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Detection of Vibrionaceae in mussels and in their seawatergrowing area
L. Croci, P. Serratore1, L. Cozzi, A.1 Stacchini2, S. Milandri1, E. Suffrediniand L. TotiIstituto Superiore di SanitaÁ, Laboratorio Alimenti, 1Centro Ricerche Interdipartimentale delle Scienze del Mare
and 2UniversitaÁ degli Studi di Roma `la Sapienza', FacoltaÁ di Economia e Commercio, Dipartimento di Scienze
Merceologiche, Rome, Italy
251/00: received 11 July 2000, revised 18 October 2000 and accepted 20 October 2000
L . CROCI , P . SERRATORE, L . COZZI , A . STACCHIN I , S . MILANDRI , E . SUFFREDINI
AND L. TOTI . 2001.
Aims: The seasonal trend and frequency of detection of Vibrionaceae in seawater samples and
in molluscs collected in the Adriatic Sea was measured.
Methods and Results: Over a 2-year period, 726 bacterial strains were isolated, of which
46á9% belonged to the Vibrio genus, 29á8% to the Aeromonas genus and the remaining 23á3%
was made up of the Pseudomonas, Flavobacterium, Pasteurella, Agrobacterium and Ochrobacterium
genera. Many of the isolated strains were shown to produce toxins.
Conclusions: The Vibrio genus, which was isolated more often than the other genera, was
particularly prevalent in summer (54á4% of the total number of bacteria isolated during this
season), while it was scarce in the winter months.
INTRODUCTION
Among the autochthonous micro¯ora of the marine envi-
ronment are various members of the Vibrionaceae family,
which includes species pathogenic, or potentially patho-
genic, to man and constituting a threat to health via raw
shell®sh. Of those species pathogenic to man, Vibriocholerae, V. parahaemolyticus and V. vulni®cus are respon-
sible for most food-borne infections.
Vibrio cholerae 01, the aetiological agent of cholera, still
causes this disease in certain developing nations (WHO 1999)
and, even in Italy, food-borne infection attributable to seafood
consumption has been reported (Maggi et al. 1997). Vibrioparahaemolyticus is of particular signi®cance in Japan, where it
accounts for 40±70% of cases of food poisoning (Hackney and
Dicharry 1988; Lipp and Rose 1997), but it has also been
recognized as the cause of gastroenteritis in other parts of the
world (Twedt 1989; CDC 1999). In Italy V. parahaemolyticushas been found in edible molluscs particularly in the summer
months (Boccia et al. 1978; Maugeri et al. 1987).
Vibrio vulni®cus, the presence of which has been well
documented in water and molluscs (O'Neill et al. 1992) can
cause fulminant septicaemia in susceptible hosts (Morris and
Black 1985; Jackson et al. 1997; Robinson 2000). Infection
has been associated with consumption of raw oysters and
contact with shell®sh (e.g. via a wound). Furthermore, other
vibrios, such as V. cholerae non-O1, have at times been
associated with human gastroenteritis and are considered
occasional pathogens (Morris et al. 1990; Klontz 1990).
Recently, a highly epidemic form of cholera-like disease has
been strongly associated with a strain of V. cholerae non-O1,
designated V. cholerae O139 Bengal (Albert et al. 1993).
A case of infection by V. cholerae O158 has been described in
Italy (Cassone, personal communication). Other vibrios
responsible for severe infections, such as Photobacteriumdamsela (formerly Listonella or Vibrio damsela) (Fraser et al.1997) are transmitted through wounds. The pathogenicity of
these bacteria is often due to their ability to secrete
extracellular products such as proteases, haemagglutinin
(Honda et al. 1987), enterotoxin (Yamamoto et al. 1982;
Spira and Fedorka-Cray 1983), haemolysin (Yamamoto et al.1984; Honda et al. 1985), cytotoxin (MacCardell et al. 19852 )
and other uncharacterized virulence factors (Nishibuchi
et al. 1983). However, in recent years other micro-organisms,
now classi®ed as Aeromonas and Plesiomonas groups (http://
www.ncbi.nlm.nih.gov/Taxonomy/tax.html), have at times
been held responsible for enterotoxic episodes (Rutala et al.1982; Merino et al. 1995). The present study, of 2 years
duration, monitored seasonal trends of the VibrionaceaeCorrespondence to: Dott.ssa L. Croci, Laboratorio Alimenti, Istituto Superiore
di SanitaÁ, Viale Regina Elena 299, 00161 Rome, Italy (e-mail: [email protected]).
