FEMS Microbiology Ecology 44 (2003) 329^334
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Bacteria associated with sardine (Sardina pilchardus) eggs in a natural environment (R|¤a de Vigo, Galicia, northwestern Spain) Beatriz M|¤guez, Mar|¤a Pilar Combarro
Area Microbiolog|¤a, Departamento de Biolog|¤a Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain Received 30 April 2002 ; received in revised form 23 December 2002 ; accepted 12 February 2003 First published online 14 March 2003
Abstract The present study was undertaken to describe the epiflora of the eggs of an important fishing species collected in a coastal zone. Microflora associated with sardine (Sardina pilchardus) eggs collected in the R|¤a de Vigo was examined from January to June 2000. The count was carried out in three different ways: a total direct count by epifluorescence, a heterotrophic bacteria count on marine agar (MA) and a total vibrio count on thiosulfate citrate bile sucrose (TCBS). It was observed that the counts of total bacteria by epifluorescence were always higher by 2^3 logarithms than the bacterial counts on MA, and by 3^4 logarithms than the count of vibrios on TCBS. In both cases the differences were statistically significant. Throughout the sampling period only a slight variation was observed in the counts undertaken, and in the measured physicochemical parameters. For the qualitative study, 250 strains isolated from MA and 81 strains recovered on TCBS were identified. Members of the genera Vibrio, Pseudoalteromonas, Pseudomonas and Moraxella were found to dominate on the culturable adherent microflora of sardine eggs and Aeromonas, Tenacibaculum (Flexibacter), Flavobacterium and Cytophaga spp. were present in minor amounts. Vibrio anguillarum and Vibrio fischeri, pathogens of fish larvae, as well as Tenacibaculum ovolyticum, a pathogen of fish eggs, were detected. 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bacterial count; Marine bacterium; Vibrio spp. ; Sardine egg; Estuarine environment
1. Introduction The sardine (Sardina pilchardus) is one of the most important ¢sh species around the Spanish coast, both economically and in terms of ¢shing stocks. In recent years, an increase in the mortality rate of this species has been observed, especially a higher mortality rate in the early stages of life, principally at the egg stage [1]. In the natural environment, the quality of eggs can be a¡ected by biotic factors such as egg and larval abundance and abiotic factors such as temperature and salinity [2]. In marine environments, plankton, particulate organic matter, and solid surfaces can be heavily colonized by bacteria [3]. The surfaces of marine ¢sh eggs form an ex-
* Corresponding author. Tel. : +34 (986) 812634/812399; Fax : +34 (986) 812556. E-mail address :
[email protected] (M.P. Combarro).
cellent substrate for colonization by bacteria [4]. On these surfaces, bacterial composition and load appear to re£ect the £ora of surrounding water, although the speci¢c adhesion to the egg surface by bacterial groups may also play an important role in the development of the egg epi£ora [5]. Taking into account that the existence of opportunistic pathogens associated with the surface of ¢sh eggs can a¡ect their mortality rate and their later development [4^9], the principal studies have focused upon rearing systems, where a high mortality rate of eggs can cause major economic losses. However, so far, studies of bacteria associated with ¢sh eggs in natural environments have not been undertaken. Furthermore, there does not exist any reference to the micro£ora associated with sardine eggs, as the sardine is not a species that can be kept in rearing systems. The purpose of this study was to carry out the qualitative and quantitative analysis of bacteria associated with the surfaces of sardine eggs collected in R|¤a de Vigo, a natural ecosystem rich in biological diversity.
