Microbiological controls across the productive cycle of

Dicentrarchus labrax L. and Sparus aurata L.: a study

from the environment to the final product

G Caruso1, G Maimone1, M Mancuso1, A Modica2, L Genovese1

1National Research Council, Istituto per l’Ambiente Marino Costiero, Section of Messina, Messina, Italy2Centro Oceanologico Mediterraneo, CEOM, Palermo, Italy

Correspondence: G Caruso, National Research Council, Istituto per l’Ambiente Marino Costiero, Section of Messina, Spianata S. Raineri

86,98122 Messina, Italy. E-mail: caruso@ist.me.cnr.it

Abstract

The microbiological quality of water, sediments andseafood products of three Sicilian marine ¢sh farms(Castellammare Gulf, Capo d’Orlando and Porto Palo)was investigated inorder to drawacomplete picture ofthe health conditions from the start (environment) tothe end (seafood product) of the productive cycle. Be-fore the beginning of ¢sh farming, lowconcentrationsof faecal contamination indicators (faecal coliforms,Escherichia coli, enterococci) were found in the waterof each examined area. Due to the enhanced organicload released fromcages, the set-up of ¢sh farming sig-ni¢cantly altered the distribution of faecal indicatorsand sometimes that of halophilic vibrios in the pelagiccompartment. Signi¢cant di¡erences in the density ofheterotrophic bacteria were sometimes recorded atthe sediment level. Despite this increase in microbialabundance, the microbiological conditions remainedacceptable for the productive process. Pathogens (Sal-monella spp.,Vibrio parahaemolyticus, Listeria monocy-togenes, Staphylococcus aureus) were mostly absentin seafood products. The study underlines that theachievement of good quality levels in aquaculturestrongly depends on the conformity of the rearing en-vironment to qualitative microbiological standards,highlighting the importance of sanitary controlsalong the di¡erent steps of the productive cycle.

Keywords: mariculture, microbiology, water,sediment, quality, sea bass, sea bream

Introduction

Aquaculture represents an important source ofhighly nutritional proteins for human feeding. The

recent expansion of productive initiatives has re-sulted in an increasing interest in the assessment oftheir potential impact on aquatic ecosystems (Bever-idge, Ross & Kelly 1994; Papoutsoglou, Costello, Sta-mou & Tziha 1996; La Rosa, Mirto, Marino, Alonzo,Maugeri & Mazzola 2001). The large biodeposition ofpseudo-faeces, faeces and feed wastes from maricul-ture may potentially produce negative e¡ects on thecomposition, structure and productivity of both pela-gic and benthic communities. For aquaculture to besuccessful, activities must not cause unacceptablechanges in water quality and not clash with otheruses (tourism, industry and urban) of the coastalareas.This is an issue extensively reported in the stu-dies aimed at evaluating the impact of ¢sh farmingon coastal zones. However, there is little informationrelating aquaculture to water quality degradation(Karakassis,Tsapakis & Pitta 2001).The health risks associated with the consumption

of low-quality seafood makes the assessment andcontrol of food safetyand qualityanother topic of glo-bal concern in aquaculture. Microbiological controlshave to be addressed ¢rstly to the environmentsdesignated to mariculture, to adequate ¢sh-farm ma-nagement practices and ¢nally to educational pro-grammes for the consumers (Liston 1990; Reilly &Kaferstein1999). Application of preventive measurescan result, in turn, in the improvement of productione⁄ciency and reduction in costs.The relationship linking the hygienic-sanitary

quality of the seafood products to the quality of therearing sites has been widely documented (Reilly &Kaferstein 1999; WHO 1999). Therefore the monitor-ing of productive areas to comply with microbiologi-cal standards has been recognized as a fundamental

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pre-requisite to obtain high-quality products (Caruso,Zaccone, Genovese & Crisa¢ 1998). The application ofseveremicrobiological inspections along the di¡erentsteps of the production line, from rearing to harvest-ing, processing and marketing of ¢sh-farm products,is imposed by the Italian directives Decreto Legislati-vo 131/92 1992a; Decreto Legislativo 530/92 1992b;Decreto Legislativo 531/92 (1992c).In the framework of a research contract funded by

