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Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation ab
a
ac
Xiaojun Zhang , Wenlong Cai , Zhen Tao
a
& Cova R. Arias
a
Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama 36849, USA b
Present address: Huaihai Institute of Technology of China, 2220055, Liamuimgang , China
c
Present address: School of Marine Sciences, Ningbo University, 315211, Ningbo, China Published online: 21 Aug 2014.
To cite this article: Xiaojun Zhang, Wenlong Cai, Zhen Tao & Cova R. Arias (2014) Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation, Journal of Aquatic Animal Health, 26:3, 190-193 To link to this article: http://dx.doi.org/10.1080/08997659.2014.922515
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:190–193, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.922515
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Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation Xiaojun Zhang,1 Wenlong Cai, Zhen Tao,2 and Cova R. Arias*
Downloaded by [Johann Christian Senckenberg] at 22:16 04 September 2014
Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama 36849, USA
Abstract The survival of Aeromonas hydrophila under low-nutrient conditions was investigated in this study. The behavior of three strains isolated from Common Carp Cyprinus carpio (China) and Channel Catfish Ictalurus punctatus (USA) was compared when cells were starved at different temperatures (4, 15, 25, and 35◦ C) over a 4week period. Temperature played a major role in cell survival, and cell viability decreased rapidly at 4◦ C. Conversely, cells stored at 15◦ C did not lose viability over time. Warmer temperatures (25◦ C and 35◦ C) decreased cell numbers by approximately one order of magnitude. Ultrastructural changes in cell morphology were observed in starved cells. Our data confirm that A. hydrophila can persist in the aquatic environment for extended periods, but survival is strongly influenced by temperature.
Aeromonas hydrophila is a gram-negative bacterium and one of the causative agents of motile Aeromonas septicemia (MAS) in fish. This pathogen affects a variety of freshwater fish species around the world (Austin and Austin 1999), including Nile Tilapia Oreochromis niloticus (Ruangpan et al. 1986), Channel Catfish Ictalurus punctatus (Defigueiredo and Plumb 1977), Goldfish Carassius auratus (Brenden and Huizinga 1986), Common Carp Cyprinus carpio (Lamers and Vanmuiswinkel 1986), and European Eel Anguilla anguilla (Esteve et al. 1994). Recently, catfish producers in Alabama have experienced severe losses due to an atypical A. hydrophila strain of unusual virulence (Hemstreet 2010). Since 2009, more than 7.5 million pounds of market-size catfish have been lost due to an epidemic of MAS that started in the heart of the catfish-producing region in western Alabama (Pridgeon and Klesius 2011). Similarly, carp production in China has been seriously affected by outbreaks of A. hydrophila which seemingly intensify over time (Nielsen et al. 2001). Currently, no preventive or palliative measures are effective against this disease (Austin and Austin 1999).
Aeromonas hydrophila is ubiquitous in natural freshwater environments, including lakes, rivers, and ponds (Rippey and Cabelli 1979), and it has been reported as part of the natural microbiota of fishes (Trust and Sparrow 1974). The vast majority of freshwater ecosystems are oligotrophic, and it is accepted that aquatic bacteria must adjust to rapid changes in nutrient availability in order to survive (Poindexter 1981). Several studies have characterized the behavior of A. hydrophila in low-nutrient water (Kersters et al. 1996; Brandi et al. 1999; Croci et al. 2001) and indicate that this bacterium can survive for extended periods even in chlorinated tap water. Moreover, A. hydrophila cells appear to enter the viable but nonculturable (VBNC) state under starvation at low temperatures (Mary et al. 2002). These studies confirm that A. hydrophila can quickly adapt to low-nutrient environments after being cultured in highnutrient media. Temperature also plays a key role in how this bacterium responds to starvation. However, the strains used in these studies were isolated from water or from human patients. Little information is available regarding the survival of fishpathogenic strains during periods of starvation. The objective of this study was to determine the culturability of three strains of A. hydrophila isolated from epizootic events in China and the USA under conditions of low-nutrient availability at different temperatures. METHODS Bacterial strains.—Three previously characterized A. hydrophila strains were used in this study. Strain ML09119, considered an atypical and highly virulent strain of A. hydrophila, was isolated from diseased Channel Catfish during a large-scale disease outbreak in Alabama in 2009 (Tekedar et al. 2013). Strain AL01 was isolated from Channel Catfish
