Experimental Pathogenicity of Aeromonas spp. for the Zebra Mussel, Dreissena polymorpha

August 4, 2017 | Autor: Gayatri Patel | Categoria: Microbiology, Medical Microbiology, Zebra Mussel
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CURRENT MICROBIOLOGY Vol. 36 (1998), pp. 19–23

An International Journal

R Springer-Verlag New York Inc. 1998

Experimental Pathogenicity of Aeromonas spp. for the Zebra Mussel, Dreissena polymorpha James S. Maki,1 Gayatri Patel,2 Ralph Mitchell2 1Marquette

University, Department of Biology, Wehr Life Sciences Building, P.O. Box 1881, Milwaukee, WI 53201-1881, USA University, Division of Engineering and Applied Sciences, Laboratory of Microbial Ecology, 40 Oxford Street, Cambridge, MA 02138, USA 2Harvard

Received: 26 March 1997 / Accepted: 7 July 1997

Abstract. Experiments were conducted to determine whether species of Aeromonas were pathogenic to the zebra mussel Dreissena polymorpha. A. jandaei, A. veronii, and A. media, identified with Biolog, were originally isolated from dead zebra mussels. When inoculated into living mussels, these bacteria resulted in the mortality of the bivalves. Two additional species, A. salmonicida salmonicida (ATCC 33678) and A. hydrophila (ATCC 7966), were also demonstrated to be pathogenic to the mussels. In addition to the pathogenicity, the data also suggest that the zebra mussels may be an important reservoir for these bacteria in freshwater environments.

Since the invasion of the Laurentian Great Lakes by the zebra mussel (Dreissena polymorpha) in the mid-1980’s [13], the range of this animal has continued to expand through inter-connected waterways and by accidental introductions into previously uninfested areas [20]. Although increasing amounts of information about the biology of this organism have become available [10, 21] there are no reports of bacterial pathogens for this organism. Recently, in a study of the heterotrophic microflora of the zebra mussel, we isolated bacteria of the genus Aeromonas from dead mussels (see below). Aeromonas spp. are well-known fish [4, 5, 15] and human pathogens [3], and their presence in zebra mussels suggested that these bacteria may also be pathogenic to the these bivalves and/or the mussels may act as an important reservoir of these pathogens of other organisms. We report here the results of tests to examine the pathogenicity of Aeromonas isolates on the zebra mussel, D. polymorpha. Materials and Methods Bacteria. Aeromonas veronii, A. jandaei, and A. media were isolated from homogenized dead zebra mussel tissue and identified by the Biolog bacterial identification system according to manufacturer’s instructions with GN microplates and a 24-h incubation (Biolog, Inc.,

Correspondence to: J.S. Maki

Hayward, CA, USA). Biolog identifications were accepted as correct if the similarity index of the genus and species name was 0.500 or greater at 24 h [17]. Cultures of A. salmonicida salmonicida (ATCC 33678) and A. hydrophila (ATCC 7966) were obtained from the American Type Culture Collection (MD, USA). An additional bacterium, Pseudomonas fragi, isolated from homogenized healthy zebra mussel tissue and identified by Biolog, was also used in the experiments as a negative control. Bacteria were maintained on either Plate Count Agar or Nutrient Agar (Difco, Detroit, MI, USA). Animals. Zebra mussels were collected from Milwaukee, WI or Buffalo, NY and transported overnight in wet paper towels on ice to Cambridge, MA. Animals were stored in aerated aquaria in artificial lake water (ALW) at 10°C. ALW is composed of Instant Ocean artificial sea water (Instant Ocean Aquarium Systems Inc., Eastlake, OH) diluted to 10% strength with distilled water and autoclaved. All mussels used in experiments were identified as zebra mussels, D. polymorpha, by known characteristics [8]. Test for pathogenicity. Bacterial cultures were grown overnight at ambient temperature (26°C) in 10 ml of Nutrient Broth (Difco) and used to inoculate 50-ml batches of Nutrient Broth. These cultures were grown at 26°C for 4 h. Cells were harvested by centrifugation (10,000 g), washed once, and resuspended in 10 ml ALW. Cells were serially diluted and used to inoculate triplicate Nutrient Agar plates for assay of colony forming units (CFU). The cell densities were 107 CFU ml21. Mussels were removed from the storage aquaria (10°C) and placed in individual sterilized beakers with 20 ml of sterile ALW. These mussels were held at 10°C overnight. Mussels were then placed in sterile Petri dishes at ambient temperature (26°C) and allowed to dry for 2 h. Three to six mussels each were then inoculated with 0.1 ml of the bacterial suspensions or with 0.1 ml of ALW as a control. Inoculations were accomplished by inserting a narrow-gauge needle dorsally just

