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doi:10.1016/j.marpolbul.2005.02.044
Occurrence of potentially pathogenic vibrios in the
marine environment of the Straits of Messina (Italy)
C. Gugliandolo a, M. Carbone b, M.T. Fera b, G.P. Irrera a, T.L. Maugeri
a
Dipartimento di Biologia Animale ed Ecologia Marina, University of Messina, Salita Sperone 31, 98166 Messina, Italy
b
Dipartimento di Patologia e Microbiologia Sperimentale, University of Messina, 98125, Italy
Vibrios are ubiquitous in marine and estuarine environments and are commonly present in or on shellfish
and other seafood. They are present in the environment
as free-living and associated with different substrata
(Tamplin et al., 1990). Epibionts are able to survive in
the natural environment longer than free-living forms,
and by means of adhesive strategies, they can adapt to
adverse conditions, e.g. organic matter limitation (Roszak and Colwell, 1987; Carman and Dobbs, 1997). The
colonisation of planktonic copepod integument by Vibrio spp. is a well-described phenomenon especially in
what concerns the attachment to the copepods in faecal
polluted and non-polluted coastal zones (Kaneko and
Colwell, 1975; Venkateswaran et al., 1989; Tamplin
et al., 1990; Carli et al., 1993; Maugeri et al., 2004). Larger numbers of V. cholerae are associated with zooplankton than are found in the surrounding water
*
a,*
Corresponding author. Tel.: +39906765523; fax: +3990393409.
E-mail addresses: [email protected] (T.L. Maugeri).
column (Huq et al., 1983). The dispersion of eggs and
faecal pellets should contribute to bacterial diffusion in
the aquatic environment.
Interest in the occurrence of potentially pathogenic
vibrios is high from an epidemiological and ecological
point of view. Vibrios able to cause human disease include the cholera toxin-producing strains of V. cholerae,
that are responsible for epidemic/pandemic cholera,
thermostable direct hemolysin-producing strains of V.
parahaemolyticus, a leading cause of gastro-enteritis
and V. vulnificus, which can cause sepsis and serious
wound infections. The transmission of V. cholerae
strains from their environmental reservoir to humans
through water sources or seafood has been demonstrated. Other non-epidemic Vibrio species, including
V. parahaemolyticus and V. vulnificus, are usually associated with the consumption of raw or undercooked
shellfish and seafood or exposure of skin wounds to
seawater (Morris, 2003). V. alginolyticus, V. fluvialis,
V. furnissii, V. hollisae and V. metschnikovii are halophilic vibrios also involved in human diseases (Farmer
and Hickman-Brenner, 1992). These species are present
Baseline / Marine Pollution Bulletin 50 (2005) 682–697
in estuarine and marine environments along with other
pathogenic and non-pathogenic species.
In Europe, the occurrence of pathogenic vibrios in the
marine environment has been well documented by several
authors (Høi et al., 1998; Hervio-Heath et al., 2002; Jores
et al., 2003). In Italy, the occurrence of potentially pathogenic vibrios in aquatic environments such as rivers
(Caldini et al., 1997), brackish (Maugeri, 1994; Maugeri
et al., 2000), estuarine (Barbieri et al., 1999) and coastal
marine sites (Carli et al., 1993; Montanari et al., 1999;
Dumontet et al., 2000) has been reported. The association of Vibrio spp. with marine plankton has also been
demonstrated in the Mediterranean area for V. alginolyticus, V. cholerae non-O1,V. vulnificus, V. parahaemolyticus and V. fluvialis in different seasons and in different
temperature and salinity conditions (Carli et al., 1993;
Pruzzo et al., 1996; Montanari et al., 1999; Dumontet
et al., 2000; Maugeri et al., 2004).
The occurrence of potentially pathogenic vibrios in
the Ionian coast of the Straits of Messina (Italy) as free
living (>0.2–64 lm) and associated with small (>64–
200 lm) and large (> 200 lm) size classes of plankton
is here studied. In order to elucidate the role of copepods, which represent the main component of zooplankton, the presence of potentially pathogenic vibrios as
firmly bound to copepods is here investigated. Finally,
we wanted to determine if there was any correlation
between the occurrence of Vibrio spp. and that of the
indicator bacteria (E. coli and enterococci) generally
used in assessing water quality.
Monthly field sampling was carried out from March
to October 2003. Seawater and plankton samples were
collected at a station located in the Straits of Messina,
ca. 50 m from the coast, Lat. 3825 0 2100 N–Long.
