APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1980, p.

907-911
0099-2240/80/11-0907/05$02.00/0

Vol. 40, No.5

Relative Effects of Bacterial and Protozoan Predators on

Survival of Escherichia coli in Estuarine Water Samples
J. McCAMBRIDGE*

AND

T. A. McMEEKIN

Faculty of Agricultural Science, University of Tasmania, Hobart, Tasmania

7001, Australia

The relative effect of protozoan and bacterial predators on the survival of

Escherichia coli in estuarine water samples was examined. Predacious protozoa
exerted their major influence on E. coli destruction during the first 2 days of a 10day-decline period. Inhibition of protozoa after day 2 had little effect on E. coli
survival. Bacterial predators also contributed to E. coli destruction but in natural
estuarine water samples were maintained at lower levels due to "grazing" by
predacious protozoa.

The survival of sewage bacteria in seawater (bottom layer, Davis agar, 1.5% [wt/vol] in estuarine
has been attributed to many factors which have water, top layer, Davis agar, 0.7% [wt/vol] in estuarine
0.5 ml of E. coli suspension [approximately
been reviewed in detail (9), and the role of water plusper
ml] and 0.5 ml of the estuarine water
1010 cells
microbial predators in the destruction of fecal sample
to be examined). In some experiments, the
bacteria has been suggested by several workers. addition of the crystalline antibiotic cycloheximide
Mitchell et al. (12) isolated two marine bacteria (The Upjohn Co.) to this top layer at a concentration
responsible for the death of Escherichia coli in of 500 mg/liter enabled the enumeration of predacious
seawater, Mitchell and Morris (10) observed the bacteria only. This compound is active against eucardominance of protozoans, and Roper and Mar- yotic organisms such as protozoa, fungi, and yeasts,
shall (13) investigated the role of a fruiting but is tolerated in the concentrations used by most
myxobacter (Polyangium sp.) and a small bacteria, including E. coli (16).
Effect of bacteria and protozoa on E. coli suramoeba (Vexillifera sp.) in this context. EnzinTo determine the effect of both protozoan and
ger and Cooper (5) studied the relative roles of vival.
acting together on the survival of
bacterial
bacteria and protozoa in the removal of E. coli E. coli in predators
estuarine water samples, an E. coli suspenfrom estuarine water and suggested that coli- sion was added to 49 ml of freshly collected estuarine
form destruction was insignificant in the absence water to give a final concentration of approximately
of protozoa and that bacterial predators were 108 cells per ml. The effect of bacterial predators alone
not important. The purpose of this study was to on the survival of E. coli was determined by the
examine the importance of both bacterial and inhibition of the protozoan predators. This was
protozoan predators and to determine the time achieved by the addition of the antibiotic cyclohexi(final concentration, 500 mg/liter) to 49 ml of
period during which the latter organisms were mide
water containing an E. coli suspension (apestuarine
most active in the decline of E. coli in estuarine proximately
108 cells per ml). Microscopic examination
water samples.
of water samples and plaques produced on doublelayer plates was carried out throughout the duration
MATERIALS AND METHODS
of the experiment. Two autoclaved estuarine water
Cultures. E. coli strain M13 (culture collection of samples containing a similar E. coli suspension were
the Department of Agricultural Science, University of
Tasmania) was used in this study. Cells were grown in
nutrient broth (Oxoid) with shaking at 22C for 24 h,
washed twice in saline (0.9% [wt/vol] NaCi), and harvested by centrifugation (4,000 x g for 20 min) to
provide a washed cell suspension (approximately 1010
cells per ml). This suspension was used as a nutrient
source for enumeration of predacious microorganisms
and, when appropriately diluted, to inoculate estuarine
water samples.
Counting methods. Viable counts of all organisms
were made at 2-day intervals by preparing a standard
decimal dilution series in saline (0.9% [wt/vol] NaCl).
E. coli cells were enumerated on MacConkey agar
(Oxoid) after incubation at 37C for 18 h. Bacterial
and protozoan predators of E. coli were counted as
plaque-forming units (PFU) on double-layer plates

