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692 Baseline / Marine Pollution Bulletin 50 (2005) 682–697 Lee, M.R., Correa, J.A., Zhang, H., 2002. Effective metal concentrations in porewater and seawater labile metal concentrations associated with copper mine tailings disposal into the coastal waters of the Atacama region of northern Chile. Marine Pollution Bulletin 44, 956–976. Lionetto, M.G., Caricato, R., Giordano, M.E., Pascariello, M.F., Marinosci, L., Schettino, T., 2003. Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area. Marine Pollution Bulletin 46, 324–330. OSPAR Commission, 2000. Commission for the Protection of the marine Environment of the North-East Atlantic-Quality Status Report. Phillips, D.J.H., 1977. The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments — a review. Environmental Pollution 13, 281–317. Rosland, E., Lund, W., 1998. Direct determination of trace metals in seawater by ICPMS. Journal of Analytical Atomic Spectrometry 13 (11), 1239–1244. Scoullos, M., Dassenakis, M., 1983. Trace metals in a tidal Mediterranean embayment. Marine Pollution Bulletin 14 (1), 24– 29. Scoullos, M., Dassenakis, M., 1986. MAP/UNEP Technical Report Series ÔBiogeochemical studies of selected pollutants in the open waters of the Mediterranean (MED POL VIII) 8, Addendum, 45–63. Storelli, M.M., Storelli, A., Marcotrigiano, G.O., 2001. Heavy metals in the aquatic environment of the Southern Adriatic sea, Italy. Macroalgae, sediments and benthic species. Environment International 26, 505–509. UNEP, 1993. Preliminary assessment of the state of pollution of Mediterranean sea by zinc, copper and their compounds and proposed measures. UNEP (OCA)/MED/WG.66/Inf.3. UNEP, 1996. The state of the marine and coastal environment in the Mediterranean Region. MAP Technical Report Series No. 100, UNEP Athens, p. 142. Viel, M., Zurlini, G., 1988. Indagine ambientale dei sistemi marini costieri: la regione Puglia. Acqua-Aria speciale, 87–98. Viel, M., Damiani, V., Zurlini, G., De Rosa, S., 1986. Geochimica degli elementi in traccia e forme del fosforo nei sedimenti della piattaforma pugliese. In: Enea Comitato Nazionale per lo sviluppo dellÕEnergia Nucleare e delle Energie Alternative. ‘‘Indagine ambientale del sistema marino costiero della regione Puglia’’. Elementi per la definizione del Piano delle coste, parte B, pp. 149– 169. 0025-326X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. 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 Barbieri, E., Falzano, L., Fiorentini, C., Pianetti, A., Baffone, W., Fabbri, A., Matarrese, P., Casiere, A., Katouli, M., Kühn, I., Möllby, R., Bruscolini, F., Donelli, G., 1999. Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast. 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