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

MICHEL J. GAUTHIER,' PATRICK M. 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

Accepted November 24, 1988

GAUTHIER, M. J., MUNRO, F'. M., and BREITTMAYER, V. A. 1989. Influence of prior growth conditions on low nutrient 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 bacteriologi- cal 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 nutri- t ive~. Par contre, certains milieux de culture complexes permettaient une production de populations bactkriennes qui s'adap- taient 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 environ- ments, 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, Stras- bourg, France.

Growth conditions Stock cultures were maintained in liquid nitrogen. The survival of

strain LTf in seawater was analysed with cells grown at 37°C 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 30°C.

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 44°C) with or without oxygen (aerobiosis, anaerobiosis) was deter- mined with cells grown oi TSB only. Growth under anaerobic condi- tions was carried out using the Gas Pak anaerobic system (BBL, Cockeysville, MD). Whatever the culture conditions, cells were har- vested 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 col- lected (pH 8.1; salinity, 37.2%,,). Each was inoculated with cells pre- viously 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 26°C).

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CAN. 1. MICROBIOL. VOL. 35, 1989

I I I I

- . 0 1 2 3 4 5 6 7 25 0 1 2 3 4 5 6 7

Hours Days

FIG. 1. Survival in seawater of E. coli previously grown in nutrient broth for different periods at 37°C. (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.

- CFU pH6 H CFU pH7

CFU p ~ 8

0 1 3 5 7 10 Days

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.

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 37°C before cfu were enumerated. In some experiments, the total bacterial number was determined by the acri- dine 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).

- AODC 44°C wCFU 37°C \ MCFU 25°C u C F U 44°C

0 1 3 5 7 10 Days

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.

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 37°C) were tested, survival also depended on physicochemical conditions used for their cul-

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MAODC AERO

GAUTHIER ET AL.

-1 0 1 3 5 7 10

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 con- ditions.

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 (44°C; 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 morphol- ogy 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 con- centration (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 sea- water, 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 supple- mented 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" condi- tion) and aerobically in CFAB (pH 7, 37°C; "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 previ- ously described (Figs. 6 and 7).

1 . , . . , , . . . , 0 1 3 5 7 10

Days

M CFU 0%.

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.

J - , . - . 0 2 4 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 supple- mented 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 physico- chemical factors on their decline in culturability. Recently, however, experimental observations have emphasized the pos- sible adaptation of E. coli to growth in seawater. Such studies - have shown an increase in E. coli survival after previous

Discussion growth in a seawater medium (Gauthier et al. 1987; Munro Most of the studies concerning the fate of enteric bacteria in et al. 1987). In this connection, results reported here suggest

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

M F U AERO

WC FU ANAERO

A A DVC .oCFU

1 . . , , > . . I

0 2 4 7 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 gener- ally 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 intra- cellular 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 pro- duced (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, differ- ences 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 sub- strates 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 signifi- cantly 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 ran- dom 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 pro- cesses 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 bac- teria 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 discus-

sions 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.

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