included as control treatments. One of these contained

cycloheximide (500 mg/liter) to determine directly the
effect of this antibiotic on E. coli. Three replications
of each treatment were incubated at 22C for 10 days.
Results for E. coli survival were analyzed by using
analysis of variance techniques.
Effect of periodic inhibition of protozoans on
E. coli survival. The time interval during which
predacious protozoa were active in E. coli destruction
in a 10-day-decline period was determined by the
exposure of E. coli to these predators for different
lengths of time. After this exposure, protozoan predators were inhibited for the remainder of the experi-

ment.
An E. coli suspension (final concentration, approximately 108 cells per ml) was added to a sample of
freshly collected estuarine water (400 ml) and incu907

908

APPL. ENVIRON. MICROBIOL.

McCAMBRIDGE AND McMEEKIN

bated at 220C. At 12-h intervals, a 50-ml sample was

withdrawn, placed in a flask containing cycloheximide
(final concentration, 500 mg/liter), and also incubated
at 220C. The survival of E. coli cells and the growth
of predators in all flasks were determined immediately
after withdrawal of the sample and at 2-day intervals
thereafter. An E. coli suspension in 400 ml of autoclaved estuarine water was included as a control.
Three replications of the complete experiment were
conducted.
This experiment was repeated on two further occasions with E. coli suspensions of approximately 108
and 10' cells per ml, respectively. In these two experiments, total PFU (protozoa and bacteria) were determined as described above. Bacterial predators alone
were counted as PFU on double-layer plates to which
cycloheximide (final concentration, 500 mg/liter) had
been added to the top layer.

RESULTS
Effect of protozoa and bacteria on E. coli
survival in estuarine water samples. E. coli
cells inoculated into natural estuarine water containing both protozoa and bacteria were reduced
from approximately 10' to less than 10 organisms
per ml in 10 days (Fig. la), compared with 104
organisms per ml in the presence of bacteria
alone. Microscopic examination of all water samTIM (DAYS)
ples containing cycloheximide and plaques produced on the corresponding double-layer plates
revealed the complete inhibition of protozoan
predators. The destruction of all microbial predators by autoclaving resulted in E. coli numbers
remaining virtually unaltered after 10 days. Similarly, the direct effect of cycloheximide on E.
coli survival, in autoclaved estuarine water, was
negligible (Fig. la). Numbers of predacious microorganisms increased in both unautoclaved
water samples (Fig. lb). On double-layer plates
inoculated with natural estuarine water, the
number of PFU (protozoa and bacteria) increased from less than 10 to a maximum of 103
PFU/ml in 10 days. Similarly, on plates incubated with estuarine water samples treated with
cycloheximide, the number of PFU (bacteria
alone) increased from less than 10 to 103 PFU/
ml in 10 days.
Effect of periodic inhibition of protozoans on E. coli survival. The effect of periodic inhibition of protozoa by cycloheximide
on the survival of E. coli in estuarine water
TIME (DAYS)
samples is shown in Table 1. In natural estuarine
FIG. 1. (a) Effect of protozoa and bacteria on E.
water, E. coli cells were reduced from 3.8 x 107
to less than 10 organisms per ml in the 10-day- coli survival. E. coli survival in: autoclaved estuarine
decline period. If protozoa were inhibited at the water (A); autoclaved estuarine waterplus cycloheximide (0); natural estuarine water (0); natural escommencement of the experiment (day 0), E. tuarine
plus cycloheximide (0). (b) Growth of
coli cells were only reduced to 1.7 x 102 organ- bacterialwater
and protozoan predators in: natural estuisms per ml. Similarly, inhibition of protozoa arine
water (/); natural estuarine water plus cycloafter 0.5, 1, and 1.5 days resulted in a signifi- heximide (-). Each point represents the mean 1
cantly increased survival of E. coli cells com- standard error of the mean.

