm,
Millipore Corp., Bedford, MA). Sterility of filtered urine was
checked by incubating an aliquot in MHB for 48 h at 37 and 30C.
The influence on survival of pH (6, 7, and 8), NaCl concentration
(0, 5, 10, 20, or 30 g/L), and incubation temperature (25, 37, and
44C) with or without oxygen (aerobiosis, anaerobiosis) was determined with cells grown oi TSB only. Growth under anaerobic conditions was carried out using the Gas Pak anaerobic system (BBL,
Cockeysville, MD). Whatever the culture conditions, cells were harvested at the end of the exponential phase of growth (5 -6 h at 37 "C).
Survival of strain LTf was also studied with cells grown for 3 , 4 , 5,
6, and 25 h a t 37OC in NB.
Microcosms
Microcosms consisted of 500-rnL Erlenmeyer flasks containing
200 mL of natural, filtered seawater (pore size 0.22 pm) freshly collected (pH 8.1; salinity, 37.2%,,). Each was inoculated with cells previously grown as described above and washed three times by
centrifugation (10 000 g, 4"C, 15 min) in artificial seawater (ASW;
Lyman and Fleming 1940). Microcosms were finally inoculated with
the resulting suspension in ASW to approximately 2 x lo6 cfu/mL.
They were then gently stirred in the dark at room temperature (23 to
26C).

CAN. 1. MICROBIOL.

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 05/10/13

For personal use only.

VOL. 35,

1989

- .
0 1 2 3 4 5 6 7
Hours

25

3
4
Days

FIG. 1. Survival in seawater of E. coli previously grown in nutrient broth for different periods at 37C. (A) Growth curve in NB; arrou
indicate the time of harvest of cells. (B) Survival of E. coli cells in seawater after growth in NB for 3, 4, 5 , 6, and 25 h, as indicated in pare]
theses.

0 1

5
Days

AODC 44C
wCFU
37C
MCFU
25C
u C F U 44C

CFU pH6
H CFU pH7
CFU p ~ 8
10

0 1

5
Days

10

FIG.2. Comparative survival in seawater of E. coli cells previously

grown in tryptic soy broth for 5 -6 h at different pH. AODC values
were identical at all pH values studied.

FIG. 3. Comparative survival in seawater of E. coli cells previous1

grown in tryptic soy broth for 5-6 h at different temperature:
AODC values were identical at all temperatures studied.

Enumeration
Culturable cells (cfu) were counted in triplicate by the membrane
filtration technique (Millipore filters, 0.45 pm pore size) on nutrient
agar (Difco). Dilutions were prepared with ASW. Plates were
incubated for 2 d at 37C before cfu were enumerated. In some
experiments, the total bacterial number was determined by the acridine orange direct count method (AODC) (Hobbie et al. 1977) and
the total direct viable cell count (DVC) by the method of Kogure
et al. (1979) modified by Quinn (1984).
The decay rate k (per day) of bacteria in different microcosms was
calculated according to the equation k = (log No - log N,) / t ,
where No is the initial concentration of bacteria (cfu/mL), N, is the
number of cfu/mL of microcosm seawater after 4 days, and t equals
4 days (Chamberlin and Mitchell 1978).

Reproducibility of results
All experiments were performed in duplicate, and the reported da
are means of replicate samples. Significance of observed differencr
was tested by an analysis of variance (Schwartz 1980).

Results
The survival of E. coli 12 in nutrient-free seawater
depended on the age of cells when they were harvested and
released in seawater. Their resistance increased rapidly with
preincubation time and was maximal during the late stationary
phase (25 h at 37OC; Fig. 1). When cells harvested at the same
period of growth (5-6 h at 37C) were tested, survival also
depended on physicochemical conditions used for their cul-

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 05/10/13

For personal use only.

GAUTHIER ET AL.

MAODC

AERO
M CFU 0%.

-1

0 1

5
Days

FIG. 4. Survival in seawater of E. coli cells previously grown in

tryptic soy broth for 5-6 h in the presence (AERO) or absence
(ANAERO) of oxygen. AODC values were identical under both conditions.

