Starvation-Survival Processes of the Bacterial Fish Pathogen Yersinia ruckeri

System. App!. Microbiol. 17, 161-168 (1994) © Gustav Fischer Verlag, Stuttgart · Jena . New York

Starvation-Survival Processes of the Bacterial Fish Pathogen

Yersinia ruckeri JESOS L. ROMALDE *, JUAN L. BARJA, BEATRIZ MAGARINOS and ALICIA E. TORANZO Departamento de Microbiologia y Parasitologia. Facultad de Biologia. Universidad de Santiago de Compostela, 15706. Spain.

Received April 15, 1994

Summary The fish pathogen Yersinia rucker; survived for more than three months in the environments studied (river, lake and estuary) .• The three strains showed similar survival dynamics, regardless of their origin or serotype. The number of culturable cells increased in the first 15 days from 1 (water microcosms) to 3-logunit (sediment microcosms), and then declined during a 100-day period. Persistence of culturable cells was greater in sediments than in waters, as well as at 6°C than at 18°C. Therefore, while a situation of long term survival could be stated in all sediments at both temperatures and in river water at 6°C, in estuary and lake waters situations of non-culturability were observed. In addition, measurement of the cellular metabolic activity showed decreases in the respiratory rates to 60-70% of the original values in the cases of sediments and river water, and to 10-15% when the cells became non-culturable. However, in all the microcosms, the acridine orange direct counts (AODC) remained nearly constant during the experimental period at values of about 106 celUml in water and 108 celUml in sediments. These findings demonstrated that Y. rucker; may undergo a dormant state under certain starvation conditions. Non-culturable cells showed marked changes in morphology and size. Slight changes in LPS patterns of dormant cells were also detected, but not in membrane proteins or plasmid profiles. Moreover, maintenance of virulence during the non-culturability state was demonstrated. Dormant cells were easily resuscitated by addition of fresh medium to the microcosms, showing the resuscitated cells levels of metabolic activity and plate counts similar to those seen prior the start of the experiment.

Key words: Yersinia ruckeri, starvation, dormancy, water and sediment microcosms.

Introduction

The enterobacterium Yersinia ruckeri is the etiological agent of the enteric redmouth disease (ERM) affecting mainly fish reared in fresh waters (Rucker 1966; Michel et aI., 1986; Daly, 1990; Romalde et aI., 1990). Sporadic cases in fish reared in marine waters (Michel et aI., 1986), as well as isolations from wild fish (Willumsen, 1989) have also been reported. This disease is of great importance due to the serious economic losses it causes in aquaculture operations throughout the world (Austin and Austin 1993). Y. ruckeri is considered to be an obligate pathogen (Poindexter, 1981) whose mode of transmission has been related to carrier wild or farmed fish and other putative vectors like aquatic invertebrates and birds (Austin and Austin, 1993; Willumsen, 1989). In addition, the presence of Y. ruckeri in the faeces of carrier fish has been * Corresponding author

demonstrated during at least two months after an ERM outbreak (Rodges, 1992; Tellervo et aI., 1992). Therefore, its ability to survive and remain infective in the aquatic environment must be taken into account as a major determinant in the spread of the disease. However, practically no studies about the persistence of Y. ruckeri in waters and sediments were performed and, therefore, the importance of the viability in the environment of this fish pathogen in the dissemination of the ERM is still unclear. States of non-culturability (dormancy) (see review of Oliver, 1993), often induced when the bacteria are exposed to adverse environmental conditions, have been described for other clinical or environmental Gram-negative organisms such as Escherichia coli, Vibrio cholerae and Salmonella enteritidis (Roszak et aI., 1984), Shigella sonnei and S. {lexneri (Colwell et aI., 1985), V. vulnificus (Linder and Oliver, 1989) and Aeromonas salmonicida

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J. L. Barja, B. Magarinos and A. E. Toranzo

(Rose et al., 1990). It has been reported that during this dormant phase, bacteria can dramatically change in several characteristics such as size, protein, lipopolysaccharide (LPS) and fatty-acid profiles (Poindexter, 1981; Austin et al., 1984; Kjellerberg et al., 1987; Linder and Oliver, 1989), and plasmid content (Byrd et al., 1992) as well as in the hydrophobicity of cells (Kjellerberg and Hermansson, 1984). However, the maintenance of the virulence in the non-culturable state of pathogenic bacteria was variable according to the bacterial species studied (Roszak and Colwell, 1987).