1560 0 2300 E. Details of sampling and samples treatment
for the collection of bacteria as free living, associated
with small and large plankton are given elsewhere (Maugeri et al., 2004).
To collect bacteria adhering to copepods, male and
female copepods were selected from each large-plankton
sample by stereoscopic microscopy examination. A total
of ten mixed specimens, each of different species (Themora stylifera, Acartia clausi, Centropages typicus and
Paracalanus parvus), were suspended in 10 ml of sterile
phosphate-buffered saline (PBS) and centrifuged
twice at 130 g for 10 min at 4 C to remove loosely attached bacteria. The pellet containing bacteria adhering
to copepods was then suspended into 10 ml of sterile
PBS.
In order to enumerate total vibrios (TV), E. coli and
enterococci (Ent) as free-living (> 0.2–64 lm), associated with small (> 64–200 lm) and large plankton
(>200 lm), and adhering to copepods, aliquots of each
sample were directly inoculated onto the appropriate
cultural medium. Plates of thiosulphate-citrate-bile
salts-sucrose medium (Difco), incubated at 37 C for
693
24 h, were used for the enumeration and isolation of
Vibrio spp. Evaluation of faecal indicator bacteria and
Vibrio spp. biochemical identification were performed
according to Maugeri et al. (2004). To confirm the phenotypic identification of V. cholerae, V. parahaemolyticus and V. vulnificus isolates, a PCR method was
performed on DNA extracted from single colonies.
Strains were grown overnight at 37 C on Bacto Marine
Agar 2216 (Difco). Colonies were picked up, suspended
in 200 ll of distilled water and bacterial cells were collected by centrifugation at 11000 g for 15 min at 4 C.
The pellet was resuspended in 100 ll of filtered, autoclaved, deionised water and boiled for 10 min. The cell
lysate (5 ll) was used as template in the PCR assay
immediately after extraction. The number of cycles of
amplification and time duration, primers annealing
and extension of each set of primers are shown in Table
1. V. cholerae non-O1 (our laboratory strain),V. parahaemolyticus ATCC 17802, Kanagawa positive strain,V.
vulnificus ATCC 27562 and V. vulnificus ATCC 33149
were used as reference strains.
Temperature, pH and salinity values ranged from 14
to 23 C, from 7.2 to 8.1 and from 37.6 to 38.0&,
respectively. The abundance of large plankton ranged
from 178 to 616.67 individuals per m3 of seawater and
was largest in August. Copepods accounted for 85.17%
of the total number on average. Cladocerans (av.
4.77%), ostracods (av. 3.02%), chaetognates (av.
2.20%) and molluscan pteropoda (av. 1.86%) represented the main groups. The greatest abundance of
small plankton was observed in April, predominantly
comprised of nauplii of crustaceans and tintinnids.
Vibrios associated with large-plankton samples ranged from 1 to 4.5 · 10 CFU l1 and their maximum
was observed in September. E. coli and enterococci varied from 1 to 3.0 · 10 CFU l1 and <1 to 3.8 ·
10 CFU l1, respectively (Fig. 1a).
Vibrios associated with small plankton ranged from
3.2 to 2.4 · 102 CFU l1 and their numbers were larger
in the spring period, from March to May. Their lowest
number was observed in October. E. coli and enterococci numbers varied from <1 to 1.7 · 103 and from
<1 to 4.0 · 103 CFU l1, respectively (Fig. 1b).
Free-living vibrios ranged from 4.5 · 10 to 5.0 ·
103 CFU l1, with larger numbers in May and again in
August. E. coli and enterococci in seawater ranged from
4.0 · 103 to 1.5 · 106 CFU l1 and from <1 to
5.5 · 104 CFU l1, respectively. E. coli numbers were
larger in May and again in August. Lower numbers of
enterococci were observed in summer and early autumn
(from July to October) (Fig. 1c).
The largest numbers of vibrios adhering to copepods
were recorded in October. No vibrios were encountered
on copepods in March and April. Adhering enterococci
and E. coli abundances per copepod were highest in
May and August, respectively (Fig. 2).
94 C 1 min
60 C 1 min
72 C 1 min
94 C 1 min
60 C 1 min
72 C 1 min
94 C 30 s
55 C 30 s
72 C 30 s
prVC-F: 5 0 -TTA AGC STT TTC RCT GAG AAT G-3 0
prVCM-R: 5 0 -AGT CAC TTA ACC ATA CAA CCC G-3 0
VP33: 5 0 - TGC GAA TTC GAT AGG GTG TTA ACC-3 0
VP32: 5 0 - CGA ATC CTT GAA CAT ACG CAG C-3 0
Vib2: 5 0 -TCT AGC GGA GAC GCT GGA-3 0
Vib3R: 5 0 -GCT CAC TTT CGC AAG TTG GCC-3 0
16S-23SrRNAISRa
pR72H conservative
fragment
16S rRNA
V. cholerae
V. parahaemolyticus
a
ISR intergenic spacer region.