*Corresponding author: [email protected] 1 Present address: Huaihai Institute of Technology of China, Liamuimgang 2220055, China. 2 Present address: School of Marine Sciences, Ningbo University, Ningbo 315211, China. Received December 13, 2013; accepted March 22, 2014
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in 2001 during a typical MAS episode, and it displayed low virulence toward Channel Catfish in artificial challenges (Arias, personal observation). Strain HC060718-1 was isolated from a Common Carp during a severe MAS outbreak in that species in Jiangsu Province, China. Bacteria were stored at −80◦ C as glycerol stocks and routinely cultured on Luria Bertani (LB) agar or broth with shaking (125 rpm) at 28 ± 2◦ C for 24 h. Microcosm inoculation.—Individual colonies from each strain were inoculated into 5 mL of LB broth and incubated at 28 ± 2◦ C overnight with shaking. An aliquot (50 µL) of a 24-h culture was inoculated into 15 mL of LB broth and incubated overnight as before. Cultures were then centrifuged at 5,000 × g for 10 min, and pellets were washed twice with 0.85% (weight : volume) NaCl sterile solution to avoid any carryover of nutrients. Cells were finally resuspended in sterile distilled water to an approximate concentration of 106 colony-forming units (CFU)/mL. Four aliquots of 10 mL each were transferred to sterile tubes and stored in the dark at 4, 15, 25, and 35◦ C, respectively. Temperatures were selected based on the upper (35◦ C) and lower limits (15◦ C) of MAS occurrence in aquaculture ponds. A lower temperature (4◦ C) was added to test whether A. hydrophila can survive winter temperatures. For statistical analysis, there were three biological replicates per temperature. Cell enumeration.—Cell counts were determined immediately after cells were resuspended in sterile distilled water and at 1, 7, 14, 21, and 28 d postinoculation. Cell suspensions were 10-fold serially diluted in sterile distilled water and 100 µL of each dilution plated on LB agar in triplicate. Colonies were enumerated after 24 h of incubation at 28◦ C. Statistical analysis.—Cell counts were converted to base10 logarithms to meet the model assumption of a normal distribution, and mean log10 CFUs/mL ± SEs were calculated. One-way analysis of variance (ANOVA) was used to determine the differences in A. hydrophila CFU/mL from the short-term survival study and Welch’s ANOVA (allowing for unequal variance) in different strains. If the results of either the ANOVA or Welch’s ANOVA were statistically significant (P < 0.05), Tukey’s and Scheff´e’s method were applied to perform post hoc, pairwise comparisons at α = 0.05 for the means of the log A. hydrophila counts or Dunnett’s T3 test (allowing unequal variance) for post hoc, pairwise comparisons for counts in different strains at α = 0.05. Ultrastructural analysis.—Changes in morphology were monitored under starvation conditions using scanning electron microscopy as previously described (Arias et al. 2012). Briefly, cells (5 µL of culture) were fixed in 2.5% glutaraldehyde (volume : volume) at 4◦ C overnight. Samples were filtered through Isopore membrane (0.2 µm GTBP 02500; Millipore) dehydrated in a graded ethanol series (50, 70, 90, and 100%), critical-point dried in CO2 in an EMS 850 (Electron Microscopy Science) and coated with gold palladium alloy in an EMS 550X (Electron Microscopy Science). The coated samples were examined using a Zeiss EVO 50.
TABLE 1. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in sterile distilled water at 4◦ C. Data were log10 transformed to ensure normality. Within columns, significantly different means (P < 0.05) are denoted by different lowercase letters; within rows, significantly different means are denoted by different uppercase letters.