909

PROTOZOAN AND BACTERIAL PREDATION ON E. COLI

VOL. 40, 1980

pared with that in natural estuarine water (Table 1). However, subsequent inhibition of the
protozoa at 2, 2.5, and 3 days did not increase E.
coli survival compared with that in the natural
sample. The logio number of E. coli cells surviving at day 10 after exposure to protozoa for
various times is also shown in Table 1. As the
time of protozoan action increased from 0 to 2
days, the logio number of E. coli surviving at day
10 gradually declined. Inhibition of predacious
protozoa after day 2, however, had no further
effect on E. coli survival. The numbers of predators developing in various treatments, determined as PFU, are shown in Table 2. The logio
number of PFU in natural estuarine water represents both bacterial and protozoan predators,
whereas those in the remaining samples are bacterial predators only. The number of PFU at
day 10 in samples in which protozoa were inhibited after 0, 0.5, 1, 1.5, and 2 days were

significantly higher than the number of PFU

present in natural estuarine water. Inhibition of
protozoa after 2.5 and 3 days, however, resulted
in no significant difference between the number
of PFU in these samples compared with that in
natural estuarine water.
When this experiment was repeated with an
initial E. coli concentration of 10' and 105 cells
per ml, the pattem of E. coli decline and
predator growth was similar to that shown in
Tables 1 and 2, respectively. With an initial E.
coli concentration of 10' cells per ml the total
PFU (protozoa and bacteria) in natural estuarine water increased from 10 to a maximum of 2
x 10' PFU/ml after 2 to 3 days and gradually
declined to the end of the incubation period.
Similarly, bacterial predators increased to reach
a maximum of 8 x 103 PFU/ml at day 2 and
then gradually declined. Inhibition of protozoa,
however, resulted in a marked increase in the

TABLE 1. E. coli survival in estuarine water after periodic inhibition ofprotozoa

Log no. of E. coli per ml' at following time of protozoan inhibitionb (days):

Log no. of

Time

E. coli
per
in natu-

(dy)mla
ral estuarine
(cdays)

0.5

1.0

1.5

2.0

2.5

3.0

water

7.58 (0.02) 7.56 (0.02)

0
7.55 (0.01)
0.5
1.0 7.62 (0.01) 7.63 (0.01) 7.58 (0.01) 7.49 (0.02)
7.55 (0.04)
1.5
2.0 7.30 (0.04) 7.42 (0.01) 7.35 (0.05) 7.41 (0.01) 7.38 (0.01) 7.28 (0.10)
6.72 (0.11)
2.5
3.0 5.33 (0.2) 7.26 (0.04) 7.00 (Q.19) 7.47 (0.06) 7.30 (0.05) 7.34 (0.07) 6.84 (0.32)
4.0 4.50 (0.11) 6.53 (0.07) 5.93 (0.21) 6.63 (0.26) 6.68 (0.10) 6.12 (0.48) 5.87 (0.85)
6.0 3.20 (0.15) 4.76 (0.45) 4.16 (0.20) 4.88 (0.37) 4.81 (0.08) 3.35 (0.26) 3.18 (0.49)
8.0 1.63 (0.11) 2.94 (0.36) 2.17 (0.09) 3.46 (0.28) 2.17 (0.09) 1.43 (0.22) 1.05 (0.55)
10.0 0.46 (0.24) 2.23 (0.13) 1.91 (0.03) 1.92 (0.33) 1.46 (0.19) 0.36 (0.36) 0.23 (0.23)
a Values represent mean of three observations; standard error is shown within parentheses.
b Time of
cycloheximide addition to sample.

5.49 (0.25)
4.34 (0.15)
3.30 (0.07)
1.28 (0.16)
0.20 (0.20)

TABLE 2. Growth of PFU in estuarine water after periodic inhibition of protozoa

Log no. of PFU per mla at following time of protozoan inhibitionb (days):

Time

(days)

Log no. of
PFU
per
nml'
in natural estuarine
water

0.5

1.0

1.5

2.0

2.5

0.79 (0.40) 1.04 (0.09)