0168-6496 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-6496(03)00070-9
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2. Materials and methods 2.1. Sampling During the sampling period, from January to June 2000, samples of sardine (S. pilchardus) eggs were collected monthly. Samples were taken from the R|¤a de Vigo in Galicia, northwestern Spain. The eggs were obtained by trawling at a depth of 10 m in the central zone and the south of the estuary, using a bongo net with a mesh of 335 Wm. The samples were placed in jars containing sterile seawater before their immediate transfer to the laboratory. At no time did transfer exceed 4 h. Once in the laboratory, they were processed immediately. Salinity and temperature were determined in situ using a CTD model seabird 25. 2.2. Enumeration and isolation of bacteria The method of Hansen and Olafsen [4] was followed to isolate the bacteria from the eggs. This entailed three successive washes of the eggs in sterile seawater before being homogenized. To facilitate the removal of surface bacteria 10 Wg ml31 of Tween 80 (Panreac) was added. Three types of counts were completed using decimal dilutions of ¢ltered and sterile seawater obtained from the homogenate: total direct count (TDC), heterotrophic bacteria count (HBC) and total vibrio count (TVC). Direct counts of bacteria were determined by epi£uorescence microscopy with acridine orange (Sigma) following the procedures of Kepner and Pratt [10]. Bacteria were counted at 1250U magni¢cation with an Olympus BH2RFC £uorescence microscope using blue light excitation. An ocular 10U10 grid was utilized and a total of 60 ¢elds were counted. The results are expressed as cells ml31 . For HBC, a 0.1-ml aliquot of each dilution was plated in triplicate on marine agar (MA, Cultimed). For TVC, a 0.1-ml aliquot of each dilution was further added to make a ¢nal volume of 10 ml of sterile seawater and ¢ltered through 0.45-Wm-pore-size membrane ¢lters (Millipore). Subsequently, the ¢lters were cultured on thiosulfate citrate bile sucrose agar (TCBS, Cultimed). After 5 days incubation at 15‡C, the numbers of colonies on both media were counted and the average number of colony-forming units (CFU) per egg was calculated. Statistical analysis was performed using the program SPSS 10.0. A Pearson correlation analysis was carried
out between each of the three types of counts and the measured physicochemical parameters. The study of the seasonal evolution and the possible signi¢cant di¡erences between the counts were determined with the non-parametric Mann^Whitney test. 2.3. Isolate characterization Bacterial isolates were taken randomly from MA and TCBS plates from each sampling. The phenotypic identi¢cation of the 250 MA and the 81 TCBS isolates was determined. The isolates were characterized to genus level following the identi¢cation schemes of Bergey’s Manual of Determinative Bacteriology [51], Muroga et al. [11], and Hansen and Bech [12]. Routine identi¢cation included the following tests : colony pigmentation ; Gram reaction [13] ; catalase and oxidase [14] ; motility [15] ; fermentative or oxidative metabolism [15] ; H2 S production; Ryu’s stain for £agella [16] ; gliding motility [17]; growth on TCBS and in 0% NaCl; susceptibility to the vibriostatic agent O/129 (150 Wg) [15]; and Thornley’s decarboxylase for arginine [18]. Vibrio and related genera were identi¢ed following the program ‘Probabilistic Identi¢cation of Bacteria’ (Bryant, 1995) and using the tests of Alsina and Blanch [19,20] : Moeller’s decarboxylases for lysine and ornithine [21] ; Thornley’s dihydrolase for arginine; growth at 4, 30, 35 and 40‡C; growth in 6, 8 and 10% NaCl; nitrite reduction, Voges^Proskauer, methyl red and indole [22] ; production of L-galactosidase [14]; acid production from L-arabinose, arbutin, myo-inositol, D-mannitol, salicin, sorbitol and sucrose [23] ; utilization as sole carbon sources of L-arabinose, citrate, D-glucose, D-glucosamine, lactose, K-ketoglutarate and D-melibiose [24] ; hydrolysis of urea, gelatin and esculin [14]; and sensitivity to the vibriostatic agent O/129 (10 Wg). Alteromonas/Pseudoalteromonas species were identi¢ed with the tests reported by Sawabe et al. [25] and Venkateswaran and Dohomoto [26]: growth at 4, 35 and 40‡C ; nitrite reduction; hydrolysis of starch [14], chitin [27], agar, Tween 80 [28] and gelatin; utilization as sole carbon sources of acetate, citrate, D-fructose, D-galactose, D-glucosamine, D-glucose, D-mannose, lactose, D-malate, maltose, D-mannitol, melibiose, D-sorbitol, succinate, sucrose, trehalose and xylose [24]. Identi¢cation of Pseudomonas species was based on the tests of Palleroni [29], Verhille et al. [30] and Nishimori et al. [31]: £uorescent
Table 1 Physicochemical parameters measured at the sampling station and bacterial counts obtained from the eggs during the sampling period Sampling month
Temperaturea (‡C)
Salinitya (x)
TDC (cells/egg)
HBC (CFU/egg)
TVC (CFU/egg)
January February March April May June
12.7 12.9 13.5 14.2 15.3 14.6
35.3 35.4 35.7 34.6 33.8 33.9
1.54U105 1U105 6.64U104 8.91U104 1.57U105 5.32U104
307 189 69 80 240 121
40 28 5 10 9 5
a
Data supplied by Dr. Guisande.
