Microb Ecol (1995) 30:183-192
MICROBIAL ECOLOGYInc. © 1995Springer-Verlag New York
Dormant/Unculturable Cells of the Fish Pathogen A e r o m o n a s salmonicida
I. Effendi, B. Austin Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh, EHI4 4AS, Scotland, U.K. Received: 23 September 1994; Revised: 14 December 1994
Abstract. Viable cells of Aeromonas salmonicida remained in experimental marine systems after plate counts indicated an absence of culturable cells. These so-called/viable but nonculturable (VBNC) cells were coccoid and smaller than their normal culturable counterparts. There was no reduction in lipopolysaccharide of the VBNC cells. There was an alteration in protein composition, however, with a decline in some (15, 70, 30, 22, and 17 kDa), but an increase in another protein (49 kDa). A significant loss of DNA occurred. The VBNC cells responded to fluorescent antibodies prepared against A. salmonicida by developing enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), and they demonstrated respiratory activity. It was concluded that A. salmonicida survived in seawater, but major morphological changes occurred with cells retaining some viability but losing pathogenicity to Atlantic salmon (Salmo salar). Introduction Aeromonas salmonicida, the causal agent of furunculosis, has a wide distribution, diverse host range, and is economically devastating to salmonids [4]. By definition, the organism is restricted to fish and is not found in surface waters [31]. Yet this definition does not help explain the spread of A. salmonicida between fish populations. There is controversy as to whether or not the organism is capable of a free-living existence in the natural environment away from the fish host. Some workers considered that cells did not survive for prolonged periods in the aquatic environment [22, 23, 33]. In contrast, others pointed to the presence of intact and apparently viable cells in water [1, 13, 26]. These cells, however, did not produce colonies on routine bacteriological media. The question to be resolved concerned whether or not such cells could be regarded as truly viable. This study has continued
Correspondence to: B. Austin, Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland, U.K.
184
I. Effendi, B. Austin
the previous work of Effendi and Austin [12, 13] by examining the characteristics of so-called viable but non-culturable (VBNC) cells of A. salmonicida. Materials and Methods
Bacterial Cultures Two virulent cultures ofA. salmonicida (256/91 and AS20), isolated from clinically diseased Atlantic salmon (Salmo salar L.) in Scotland during 1991, were maintained on tryptone soya agar (TSA; Oxoid, Basingstoke, England) at 4°C, with subcultufing every 7-14 days. Stock cultures were freeze dried. Cultures produced three different colony types, termed "rough", "smooth", and "G-phase", which are normally associated with high, low, and intermediate virulence, respectively [4].
VBNC Cells VBNC cells were established, as described elsewhere [13]. Essentially, 48- to 72-h cultures of A. salmonicida (AS20 and 256/91) on TSA plates were washed three times in sterile (121°C/15 min) 0.85% (w/v) saline before suspension in 100 ml of sterile (121°C/15 min) seawater (salinity = 25 %0) in conical flasks to achieve approximately 107-108 cells/per milliliter. The flasks were covered with aluminium foil, to prevent possible interference from light, and incubated at 18°C. The presence of culturable ceils was examined daily by spreading 0.1-ml samples on triplicate plates of TSA and L-form (L-F) medium [24], with incubation at 22°C for 3-4 days. The inoculated media were also examined at a magnification of x40 and X 100 with a Kyowa (Tokyo, Japan) stereomicroscope to determine the presence of microcolonies. This process was repeated until the colony count reached zero. The system was then kept for 3 weeks before further examination, when the entire contents of duplicate flasks were inoculated into equal volumes of double-strength tryptone soya broth (TSB; Oxoid) and incubated at 18°C for 7 days. The absence of turbidity was used as a final indicator for the lack of culturability. Parallel systems, deemed to be devoid of culturable cells, were filtered (0.45~m Millipore [Edinburgh, Scotland] Millex porosity filters), and the filtrate was retained for the examination of filterable cell forms.
Transmission Electron Microscopy (TEM) Bacterial suspensions were fixed overnight in 0.2% (v/v) buffered (pH 7.4) glutaraldehyde (EMscope Watford, England). One drop (0.05 ml) was pipetted onto a plastic (Formvar; Merck, Poole, England) coated copper electron microscope grid, and stained with an equal volume of 1% (w/v) phosphotungstic acid (EMscope). The grids were placed on filter paper and air dried at room temperature. The specimens were examined promptly in an AEI (Manchester, England) EM6G transmission electron microscope.
Determination of Viability The determination of viability included use of the indirect fluorescent antibody technique (iFAT), the development of enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), epifluorescence microscopy following staining with acridine orange, the assessment of respiratory activity by the reduction of tetrazolium, and culturability on TSA and L-F medium. Cells killed by autoclaving (121°C/15 min) or formalin (0.5% v/v for 15 min) were used for controls. All specimens for microscopy were examined at magnifications of x400 and x 1000 on a Carl Zeiss (Welwyn Garden City, England) Axiophot microscope. The iFAT technique described by Schill et al. [35] was used. Thus, 100-~1 volumes of seawater containing A. salmonicida were air dried onto microscope slides and mixed with 20-~1 amounts of a
Unculturable Cells of Aeromonas salmonicida
185
diluted (1:200 dilution in phosphate-buffered saline; PBS, Oxoid) polyclonal antiserum to A. salmonicida (titer by whole cell agglutination = 1:2,048), which was produced in female New Zealand white rabbits. Incubation was at 37°C for 30 rain in a humid chamber. Following two washings in PBS, the slides were air dried before the addition of 20-tzl amounts of a 1:20 dilution (in PBS) of fluoresceinlabeled horseradish peroxidase conjugated to donkey antirabbit immunoglobulin G (IgG) (SAPU; Scottish Antibody Production Unit Carluke, Scotland) with incubation for a further 30 rain in a darkened chamber. After thorough washing with PBS, the slides were mounted in carbonate-buffered glycerol (Sigma, St. Louis, Mo). Direct viable counts were assessed by the method of Kogure et al. [19]. Seawater samples containing A. salmonicida were mixed with 0.025% (w/v) yeast extract (Oxoid) to permit cell growth and 0.002% (w/v) nalidixic acid (Sigma), which stopped cell division. After incubation for 6 h at room temperature, viable cells were observed to enlarge and develop bizarre shapes. The epifluorescence microscopy technique outlined by Morgan et al. [26] was used. Acridine orange (CI 46005, Sigma) was mixed with the marine samples in the ratio of 1:99 and incubated in a dark chamber for 5 min before transfer to microscope slides. The samples were examined to determine the presence and nature (green or orange) of the fluorescence. Respiratory activity was measured using the procedures of Austin [3] and Zimmerman et al. [39], and involved use of 0.2% (w/v) 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolinm bromide (MTT; Sigma) or 0.2% (w/v) 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT; Sigma) prepared in filtered (0.22-txm pore size Millipore Millex porosity filters) seawater. MTT and INT were mixed separately with equal volumes of seawater containing A. salmonicida and examined after incubation for 5 rain at room temperature in a darkened chamber. Respiratory activity was assessed by the deposition of insoluble formazan, as granules, within the bacterial cells.