tivation. It was significantly lowered when they were grown

in a medium with an acid or alkaline pH ( a = 0.01 ; Fig. 2),
at high temperature (44C; a = 0.001; Fig. 3), or in the
absence of oxygen (cr = 0.0001 ; Fig. 4). The effect of sodium
chloride was more complex (Fig. 5). Although cell morphology remained unchanged whatever the salt concentration their
ability to grow on a bacteriological medium, as observed by
the AODC method, was lowered ( a = 0.05) when they were
previously grown in a medium with an intermediate salt concentration (10 g NaClIL) even though a protective effect was
observed after growth on a medium with a higher salt content
(30 g NaClIL) ( a = 0.05).
The composition of the medium also influenced the growth
of cells in seawater microcosms (Fig. 6), but it affected only
the final concentration of culturable cells after 10 days in seawater, and not the initial decay rate which was constant (k =
1.1 d-') for cells grown in the 6 different media. Among
complex bacteriological media, CFAB and MHB gave rise to
more resistant cells. Those grown in the mineral base supplemented with glucose as sole source of carbon (medium M 9)
were significantly ( a = 0.05) less resistant than cells grown
in CFAB or MHB but were more resistant than those produced
in NB, TSB, and m-FCB. The most sensitive cells (k =
2.8 d-l) were obtained from urine, and the incubation of this
medium under anaerobic conditions still further increased
their sensitivity to seawater (k = 4.15 d-I; Fig. 7). The
simultaneous analysis of decrease in cfu, AODC, and DVC of
cells previously
anaerobically in urine ("worst" condition) and aerobically in CFAB (pH 7, 37C; "best" condition;
Fig. 8) showed that the number of viable cells remained very
high (at least half of the initial population) in both cases during
the first 4 d in seawater, then decreased more rapidly for cells
grown in urine. Variations of cfu were identical to those previously described (Figs. 6 and 7).

Discussion
Most of the studies concerning the fate of enteric bacteria in

. .

0 1

10

. .

5
Days

10

FIG. 5. Comparative survival in seawater of E. coli cells previously

grown for 5-6 h in tryptic soy broth supplemented with various
amounts of sodium chloride (%, parts per thousand). AODC values
were identical under all conditions studied.

.
7

10

Days
FIG. 6. Comparative survival in seawater of E. coli cells previously
grown for 5-6 h in different complex or minimal media. CFA,
colonization factor antigen broth; MHB, Mueller -Hinton broth; NB,
nutrient broth; TSB, tryptic soy broth; M9, minimal medium supplemented with glucose; m-FCB, specific broth for enumeration of
coliforms.

seawater carried out in laboratory microcosms or in field

experiments with diffusion chambers (Vasconcelos and Swartz
1976; Anderson et al. 1983; Lessard and Sieburth 1983) were
developed to analyse the influence of environmental physicochemical factors on their decline in culturability. Recently,
however, experimental observations have emphasized the posin seawater. Such studies
sible adaptation of E. coli to growth
have shown an increase in E. coli survival after previous
growth in a seawater medium (Gauthier et al. 1987; Munro
et al. 1987). In this connection, results reported here suggest

CAN. J. MICROBI(3L. VOL. 35, 1989

382

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 05/10/13

For personal use only.

WC

U AERO
FUANAERO

A A DVC
.oCFU

. .

>

10

Days
FIG.7. Survival in seawater of E. coli cells previously grown in
urine in the presence (AERO) or the absence (ANAERO) of oxygen.

that the ability of E. coli cells to grow in seawater more generally depends on how and how long they are grown before their
incubation into seawater. The increased resistance of cells
during the second half of the exponential phase of growth and
the stationary phase could result from the progressive intracellular accumulation of organic or inorganic substances
subsequently used as osmoprotectants. Many other structural
and (or) metabolic changes could, however, account for this
evolution since the chemical composition of E. coli cells is
largely modulated by growth rate (Bremer and Dennis 1987).
Other parameters such as high temperature and the absence
of oxygen during prior growth in a complex bacteriological
medium were the most influential environmental factors and
increased the sensitivity of cells. This could result from the
addition of salinity and low nutrient effects and, possibly, the
subsequent production of cells with a modified structure and
a lower energy charge. At high temperature, the fatty acid
composition of E. coli membrane lipids changes markedly,
with a decrease of the unsaturated fraction, thus maintaining
the fluidity of membranes at an optimal level (Ingraham 1987).
Amounts and (or) activity of proteins and (or) enzymes are
also adjusted, and some specific heat-shock proteins are produced (Ingraham 1987). On the other hand, it is a well-known
fact that ATP generation is considerably lowered under
anaerobic conditions (Moat 1979). In the same way, differences in the ability of cells to grow in seawater after growth
in different bacteriological media could be explained by (i) the
variable intracellular accumulation of reserve energy materials
(Kjelleberg et al. 1987), (ii) the accumulation of different substrates favouring the setting and functioning of osmoregulation
processes (Tempest et al. 1970; Brown 1974; Measures 1975;
Roller and Anagnostopoulos 1982), and (or) (iii) the presence
or absence of cell constituents such as binding proteins
(Weiner and Heppel 1971; Ames and Lever 1972; Rosen
1973; Rosen and Heppel 1973) which could allow or facilitate
substrate capture and subsequent adaptation to seawater.
Whatever the mechanism, the most striking fact was that
growth on different media led to the production of bacteria
with the same initial rate of decline in cfu but with significantly different numbers of cells able to grow in seawater.