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Fig. 1. Variation of the di¡erent bacterial counts obtained from the epi£ora of sardine eggs during the sampling period. 8, TDC; F, HBC; R, TVC.
pigments [32] ; growth at 4 and 40‡C ; hydrolysis of gelatin and Tween 80; utilization as sole carbon sources of L-arabinose, L-citrulline, myo-inositol, 2-ketogluconate, L-lysine, D-malate, D-mannose, D-mannitol, phenyl acetate, sucrose, m-tartrate, trehalose, L-tryptophan and xylose [24]. Tenacibaculum (Flexibacter) species were identi¢ed using the tests reported by Reichenbach [33], Hansen et al. [6], and Suzuki et al. [34] : degradation of agar, cellulose [14], chitin, DNA [28] and starch; production of L-galactosidase; nitrite reduction; and growth at 4‡C.
3. Results A total of six samples were examined from January to June 2000, the period of greatest abundance of sardine eggs. During this time, average temperature ranged from 12.7‡C in January to 15.3‡C in May, and salinity ranged from 33.8 to 35.7x (Table 1). The total bacterial count oscillated between 5.32U104 cells/egg in June and 1.57U105 cells/egg in May. The highest counts of heterotrophic bacteria and total vibrios were, in both cases, recorded in January, whilst the lowest counts were in March (Table 1). In all cases, the values obtained for total bacterial counts by epi£uorescence were 2^3 logarithms higher than the counts using MA, and 3^4 logarithms higher than those obtained by TCBS (Fig. 1). In both cases the di¡erences were statistically signi¢cant (P 6 0.005). These counts remained relatively constant throughout the sampling period and there were no signi¢cant di¡erences between the counts observed in the winter and those recorded in the spring. The only correlation observed was that between the TDC and HBC on MA (0.037; P 6 0.05), and there was no correlation between the bacterial counts and measured physicochemical parameters. Members of the genera Vibrio, Alteromonas/Pseudoalteromonas, Pseudomonas and Moraxella, which constituted 75.2% of total MA isolates (Table 2), dominated the culturable adherent micro£ora of sardine eggs. These genera showed a uniform distribution throughout the sampling
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period, although Vibrio displayed a higher percentage of isolates in April, with this number diminishing in May and June. Flavobacterium, Tenacibaculum (Flexibacter), Aeromonas and Cytophaga-like bacteria were detected as minor components of the epi£ora. Of these genera, only Flavobacterium revealed a uniform distribution during the sampling period, whereas the presence of Tenacibaculum (Flexibacter) was not detected in April and neither was Aeromonas in January and June, nor Cytophaga-like bacteria in February and April. The species belonging to Vibrio and related genera were identi¢ed using the program ‘Probabilistic Identi¢cation of Bacteria’ with a probability higher than 0.9. V. splendidus I accounted for 30% of total MA isolates within this group of bacteria, whilst V. £uvialis (10%), V. aestuarianus (10%), V. ¢scheri (8.6%) and V. anguillarum (8.6%) were isolated at lower levels. V. mediterranei, V. pelagius and V. nereis were detected in minor amounts. All isolates from TCBS plates corresponded to members of the Vibrionaceae family. A very similar incidence of Vibrio species was obtained on TCBS as on MA plates. With reference to seasonal distribution, a greater abundance of species was observed during spring and V. mediterranei was only detected during this season, whilst V. aestuarianus and V. ¢scheri were then detected at their highest percentage. By contrast, the dominance of V. splendidus I was restricted to samples taken in winter (Fig. 2). The species included in the group Alteromonas/Pseudoalteromonas were Ps. elyakovii (24.4%), Ps. piscicida (15.5%), A. macleodii (8.8%) and Ps. haloplanktis (6.7%). Of the isolates included initially in the genus Pseudomonas, 21.9% were identi¢ed as P. putida, and 9.8% as P. chloroTable 2 Generic and species distribution of bacteria obtained from the egg samples on MA plates Genera (250 strains)
