Microbiological controls in polyculture farming: A pilot case study in the Castellammare Gulf (Sicily)

Citation: Caruso G, Caruso R, Sarà G (2020) Microbiological controls in polyculture farming: a pilot case study in the Castellammare Gulf (Sicily). J Clin Microbiol Biochem Technol 6(1): 014-028. DOI: https://dx.doi.org/10.17352/jcmbt.000039 economic growth, that can lead to social benefi ts in terms of new occupational perspectives [1]. Productive activities, however, have well-known impacts on the environment, mainly due to the release of feed wastes, chemical and pharmaceuticals (i.e. Introduction

The severity of aquaculture impacts varies in relation to the reared species, local environmental conditions, farming and management techniques. Future developments in the aquaculture fi eld depend on the selection of rearing practices able to meet the principles of environmental sustainability [3,4]. In this context, particular interest has recently been addressed to to Integrated Multi-Trophic Aquaculture (IMTA) systems, where organisms belonging to different trophic levels and inhabiting different ecological niches are co-cultured. IMTA includes several aquaculture practices based on the complementarity of different productive compartments. By IMTA, inorganic and organic wastes from fi nfi sh are assimilated respectively by autotrophic (i.e. phytoplankton, macroalgae, plants) and heterotrophic species (e.g. oysters, mussels, sea cucumbers) that are co-cultured with the target reared species. It can represent a key alternative in the evolution of aquaculture systems, allowing to face a double challenge: sustaining the growing demand of aquatic products and preserving the environment by reducing wastes [5 ,6]. IMTA has also been suggested as a possible ecofriendly strategy to mitigate the effects of multiple stressors [7].
In Asian Countries, the integrated cultivation of fi sh with organisms of different trophic levels, as well as the rearing of both shellfi sh and seaweed in lagoons or bays close to fi sh farming structures is an old practice [8,9]. In China, the polyculture systems with the macroalga Eucheuma gelatinae and the bivalve Gafrarium tumidum provided a suitable tool for the purifi cation of eutrophic seawater and control of algal bloom [10]. In other Countries, such as Canada, Reid, et al. [11], reviewed shellfi sh production in the context of open-water IMTA; later, Chopin [12] underlined the advantages of IMTA systems, able to increase economic profi tability per cultivation unit through co-cultivation of many species (fi sh, seaweeds, invertebrates), characterized by environmental sustainability and societal acceptability. More recently, Buck, et al. [13], have provided an in-depth review of the main variables affecting IMTA and the potential benefi ts/limitations of offshore mariculture plants. The feasibility of integrated fi sh, shellfi sh and/or seaweed aquaculture within offshore wind farming areas has been reported to be affected by factors such as biological feasibility, technological implementation, environmental sustainability and economic feasibility of the farming systems.
In Europe, Alexander, et al. [14], studied the main incentives and barriers to the development of IMTA, highlighting that moving from IMTA at a pilot scale to commercial scale developments requires changes in policy and legislation to promote innovation and technologies for aquaculture and simplifi cation of the regulations regarding licensing or spatial planning for aquaculture. Although attention to IMTA systems is more recent compared to Asia, in Europe integrated polyculture production still faces inherent diffi culties which limit the adoption of this practice across this Continent [15]. In Italy integrated polyculture of fi sh and low-trophiclevel organisms such as shellfi sh is a still scarcely exploited practice [16]. Among shellfi sh, mussels, oysters and clams are organisms highly appreciated by consumers; oysters are commonly cultured in shallow waters or in intertidal zones near estuaries, while mussels and clams are cultured also in brackish inland ponds. Although the impacts on the environment of bivalve cultivation are well known in terms of local benthic (i.e. physical disturbance, changes in sediment topography and sedimentation, accumulation of debris, biodeposition) and water column effects (alteration in water quality and nutrient cycling) as well as of wider ecological effects (on fi sh, seabirds, transmission of diseases) [17], the effects of bivalve cultivation in polyculture have been underestimated [18]. The limited knowledge of growth performance of the reared organisms explains the delayed success of IMTA experiences and of the economic performance of this practice. Polyculture experiences have proliferated in recent years [15], nevertheless studies regarding the microbiological implications of polyculture systems are not available yet. Microbiological controls in aquaculture systems play a crucial role to prevent possible transmission to man of pathogenic bacteria or toxins via food consumption [19][20][21][22][23]. Compliance to high quality microbiological criteria is particularly important in shellfi sh farming areas, in relation to the fi ltering-feeding behavior of these organisms that results in the concentration of bacteria and polluting substances inside the animal body. Consequently, hygienico-sanitary monitoring of shellfi sh microbiological quality must be extended also to the waters of the farming environments [24][25][26][27].
The Gulf of Castellammare (Trapani, Sicily) is a 70 Km wide coastal area that extends along the Tyrrhenian coast between Cape S. Vito and Terrasini. Thanks to its particular morphological and hydrological characteristics -which ensures an effi cient water exchange and low urban and industrial loading -this area has traditionally been designed for mariculture purposes. Some reports on the microbiological conditions of this ecosystem are those available from Caruso, et al. [28][29][30]; nevertheless, knowledge of the health risks related to aquaculture farming is still scarce. In the framework of a pilot IMTA experiment of mussels and fi sh co-cultivation, an investigation of the bacteriological quality of shellfi sh products was undertaken. With the aim of assessing the microbiological quality of both shellfi sh and environment, the bacterial indicators of faecal pollution (faecal coliforms, Escherichia coli and Salmonella spp.) as well as heterotrophic microfl ora (marine and not marine bacteria), autochthonous bacteria (Vibrio spp. and their potentially pathogenic members) were estimated. In addition, the expression of hydrolytic enzymes (caseinolytic, lipolytic, haemolytic and lecithinolytic activities) as presumptive virulence factors involved in the pathogenicity mechanisms of bacteria, was also studied.

