Relationships among virulence for fish, enterotoxigenicity, and phenotypic characteristics of motile Aeromonas

Aquucutture, 67 (1987) 29-39 Elsevier Science ~blishe~ B.V., Amsterdam -

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Printed in The Netherlands

Relationships Among Virulence for Fish, Enterotoxigenicity, and Phenotypic Characteristics of Motile Aeromonus YSABEL SANTOS*, ALICIA E. TORANZO, CARLOS P. DOPAZO, TERESA P. NIETO and JUAN L. BARJA ~e~artamento de Microbiology y ~ar~ito~gio, de Compostela, Santiago 15703
F~cult~d de ~io~og~~, Uniuersid~d de Santiago

“To whom correspondence should be addressed. (Accepted 22 January 1987)

ABSTRACT Santos, Y., Toranzo, A.E., Dopazo, C.P., Nieto, T.P. and Barja, J.L., 1987. Relationships among virulence for fish, enterotoxigenicity, and phenotypic characteristics of motile Aeromonas. Aquaculture, 67: 29-39. In this study we have analysed the biochemical, enzymatic, and cell surface properties of 59 Aeromonas strains isolated from fish culture systems, with the aim of establishing the possible relationships among some of these phenotypic characters and pathogenicity. The cytotoxic activity of extracellular products was also evaluated. Virulence assays showed that 64.3% of the strains were pathogenic for fish. However, both pathogenic and non-pathogenic Aeromonas isolates were able to produce enterotoxins. The majority of the strains were proteolytic, amylolytic and produced DNAase. Nevertheless, elastase and staphylolytic activities were present only in A. hydrophilu. Although 96% of the isolates produced haemolysins, a clear specificity toward trout or human erythrocytes was not found in the pathogenic strains. Similarly there was no specificity in the haemagglutinating activity. Statistical analysis of the association between virulence and phenotypic traits revealed a positive relationship among virulence for fish and arabinose and sucrose fermentation, elastase and haemolysis of human erythrocytes. However, only the LDC test showed a significant relation with enterotoxin production. These findings suggest that different mechanisms are involved in the invasion by Aeromonas of their poikiIotherm and homeotherm hosts. Extracellular products of the Aeromonas strains displayed cytotoxicity on fish cell-lines regardless of the virulence capacities of the strains, which indicates that cytotoxic activity is not an adequate criterion of pathogenicity.

INTRODUCTION

Motile Aeromonm are considered autochthonous inhabitants of aquatic environments and have been documented as opportunistic pathogens for fresh-

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0 1987 Elsevier Science Publishers B.V.

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water fishes and other poikilotherm and homeotherm animals (Boulanger et al., 1977; De Figueiredo and Plumb, 197’7; Davis et al., 1978; Hazen et al., 1978; Kaper et al., 1981; Toranzo et al., 1985). The Aeromonas species can produce several extracellular toxins or enzymes such as proteases, haemolysins, cytotoxins and enterotoxins, but their role in fish and human diseases has not been determined. Several attempts have been made to correlate biochemical, enzymatic and cell surface characteristics with the toxigenicity of Aeromonas isolates (I&u et al., 1981; Kaper et al., 1981; Wakabayashi et al., 1981; Burke et al., 1984; ~rnbull et al., 1984; Morgan et al., 1985). However, the usefulness of these characters in defining strains virulent for fish or enterotoxigenic for homeotherms is the subject of controversy. In this work we analysed several virulence factors present in Aeromonas strains isolated from fish culture systems with the aim of establishing their possible relationship with pathogenicity. MATERIAL AND METHODS