Species a
Vibrio
70 (28.1)
Alteromonas/ Pseudoalteromonas
45 (18)
Pseudomonas
41 (16.4)
Moraxella Flexibacter
31 (12.4) 17 (6.8)
Flavobacterium Aeromonas Cytophaga
16 (6.4) 15 (6) 15 (6)
a
V. splendidus V. £uvialis V. aestuarianus V. ¢scheri V. anguillarum V. mediterranei V. pelagius I V. nereis Ps. elyakovii
21(30) 7(10) 7(10) 6(8.6) 6(8.6) 4(5.7) 3(4.3) 2(2.9) 11(24.4)
Ps. piscicida A. macleodii Ps. haloplanktis P. putida P. chlororaphis
7(15.5) 4(8.8) 3(6.7) 9(21.9) 4(9.8)
F. maritimus F. ovolyticus
7(41.2) 4(23.6)
Number of isolates (relative frequencies).
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Fig. 2. Distribution of Vibrio spp. obtained from the egg samples on MA plates during the sampling period.
raphis. Two species of the genus Tenacibaculum (Flexibacter) were identi¢ed: T. maritimum (41.2%) and T. ovolyticum (23.6%) (Table 2).
4. Discussion Hitherto, no study has been undertaken about the micro£ora associated with ¢sh eggs in natural environments. In this work, focused on sardine eggs and carried out in a natural environment, the results of previous studies of micro£ora associated with eggs in rearing systems have been taken as a reference point. Viable counts of the epi£ora of sardine eggs oscillated between 6.9 CFU/egg and 3.07U102 CFU/egg. These counts are noticeably lower than those obtained in rearing systems, where viable counts vary between 2U102 and 8U104 CFU/egg in halibut (Hippoglossus hippoglossus) eggs [5] and 3.7U106 CFU/egg in turbot (Scophthalmus maximus) eggs [7]. These lower counts in a natural environment can be explained by the special conditions that exist in intensive rearing systems, which facilitate higher levels of bacterial colonization. In this context, Hameed [35] observed a positive correlation between the bacterial counts and the incubation time of the eggs of the white shrimp (Penaeus indicus). Furthermore, in a review article of 1999, Hansen and Olafsen [5] pointed out that in previous studies (Hansen and Olafsen, unpublished data; [7]) about bacterial quanti¢cation on ¢sh eggs in rearing systems, a higher bacterial colonization had been observed after the incubation of the eggs. From this, they deduced that this colonization was possibly greater in rearing systems than in natural ecosystems. The TDCs by epi£uorescence have always been signi¢cantly higher than those on plates. In this context, Colwell et al. [36] found that less than 1% of the total number of
bacteria present in marine ecosystems are capable of growing in arti¢cial media. On the other hand, the positive correlation found between TDC by epi£uorescence and HBC on MA demonstrates that the percentage of viable and culturable bacteria has remained relatively constant during sampling. Neither marked £uctuations in the bacterial counts nor the existence of any correlation between the level of these counts and the measured physicochemical parameters were detected. This could be explained by the fact that, during the sampling period, the variation of these parameters was only marginal. Our results do not contradict previous studies that found a correlation between MA and TCBS counts and temperature [37], or between some Vibrio species and temperature [38^43] and salinity [40,41]. This is because in all these studies the variations in temperature and salinity during the sampling period were always greater than 9‡C and 13x respectively, whereas in our case the variations in temperature and salinity were only 2.6‡C and 1.9x respectively, which should not in£uence the bacterial count. Although a seasonal variation was not detected in Vibrio spp., a larger number of species was observed in spring. This increase could be related to the slight rise in temperature during that period, which could stimulate the development of a greater number of mesophilic species such as V. mediterranei, only detected in spring. This may also explain why a psychrotrophic species, such as V. splendidus I, was mainly detected in winter. Thus, our results agree with those of Pujalte et al. [37], who found a clear predominance of V. splendidus in colder months and a greater variety of Vibrio species associated with Mediterranean oysters during the warmer periods of the year. Major bacterial genera present in the epi£ora of sardine eggs were Vibrio, Pseudoalteromonas, Pseudomonas and Moraxella. Some of these genera have also been found