Day 0 1 7 14 21 28
ML09-119 6.602 6.648 4.958 4.679 4.726 3.131
± ± ± ± ± ±
AL01
0.008 zZ 0.044 zZ 0.194 yZ 0.211 yZ 0.352 yZ 0.082 xZ
± ± ± ± ± ±
6.230 6.318 4.763 4.600 4.034 3.071
0.005 zZ 0.073 zY 0.348 yZ 0.372 yZ 0.523 yZ 0.103 xZ
HC060718-1 6.556 6.603 5.382 4.816 4.784 3.558
± ± ± ± ± ±
0.002 zZ 0.055 zZ 0.207 yZ 0.188 xZ 0.158 xZ 0.361 wZ
RESULTS AND DISCUSSION The behavior of A. hydrophila under conditions of starvation at different temperatures was determined over a 4-week period. Table 1 summarizes cell survival at 4◦ C. Initial cells counts (approximately 106 CFU/mL) were statistically identical in all strains. The first significant reduction was observed at day 7 in all strains. Cell numbers remained constant for 2 weeks in strains ML09-119 and AL01 but dropped significantly by day 28. Significant reductions in cell counts in strain HC060718-1 were observed on days 7, 14, and 28. By the end of the experiment (day 28), all strains had experienced a reduction of three orders of magnitude in culturability. Our results are in agreement with those of Mary et al. (2002), in which a sharp decrease in cell culturability of A. hydrophila type strain ATCC 7966 was observed within the first few days of incubation at 4◦ C. According to the Mary et al. (2002) study, A. hydrophila entered the VBNC state at low temperatures because cells appeared to remain viable (as per the LIVE/DEAD BacLight Bacterial Viability kit), but they could no longer be cultured on media. However, raising the temperature to 25◦ C did not reverse the VBNC stage, bringing into question the true nature of those VBNC forms. Whether A. hydrophila can survive as VBNC cells at low temperatures remains uncertain. Cell viability at 15◦ C slightly varied between strains throughout the 4-week study (Table 2). Overall, cells maintained viability over time, and no significant decrease in cell counts was TABLE 2. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in sterile distilled water at 15◦ C. See Table 1 for additional information.
Day 0 1 7 14 21 28
ML09-119 6.602 6.777 6.685 6.476 6.492 6.160
± ± ± ± ± ±
0.007 zZ 0.034 zY 0.103 zZ 0.114 zZ 0.077 zZ 0.213 yZ
AL01 6.230 6.598 6.734 6.144 6.479 6.408
± ± ± ± ± ±
0.003 xw,Z 0.006 zy,X 0.091 zZ 0.056 wY 0.189 zyx,Z 0.210 yxw,Z
HC060718-1 6.556 6.899 6.733 6.469 6.129 6.416
± ± ± ± ± ±
0.009 yx,Z 0.006 zZ 0.097 zy,Z 0.130 xZ 0.156 wZ 0.082 xZ
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TABLE 3. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in ultrapure water at 25◦ C. See Table 1 for more information.
Day
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0 1 7 14 21 28
ML09-119 7.146 6.500 6.219 5.476 4.964 5.030
± ± ± ± ± ±
0.004 zZ 0.209 yZ 0.194 yZ 0.044 xY 0.236 wZ 0.333 xw,Z
AL01 6.845 6.682 6.260 5.925 5.373 5.211
± ± ± ± ± ±
0.007 zZ 0.102 zZ 0.170 zy,Z 0.134 yZ 0.378 xZ 0.376 xZ
HC060718-1 6.663 6.627 6.287 6.114 5.514 5.076
± ± ± ± ± ±
0.003 zZ 0.111 zZ 0.178 zy,Z 0.163 zy,Z 0.487 yx,Z 0.655 xZ
observed during the experiment. Conversely, at 25◦ C there was a stronger effect on cell survival, with cell counts decreasing over time to approximately 105 CFU/mL (Table 3). The initial and final counts did not differ between strains, but ML09-119 presented fewer culturable cells than the other two strains at day 14. Previous studies compared the survival of A. hydrophila at 10◦ C and 20◦ C (Croci et al. 2001) and room temperature (Kersters et al. 1996), with similar results. Lower temperatures (10–15◦ C) favored the survival of A. hydrophila cells regardless of the bacterial strain or water chemistry used. By contrast, higher temperatures (≥20◦ C) reduced culturability over time. At 35◦ C, all strains showed a significant decrease in cell numbers at day 7, after which they remained constant for the duration of the study (Table 4). To the best of our knowledge, these are the first data on A. hydrophila survival at 35◦ C. Although 35◦ C might seem excessively high for natural aquatic environments, catfish ponds typically reach (and could exceed) this temperature during the summer months. The morphology of starved cells at day 21 was compared with that of fresh (24-h) cultures. As can be seen in Figure 1, the morphology of A. hydrophila cells dramatically changed during starvation, with cells becoming shorter. Cell length decreased significantly, from 2.0 ± 0.2 µm in fresh cultures to 1.0 ± 0.2 µm in starved cells. The observed changes were similar in all three strains examined. “Rounding up” is a common phenomenon in cells subjected to starvation and has previously been described in several gram-negative species (Wai et al. 1999). Starved cells were covered by a “veil of secreted slime” that has also been observed in bacterial cells TABLE 4. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in ultrapure water at 35◦ C. See Table 1 for additional information.