Examination o f Subcellular Components Whole cell proteins, outer membrane proteins (OMP), chromosomal DNA, and lipopolysaccharide profile (LPS) of VBNC cells were examined by the methods of Loghothetis and Austin [20]. Proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4% stacking gel and 12% separating gel on a Biorad (Hemel Hempstead, England) Wide Mini Sub Cell model 1,000/500 electrophoresis unit. Samples (10 ixl) were mixed with an equal volume of loading buffer, and volumes of 10-20 ~1 were loaded into tile gel and run for 3 h at a constant voltage of 150 V, using reservoir buffer, that is, Tris-glycine (pH 8.3) containing 0.1% SDS. After electrophoresis, the protein samples were stained with Coomassie brilliant blue R-250 (Boehringer Mannheim, Lewes, England). Phage X DNA digested with Hind 111was used as a molecular weight marker (NBL Gene Sciences, Cramlington, England; Ref. No. 030204) to determine the size of the protein bands. LPS, extracted by the methods of Schill et al. [35], was analysed by SDS-PAGE using a 4% stacking gel and 14% separating gel on a Biorad Wide Mini Sub Cell model 1,000/500 electrophoresis unit. Samples (10 Ixl) of LPS were mixed with an equal volume of loading buffer, and 10- to 20-1xl amounts were run for 3 h at a constant voltage of 150 V, using reservoir buffer, that is, Tris-glycine (pH 8.3) containing 0.1% SDS. After electrophoresis, the LPS was stained with silver [38]. Molecular weight markers were included, as before. DNA was extracted using the alkaline SDS methods of Kado and Liu [17] and Portnoy et al. [32]. The cells were washed three time with riffs buffer (50 mM-Tris, HC1, 0.1 M NaC1; pH 8.0); resuspended in 100 txl of lysis buffer (50 mM-Tris acetate, 3% SDS; pH 12.4); and incubated at 95°C for 5 rain. The suspension was briefly extracted with equal volumes of phenol/chloroform (1:1), and the emulsion was separated by centrifugation (15,000 rpm/10 rain), with the aqueous phase removed. Equal volumes of isopropanol and 15% (w/v) sodium acetate were added and mixed gently. The preparation was then kept in -70°C for 15 rain, centrifuged (14,000 rpm/10 rain), washed with 70% ethanol, recentrifuged (14,000 rproJl0 rain), and the pellet was vacuum dried. DNA samples were diluted with distilled water, mixed with loading buffer (4/1; 50% glycerol, 1 mM ethylenediamine tetraacetic acid (EDTA), 0.05% bromophenol blue, 0.05% xylene cyanol FF), and 20-1,1 volumes were separated by electrophoresis on a Biorad Wide Mini Sub Cell model 1,000/500 electrophoresis unit through 0.8% agarose (BioRad) in distilled water and buffer (50:1; 89 rnM Tris, 2.5 m~ NaiEDTA, and 89 mM boric acid, pH
186
I. Effendi, B. Austin
8.2) at 15 V for 12 h. The gel was stained with ethidium bromide and examined at 302 nm with an ultraviolet Transilluminator (UV P Inc., San Gabriel, Ca model TM36). Reference plasmids of 0.564, 2.0, 2.3, 4.3, 6.5, and 9.4 kb (NBL Gene Sciences) were included for comparison.
Fish Experiments Atlantic salmon (in groups of six) of approximately 20 g each were maintained in fiberglass tanks (100 × 100 × 25 cm) containing aerated seawater (salinity = 25%0) at 16-18°C. Portions (0.1 ml) were taken from filtered (0.45-tzm Millipore, Millex porosity filters) and VBNC cell systems and injected intramuscularly into the fish. The animals were observed daily for 2 weeks. The kidney and spleen of any dead or moribund animals or representatives of the survivors after 14 days were examined by plating a loopful of material onto TSA and L-F medium with incubation at 22°C for 3-4 d. To determine the possible presence of carriers of A. salmonicida, the survivors from duplicate groups were injected intramuscularly with the immunosuppressant prednisolone acetate (20 mg/kg body weight; Boots Deltastab, Nottingham, England). The fish were then returned to the aquarium and observed for another 14 days. The presence of culturable A. salmonicida was examined, as above.
Immunohistochemistry The presence ofA. saImonicida cells in spleen and kidney was also examined by immunohistochemical techniques. Thus, --0.05-g samples of tissue were removed; fixed in 3-ml volumes of freshwater Davidson's tissue fixer [36] (20% [v/v] formalin, 20% [v/v] glycerol, 10% [v/v] glacial acetic acid, 30% [v/v] absolute alcohol, and 30% [v/v] distilled water); and incubated at room temperature for 24-48 h. Thereafter, the tissue fixative was removed and replaced by 3 ml of 70% (v/v) ethanol, and kept at 5°C until processed using the technique described by Loghothetis and Austin [20]. Polyclonal rabbit antiserum to whole cells and lysozyme-induced L-forms of A. salmonicida was diluted 1:200 [25]. Tissue samples were incubated with this antiserum for 30 rain in a humid chamber. After washing three times in PBS, the secondary antibody, a horseradish-peroxidase goat anti-rabbit IgG conjugate (SAPU), diluted 1:1,000, was applied with incubation for 30 min. The sections were washed three times in PBS, then incubated with the chromogenic substrate 3,3'diaminobenzidine tetrahydrochloride (DAB; Boehringer Mannheim). After three washings in PBS, the sections were counterstained with Harris hematoxylin for 2 rain and dehydrated through a series of ethanol solutions: 50%, 75%, 96%, and 100% (v/v) followed by a 30-s dip in Histosol No. 2. Coverslips were mounted using Eukitt. The slides were dried on a hot plate, then examined microscopically, as before.
Results O n c e cells b e c a m e nonculturable, they were o b s e r v e d to be c o c c o i d and surrounded b y extracellular material (Fig. 1). C o m p a r e d to culturable cells (Fig. 2), these forms d e m o n s t r a t e d diffuse edges and, b y T E M , were less electron dense. T h e cocci were a p p r o x i m a t e l y 20% o f the v o l u m e o f n o r m a l r o d - s h a p e d cells (Fig. 2). In comparison, cells that had p a s s e d through the pores o f 0.45-txm p o r e - s i z e filters, were also coccoid, albeit with irregular edges, being a p p r o x i m a t e l y 50% o f the size o f n o r m a l culturable cells. By T E M , these cells a p p e a r e d to have central electron-dense sunken areas. Generally, an overall reduction o f protein content o f the V B N C c o m p a r e d to the n o r m a l cells o f both strains (Fig. 3) was observed. In the V B N C cells, there was a decrease in proteins with m o l e c u l a r weights o f 115, 70, 30, 22, and 17 kDa. In contrast, however, the expression o f the 4 9 - k D a protein a p p e a r e d to be increased, that is, the A - l a y e r protein [4] (Fig. 3).