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as dominant members of the micro£ora adhered to ¢sh eggs in rearing systems. For instance, Hansen and Olafsen [4] and Morrison and MacDonald [44] reported that Pseudomonas, Alteromonas, Aeromonas, and FlavobacteriumCytophaga spp. are dominant on cod (Gadus morhua) and halibut (H. hippoglossus) eggs, whilst Keskin et al. [7] found that Aeromonas, Moraxella and Pseudomonas spp. were predominant on turbot (S. maximus) eggs. The genus Vibrio was present only in minor amounts on cod eggs [4], yet Vibrio is one the most abundant genera on sardine eggs. In our view, the proliferation of this bacterium in association with ¢sh eggs in a natural ecosystem is logical, considering that the vibrios are natural inhabitants of the aquatic environment, with mainly marine and estuarine habitats [45^47]. Furthermore, the members of the Vibrio genera are excellent colonizers of mucous surfaces [48] and can easily colonize the surface of the eggs [49]. The detection of Cytophaga-like bacteria, Flavobacterium and Aeromonas present, as minor genera, on sardine eggs is another important di¡erence between this study and those previously undertaken in rearing systems. It is possible that the di¡erent conditions in rearing systems favor the predominance of certain groups of bacteria. However, the genus Tenacibaculum (Flexibacter) has been detected in a low frequency in this study, as it was previously in rearing systems [4]. Among the species of the genus Vibrio, two stand out as being most relevant. These are V. anguillarum, an important ¢sh pathogen [50], and V. ¢scheri, previously detected on cod and halibut eggs in rearing systems [4]. Both species have been used in experimental infections during the incubation of halibut eggs [49] without showing an important increase in the mortality rate of eggs, but certainly with an increase amongst the larvae that are infected posthatch. In this context, Bergh et al. [49] reported that Vibrio spp. do not possess the enzymatic capability needed to penetrate the eggshell. In the present study, some important ¢sh pathogens, such as Ps. piscicida, P. chlororaphis, P. putida and T. maritimum (F. maritimus), have been identi¢ed, but, to date, none of these bacteria have been directly related with mortality in marine ¢sh eggs. Of greater interest is the detection of T. ovolyticum (F. ovolyticus) on sardine eggs, since the correlation between this species and the mortality rate of reared halibut eggs [6,49] and larvae [6] has already been shown. It has been demonstrated that the exoproteolytic activity of this species causes ulceration at the egg surface [49]. However, in this study it is possible that T. ovolyticum (F. ovolyticus) has not had a decisive in£uence upon the mortality rate of the sardine eggs, as the percentage in the associated micro£ora is very low and, so far, it has only been demonstrated as pathogenic when it constitutes the major part of bacterial epi£ora [6]. Nevertheless, its detection should not be underestimated, bearing in mind that oceanographic conditions can cause a high stability in the area, which could
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help this species to proliferate and then to a¡ect the survival of the eggs. It is di⁄cult to evaluate the quality of sardine eggs or the possible e¡ect of opportunistic pathogens, when the sardine is a species that is not kept in rearing systems. However, considering the slight microbiota isolated in this study, in comparison to that obtained for other species in rearing systems and the di¡erent factors to which these eggs can be subjected in a natural environment, it is appropriate to surmise that, in this case, the associated microbiota may not be a predominant factor in the mortality rate of sardine eggs.