Study site
The Castellammare Gulf was chosen as a polyculture site due to the intense aquaculture practiced in this area; the wastes released by productive plants such as faecal pellets and food residuals cause the organic enrichment of the waters [29].
Citation: Caruso  In this context, shellfi sh farming could represent a suitable tool to convert the trophic inputs into biomass production. The study area was located 3 miles offshore Balestrate, where the polyculture pilot plant was set up (Figure 1). The geographical coordinates of the sampling sites are reported in Table 1.
The experimental design of integrated cultivation consisted of fi ve submersible cages (Farmocean, Sweden; volume = 4,500 m 3 ) and 6 smaller cages (volume = 1,000 m 3 ) fi lled with seabass (Dicentrarchus labrax) and seabream (Sparis aurata) for a total annual production of about 600 tons of biomass. All the species selected for the study were bivalves highly appreciated due to their high nutritional value: Japanese oyster (Cassostrea gigas), European oyster (Ostrea edulis), common mussel (Mytilus galloprovincialis), Philippine clam (Tapes philippinarum), which belong to Ostreidae (Ostrea genus), Mytilidae (Mytilus genus) and Veneridae (Tapes genus) families, respectively. Mussel seed was cultivated in tight nylon net bags for 12 months in sites close to fi sh cages (hereafter indicated as Impact sites) and 1 Km far from fi sh cages (hereafter indicated as Controls).

Collection of shellfi sh and water samples
Shellfi sh and surface waters were collected at both Impact and Control sites, during two different samplings, performed in spring and autumn. Surface seawater samples were drawn using sterile 10 litres Niskin bottles. Both seawater and shellfi sh samples were stored at +5°C in fridge containers until their analysis at the CNR laboratory. They were examined within 4 hours of sampling.

Physico-chemical parameters
The main physico-chemical parameters, temperature and salinity, were recorded using a multiparametric probe.

Shellfi sh samples
The bacteriological analysis of shellfi sh was carried out

Assays for enzymatic activities as proxies of virulence factors
All the Vibrio spp. isolates were screened for the presence of enzymes involved in virulence. Before such assays, bacterial strains were grown overnight in Tryptic Soy agar with 2% NaCl at 35° C, and further inoculated into different solid culture media according to Garcıa Moreno and Landgraf [31]. In detail, protease (caseinolytic) activity was determined by incubation at 35°C in skim milk agar plates (2% final concentration).
Bacterial isolates able to produce clearing zones into the medium containing the casein substrate were recorded as positive.
Lecithinolytic activity was determined by streaking bacteria onto a nutrient basal agar containing 10% (v/v) egg yolk emulsion, further incubated at 35°C for up to 7 days. Zones of precipitation surrounding the colonies indicated the production of lecithinase.
Lipase production was tested by inclusion into a nutrient basal agar of Tween 80 (polyoxyethylene sorbitan mono-oleate, 1% final concentration) as a substrate for bacterial growth.
The production of haemolysins (i.e. Kanagawa phenomenon) was assayed on plates of Columbia agar base added with a 5% suspension of sheep red blood cells. The colonies able to produce haemolysis after incubation at 35°C for 24 hours were considered as positive. Pearson's correlation was used to assess whether the abundance of bacteria was related to the environmental parameters.