Bacterial strains In this study we used 49 motile Aeromonas isolated from diseased rainbow trout (SaEmo gu~rd~er~) and water tanks. Ten reference strains from the American Type Culture Collection (ATCC) and particular donors were also included. All strains were subjected to taxonomical analysis (Toranzo et al., 1986), and identified as A. ~ydrop~~la (39)) A. sobria (14)) A. cauiae f 3) and Akromonas spp. (3) following the schemes of Popoff and V&on (1976) and Popoff (1984). The strains were routinely cultured on trypticase soy agar (TSA) or broth (TSB ) (Difco) at 25” C for 24 to 48 h. The stock cultures were maintained on TSA slants at 15’ C under mineral oil and - 70” C with 15% ( u/u) glycerol. Virulence for fish The assays of pathogenicity were conducted at 18”C-20°C by intraperitoneal inoculation of fingerling rainbow trout as previously described (Nieto et al., 1985). The degree of virulence, expressed as LD,, (50% mean lethal dose) was calculated by the Reed and MGench method (1938). Enterotoxin test The enterotoxigenic capacity for our Aeromonas isolates was measured by the suckling mouse assay, following basically the Burke et al. (1984) procedures. Mice (2 to 4 days old) were inoculated with 0.1 ml of cell-free supernatants of each strain. After incubation for 3 h at 30” C, the ratio of intestinal weight to remaining body weight ( IW/BW ) was measured. Ratios above 0.08 were scored as positive response. The stability of Aeromon~ enterotoxin was determined after heat treatment (100’ C for 30 min) of some positive samples.

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Detection of extracelluEarenzymes and haemolytic activity Proteolytic (caseinase and gelatinase) , lipolytic (lipase and phospholipase) , amylolytic, aesculin hydrolysis and DNAase activities were evaluated by plating according to procedures of West and Colwell (1984) and Wakabayashi et al. (1981). Bacteriolytic capacity was assayed using as substrate heatkilled Staphylococcus aureus cells. A modification of the Scharman method (Hsu et al., 1981) was used in elastase determination. Haemolysin production was determined on TSA plates containing poikilotherm (salmon and trout) or homeotherm (human, sheep and guinea pig) red blood cells at 5% (u/v) final concentration. Agglutination assays and inhibition of haemagglutination The adherence of Aeromonas to host cells was tested by agglutination of fish and human erythrocytes and yeast cells, as previously described (Toranzo et al.., 1983). Reactions were considered negative if agglutination had not occurred within 10 mini The minimum haemagglutinating dose (MI-ID ) was defined as the -smallestnumber of bacterial cells/ml that gave visible agglutination in 10 min, and the haemagglutinatingpower ( AP) was calculated as lOll/MHD (Duguid, 1959). Inhibition of haemagglutination was performed essentially as the agglutination test using I% (u/v) solution in PBS of D-mannose, L-fucose and Dgalactose. Stability after boiling and acriflavine test These eel1 surface characteristics were determined according to Mittal et al. (1980) procedures. Cell precipitation was verified after boiling an 18-h-old culture in brain-heart infusion (BHI) (Difco) broth for 1 h. Agglutination in acriflavine (0.2% ) was conducted on slides using colonies from blood agar plates. ~ytotoxicity assays Cultures in TSB from a seleeted group of Aeromon~ strains were incubated at 20’ C for 24 h in a rotary shaker, and centrifuged for 10 min at 4 oC. Supernatants were filtered and the cytotoxicity of extracellular products was measured as previously described (Toranzo et al., 1983) using three fish cell-lines: Chinook Salmon Embryo (CHSE-214)) Fathead Minnow peduncle (FHM) and Epithelioma Papullosum of Carp (EPC ) . Cells were grown as monolayers in 24-well culture plates at 16°C (CHSE214) or 25°C (FHM and EPC), using L-15 medium supplemented with 10% foetal calf serum. For the toxicity tests, the different ceil-lines were inoculated with 0.1 ml of serial dilutions of each culture filtrate. Non-inoculated monolayers as well as-cells added to fresh TSB were used as controls. Plates were

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Virulent

’

Weokly

Aviruleol

Virulent

Fig. 1. Percentage of Aeromonas strains within each level of virulence displaying negative agglutination in acriflavine (shaded columns) and instability after boiling (black columns).

incubated at 18”C, and wells showing totally or partially destroyed monolayers in a 3-day period were scored as positive cytotoxic responses.