Acknowledgements We thank Dr. C. Guisande (University of Vigo) for providing the samples and the oceanographic data. This study was supported by the project Mar99-0328-C03-01 (CICYT).
References [1] Southward, A.J. and Demir, N. (1973) Seasonal changes in dimensions and viability developing eggs of the Cornish Pilchard (Sardina pilchardus Walb) o¡ Plymouth. Proceedings of a Symposium on the Early Life History of Fish, Oban, May. [2] Guisande, C., Riveiro, I., Sola¤, A. and Valde¤s, L. (1998) E¡ect of biotic and abiotic factors on the biochemical composition of wild eggs and larvae of several ¢sh species. Mar. Ecol. Prog. Ser. 163, 53^61. [3] Sieburth, J.M. (1979) Sea Microbes. Oxford University Press, New York. [4] Hansen, G.H. and Olafsen, J.A. (1989) Bacterial colonization of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus) eggs in marine aquaculture. Appl. Environ. Microbiol. 55, 1435^1446. [5] Hansen, G.H. and Olafsen, J.A. (1999) Bacterial interactions in early life stages of marine cold water ¢sh. Microb. Ecol. 38, 1^26. [6] Hansen, G.H., Bergh, O., Michaelsen, J. and Knappskog, D. (1992) Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of atlantic halibut, Hippoglossus hippoglossus L. Int. J. Syst. Bacteriol. 42, 451^458. [7] Keskin, M., Keskin, M. and Rosenthal, H. (1994) Pathways of bacterial contamination during egg incubation and larval rearing of turbot, Scophthalmus maximus. J. Appl. Ichthyol. 10, 1^9. [8] Oppenheimer, C.H. (1955) The e¡ect of marine bacteria on the development and hatching of pelagic ¢sh eggs and the control of such bacteria by antibiotics. Copeia 1, 43^49. [9] Shelbourne, J.E. (1963) A marine ¢sh rearing experiment using antibiotics. Nature 198, 74^75. [10] Kepner, J.R. and Pratt, J.R. (1994) Use of £uorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol. Rev. 58, 603^615. [11] Muroga, K., Higashi, M. and Keitoku, H. (1987) The isolation of intestinal micro£ora of farmed red seabream (Pagrus major) and black seabream (Acanthopagrus schlegeli) at larval and juvenile stages. Aquaculture 65, 79^88. [12] Hansen, B. and Bech, G. (1996) Bacteria associated with a marine planktonic copepod in culture. Bacterial genera in seawater, body surfaces, intestines and fecal pellets and succession during fecal pellet degradation. J. Planktonic Res. 18, 257^273.
