Journal of Applied Bacteriology 1990,68, 189-198 3042/03/89

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems

I . BARCINA*, J . M . GONZALEZ, J . I R I B E R R I & L. EGEA Departmento de Microbiologia e lnmunologia, Facultad de Ciencias, Universidad del Pais Vasco, Apdo 644, 48080-Bilba0, Spain

Accepted 8 June 1989

BARCINA, I., GONZALEZ, J.M., I R I B E R R I , J. & EGEA, L. 1990. Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems. Journal of Applied Bacteriology 68, 189-198.

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were sig- nificantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([14C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh- and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We con- cluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes

Enteric bacteria in natural aquatic ecosystems are affected by different factors. Bacterial com- petition (Jannasch 1968), predation (Enzinger & Cooper 1976; McCambridge & McMeekin 1979, 1981; Barcina et al. 1986a), temperature (McFeters & Stuart 1972; Davenport et al. 1976; Anderson et al. 1983; Gameson 1984; Barcina et al. 1986a), nutrient concentration (Hendricks & Morrison 1967; Hendricks 1972; Barcina et al. 1986b), light and other physical and chemical parameters have been cited as responsible factors for decrease in colony counts in aquatic ecosystems.

Physical and chemical parameters have been cited by some authors (Sieburth & Pratt 1962;

* Corresponding author.

Verstraete & Voets 1975; Rhodes et al. 1983; Munro et al. 1987; Gauthier et al. 1987) and their effects have been studied mainly by moni- toring the evolution of enteric bacterial colony counts during an incubation period of several days. Other methods, however, such as indirect activity measurements, are not usually used on enteric bacteria in natural aquatic media.

Visible light has been mentioned as an impor- tant factor affecting enteric bacteria survival in natural waters (Gameson & Saxon 1967; Jagger 1975; Krinsky 1976; Grigsby & Calkins 1979; Kapuscinski & Mitchell 1981; McCambridge & McMeekin 1981; Fujioka et al. 1981; Barcina et al. 1986b). Until now, most of the experiments have compared only the changes in numbers of colony-forming units (cfu) of enteric bacteria.

I . Barcina et al. Recently, however, direct count techniques

such as acridine orange direct count (AODC method) (Hobbie et al. 1977), have been shown that cfu counts do not give good estimates of the numbers of enteric bacteria present in natural aquatic systems where different stressful factors can induce an inability to form colonies on standard bacteriological media (Xu et al. 1982; Grimes et al. 1986; Roszak & Colwell 1987; Lopez-Torres et al. 1988). Visible light is one of those factors (see above) and there may, therefore, be an important fraction of non- culturable enteric bacteria present in those natural aquatic systems.

Roszak & Colwell (1987) studied the meta- bolic effect of a natural aquatic system on Escherichia coli and Salmonella enteritidis and defined the viviform population as the whole of bacteria counted by a direct count method (the AODC method). In that viviform population they distinguished two major groups of cells: culturable cells and somnicells. The former are formed by cells which were able to form colo- nies on standard bacteriological media and the somnicells are non-culturable cells. Barcina et al. (1989a) studied E . coli survival under visible light illumination in freshwater and showed that after 72 h most of E . coli cells were in a somni- cell stage; that is, they were non-culturable cells.

Activity measurements have been little used to show physiological behaviour of enteric bac- teria in illuminated natural aquatic media by visible light. Indirect activity measurements were proposed by Barcina et al. (1989b) to test the effect of visible light on E. coli cells in fresh- water, but we know of no studies that compared these techniques with the effects of visible light on different species of enteric bacteria in differ- ent natural aquatic systems.

The aim of this work was to study the effects of visible light on enteric bacteria in natural aquatic systems in respect of their ability to form colonies on suitable culture media, and their ability to metabolize glucose in these environmental conditions. Indirect activity mea- surements were also used to check the effect of freshwater and seawater on enteric bacteria.

Materials and Methods

This study was carried out with natural water samples from the Butron river (Spain) and La Salvaje beach, 500m offshore (Spain). All samples were collected from the surface.

M I C R O - O R G A N I S M S

Bacterial strains employed in this study were Escherichia coli ATCC 11775 and Enterococcus faecalis ATCC 19433.

I N O C U L A P R E P A R A T I O N

The organisms were grown in nutrient broth at 28°C for 8 h. Cells from the exponential phase were harvested by centrifugation (3000 g for 15 min) and washed three times with sterile saline solution (0.9% w/v). The pellets were suspended in saline solution, inoculated in fresh nutrient broth, and incubated at 28°C for 18 h. The cells were harvested at stationary phase as described above. The final suspensions were inoculated in the freshwater or seawater samples to give a final density of approximately 10' cells/ml.