The associations among enterotoxigenicity, virulence for fish and several biochemical, enzymatic and cell surface characteristics of our isolates were assessed using the chi-square (x2) test and a reciprocal average analysis.

RESULTS

Fish puthogen~c~ty The virulence assays showed that 36 of 56 (64.3% ) Aerosol strains tested (the three Aerosols spp. were not considered) were pathogenic for fingerling rainbow trout with an LDeOranging between 104-lo5 (22 virulent strains) and 106-lo7 (14 weakly virulent strains). The remaining strains, displaying an LDhO> lo* belonged to the avirulent category (Fig. 1) . We found that the percentages of virulent strains were comparatively higher in A. hydrophila (71.4% ) than in A. sobria (42.8% ) isolates. Enterotoxigenic capacity of Aeromonas Twenty-one of 34 (62% ) randomly selected fish pathogenic and non-pathogenic strains were able to produce enterotoxins. Similar percentages of virulent strains in both A. ~ydrophi~ (59% ) and A. sobria (60% ) isolates displayed a positive response in the suckling mouse test. Nevertheless, a greater number of avirulent A. hydrophila than A. sobria strains were enterotoxigenic (data not shown).

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Produ~t~an of extra~e~lularenzymes and ~mo~ysins The analysis of extracellular enzymatic activities indicated that the majority of strains (more than 96% ) were amylol~ic and hydrolized casein, gelatin and DNA. A smaller number of isolates displayed aesculin hydrolysis ( 76.2% ) , lipase (81.3%) and phospholipase (69.4% > activities. Interestingly, elastase and staphylolytic enzymes were only produced by A. hydrup~iZuspecies. Haemolytic assays demonstrated that 57 of 59 (96%) strains showed lytic activity against some of the erythrocytes used. Moreover, although both A. hydrophila and A. sobria produced haemolysins, A. hydrophila isolates included a higher percentage of haemolytic strains. On the other hand, 37 of 39 fish pathogenic strains showed the capacity to haemolyze trout erythrocytes. Furthermore, six of these positive strains showed specific activity towards trout blood cells only. Similar specificity against human erythrocytes was not observed within the enterotoxigenic strains. Agglutinating activity of Aeromonas strains The assays of agglutination, performed to explore the adherence ability of our isolates, showed that 68% of strains produced agglutinins. If we analyse the data at the species level, a greater number of A. sobria than A. ~ydrophi~ displayed haemagglutinating capacity. The agglutinating activity for fish or human e~hroc~es were q~a~titated in 25 positive haemagglutinator isolates. The haemagglutinating power of the majority of strains ranged between 10’ and 103. The sugar inhibition studies revealed that, although the patterns of inhibition were different according to the source of erythrocytes, in general the haemagglutinations were sensitive to inhibition by D-mannose, usually in association with L-fucose. Additional tests related to cell surface characteristics Fig. 1 illustrates the number of Aeromonas strains included in each level of virulence showing a negative agglutination in acriflavine and precipitation after boiling. Virulent and non-virulent strains displayed similar patterns for these activities. Cytotoxic activity on fish cell-lines The cytotoxic assays showed that both pathogenic and non-pathogenic Aeromo~ strains gave positive responses on some of the fish cell-lines tested. Degenerative changes started within 3 h or 6 h after inoculation of the extracellular products. In general, all the samples produced rounding, shrinking and dendritic elongations (Fig. 2B). Finally, all the cells became round and detached from the wells (Fig. 2C).Heat treatment of the culture filtrates resulted in a loss of cytotoxicity.

Fig. 2. Cytotoxic activity produced by the extracellular products ofA. hy&ophilaB-32, on the f&h cell-line FHM: (A) control cells (inoculated with sterile broth1); (B) initial changes on theI cell monolayer 3 h after exposure to culture filtrate; (C) final cytotcJxic effects showing rolunding and loss of cell adherence.