Unculturable Cells of Aeromonas salmonicida
187
Fig. 1. Transmission electron micrograph of a VBNC cell of A. salmonicida 256/91. Bar = 0.4 ~m.
Fig. 2. Transmission electron micrograph of a "normal" culturable cell of A. salmonicida 245/91. Bar = 1 p~m.
From SDS-PAGE, it was determined that there was no change in LPS profiles between normal and VBNC cells, insofar as two LPS bands of 99 and 50 kDa were observed in both cell types (Fig. 4). However, a significant loss of D N A was observed in the VBNC cells (Fig. 5). Certainly, the data revealed that neither filtered cells nor those from systems considered to be devoid of colony-forming units, that is, those containing VBNC cells, produced visible or even microcolonies on TSA or L-F medium. Moreover, no evidence was found for the presence of morphological variants, such as L-form colonies on L-F medium. Further examination determined that these cells responded like normal cells to iFAT, the direct viable count technique in which enlarged and bizarre-shaped cells developed, and respiratory activity, which was indicated by the deposition of colored formazan from the reduction of the tetrazoliums. Therefore, the evidence suggests that the cells are intact and seemingly viable. However, it was not determined whether or not the cells were actually damaged.
188
I. Effendi, B. Austin
Fig. 3. SDS-PAGEof whole cell proteins of normal and VBNC cells of, A, A. salmonicida 256/91 and, B, AS20. The protein samples are, a, lanes 1-4, culturable cells of 256/91; lane 5, molecular weight markers; lane 6-10, VBNC cells of 256/91; and, b, lane 1-4, culturable cells of AS20; lane 5, molecular weight markers; lane 6-10, VBNC cells of AS20. Injection of filtered and unculturable cells ofA. salmonicida into Atlantic salmon did not result in clinical disease. In fact, disease did not develop even after the use of prednisolone acetate. Moreover, bacterial cells were not recovered from the kidney and spleen of these fish. Supporting evidence was provided by the examination of kidney and spleen by immunohistochemistry, when chromogenic reactions for A. salmonicida were not observed in the tissues. Therefore, no evidence was found for the persistence of these cell types in fish themselves.
Discussion The existence of so-called dormant cells of A. salmonicida was first described by Allen-Austin et al. [1]. Yet, this concept of dormancy/nonculturability has been
Unculturable Cells of Aeromonas salmonicida
189
Fig. 4. SDS-PAGE of LPS profiles of normal and VBNC cells ofA. salmonicida 256/91 and AS20. The LPS samples are, as follows: lane 1, culturable cells of 256/91; lanes 2 and 3, VBNC cells of 256/91; lane 4, molecular weight markers; lanes 5 and 6, VBNC cells of AS20; and lanes 7 and 8, culturable cells of AS20.
Fig. 5. DNA profiles of normal and VBNC ceils of A. salmonicida 256/91 and AS20. The DNA samples are, as follows: lanes 1 and 2, culturable cells of 256/91; lanes 3 and 4, VBNC cells of 256/91; lane 5, culturable cells of AS20; lanes 6 and 7, VBNC cells of AS20; lane 8, molecular weight markers.
contested b y s o m e researchers. R o s e et al. [33] c o n t e n d e d that it w o u l d be difficult to p r o v e c o n c l u s i v e l y that culturable cells were really absent f r o m aquatic samples. Thus, resuscitation, as d e s c r i b e d p r e v i o u s l y [1], was c o n s i d e r e d to be attributed to the p r e s e n c e o f a small r e s i d u u m o f culturable cells. Similarly, M o r g a n et al. [26] i n d i c a t e d that the presence o f low n u m b e r s o f viable cells within samples c o u l d be r e s p o n s i b l e for apparently m i s l e a d i n g results. Certainly, w e have shown that V B N C cells o f A . s a l m o n i c i d a r e m a i n e d in e x p e r i m e n t a l systems after culturing
190
I. Effendi,B. Austin
indicated a total absence of colony-forming units. This is in accordance with the view of Morgan et al. [27], who reported the presence of such VBNC cells of A. salmonicida in a lake water system. These workers used rhodamine as an indicator of membrane potential and showed that the absence of culturability was associated with retention of viability. The VBNC cells have been shown to be intact and contain DNA. This indicates that they are more than just empty cells [26]. However, the precise physiological state of the cells has not been clearly resolved from the methods used to date. The data in this study indicated that the VBNC cells of A. salmonicida remained intact and responded like culturable cells to iFAT. Yet, a disadvantage is that iFAT cannot differentiate between truly viable cells, which will grow in standard bacteriological media, VBNC, freshly dead cells, or even cellular debris [7, 10]. This problem was addressed by Kogure et al. [19], who devised the direct viable count technique to identify viable cells in mixed microbial communities. In this study, the VBNC cells of A. salmonicida reacted by developing enlarged and bizarre shapes after the addition of yeast extract and nalidixic acid. This is indicative of viability. Similarly, the response of the cells to tetrazolium dyes also suggest viability [6, 30, 34]. However, the cells did not produce colonies on TSA or L-F medium. Thus, doubt remains about whether the cells could be dying and, therefore, incapable of resuscitation. The significance of VBNC cells ofA. salmonicida would be clearly established if, like with Vibrio cholerae [10], there was a clearly defined role in pathogenicity. Yet in this study, injection of VBNC cells of A. salmonicida into Atlantic salmon did not result in clinical disease, even after the use of an immunosuppressant. Certainly, other investigators have also questioned the role in pathogenicity of such forms of A. salmonicida [26, 33]. An assumption could be made that the cells were destroyed by the defense system of the fish. It is indeed questionable whether or not VBNC cells of A. salmonicida in the marine environment could be involved in outbreaks of furunculosis. Of course, it is possible that the cells could regain virulence. In this respect, osmotically fragile cells, that is, L-forms of A. salmonicida, which also failed to grow on conventional bacteriological media, have been shown to revert to normal culturable cells, which were virulent [25]. The reduction in size of the VBNC cells ofA. salmonicida to a coccoid morphology is in agreement with changes of aquatic bacteria associated with nutrient deprivation, that is, starvation-survival [28]. Other fish pathogens, notably Yersinia ruckeri [37] and Pasteurella piscicida [21] have also been reported to assume a coccoid morphology during starvation. It is interesting that the VBNC cells of A. salmonicida maintained a rough appearance, with an external protein matrix attached to the cell surface [26]. This coincides with previous work, which found external protein matrices on marine Vibrio [5, 11, 18]. The synthesis or alteration of expression of certain proteins is considered essential for the long-term survival strategy of starving bacteria [ 18]. For example, Nystrom et al. [29] observed the synthesis of protein in Vibrio sp. during the initial 2-4 h and after 1 week of carbon starvation. Therefore, the results with A. salmonicida are not surprising. Similarly, the lack of any substantive change in LPS profiles is in line with observations of other nonculturable fish pathogens, such as P. piscicida [21]. The reduction of DNA in the nonculturable cells of A. salmonicida is in line
Unculturable Cells of Aeromonas salmonicida
191