Results
Temperature values ranged from a minimum of 14

Shellfi sh samples
The results obtained in the examined shellfi sh are shown in Table 2a,b.
Regarding the qualitative composition of vibrios (   VP counts showed the constant presence of these microorganisms with densities in the order of 10 2 -10 3 CFU/g; the highest microbial concentrations were recorded in the mussel sample collected from the Impact sites, while the lowest ones occurred in one sample of the oyster C.gigas (10 2 CFU/g from both the Control and Impact sites). VPP were mostly absent in the shellfi sh collected during autumn (data not shown).
The qualitative analysis of vibrios (

Water samples
The results obtained from the seawater samples are reported in Table 5a,b and shown in Figures 2 and 3.
In spring (Table 5a and Figure 2) the examined area did not show signifi cant faecal contamination inputs; the abundance of FC was comprised between 2×10 0 and 1.47×10 3 CFU/100 ml; the maximum value was recorded at the station 1. The abundance of not marine bacteria ranged from 1.5×10 1 to 1.24×10 3 CFU/100 ml, recorded at stations 18 and 1, respectively.
Marine bacteria showed concentrations comprised between a minimum of 3.0×10 2 (station 9) and a maximum of 1.27×10 3 CFU/100 ml recorded at station 1.
The qualitative study of the vibrios community highlighted that Vibrio spp., V. vulnifi cus and V. alginolyticus were predominant at the Impact sites, while V. vulnifi cus was not detected at the Control sites (Table 3).
In autumn (Table 5b and Figure 3 E. coli was generally absent, except for the station 1, where     The qualitative study of vibrios isolates (

Virulence factors of Vibrio spp
The results of the screening for enzymatic activities as proxies of potential virulence of Vibrio spp. isolates are shown in

Discussion
The sustainable use and a correct management of resources, also through the application of eco-friendly production methods [32], could represent the winning strategies for future innovation in aquaculture sector, that is now considered a pillar for Blue Growth [33]. Accurate marine planning of the space dedicated to sea farming, together with the application of good practices for animal welfare, are priority measures to ensure sustainable aquaculture growth.
To date, European aquaculture relies on the farming of a few major species, such as sea bass, sea bream, oyster, mussel, salmon, and others minor productions, such as carp [34]. In Italy shellfi sh farming represents an important sector of aquaculture, with clams and mussels representing the 94.2% and 70.8% of the European aquaculture productions, respectively. In Sicily, however, the production of mussels (about 700 tons, equivalent to 0.5% of national production) is still low. Polyculture has now been regarded as an environmentally sustainable practice to increase local mussel production [16]. For shellfi sh farmers, however, microbiological quality of the products is often a cause of concern, causing severe economic losses. In this context, this study has aimed at determining the potential   [27]; range: 2 -94 MPN/ 100 ml), but signifi cantly lower than those measured by Zaccone. et al.

Faecal pollution indicators: spatial and temporal distribution
(log FC: 2.8 CFU/ml) [22]. Low concentrations of ENT (range: 0.55-10.70 CFU/g) were also found in the sediments of a mussel-farming area of the Gulf of Gaeta [26].