Day 0 1 7 14 21 28
ML09-119 7.146 6.264 4.457 4.839 4.659 5.075
± ± ± ± ± ±
0.006 zZ 0.093 yZ 0.523 xZ 0.329 xZ 0.475 xZ 0.112 xZ
AL01 6.845 6.169 4.243 4.629 4.019 5.108
± ± ± ± ± ±
0.009 zZ 0.148 zZ 0.656 yZ 0.456 yZ 0.541 yZ 0.247 yZ
HC060718-1 6.662 5.759 4.248 4.631 3.892 4.793
± ± ± ± ± ±
0.005 zZ 0.432 zy,Z 0.652 yx,Z 0.416 yx,Z 0.398 xZ 0.122 yx,Z
FIGURE 1. Morphological changes to Aeromonas hydrophila cells during starvation in sterilized distilled water as determined by scanning electronic microscopy. Panel (A) shows 24-h-old cells immediately prior to inoculation into microcosms, panel (B) cells maintained under starvation for 28 d at 25◦ C. Bars = 1 µm.
maintained under low-nutrient conditions (Arias et al. 2012). Such a drastic change in their ultrastructure is likely to affect the attachment and colonization capabilities of this fish pathogen, but further studies on its infectivity and pathogenicity are needed to test this hypothesis. In summary, our data show that all three strains of A. hydrophila behaved very similarly under starvation and that all can survive for extended periods at moderate and warm temperatures. Based on our study and others (Croci et al. 2001), the threshold between survival and loss of culturability occurs between 4◦ C and 10◦ C; above 15◦ C, cells lose some viability over time but can survive in water without nutrients for several weeks. Our results support the notion that water can act as a reservoir and serve as a dispersal mechanism for this pathogen. Future studies should assess the virulence of starved A. hydrophila cells to better define the life cycle of this species in the aquatic environment. ACKNOWLEDGMENTS We thank Michael Miller (Advanced Microscopy and Imaging Laboratory, Auburn University) for his help with scanning microscopy. This research was funded by the USDA– ARS/Auburn University Specific Cooperative Agreement Prevention of Diseases of Farmed Raised Fish and USDA–ARS CRIS project 6420-32000-022-00D. Xiaojun Zhang is the recipient of a fellowship from the Jiangsu Overseas Research and Training Program for University Prominent Young and MiddleAged Teachers and Presidents. Wenlong Cai thanks Shanghai Ocean University for partially supporting his research fellowship. Zhen Tao is the recipient of a graduate research fellowship funded by the Chinese Scholarship Council and Qingdao Ocean University. REFERENCES Arias, C. R., S. LaFrentz, W. Cai, and O. Olivares-Fuster. 2012. Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes. BMC Microbiology [online serial] 12:266.
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COMMUNICATION Austin, B., and D. A. Austin 1999. Bacterial fish pathogens: diseases of farmed and wild fish. Springer, New York. Brandi, G., M. Sisti, F. Giardini, G. F. Schiavano, and A. Albano. 1999. Survival ability of cytotoxic strains of motile Aeromonas spp. in different types of water. Letters in Applied Microbiology 29:211–215. Brenden, R. A., and H. W. Huizinga. 1986. Pathophysiology of experimental Aeromonas hydrophila infection in Goldfish, Carassius auratus. Journal of Fish Diseases 9:163–167. Croci L., S. De Pasquale, L. Cozzi, and L. Toti. 2001. Behavior of Aeromonas hydrophila in bottled mineral water. Journal of Food Protection 64:1836– 1840. Defigueiredo, J., and J. A. Plumb. 1977. Virulence of different isolates of Aeromonas hydrophila in Channel Catfish. Aquaculture 11:349–354. Esteve, C., C. Amaro, and A. E. Toranzo. 1994. O-serogrouping and surface composition of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels. FEMS Microbiology Letters 117:85–90. Hemstreet, B. 2010. An update on Aeromonas hydrophila from a fish health specialist for summer 2010. Catfish Journal 24:4. Kersters, I., G. Huys, H. Van Duffel, M. Vancanneyt, K. Kersters, and W. Verstaete. 1996. Survival potential of Aeromonas hydrophila in freshwaters and nutrient-poor waters in comparison with other bacteria. Journal of Applied Bacteriology 80:266–276. Lamers, C. H. J., and W. B. Vanmuiswinkel. 1986. Natural and acquired agglutinins to Aeromonas hydrophila in carp (Cyprinus carpio). Canadian Journal of Fisheries and Aquatic Sciences 43:619–624. Mary, P., N. E. Chihib, O. Charafeddine, C. Defives, and J. P. Hornez. 2002. Starvation survival and viable but nonculturable states in Aeromonas hydrophila. Microbial Ecology 43:250–258.