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CURRENT MICROBIOLOGY Vol. 36 (1998)

Table 1. Results of the inoculation of zebra mussels with Aeromonas jandaei and A. veronii after incubation for 5 days No. mussels inoculated

No. dead mussels

Control Pseudomonas fragi Aeromonas jandaei

3 3 3

0 0 3

Aeromonas veronii

3

2

Treatment

between the valves and injecting the cell suspensions or ALW slowly to avoid dripping of the material outside the shells. Mussels were incubated in the dry sterile Petri dishes overnight and then placed back into beakers with 20 ml ALW and incubated at 10°C for between 5 and 8 days. Mussels were examined every day and judged to be alive if their siphons were exposed and they responded to stimuli (touching with a sterile glass rod) by closing their shells [18]. Dead mussels most often appeared with their shells open and did not respond to any stimulus. Confirmation of pathogenicity. The action of the Aeromonas spp. as the agent of mortality was confirmed by satisfying Koch’s postulates with dead mussels. The tissues of each dead mussel were individually scraped into a sterile tared container with sterile scalpel for determination of wet tissue weight. Tissues from each dead mussel were homogenized in a tissue grinder with sterile ALW, serially diluted, and used to inoculate Plate Count Agar (PCA, Difco). Plates were incubated for 5–7 days at 10°C before CFU were counted. The mean and standard deviations of CFU from each sample were determined. Bacterial strain diversity of the resulting colonies was estimated by use of colony morphology as an indicator of strain type [19, 25] and the Shannon Index [26]. The predominant bacterial types were isolated, streaked for purity, and characterized and/or identified by the Biolog bacterial identification system and a 24-h incubation according to manufacturer’s instructions. Statistical analyses. In order to statistically examine the pathogenicity of the Aeromonas spp., the percentage of mussels that died after inoculation with each treatment (i.e., ALW, Pseudomonas fragi, Aeromonas spp.) in each experiment was determined. The data were pooled, and the mean and standard deviations of these percentages was calculated. Percentages were normalized with an arcsin transformation and analyzed for significance by analysis of variance (ANOVA) and the Tukey test [26].

Results The initial isolations of the Aeromonas species were from a dead mussel that had been stored for 5 weeks at 4°C in an aerated aquarium containing sterile ALW. The ALW had been changed every 2 weeks. The mussel’s shell was wide open and the organism did not respond to being touched by a sterile glass rod. After incubation of serially diluted homogenized tissue on PCA plates, it was determined that the tissue contained 5.2 (61.2) 3 109 CFU g21 tissue. In total, 108 colonies were examined for their morphology, and the Shannon Index for diversity was equal to 0.538. A healthy mussel has a diversity of about

Tissue mass (g)

CFU (6SD) g21 tissue

(a) 0.225 (b) 0.366 (c) 0.367 (a) 0.372 (b) 0.244

7.5 (0.2) 3 107 8.9 (1.2) 3 107 1.4 (0.5) 3 108 3.1 (0.9) 3 108 4.6 (0.1) 3 108

Identification of predominant colony type

Aeromonas media Aeromonas media Aeromonas jandaei Aeromonas veronii Aeromonas veronii

0.9 (Maki, unpublished). Two colony types constituted 85% of the colonies. They were identified as Aeromonas veronii (Biolog similarity index 5 0.593) and Aeromonas jandaei (Biolog similarity index 5 0.586). Cultures of these two bacteria, a culture of Pseudomonas fragi (Biolog similarity 5 0.724) as one control, and ALW as a second control were used to inoculate three mussels each. After 5 days of incubation, all mussels in the control groups were alive, while all three mussels inoculated with A. jandaei and two of the mussels inoculated with A. veronii had died (Table 1). The indices for diversity based on colony morphology of the isolates from the mussels inoculated with A. jandaei were between 0.179 and 0.674. However, in two out of the three mussels, Aeromonas media, colony morphology representing 61 (613) % of 217 total colonies from the two mussels, was identified by Biolog as the predominant colony type (Table 1, Biolog similarity 5 0.773). In the third mussel, A. jandaei, colony morphology representing 88% of 59 total colonies, was identified (Biolog similarity 5 0.567). In the mussels that died after being inoculated with A. veronii, the diversity indices were 0.043 and 0.202, and A. veronii was identified by Biolog as the predominant colony type (colony morphology represented 93 6 6% of 134 total colonies from the two mussels) in both cases (Biolog similarity 5 0.676). The isolates of A. jandaei, A. veronii, and A. media obtained in the first experiment were used to inoculate a new set of mussels along with the same controls. For controls and A. jandaei, three mussels were inoculated, while for A. veronii and A. media, six mussels were inoculated (Table 2). One mussel inoculated with P. fragi died after 3 days, and P. fragi was the predominant bacterial type isolated from this mussel (Table 2). No other control mussels died. By eight days all the mussels inoculated with Aeromonas spp. had died, and the predominant bacterial types isolated from each dead mussel corresponded to the bacterium the mussels were inoculated with (Table 2). For A. veronii, colony morphology represented 88 (68)% of 60 (612) colonies per