Distribution patterns of heterotrophic marine and notmarine bacteria
Bacteria have always been considered as the normal inhabitants of molluscan microfl ora; in oysters the presence of heterotrophic bacteria has implications not only for hatchery production but also for environmental and human health [39].
In the polyculture experiment, heterotrophic bacteria in shellfi sh collected at the Impact sites exhibited in both samplings almost doubled abundances than those at the Control sites, although these quantitative variations were not statistically signifi cant. This fi nding could be explained by a stimulation of heterotrophic bacterial growth by feed wastes and excretions released by fi sh farming practices, that enriched waters with nutrients and organic substrates. At polyculture sites, mussel and clam were the species concentrating inside their body the highest bacterial abundance in spring, while in autumn O. edulis was more rich in heterotrophic bacteria.
At impact sites in spring allochthonous not-marine bacteria correlated with E. coli only weakly (r= 0.56, P<0.05), suggesting that microorganisms other than E. coli contributed to the bacterial fl ora present in these sites. In this season, inverse signifi cant relationships between marine and notmarine bacteria were found, as expected (r= -0.72, P<0.01), while in autumn marine and allochtonous not-marine bacteria were directly correlated (r= 0.78, P<0.01), indicating that the reciprocal relationships between these microorganisms was modulated not only by fi sh farming wastes but also by other organic inputs, such as continental terrigenous matter.
Like in shellfi sh, in water heterotrophic bacterial abundances reached their highest values during the spring than the autumn period; bacterial growth was supported by the favorable temperature conditions (r= 0.54, P<0.05, between T and marine bacteria). In spring, marine and notmarine bacterial concentrations in water were unaffected by polyculture activities, as shown by the similar values recorded at both Impact and control sites. In autumn, higher bacterial abundances (marine and not-marine) measured at control than at impact sites were probably related to their different geographical location, being the control sites located in proximity of the coast, and therefore affected by organic inputs introduced into the marine environment.

Distribution patterns of Vibrio spp.
Members belonging to Vibrio genus are an ubiquitous component of the heterotrophic microfl ora autochthonous of marine and brackish environments. In shellfi sh collected from Castellammare, these microorganisms in spring accounted for an average percentage of the 43.2% and 11.5% of the heterotrophic bacteria at Impact and Control sites, respectively, while in autumn for the 37.0% and 29.3% of the heterotrophic Citation: Caruso  bacteria at Impact and Control sites, respectively. Percentages close to 70% and 90% of heterotrophic bacteria were reached in C. gigas and M. galloprovincialis in spring, while in autumn in the same species they approached the 50% of the heterotrophic bacteria. These fi ndings confi rmed the widespread occurrence of pathogenic vibrio species that has been documented in shellfi sh and water samples from several European Countries [24,[27][28]40,41]. Bivalves have been regarded as an important ecological niche for vibrios, since these bacteria can persist inside molluscan tissues even after their depuration process [42]. Indeed, molluscs normally host a diversifi ed microbial community that varies in relation with environmental conditions, in agreement with the fi ltering feeding capacity of these organisms [43]. In the shellfi sh samples examined in our study, signifi cantly higher concentrations than those recorded in water were observed for all microbial parameters (for VP as well as for FC, E. coli, not-marine and marine bacteria); in summer, this concentration ability was more relevant for E. coli, not-marine and marine bacteria (from 5.55 to 31.8 times), while in autumn for FC and marine bacteria (up to 5 orders of magnitude and 3 times, respectively).
Vibrio species are very frequent in aquatic ecosystems [21,[44][45][46][47][48][49], and in coastal environments their concentrations can reach values higher than 10 3 CFU/100 ml, supported by the availability of organic substrates. The abundance of vibrios found in Castellammare waters was in the same range of magnitude as reported in a previous study in the same area [30], namely of 10 3 CFU/100 ml and 10 2 CFU/100 ml for VP and VPP respectively. Vibrio spp. concentrations of 10 4 CFU/100 ml were detected in seawater samples collected at a hatchery plant of Ostrea edulis located off the Mediterranean Spanish coast [50]. Higher concentrations of VP (up to 1.64x10 4 CFU/g) were reported in the sediments of a mussel farm in the Gulf of Gaeta (Tyrrhenian Sea) [22]. Spatial and temporal variations characterized the distribution of halophilic vibrios in shellfi sh produced in the Castellammare Gulf; in spring vibrios were about 6 times more abundant at impact than at control stations, while in autumn their spatial variations became more narrow (about 2 times between impact and control stations). In both samplings, however, ANOVA suggested that their quantitative variations in shellfi sh samples were not related to polyculture activities.
Seasonal fl uctuations in the abundance of halophilic vibrios, with higher concentrations in summer compared to autumn, refl ected the trends observed in the distribution of total heterotrophic bacteria and were consistent with the typical ecology of these bacteria, whose growth is supported by warm temperature and high nutrient availability [51]. The concentrations measured at the most coastal stations refl ected the preferential distribution of vibrios in coastal habitats rich in organic matter [28]. Moreover, in autumn at Impact sites  [28,30]. The good bacteriological quality of farming waters is a basic requisite for productive activities, that is required also in order to limit the massive use of antibiotics and prevent possible related antibiotic resistance phenomena in aquaculture [52].

