Ecology of Vibrios in the Oliveri-Tindari Lagoon (Messina), Two-Year Study

P.S.Z.N. I: Marine Ecology, 13 (2): 149-161 (1992) Q 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0173-9565

Accepted: June 27,1991

Ecology of Vibrios in the Oliveri-Tindari Lagoon (Messina), Two-Year Study RENATA ZACCONE, ERMANNO CRISAFI & LUCREZIA GENOVESE

Istituto Sperimentale Talassografico CNR, Spianata S. Raineri 86, 1-98122 Messina, Italy.

With 3 figures and 2 tables

Key words: Brackish water, ecology, Vibrio cholerae, V. parahaemolyticus.

Abstract. A two-year ecological study in the Oliveri-Tindari (Messina) brackish water ponds evaluated the quantitative and qualitative distribution of Vibrio species in relation to the physico- chemical conditions of the ponds.

The evaluation of 204 samples shows a general relationship between the halophilic vibrios and water temperature. In particular this relationship is stronger for the Vibrio fraction able to grow at 3 7 T , i. e., those species related to human infections. On the other hand, the halophilic vibrios show no relation with the index of fecal contamination.

The ecology of V. parahaemolyticus is discussed in relation to the salinity of Marinello pond. The distribution of V. cholerae non 01, isolated from all ponds, shows that this microorganism

can be considered as autochthonous in coastal environments.

Problem

The ecology of halophilic vibrios has stimulated great interest since a close relationship between the consumption of raw seafood and the occurrence of gastroenteritis or alimentary toxic infection episodes attributed to Vibrio cholerae and V. parahaemolyticus has been observed. Besides these two vibrios, whose pathogenicity is well established, the marine and brackish environments hold other potentially pathogenic vibrio species such as V. fluvialis, V. vul- nificus, V. alginolyticus, and V. cholerae non 0 1 . BLAKE ef al. (1980) extensively reviewed the vibrios other than V. cholerae that are related in some way to human infections.

V. cholerae survives longer in both marine and estuarine waters than E. coli (HOOD & NESS, 1982). Studies on the ecology of V.parahaemo1yticu.s and V. cholerae in the Chesapeake Bay point out the relationship between bacteria presence and water temperature and salinity (KAPER et al., 1979; COLWELL et al., 1984). According to most authors, no relationship exists with fecal pollution. For this reason, the traditional indices of fecal contamination alone are not reliable indicators of the quality of either water or edible molluscs (CRISAFI et al., 1985 b; ZACCONE et al., 1985).

U. S. Copyright Clearance Center Code Statement: 0173-9565’9211302-0149$02.50/0

150 ZACCONE, CRISAFI & GENOVESE

The Oliveri-Tindari lagoon area is located along the Tyrrhenian coast in the province of Messina (Fig. 1). This lagoon area has recently formed from the closing of sand banks; at present, it consists of six small, shallow brackish ponds. Their morphology is subject to a considerable dynamism, particularly in the case of the most recently formed ponds (ABBRUZZESE & A R I C ~ , 1955; CRISAFI et al., 1981). After a positive evaluation in terms of fecal contamination the possibility of mussel cultures here had been suggested (FARANDA & PERNICE, 1974). Since a possible utilization for aquaculture remains valid, a study was carried out in 1987/88 in order to evaluate the chemico-physical and biological parameters of the ponds.

The aim of this research was to obtain further information on the ecology of Vibrio species; these brackish ponds represent a distinctive environment because of their unique geomorphological and biological features.

Material and Met hods

Water samples were taken in March, June, September, and December 1987 from 18 stations distributed in the six ponds (Fig. 1); for each station, two samples were taken, one from the surface layer and the other from the bottom layer (varying between 1.5 and 3.5 m depth). During the second year (1988), samples were taken monthly from the three oldest ponds (Marinello, Mergolo, Verde) and from Port0 pond, which recently separated from the sea.

A total of 204 samples were taken, transported to the laboratory within a few hours, and concentrated on a 0.45 pm Millipore filtering membrane. The membrane was subsequently put on TCBS Agar (Difco) (3 % NaCl final concentration) and incubated at 24°C for 48 h and at 37°C for 24 h. The two incubation temperatures were selected to distinguish between the group of halophilic vibrios characteristic of the marine environment (VP total vibrios presumptive) and the fraction able to grow at 37°C (VPP V.parahaemolyticus presumptive); the latter mainly consists of species related to human infections. At the same time the total and fecal coliforms and enterococci were estimated by means of the filtering membrane technique with m-Endo broth, m-FC broth, and m- Enterococcus agar (Difco) media.