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Nielsen, M. E., L. Hoi, A. A. Schmidt, D. Qian, T. Shimada, J. Y. Shen, and J. L. Larsen. 2001. Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Diseases of Aquatic Organisms 46:23–29. Poindexter, J. S. 1981. Oligotrophy: fast and famine existence. Advances in Microbial Ecology 5:63–89. Pridgeon, J. W., and P. Klesius. 2011. Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected Channel Catfish during a disease outbreak in west Alabama (USA) in 2009. Diseases of Aquatic Organisms 94:249–253. Rippey, S. R., and V. J. Cabelli. 1979. Membrane filter procedure for enumeration of Aeromonas hydrophila in freshwaters. Applied and Environmental Microbiology 38:108–113. Ruangpan, L., T. Kitao, and T. Yoshida. 1986. Protective efficacy of Aeromonas hydrophila vaccines in Nile Tilapia. Veterinary Immunology and Immunopathology 12:345–350. Tekedar, H. C., G. C. Waldbieser, A. Karsi, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegrad, M. Hossain, S. C. Schroeder, L. Khoo, and M. L. Lawrence. 2013. Complete genome sequence of Channel Catfish epidemic isolate, Aeromonas hydrophila strain Ml09-119. Genome Announcements [online serial] 1(5):e00755-13. Trust, T. J., and A. H. Sparrow. 1974. The bacterial flora in the alimentary tract of freshwater salmonid fishes. Canadian Journal of Microbiology 20:1219– 1228. Wai, S. N., Y. Mizunoe, and S. Yoshida. 1999. How Vibrio cholerae survive during starvation. FEMS Microbiology Letters 180: 123–131.
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation ab
a
ac
Xiaojun Zhang , Wenlong Cai , Zhen Tao
a
& Cova R. Arias
a
Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama 36849, USA b
Present address: Huaihai Institute of Technology of China, 2220055, Liamuimgang , China
c
Present address: School of Marine Sciences, Ningbo University, 315211, Ningbo, China Published online: 21 Aug 2014.
To cite this article: Xiaojun Zhang, Wenlong Cai, Zhen Tao & Cova R. Arias (2014) Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation, Journal of Aquatic Animal Health, 26:3, 190-193 To link to this article: http://dx.doi.org/10.1080/08997659.2014.922515
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:190–193, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.922515
COMMUNICATION
Survival of Fish-Pathogenic Strains of Aeromonas hydrophila under Starvation Xiaojun Zhang,1 Wenlong Cai, Zhen Tao,2 and Cova R. Arias*
Downloaded by [Johann Christian Senckenberg] at 22:16 04 September 2014
Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama 36849, USA
Abstract The survival of Aeromonas hydrophila under low-nutrient conditions was investigated in this study. The behavior of three strains isolated from Common Carp Cyprinus carpio (China) and Channel Catfish Ictalurus punctatus (USA) was compared when cells were starved at different temperatures (4, 15, 25, and 35◦ C) over a 4week period. Temperature played a major role in cell survival, and cell viability decreased rapidly at 4◦ C. Conversely, cells stored at 15◦ C did not lose viability over time. Warmer temperatures (25◦ C and 35◦ C) decreased cell numbers by approximately one order of magnitude. Ultrastructural changes in cell morphology were observed in starved cells. Our data confirm that A. hydrophila can persist in the aquatic environment for extended periods, but survival is strongly influenced by temperature.
Aeromonas hydrophila is a gram-negative bacterium and one of the causative agents of motile Aeromonas septicemia (MAS) in fish. This pathogen affects a variety of freshwater fish species around the world (Austin and Austin 1999), including Nile Tilapia Oreochromis niloticus (Ruangpan et al. 1986), Channel Catfish Ictalurus punctatus (Defigueiredo and Plumb 1977), Goldfish Carassius auratus (Brenden and Huizinga 1986), Common Carp Cyprinus carpio (Lamers and Vanmuiswinkel 1986), and European Eel Anguilla anguilla (Esteve et al. 1994). Recently, catfish producers in Alabama have experienced severe losses due to an atypical A. hydrophila strain of unusual virulence (Hemstreet 2010). Since 2009, more than 7.5 million pounds of market-size catfish have been lost due to an epidemic of MAS that started in the heart of the catfish-producing region in western Alabama (Pridgeon and Klesius 2011). Similarly, carp production in China has been seriously affected by outbreaks of A. hydrophila which seemingly intensify over time (Nielsen et al. 2001). Currently, no preventive or palliative measures are effective against this disease (Austin and Austin 1999).