T E C H N I C A L M E T H O D S

All experiments were carried out in 2 1 flasks containing 500 ml of sterile subsamples, obtained by filtering natural water through membrane filters (0.2 pm; Millipore). The incu- bation of inoculated subsamples was done at 20°C with shaking at 180 rev/min in an orbital incubator with an illumination system consist- ing of seven Sylvania F30 W/T8/D lamps. The luminous spectrum of these lamps is shown in Fig. 1. The following parameters were studied both in illuminated and in non-illuminated sub- samples.

1 I I 1

10

8

: ~6 3

4

2

0 350 450 550 650 750

nm

Fig. 1. Luminous spectrum of the lamps employed.

Survival strategy of enterobacteria 191 PLATE C O U N T S

Colony forming units (cfu) of E. coli and Ent. faecalis were enumerated on Trypticase Soy Agar (BBL) supplemented with 0.3% yeast extract and 0.5% glucose (TSY) (Rhodes et al. 1983) incubated at 37°C for 24 h. The areas of the colonies of E. coli and Ent. faecalis on TSY after incubation at 37°C for 24 h were calcu- lated by measuring colony diameters and assuming that the colonies were all circular.

A C R I D I N E O R A N G E DIRECT C O U N T S

The total number of cells in the subsamples was estimated by the AODC method of Hagstrom et al. (1979). Subsamples were preserved in 2% formaldehyde (final concentration).

I N D I R E C T ACTIVITY MEASUREMENTS

Indirect activity measurements were done with [U-'4C]glucose (250 mCi/mmol; Radiochemical Centre, Amersham, England) at a saturated con- centration (100 pgC/l). Three 5 ml subsamples were analysed for each uptake measurement, and they were incubated for 30 min in the dark at 20°C with shaking. Forty p1 of 2 N H2S04 were then injected into the flasks and the sub- samples were further incubated for 1 h to trap the 14C0, released on a filter paper impreg- nated with 1-phenetilamine (Hobbie & Craw- ford 1969). After both incubation periods, the entire volume of each subsample was filtered through membrane filters (0.2 pm; Millipore). The filters were rinsed three times with 5 ml of filtered water (0.2 pm membrane filters), placed in Unisolve- 1 (Hispanoland, Barcelona, Spain), and radioassayed by liquid scintillation counting. Quench curves were computed by the channel ratio method. Controls for abiotic absorption were prepared.

Total ['4C]glucose uptake was defined as the sum of assimilation and respiration of [14C]gIucose. Percentage of total [14C]glucose uptake with respect to the [14C]glucose added was calculated by the following expression :

Total [ '4C]glucose uptake [14C]glucose added

x 100

Assimilated and respired fractions were defined as Wright & Burnison (1979). Respir- ation (or mineralization) is defined as the pro-

duction of 14C0, and assimilation as the substrate retained in the cell (that is, substrate taken up but not respired). Percentages of assimilation and respiration with respect to total [14C]glucose uptake were obtained by dividing the assimilated or respired fraction by the total ['4C]glucose uptake and then multi- plying by 100.

STATISTICAL A N A L Y S I S

Colony areas were compared by Student's t-test according with Sokal & Rohlf (1969). Regres- sion analyses between the different parameters and incubation times were used throughout this study, and slopes, if different from zero, were compared to see if there were any differences between the effect of visible light on different parameters, natural media and enteric bacteria species. Any probability (P) less than 0.05 was considered significant.

Results

The following results correspond to means of three experiments carried out for E. coli and Ent. faecalis in both natural aquatic media.

DIRECT A N D CfU C O U N T S

In all cases both direct and cfu counts carried out in non-illuminated systems did not show any significant changes during the experiments. In illuminated systems, however, cfu counts of both E. coli and Ent. faecalis decreased during the incubation period although total number of bacteria (AODC method) remained constant during the experiments done in illuminated systems. Culturable and somnicell fractions of viviform population for E . coli and Ent. faecalis in both aquatic media for illuminated and non- illuminated systems are compared in Fig. 2.

There was a decrease in the ratios of cfu of E . coli to cfu of Ent. faecalis throughout the experi- ments in illuminated systems (Table 1). For sea- water, this was by a factor of 2 at 24 h and 7 at 48 h, and for freshwater, by a factor of 30 and 229, for 24 and 48 h respectively. However, in non-illuminated systems very small differences were observed (Table 1).