Centro OceanologicoMediterraneo-Palermo, amulti-disciplinary studyhas been carried out by the Istitutoper l’Ambiente Marino Costiero-Messina on three Si-cilian areas [Gulf of Castellammare (Northern Tyr-rhenian Sea), Capo d’Orlando (Tyrrhenian coast,between Messina and Palermo) and Porto Palo(south-eastern side of the Sicily)] designated for mar-iculture, in order to verify themicrobiological qualityof the environment and of ¢nal products ready fordistribution. The only available report on environ-mental health conditions in the studied areas con-cerned the Gulf of Castellammare, where a 3-yearinvestigation (Caruso et al. 1998; Genovese, Caruso,La Ferla, Leonardi, Giacobbe, Maimone, Micale & Zac-cone 1998) showed the presence of low levels ofmicrobial contamination with respect to faecal indi-cator and potentially pathogenic vibrios. In contrast,for the other ¢sh-farming areas included in this in-vestigation, there is a lack of speci¢c informationabout the microbiological conditions of the watersand on the extent of the potential hazard for humanhealth consequent to their use as aquaculture sites.

Materials and methods

Water

Surface samples were collected from a total of eightstations, of which four were indicated as cage sites,and the others as control blanks, located at about1km from the ¢sh farm. A total number of 48,30 and16 samples were drawn from Castellammare Gulf,Capo d’Orlando and Porto Palo respectively. One litreof each sample, aseptically collected with a Niskinbottle, was used to enumerate faecal coliforms (FCs)and enterococci (ENT), by membrane ¢ltration of sui-table volumes (10, 100mL) of sample and incubationat 44.5 1C for 24 handat 35 1C for 48 h into the selec-tive culture media (m-FC added with 1.5% agar andSlanetz and Bartleymedium (Oxoid, Hampshire, Eng-land) respectively). Colonies grown on m-FC agarwere con¢rmed to be Escherichia coli by indole test(Decreto Presidente della Repubblica 470/821982).

The search for Pseudomonas aeruginosa and Aero-monas hydrophila strains was carried out by ¢ltering10 and100mL of sample on 0.45-mmMillipore mem-branes, and by incubating the ¢lter on the respectivemedia P. aeruginosa selective agar (Oxoid) and A. hy-drophila medium (Oxoid) added with Ampicillin se-lective supplement (Oxoid) at 35 1C for 24^48 h. Asan indicator of the trophic conditions of the environ-ment, the viable heterotrophic aerobic £ora able togrow at 20 1C and 35 1C was estimated on Marineagar (Difco, BD Franklin Lakes, NJ, USA) platesincubated for 7 days. The density of total halophilicvibrios growing at 20 1C was determined by mem-brane ¢ltration and incubation of the ¢lters on TCBSagar (Difco) plates added with 2% sodium chloride.

Sediment

From the same stations described above, at the sametime as water sampling, sediment samples were col-lected using a Box corer; surface subsamples weretaken with a sterile polypropylene container andmaintained at15 1C until assayed. The treatment ofthe sediments for the bacteriological analyses in-cluded the homogenization of 1g of each sample insterile seawater (1:10w/v) and seeding into culturemedia of the supernatant obtained. The density ofheterotrophic aerobic bacteria was determined byspreading into Marine agar plates (Difco) incubatedat 20 1C for 7 days (Cavallo, Rizzi, Vozza & Stabili1999). For the determination of Clostridium perfrin-gens, suitable volumes (0.1mL) of serial dilutions fromthe initial supernatant were poured into the selectivemedium, Perfringens selective base (Oxoid), addedwith supplements A and B (Oxoid), and allowed toresolidify. Plates were incubated at 35 1C for 24 and48 h in AnaeroGen Compact System (Oxoid). Countswere expressed in terms of colony forming units(CFU) per gram of sediment (CFUg�1), expressed indry weight.