with previous studies. For example, a decline of DNA content of Vibrio during starvation has been reported [2, 15, 16]. Conversely, plasmid DNA was maintained in VBNC cells of E. coli [8, 9, 14] and P. piscicida [21]. Thus, it may be concluded that, in similarity with previous work in freshwater systems [26, 27], A. salmonicida survives in the marine environment after plate counts have declined to zero. The surviving cells undergo major morphological change, retain some viability, but lose pathogenicity. References 1. Allen-Austin D, Austin B, Colwell RR (1984) Survival ofAeromonas salmonicida in fiver water. FEMS Microbiol Lett 21:143-146 2. Amy PS, Pauling C, Morita RY (1983) Starvation-survival processes of a marine vibrio. Appl Environ Microbiol 45:1041-1048 3. AustinB (1987) A rapid method for the determination of antibiotic resistance in baeterial pathogens within diseased specimens. FEMS Microbiol Lett 43:295-300 4. Austin B, Austin DA (1993) Bacterial fish pathogens disease of farmed and wild fish, 2nd ed. Simon & Schuster, Chichester 5. Baker DA, Park RWA (1975) Changes in morphology and cell wall structure that occur during growth of Vibrio sp. NCTC44716 in batch culture. J Gen Microbiol 86:12-28 6. Bej AK, Mahbubani MH, Atlas RM (1991) Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl Environ Microbiol 57:597-600 7. Brayton PR, Tamplin ML, Huq A, Colwell RR (1987) Enumeration of Vibrio cholerae 01 in Bangladesh waters by fluorescent-antibody direct viable count. Appl Environ Microbiol 53:2862-2865 8. Byrd JJ, Colwell RR (1990) Maintenance of plasmids pBR322 and pUC8 in non-culturable Escherichia coli in the marine environment. Appl Environ Microbiol 56:2104-2107 9. Caldwell BA, Ye C, Griffiths RP, Moyer CL, Morita RY (1989) Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water. Appl Environ Microbiol 55:1860-1864 10. Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM (1985) Viable but nonculturable Vibrio cholerae and related pathogens in the environment: implication for release of genetically engineered microorganisms. Bio/Technol 3:817-820 11. Dawson MP, Humphrey BA, Marshall KC (1981) Adhesion: a tactic in the survival strategy of marine vibrio during starvation. Curr Microbiol 6:195-199 12. Effendi I, Austin B (1991) Survival of Aeromonas salmonicida in seawater. FEMS Microbiol Lett 84:103-106 13. Effendi I, Austin B (1994) Survival of the fish pathogen Aeromonas salmonicida in the marine environment. J Fish Dis 17:375-385 14. Flint KP (1987) The long-term survival of Escherichia coli in river water. J Appl Bacteriol 63:261-270 15. Hoff KA (1989) Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities. Appl Environ Microbiol 55:1775-1786 16. Hood MA, Gujkert JB, White DC, Deck F (1986) Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA and protein levels in Vibrio cholerae. Appl Environ Microbiol 52:788-793 17. Kado CI, Liu S (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365-1373 18. Kjelleberg S, Humphrey BA, Marshall KC (1982) Effect of interfaces on small, starved marine bacteria. Appl Environ Microbiol 43:1166-1172 19. Kogure K, Simidu U, Taga N (1979) A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 25:415-420 20. Loghothetis PN, Austin B (1994) Immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila. Fish Shellfish Immunol 4:239-254
192
I. Effendi, B. Austin
21. Margarinos B, Romalde JL, Barja JL, Toranzo AE (1994) Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment. Appl Environ Microbiol 60:180-186 22. McCarthy DH (1977) Some ecological aspects of the bacterial fish pathogen Aeromonas salmon# cida. Aquat Microbiol 6:299-324 23. McCarthy DH, Roberts RJ (1980) Furunculosis of fish--the present state of our knowledge. In: Droop MR and Jannasch HW (eds) Advances in aquatic microbiology, Academic Press, London, pp 293-341 24. McIntosh D, Austin B (1988) Comparison of methods for the induction, propagation and recovery of L-phase variants of Aeromonas spp. J Diarrh Dis Res 6:131-136 25. McIntoshD, Austin B (1990) Recovery of cell wall deficient forms (L-forms) ofthefishpathogens Aeromonas salmonicida and Yersinia ruckeri. Syst Appl Microbiol 13:378-381 26. Morgan JAW, Cranwell PA, Pickup RW (1991) Survival ofAeromonas salmonicida in Lake water. Appl Environ Microbiol 57, 1777-1782 27. Morgan JAW, Rhodes G, Pickup RW (1993) Survival of nonculturable Aeromonas salmonicida in lake water. Appl Environ Microbiol 59:874-880 28. Morita RY (1982) Starvation-survival of heterotrophic bacteria in the marine environment. Adv Microb Ecol 6:171-198 29. Nystrom T, Flardh K, Kjelleberg S (1990) Responses to multiple-nutrient starvation in a marine Vibrio sp. strain CCUG15956. J Bacteriol 172:7085-7097 30. Oliver JD, Nilson L, Kjelleberg S (1991) Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl Environ Microbiol 57:2640-2644 31. Popoff M (1984) Genus III. Aeromonas Kluyver and Van Niel 1936, 398 AL. In: Krieg NR, Holt JG (eds) Bergey's manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, pp 545-548 32. Portnoy DA, Moseley SL, Falkow S (1981) Characterization of plasmids and plasmid-association determinants of Yersinia enterocolitica pathogenesis. Infect Immun 31:775-782 33. Rose AS, Ellis AE, Munro ALS (1993) The survival ofAeromonas salmonicida subsp, salmonicida in sea water. J Fish Dis 13:205-214 34. Roslev P, King GM (1993) Application of tetrazolium salt with a water soluble formazan as indicator of viability in respiring bacteria. Appl Environ Microbiol 59:2891-2896 35. Schill WB, Bullock GL, Anderson DP (1989) Serology. In: Austin B, Austin DA (eds) Methods for the microbiological examination of fish and shellfish. Ellis Horwood, Chichester pp 98-140 36. Shaw BL, Battle HI (1957) The gross and microscopic anatomy of the digestive tract of the oyster, Crassostrea virginica Gmelin. Can J Zool 35:324-347 37. Thorsen BK, Enger O, Norland S, Hoff KA (1992) Long-term starvation survival of Yersinia ruckeri at different salinities studied by microscopical and flow cytometric methods. Appl Environ Microbiol 58:1624-1628 38. Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115-119 39. Zimmerman R, Iturriaga R, Becker-Birck J (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl Environ Microbiol 3:926-935
MICROBIAL ECOLOGYInc. © 1995Springer-Verlag New York
Dormant/Unculturable Cells of the Fish Pathogen A e r o m o n a s salmonicida
I. Effendi, B. Austin Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh, EHI4 4AS, Scotland, U.K. Received: 23 September 1994; Revised: 14 December 1994
Abstract. Viable cells of Aeromonas salmonicida remained in experimental marine systems after plate counts indicated an absence of culturable cells. These so-called/viable but nonculturable (VBNC) cells were coccoid and smaller than their normal culturable counterparts. There was no reduction in lipopolysaccharide of the VBNC cells. There was an alteration in protein composition, however, with a decline in some (15, 70, 30, 22, and 17 kDa), but an increase in another protein (49 kDa). A significant loss of DNA occurred. The VBNC cells responded to fluorescent antibodies prepared against A. salmonicida by developing enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), and they demonstrated respiratory activity. It was concluded that A. salmonicida survived in seawater, but major morphological changes occurred with cells retaining some viability but losing pathogenicity to Atlantic salmon (Salmo salar). Introduction Aeromonas salmonicida, the causal agent of furunculosis, has a wide distribution, diverse host range, and is economically devastating to salmonids [4]. By definition, the organism is restricted to fish and is not found in surface waters [31]. Yet this definition does not help explain the spread of A. salmonicida between fish populations. There is controversy as to whether or not the organism is capable of a free-living existence in the natural environment away from the fish host. Some workers considered that cells did not survive for prolonged periods in the aquatic environment [22, 23, 33]. In contrast, others pointed to the presence of intact and apparently viable cells in water [1, 13, 26]. These cells, however, did not produce colonies on routine bacteriological media. The question to be resolved concerned whether or not such cells could be regarded as truly viable. This study has continued
Correspondence to: B. Austin, Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland, U.K.