During these experiments, colony areas of cfu on TSY were measured and there was a pro- gressive diminution in illuminated as opposed to non-illuminated systems for both E . coli and

192 I . Barcina et al.

Fig. 2. Fractions of somnicells (0) and culturable cells (m) from the viviform population of Escherichia coli and Enterococcusfaecalis in non-illuminated (a) and illuminated (b) systems. A. Escherichia coli in seawater; B. Entero- coccusfaecalis in seawater; C . Escherichia coli in freshwater; D. Enterococcusfaecalis in freshwater.

Ent. faecalis in fresh- and seawater. These areas of illuminated and non-illuminated results are shown in Table 2 as percentages of systems. Thus there were significant differences illuminated to non-illuminated systems and at 12 h for E . coli (P < 0.001) and Ent. faecalis indicate a decrease from ca 100% to about (P < 0.05) in freshwater, and at 24 h in seawater 32-34% for E. coli and 4&50% for Ent.faecalis. (P < 0.001 and P < 0.01 for E . coli and Ent. fae- There were also differences between the colony calis respectively) (Table 2).

Table 1. Ratios of Escherichia coli to Enterococcusfaecalis during illuminated and non-illuminated experiments in fresh and marine systems

In seawater In freshwater Time

(h) Illuminated Non-illuminated Illuminated Non-illuminated

0 5.11 3.52 3.32 3.52 12 4.03 2.68 1 . 1 1 6.92 24 2.82 2.54 0.19 3.91 36 1.46 2.90 0.01 2.70 48 0.70 1.70 0.01 3.19

Survival strategy of enterobacteria 193 Table 2. Percentages of colony areas for illuminated cells with respect to non-illuminated cells during the experi-

ments carried out in fresh- and seawater In seawater In freshwater

Escherichia coli Enterococcus fuecalis Escherichia coli Enterococcus faecalis Times

(h) Percentage P* Percentage P* Percentage P* Percentage P*

0 100.52 NS 101.51 NS 95.01 NS 92.39 NS 12 87.77 NS 96.27 NS 79.03 0.001 94.07 0.05 24 58.16 0.001 85.90 0.01 68.21 0.0 1 92.47 0.0 1

- 54.22 0.001 36 - - 67.63 0.001 -

48 34.30 0.001 39.99 0.001 32.36 0.00 1 50.31 0~001

* Significance level.

I N D I R E C T A C T I V I T Y M E A S U R E M E N T S

Table 3 shows total uptake of ['4C]glucose for illuminated and non-illuminated systems. For illuminated cells, there was a sharp decrease in the total uptake of glucose. In non-illuminated cells the decreases were smaller or there was no change.

It is important to note that E . coli cells in freshwater took up much more [14C]glucose (about 2.4 x lo6 cpm) than in seawater (about lo5 cpm) at the beginning of the experiments. However, Ent. faecalis took up similar quan- tities of [ 14C]glucose in both natural aquatic media (about 5-6 x lo5 cpm). Percentages of glucose uptake are given in Table 3.

The results of assimilation and respiration percentages with respect to the total glucose uptake are shown in Table 4. This shows decreases of assimilation percentages and increases of respiration percentages in all illumi- nated systems studied which were not observed in the non-illuminated systems.

There were differences in the glucose assimi- lation and respiration percentages of E . coli at the beginning of the experiments carried out in freshwater but not in seawater (Table 4). For E . coli cells in freshwater the percentages of glucose assimilation and respiration were about 86% and 14%, respectively at 0 h and E . coli in seawater presented percentages about 24% and 76%, respectively, also at 0 h. On the other hand, Ent . faecalis did not give such high differ- ences as E . coli.

D I F F E R E N C E S B E T W E E N P A R A M E T E R S ,

S P E C I E S A N D N A T U R A L W A T E R S

In illuminated systems, it was noted tha: semi- log representations of decreases of cfu numbers,

total uptake of glucose, and assimilated and respired fractions of glucose taken up vs time were linearly related ( P < 0.05). Slopes of these were significantly different from zero ( P < 0.05) and were used to compare decreases of the parameters studied. Slopes for non-illuminated systems and direct counts (AODC method) were not significantly different from zero.

Significant differences between cfu number decrease and the corresponding decreases of total uptake ( P < 0.001), assimilation ( P < 0.025) and respiration ( P < 0.001) of glucose were observed in all cases except for Ent. faecalis in illuminated marine systems; in these we observed only significant differences between cfu numbers and assimilation of glucose decreases ( P < 0.05).