Fish

Five specimens of ¢sh (Dicentrarchus labrax L. andSparus aurata L.) achieving the commercial size werealso sacri¢ced. Precautions were taken in asepticalsurgery of each sample to avoid contamination of ¢l-lets with bacteria coming from other sites (skin andintestine). After homogenization for15min in sterilephysiological saline solution of small amounts (from2 to 5 g) of sample through Stomacher Lab Blender80 (Seward Medical Uac House, London, England),

Aquaculture Research, 2004, 35, 184^193 Microbiological controls in Sicilian ¢sh farming G Caruso et al.

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decimal dilutions in the same liquid medium wereperformed and inoculated in di¡erent media accord-ing to the parameter to be tested, as reported below.Total bacterial counts were estimated after inocu-

lation by spreading of Plate Count Agar (Oxoid) platesand incubation at 20 1C and 32 1C.To estimate the E. coli density, the Petri¢lm System

(3M Microbiology Products, St Paul, MN, USA) wasused (De W. Blackburn, Baylis & Petitt 1996); seeded¢lms were further incubated at 35 1C for 24 h.The qualitative search of Salmonella spp. was car-

ried out by pre-enriching 25 g of sample in bu¡eredalkaline peptone water at 35 1C for 18^24 h, and en-riching in Rappaport Vassiliadis Broth (Oxoid) andTetrationate Broth (Oxoid) addedwith an iodine solu-tion (ICN Pharmaceuticals, Costa Mesa, CA, USA) in-cubated for 24^48 h at 42 1C and 35 1C respectively.Isolation was performed on plates of Salmonella-Shi-gella (SS, Oxoid) and Hektoen enteric agar (Oxoid) se-lective culturemedia. All the colonies presumed to beSalmonella spp. (lactose not fermenting and hydrogensulphide producing) were biochemically character-ized through inoculation in Triple Sugar Iron agar(Difco) tubes. Typical strains were further identi¢edby using API 20E (Biomerieux, Marcy l’Etoile,France) strips incubated at 35 1C for 24 h.To enumerate Staphylococcus aureus, plates of se-

lective Baird Parker medium (Oxoid) containing egg-yolk tellurite emulsion were inoculated in duplicatewith decimal dilutions of the sample homogenized,and incubated at 35 1C for 24^48 h; all the grey-black, tellurite-reducing colonies surrounded by aclear zone were tested for coagulase production byusing the agglutination Staphylase (Oxoid) test.Presence of strains belonging to the speciesVibrio

parahaemolyticus was detected by both the enrich-ment of 25 g of sample in alkaline peptone water (pH8.6) incubated at 35 1C for 6^18 h and the inocula-tion of selective Salt Polymixin Broth (De Medici, Fe-nicia, Ore¢ce & Stacchini1996) incubated at 35 1C for8 h. Isolation on TCBS agar (Difco) plates added with2% NaCl at 35 1C yielded to presumedV. parahaemo-lyticus colonies, of which at least four per plate weretested for sensitivity to the vibriostatic agent O129(2,4-diamino 6,7-di-isopropylpteridine, 150-mg diag-nostic discs, Oxoid) in order to distinguishVibrio fromAeromonas belonging strains.To determinate Listeria monocytogenes, serial dilu-

tions of the sample homogenized were enriched byinoculation in triplicate tubes of the selective Fraserbroth (Oxoid) in the most probable number (MPN)method, according to the procedure reported by the

Ordinanza Ministeriale 7/12/93 (1993) for the quan-titative determination of L. monocytogenes. After in-cubation at 35 1C for 24^48 h, the presumed positivecultures were further inoculated on Oxford agar (Ox-oid) plates incubated at 35 1C for 24 h, to isolate thepathogen. Among the suspected colonies showing adark pigmentation ¢ve were randomly selected andassayed for hemolysin production on Columbia agarbase (Oxoid) added with 5% sheep erythrocytes (ICNPharmaceuticals). Strains positive for Gram staining,beta hemolysis and catalase production, and negativefor oxidase, were further subjected to biochemicalidenti¢cation using API Listeria (Biome¤ rieux) strips.In order to follow microbiological alterations oc-

curring during refrigeration, the above-cited micro-biological analyses were repeated at selected timeintervals, namely at 1, 3, 9, 12, 15 days after sampleconservation at15 1C.