Aeromonas hydrophila is ubiquitous in natural freshwater environments, including lakes, rivers, and ponds (Rippey and Cabelli 1979), and it has been reported as part of the natural microbiota of fishes (Trust and Sparrow 1974). The vast majority of freshwater ecosystems are oligotrophic, and it is accepted that aquatic bacteria must adjust to rapid changes in nutrient availability in order to survive (Poindexter 1981). Several studies have characterized the behavior of A. hydrophila in low-nutrient water (Kersters et al. 1996; Brandi et al. 1999; Croci et al. 2001) and indicate that this bacterium can survive for extended periods even in chlorinated tap water. Moreover, A. hydrophila cells appear to enter the viable but nonculturable (VBNC) state under starvation at low temperatures (Mary et al. 2002). These studies confirm that A. hydrophila can quickly adapt to low-nutrient environments after being cultured in highnutrient media. Temperature also plays a key role in how this bacterium responds to starvation. However, the strains used in these studies were isolated from water or from human patients. Little information is available regarding the survival of fishpathogenic strains during periods of starvation. The objective of this study was to determine the culturability of three strains of A. hydrophila isolated from epizootic events in China and the USA under conditions of low-nutrient availability at different temperatures. METHODS Bacterial strains.—Three previously characterized A. hydrophila strains were used in this study. Strain ML09119, considered an atypical and highly virulent strain of A. hydrophila, was isolated from diseased Channel Catfish during a large-scale disease outbreak in Alabama in 2009 (Tekedar et al. 2013). Strain AL01 was isolated from Channel Catfish
*Corresponding author: [email protected] 1 Present address: Huaihai Institute of Technology of China, Liamuimgang 2220055, China. 2 Present address: School of Marine Sciences, Ningbo University, Ningbo 315211, China. Received December 13, 2013; accepted March 22, 2014
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in 2001 during a typical MAS episode, and it displayed low virulence toward Channel Catfish in artificial challenges (Arias, personal observation). Strain HC060718-1 was isolated from a Common Carp during a severe MAS outbreak in that species in Jiangsu Province, China. Bacteria were stored at −80◦ C as glycerol stocks and routinely cultured on Luria Bertani (LB) agar or broth with shaking (125 rpm) at 28 ± 2◦ C for 24 h. Microcosm inoculation.—Individual colonies from each strain were inoculated into 5 mL of LB broth and incubated at 28 ± 2◦ C overnight with shaking. An aliquot (50 µL) of a 24-h culture was inoculated into 15 mL of LB broth and incubated overnight as before. Cultures were then centrifuged at 5,000 × g for 10 min, and pellets were washed twice with 0.85% (weight : volume) NaCl sterile solution to avoid any carryover of nutrients. Cells were finally resuspended in sterile distilled water to an approximate concentration of 106 colony-forming units (CFU)/mL. Four aliquots of 10 mL each were transferred to sterile tubes and stored in the dark at 4, 15, 25, and 35◦ C, respectively. Temperatures were selected based on the upper (35◦ C) and lower limits (15◦ C) of MAS occurrence in aquaculture ponds. A lower temperature (4◦ C) was added to test whether A. hydrophila can survive winter temperatures. For statistical analysis, there were three biological replicates per temperature. Cell enumeration.—Cell counts were determined immediately after cells were resuspended in sterile distilled water and at 1, 7, 14, 21, and 28 d postinoculation. Cell suspensions were 10-fold serially diluted in sterile distilled water and 100 µL of each dilution plated on LB agar in triplicate. Colonies were enumerated after 24 h of incubation at 28◦ C. Statistical analysis.—Cell counts were converted to base10 logarithms to meet the model assumption of a normal distribution, and mean log10 CFUs/mL ± SEs were calculated. One-way analysis of variance (ANOVA) was used to determine the differences in A. hydrophila CFU/mL from the short-term survival study and Welch’s ANOVA (allowing for unequal variance) in different strains. If the results of either the ANOVA or Welch’s ANOVA were statistically significant (P < 0.05), Tukey’s and Scheff´e’s method were applied to perform post hoc, pairwise comparisons at α = 0.05 for the means of the log A. hydrophila counts or Dunnett’s T3 test (allowing unequal variance) for post hoc, pairwise comparisons for counts in different strains at α = 0.05. Ultrastructural analysis.—Changes in morphology were monitored under starvation conditions using scanning electron microscopy as previously described (Arias et al. 2012). Briefly, cells (5 µL of culture) were fixed in 2.5% glutaraldehyde (volume : volume) at 4◦ C overnight. Samples were filtered through Isopore membrane (0.2 µm GTBP 02500; Millipore) dehydrated in a graded ethanol series (50, 70, 90, and 100%), critical-point dried in CO2 in an EMS 850 (Electron Microscopy Science) and coated with gold palladium alloy in an EMS 550X (Electron Microscopy Science). The coated samples were examined using a Zeiss EVO 50.