184
I. Effendi, B. Austin
the previous work of Effendi and Austin [12, 13] by examining the characteristics of so-called viable but non-culturable (VBNC) cells of A. salmonicida. Materials and Methods
Bacterial Cultures Two virulent cultures ofA. salmonicida (256/91 and AS20), isolated from clinically diseased Atlantic salmon (Salmo salar L.) in Scotland during 1991, were maintained on tryptone soya agar (TSA; Oxoid, Basingstoke, England) at 4°C, with subcultufing every 7-14 days. Stock cultures were freeze dried. Cultures produced three different colony types, termed "rough", "smooth", and "G-phase", which are normally associated with high, low, and intermediate virulence, respectively [4].
VBNC Cells VBNC cells were established, as described elsewhere [13]. Essentially, 48- to 72-h cultures of A. salmonicida (AS20 and 256/91) on TSA plates were washed three times in sterile (121°C/15 min) 0.85% (w/v) saline before suspension in 100 ml of sterile (121°C/15 min) seawater (salinity = 25 %0) in conical flasks to achieve approximately 107-108 cells/per milliliter. The flasks were covered with aluminium foil, to prevent possible interference from light, and incubated at 18°C. The presence of culturable ceils was examined daily by spreading 0.1-ml samples on triplicate plates of TSA and L-form (L-F) medium [24], with incubation at 22°C for 3-4 days. The inoculated media were also examined at a magnification of x40 and X 100 with a Kyowa (Tokyo, Japan) stereomicroscope to determine the presence of microcolonies. This process was repeated until the colony count reached zero. The system was then kept for 3 weeks before further examination, when the entire contents of duplicate flasks were inoculated into equal volumes of double-strength tryptone soya broth (TSB; Oxoid) and incubated at 18°C for 7 days. The absence of turbidity was used as a final indicator for the lack of culturability. Parallel systems, deemed to be devoid of culturable cells, were filtered (0.45~m Millipore [Edinburgh, Scotland] Millex porosity filters), and the filtrate was retained for the examination of filterable cell forms.
Transmission Electron Microscopy (TEM) Bacterial suspensions were fixed overnight in 0.2% (v/v) buffered (pH 7.4) glutaraldehyde (EMscope Watford, England). One drop (0.05 ml) was pipetted onto a plastic (Formvar; Merck, Poole, England) coated copper electron microscope grid, and stained with an equal volume of 1% (w/v) phosphotungstic acid (EMscope). The grids were placed on filter paper and air dried at room temperature. The specimens were examined promptly in an AEI (Manchester, England) EM6G transmission electron microscope.
Determination of Viability The determination of viability included use of the indirect fluorescent antibody technique (iFAT), the development of enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), epifluorescence microscopy following staining with acridine orange, the assessment of respiratory activity by the reduction of tetrazolium, and culturability on TSA and L-F medium. Cells killed by autoclaving (121°C/15 min) or formalin (0.5% v/v for 15 min) were used for controls. All specimens for microscopy were examined at magnifications of x400 and x 1000 on a Carl Zeiss (Welwyn Garden City, England) Axiophot microscope. The iFAT technique described by Schill et al. [35] was used. Thus, 100-~1 volumes of seawater containing A. salmonicida were air dried onto microscope slides and mixed with 20-~1 amounts of a
Unculturable Cells of Aeromonas salmonicida
185
diluted (1:200 dilution in phosphate-buffered saline; PBS, Oxoid) polyclonal antiserum to A. salmonicida (titer by whole cell agglutination = 1:2,048), which was produced in female New Zealand white rabbits. Incubation was at 37°C for 30 rain in a humid chamber. Following two washings in PBS, the slides were air dried before the addition of 20-tzl amounts of a 1:20 dilution (in PBS) of fluoresceinlabeled horseradish peroxidase conjugated to donkey antirabbit immunoglobulin G (IgG) (SAPU; Scottish Antibody Production Unit Carluke, Scotland) with incubation for a further 30 rain in a darkened chamber. After thorough washing with PBS, the slides were mounted in carbonate-buffered glycerol (Sigma, St. Louis, Mo). Direct viable counts were assessed by the method of Kogure et al. [19]. Seawater samples containing A. salmonicida were mixed with 0.025% (w/v) yeast extract (Oxoid) to permit cell growth and 0.002% (w/v) nalidixic acid (Sigma), which stopped cell division. After incubation for 6 h at room temperature, viable cells were observed to enlarge and develop bizarre shapes. The epifluorescence microscopy technique outlined by Morgan et al. [26] was used. Acridine orange (CI 46005, Sigma) was mixed with the marine samples in the ratio of 1:99 and incubated in a dark chamber for 5 min before transfer to microscope slides. The samples were examined to determine the presence and nature (green or orange) of the fluorescence. Respiratory activity was measured using the procedures of Austin [3] and Zimmerman et al. [39], and involved use of 0.2% (w/v) 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolinm bromide (MTT; Sigma) or 0.2% (w/v) 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT; Sigma) prepared in filtered (0.22-txm pore size Millipore Millex porosity filters) seawater. MTT and INT were mixed separately with equal volumes of seawater containing A. salmonicida and examined after incubation for 5 rain at room temperature in a darkened chamber. Respiratory activity was assessed by the deposition of insoluble formazan, as granules, within the bacterial cells.