0
1.03 (0.03)
0.5
1.79 (0.14)
1.0
1.86 (0.06)
1.5
2.0 1.73 (0.17) 1.91 (0.22) 2.04 (0.20) 2.10 (0.19) 2.05 (0.14) 1.75 (0.13)
1.74 (0.09)
2.5
3.0
4.0 2.50 (0.09) 2.70 (0.30) 3.12 (0.26) 3.00 (0.14) 3.01 (0.08) 2.72 (0.26) 2.71 (0.15)
6.0 2.70 (0.05) 3.53 (0.10) 3.51 (0.15) 2.71 (0.35) 2.77 (0.02) 2.76 (0.23) 2.79 (0.15)
8.0 2.96 (0.13) 3.63 (0.07) 3.30 (0.18) 3.25 (0.07) 3.42 (0.15) 3.12 (0.13) 3.09 (0.09)
10.0 2.61 (0.18) 3.26 (0.06) 3.20 (0.13) 3.31 (0.14) 3.38 (0.07) 3.42 (0.08) 2.81 (0.10)
a
Values represent mean of three observations; standard error is shown within parentheses.
b Time of cycloheximide addition to sample.

3.0

2.44
2.62
2.71
2.98
2.43

(0.05)
(0.09)
(0.03)
(0.09)
(0.13)

910

McCAMBRIDGE AND McMEEKIN

APPL. ENVIRON. MICROBIOL.

number of bacterial predators compared with indicating that the former "graze" not only on
those present in natural estuarine water, reach- E. coli cells but also on bacterial predators,
ing a maximum level of 5 x 104 PFU/ml after 5 thereby maintaining them at relatively low levdays. When an initial E. coli concentration of els in the natural sample. Periodic inhibition of
105 cells per ml was used, the total number of the protozoan predators showed that their major
PFU which developed was reduced to 35 PFU/ effect on bacterial predators (as for E. coli) is
ml after 2 to 3 days, and the number of bacterial also during the first 2 days of the 10-day-decline
PFU was reduced to 25 PFU/ml. Inhibition of period.
protozoa again resulted in a marked increase in
It has been suggested by several workers (1, 3,
bacterial PFU to 2.5 x 103 PFU/ml after 5 to 6 7) that protozoan multiplication and bacterial
days. Microscopic examination of all plaques decline stop when the prey density falls to a
produced on plates containing cycloheximide re- critical cell density of 106 to 107 cells per ml. At
vealed the presence of bacterial predators only. this level, the energy used by the protozoan
predator in searching for the prey equals that
DISCUSSION
obtained from feeding (4). Bacterial prey also
The role of the natural microbial population, persist due to the development of predationparticularly predacious microorganisms, in the resistant organisms (6) and the evolution of
destruction of bacteria of sewage origin in estu- avoidance strategies (15). This latter phenomearine water, has been well documented (5, 8, 10, non even occurs in experimental systems, where
13). Although light-induced cell damage has re- sufficient heterogeneity appears to exist (15),
cently been considered as the principle agent in and in natural ecosystems, where clay particles,
coliform decay in seawater (2), the possible in- for example, may completely inhibit predatorteraction between indigenous microbial preda- prey interactions (14). Thus, protozoan predators and light injury has also been suggested. tors acting in natural estuarine water samples
Enzinger and Cooper (5) examined the effect of would be expected to only reduce an influx of
predacious protozoans on the survival of E. coli sewage bacteria to this critical level. In fact,
in estuarine water by using an antibiotic-resist- bacterial numbers may be reduced to less than
ant E. coli mutant and subsequently destroying 10 cells per ml in 10 days (Fig. la) and to 0 in 8
the bacterial predators present in the water sam- days (11), indicating that predators other than
ple. They found that survival of E. coli was protozoa may also be exerting an influence on
dependent on the presence of protozoan preda- the E. coli prey population. Further, the exertion
tors and not on the presence of lytic bacteria. by protozoa of their major effect during the first
However, if protozoa alone were responsible for 2 days of a 10-day-decline period and the supthe destruction of the E. coli prey, then inhibi- pression of bacterial predators by these protozoa
tion of this predacious group should result in suggest that there may be interacting microbial
prey survival similar to that which occurs in predators each playing a distinct role in the
autoclaved estuarine water. In this study, the destruction of fecal bacteria after their introducsurvival of E. coli in estuarine water was in- tion into estuarine water samples.
creased when the predacious protozoan population was inhibited by the use of cycloheximide
ACKNOWLEDGMENTS
(Fig. la), but the survival was less than that in
This work was supported by the Rural Credits Developthe autoclaved sample. This suggests that bac- ment Fund of the Reserve Bank of Australia.
We thank M. M. Roper for helpful discussion and R. K.
terial predators are also important in the decline
Lowry (CSIRO) for statistical analysis.
of E. coli in estuarine water samples.
When protozoan predators were inhibited
LITERATURE CITED
after 2 days of incubation, survival of E. coli was
similar to that in the natural sample, suggesting 1. Berk, S. G., R. R. Colwell, and E. B. Small. 1976. A
study of feeding responses to bacterial prey by estuarine
that these predators exert their major influence
ciliates. Trans. Am. Microsc. Soc. 95:514-520.
on the E. coli prey population during this period. 2. Chamberlin,
C. E., and R. Mitchell. 1978. A decay
Increasing the exposure time of the E. coli prey
model for enteric bacteria in natural waters, p. 325-348.
In R. Mitchell (ed.), Water pollution microbiology, vol.
to the protozoan predators beyond 2 days had
2. John Wiley and Sons, New York.
no greater effect on the number of prey cells
S. K. A., and M. Alexander. 1975. Regulation of
surviving after 10 days (Table 1). Bacterial pred- 3. Danso,
predation by prey density: the protozoan-Rhizobium
ators increased in numbers in the natural sample
relationship. Appl. Microbiol. 29:515-521.
after the introduction of E. coli into estuarine 4. Danso, S. K. A., S. 0. Keya, and M. Alexander. 1975.
Protozoa and the decline of Rhizobium populations
water. When the protozoan predators were inadded to soil. Can. J. Microbiol. 21:884495.
hibited, however, the bacterial predators 5. Enzinger,
R. M., and R. C. Cooper. 1976. Role of
reached and were maintained at higher levels,
bacteria and protozoa in the removal of Escherichia