Results

Water

The mean values of bacteriological parameters re-corded before and after the starting of maricultureexperiment, at cage and control stations separately,are shown in Figs 1^3. In the Castellammare Gulf(Fig. 1a), after the introduction of ¢sh into the cages(March, May and July 2001), an increase in the con-centration of all faecal indicators, dependent on therelease of faecal pellets, was recorded at cage stationsonly, where all faecal bacterial indicators were no-ticed at very low densities before ¢sh farming (March2000). Statistical analysis by ANOVA test con¢rmedthat highly signi¢cant di¡erences occurred in rela-tion to time (before and after ¢sh farming) and site(cage and control stations) for FCs (F57.65 and7.81, Po0.05 respectively), E. coli (F57.75 and 7.93,Po0.05 respectively) and ENT (F520.76 and 25.48,Po0.01respectively).Compared with the values obtained before the be-

ginning of productive processes, the abundances ofheterotrophic bacteria (Fig. 1b) also increased, al-though not signi¢cantly, both in the fractiongrowingat 20 1C (from 2.22 � 103 to 2.24 � 103 CFUmL�1,at cages, and from1.17 � 103 to1.48 � 103 CFUmL�1

at control stations) aswell as at 35 1C (from1.51 � 103

to1.59 � 103 CFUmL�1at cage stations only).Mariculture activity a¡ected also the fraction of

halophilic vibrios (Fig. 1c), although temporal andspatial variations in their mean values were notstatistically signi¢cant. After ¢sh rearing (July 2001)

Microbiological controls in Sicilian ¢sh farming G Caruso et al. Aquaculture Research, 2004, 35, 184^193

186 r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 184^193

their initial mean concentrations (9.19 � 102 and2.26 � 102 CFU100mL�1 at cage and control sta-tions, respectively, before ¢sh farming) had increased,reaching 1.08 � 103 and 7.06 � 102 CFU100mL�1

at cage and control stations respectively.Vibrio genusrepresented a considerable fraction within the bac-terial heterotrophic £ora, ranging on average from

65.74 to 98.38% and from 33.50 to 86.87% before andafter cage installation respectively. Microorganismspresumed to be P. aeruginosa and A. hydrophila

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Figure 1 Water samples. Mean values of: (a) faecal pollu-tion indicators (faecal coliforms, FCs; Escherichia coli, E.coli; enterococci, ENT), (b) heterotrophic bacteria (MA)and (c) presumptive vibrios (VPP) and bacteria potentiallypathogenic to reared ¢sh (Aeromonas hydrophila, Pseudo-monas aeruginosa) found in Castellammare Gulf.

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Figure 2 Water samples. Meanvalues of: (a) faecal pollu-tion indicators (faecal coliforms, FCs; Escherichia coli, E.coli; enterococci, ENT), (b) heterotrophic bacteria (MA)and (c) presumptive vibrios (VPP) and bacteria potentiallypathogenic to reared ¢sh (Aeromonas hydrophila, Pseudo-monas aeruginosa) found in Capo d’Orlando.

Aquaculture Research, 2004, 35, 184^193 Microbiological controls in Sicilian ¢sh farming G Caruso et al.

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(Fig. 1c) were present throughout the study periodwith a mean density of about102 CFU100mL�1.In the waters of Capo d’Orlando, following ¢sh in-

troduction, both FCs and E. coli (Fig. 2a) displayed

only a slight increase with respect to that recordedin Castellammare Gulf, but di¡erences between cageand control stations were statistically signi¢cant(F55.39, Po0.05). Di¡erences between stationswere detected by ANOVA values before mariculturestarting only (F511.14, Po0.01).After mariculture experiments, mean concentra-

tions of heterotrophic bacteria (Fig. 2b), one order ofmagnitude lower than those of Castellammare area,increased not only at cage (from 1.6 � 102 to4.91 � 102 CFUmL�1and from1.36 � 102 to 2.61 �102 CFUmL�1, for the fraction growing at 20 1C and35 1C respectively) but also at control stations (from2.66 � 102 to 5.74 � 102 CFUmL�1and from 2.26 �102 to 6.86 � 102 CFUmL�1).The halophilic vibrios (Fig. 2c), initially occurring