Examination o f Subcellular Components Whole cell proteins, outer membrane proteins (OMP), chromosomal DNA, and lipopolysaccharide profile (LPS) of VBNC cells were examined by the methods of Loghothetis and Austin [20]. Proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4% stacking gel and 12% separating gel on a Biorad (Hemel Hempstead, England) Wide Mini Sub Cell model 1,000/500 electrophoresis unit. Samples (10 ixl) were mixed with an equal volume of loading buffer, and volumes of 10-20 ~1 were loaded into tile gel and run for 3 h at a constant voltage of 150 V, using reservoir buffer, that is, Tris-glycine (pH 8.3) containing 0.1% SDS. After electrophoresis, the protein samples were stained with Coomassie brilliant blue R-250 (Boehringer Mannheim, Lewes, England). Phage X DNA digested with Hind 111was used as a molecular weight marker (NBL Gene Sciences, Cramlington, England; Ref. No. 030204) to determine the size of the protein bands. LPS, extracted by the methods of Schill et al. [35], was analysed by SDS-PAGE using a 4% stacking gel and 14% separating gel on a Biorad Wide Mini Sub Cell model 1,000/500 electrophoresis unit. Samples (10 Ixl) of LPS were mixed with an equal volume of loading buffer, and 10- to 20-1xl amounts were run for 3 h at a constant voltage of 150 V, using reservoir buffer, that is, Tris-glycine (pH 8.3) containing 0.1% SDS. After electrophoresis, the LPS was stained with silver [38]. Molecular weight markers were included, as before. DNA was extracted using the alkaline SDS methods of Kado and Liu [17] and Portnoy et al. [32]. The cells were washed three time with riffs buffer (50 mM-Tris, HC1, 0.1 M NaC1; pH 8.0); resuspended in 100 txl of lysis buffer (50 mM-Tris acetate, 3% SDS; pH 12.4); and incubated at 95°C for 5 rain. The suspension was briefly extracted with equal volumes of phenol/chloroform (1:1), and the emulsion was separated by centrifugation (15,000 rpm/10 rain), with the aqueous phase removed. Equal volumes of isopropanol and 15% (w/v) sodium acetate were added and mixed gently. The preparation was then kept in -70°C for 15 rain, centrifuged (14,000 rpm/10 rain), washed with 70% ethanol, recentrifuged (14,000 rproJl0 rain), and the pellet was vacuum dried. DNA samples were diluted with distilled water, mixed with loading buffer (4/1; 50% glycerol, 1 mM ethylenediamine tetraacetic acid (EDTA), 0.05% bromophenol blue, 0.05% xylene cyanol FF), and 20-1,1 volumes were separated by electrophoresis on a Biorad Wide Mini Sub Cell model 1,000/500 electrophoresis unit through 0.8% agarose (BioRad) in distilled water and buffer (50:1; 89 rnM Tris, 2.5 m~ NaiEDTA, and 89 mM boric acid, pH
186
I. Effendi, B. Austin
8.2) at 15 V for 12 h. The gel was stained with ethidium bromide and examined at 302 nm with an ultraviolet Transilluminator (UV P Inc., San Gabriel, Ca model TM36). Reference plasmids of 0.564, 2.0, 2.3, 4.3, 6.5, and 9.4 kb (NBL Gene Sciences) were included for comparison.
Fish Experiments Atlantic salmon (in groups of six) of approximately 20 g each were maintained in fiberglass tanks (100 × 100 × 25 cm) containing aerated seawater (salinity = 25%0) at 16-18°C. Portions (0.1 ml) were taken from filtered (0.45-tzm Millipore, Millex porosity filters) and VBNC cell systems and injected intramuscularly into the fish. The animals were observed daily for 2 weeks. The kidney and spleen of any dead or moribund animals or representatives of the survivors after 14 days were examined by plating a loopful of material onto TSA and L-F medium with incubation at 22°C for 3-4 d. To determine the possible presence of carriers of A. salmonicida, the survivors from duplicate groups were injected intramuscularly with the immunosuppressant prednisolone acetate (20 mg/kg body weight; Boots Deltastab, Nottingham, England). The fish were then returned to the aquarium and observed for another 14 days. The presence of culturable A. salmonicida was examined, as above.
Immunohistochemistry The presence ofA. saImonicida cells in spleen and kidney was also examined by immunohistochemical techniques. Thus, --0.05-g samples of tissue were removed; fixed in 3-ml volumes of freshwater Davidson's tissue fixer [36] (20% [v/v] formalin, 20% [v/v] glycerol, 10% [v/v] glacial acetic acid, 30% [v/v] absolute alcohol, and 30% [v/v] distilled water); and incubated at room temperature for 24-48 h. Thereafter, the tissue fixative was removed and replaced by 3 ml of 70% (v/v) ethanol, and kept at 5°C until processed using the technique described by Loghothetis and Austin [20]. Polyclonal rabbit antiserum to whole cells and lysozyme-induced L-forms of A. salmonicida was diluted 1:200 [25]. Tissue samples were incubated with this antiserum for 30 rain in a humid chamber. After washing three times in PBS, the secondary antibody, a horseradish-peroxidase goat anti-rabbit IgG conjugate (SAPU), diluted 1:1,000, was applied with incubation for 30 min. The sections were washed three times in PBS, then incubated with the chromogenic substrate 3,3'diaminobenzidine tetrahydrochloride (DAB; Boehringer Mannheim). After three washings in PBS, the sections were counterstained with Harris hematoxylin for 2 rain and dehydrated through a series of ethanol solutions: 50%, 75%, 96%, and 100% (v/v) followed by a 30-s dip in Histosol No. 2. Coverslips were mounted using Eukitt. The slides were dried on a hot plate, then examined microscopically, as before.
Results O n c e cells b e c a m e nonculturable, they were o b s e r v e d to be c o c c o i d and surrounded b y extracellular material (Fig. 1). C o m p a r e d to culturable cells (Fig. 2), these forms d e m o n s t r a t e d diffuse edges and, b y T E M , were less electron dense. T h e cocci were a p p r o x i m a t e l y 20% o f the v o l u m e o f n o r m a l r o d - s h a p e d cells (Fig. 2). In comparison, cells that had p a s s e d through the pores o f 0.45-txm p o r e - s i z e filters, were also coccoid, albeit with irregular edges, being a p p r o x i m a t e l y 50% o f the size o f n o r m a l culturable cells. By T E M , these cells a p p e a r e d to have central electron-dense sunken areas. Generally, an overall reduction o f protein content o f the V B N C c o m p a r e d to the n o r m a l cells o f both strains (Fig. 3) was observed. In the V B N C cells, there was a decrease in proteins with m o l e c u l a r weights o f 115, 70, 30, 22, and 17 kDa. In contrast, however, the expression o f the 4 9 - k D a protein a p p e a r e d to be increased, that is, the A - l a y e r protein [4] (Fig. 3).