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J.S. Maki et al.: Aeromonas Pathogenicity to Mussels Table 2. Results of inoculation of zebra mussels with Aeromonas jandaei, A. veronii, and A. media after incubation for 8 days No. mussels inoculated

No. dead mussels

Control Pseudomonas fragi Aeromonas jandaei

3 3 3

0 1 3

Aeromonas veronii

6

6

Aeromonas media

6

6

Treatment

Tissue mass (g)

CFU (6SD) g21 tissue

0.115 (a) 0.350 (b) 0.507 (c) 0.426 (a) 0.531 (b) 0.529 (c) 0.081 (d) 0.572 (e) 0.355 (f) 0.358 (a) 0.267 (b) 0.133 (c) 0.193 (d) 0.244 (e) 0.167 (f) 0.248

8.5 (0.6) 3 108 4.3 (0.3) 3 109 6.6 (1.7) 3 108 3.7 (0.2) 3 108 2.4 (0.1) 3 109 2.3 (0.2) 3 109 4.7 (0.7) 3 109 1.9 (0.4) 3 108 1.2 (0.2) 3 108 8.2 (1.6) 3 107 6.8 (1.2) 3 109 3.3 (0.9) 3 109 7.4 (0.4) 3 109 4.6 (0.3) 3 109 8.3 (1.6) 3 109 4.4 (1.6) 3 109

Identification of predominant colony type

Pseudomonas fragi Aeromonas jandaei Aeromonas jandaei Aeromonas jandaei Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas media Aeromonas media Aeromonas media Aeromonas media Aeromonas media Aeromonas media

Table 3. Results of inoculation of zebra mussels with Aeromonas salmonicida salmonicida and A. hydrophila after incubation for 6 days No. mussels inoculated

No. dead mussels

Control Pseudomonas fragi Aeromonas salmonicida salmonicida

6 6 6

1 0 6

Aeromonas hydrophila

6

6

Treatment

mussel. For A. media, colony morphology represented 93 (612)% of 106 (637) colonies per mussel. For A. jandaei, colony morphology represented 94 (68)% of 62 (623) colonies per mussel. All Biolog similarities were greater than 0.500 after 24 h incubation. The third experiment involved two cultures obtained from the American Type Culture Collection: Aeromonas salmonicida salmonicida and Aeromonas hydrophila. With the same controls (ALW and P. fragi), these two bacteria were inoculated into six mussels each. After 6 days all six mussels inoculated with Aeromonas spp. had died (Table 3). One mussel in the ALW control group died after 3 days. No other control mussels died. For each mussel inoculated with A. salmonicida salmonicida, the resulting predominant bacteria, with colony morphology

Tissue mass (g)

CFU (6SD) g21 tissue

Identification of predominant colony type No Predominant Bacterium

(a) 0.341 (b) 0.421 (c) 0.312 (d) 0.292 (e) 0.142 (f) 0.411 (a) 0.215 (b) 0.139 (c) 0.131 (d) 0.349 (e) 0.112 (f) 0.214

1.9 (0.1) 3 108 9.2 (2.2) 3 107 1.0 (0.2) 3 108 5.5 (1.4) 3 108 1.6 (0.3) 3 108 4.9 (1.7) 3 108 8.5 (2.5) 3 107 5.4 (0.5) 3 108 3.7 (1.0) 3 1010 8.4 (2.9) 3 107 4.5 (1.0) 3 109 6.4 (1.8) 3 107

Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila

representing 91 (65)% of 59 (611) colonies per mussel, were only identified to the genus Aeromonas by Biolog (similarities 5 0.379 to 0.421). The predominant colony type from each mussel inoculated with A. hydrophila, colony morphology representing 87 (66)% of 53 (613) colonies per mussel, were all identified as A. hydrophila by Biolog (similarities 5 0.532 to 0.729). No predominant type of bacterium was found in the control mussel that died. The pooled data showed that overall the percentage of mussels that died after inoculation with Aeromonas spp. was much higher than in the ALW and P. fragiinoculated mussels (Fig. 1). The percentages of mussel mortality for the Aeromonas spp. and the two controls were different (P , 0.003, ANOVA). The difference

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Fig. 1. Comparison of the mean percentage zebra mussel mortality after inoculation with ALW, Pseudomonas fragi, and Aeromonas spp. over the three experiments. Mortality after inoculation with Aeromonas spp. was different from the other two treatments (see text). Bars denote 1 standard deviation.

between treatments was as follows: mortality from Aeromonas spp.-treated mussels . mortality from P. fragi-treated mussels 5 mortality in ALW-treated mussels (P , 0.005, Tukey test).