Comparison of slopes of faecal indicator bac- teria in different natural aquatic media showed that the decrease of E . coli cfu numbers was sig- nificantly lower ( P < 0.001) in illuminated-fresh than in illuminated-marine waters. For Ent. fae- calis in illuminated systems, decrease of cfu numbers was not significantly different in fresh- and seawater but, in this case, it was seen that slopes of total uptake ( P < 0.001) and respir- ation ( P < 0.001) of glucose were significantly lower in sea- than in freshwaters. No differences were found, either for E. coli or for Ent. faecalis, with respect to glucose assimilation decrease.

When slopes between E . coli and Ent . faecalis in a single natural aquatic medium were com- pared, there were significant differences in cfu number decrease in illuminated-seawater ( P < 0.001) and in illuminated-freshwater ( P < 0905) systems, descending faster with E . coli than with Ent. faecalis. Respiration of glucose decrease was significantly different ( P < 0.001) between E . coli and Ent. faecalis in both illuminated systems and decrease of total

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12.9

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14.9

8 37

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88.7

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7.07

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36

35.6

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7 85

.93

Survival strategy of enterobacteria 195

glucose uptake was in fresh- ( P < 0.025) and in seawater ( P < 04)05) illuminated systems. However, respiration and total uptake of glucose of E . coli decreased faster than Ent. fae- calis in illuminated seawater but this was opposite in illuminated freshwater. No differ- ences for glucose assimilation were found between E. coli and Ent. faecalis in illuminated fresh- or seawater.

Discussion

DIRECT A N D CfU C O U N T S

Differences between direct (AODC method) and cfu counts provide information about the frac- tions of somnicells and culturable cells which constitute the viviform populations in our aquatic systems. Figure 2 shows these fractions and it can be seen that visible light induces a progressive dormancy of both E. coli and Ent. faecalis in fresh- and in seawater, detected because most of the cells lost their ability to form colonies on a suitable culture medium. Such organisms are called somnicells. In illumi- nated systems, fractions of culturable cells from viviform populations are very small and stan- dard bacteriological media should not therefore be used to detect numbers of enteric bacteria in natural waters exposed to visible light because this procedure would give an important under- estimation of enteric bacteria real numbers.

Significant differences ( P < 0.001) in colony areas (Table 2) have been found throughout the experimental period between illuminated and non-illuminated systems. This would represent a stress effect of visible light on both E . coli and Ent. faecalis in natural waters and could be a first step towards the loss of ability to form colonies on standard bacteriological media, i.e. towards the somnicell stage.

It is concluded, therefore, that visible light has a stressful effect on enteric bacteria in natural aquatic media. However, Fujioka e t al. (1981) indicated, for seawater, that visible spectrum of sunlight 'has a killing rather than a stressful effect on the indicator bacteria' (faecal coliforms and faecal streptococci). Other authors (Hollaender 1943; Gameson & Gould 1974; Krinsky 1976) used the terms lethal and death when referring to decrease in cfu number. Nevertheless, these authors did not make direct counts to determine total number of enteric bac-

teria (viviform population) in their experiments. Therefore, the terms killing, lethal and death have been interpreted by them as a decrease in cfu numbers and they did not consider them to be a decrease in total cell numbers of a viviform population. At present, according with several authors (Grimes et al. 1986; Roszak & Colwell 1987; Barcina e t al. 1989) these terms (killing, lethal and death) are understood as a decrease in bacterial total number. Caution is therefore necessary in interpreting existing literature, par- ticularly that published before eighties.

A comparison of the decrease of cfu numbers of E. coli and Ent. faecalis during our experi- ments shows a more rapid inactivation of E . coli as opposed to Ent. faecalis both in marine and in freshwater exposed to visible light. This results in a rapid reduction of the E . coli to Ent. faecalis cfu ratio (Table 1) in illuminated systems although no important decline in the ratio of E . coli to Ent. faecalis in non- illuminated systems was observed. These results indicate that the significance of the faecal coli- forms to faecal streptococci ratio established by Geldreich & Kenner (1969) for 24 h after input of faecal indicator bacteria in natural waters is not valid in natural aquatic ecosystems where visible light can affect enteric bacteria survival.

INITIAL EFFECT OF SEAWATER VERSUS FRESHWATER

Several authors (Zobell 1936; Carlucci & Pramer 1959; Vasconcelos & Swartz 1976; Rhodes et al. 1983; Munro et al. 1987) have observed a toxic effect of seawater on enteric bacteria. They showed, that in the absence of natural microflora, there was a decrease in numbers of enteric bacteria in seawater during their incubation periods.