Some species known to be pathogenic for humans were isolated from the ponds (a total of 68 samples were examined) by using enrichment media followed by growth on TCBS Agar at 37 "C. The following media were used for the isolation of pathogens:

- AP (alkaline peptone water) for V. cholerae (KAPER el al., 1979); - VPSM (V.parahaemolyticus salt meat) broth (OMS, 1977) for V. parahuemolyticus; - PPC (phytone peptone carbenicilline) for V. parahaemolyricus prepared according to TOTI et

- FEM (V. fluvialis enrichment medium) broth as suggested by NISHIBUCHI el al. (1983) for

VPSM and FEM broths did not prove to be selective for the researched species and were omitted in the second year of this study.

The isolated colonies were maintained on Marine Agar 2216 (Difco) and identified on the basis of their morphological, cultural, and biochemical characteristics as previously described by CRISAFI et al. (1985a). Strains biochemically similar to V. cholerae were tested for agglutination on slides with a 01 polyvalent antiserum (Difco). They were also tested for the production of the choleric toxin by means of a latex reagent (VET-RLA Oxoid). The Kanagawa test was performed on V. parahaemolyricus strains with horse erythrocytes.

al. (1983);

V. fluviah.

Vibrios in brackish waters 15 1

A: Marinello B: Mergolo C: Verde D: Port0 E: Nuovo F: Piccolo

N

Fig. 1. Examined area and stations.

Results

The main physico-chemical characteristics of the ponds - temperature, salinity, dissolved oxygen, and ammonia - are reported in Fig. 2 a, b. The results of the comprehensive study obtained in the first year are documented in a Report (Istituto Sperimentale Talassografico CNR Messina, 1990).

The quantitative analyses of halophilic vibrios demonstrated a certain homo- geneity within each pond; for this reason, we report the mean values of VP and VPP in the graphic plot representations relative to each basin (Fig. 3). Previous studies show that VP bacterial concentrations gradually increase with tempera- ture during summer and are higher than those in seawater samples taken near the coast (CRISAFI et al., 1985 a; CRISAFI, 1988).

152

40

30

0 I $ 2 0 I=

10

0

40

30

0 I f " K

10

0

ZACCONE. CRISAFI & GENOVESE

Marinello pond

Dec WBB Jun Sep Dec

im

140

120

im 8

m

60

40

160

140

120

100 g

m

60

40

Fig.2a. Variation of the physico-chemical parameters in Marinello and Verde ponds (T = temperature in "C; S = salinity in L; NH3 = ammonia in pg-a . I-l; 02% = oxygen saturation in %).

Vibrios in brackish waters 153

10

Mnr'87 Jun Sep Dec Mar'@ Jun sep Dee

-&- 9% - NHj -4 S -& T

Fig. 2 b. Variation of the physico-chemical parameters in Mergolo and Porto ponds (T = tempera- ture in "C; S = salinity in %o; NH3 = ammonia in pg-a . I - ] ; 0 2 % = oxygen saturation in %).

154 ZACCONE, CRISAFI & GENOVESE

Marinello pond 5

4

r

E E 3 i i '

1

0

Verde pond 5

Jun sep Jun sep Dee

Fig. 3 a. Bacterial density in vibrios presumptive (VP) and in V. paruhaemolyricus presumptive (VPP) in Marinello and Verde ponds (average between the surface and bottom).

Vibrios in brackish waters 155

5

4

.; ii r 8 3 X z 2 2 B

1

0

5

4

r

; 3

$ 7

X

2 2 - B

1

0

Mergolo pond

Jun Sap Dec T

W B ( I Jun Sep Dec

IliillmwJ IZ9w

Porto pond

Mar'87

i Jun Sep Dec w m Jun sep Dec

Fig. 3 b. Bacterial density in vibrios presumptive (VP) and in V. paruhaemolyticus presumptive (VPP) in Mergolo and Porto ponds (average between the surface and bottom).

156 ZACCONE, CRISAFI & GENOVESE

VPP concentrations show a similar trend, although with a stronger correlation with temperature. A sharp decrease in the bacterial concentration occurred at the lowest temperature (7.8"C) recorded in the two years. The maximum values were recorded in the summer months, as expected. The salinity of each basin shows a seasonal variation as well.