The studies reported here were done to determine the survival period of Y. ruckeri in different types of water and sediment. In addition, we evaluate the presence of a dormant state, and the possible cellular changes of this fish pathogen under starvation conditions. The capacity of the dormant forms to retain the infectivity for fish was also assessed.

Materials and Methods Bacterial strains and environmental samples. Three strains of Y. ruckeri, representatives of the predominant serotypes throughout the world (Davies, 1990; Rodges, 1992) were included in this study. Strain PP31 belongs to the serotype 01a (former serovar I) and was isolated from rainbow trout (Oncorhynchus mykiss) in Spain, strain RS54 of the serotype 01b (former serovar III) isolated from Dolly Varden trout (Salvelinus malma) in Canada, and strain 11.29 (serotype 02b) isolated in USA from pacific salmon (0. tshawytcha).

Survival experiments were conducted in three different types of water and sediment: river, lake and estuary. Samples were collected from Tambre river, Lake Portodemouros and Ria de Muros (15% NaCl) (Galicia, North West Spain) and transported to the laboratory in cold-storage containers. The physico-chemical characteristics of the samples were analyzed and the results are shown in Table 1. Waters were filtered through 0.2-I-tm-poresize membranes (Millipore, Madrid, Spain) and sediments were autoclaved at 120°C for 30 min. Microcosms conditions. The experimental systems were constructed by placing 100 ml of sterile water or sediment in 250 ml Erlenmeyer flasks capped with cotton wool bungs. Twenty ml of the corresponding water were added to each sediment flask. All the microcosms were prepared in duplicate to assay different incubation temperatures (6 and 18°C). The 36 microcosms (resulting from the combination of 3 strains, 3 waters, 3 sediments, and 2 temperatures) were inoculated to a final concentration of approximately 104-10 5 cells per ml and incubated in the dark at 40 rpm on a rotary shaker. Bacterial counts and Microscopic observations. To determine the evolution of culturable cells in each microcosm, samples of 0.5 ml were taken at 0, 6, and 24 H and then daily for a period of three months. Samples were serial-diluted in phosphate buffered saline (PBS; pH 7.4) and plated in duplicate on both tryptone soy agar (TSA; Difco Laboratories. Detroit, Mich.) and 1110 diluted TSA in order to obtain a low-nutrient medium. When culturable bacteria could not be recovered, samples of 1 ml taken directly from the respective flasks were seeded onto TSA and diluted TSA plates to obtain a detection limit of 1 CFU/ml. During the incubation period of the microcosms, microscopical observations of the cells were performed periodically, and the changes in morphology and size were monitored. The number of total bacteria was evaluated by epifluorescence

Table 1. Physico-chemical characteristics of the water and sediment samples. Characteristic (units) Aluminium (ppm) Calcium (ppm) Copper (ppm) Iron (ppm) Manganese (ppm) Magnesium (ppm) NOi (ppm) NO) (ppm) NHt (ppm) Phosphorus (ppm) Potassium (ppm) S04= (ppm) Sodium (ppm) Zinc (ppm) pH Conductivity (l-tS/cm) Safinity (%0) Total Carbon (%) Organic matter (%) Total nitrogen (%) C/N relation CCEb

Water

Sediment"

River

Estuary

0.02 3.30

0.08 4.67

0.06 2.75

0.36

0.00

0.00

Lake

3.52 3.27 x 10- 3 0.70 0.25

2.75 6.13 x 10- 3 0.41 0.26

6.38 0.07 104.53 0.25

0.60 1.30 5.00

430.00 163.69 11,000

0.40 28.48 5.00

6.72 62.64 0.00

7.83 23.96 15.00

6.08 64.72 0.00

" Analysis of the fraction minor than 2 mm. b CCE, Capacity of Cationic Exchange.