Unculturable Cells of Aeromonas salmonicida
187
Fig. 1. Transmission electron micrograph of a VBNC cell of A. salmonicida 256/91. Bar = 0.4 ~m.
Fig. 2. Transmission electron micrograph of a "normal" culturable cell of A. salmonicida 245/91. Bar = 1 p~m.
From SDS-PAGE, it was determined that there was no change in LPS profiles between normal and VBNC cells, insofar as two LPS bands of 99 and 50 kDa were observed in both cell types (Fig. 4). However, a significant loss of D N A was observed in the VBNC cells (Fig. 5). Certainly, the data revealed that neither filtered cells nor those from systems considered to be devoid of colony-forming units, that is, those containing VBNC cells, produced visible or even microcolonies on TSA or L-F medium. Moreover, no evidence was found for the presence of morphological variants, such as L-form colonies on L-F medium. Further examination determined that these cells responded like normal cells to iFAT, the direct viable count technique in which enlarged and bizarre-shaped cells developed, and respiratory activity, which was indicated by the deposition of colored formazan from the reduction of the tetrazoliums. Therefore, the evidence suggests that the cells are intact and seemingly viable. However, it was not determined whether or not the cells were actually damaged.
188
I. Effendi, B. Austin
Fig. 3. SDS-PAGEof whole cell proteins of normal and VBNC cells of, A, A. salmonicida 256/91 and, B, AS20. The protein samples are, a, lanes 1-4, culturable cells of 256/91; lane 5, molecular weight markers; lane 6-10, VBNC cells of 256/91; and, b, lane 1-4, culturable cells of AS20; lane 5, molecular weight markers; lane 6-10, VBNC cells of AS20. Injection of filtered and unculturable cells ofA. salmonicida into Atlantic salmon did not result in clinical disease. In fact, disease did not develop even after the use of prednisolone acetate. Moreover, bacterial cells were not recovered from the kidney and spleen of these fish. Supporting evidence was provided by the examination of kidney and spleen by immunohistochemistry, when chromogenic reactions for A. salmonicida were not observed in the tissues. Therefore, no evidence was found for the persistence of these cell types in fish themselves.
Discussion The existence of so-called dormant cells of A. salmonicida was first described by Allen-Austin et al. [1]. Yet, this concept of dormancy/nonculturability has been
Unculturable Cells of Aeromonas salmonicida
189
Fig. 4. SDS-PAGE of LPS profiles of normal and VBNC cells ofA. salmonicida 256/91 and AS20. The LPS samples are, as follows: lane 1, culturable cells of 256/91; lanes 2 and 3, VBNC cells of 256/91; lane 4, molecular weight markers; lanes 5 and 6, VBNC cells of AS20; and lanes 7 and 8, culturable cells of AS20.
Fig. 5. DNA profiles of normal and VBNC ceils of A. salmonicida 256/91 and AS20. The DNA samples are, as follows: lanes 1 and 2, culturable cells of 256/91; lanes 3 and 4, VBNC cells of 256/91; lane 5, culturable cells of AS20; lanes 6 and 7, VBNC cells of AS20; lane 8, molecular weight markers.
contested b y s o m e researchers. R o s e et al. [33] c o n t e n d e d that it w o u l d be difficult to p r o v e c o n c l u s i v e l y that culturable cells were really absent f r o m aquatic samples. Thus, resuscitation, as d e s c r i b e d p r e v i o u s l y [1], was c o n s i d e r e d to be attributed to the p r e s e n c e o f a small r e s i d u u m o f culturable cells. Similarly, M o r g a n et al. [26] i n d i c a t e d that the presence o f low n u m b e r s o f viable cells within samples c o u l d be r e s p o n s i b l e for apparently m i s l e a d i n g results. Certainly, w e have shown that V B N C cells o f A . s a l m o n i c i d a r e m a i n e d in e x p e r i m e n t a l systems after culturing
190
I. Effendi,B. Austin
indicated a total absence of colony-forming units. This is in accordance with the view of Morgan et al. [27], who reported the presence of such VBNC cells of A. salmonicida in a lake water system. These workers used rhodamine as an indicator of membrane potential and showed that the absence of culturability was associated with retention of viability. The VBNC cells have been shown to be intact and contain DNA. This indicates that they are more than just empty cells [26]. However, the precise physiological state of the cells has not been clearly resolved from the methods used to date. The data in this study indicated that the VBNC cells of A. salmonicida remained intact and responded like culturable cells to iFAT. Yet, a disadvantage is that iFAT cannot differentiate between truly viable cells, which will grow in standard bacteriological media, VBNC, freshly dead cells, or even cellular debris [7, 10]. This problem was addressed by Kogure et al. [19], who devised the direct viable count technique to identify viable cells in mixed microbial communities. In this study, the VBNC cells of A. salmonicida reacted by developing enlarged and bizarre shapes after the addition of yeast extract and nalidixic acid. This is indicative of viability. Similarly, the response of the cells to tetrazolium dyes also suggest viability [6, 30, 34]. However, the cells did not produce colonies on TSA or L-F medium. Thus, doubt remains about whether the cells could be dying and, therefore, incapable of resuscitation. The significance of VBNC cells ofA. salmonicida would be clearly established if, like with Vibrio cholerae [10], there was a clearly defined role in pathogenicity. Yet in this study, injection of VBNC cells of A. salmonicida into Atlantic salmon did not result in clinical disease, even after the use of an immunosuppressant. Certainly, other investigators have also questioned the role in pathogenicity of such forms of A. salmonicida [26, 33]. An assumption could be made that the cells were destroyed by the defense system of the fish. It is indeed questionable whether or not VBNC cells of A. salmonicida in the marine environment could be involved in outbreaks of furunculosis. Of course, it is possible that the cells could regain virulence. In this respect, osmotically fragile cells, that is, L-forms of A. salmonicida, which also failed to grow on conventional bacteriological media, have been shown to revert to normal culturable cells, which were virulent [25]. The reduction in size of the VBNC cells ofA. salmonicida to a coccoid morphology is in agreement with changes of aquatic bacteria associated with nutrient deprivation, that is, starvation-survival [28]. Other fish pathogens, notably Yersinia ruckeri [37] and Pasteurella piscicida [21] have also been reported to assume a coccoid morphology during starvation. It is interesting that the VBNC cells of A. salmonicida maintained a rough appearance, with an external protein matrix attached to the cell surface [26]. This coincides with previous work, which found external protein matrices on marine Vibrio [5, 11, 18]. The synthesis or alteration of expression of certain proteins is considered essential for the long-term survival strategy of starving bacteria [ 18]. For example, Nystrom et al. [29] observed the synthesis of protein in Vibrio sp. during the initial 2-4 h and after 1 week of carbon starvation. Therefore, the results with A. salmonicida are not surprising. Similarly, the lack of any substantive change in LPS profiles is in line with observations of other nonculturable fish pathogens, such as P. piscicida [21]. The reduction of DNA in the nonculturable cells of A. salmonicida is in line