Tables 1 and 2 show the distribution of isolated vibrios. Among the different species, V. afginofyticus was the most frequently found, as previously observed by CRISAFI & MAUGERI (1990) and ZACCONE et af . (1988) in different marine and brackish environments.

V. choferae non 01 was isolated from 4 of the 6 ponds during the first year (12 strains). In these ponds, salinity ranged from 21.50 to 38.57 X and tempera- ture from 14.1 to 28.8"C. This species was also isolated from Marinello and Mergolo ponds during the second year of analyses. V. choferae non 01 strains were isolated at every survey, with no apparent relation to season, water temperature, or salinity.

The agglutination tests on slides with a 01 polyvalent antiserum was negative, as were the toxinogenesis tests with the Oxoid Kit.

V. parahuemolyticus was isolated nearly constantly from the Marinello pond in every season (Table 1). The salinity here was always somewhat lower than in the other ponds because of an inflow of continental waters and a stronger isolation from the sea (minimum 16.14 %, maximum 28.46 '%) (Fig. 2). The occurrence of V. parahuemolyticus here supports the findings of KANEKO & COLWELL (1973) on the ecology (ambient salinity) of this microorganism. During 1988, it was also isolated from the Porto and Verde ponds in the summer months (Table2). The Kanagawa test using horse erythrocytes was positive for 43 % of the V . parahuemolyticus strains.

Moreover, 5 strains ascribable to the V.fluvialis species were isolated from the Marinello (3 strains), Mergolo, and Porto ponds; V. metschnikovii, charac- terized by the negative reaction to the Oxidase test, was occasionally found in four ponds only (Tables 1 and 2).

V. vufnificus could not be isolated from any sample. Such lactose-fermenting vibrios were isolated from marine samples from North Carolina and Georgia (OLIVER et af . , 1982).

Total and fecal coliforms were nearly always absent; this observation con- firms the results of various authors showing the lack of correlation between the presence of V. choferae and V. parahuemolyticus and the bacteriological values of fecal pollution. Occasionally, 20-10 CFU/100 ml of fecal streptococci, undoubtably not of human origin, were found.

The AP broth proved to be the best enrichment medium. The other media showed a low selectivity for the pathogenic species since they allowed a strong growth of V. alginofyticus, which often prevented isolation of the other species.

The TCBS medium amended with 2 % NaCl seems to favour the isolation of V. parahaemolyticus over V. cholerae; TCBS with a lower NaCl concentration was more appropriate for V.choferae. At any rate, it represents the best isolating medium for halophilic vibrios (BOLINCES et a f . , 1988).

Vibrios in brackish waters 157

Table 1. Results of isolation of halophilic vibrios during 1987.

pond enrichment isolation April June September December medium medium

Marinello VPSM

Mergolo

Verde

Porto

Nuovo

Piccolo

AP

FEM

VPSM AP

FEM

VPSM AP

FEM PPC

Diretta VPSM AP

FEM PPC

VPSM AP

FEM

VPSM AP

FEM

TCBS + NaCl V.P., V.a. V.a.

TCBS + NaCl V.p., V.m. V.a. TCBS + NaCl V.P., V.a. V.a. V.a.

TCBS V.P., V.f. v.p. V.P., V.f.

TCBS + NaCl TCBS V.a., Ent. TCBS + NaCl Ent., V.m. V.a., V.f. TCBS + NaCl V.a. V.a.

V.a. V.a.

V.m.

TCBS + NaCl V.a. V.a. V.a. TCBS V.a. V.ch., V.a., Ps. TCBS + NaCl Ent. V.ch., V.a. TCBS + NaCl V.a. V.a. V.a. TCBS + NaCl V.a.

TCBS + NaCl Ent., V.f. V.a.

TCBS Ent., V.ang. V.a. V.ch., Ent., Ps. V.ch., V.a., Ps., Ent.

TCBS + NaCl V.ch., V.a. V.a. TCBS + NaCl V.ch. V.a. V.a., Ent. V.a., V.m. TCBS + NaCl V.a.

V.a.

TCBS + NaCl TCBS Ps., V.a. V.ch., Ent. TCBS + NaCl V.a. V.a. TCBS + NaCl V.a.

TCBS + NaCl Ent. V.a. TCBS V.a. V.ch., Ent. TCBS + NaCl Vsh., V.a. TCBS + NaCl V.a. V.a., Ent.

