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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
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
CAN. 1. MICROBIOL.
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
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-
GAUTHIER ET AL.
MAODC
AERO
M CFU 0%.
-1
0 1
5
Days
Discussion
Most of the studies concerning the fate of enteric bacteria in
. .
0 1
10
. .
5
Days
10
.
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.
382
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.
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.
GAUTHIER ET
AL.
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