35 TABLE 1 Enterotoxin production and virulence for fish of Aeromonas isolates Characters

in relationto differentphenotypiccharacteristics

A~rorno~~ strains n=34

n=59

Voges-Proskauer Lysine decarboxylase Arabinose fermen~tion Sucrose fermentation Elastase Haemolysis (trout erythrocytes ) Haemolysis (human erythrocytes) Haemagglutination (trout e~hr~~es ) Haemagglutination (human erythrocytes) Virulence for fish

Vir+ (n=39)

Vir(n=20)

Ent” (n=21)

Ent(n=13)

25 37 36 33 20 37

14 19 13 13 5 18

14 21 18 16 10 20

6 10 11 12 6 12

(64.1)’ (94.9) (92.3) (84.6) (51.3) (94.9)

30 (76.9) -

(70) (95) (65) (65) (25) (90)

Il(55)

(66.6) (100) (%!7) (76.2) (47.6) (95.2)

(46.1) (76.9) (84.6) (92.3) (46.1) (92.3)

14 (66.6)

8 (61.5)

13 (33.3)

5 (25)

6 (28.5)

6 (46.1)

21 (53.8)

11 (55)

10 (47.6)

9 (69.2)

-

-

13 (61.9)

10 (76.9)

‘Percentages are based on the total number of strains tested in each category of virulence or enterotoxigenicity. Vir+, strains virulent for trout; Vir-, non-virulent strains; Ent+, suckling-mouse positive strains; Ent-, suckling-mouse negative strains. Percentages underlined indicate a significant difference (PC 0.1, x2 test).

Relationships between pathogenicity and phenotypic traits Table 1 shows the positive relationships found among virulence for fish and fermentation of arabinose and sucrose, elastase, and production of haemolysins toward human erythrocytes (PC 0.1, x2 test). As shown in Fig. 3, similar associations were observed using a reciprocal average analysis. The majority of the virulent strains associated with the above-described characters are situated on the upper region of the axes, whereas most of the non-pathogenic Aeromonas associated with a VP-positive reaction are grouped in the bottom area. The x2 test was also applied to establish positive or negative relationships among enterotoxigenic capacity and biochemical, enzymatic and haemagglutinating properties. The overall results indicated that only the lysine decarboxylase reaction showed a significant positive relation with enterotoxin production.

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* ***

. Vir

*

* *

***

* SW:

0

4x!

**

-.

*

“gl? .

&It

3-h

Ht Gel

.t.h&=

‘*so,~~***-*-*---ES

ADH

*

LDC Ggl .

Elr Stph

bbb

bb . * l

. VP

Fig. 3. Distribution of Aeromonasstrains and phenotypic characters, determined by reciprocal average analysis. Virulent strains (*) ; Non-virulent strains ( 0 ) . Characters. Vir, virulence for fish; Hgt, haemagglutination of trout erythrocytes; Hgh, haemagglutination of human erythrocytes; Ht, haemolysis of trout erythrocytes; Hh, haemolysis of human erythrocytes; VP, Voges-Proskauer; LDC, lysine decarboxylase; ADH, adenine dihydrolase; Ggl, gas from glucose; Sue, sucrose fermentation; Ara, arabinose fe~en~tion; Cas, caseinase; Gel, gelatinase; Els, elastase; Stph, staphylolysis; Es, aesculin hydrolysis. DISCUSSION

Motile Aeromonas species are a complex group with a wide variation in pathogenicity for humans and fish (De Figueiredo and Plumb, 1977; Wakabayashi et al., 1981; Toranzo et al., 1985). Hence, it is important from etiological and epidemiologi~al studies to establish whether any of their phenot~ic characteristics, either singly or in combination, can be related to the virulence of these organisms. Some biochemical, enzymatic and cell surface properties have been reported as potential indicators of virulence in Aeromonas strains (Hsu et al., 1981; Kaper et al., 1981; Wakabayashi et al., 1981; Turnbull et al., 1984; Morgan et al., 1985)) but their role in the disease process still remains unknown. Similarly to Wakabayashi et al. (1981) and Hsu et al. (1981) , we have found in Aeromonas isolates a significant relationship between fish pathogenicity and elastase and staphylol~ic activities (Table 1, Fig. 3 ) . Neve~heless, the fact that in our study these activities were only observed in some virulent and avirulent A. hydrophila isolates but not in the other Aeromonas species seems to indicate that these enzymes are not a prerequisite of virulence in all motile Aeromonas strains. Moreover, in agreement with Wakabayashi et al. (1981)) we consider that elastase and staphylolysis together with aesculin hydrolysis are valuable criteria for the identification of A. hydrophila species.

