Download Enhancement of Edwardsiella tarda and Aeromonas salmonicida

FEMS Microbiology Letters 51 (1988) 95-100
Published by Elsevier
95
FEM 03191
Enhancement of Edwardsiella tarda and A eromonas salmonicida
through ingestion by the ciliated protozoan
Tetrahymena pyriformis
C h r i s t o p h e r H. K i n g a n d E m m e t t B. Shotts
Department of Medical Microbiology, University of Georgia, Athens, GA, U.S.A.
Received 9 February 1988
Accepted 16 February 1988
Key words: Coculture; Enhancement; Survival; Chlorination; Intracellular bacterium
1. SUMMARY
When two common bacterial fish pathogens
were cocultured with a ciliated protozoan, enhancement of each bacterial species was observed
over time. Enhancement was hypothesized to be
related to the uptake of intracellular nutrients by
bacteria which survived protozoan ingestion. To
test this ingestion/survival phenomenon, we developed a technique of chlorination and sonication
of cocultures which showed that viable cells of
both bacteria were contained within the protozoa.
This implicated the importance of ingestion and
survival from digestive processes for the increased
growth of each bacterium.
2. I N T R O D U C T I O N
Obligate pathogenic bacteria normally do not
thrive in environments outside their primary host.
Therefore, studies of the etiology of bacterial
Correspondence to: C.H. King, Department of Medical Microbiology, University of Georgia, Athens, GA 30605, U.S.A.
pathogens have been directed toward examining
the mechanisms for survival of organisms when
they are not associated with their host.
To survive, pathogenic bacteria must find a
carbon and energy source of suitable quality and
concentration outside the host organism. A common mechanism used to obtain carbon and energy
sources is through close physical association with
other microorganisms. Enhancement of natural
bacteria through interspecies interactions is common in aquatic ecosystems [1-3]. Recently, enhancement of the pathogenic bacterium Vibrio
cholerae by attachment to crustacean zooplankton
has been documented in marine systems [4]. Vibrio
parahaemolyticus has also been isolated from zooplankton collected in freshwaters [5].
Another example of microbial-microbial interactions was demonstrated by a study in which
Legionella pneumophila were shown to proliferate
within cyanobacterial mats in fresh water [6].
Given the fastidious nature of this bacterium, this
microbial interaction was believed to be advantageous to the survival of L. pneurnophila in this
freshwater system. Recent studies on the role of
protozoa in the spread of Legionella have shown
that this bacterium is capable of infecting proto-
0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies
96
zoa, with subsequent enhancement through this
association [7-9]. Intracellular growth in ciliated
and amoeboid forms of protozoa was described as
a possible mechanism for survival of Legionella in
natural waters.
Microbial survival in surface waters as it applies to bacterial-protozoan ecology is well documented. Ecological data support the role that
bacterial-protozoan interactions play in aquatic
food webs [10-12]. Protozoan predation on
bacterial populations serves as a catalyst for the
remineralization and recycling of elements essential for microbial growth [13]. Studies have also
shown seasonal growth of certain protozoan populations, with concurrent increases in bacterial
populations [14].
In this study, the effects of static coculturing on
the survival of two common bacterial pathogens of
freshwater fish (Aeromonas salmonicida and
Edwardsiella tarda) with a freshwater ciliated protozoan (Tetrahymena pyriformis) were investigated. T. pyriformis was selected because it is
ubiquitous in fresh water and feeds primarily on
bacteria [15]. Bacteria were also cultured in lysed
cell suspensions and supernatant of fractured cells
of T. pyriformis. A new method is described for
the isolation of viable intracellular bacteria from
their ciliated protozoan host using sodium hypochlorite. In addition, the importance of these interactions to the survival and enhancement of
aquatic bacterial pathogens in nutrient-poor
freshwater ecosystems is discussed.
3. MATERIALS A N D M E T H O D S
Bacterial isolates used in this study were obtained from bacterial stocks used in ongoing virulence studies in this laboratory. Aeromonas
salmonicida were maintained and enumerated on
RS medium [16], and Edwardsiella tarda on blood
agar medium. Bacterial coculture inocula were
prepared by growing each species in BHI medium
(Scott Lab., Inc.) at 37 ° C for 24 h, pelleting by
centrifugation ( 4 0 0 0 × g ) for 10 min at 4 ° C ,
washing 3 times with sterile saline solution (0.85%),
and resuspending in saline solution. Bacterial
numbers were adjusted to 103 cfu/rnl using spec-
trophotometric absorbance measurements (wavelength, 660 nm) with a Spectronic 2000 (Bausch
and Lomb). Serial ten-fold dilutions in sterile
saline solution were spread-plated on agar medium
for enumeration of colony forming units (cfu).
Tetrahymena pyriformis (Strain ATCC No.
30327) broth cultures were grown in 100 ml of
Elliotts Medium No. 2 at 2 5 ° C for 48 h [16]. Cells
were prepared for coculture by gravity filtration
through 0.8 /~m filters (Millipore Corp.). Cells
were then resuspended in a 1 : 1 solution of broth
and saline solution at 25 ° C for 24 h to reduce
osmotic shock before filtration and resuspension
into 100% saline solution. Protozoa were enumerated by adding a drop of 37% formaldehyde to
a 1 ml portion of cell suspension, counting fixed
cells on a hemocytometer, and adjusting to 10 4
cells/ml. Lysed washed cell suspensions and
filtered cellular supernatant were prepared in
saline solution [9].