V. vulnificus
PCR
conditions
Primer sequences
Target genes
Vibrio species
Table 1
List of target genes, oligonucleotide primers and PCR conditions used for confirmation of Vibrio species identification
35
30
30
No. of
cycles
273
387
300
Amplicon
size (bp)
Kim and Jeong (2001)
Lee et al. (1995)
Chun et al. (1999)
Source
X74726
AF378706–AF378713
AF114721
Accession
number
694
Baseline / Marine Pollution Bulletin 50 (2005) 682–697
Fig. 1. Abundance of total vibrios (TV) and faecal indicators in the
Straits of Messina from March to October, 2003, and the variation in
water temperature: (a) bacteria associated with large plankton, (b)
bacteria associated with small plankton, (c) free-living bacteria in
seawater samples.
Fig. 2. Abundance (CFU/copepod) of total vibrios (TV) and faecal
indicators adhering to copepods from March to October, 2003, and the
variation in water temperature.
A total of 275 strains were isolated of which 49.81%
(137/275) were identified as vibrios, 22.55% (62/275) as
Baseline / Marine Pollution Bulletin 50 (2005) 682–697
695
Table 2
Number of potentially pathogenic Vibrio species, and their relative percentages in each class, as associated with large and small plankton, free living,
and adhering to copepods, isolated from the marine environment of the Straits of Messina
Vibrios associated
with large plankton
Vibrios associated
with small plankton
Free-living vibrios
Vibrios adhering
to copepods
V. alginolyticus
V. cholerae
V. fluvialis
V. metschinikovii
V. parahaemolyticus
V. vulnificus
Vibrio spp.
18/38 (47.37%)
8/38 (21.05%)
–
–
2/38 (5.26%)
4/38 (10.53%)
6/38 (15.79%)
10/28 (35.71%)
2/28 (7.14%)
2/28 (7.14%)
–
–
6/28 (21.43%)
8/28 (28.57%)
15/27 (59.26%)
4/27 (14.81%)
1/27 (3.70%)
1/27 (3.70%)
–
1/27 (3.70%)
5/27(18.53%)
31/44 (70.45%)
3/44 (6.82%)
1/44 (2.27%)
–
–
2/44 (4.55%)
7/44 (15.91%)
Total (137)
38/137 (27.74%)
28/137 (20.44%)
27/137 (19.70%)
44/137 (32.12%)
Aeromonas spp. and the remaining 27.64% (76/275) were
found to belong to other genera (Flavobacterium, Pasteurella and Pseudomonas).
Of the total 137 isolated vibrios, 27 were free living,
28 associated with small plankton, 38 associated with
large plankton, and 44 adhering to copepods. The numbers of strains of Vibrio species isolated from samples
are shown in Table 2. V. alginolyticus was the predominant species, followed by V. cholerae,V. vulnificus, V. fluvialis, V. parahaemolyticus and V. metschnikovii. The
remaining strains, grouped into Vibrio spp., were referred to V. campbellii, V. cincinnatiensis, V. furnissi,
V. pelagius and V. mediterranei.
V. alginolyticus was isolated from March to October
as free living, and from June to October as associated
with small and large plankton. V. alginolyticus was more
abundant in large than in small plankton.
V. cholerae was recovered as free living in August and
September, from August to September as associated
with large plankton and only in September as associated
with small plankton. V. cholerae was more abundant in
larger plankton than in the other samples. The PCR
assay, using the two primers, prVC-F and prVCM-R,
confirmed the identification of the 17 V. cholerae strains.
These isolates were serotyped V. cholerae non-O1/nonO139.
V. vulnificus was isolated from small plankton samples in September and from all samples collected in
October. Its occurrence reached the highest percentage
in small plankton in comparison with that in seawater
and large-plankton samples. All strains, identified as
V. vulnificus by the API 20E system, were arginine dihydrolase, lysine decarboxylase+ and ornithine decarboxylase (ODC), except one, which was ODC+. All
strains of V. vulnificus belonged to biotype 1 (indole positive) that are known to be pathogenic for humans (Biosca et al., 1996). Amplification of 16S rRNA from these
isolates with primers Vib2 and Vib3R confirmed their
identification as V. vulnificus strains.