River

Estuary

Lake

4.50 576.00 1.00 151.00 30.50 13.56

0.00 166.00 0.50 20.50 0.50 5.04

19.80 120.00 1.00 43.50 117.50 4.32

65.20 3.51

5.70 7.02

3.72 4.68

1.38 5.00

60.03 1.00

1.61 0.50

1.34 2.32 0.14 9.57 3.44

1.59

2.75 0.03 53.00 4.75

1.93 3.33 0.05 38.60 1.43

Survival of Y. ruckeri in aquatic environments microscopy employing the acridine orange method (Hobbie et al., 1977). Samples, taken approximately at 10 days intervals, were diluted, filtered onto 0.20 !-1m Nucleopore filters (Nucleopore Corp., Pleasanton, USA), prestained with irgalan black, and stained with 0.01 % acridine orange. After 5 min. staining, filters were washed twice, and the number of bacteria calculated. Metabolic activity and resuscitation studies. The metabolic activity of the cells in the different microcosms was determined by measuring the A600 with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) following the procedures of Peck (1985). This method is based on the reduction of the compound MTT by the bacterial dehydrogenases in direct proportion to the number of viable bacteria. The results were expressed as the percentage of the remaining respiratory activity, taking as 100% the value at zero time. After 11 0 days of starting the survival studies, tryptone soy broth (TSB; Difco) was added to a final concentration of 10% to each microcosm in order to evaluate the reactivation capacity of the Y. ruckeri strains. Samples were withdrawn at 24 and 48 h and analyzed as above. Respiratory rates after resuscitation were also determined. Analysis of cellular changes. To determine if cellular changes occurred during the starvation period, 50 ml of the corresponding water microcosm were centrifuged when culturable bacteria were below the detection limit (1 CFU/ml). The number of dormant cells present was then calculated on the basis of the AODC, and the appropriate dilutions for each assay were prepared. The possible biochemical and physiological alterations during the

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163

starvation period were studied by using the miniaturized systems API 20E and API ZYM (bioMerieux S.A., Madrid, Spain). In order to assess the possible antigenic changes, slide agglutinations of the dormant cells were performed using the "0" antigens and the correspondent antisera raised in New Zealand rabbits against the three strains included in this study. To determine alterations in the components of the bacterial cell wall, we analyzed the LPS as well as the total and outer membrane proteins profiles by the methods of Hitchcook and Brown (1983) and Crosa and Hodges (1981) respectively. In addition, the plasmid profiles of the isolates during the dormant state were compared with those of the original strains following the procedures of Birnboim and Doly (1979). The infectivity and virulence of the uncluturable Y. ruckeri were examined in fingerling rainbow trout (6-8 g) as previously described by Toranzo et al., (1983). Fish were inoculated intraperitoneally with doses ranging from 106 to 103 CFU and maintained at 18°C with continous fresh water flow. The original strains were also inoculated as positive controls. The degree of virulence (LDso) was determined after a 7-day period by the method of Reed and Muench (1938).

Results Survival in the different microcosms

The results obtained in the bacterial counts for the different microcosms reflected similar survival dynamics for

106 104 102 10° 108 106

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104 102 10°

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20

40

60

80 100 120 140

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40

60

80

100 120 140

Days

Fig. 1. Survival of Y. ruckeri in river water and sediment. A, water at 6°C; B, water at 18°C; C, sediment at 6°C; D, sediment at 18 ~c. - - , strain PP31; - , strain 11.29; ..... , strain RS54. Arrowheads indicate the addition of fresh medium to the microcosms. Counts from TSA medium.