-1
0 1 2 3 4 5 6 7 8 9 1 0

Days
FIG. 8. Survival in seawater of E. coli cells previously grown
anaerobically in urine (closed symbols) or aerobically in CFAB (open
symbols), and analysed by AODC, DVC, and cfu counting methods.

This situation seems rather similar to that of marine bacteria

exposed to oligotrophic oceanic environments, for which it has
been assumed that only a small part of the initial population
can survive and adapt, depending on available nutrients. Then,
as stated by Morita (1982), "the question still remains as to
what dictates which few cells will survive . . . Is there a random or genetic selection of cells that will survive?"
The protective influence of sodium chloride, even at rather
high concentration (30 g/L) seemed less important than that of
natural seawater (Gauthier et al. 1987). Furthermore, there
was probably a threshold concentration below which sodium
chloride exerted an unfavourable effect on the following
starvation survival of cells.
Growth of E. coli under conditions closely related to those
found in vivo, namely anaerobic growth in urine, led to the
production of cells far more sensitive to seawater than those
grown in bacteriological media. In both cases, however, an
important fraction of cells remained viable in seawater at least
during 7 days. This observation shows that the rapidity of processes responsible for the evolution toward the unculturable
stage probably depends on the structural and metabolic state
of cells before their release in seawater. Although additional
studies are necessary to confirm such hypersensitivity of
in vivo grown cells, this observation suggests that enteric bacteria grown under laboratory conditions, e.g., aerobically in
complex media, could sufficiently differ from those grown in
the mammalian intestine or urinary tract to preclude their use
for laboratory tests aiming to analyse their ability to grow and
reproduce in marine environments.

Acknowledgements
We would like to thank Dr. R. Colwell for helpful discussions on this work and correction of the manuscript. We also
thank B. Chabanne, H. Olagnero, and C. Minghelli (Service
de photographie de 1'Institut national de la sant6 et de la
recherche mkdicale, ADR-PACA, Nice) for their technical
assistance. This work was partly supported by grant Fral6(K)
from the World Health Organization, Geneva.

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 05/10/13

For personal use only.

GAUTHIER ET

AMES,G. F., and LEVER,J. 1972. The histidine-binding protein J is a

component of histidine transport. J. Biol. Chem. 247: 4309-4316.
ANDERSON,
I. C., RHODES,M. W., and KATOR,H. I. 1979. Sublethal
stress in Escherichia coli: a function of salinity. Appl. Environ.
Microbiol. 38: 1147- 1152.
1983. Seasonal variation in survival of Escherichia coli
exposed to "in situ" membrane diffusion chambers containing
filtered and nonfiltered estuarine waters. Appl. Environ.
Microbiol. 45: 1877 - 1883.
BREMER,H., and DENNIS,P. P. 1987. Modulation of chemical composition and other parameters of the cell by growth rate. In
Escherichia coli and Salmonella typhimunum. Cellular and
molecular biology. Edited by F. C. Neidhardt, J. Ingraham, K. B.
Low, B. Magasanik, M. Schaechter, and H. E. Umbarger. American Society for Microbiology, Washington, D.C. pp. 1527 - 1542.
BROWN,A. D. 1974. Microbial water relations: features of the intracellular composition of sugar-tolerant yeasts. J. Bacteriol. 118:
769 -777.
CHAMBERLIN,
C. E., and MITCHELL,R. 1978. A decay model for
enteric bacteria in natural waters. In Water pollution microbiology.
Edited by R. Mitchell. John Wiley & Sons, New York. pp.
325 - 348.
DAWE,L. L., and PENROSE,W. R. 1978. "Bactericidal" property of
seawater: death or debilitation? Appl. Environ. Microbiol. 35:
829-833.
EVANS,D. G., EVANS,D. J., and ~ O AW., 1977. Hemagglutination
of human group A erythrocytes by enterotoxigenic Escherichia coli
isolated from adults with diarrhea: correlation with colonization
factor. Infect. Immun. 18: 330- 337.
FAUST,M. A,, AOTAKY,
A. E., and HARGADON,
M. L. 1975. Effect
of physical parameters on the "in situ" survival of Escherichia coli
MC-6 in an estuarine environment. Appl. Microbiol. 30:
800 -806.
GAUTHIER,
M. J., MUNRO,P. M., and MOHAJER,S. 1987. Influence
of salts and sodium chloride on the recovery of Escherichia coli
from seawater. Curr. Microbiol. 15: 5 - 10.
GUTHRIE,
R. K., and SCOVILL,
M. A. 1984. Recovery of Escherichia
coli and Vibrio cholerae from aquatic microcosms. Water Res. 18:
1055 - 1057.
HOBBIE,J. E., DALEY,R. J., and JASPER,S. 1977. Use of nucleopore
filters for counting bacteria by fluorescence microscopy. Appl.
Environ. Microbiol. 33: 1225 - 1228.
HOOD,M. A., and NESS,G. E. 1982. Survival of Vibrio cholerae and
Escherichia coli in estuarine waters and sediments. Appl. Environ.
Microbiol. 43: 578-584.
INGRAHAM,
J. 1987. Effect of temperature, pH, water activity, and
pressure on growth. In Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. Edited by F. C. Neidhardt,
J. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E.
Umbarger. American Society for Microbiology, Washington,
D.C. pp. 1543- 1554.
KJELLEBERG,
S., HERMANSON,
M., and MARDEN,
P. 1987. The transient phase between growth and nongrowth of heterotrophic bacteria with emphasis of the marine environment. Annu. Rev.
Microbiol. 41: 25 -49.
KOGURE,K., SIMIDU,U., and TAGA,N. 1979. A tentative direct
microscopic method for counting living marine bacteria. Can. J.