Unculturable Cells of Aeromonas salmonicida
191
with previous studies. For example, a decline of DNA content of Vibrio during starvation has been reported [2, 15, 16]. Conversely, plasmid DNA was maintained in VBNC cells of E. coli [8, 9, 14] and P. piscicida [21]. Thus, it may be concluded that, in similarity with previous work in freshwater systems [26, 27], A. salmonicida survives in the marine environment after plate counts have declined to zero. The surviving cells undergo major morphological change, retain some viability, but lose pathogenicity. References 1. Allen-Austin D, Austin B, Colwell RR (1984) Survival ofAeromonas salmonicida in fiver water. FEMS Microbiol Lett 21:143-146 2. Amy PS, Pauling C, Morita RY (1983) Starvation-survival processes of a marine vibrio. Appl Environ Microbiol 45:1041-1048 3. AustinB (1987) A rapid method for the determination of antibiotic resistance in baeterial pathogens within diseased specimens. FEMS Microbiol Lett 43:295-300 4. Austin B, Austin DA (1993) Bacterial fish pathogens disease of farmed and wild fish, 2nd ed. Simon & Schuster, Chichester 5. Baker DA, Park RWA (1975) Changes in morphology and cell wall structure that occur during growth of Vibrio sp. NCTC44716 in batch culture. J Gen Microbiol 86:12-28 6. Bej AK, Mahbubani MH, Atlas RM (1991) Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl Environ Microbiol 57:597-600 7. Brayton PR, Tamplin ML, Huq A, Colwell RR (1987) Enumeration of Vibrio cholerae 01 in Bangladesh waters by fluorescent-antibody direct viable count. Appl Environ Microbiol 53:2862-2865 8. Byrd JJ, Colwell RR (1990) Maintenance of plasmids pBR322 and pUC8 in non-culturable Escherichia coli in the marine environment. Appl Environ Microbiol 56:2104-2107 9. Caldwell BA, Ye C, Griffiths RP, Moyer CL, Morita RY (1989) Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water. Appl Environ Microbiol 55:1860-1864 10. Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM (1985) Viable but nonculturable Vibrio cholerae and related pathogens in the environment: implication for release of genetically engineered microorganisms. Bio/Technol 3:817-820 11. Dawson MP, Humphrey BA, Marshall KC (1981) Adhesion: a tactic in the survival strategy of marine vibrio during starvation. Curr Microbiol 6:195-199 12. Effendi I, Austin B (1991) Survival of Aeromonas salmonicida in seawater. FEMS Microbiol Lett 84:103-106 13. Effendi I, Austin B (1994) Survival of the fish pathogen Aeromonas salmonicida in the marine environment. J Fish Dis 17:375-385 14. Flint KP (1987) The long-term survival of Escherichia coli in river water. J Appl Bacteriol 63:261-270 15. Hoff KA (1989) Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities. Appl Environ Microbiol 55:1775-1786 16. Hood MA, Gujkert JB, White DC, Deck F (1986) Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA and protein levels in Vibrio cholerae. Appl Environ Microbiol 52:788-793 17. Kado CI, Liu S (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365-1373 18. Kjelleberg S, Humphrey BA, Marshall KC (1982) Effect of interfaces on small, starved marine bacteria. Appl Environ Microbiol 43:1166-1172 19. Kogure K, Simidu U, Taga N (1979) A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 25:415-420 20. Loghothetis PN, Austin B (1994) Immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila. Fish Shellfish Immunol 4:239-254
192
I. Effendi, B. Austin
21. Margarinos B, Romalde JL, Barja JL, Toranzo AE (1994) Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment. Appl Environ Microbiol 60:180-186 22. McCarthy DH (1977) Some ecological aspects of the bacterial fish pathogen Aeromonas salmon# cida. Aquat Microbiol 6:299-324 23. McCarthy DH, Roberts RJ (1980) Furunculosis of fish--the present state of our knowledge. In: Droop MR and Jannasch HW (eds) Advances in aquatic microbiology, Academic Press, London, pp 293-341 24. McIntosh D, Austin B (1988) Comparison of methods for the induction, propagation and recovery of L-phase variants of Aeromonas spp. J Diarrh Dis Res 6:131-136 25. McIntoshD, Austin B (1990) Recovery of cell wall deficient forms (L-forms) ofthefishpathogens Aeromonas salmonicida and Yersinia ruckeri. Syst Appl Microbiol 13:378-381 26. Morgan JAW, Cranwell PA, Pickup RW (1991) Survival ofAeromonas salmonicida in Lake water. Appl Environ Microbiol 57, 1777-1782 27. Morgan JAW, Rhodes G, Pickup RW (1993) Survival of nonculturable Aeromonas salmonicida in lake water. Appl Environ Microbiol 59:874-880 28. Morita RY (1982) Starvation-survival of heterotrophic bacteria in the marine environment. Adv Microb Ecol 6:171-198 29. Nystrom T, Flardh K, Kjelleberg S (1990) Responses to multiple-nutrient starvation in a marine Vibrio sp. strain CCUG15956. J Bacteriol 172:7085-7097 30. Oliver JD, Nilson L, Kjelleberg S (1991) Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl Environ Microbiol 57:2640-2644 31. Popoff M (1984) Genus III. Aeromonas Kluyver and Van Niel 1936, 398 AL. In: Krieg NR, Holt JG (eds) Bergey's manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, pp 545-548 32. Portnoy DA, Moseley SL, Falkow S (1981) Characterization of plasmids and plasmid-association determinants of Yersinia enterocolitica pathogenesis. Infect Immun 31:775-782 33. Rose AS, Ellis AE, Munro ALS (1993) The survival ofAeromonas salmonicida subsp, salmonicida in sea water. J Fish Dis 13:205-214 34. Roslev P, King GM (1993) Application of tetrazolium salt with a water soluble formazan as indicator of viability in respiring bacteria. Appl Environ Microbiol 59:2891-2896 35. Schill WB, Bullock GL, Anderson DP (1989) Serology. In: Austin B, Austin DA (eds) Methods for the microbiological examination of fish and shellfish. Ellis Horwood, Chichester pp 98-140 36. Shaw BL, Battle HI (1957) The gross and microscopic anatomy of the digestive tract of the oyster, Crassostrea virginica Gmelin. Can J Zool 35:324-347 37. Thorsen BK, Enger O, Norland S, Hoff KA (1992) Long-term starvation survival of Yersinia ruckeri at different salinities studied by microscopical and flow cytometric methods. Appl Environ Microbiol 58:1624-1628 38. Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115-119 39. Zimmerman R, Iturriaga R, Becker-Birck J (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl Environ Microbiol 3:926-935