in low concentrations (5.3 � 101 and 4.8 � 101

CFU100mL�1 at cage and control stations respec-tively), exceeded 102 CFU100mL�1 at both sitesafter cage installation. The low values recorded fortrophic-related bacterial parameters (heterotrophicbacteria and vibrios) suggested the low availabilityof organic matter in this area with respect to the for-mer; in thesewaters vibrios represented a moderatelyabundant fraction of the total heterotrophic £ora,ranging on average from11.40 to 50.69%, before ¢shfarming, and from17.59 to 75.07% after ¢sh farming.ANOVA results showed that ‘site’ (cages versus controlsF59.84, Po0.05) and ‘time’ (before versus after,F519.11, Po0.01) variables accounted for variancein the distribution of halophilic vibrios at cage sta-tions. Like heterotrophic bacteria, temporal di¡er-ences were also extended to control stations (F5

8.40, Po0.05).Presumed P. aeruginosa strains were recovered in

concentrations lower than those observed in Castel-lammare waters, without any signi¢cant temporalvariation (Fig. 2c). A. hydrophila displayed signi¢cantvariations in relation to the site sampled (F523.89,Po0.01).The waters of Porto Palo were characterized in

both samplings (before, July 2000, and after ¢shfarming, July 2001) by mostly negative values of FCsand E. coli, while ENT did not exceed 15 CFU100mL�1 (Fig. 3a). After ¢sh farming, mean densi-ties of heterotrophic bacteria growing at 20 1C in-creased both at cage and control stations (from1.01 � 103 to 1.59 � 103 CFUmL�1 and from 1.13 to2.76 � 103 CFUmL�1, respectively, Fig. 3b). Also ha-lophilic vibrios (Fig. 3c) almost doubled in number,reaching mean values of 6.25 � 102 and 1.23 �103 CFU100mL�1 at cage and control stations

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Figure 3 Water samples. Meanvalues of: (a) faecal pollu-tion indicators (faecal coliforms, FCs; Escherichia coli, E.coli; enterococci, ENT), (b) heterotrophic bacteria (MA)and (c) presumptive vibrios (VPP) and bacteria potentiallypathogenic to reared ¢sh (Aeromonas hydrophila, Pseudo-monas aeruginosa) found in Porto Palo.

Microbiological controls in Sicilian ¢sh farming G Caruso et al. Aquaculture Research, 2004, 35, 184^193

188 r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 184^193

respectively. They constituted 3.60^96.91% and1.15^100% of the total heterotrophic £ora, respectively, be-fore and after ¢sh farming. After mariculture begin-ning, the abundance of P. aeruginosa decreased (Fig.3c), with signi¢cant temporal di¡erences at controlsites (F512.31, Po0.01). At cages, the concentrationof A. hydrophilawas low and similar to that recordedin Capo d’Orlando (Fig.3c).

Sediment

Before mariculture, heterotrophic bacteria in the se-diments (Fig. 4a) showed mean concentrations vari-able from 9.43 � 103 (control)^1.54 � 104 (cage)CFUg�1 found in Castellammare Gulf to 3.53 � 103

(control)^1.85 � 103 (cage) CFUg�1 in Capo d’Orlan-do and to 3.65 � 103 (control)^3.0 � 103 (cage)CFUg�1 in Porto Palo.With the beginning of mari-culture activity, their concentrations increased byabout four times under the cages (cages versus con-trols, F528.57, Po0.01) in Castellammare Gulf,

while no signi¢cant increases were recorded in Capod’Orlando (on average, from1.85 � 103 to 6.03 � 103

CFUg�1). As previously observed in waters, in thisarea bacterial densities were initially one order ofmagnitude lower than that recorded in Castellam-mare Gulf, re£ecting a lower trophic content.Microorganisms identi¢ed to be C. perfringens (Fig.

