Vibrio

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


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.

REFERENCES11

Albert, M.J., Sindique, A.K., Islam, M.S., Faruque, A.S.G., Ans-

aruzzaman, M., Faruque, S.M. and Sack, R.B. (1993) Large

outbreak of clinical cholera due to Vibrio cholerae non-01 in

Bangladesh. Lancet 341, 704.

Boccia, A., Montanaro, D., Annino, I. and Schioppa, F. (1978)

Isolamento di vibrioni alo®li riferibili a V parahaemolyticus e V.

alginolyticus. da molluschi bivalvi e da campioni di acqua della zona

costiera della cittaÁ di Napoli. Igiene Moderna12 71, 894±905.

Center for Disease Control and Prevention (1999) Outbreak of Vibrio

parahaemolyticus infection associated with eating raw oysters and

clams harvested from Long Island Sound ± Connecticut, New Jersey

and New York, 1998. Journal of the American Medical Association

281, 603±604.

Colwell, R.R. and Hug, A. (1994) Vibrios in the environment: viable

but nonculturable Vibrio cholerae. In Vibrio cholerae and Cholera:

Molecular to Global Perspectives ed. Wachsmuth, I.K., Blake, P.A.

and Olsvik, O. pp. 117±130. Washington, DC: American Society for

Microbiology.

Fraser, S.L., Purcell, B.K., Delgado, B. Jr, Baker, A.E. and Whelen,

A.C. (1997) Rapidly fatal infection due to Photobacterium (Vibrio)

damsela. Clinical Infection and Disease 25, 935±936.

Hackney, C.R., Ray, B. and Speck, M.L. (1980) Incidence of Vibrio

parahaemolyticus and the microbiological quality of seafood in North

Carolina. Journal of Food Protection 43, 763 ±769.

Hackney, C.R. and Dicharry, A. (1988) Seafood-borne bacterial

pathogens of marine origin. Food Technology 42, 104±109.

Honda, T., Arita, M., Takeda, T., Youh, M. and Miwatani, T. (1985)

Non-01 Vibrio cholerae produces two newly identi®ed toxins related

to Vibrio parahaemolyticus haemolysin and Escherichia coli heat stable

enterotoxin. Lancet ii, 163±164.

Honda, T., Booth, B.A., Boesman-Finkelstein, M. and Finkelstein,

R.A. (1987) Comparative study of Vibrio cholerae non-O1 protease

and soluble hemagglutinin with those of Vibrio cholerae O1. Infection

and Immunity 55, 451±454.

Jackson, J.K., Murphree, R.L. and Tamplin, M.L. (1997) Evidence

that mortality from V. vulni®cus infection results from single strains

60

50

40

30

20

10

0Vibrios Aeromonas Pseudomonas Others

%

Fig. 2 Relative percentages of isolates of

the various genera with respect to the total

number of bacteria isolated per season.

, Winter; , spring; , summer; u, autumn

60 L. CROCI ET AL .


among heterogeneous populations in shell®sh. Journal of Clinical

Microbiology 35, 2098±2101.

Klontz, K.C. (1990) Fatalities associated with V. parahaemolyticus and

V. cholerae non-01 infections in Florida (1981±88). Southern

Medicine Journal 83, 500±502.

Lee, K.K. and Yii, K.C. (1996) Comparison of three methods for

assaying hydrophobicity of pathogenic vibrios. Letters in Applied


Lipp, E.K. and Rose, J.B. (1997) The role of seafood in foodborne

diseases in the United States of America. Review of Science

Technology 16, 620±640.

MacCardell, B.A., Madden, J.M. and Singh, D.B. (1985) Isolation and

characterization of a cytolysin produced by Vibrio cholerae serogroup

non 0-1. Canadian Journal of Microbiology 31, 711±720.

Maggi, P., Carbonara, S., Fico, C., Santantonio, T., Romanelli, C.,

Sforza, E. and Pastore, G. (1997) Epidemiological, clinical and

therapeutic evaluation of the Italian cholera epidemic in 1994.

European Journal of Epidemiology 13, 95±97.

Marino, A., Crisa®, G., Maugeri, T.L., Nostro, A. and Alonzo, V.

(1999) Uptake and retention of Vibrio cholerae non-O1, Salmonella

typhi, Escherichia coli and Vibrio harvey by mussels in seawater.


Maugeri, T.L., Crisa®, E. and Zaccone, R. (1987) Halophilic vibrios in

cultivated molluscs. Microbiologie Aliments Nutrition13 5, 135±139.

Merino, S., Rubires, X., Knochel, S. and Tomas, J.M. (1995)

Emerging pathogens: Aeromonas spp. International Journal of Food


Morris, J.G. Jr and Black, R.E. (1985) Cholerae and other vibrioses in

the United States. New England Journal of Medicine 312, 343±350.

Morris, J.G. Jr, Takeda, T., Tall, B.D., Losonsky, G.A., Battacharya,

S.K., Forrest, B.D., Kay, B.A. and Nishibuchi, M. (1990) Experi-

mental non-0 group 1 Vibrio cholerae gastroenteritis in humans.

Journal of Clinical Investigations 85, 697±705.

Nishibuchi, M., Siedler, R.J., Rollins, D.M. and Joseph, S.W. (1983)

Vibrio factors cause rapid ¯uid accumulation in suckling mice.

Infection and Immunity 40, 1083±1091.

O'Neill, K.R., Jones, S.H. and Grimes, D.J. (1992) Seasonal incidence

of V. vulni®cus in the Great Bay estuary of New Hampshire and

Maine. Applied Environmental Microbiology 58, 3257±3262.

Robinson, R.K. (ed. in chief) (2000) Encyclopedia of Food Microbiology.

London: Academic Press.

Rutala, W.A., Sarulli, F.A.J., Finch, C.S., MacCormack, J.N.

and Steinkraus, G.E. (1982) Oyster-associated outbreack of

diarrheal disease possible caused by Plesiomonas shigelloides. Lancet

1, 739.

Spira, W.M. and Fedorka-Cray, P.J. (1983) Production of cholerae

toxin-like toxin produced by Vibrio mimicus and non-01 Vibrio

cholerae: batch culture conditions for optimum yelds and isolation of

hypertoxigenic lincomycin-resistant mutants. Infection and Immunity

42, 501±509.

Twedt, R.M. (1989) Vibrio parahaemolyticus. In Foodborne Bacterial

Pathogens ed. Doyle, M.P. New York: Marcel Dekker.

Varnam, A.H. and Evans, M.G. (1991) Foodborne Pathogens. An

Illustrated Text pp. 185±200. London: Wolfe.

WHO (1999) Cholera, 1998. Weekly Epidemiological Record 74,

257±264.

Yamamoto, K., Takeda, Y., Miwatani, T. and Craig, J.P. (1982)

Puri®cation and some properties of a non-01 Vibrio cholerae

enterotoxin that is identical to cholera enterotoxin. Infection and

Immunity 39, 1128±1135.

Yamamoto, K., Al-Omani, M., Honda, T., Takeda, Y. and Miwatani,

T. (1984) Non-01 Vibrio cholerae hemolysin: puri®cation, partial

characterization, and immunological relatedness to El Tor hemol-

ysin. Infection and Immunity 45, 192±196.

VIBRIOS IN MUSSELS AND SEAWATER 61


Documents

Vibrio