FEMSEC 1508 29-4-03
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[13] Buck, J.D. (1982) Nonstaining (KOH) method for determination of Gram reactions of marine bacteria. Appl. Environ. Microbiol. 44, 992^993. [14] Smibert, R.M. and Krieg, N.R. (1981) General characterization. In: Manual of Methods for General Bacteriology (Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R. and Phillips, G.B., Eds.), pp. 409^443. American Society for Microbiology, Washington, DC. [15] Baumman, P., Baumman, L. and Mandel, M. (1971) Taxonomy of marine bacteria: the genus Beneckea. J. Bacteriol. 107, 268^294. [16] Kodaka, H., Arm¢eld, A.Y., Lombard, G. and Dowell, V.R. (1982) Practical procedure for demonstrating bacterial £agella. J. Clin. Microbiol. 16, 948. [17] Allen, D.A., Austin, B. and Colwell, R.R. (1983) Aeromonas media, a new species isolated from river water. Int. J. Syst. Bacteriol. 33, 599^ 604. [18] Thornley, M.J. (1960) The di¡erenciation of Pseudomonas from other gram negative bacteria on the basis of arginine metabolism. J. Appl. Bacteriol. 23, 37^52. [19] Alsina, M. and Blanch, A.R. (1994) A set of keys for biochemical identi¢cation of environmental Vibrio species. J. Appl. Bacteriol. 76, 79^85. [20] Alsina, M. and Blanch, A.R. (1994) Improvement and update of a set of keys for biochemical identi¢cation of Vibrio species. J. Appl. Bacteriol. 77, 719^721. [21] Lee, J.V. and Donovan, T.J. (1985) Vibrio, Aeromonas and Plesiomonas. In: Isolation and Identi¢cation of Microorganisms of Medical and Veterinary Importance (Collins, C.H. and Grange, J.M., Eds.), pp. 13^33. Society of Applied Bacteriology, Academic Press, London. [22] Hansen, G.H. and So«rheim, R. (1991) Improved method for phenotypical characterization of marine bacteria. J. Microbiol. Methods 13, 231^241. [23] Esteve, C. (1995) Numerical taxonomy of Aeromonadaceae and Vibrionaceae associated with reared ¢sh and surrounding fresh and brackish water. Syst. Appl. Microbiol. 18, 391^402. [24] Baumman, P. and Baumman, L. (1981) The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In: The Prokaryotes. A Handbook on Habitats, Isolation, and Identi¢cation of Bacteria (Starr, M.P., Stolp, H., Tru«per, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 1302^1331. Springer-Verlag, New York. [25] Sawabe, T., Tanaka, R., Iqbal, M.M., Tajima, K., Ezura, Y., Ivanova, E.P. and Christen, R. (2000) Assignment of Alteromonas elyakovii KMM 162T and ¢ve strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species. Int. J. Syst. Evol. Microbiol. 50, 265^271. [26] Venkateswaran, K. and Dohmoto, N. (2000) Pseudoalteromonas peptidolytica sp. nov., a novel marine mussel-thread-degrading bacterium isolated from the Sea of Japan. Int. J. Syst. Evol. Microbiol. 50, 565^ 574. [27] Gauthier, M.J., Aubert, J. and Aubert, M. (1982) Numerical analysis of the heterotrophic bacterial £ora from necroses of marine ¢shes. Rev. Int. Oceanogr. Me¤d. LXVI^LXVII. [28] Sierra, G. (1957) A simple method for the detection of lipolytic activity of microorganisms and some observations on the in£uence of the contact between cells fatty substrates. Antonie van Leewenhoek J. Microbiol. Serol. 23, 15^22. [29] Palleroni, N.J. (1981) Introduction to the family Pseudomonadaceae. In: The Prokaryotes. A Handbook on Habitats, Isolation, and Identi¢cation of Bacteria (Starr, M.P., Stolp, H., Tru«per, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 3072^3085. Springer-Verlag, New York. [30] Verhille, S., Ba|«da, N., Dabboussi, F., Hamze, M., Izard, D. and Leclerc, H. (1999) Pseudomonas gessardii sp. nov. and Pseudomonas migulae sp. nov., two new species isolated from natural mineral waters. Int. J. Syst. Evol. Microbiol. 49, 1559^1572.