4b), which grow well in environments with low Ehredox values, occurred at higher densities in Castel-lammare area (mean 5.5 � 101, at control,1.38 � 102

CFUg�1, at cage) and showed a signi¢cant decrease(F59.56, Po0.05) after ¢sh farming. In Capo d’Or-lando and Porto Palo they were detected in lowerconcentrations, ranging in the same order of magni-tude (0.4 � 101, cage ^ 3.6 � 101CFUg�1, control;0, control ^ 3.4 � 101CFUg�1, cage respectively).Increases recorded in Porto Palo in the counts of

heterotrophic bacteria (from 3.0 � 103 to 4.1 � 103

CFUg�1) and C. perfringens (from 3.4 � 101to 2.14 �102 CFUg�1) were not statistically signi¢cant.

Fish

Results of quantitative determinations of the micro-bial £ora present in the samples of food examinedare shown in Table 1. They refer to two di¡erent spe-cies (D. labrax L. and S. aurata L.) and to two sites only,Castellammare Gulf and Porto Palo, as productiveprocesses were stopped in the third site (Capo d’Or-lando). In sea bass (D. labrax) reared in Castellam-mare, the total microbial £ora was in the order of103 CFUg�1 during the ¢rst day of sampling, whilelower densities (102 CFUg�1) were found in the spe-cimens reared at Porto Palo both at 20 1C and 35 1C.Values obtained suggested that the microbiologicalquality levels of the sea bass specimens reared at Cas-tellammare were acceptable within the ninth dayfrom sampling. In contrast, in the specimens of seabass reared at Porto Palo, the deterioration of the mi-crobiological quality had occurred earlier, by thethird day. Bacterial concentrations increased to 108

and109 CFUg�1after12 days of storage.In the specimens of sea bream (S. aurata) reared in

Castellammare Gulf, total bacteria counts werehigherthan in sea bass of the same site, displayingonthe ¢rstsampling daydensities equal to104 and103 CFUg�1offood at 20 1C and 35 1C respectively. During samplestorage at 15 1C, bacterial concentrations increasedprogressively, reaching peaks of 108 CFUg�1 of food,both at 20 1C and 35 1C, after15 days of refrigeration.Deterioration in quality levels occurred mainly be-tween the third and the ninth day of storage.

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Aquaculture Research, 2004, 35, 184^193 Microbiological controls in Sicilian ¢sh farming G Caruso et al.

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No strains of E. coli, V. parahaemolyticus and L.monocytogenes were isolated from the sea bass andbream specimens analysed. In ¢sh samples collectedfrom Castellammare, no S. aureus and Salmonella spp.strains were isolated.In the samples coming from Porto Palo rearings,

negative results were obtainedwith respect to Salmo-nella, while two strains identi¢ed as S. aureus wereisolated after15 days of storage. During the ¢rst sam-pling, two strains con¢rmed to be L. monocytogeneswere also isolated, and the recovery of a higher num-ber (seven strains) of this microorganism was re-corded after15 days of refrigeration only.The low levels of saprophytes recorded and the

non-recovery of bacteria such as Enterobacteriaceaeand S. aureus suggested that samples examined didnot represent potential risks for consumers with re-spect to current regulations in microbiology of ¢sh-ery products. These indicate the guideline values of102 CFUg�1for S. aureus,11g�1for L. monocytogenesand the absence of Salmonella strains.

Discussion

The values of the microbial variables obtained beforethe set-up of ¢sh farming showed that the selected

areas were in compliance with the microbiologicalwater quality standards for productive purposes.Mariculture activity was responsible for a signi¢cantincrease in faecal pollution indicators (FCs, E. coli andENT) in the waters; despite this, the samples exam-ined showed bacterial counts that fell within thethreshold levels (E. coli: 2 CFU100mL�1) recom-mended by Decreto Legislativo 131/92 (1992a) forthe water considered as ‘acceptable’ for shell¢sh cul-ture. The low densities of P. aeruginosa found in thewater also con¢rmed that ¢sh farming caused limitedalterations in environmental quality.The impact of ¢sh rearing, even if not signi¢cant,

was sometimes extended not only to cage stations,but also to the control ones; this suggested awide im-pact of mariculture activity over the area or the pre-sence of local organic inputs in sites di¡erent fromthose directly concerned with the cage.The viable bacterial concentrations found in the

waters examined varied in the same range asreported in oligotrophic ecosystems (102^103 CFUmL�1); these low bacterial densities depended alsoon the geographic position of sampled sites, far fromthe organic inputs drawn from coastal zone. In parti-cular, Pseudomonas spp., common both in seawaterand sediments of temperate or tropical zones (Paller-oni 1984; Stolp 1988; Cavallo et al. 1999), are charac-