In our experiments we noted that E . coli in freshwater took up much more ['4C]glucose than in seawater (Table 3) . However, Ent. fae- calis showed similar incorporation of ['4C]glucose in freshwater and in seawater (Table 3). That means that seawater has a greater effect on transport of glucose by E. coli cells than does freshwater. Moreover, percent- ages of assimilation of E . coli in freshwater at 0 h were very high compared with those in sea- water (Table 4). However, for Ent. faecalis differ- ences in assimilation and respiration percentages were not as important as for E. coli

196 I . Barcina et al. (Table 4). By that, we can deduce E. coli is more sensitive to natural waters than is Ent. faecalis, and that natural waters can affect enteric bac- teria metabolism. Moreover, these effects were produced immediately after input of enteric bac- teria in natural waters. In spite of these results, cfu numbers of both E . coli and Ent. faecalis were not affected by natural fresh- and seawater at the beginning of the experiments because the density of these enteric bacteria were similar.

By these facts we can conclude that seawater has several effects on E. coli cells which can be shown metabolically and immediately (without long incubation periods) by using indirect activ- ity measurements, and not only by counts throughout a long incubation period (usually several days) as other authors (Zobell 1936; Carlucci & Pramer 1959; Rhodes et al. 1983; Munro el al. 1987) have shown. For E . coli in seawater with respect to freshwater, these effects are a decrease of total glucose uptake and a use of that glucose taken up mainly by degradative processes (respiration). This could be interpreted as a mechanism of resistance of E . coli to sea- water.

I N D I R E C T A C T I V I T Y M E A S U R E M E N T S

U N D E R I L L U M I N A T I O N

Progressive decrease of total glucose uptake by illuminated cells (Table 3), both in fresh- and seawater, indicates an inactivation of glucose transport into the cells as a consequence of visible light action.

If we analyse percentages of assimilation and respiration with respect to total glucose uptake (Table 4) we can observe a progressive inac- tivation of biosynthetic process throughout the illuminated experiments for both E . coli and Ent. faecalis in fresh- and seawater. This means that the glucose taken up is mainly used in degradative process, that is, enteric bacteria in illuminated natural waters become dormant cells which are characterized by using substrates taken up for maintenance functions and by inactivating their biosynthetic processes.

Roszak & Colwell (1987) studied evolution of E . coli and Salmonella enteritidis in marine dark systems and observed throughout their incu- bation period (1 or 2 months) an increase in the number of cells which incorporated substrates for maintenance functions but not for growth.

From these results we see that visible light lead to progressive dormancy of enteric bacteria more rapidly than occurs in natural aquatic media in the dark, and therefore we can aftirm that the effects of visible light on enteric bacteria are additional to those of natural aquatic media.

From the results so far discussed, we can deduce that visible light stimulates in E . coli and Ent. faecalis cells in natural aquatic media dif- ferent metabolic effects which lead to a progres- sive dormancy. This progressive dormancy lead enteric bacteria to a somnicell stage which can be defined and characterized by (1) inability to form colonies on standard bacteriological media, (2) incapacity to incorporate substrates, and (3) inactivation of biosynthetic processes and a metabolism of maintenance.

D I F F E R E N C E S B E T W E E N P A R A M E T E R S , SPECIES A N D N A T U R A L W A T E R S

We have found differences between enteric bac- terium species and between each species in the two ecosystems. This means that visible light effects depend on the natural aquatic ecosystem studied and on enteric bacteria species used. Differences in the effect of visible light on the decrease in cfu for different enteric bacteria and natural aquatic media have already been noted by Fujioka et al. (1981). In our study, we point out differences both for the decrease in cfu and for indirect activity measurements (respiration and total uptake of glucose). With respect to these indirect activity measurements it is impor- tant to note that no differences have been obtained for glucose assimilation decrease between E . coli and/or Ent. faecalis in fresh and/or seawater. By this fact, we can deduce that the effect of visible light on assimilative processes of enteric bacteria in natural waters could be independent of the enteric bacteria species and the natural aquatic media studied.

These facts suggest that visible light affects the parameters studied with different intensity in each case (excepting glucose assimilation) and bacteria of enteric origin may respond to illumi- nated natural aquatic systems in a species- specific manner. In spite of those differences, enteric bacteria show a general survival strategy characterized by reaching the somnicell stage as defined above.

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