Separate cocultures of each bacterium with T.
pyriformis were prepared in 100 ml jars using 2.5
ml of each bacterial species (103 cfu/ml) with 22.5
ml of T. pyriformis (10 4 cells/ml) in saline solution. Controls were established by inoculating 2.5
ml of each bacterium (103 c f u / m l ) into 22.5 ml of
saline solution, 22.5 ml of lysed washed cells suspension, and 22.5 ml of filtered lysed cell supernatant, respectively. Bacteria were enumerated
(cfu) in samples each 24 h for 3 days.
Sodium hypochlorite experiments were designed to expose extracellular bacteria to lethal doses
of chlorine residuals and to prevent disruption of
cells containing ingested bacteria. Separation of
intracellular bacteria in each coculture was performed using a 5% solution of sodium hypochlorite (Aldrich Chemical Co.). Diluted chlorine residuals (in saline solution) were measured using
the DPD colorimetric method [17]. The chlorine
demand of the saline solution was measured to
facilitate correct chlorine residuals in treated samples.
Treatments consisted of inoculating 2 ml of 3 h
coculture suspensions into 19 ml of a 4 m g / l free
available chlorine solution (pH 7.0, 25 ° C). These
samples were then slowly shaken on a Model G-2
gyratory shaker (New Brunswick Scientific Co.)
for 10 min, neutralized with 2 ml of sodium thio-
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sulfate, and filtered through a 0.45/zm filter (Millipore) to concentrate protozoan cells. Samples
were monitored microscopically for viable or intact protozoan cells, and then sonicated for 10 s at
40 watts using a microtip probe (Heat-SystemsUltrasonics, Inc.). A separate aliquot of cells was
sonicated first, then chlorinated to serve as a
control. All suspensions were then spread-plated
onto agar medium (as above) for the detection of
viable bacteria.
Scanning electron micrographs were prepared
using cells taken from 3 h cocultures. Specimens
were agar-embedded, ethanol-infiltrated, and
cryofractured [18]. Cryofractured cells were viewed
with a Phillips 505 scanning electron microscope.
4. RESULTS A N D DISCUSSION
Upon the addition of 103,E. tarda to the saline
control, the numbers declined by more than two
magnitudes within three days (Fig. 1A). However,
when inoculated into saline solution with T. pyriformis, the number of E. tarda (cfu/ml) increased
by 2-3-fold. These data suggested that abiotic
factors may influence the enhancement of E. tarda
when in coculture with T. pyriformis, so we ex-
A
amined possible nutritional factors that the protozoa might supply to E. tarda. The average number
of E. tarda remained constant when incubated
with filtered, washed, lysed cells, but increased an
average of 2-fold when incubated with the filtered
supernatant of T. pyriformis (Fig. 1A).
Aeromonas sahnonicida were shown to increase
two fold over the initial inoculum when in coculture with T. pyriformis (Fig. 1B). Saline controls
did not support the growth of these bacteria. We
observed enhancement of A. salmonicida when
incubated with lysed cells or supematant of lysed
cells, with the latter incubation producing the
most efficient bacterial growth system. Electron
microscopic examination of T. pyriformis cells in
coculture with each bacterium showed vacuolation
of bacteria, with large numbers of bacteria in each
vacuole. The SEM cryofracture preparations of
protozoan ceils before sonication showed protozoan cells with bacteria-packed vacuoles (Fig. 2).
Experiments designed to isolate viable intracellular bacteria using sodium hypochorite were successful. We showed that intact T. pyriformis cells
could be cultured in Elliotts broth (when washed
in saline solution) after treatment with 4 mg/1 free
chlorine residuals. Studies showed that both
bacterial isolates were killed when protozoan cells
from coculture were sonicated first and then chlo-
B
_'l
'l
.
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Fig. 2. Electron photomicrograph of a cryofractured cell of Tetrahymenapyriformis showing vacuoles containing bacteria (B). × 5400.
rinated. When coculture protozoa from each experiment were chlorinated, neutralized, and sonicated, we observed growth of E. tarda and A.
salmonicida upon subsequent plating, indicative of
the recovery of viable intracellular bacteria from
T. pyriformis.
The presence of viable intracellular bacteria
could indicate a possible survival of these species
from protozoan digestive processes, and might
point to an enhancement mechanism for the
growth of these and possibly other bacteria
through protozoan ingestion. The enhancement of
E. tarda and A. salmonicida when cultured with
filtered supernatant of T. pyriformis shows that
bacterial growth may have been stimulated by
intracellular components. In the E. tarda experiments, this was further shown by the inability of
washed, lysed cell debris to support bacterial
growth. Previous studies in our laboratory (data
not shown) have shown no enhancement of either
bacterium when incubated in 0.22 /~m filtered
(Millipore) saline solution from saline-grown T.
pyriformis with and without killed bacteria. This
indicates that enhancement of these bacteria was
not due to excretion of protozoan extracellular
nutrients alone.
Our study documents the enhancement of E.
tarda and A. salmonicida through association with
T. pyriformis. In the case of E. tarda, there seemed
to be an intracellular requirement for enhancement. These studies support other work [19] which
has suggested that freshwater protozoan feeding of
bacterial populations may enhance the growth of
the bacteria. Our studies also provide a new technique for the separation of intracellular and extracellular bacteria from ciliated protozoans using
free chlorine residuals. These techniques can be
applied to initial studies of bacterial-protozoan
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i n t e r a c t i o n s r e l a t e d to v i a b l e i n t r a c e l l u l a r b a c t e r i a l
multiplication.
ACKNOWLEDGEMENT
T h i s s t u d y was s u p p o r t e d b y U n i t e d States
D e p a r t m e n t of A g r i c u l t u r e g r a n t n u m b e r 10-21R R 2 1 1 - 0 5 1 a n d is C o n t r i b u t i o n N u m b e r 2688 of
the V e t e r i n a r y M e d i c i n e E x p e r i m e n t a l S t a t i o n .
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