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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- 97 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 . 98 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 99 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 . REFERENCES [1] Mikhayenko, L.Y. and Kulikova, I.Y. (1973) Hydrobiol. J. 9, 32-38. [2] Caldwell, D.E. and Caldwell, S.J. (1978) Can. J. Microbiol. 24, 922-931. [3] Gallucci, K.K. and Paerl, H.W. (1983) Appl. Environ. Microbiol. 45, 557-562. [4] Hug, A., Small, E.B., West, P.A., Huq, M.I., Rahman, R. and Colwell, R.R. (1983) Appl. Environ. Microbiol. 45, 275-283. [5] Sarkar, B.L., Balakrish, G., Sircar, B.K. and Pal, S.C. (1983) Appl. Environ. Microbiol. 46, 288-290. [6] Tinson, D.L., Pope, D.H., Cherry, W.B. and Flierman, C.B. (1980) Appl. Environ. Microbiol. 39, 456-459. [7] Rowbotham, T.J. (1980) J. Clin. Pathol. 33, 1179-1183. [8] Tyndall, R.L. and Dominque, E.L. (1982) Appl. Environ. Microbiol. 44, 954-959. [9] Fields, B.S., Shotts, E.B., Feeley, J.C., Gorman, G.W. and Martin, W.T. (1984) Appl. Environ. Microbiol. 47, 467-471. [10] Pace, M.L. and Orcutt, J.D., Jr. (1981) Limnol. Oceanogr. 26, 822-830. [11] Porter, K.G. (1984) in Current Perspectives in Microbial Ecology (Klug, M.J. Reddy, C.A., Eds.) pp. 340-345. American Society of Microbiology, Washington. D.C. [12] Pratt, J.D. and Cairns, J., Jr. (1985) J. Protozool. 32, 415-423. [13] McCambridge, J. and McMeekin, T.A. (1980) Appl. Environ. Microbiol. 40, 907-911. [14] Orcutt, J.D. and Porter, K.G. (1983) Limnol. Oceanogr. 28, 720-730. [15] Elliott, A.M. (1983) Biology of Tetrahymena, pp. 1-380. Dowden, Hutchison, and Ross, Stoudsburg, PA. [16] Shotts, E.B. and Rimler, R. (1973) Appl. Environ. Microbiol. 26, 550-553. [17] American Public Health Association (1980) Standard Methods for the Examination of Water and Wastewater. 15th Edn. pp. 290-293. Am. Pub. Health Assoc., Washington, DC. [18] Humphreys, W.J., Spurlock, B.O. and Johnson, J.S. (1974) in Scanning Electron Microscopy (Corvin, I., Ed.) pp. 707-734, IITRI, Chicago, IL. [19] Sieburth, J.M. and Davis, P.G. (1982) Ann. Inst. Oceanogr. 58, 285-296.