TABLE 1. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in sterile distilled water at 4◦ C. Data were log10 transformed to ensure normality. Within columns, significantly different means (P < 0.05) are denoted by different lowercase letters; within rows, significantly different means are denoted by different uppercase letters.
Day 0 1 7 14 21 28
ML09-119 6.602 6.648 4.958 4.679 4.726 3.131
± ± ± ± ± ±
AL01
0.008 zZ 0.044 zZ 0.194 yZ 0.211 yZ 0.352 yZ 0.082 xZ
± ± ± ± ± ±
6.230 6.318 4.763 4.600 4.034 3.071
0.005 zZ 0.073 zY 0.348 yZ 0.372 yZ 0.523 yZ 0.103 xZ
HC060718-1 6.556 6.603 5.382 4.816 4.784 3.558
± ± ± ± ± ±
0.002 zZ 0.055 zZ 0.207 yZ 0.188 xZ 0.158 xZ 0.361 wZ
RESULTS AND DISCUSSION The behavior of A. hydrophila under conditions of starvation at different temperatures was determined over a 4-week period. Table 1 summarizes cell survival at 4◦ C. Initial cells counts (approximately 106 CFU/mL) were statistically identical in all strains. The first significant reduction was observed at day 7 in all strains. Cell numbers remained constant for 2 weeks in strains ML09-119 and AL01 but dropped significantly by day 28. Significant reductions in cell counts in strain HC060718-1 were observed on days 7, 14, and 28. By the end of the experiment (day 28), all strains had experienced a reduction of three orders of magnitude in culturability. Our results are in agreement with those of Mary et al. (2002), in which a sharp decrease in cell culturability of A. hydrophila type strain ATCC 7966 was observed within the first few days of incubation at 4◦ C. According to the Mary et al. (2002) study, A. hydrophila entered the VBNC state at low temperatures because cells appeared to remain viable (as per the LIVE/DEAD BacLight Bacterial Viability kit), but they could no longer be cultured on media. However, raising the temperature to 25◦ C did not reverse the VBNC stage, bringing into question the true nature of those VBNC forms. Whether A. hydrophila can survive as VBNC cells at low temperatures remains uncertain. Cell viability at 15◦ C slightly varied between strains throughout the 4-week study (Table 2). Overall, cells maintained viability over time, and no significant decrease in cell counts was TABLE 2. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in sterile distilled water at 15◦ C. See Table 1 for additional information.
Day 0 1 7 14 21 28
ML09-119 6.602 6.777 6.685 6.476 6.492 6.160
± ± ± ± ± ±
0.007 zZ 0.034 zY 0.103 zZ 0.114 zZ 0.077 zZ 0.213 yZ
AL01 6.230 6.598 6.734 6.144 6.479 6.408
± ± ± ± ± ±
0.003 xw,Z 0.006 zy,X 0.091 zZ 0.056 wY 0.189 zyx,Z 0.210 yxw,Z
HC060718-1 6.556 6.899 6.733 6.469 6.129 6.416
± ± ± ± ± ±
0.009 yx,Z 0.006 zZ 0.097 zy,Z 0.130 xZ 0.156 wZ 0.082 xZ
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TABLE 3. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in ultrapure water at 25◦ C. See Table 1 for more information.