Discussion Our results indicate that Aeromonas jandaei, A. veronii, A. media, A. salmonicida salmonicida, and A. hydrophila can be pathogens for the zebra mussel Dreissena polymorpha in laboratory assays. A. jandaei and A. veronii, originally isolated from a dead mussel, were used to inoculate living mussels and were reisolated from these mussels when the animals died (Table 1). Subsequently they were used to inoculate a second batch of living mussels, and finally reisolated again from these mussels when they died (Table 2). A. media was isolated from dead mussels (Table 1), used to inoculate living mussels, and reisolated when these animals died (Table 2). Although A. salmonicida salmonicida and A. hydrophila were not isolated from dead mussels, when they were inoculated into living mussels, the bivalves died (Table 3). A. salmonicida salmonicida was not specifically identified by the Biolog system from these mussels, only the genus Aeromonas was, but A. hydrophila was identified as the predominant colony type from dead mussels it was inoculated into (Table 3). The mortality of the zebra mussels after being inoculated with Aeromonas spp. was significantly greater than with either of the controls (Fig. 1). The habitat for members of the genus Aeromonas is known to be aquatic environments [3, 5, 11, 15]. Thus, it

CURRENT MICROBIOLOGY Vol. 36 (1998)

is not surprising that they should be found in zebra mussels, which are filter feeders and have been demonstrated to be able to consume bacteria [9, 23]. The temperature at which the experiments were conducted as well within the range of growth for this genus [2]. A. salmonicida salmonicida and A. hydrophila are wellknown fish pathogens [4, 5, 16]. A. jandaei, A. veronii, and A. hydrophila have been found in clinical specimens [2, 7, 14, 16]. A. media, originally isolated from river water [1], has not been found in clinical specimens [2]. The mechanisms of pathogenicity of Aeromonas spp. are complex. The best studied are those of A. salmonicida salmonicida and A. hydrophila in a number of other animals, primarily fish [5, 12]. The pathogenicity of these species involves cell surface layers that may aid in the adhesion of the bacteria to host cells and, presumably, the bacterial colonization of the infected organism. In addition, Aeromonas spp. produce extracellular products including proteases, hemolysins, and toxins, which result in host tissue damage and may aid the spread of the bacteria to new tissues [3–5, 11, 12]. Challenging oysters with an Aeromonas isolate resulted in the mortality of the bivalves after the bacterium had invaded the hemolymph and the soft tissues [22]. This suggests for zebra mussels that the adhesion of the bacteria in colonization, and their production of extracellular proteases and toxins that may be involved in internal spreading, are likely candidates for the mechanisms of pathogenicity and need to be examined as they have been in other organisms. In any case, multiple factors are probably involved, and different strains may produce various products with similar activities [3]. Overall the colony morphology of the Aeromonas spp. represented 88 (611)% of the colonies cultured from the dead mussels. Other bacterial genera found in freshly collected zebra mussels identified with Biolog include Pseudomonas, Acinetobacter, Comamonas, and Sphingobacterium (Maki, unpublished). The data indicate that the bacteria present in the mussels when they were inoculated with Aeromonas spp. did not prevent the pathogenicity of the latter. However, investigations with fish have shown that some bacteria can have an inhibitory effect on Aeromonas pathogenicity either through competition for nutrients or production of potentially harmful metabolites [6, 24]. Thus, it is also possible that some of the microbiota of zebra mussels may be able to provide some similar protection against Aeromonas infection, although we have no evidence to support this. We did find the presence of two different species of Aeromonas in some of the zebra mussels that were examined. This suggests that the presence of more than one Aeromonas spp. in a zebra mussel may result in a synergistic pathogenicity. The data indicate that a number of Aeromonas

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J.S. Maki et al.: Aeromonas Pathogenicity to Mussels

species can be pathogenic to zebra mussels. Because some of these species were originally isolated from the mussels themselves, this suggests that the bacteria naturally occur in these bivalves, and the mussels may act as a reservoir for these pathogenic bacteria. Currently, there is no information on how widely distributed these bacteria are in zebra mussels. However, the fact that some are also well-known fish and/or human pathogens may suggest an additional effect of zebra mussels in areas of fish aquaculture and/or human contact. ACKNOWLEDGMENTS The authors thank K.D. Noel for his comments on the manuscript. Research was supported in part by grants from Ontario Hydro, The Ontario Ministry of the Environment, the NOAA National Sea Grant College Program to Harvard University, and by Marquette University.

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