AL.

383

Microbiol. 25: 415 -420.

LESSARD,
E. J., and SIEBURTH,
J. M. 1983. Survival of natural sewage
population of enteric bacteria in diffusion and batch chambers in
the marine environment. Appl. Environ. Microbiol. 45: 950 -959.
LYMAN,J., and FLEMING,R. H. 1940. Composition of seawater. J.
Mar. Res. 3: 134- 146.
MANIATIS,T., FRITSCH,E. F., and SAMBROOK,
J. 1982. Molecular
cloning. A laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor. p. 68.
MEASURES,
J. C. 1975. Role of amino acids in osmoregulation of nonhalophylic bacteria. Nature (London), 257: 398-400.
MOAT,A. G. 1979. Microbial physiology. John Wiley & Sons, New
York. pp. 163-173.
MORITA,R. Y. 1982. Starvation-survival of heterotrophs in the
marine environment. In Advances in microbial ecology. Vol. 6.
Edited by K. C. Marshall. Plenum Press, New York. pp.
171 - 198.
MUNRO,P. M., GAUTHIER,M. J., and LAUMOND,F. M. 1987.
Changes in Escherichia coli starved in seawater or grown in
seawater-wastewater mixtures. Appl. Environ. Microbiol. 53:
1476- 1481.
QUINN,J. P. 1984. The modification and evaluation of some cytochemical techniques for the enumeration of metabolically active
heterotrophic bacteria in aquatic environment. J. Appl. Bacteriol.
57: 51-57.
RHODES,M. W., ANDERSON,
I. C., and KATOR,H. I. 1983. "In situ"
development of sublethal stress in Escherichia coli: effects on
enumeration. Appl. Environ. Microbiol. 45: 1870- 1876.
ROLLER,S. D., and ANAGNOSTOPOULO~,
G. D. 1982. Accumulation
of carbohydrates by Escherichia coli Br/r/l during growth at low
water activity. J. Appl. Bacteriol. 52: 425 -434.
ROSEN,B. P. 1973. Basic amino-acid transport in Escherichia coli 11.
Purification and properties of an arginine-binding protein. J. Biol.
Chem. 248: 1211 -1218.
ROSEN,B. P., and HEPPEL,L. A. 1973. Present status of binding proteins that are released from Gram-negative bacteria by osmotic
shock. In Bacterial membranes and walls. Edited by L. Lieve.
Marcel Dekker, Inc., New York. pp. 209-239.
ROSZAK,D. B., GRIMES,D. J., and COLWELL,
R. R. 1984. Viable but
nonrecoverable stage of Salmonella enteritidis in aquatic systems.
Can. J. Microbiol. 30: 334-338.
SCHWARTZ,
D. 1980. MBthodes statistiques h l'usage des mdecins et
des biologistes. Flarnmarion, Paris. pp. 173-187.
TEMPEST,D. W., MEERS,J. L., and BROWN,C. M. 1970. Influence
of the environment on the content and composition of microbial
free amino acid pools. J. Gen. Microbiol. 64: 17 1 - 185.
VASCONCELOS,
G. J., and SWARTZ,R. G. 1976. Survival of bacteria
in seawater using a diffusion chamber apparatus "in situ". Appl.
Environ. Microbiol. 31: 913 -920.
WEINER,J. H., and HEPPEL,L. A. 1971. A binding protein for glutamine and its relation to active transport in Escherichia coli. J. Biol.
Chem. 246: 6933 -6941.
Xu, H.-S., ROBERTS,
N., SINGLETON,
F. L., ATWELL,R. W., GRIMES,
D. J., and COLWELL,R. R. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and
marine environment. Microb. Ecol. 8: 313 - 323.