VOL. 40, 1980

6.
7.

8.
9.

10.

PROTOZOAN AND BACTERIAL PREDATION ON E. COLI

coli from estuarine waters. Appl. Environ. Microbiol.

31:758-763.
Gude, H. 1979. Grazing by protozoa as selection factor for
activated sludge bacteria. Microb. Ecol. 5:225-237.
Habte, M., and M. Alexander. 1978. Protozoan density
and the coexistence of protozoan predators and bacterial prey. Ecology 59:140-146.
McCambridge, J., and T. A. McMeekin. 1979. Protozoan predation of Escherichia coli in estuarine waters.
Water Res. 13:659-664.
Mitchell, R., and C. Chamberlin. 1975. Factors influencing the survival of enteric microorganisms in the
sea: an overview, p. 237-257. In A. L. H. Gameson (ed.),
Discharge of sewage from sea outfalls. Pergamon Press,
London.
Mitchell, R., and J. C. Morris. 1969. The fate of intestinal bacteria in the sea, p. 811-821. In S. H. Jenkins
(ed.), Advances in water pollution research. Pergamon

911

Press, Oxford.
11. Mitchell, R., and S. Yankofsky. 1969. Implication of a
marine ameba in the decline of Escherichia coli in
seawater. Environ. Sci. Technol. 3:574-576.
12. Mitchell, R., S. Yankofsky, and H. W. Jannasch. 1967.
Lysis of Escherichia coli by marine microorganisms.
Nature (London) 215:891-893.
13. Roper, M. M., and K. C. Marshall. 1978. Biological
control agents of sewage bacteria in marine habitats.
Aust. J. Mar. Freshwater Res. 29:335-343.
14. Roper, M. M., and K. C. Marshall. 1978. Effects of a
clay mineral on microbial predation and parasitism of
Escherichia coli. Microb. Ecol. 4:279-289.
15. van Den Ende, P. 1973. Predator-prey interactions in
continuous culture. Science 181:562-564.
16. Whiffen, A. J. 1948. The production, assay, and antibiotic
activity of actidione, an antibiotic from Streptomyces
griseus. J. Bacteriol. 56:283-291.