Day
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0 1 7 14 21 28
ML09-119 7.146 6.500 6.219 5.476 4.964 5.030
± ± ± ± ± ±
0.004 zZ 0.209 yZ 0.194 yZ 0.044 xY 0.236 wZ 0.333 xw,Z
AL01 6.845 6.682 6.260 5.925 5.373 5.211
± ± ± ± ± ±
0.007 zZ 0.102 zZ 0.170 zy,Z 0.134 yZ 0.378 xZ 0.376 xZ
HC060718-1 6.663 6.627 6.287 6.114 5.514 5.076
± ± ± ± ± ±
0.003 zZ 0.111 zZ 0.178 zy,Z 0.163 zy,Z 0.487 yx,Z 0.655 xZ
observed during the experiment. Conversely, at 25◦ C there was a stronger effect on cell survival, with cell counts decreasing over time to approximately 105 CFU/mL (Table 3). The initial and final counts did not differ between strains, but ML09-119 presented fewer culturable cells than the other two strains at day 14. Previous studies compared the survival of A. hydrophila at 10◦ C and 20◦ C (Croci et al. 2001) and room temperature (Kersters et al. 1996), with similar results. Lower temperatures (10–15◦ C) favored the survival of A. hydrophila cells regardless of the bacterial strain or water chemistry used. By contrast, higher temperatures (≥20◦ C) reduced culturability over time. At 35◦ C, all strains showed a significant decrease in cell numbers at day 7, after which they remained constant for the duration of the study (Table 4). To the best of our knowledge, these are the first data on A. hydrophila survival at 35◦ C. Although 35◦ C might seem excessively high for natural aquatic environments, catfish ponds typically reach (and could exceed) this temperature during the summer months. The morphology of starved cells at day 21 was compared with that of fresh (24-h) cultures. As can be seen in Figure 1, the morphology of A. hydrophila cells dramatically changed during starvation, with cells becoming shorter. Cell length decreased significantly, from 2.0 ± 0.2 µm in fresh cultures to 1.0 ± 0.2 µm in starved cells. The observed changes were similar in all three strains examined. “Rounding up” is a common phenomenon in cells subjected to starvation and has previously been described in several gram-negative species (Wai et al. 1999). Starved cells were covered by a “veil of secreted slime” that has also been observed in bacterial cells TABLE 4. Total number per milliliter (mean ± SE) of colony-forming units of three strains of Aeromonas hydrophila obtained when cells were maintained in ultrapure water at 35◦ C. See Table 1 for additional information.
Day 0 1 7 14 21 28
ML09-119 7.146 6.264 4.457 4.839 4.659 5.075
± ± ± ± ± ±
0.006 zZ 0.093 yZ 0.523 xZ 0.329 xZ 0.475 xZ 0.112 xZ
AL01 6.845 6.169 4.243 4.629 4.019 5.108
± ± ± ± ± ±
0.009 zZ 0.148 zZ 0.656 yZ 0.456 yZ 0.541 yZ 0.247 yZ
HC060718-1 6.662 5.759 4.248 4.631 3.892 4.793
± ± ± ± ± ±
0.005 zZ 0.432 zy,Z 0.652 yx,Z 0.416 yx,Z 0.398 xZ 0.122 yx,Z
FIGURE 1. Morphological changes to Aeromonas hydrophila cells during starvation in sterilized distilled water as determined by scanning electronic microscopy. Panel (A) shows 24-h-old cells immediately prior to inoculation into microcosms, panel (B) cells maintained under starvation for 28 d at 25◦ C. Bars = 1 µm.
maintained under low-nutrient conditions (Arias et al. 2012). Such a drastic change in their ultrastructure is likely to affect the attachment and colonization capabilities of this fish pathogen, but further studies on its infectivity and pathogenicity are needed to test this hypothesis. In summary, our data show that all three strains of A. hydrophila behaved very similarly under starvation and that all can survive for extended periods at moderate and warm temperatures. Based on our study and others (Croci et al. 2001), the threshold between survival and loss of culturability occurs between 4◦ C and 10◦ C; above 15◦ C, cells lose some viability over time but can survive in water without nutrients for several weeks. Our results support the notion that water can act as a reservoir and serve as a dispersal mechanism for this pathogen. Future studies should assess the virulence of starved A. hydrophila cells to better define the life cycle of this species in the aquatic environment. ACKNOWLEDGMENTS We thank Michael Miller (Advanced Microscopy and Imaging Laboratory, Auburn University) for his help with scanning microscopy. This research was funded by the USDA– ARS/Auburn University Specific Cooperative Agreement Prevention of Diseases of Farmed Raised Fish and USDA–ARS CRIS project 6420-32000-022-00D. Xiaojun Zhang is the recipient of a fellowship from the Jiangsu Overseas Research and Training Program for University Prominent Young and MiddleAged Teachers and Presidents. Wenlong Cai thanks Shanghai Ocean University for partially supporting his research fellowship. Zhen Tao is the recipient of a graduate research fellowship funded by the Chinese Scholarship Council and Qingdao Ocean University. REFERENCES Arias, C. R., S. LaFrentz, W. Cai, and O. Olivares-Fuster. 2012. Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes. BMC Microbiology [online serial] 12:266.
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