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Several authors have implicated haemolysin production in infections caused by Aeromonas strains (Allan and Stevenson, 1981; Rahim et al., 1984). Although in this work the majority of strains (96% ) were haemolytic, both pathogenic and non-pathogenic strains (either for fish or mouse) were able to lyse some of the erythrocytes assayed. Thus, it is not possible to determine what role the haemolysins play in the diseases process. The possible role of bacterial adherence in the establishment of fish and human infection is controversial (Atkinson and Trust, 1980; Larsen and Mellergard, 1984; Burke et al., 1984; Morgan et al., 1985). In our study, 68% of strains displayed agglutinating activity regardless of their virulence capacity. Furthermore, neither fish pathogenic nor enterotoxigenic strains showed specific haemagglutination toward trout or human red blood cells. Therefore, the ability to recognize and attach the receptors on the host cell surface does not appear to be a prerequisite for a successful invasion in all motile Aeromonas. Similar results were found by Toranzo et al. (1983) and Larsen and Mellergard (1984) in environmental vibrios, as well as by Morgan et al. (1985) in clinical Ae~omon~ isolates. The majority of haemagglutination reactions in our Ae$omo~s strains were inhibited by D-mannose, which indicates that this carbohy~ate or an analogue form part of a receptor on the erythrocytes recognized by the bacterial adhesins. Another pattern usually detected was the inhibition by D-mannose and L-fucose (M/F) (data not shown). Interestingly, Burke et al. (1984) reported that this M/F pattern was more frequent in environmental motile Aeromenus than in clinical isolates. The usefulness of the other cell surface characteristics such as negative agglutination in acriflavine and instability after boiling has been reported by Mittal et al. (1980) for screening Aeromonas strains virulent for fish. However, we have not observed differences between the number of fish pathogenic and non-pathogenic strains showing these properties (Fig. 1) . Hence, we consider that these factors are not suitable as presumptive criteria of pathogenicity for fish. On the other hand, there is a controversy about the existence of a relationship between cytotoxicity and virulence. In this study, both virulent and nonvirulent strains produced a cytotoxic response in the fish cell-lines tested (Fig. 2 ) , which demonstrates that cytotoxicity “in vitro” is not necessarily indicative of the pathogenicity of motile Aeromonas. We have detected a similar lack of correlation between cytotoxin production and virulence for fish in pathogenic V. u~u~ZZurumstrains isolated on the American and European Atlantic coasts (Toranzo et al., 1983,1987). Several attempts have been made to correlate biochemical traits with virulence properties in clinical and environmental Aeromonus isolates (Kaper et al., 1981; Morgan et al., 1985). Nevertheless, few similar studies have been conducted in fish isolates (Wakabayashi et al., 1981) . Our results (Table 1)

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show that the associations between phenotypic characters and virulence differ according to the poikilotherm or homeotherm systems used in the assays. In addition, precautions must be taken in establishing such correlations since a number of non-pathogenic strains also possess some virulence factors. ACKNOWLEDGEMENTS

This work was supported by Grant AQ-0018/84 from the Comisicin Asesora de Investigation Cientifica TQcnica (CAICYT) , Ministerio de Education y Ciencia, Spain, and by Grant CCB85-09013 from the Comite Conjunto Hispano-Norteamericano para la Cooperation Cientifica y Tecnologica. Y. Santos and C.P. Dopazo acknowledge the Ministerio de Education y Ciencia, and T.P. Nieto the Xunta de Galicia for fellowships. We thank Dr. Frank M. Hetrick, Department of Microbiolo~, University of Maryland, for the kind supply of the fish cell-lines.

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