164

J. L. Romalde, J. L. Barja, B. Magarinos and A. E. Toranzo

the three strains regardless of their origin, a persistence of longer than three months being observed in all the experimental systems assayed (Fig. 1). In general, during the first 15 days, the number of culturable bacteria increased by 1log-unit in water microcosms and 2 to 3 log-units in sediment systems, and then gradually decreased to levels berween < 1 and 10 4 CFU/ml depending on the experimental system and conditions (Fig. 1 and 2). In sediments and river water, a high persistence of culturable cells was recorded, with values of CFU after three months of starvation of approximately 104 bacteria/ml. In contrast, in the other water microcosms (estuary and lake), situations of non-culturability were observed after one to rwo months (with less than 1 CFU/ml) . A clear influence of the temperature on the survival of Y. ruckeri was achieved, with the number of culturable bacteria greater at 6 °C than at 18°C for each strain and microcosm (Fig. 2). The differences observed in the CFU values for each microcosm berween both temperatures ranged from 1- to 3-log-units, with the exception of lake microcosms in which similar titers were obtained at the two temperature conditions (data not shown). There was a clear influence of the salinity on the survival of Y. ruckeri. Persistence of each strain was always higher in river than in estuarine microcosms, with dif108

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ferences of 3 to 4-log-units berween them. Fig. 2 shows the survival pattern of the representative strain PP 31. A special case was that of the lake microcosm. Despite of being a fresh water environment, values of bacterial recovery in these microcosms at 6°C were closer to those of the saline systems (Fig.2A, C). This could be due to the effect of high concentrations of some water and sediment components such as SO" ions and manganese in this eutrophic environment (Table 1). The stability of each Y. ruckeri strain was higher in sediment than in water in all the environments studied, with differences of 2 to 3 orders of magnitude in the CFU numbers (Fig. 1, 2). The great persistence in river sediment is noteworthy in which the strain PP31 remained viable for a period of longer than 7 months with titers of 104 cells/ml (data not shown). Except during the first period (15 to 20 days) of the survival experiment, the number of CFU recovered in the diluted TSA was higher, in 1- or 2-log-unit, than those obtained in TSA at normal concentrations (Fig. 3). Evidence of dormancy and cell resuscitation

By epifluorenscence microscopy, it was found that after an initial increase in the AODCs of 1 to 3-log-units for

A

106 104 102

~

10°

108

E

~U

C

106 104 102

10°

+---------. ..o 20

40

60

80

Days

100 120 140

o

20

40

60

80

100 120 14(

Days

Fig. 2. Survival of the strain PP31 in the different waters and sediments. A, waters at 6°C; B, waters at 18°C; C, sediments at 6°C; D, sediments at 18°C. - , river; .... , lake; - - , estuary. Arrowheads indicate the addition of fresh medium to the microcosms. Counts from TSA medium.

Survival of Y. ruckeri in aquatic environments

water and sediment microcosms respectively, the total counts remained nearly constant along the experimental period, at values of approximately 106 bacteria/ml in water and 108 bacteria/ml in sediment systems, even when the number of culturable Y. ruckeri declined to levels below the detection limits (Fig. 3). Regarding the metabolic activity in the microcosms where long term survival was observed, the respiratory rates were maintained arround 60 to 70% of initial values. On the other hand, in the microcosms of lake and estuary water, where non-culturability was recorded, metabolism decreased by more than 80%. These results indicate that, depending on the environmental factors, Y. ruckeri may enter into a dormant phase after one to two months under starvation conditions. Strains could be resuscitated from the dormant state by addition of fresh TSB medium to the experimental systems (indicated by arrowheads in all the Figures). In fact, the bacterial counts obtained 48 h after the supplementation of the microcosms with TSB reached the original numbers ranging between 106 (water microcosms) and 10 8 (sediment microcosms) CFU/ml. In addition, the resuscitated cells showed the same level of metabolic activity as was

165

seen prior to the initiation of the starvation. Only following complete resuscitation were the plate counts seen to increase slightly compared with the initial values (data not shown).

Morphological and physiological changes In all the microcosms the average cell size of the Y. ruckeri cells decreased during the dormant state, looking finally like cocci of about 1 !lm in diameter. This decrease means a reduction to 113 or 114 of the original cell length. Consequently, the cell volume was reduced by about 65% during this phase. After resuscitation, cells recovered their original size and morphology (bacilli of 3-4 !lm diameter). In the water microcosms where a dormant state was observed, bacteria were harvested by centrifugation after a period of non-culturability of 15 days, and possible cellular alterations analyzed. No changes in the 40 biochemical and physiological tests assayed with the API 20E and API ZYM strips were observed. In addition, the dormant cells showed the same membrane protein profiles (ranging between 14 and 90 kd) with major outer protein bands of

A

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10° +....,~-+--.,............-...,....I...t~..,

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20

40

60

80

Days

100 120 140

......

