FEMS Microbiology Ecology 44 (2003) 329^334
www.fems-microbiology.org
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
FEMSEC 1508 29-4-03
B. M|¤guez, M.P. Combarro / FEMS Microbiology Ecology 44 (2003) 329^334
330
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.
FEMSEC 1508 29-4-03
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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
331
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).
FEMSEC 1508 29-4-03
332
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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
FEMSEC 1508 29-4-03
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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
333
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).
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