ã 2001 The Society for Applied Microbiology
Letters in Applied Microbiology 2001, 32, 57±61
family in samples of seawater and molluscs collected in the
Adriatic Sea and the possible presence of species having
pathogenic characteristics.
MATERIALS AND METHODS
Samples
Samples of shell®sh (Mytilus galloprovincialis, Tapes decuss-atus and Crassostrea virginica) and seawater, collected from
two coastal areas (Cesenatico and Goro) of the Adriatic Sea,
were used. The shell®sh and seawater samples were
refrigerated at 4 °C until shipped to the laboratory, where
they were analysed within 4 h of collection.
Isolation of bacteria
Shell®sh. Shell®sh specimens were scrubbed and washed in
water containing 50 p.p.m. active Cl2 and rinsed with sterile
distilled water; bodies and intervalve water were aseptically
removed and collected into a sterile beaker with an equal
volume of sterile 3% saline. The samples were homogenized
with a Sterilmixer (PBI International, Milano, Italy3 ) at
11 000 rev min±1 for 20 s and diluted in the above diluent.
Dilutions of mussel extracts (0á1 ml), prepared as des-
cribed above, were streaked onto thiosulfate citrate bile salts
sucrose (TCBS)4,5 agar (Difco4,5 , Detroit, MH, USA) containing
3% NaCl.
Seawater. Volumes (100 and 10 ml) of the seawater samples
were ®ltered through 0á45-lm Millipore6 membranes (Vimo-
drone (MI) Italy) and a further sample was diluted in sterile
3% saline (1:10, 1:100 and 1:1000). The Millipore mem-
branes were placed on TCBS agar and 0á1 ml of water and of
the above dilutions were streaked onto TCBS agar. All
TCBS agar plates were incubated at 20 °C for 5 d.
Identi®cation of bacterial species
The green and yellow colonies isolated on TCBS agar were
transferred to fresh TCBS agar plates and Tryptone Soya
Agar (TSA; Oxoid, Hampshire, UK7 ) with 3% NaCl and
0á2% Yeast Extract (Oxoid) was added. After incubation at
20 °C for 5 d the isolates were subjected to the following
identi®cation tests: oxidative and fermentative glucose
utilization; oxidase; citrate utilization; indole production;
nitrate reduction and API 20 NE with added 3% NaCl
(Varnam and Evans 1991).
The Aeromonas strains were subjected to suicide tests to
distinguish Aeromonas hydrophila from Aerom. caviae(Varnam and Evans 1991).
Test on mouse
Isolates belonging to the genera Vibrio and Aeromonas were
subjected to a mouse test to reveal any production of toxic
substances.
Table 1 Bacteria isolated from mussels and seawater samples
Genus No. Frequency (%)* Species No. Frequency (%) Frequency (%)*
Vibrio 340 46á86 V. alginolyticus 231 67á95 31á82
Photobacterium damsela 37 10á88 5á10
V. parahaemolyticus 34 10 4á68
V. vulni®cus 34 10 4á68
V. cholerae non 01 4 1á17 0á55
Aeromonas 216 29á75 Aerom. salmonicida 125 57á87 17á22
Aerom. hydrophila/caviae 84 38á88 11á57
Aerom. sobriae 7 3á24 0á96
Pseudomonas 50 6á88 Ps. paucimobilis 25 50 3á44
Ps. putrefaciens 15 30 2á07
Ps. vesicularis 10 20 1á38
Flavobacterium 43 5á92 Fl. indologenes 19 44á18 2á62
Fl. multivorum 16 37á21 2á20
Fl. odoratum 8 18á61 1á10
Pasteurella 28 3á85 Past. spp. 23 2á14 3á17
Past. multocida 5 17á86 0á69
Ochrobacterium 28 3á85 O. anthropi 28 100 3á86
Agrobacterium 21 2á89 Ag. radiobacter 21 100 2á89
Total 726 100 726 100 100
*Percentage with respect to the total isolates (726).
Percentage with respect to the total isolates belonging to the same genus.
58 L. CROCI ET AL .
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 57±61
Bacterial strains, kept on TSA slants at room temperature,
were cultured in Tryptone Soya Broth with 3% NaCl at
37 °C for 24 h and subcultured in Bacto8 Peptone (Difco)
(pH 8á5) with 3% NaCl at 25 °C for 4 d.
Bacterial cells were extracted, after centrifugation at
3500 rev min±1 for 15 min, with 100 ml ethanol containing
1% acetic acid. After removal of ethanol by evaporation,
an aliquot of the extract was dissolved in 4 ml distilled
water.
A further sample of the extract was heated at 90 °C for
15 min, after dissolution in 4 ml distilled water.