[31] Nishimori, E., Kita-Tsukamoto, K. and Wakabayashi, H. (2000) Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis. Int. J. Syst. Evol. Microbiol. 50, 83^89. [32] Palleroni, N.J. (1981) Human an animal pathogenic pseudomonads. In: The Prokaryotes. A Handbook on Habitats, Isolation, and Identi¢cation of Bacteria (Starr, M.P., Stolp, H., Tru«per, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 3086^3103. Springer-Verlag, New York. [33] Reichenbach, H. (1984) Genus Flexibacter. In: Bergey’s Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., Eds.), pp. 2061^2071. Williams and Wilkins, Baltimore, MD. [34] Suzuki, M., Nakagawa, Y., Harayama, S. and Yamamoto, S. (2001) Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria : proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimun comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov. Int. J. Syst. Evol. Microbiol. 51, 1639^1652. [35] Hameed, A.S. (1997) Quality of eggs produced from wild and captive spawners of Penaus indicus H. Milne Edwards and their bacterial load. Aquat. Res. 28, 301^303. [36] Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganims. Biotechnology 3, 817^820. [37] Pujalte, M.J., Ortigosa, M., Macia¤n, M.C. and Garay, E. (1999) Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. Int. Microbiol. 2, 259^266. [38] Colwell, R.R., Kaper, J.B. and Joseph, S.W. (1977) V. cholerae and V. parahaemolyticus and other vibrios. Occurrence and distribution in Chesapeake Bay. Science 198, 394^396. [39] Kaneko, T. and Colwell, R.R. (1973) Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J. Bacteriol. 113, 24^31. [40] Kaneko, T. and Colwell, R.R. (1978) The annual cycle of Vibrio parahaemolyticus in Chesapeake Bay. Microb. Ecol. 4, 135^155. [41] Kelly, M.T. (1982) E¡ect of temperature and salinity on Vibrio (Benekea) vulni¢cus occurrence in a Gulf Coast environment. Appl. Environ. Microbiol. 44, 820^824. [42] Montanari, M.P., Pruzzo, C., Pane, L. and Colwell, R.R. (1999) Vibrios associated with plankton in coastal zone of the Adriatic Sea (Italy). FEMS Microbiol. Ecol. 29, 241^247. [43] Oliver, J.D., Warner, R.A. and Cleland, D.R. (1983) Distribution of Vibrio vulni¢cus and other lactose-fermenting vibrios in marine environment. Appl. Environ. Microbiol. 45, 985^988. [44] Morrison, C.M. and MacDonald, C.A. (1995) Normal and abnormal jaw development of the yolk sac larvae of Atlantic halibut Hippoglossos hippoglossus. Dis. Aquat. Org. 22, 173^184. [45] Caruso, G., Zaccone, R. and Crisa¢, E. (1996) Distribution and numerical taxonomy of Vibrionaceae in waters of the straits of Messina. Microbiologica 19, 155^166. [46] Huq, A. and Colwell, R.R. (1995) Vibrios in the marine and estuarine environments. J. Mar. Biotechnol. 3, 60^63. [47] Matte¤, G.R., Matte¤, M.H., Rivera, I.G. and Martins, M.T. (1994) Distribution of potentially pathogenic vibrios in oysters from tropical region. J. Food Protect. 10, 870^873. [48] Strom, E. and Olafsen, J.A. (1990) The indigenous micro£ora of wild-captured juvenile cod in net-pen rearing. In: Microbiology in Poikilotherms (Le¤sel, R., Ed.), pp. 181^185. Elsevier Science, Amsterdam. [49] Bergh, O., Hansen, G.H. and Taxt, R.E. (1992) Experimental infection of eggs and yolk sac larvae of halibut (Hippoglossus hippoglossus). J. Fish Dis. 15, 379^391. [50] Colwell, R.R. (1984) Biotechnology in the marine sciences. In: Biotechnology in the Marine Sciences (Colwell, R.R., Praiser, E.R. and Sinskeym, A.J., Eds.), pp. 1^27. John Wiley and Sons, New York. [51] Krieg, N.R. and Holt, J.G. (Eds.) (1984) Bergey’s Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore, MD.
FEMSEC 1508 29-4-03