Table 1 Periodical microbiological seafood controls performed on sea bass (Dicentrarchus labrax) and sea bream (Sparusaurata) specimens of Castellammare Gulf and Porto Palo

Days a b c d e f g

Castellammare Gulf

D. labrax

1 2.22E103 2.00E103 0 0 0 0 0

3 3.20E103 1.00E103 0 0 0 0 0

9 3.86E107 4.03E107 0 0 0 0 0

12 1.61E108 1.97E108 0 0 0 0 0

15 1.85E108 2.01E108 0 0 0 0 0

S. aurata

1 1.63E104 1.17E103 0 0 0 0 0

3 1.01E105 1.57E104 0 0 0 0 0

9 7.85E107 6.69E107 0 0 0 0 0

12 4.55E107 1.07E108 0 0 0 0 0

15 1.65E108 1.17E108 0 0 0 0 0

Porto Palo

D. labrax

1 6.40E102 4.00E102 0 0 1 0 0

3 2.61E106 2.85E107 0 0 0 0 0

9 4.60E106 6.30E106 0 0 0 0 0

12 2.86E108 1.31E108 0 0 0 0 0

15 1.19E109 5.65E108 0 2 7 0 0

Total bacterial counts, as estimated on Plate Count Agar at 20 1C (a) and 32 1C (b), and results of the search for Escherichia coli (c),Staphylococcus aureus (d), Listeria monocytogenes (e), Salmonella spp. (f) andVibrio parahaemolyticus (g) are reported.

Microbiological controls in Sicilian ¢sh farming G Caruso et al. Aquaculture Research, 2004, 35, 184^193

190 r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 184^193

terized by a wide metabolic versatility and toleranceto a wide variety of physical conditions, thus beingable to colonize ecological niches where nutrientsare limited. Some strains belonging to this genus areopportunistic pathogens causing disease in suscepti-ble (immunocompromised) ¢sh (Austin, Garges &Conrad1979).Besides the species of Vibrio that are saprophytes

and usual inhabitants of the marine environment(Montanari, Pruzzo, Pane & Colwell 1999), some spe-cies are con¢rmed pathogens, being involved in ¢sh(V. alginolyticus and V. anguillarum) and human (V.cholerae non-O1,V. parahaemolyticus,V. vulni¢cus) dis-eases (De Paola, Hopkins, Peeler,Wentz & McPhear-son 1990). The frequent occurrence of potentiallypathogenic species in marine environments (Bar-bieri, Falzano, Fiorentini, Pianetti, Ba¡one, Fabbri,Matarrese, Casiere, Katouli, Kˇhn, M˛llby, Bruscolini& Donelli 1999; Ripabelli, Sammarco, Grasso, Fanelli,Caprioli & Luzzi 1999) whose abundance does notcorrelate with FC concentrations (Caruso et al. 1998)support the need to include the qualitative analysis ofthese bacteria in monitoring programmes of aqua-culture sites, to achieve a more complete evaluationof microbiological quality.Microbiological controls in aquaculture have to be

addressed also to some motile Aeromonas species,such as A. hydrophila, that in recent years have beeninvolved in the transmission of diseases to reared ¢sh(Bartoli, Monetti & Licciardi 1997). In contrast, risksfor human health coming from Aeromonas spp. infarmed ¢sh are considered low (WHO1999).With respect to the benthic environment, the sig-