(V. a. = V. alginolyticus, V. p. = V. parahaemolyticus, V. m. = V. metschnikovii, V. f. = V. fluvialis, V. ch. = V. cholerae non 01, V. ang. = V. anguillarum, Ps. = Pseudomonas sp., Ent. = Enterobac- teriaceae) .

158 ZACCONE, CRISAFI & GENOVESE

Table 2. Results of isolation of halophilic vibrios during 1988/89.

pond enrichment isolation February March April May medium medium

Marinello AP

PPC

Mergolo AP

PPC

Verde AP

PPC

Port0 AP

PPC

[ + NaCl TCBS + NaCl

{ + NaCl TCBS + NaCl

[ + NaCl TCBS + NaCl

[ :::+ NaCl TCBS + NaCl

V.ch.

V.a., Ent. V.ch. 3V.a. V.ch.

Ent.

Ent. V.a.

V.a. Ent. Ent., V.m. V.a.

Ps .

V.ch.

V.P., V.a. V.P., V.a.

V.a.

2V.a.

3V.a.

2V.a. V.a.

Ent.

2v.p.

Ps .

V.m.

V.ch.,V.a.

2V.a.

V.m.

Discussion

The present investigations contribute to our knowledge of the vibrio ecology in brackish environments.

In the examined ponds, no quantitative difference in the total bacterial concentration - observed by direct counting under the epifluorescence micro- scope (LA FERLA et al., 1990) - was found. On the other hand, the Vibrionaceae distribution reflects the qualitative differences of the various environments.

The isolation of the V. parahaemolyticus species in almost exclusively one pond (Marinello) during the investigation could be explained by the lowest salinity here (range 16.14-28.46%0). According to ROBERTS et al. (1982), tem- peratures above 15°C and salinities above 8 % are appropriate conditions for V. parahaemolyticus. This organism is typical for coastal and estuarine environ- ments with a high content of organic substances. In fact, it was found during previous studies in Sicilian waters only in coastal samples or in cultured molluscs (CRISAFI & MAUGERI, 1990).

In our investigations, V. cholerae non 01 was isolated from all ponds, even those whose salinity was similar to that of seawater. This strain was occasionally isolated in the port of Milazzo (Messina, Italy) at the highest seawater tem- peratures (Istituto Sperimentale Talassografico CNR, 1986) and from the Strait of Messina (BRUNI et al., 1987). However, this phenomenon very probably also extends to other Italian and foreign coastal and lagoon zones (DUMONTET, 1990). V. cholerae non 01 is considered, along with the other vibrios, to be an autochthonous component of the aquatic environment all over the world (MARCHAND, 1986; TISON et al., 1986; VENKATESWARAN et al., 1989).

Vibrios in brackish waters 159

June August Sep- October December January tember

V.p., V.a.

v.p. V.a.

2V.ch.

Vm. , V.a. 2V.a.

V.a.

2V.p., V.a. V.a.

V.ch.

v.p. V.P., V.ch. 2V.a. V.a.

V.ch.

V.ch. V.a.

2V.ch. 2V.a.

V.a.

V.a. V.a.

V.a.

V.a. V.a.

V.ch., V.a. V.p., V.ch.

V.p., V.a. Vp. V.ang.

2V.p., V.a.

V.ch., 2V.ang. V.ch.

V.m. V.m.

V.ch., V.ang. V.a.

V.m. V.m. V.a. V.a.

V.a., V.ch. 2V.ang.

2V.a., V.ang. V.a. 2V.a., V.m. 2V.a.

v.p.

2V.p., V.m. V.a.

V m . , Ent.

V.ch., V.p.

V.ch. V.ang.

V.a., Ps. V.a.

~~ ~ ~

(V. a. = V. alginolyticus, V. p. = V. parahaemolyticus, V. m. = V. metschnrkovii, V. f . = V. jluvialrs, V. ch = V. cholerae non 01, V. ang. = V. anguillarum, Ps. = Pseudomonas sp., Ent. = Enterobac- teriaceae) .

According to some authors it prefers lower salinities, although sodium has been demonstrated to be necessary for growth (min. 0.02M NaCl). This ion cannot be substituted by other salts (COLWELL, 1986). Although the pathogenic- ity of the strains isolated from the marine environment remains unclear, there are apparently certain differences in the toxicity of marine strains and those isolated from humans (BLAKE et al., 1980; ROBERTS et al., 1982). V. cholerae non 01 has recently been demonstrated to have two toxins: a thermostabile enterotoxin (NAG-ST) and a hemolysin (NAG-rTDH) correlated with the thermostabile hemolysin of V. parahaemolyticus (HONDA et al., 1986). The research on the toxin and on the attachment factors of the duodenal cells attest to a true pathogenic strain (DODIN et al., 1986).