Mouse injection. Each of the above extracts was injected
intraperitoneally (1 ml mouse±1) into three Swiss mice
(18±20 g). The mice were observed for 48 h and the
survival time rated.
RESULTS
Table 1 shows the numbers of various bacterial species
isolated and their frequency, expressed as the percentage of
total isolates and of bacteria belonging to the same genus.
The strains isolated from seawater and mussels were
principally of the Vibrio (46á9%) and Aeromonas (29á8%)
genera. In the Vibrio genus the most frequently isolated
species was V. alginolyticus (68á0%); pathogenic species such
as V. parahaemolyticus and V. vulni®cus were each found in
10% of cases and Ph. damsela in 10á9% of cases. In the
Aeromonas genus 57á9% were identi®ed as Aerom. salmon-icida and 38á9% as Aerom. hydrophila/caviae. Subsequently,
the suicide test showed the same numbers of Aerom.hydrophila and Aerom. caviae. The9 remaining 23á3%
belonged to the genera Pseudomonas (6á9%), Flavobacterium(5á9%), Pasteurella (3á8%), Ochrobactrum (3á8%) and
Agrobacterium (2á9%)10 .
Strains belonging to the Vibrio and Aeromonas genera were
mostly toxic to mice, except for the V. cholerae non-O1
strains which were consistently non-toxic. Toxic substances
produced by V. parahaemolyticus and Aerom. hydrophilastrains were thermostable (Table 2).
Regarding seasonal trends, Fig. 1 shows the number of
bacteria in the various genera found during different seasons
of the year, from which it appears that higher ®gures were
found in summer and autumn. Figure 2 reports the relative
percentages of isolates of the various genera with respect to
the total number of strains isolated per season; such
percentages remained almost constant during the various
seasons: 47á0 � 5á1% for Vibrio (only in spring did it reach
54á4%), 29á5 � 3á3% for Aeromonas, 7á4 � 2á3% for Pseudo-monas and 16á1 � 3á6% for other genera.
DISCUSSION
Amongst the autochthonous micro¯ora of the sea, bacteria
listed as potentially pathogenic are readily found. Such
Table 2 Percentage of bacteria producing thermostable and/or
thermolabile toxic substances
% of strains producing toxic substances
Species Thermostable Thermolabile
Photobacterium damsela 0 80
Vibrio alginolyticus 0 30
Vibrio parahaemolyticus 100 100
Aeromonas salmonicida 20 20
Aeromonas hydrophila 30 30
Vibrio cholerae non O1 0 0
Vibrio vulni®cus 0 100
Winter Spring Summer Autumn
140
120
100
80
60
40
20
0
No.
of i
sola
tes
Fig. 1 Number of isolates from molluscs
and seawater in various seasons.
, Pseudomonas; , vibrios; , Aeromonas;
u, others
VIBRIOS IN MUSSELS AND SEAWATER 59
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 57±61
micro-organisms, when present in molluscs, may be im-
portant causes of human disease, especially gastroenteritis of
unknown origin. However, it is commonly recognized that
there is no correlation between the presence of such bacteria
in the sea and indicators of faecal contamination (Hackney
et al. 1980), which are normally used to establish the
suitability for consumption of edible molluscs. Even during
depuration processes, to which molluscs are subjected before
sale, micro-organisms of the Vibrionaceae family show a
different behaviour to that of conventional bacterial indica-
tors of faecal contamination, as they may bind to mollusc
tissues more tenaciously than other bacteria making their
removal dif®cult (Lee and Yii 1996; Marino et al. 1999).
Furthermore, the seasonal trends (Fig. 1) showed that the
Vibrio genus was more prevalent in summer than autumn
and spring whilst it was scarce in winter. However, there
were no substantial variations in the percentage of isolates
compared with the total isolates from the same season
(Fig. 2). The limited recovery of Vibrio species in winter
may be due to their ability to remain quiescent, in a viable
but non-culturable state, whilst retaining their virulence
(Colwell and Hug 1994).
From the above it is clearly important to understand the
distribution of particular bacterial species in the marine
environment, especially opportunistic pathogens, so as to be
able to prevent bacterial gastroenteritis transmitted by
seafood, as well as developing methodologies for detecting
viable but non-culturable forms of these organisms.
ACKNOWLEDGEMENTS
This work, carried out under the National Programme
`Prisma 2, subproject no. 6, Human Health', was supported
by a grant from the Italian Ministry of University and
Scienti®c and Technological Research.
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VIBRIOS IN MUSSELS AND SEAWATER 61
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 57±61