ni¢cant increase in heterotrophic bacteria detectedin the sediments of most of ¢sh-farming sites, sup-ported their potential as a sensitive biomarker of or-ganic enrichment. The stimulating e¡ect of organicloads over the heterotrophic fraction of bacterio-plankton has also been shown by La Rosa, Mirto, Fa-valoro, Savona, SaraØ , Danovaro & Mazzola (2002) inthe sediments of a ¢sh-farming coastal area of theTyrrhenian Sea. In Castellammare Gulf, the higherincrease in heterotrophic bacterial counts with re-spect to that found in water suggested that thebenthic environment su¡ered the major impact ofthe modi¢ed environmental conditions, in conse-quence of a heavier organic enrichment of theecosystem. In comparison with the other areasexamined, the higher numbers of C. perfringensdetected in Castellammarewas indicative of a remotepollution. In Capo d’Orlando sediments, the lack ofsigni¢cant di¡erences due to ¢sh farming in the

abundance of both heterotrophic bacteria and C. per-fringens led us to conclude that the mariculture ex-perience had not favoured bacterial growth in thisarea, which kept a low trophism condition.As far as the quality of ¢sh-farming products is

concerned, it is known that the greatest number ofseafood-associated illnesses are associated with con-sumption of raw ¢sh harvested in waters contami-nated with raw or poorly treated human wastes(Ahmed 1991; Hackney & Pierson 1994); the qualita-tive pro¢le of micro£ora associated to reared ¢shundergoes normal changes during storage. Fishspoilage may be slowed down through maintenanceat low temperature, on ice or in refrigerating contain-ers, but good practice procedures from the moment¢sh are captured up to their ¢nal marketing repre-sent the best tool for food preservation. In thesamples of ¢sh examined, total bacterial density in-creased with time, probably as a consequence of theremarkable growth rate that vibrios are known to ex-hibit in raw ¢sh, even kept at low temperatures. Va-lues recorded in our investigation, however, fellwithin the International Commission on MicrobialSpeci¢cations for Foods (ICMSF 1986) standard va-lues for fresh ¢sh products (5 � 105^107 CFUg�1).Sowe considered the quality of aquaculture productswithin 6 days of storage at 5 1C acceptable. After theninth day of refrigeration, total bacterial concentra-tions increased, with evident organoleptic (taste and£avour) changes. For the enumeration of E. coli, thePetri¢lm System, alreadyaccepted by theAssociationof O⁄cial Analytical Chemists (AOAC 1995) for foodanalysis, con¢rmed to be a reliable alternative to tra-ditional standard techniques.The negative result obtained in the ¢rst sampling

with the search for L. monocytogenes andV. parahae-molyticus strains con¢rmed the good microbiologicalstandards of farmed products and the limited distri-bution of such a species in coastal Sicilian waters, aspreviously reported by Scoglio, Di Pietro, Mauro,Picerno, LaganaØ & Delia (2000).

Conclusions

Our study underlines the importance of environmen-tal monitoring as a fundamental step in aquaculture,providing assurance of good quality of seafood pro-ducts. In Italy the problem of the safety of seafoodproducts has only recently received adequate atten-tion and, in particular, the need to de¢ne qualitystandards for national ¢sh production has become

Aquaculture Research, 2004, 35, 184^193 Microbiological controls in Sicilian ¢sh farming G Caruso et al.

r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 184^193 191

more and more urgent (Giulini, Mietti, Rambaldi,Priore & Serra 1998). Contamination of aquacultureproducts by pathogens may be due to the indigenousbacterial £ora of the environment (A. hydrophila,V.parahaemolyticus,Vibrio vulni¢cus, L. monocytogenes)or to bacteria from allochthonous inputs (entericbacteria introduced into the sea fromanimal and hu-man wastes) (Huss 1994). Hazards may also arisefrom the bacterial contamination of food as a resultof poor hygienic standards during handling and pro-cessing (WHO1999).The low contamination found in our seafood sam-

ples re£ect the global quality of water ¢sh farms;therefore, our study con¢rms that good ¢sh-farmmanagement practices may control many of the po-tential hazards at the production level and conse-quently reduce the risks of outbreaks of human and¢sh diseases.

Acknowledgments

This work has been supported by Parco Scienti¢co eTecnologico della Sicilia funds in the framework ofthe CEOM Project 5022 SPE-A120-001.0 (‘Develop-ment of techniques and technologies of maricoltureo¡-shore. Environmental impact assessment. Sea-food quality’).

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