The in vitro tests (research on the choleric toxin and agglutination on slides) on V. cholerae non 01 strains lead us to consider such strains as not toxigenic; it is not advisable, however, to use the examined ponds for mussel cultures.

Summary

The present paper reports the qualitative and quantitative distribution of the total halophilic vibrios and of those Vibrio species related to alimentary toxin- fections (V. cholerae, V. parahaemolyticus, V. vulnificus, V. fluvialis) recovered in the Oliveri-Tindari lagoon; this system consists of six small brackish ponds previously designated as being suitable for mussel culture.

160 ZACCONE, CRISAFI & GENOVESE

Bacterial density was related to temperature and salinity over the two-year study period.

The constant presence of V. parahuemolyticus in the Marinello pond, which shows certain special features (a greater isolation from the sea and the inflow of continental waters), confirms what is already known about the ecology of this species: it prefers coastal and estuarine waters with a high content of organic matter.

V. cholerae non 01 is an autochthonous organism because it could be isolated from all ponds independent of salinity.

References

ABBRUZZESE, D. & F. A R I C ~ , 1955: Osservazioni geomorfologiche e fisico-chimiche sui laghi di Oliveri-Tindari. Boll. Pesca Piscic. Idrobiol., 10: 1-23.

BLAKE, P. A., R. E. WEAVER & D. C. HOLLIS, 1980: Diseases of humans (other than cholera) caused by vibrios. Annu. Rev. Microbiol., 3 4 341-367.

BOLINCES, J., J . L. ROMALDE & A. E. TORANZO, 1988: Evaluation of selective media for isolation and enumeration of vibrios from estuarine waters. J. Microbiol. Methods, 8: 151-160.

BRUNI, V., E. CRISAFI & T. L. MAUGERI, 1987: Preliminary data on halophilic vibrios in the Strait of Messina. Rev. Int. Octanogr. MBd., 87-88: 67-73.

COLWELL, R.R. , 1986: Vibrio cholerae and related vibrios in the aquatic environment - an ecological paradigm. In: F. MEGUSAR & M. GAUTOR (Eds.), Perspectives in Microbial Ecology. Proc. IV ISME; Ljubljana, 24-29 August, 1986: 426-434.

--, P. A. WEST, D. MANEVAL, E. F. REMMERS, E. L. ELLIOT & N. E. CARLSON, 1984: Ecology of pathogenic vibrios in Chesapeake bay. In: R. R. COLWELL (Ed.), Vibrios in the environment. John Wiley & Sons, New York: 367-387.

CRISAFI, E., 1988: Distribution of presumed vibrios in coastal and pelagic waters of Sicily. Microbiologica, 1I: 151-157.

- -, L. GENOVESE, R. LA FERLA, T. L. MAUGERI & R. ZACCONE, 1985 a: Vibrioni isolati da acque ioniche e tirreniche. Mem. Biol. Mar. Oceanogr., 15: 136-144.

- -, S . GIACOBBE & M. LEONARDI, 1981: Nuove ricerche idrobiologiche nell’area lagunare di Oli- veri-Tindari. Mem. Biol. Mar. Oceanogr., 11: 139-186.

- -, T. L. MAUGERI & R. ZACCONE, 1985 b: Controlli batteriologici sull’acqua di alimentazione di un impianto di acquacoltura. Ig. Mod., 84: 447-464.

- - & T. L. MAUGERI, 1990: Ecologia delle Vibrionaceae in ambienti costieri marini e salmastri della Sicilia. Oebalia, XVI-1 (Suppl.): 507-514.

DODIN, A., M. Dosso & M. GUILLOU, 1986: Ecologie des vibrions pathogtnes. GERBAM - 2tme Colloque International de Bacttriologie marine. CNRS, Brest, 1-5 Octobre 1984. IFREMER, Actes de Colloques, 3: 499-505.

DUMONTET, S., 1990: Sul ritrovamento di V. cholerue nel lago di Fusaro. Biologi Italiani, XX (7/8): 5-6.