V. parahaemolyticus was isolated only from large
plankton in June and October. V. parahaemolyticus
strains generated amplification with the primers VP33
and VP32. None of the isolates of V. parahaemolyticus
showed haemolytic activity on Wagatsuma agar (Sakazaki, 1973).
V. fluvialis was recovered as free living in May and
associated with small plankton in May and July. V.
metschnikovii was isolated only as free living in
September.
Among the 44 strains firmly adhering to copepods, V.
alginolyticus was the species most abundant followed by
V. cholerae, V. vulnificus and V. fluvialis. V. alginolyticus
was the predominant species in May, June, July
and October. V. cholerae was isolated from August
to October. V. vulnificus strains were isolated only in
October and V. fluvialis only in May. Within the group
of Vibrio spp., two isolates were identified as V.
campbellii.
The investigated area is subject to a wide spatial fluctuation of faecal contamination, in response to the
introduction of untreated sewage into the sea. Abundances of free-living and small-plankton associated
enterococci showed higher fluctuations than E. coli.
Free-living vibrios abundance was found directly correlated with seawater temperature, while vibrios associated with small plankton showed a negative relationship
with temperature, indicating a different seasonal behaviour. While a direct relationship was found between
densities of free-living faecal bacteria and those associated with plankton, the lack of correlation between
free-living and planktonic-associated vibrios leads to
the conclusion that Vibrio spp. in these habitats are
independent. Finally, free-living vibrios were present in
both high and low faecal-contaminated periods showing their independence from seawater pollution
(Table 3).
The major free-living Vibrio species identified in seawater over the study was V. alginolyticus which is considered a pathogenic Vibrio species, particularly of
wounds and ear infections in patients exposed to the
marine environment (Farmer and Hickman-Brenner,
1992). This species showed higher salinity adaptation
than V. fluvialis V. parahaemolyticus and V. vulnificus,
and a different habitat. V. parahaemolyticus was isolated
1
0.71
*
1
0.72
*
0.89**
1
1
0.73
*
0.76
*
0.91**
1
0.71
1
0.74*
*
0.74*
1
0.78*
1
1
0.78
*
0.91*
0.76*
*
1
0.77
P < 0.05.
P > 0.01.
Z-abund
Zooplanktonic abundance
**
F-TV
F-E. coli
F-Ent
Free-living bacteria
*
S-TV
S-E. coli.
S-Enter
Bacteria associated with small plankton
0.76
L-TV
L-E. coli
L-Ent
Bacteria associated with large plankton
0.71*
0.75
*
T
pH
Sal
1
0.74*
*
Sal
0.74
1
pH
References
Physical parameters
T
only from large plankton, but not from copepods. V. fluvialis was isolated more frequently from small plankton
than from seawater and was recovered as adhering to
copepods. V. vulnificus strains were occasionally isolated
from seawater and in greater numbers from small and
large plankton. It was also recovered as firmly bound
to copepods in September and October.
It is likely that Vibrios species, except for V. metschnikovii, isolated only as free living, derived from the
environment and did not permanently establish a relationship with copepods, but can move through different
habitats. They may colonise copepods when they find
suitable environmental conditions.
The association with plankton has been considered a
tool for the survival and distribution of bacteria in the
aquatic environment (Carman and Dobbs, 1997). In this
study, the demonstration of higher percentages of vibrios associated with small and large plankton in March
(76.44%) and April (47.54) than those of free-living, supports the hypothesis that plankton provides an overwintering site for vibrios and, to a lesser degree, for E. coli.
The behaviour of associated vibrios appears opposite in
respect to enterococci. The first showed higher percentages in colder months while associated enterococci,
mainly with large plankton, were higher in warmer
months.
These results support previous suggestions that water
quality of coastal areas should include the search for
free-living and plankton-associated Vibrio spp., because
of their independence from bacterial faecal indicators.
Our findings led to the conclusion that Vibrio spp. have
a competitive advantage in the chitinous exoskeleton
microenvironment of copepods.
1
0.89**
0.71*
0.73*
F-E. coli
0.76*
0.77*
F-TV
S-Ent
0.71*
S-E. coli
0.76*
S-TV
L-Ent
L-E. coli
L-TV
Free-living bacteria
Bacteria associated with
small plankton
Bacteria associated with
large plankton
Physical parameters
Table 3
Correlation coefficients, obtained using the Pearson method, among physical parameters, free-living and associated bacteria, and zooplanktonic abundance
F-Ent
0.72*
0.71*
Z-abund
Baseline / Marine Pollution Bulletin 50 (2005) 682–697
Zooplanktonic
abundance
696
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