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20

40

60

80

100 120 140

Days

Fig. 3. Comparison between the bacterial counts obtained in TSA medium (-), in the 1/10 diluted TSA (..... ). A, strain PP31 in estuary water at 18°C; B, strain 11.29 in lake water at 18°C; C, strain PP31 in lake sediment at 6°C; D, strain RS54 in lake sediment at 18°C. (_._._._), acridine orange direct counts (AODC). Arrowheads indicate the addition of fresh medium to the microcosms.

166

J. L. Romalde, J. L. Barja, B. Magarinos and A. E. Toranzo

about 28, 30 and 35 kd of molecular mass (Fig.4B), and harbored the typical plasmids of the original strains (Fig.4C). Only slight differences could be observed in the LPS profile of the dormant cells, with an increase in the concentration of the high molecular mass "0" chain region (Fig.4A). Interestingly, the Y. ruckeri isolates during the non culturability phase conserved their virulence capacities, showing LDso very similar to those obtained with the original strains (Table 2). Table 2. Evaluation of the virulence capacities of the Y. ruckeri strains during the nonculturable state. Strain

Serotypea

PP31 11.29 RS54

01a 02b 01b

normal state

dormant state

1.0 x 104 8.0 x 10 4 2.0 x 10 6

2.5 7.8 1.0

X X X

104 104 10 6

Serotype nomenclature following Romalde (1992) LD50, 50% mean lethal dose expressed as number of bacteria needed to kill 50% of inoculated fish in a 7-day period.

a

b

A

1

2

B

Discussion Survival has been defined as maintenance of viability under adverse circumstances. The maintenance in low-nutrient concentrations is considered as one of the disadvantegeous conditions (Rozak and Colwell, 1987). Under these situations, bacteria can develop different survival strategies. Therefore, there are cases of long-term starvation survival (i. e. V. anguillarum and V. salmonicida) (Hoff, 1989), formation of ultra microbacteria (Roszak and Colwell, 1987) or development of a non-culturable but viable or dormant status (Stevenson, 1978). In general, during starvation bacterial sizes and activities decrease until favourable conditions return. The data presented in this work demonstrate that Y. ruckeri is able to survive more than three months in fresh and marine waters and sediments. These findings could explain the appearance of the pathogen in widely dispersed locations where there have been no previous cases of ERM outbreaks (Bullock et al., 1978), as well as the recovery of Y. ruckeri several km away from an infected farm (Romalde, 1992). Similar results were obtained by Wedemeyer and Nelson (1977) who found constant numbers of culturable bacteria for at least one month in untreated lake water. Moreover, the

123

c

1 23

Fig.4. Comparison of the LPS (A), outer membrane proteins (B) and plasmid (C) profiles between the original and the dormant Y. ruckeri PP31 strains. 1, original strain; 2, dormant strain; 3, molecular weight markers. In the analysis of plasmid content, plasmids from Escherichia coli V S17 (ranging from 1.4 to 36 Md) were used as markers.