FARANDA, F. & A. PERNICE, 1974: Possibile mitilicoltura nei laghi di Oliveri-Tindari. Atti SOC. Peloritana, u): 3-24.

HONDA, T., M. NISHIBUCHI, T. MIWATANI & J. B. KAPER, 1986: Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in V. cholerae non 0 - 1 strains. Appl. Environ. Microbiol., 52 (11): 1218-1220.

HOOD, M. A. & G. E. NESS, 1982: Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments. Appl. Environ. Microbiol., 43: 578-584.

Istituto Sperimentale Talassografico, C.N.R. Messina (Ed.), 1986: Indagine oceanografica nel golf0 di Milazzo (Messina). Qualith delle acque e studio preliminare dei sedimenti per una ipotesi di acquacoltura, 1984. Rapporti, 1; 37 pp.

Istituto Sperimentale Talassografico, C.N.R. Messina (Ed.), 1990: Indagine interdisciplinare sul sistema degli stagni salmastri costieri di Oliveri-Tindari (Messina). I. Risultati delle campagne stagionali del 1987. Rapporti, 4; 34 pp.

Vibrios in brackish waters 161

KANEKO, T. & R. R . COLWELL, 1973: Ecology of Vibrio pnrahaemolyticus in Chesapeake Bay. J . Bacteriol., 113: 24-32.

&PER, J., H. LOCKMAN, R. R. COLWELL & S . W. JOSEPH, 1979: Ecology, serology and enterotoxin production of Vibrio cholerae in Chesapeake Bay. Appl. Environ. Microbiol., 37: 91-103.

LA FERLA, R., E. CRISAFI, L. GENOVESE, 1990: Primi dati sulla distribuzione del batterioplancton nell’area lagunare di Oliveri-Tindari. Oebalia, XVI-1 (Suppl.): 457-466.

MARCHAND, M., 1986: Etude tcologique des vibrions du bassin d’Arcachon. GERBAM - 2kme Colloque International de BactCriologie marine. CNRS, Brest, 1-5 Octobre 1984. IFREMER, Actes de Colloques, 3: 483-489.

NISHIBUCHI, M., N. C. ROBERTS, H. B. BRADFORD, Jr. & R. J. SEIDLER, 1983: Broth medium for enrichment of Vibrio fluvialis from the environment. Appl. Environ. Microbiol., 46: 425-429.

OLIVER, J. D., R. A. WARNER & D. R. CLELAND, 1982: Distribution and ecology of Vibrio uulnificus and other lactose-fermenting marine vibrios in coastal waters of the southeastern United States. Appl. Environ. Microbiol., 44: 1404-1414.

OMS, 1977: Directives applicables B la surveillance sanitaire de la qualitt des eaux littorales. OMS, Bureau rCgional de ]’Europe; Copenhague, 1977: 1-113.

ROBERTS, N. C., R. J . SIEBELING, J . B. KAPER & H. B. BRADFORD, Jr., 1982: Vibrios in the Louisiana Gulf coast environment. Microb. Ecol., 8 299-312.

TISON, D. L., M. NISHIBUCHI, R. J . SEIDLER & R. J. SIEBELING, 1986: Isolation of non-01 Vibrio cholerae serovars from Oregon coastal environments. Appl. Environ. Microbiol., 51: 444-445.

TOTI, L., L. CROCI, G . DE FELIP, L. VOLTERRA & F. TAGGI, 1983: Selective medium for isolation of Vibrio pavahaemolyficus WHO/ZOON/83.161.

VENKATESWARAN, K., T. TAKAI, I. M. NAVARRO, H . NAKANO, H. HASHIMOTO & R. J. SIEBELING, 1989: Ecology of V. cholerae non-Ol and Salmonella spp. and role of zooplankton in their seasonal distribution in Fukuyama coastal waters, Japan. Appl. Environ. Microbiol., 55: 1591-1598.

ZACCONE, R., E. CRISAFI & T. L. MAUGERl, 1985: Indagini batteriologiche su Ostrea edulis e Crussostreu gigus in un allevamento sperimentale (Trapani-Sicilia). Ig. Mod., 83: 754-764.

- -, T. L. MAUGERI, E. CRISAFI, L. GENOVESE & R. LAFERLA, 1988: Premikres donnCes sur I’tcoiogie des vibrions dans la zone lagunaire de Oliveri-Tindari (Messine, Italie). Rapports et procbs-verbaux des rtunions de la CIESM, 31 (2): 178.