Survival of Y. ruckeri in aquatic environments three Y. ruckeri strains showed the same survival pattern, regardless of their serotype or origin. In general, persistence of Y. ruckeri is longer in sediment than in water microcosms and it is influenced by temperature and salinity. Therefore, survival is favoured in fresh water environments (river microcosms) as well as by low temperatures (6°C). On the basis of plate counts and metabolic activity two different behaviours were observed depending on the microcosm and experimental conditions. In the cases of longterm survival, the different strains remained culturable after three months of starvation showing values of approximately 60% of the initial metabolic activity. In contrast, in the situations of dormancy, the loss of culturability was accompanied by a reduction of the respiratory rate by more than 80%. On the other hand, AODC counts were almost constant during all the experiment at very high levels (10 6 cells/ml in waters and 10 8 in sediments). A reduction in the size of Y. ruckeri cells was observed as a response to minimize cell requirements under starvation conditions. This reduction was more pronounced in the cases of non-culturability. It has been reported in several ecological studies with marine bacteria, that the miniaturization of cells increase the surface-to-volume ratio (Amy and Morita, 1983), and this fact may aid cells in obtaining substrates from a nutrient-poor environment (Morita, 1982) and in the starvation survival process (Novitsky and Morita, 1978). Similar findings have been described for fish and human pathogens such as A. salmonicida (Austin et aI., 1984; Rose et aI., 1990; Morgan et aI., 1991) and V. vulnificus (Nilsson et aI., 1991; Oliver, 1993), as well as for enterobacteria like E. coli (Xu et aI., 1982; Barcina et al., 1990), S. enteritidis and V. cholerae (Roszak et aI., 1984) and S. sonnei (Colwell et al., 1985). Y. ruckeri is a copiotropic microorganism because of its ability to grow rapidly on standard complex media (Poindexter, 1981). 'Tpis type of organism, after undergoing nutrient starvatidn, are often sensitive to media with high nutrient content (Amy and Morita, 1983; Dawes 1985). This fact in~s the recuperation on standard media. The results obtained in this work support this fact, since Y. ruckeri was more easily recovered in diluted media (1110 TSA) than in standard media (TSA) with differences of about 1 to 2 log units in the CFU counts. As reported for S. enteritidis (Roszak et al., 1984) and A. salmonicida (Austin et al., 1984) bacteria could be resusciated by adding nutrient media at low concentrations to the microcosms. On resuscitation, an increase in plate counts from less than 1 cells/ml to the original density was observed to occur. The metabolic activity also achieved the initial values. Variable results for the maintainance of the virulence of several pathogenic bacterial species during the dormant state were reported. In general, enterobacteria such as E. coli and S. enteritidis (Roszak and Colwell, 1987), as well as some fish pathogens like Pasteurella piscicida (Magarifios et al., 1994) retained their virulence. In contrast, other fish and human pathogens like A. salmonicida and V. vulnificus biotype 1 lost this capacity (Linder and Oliver, 1989; Rose et aI., 1990). Our results clearly de-

167

monstrate that dormant Y. ruckeri maintain their virulence for fish with LDso-values similar to those of the culturable state. Moreover, although variations in nucleic acid concentration, protein composition, plasmid content and fatty acid and LPS profiles have been described for dormant cells with respect to the normal state (Stevenson, 1978; Coleman and Leive, 1979; Amy and Morita, 1983, Guckert et aI., 1986; Hood et aI., 1986; Jaan et aI., 1986; Kramer and Singleton, 1992; Nystrom et al., 1992), in this study we have not found differences in the investigated enzymatic activity, membrane proteins or plasmid content. Only slight variations were detected in the high molecular mass "0" chain region of LPS. The putative role of these differences in the stat:vation process is currently under investigation in our laboratory. In conclusion, the high persistence of the fish pathogen Y. ruckeri in the environment (especially in sediments) and the pathogenicity of the dormant cells are of great importance from the epidemiological point of view. It implies a potential risk of contamination and spread of the disease among pisciculture facilities placed in the same geographical area. Accordingly adequate preventive control programs against ERM have to be developed. Acknowledgements. We thank Dr. R. M. W. Stevenson, University of Guelph (Canada) and Dr. T. M. Cook, University of Maryland (USA) for the kindly supply of the strains, and B. R. Lobelle and C. Moares, University of Santiago, for their help in the analysis of water and sediment samples. j. L. Romalde and B. Magariiios thank Xunta de Galicia (Spain) for research fellowships. This work was supported in part by Grant MAR-91-1133C02-01 from the Comision Interministerial de Ciencia y Tecnologia (CICYT), Spain.

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jesus L. Romalde, Departamento de Microbiologia y Parasitologia, Facultad de Biologia, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain. Tel.: 34-81-563100 (# 3255), Fax: 34-81-596904.