Qualitative study of Vibrio community and virulence factors
Several studies have addressed the study of Vibrio biodiversity in shellfi sh, given their relevance as aetiological agents of foodborne diseases [53]. In fact, other than the wellknown pathogenic species V. cholerae, some vibrios species such as V. parahaemolyticus, V. vulnifi cus, are primary or opportunistic pathogens responsible for severe gastroenteritis usually associated with the consumption of raw or under-cooked seafood that, through their fi ltering activity, concentrate these microorganisms inside their body [21,[23][24][25]27,54]. Also, vibrios species such as V. vulnifi cus are aetiological agents of wound infections and lethal septicemia [53,55] and other species (i.e. V. alginolyticus, V. anguillarum) are opportunistic pathogens responsible for animal diseases [56]. V. alginolyticus is a common member of microfl ora of temperate and tropical marine environments and has been isolated from guts of different marine organisms and seafood [57]; however, it has been found to act as an opportunistic pathogen for aquatic organisms being the aetiological agent of infectious diseases in marine fi sh cultured in the Mediterranean Sea and of high mortality episodes in bivalve larvae [56]. V. alginolyticus was the dominant Vibrio species in several Mediterranean waters [44,45]; it is favored by haline conditions, but it can tolerate up to salinity levels of 11 [58,59].
In the Castellammare waters V. alginolyticus and V. vulnifi cus predominated within the Vibrio community, especially in autumn. V. vulnifi cus, probably favored by the low temperature compared to spring, includes three biotypes, all able to cause human infection; biotype 1 is of greatest importance to oyster producers and consumers, biotype 2 infects eels, while biotype 3 has been isolated in Israel in association with tilapia fi sh [60]. Infection by V. vulnifi cus produces pathological changes of bacterial hemorrhagic septicaemia similar to V. anguillarum in Japanese eels in Japan, cobia, seabass, rainbow trout and European eels in England, Spain, Denmark and the Netherlands [61].
V. vulnifi cus is often recovered from Mediterranean seawaters [62]; temperatures outside the range of 13 to 22°C and salinities greater than 25 ppt reduce the survival of this bacterium in seawater [63,64]. In Mexico, it was isolated from seawater and sediments of the Gulf of Mexico estuary [65] as Citation: Caruso  V. parahaemolyticus is a natural inhabitant of coastal marine and estuarine environments; there is no correlation between its distribution and faecal pollution [67][68][69]. This species is involved in the outbreak of acute gastroenteritis associated with the consumption of raw contaminated seafood [53]. V.
parahaemolyticus is also responsible for serious infections in fi sh species [61]. High incidence of V. parahaemolyticus was documented in oyster specimens from various Countries, including Brazil [70], Mexico [71] and USA [72]. It was also commonly isolated from shellfi sh (i.e. oyster and clam) collected from several Asian [73][74][75][76][77] and European Countries [41]. In shellfi sh collected from coastal areas of Southern Italy strains of V. parahaemolyticus accounted for percentages of 5.2-6.2% of the total Vibrio community [27,78].
Another emerging human foodborne pathogen, responsible for sporadic extraintestinal diseases [79], found in the V. fl uvialis was isolated from mussels from Brazil [58] and from bivalves from Costa Rica [81]; in Italy, Ripabelli, et al. [45] found that 11%-27% of the shellfi sh and shrimps contained V.
fl uvialis without any association between this pathogen and conventional fecal pollution indicators. Commonly found in coastal marine, estuarine and brackish environments [82], V. fl uvialis was isolated in high percentages (29% of the total fl ora) in the Toulon harbor (France) [83], as well as in suburban effl uents of South Africa (41.4% of the total [84]), where a positive relationship with seawater temperature, salinity and dissolved oxygen was observed.
The Vibrio community biodiversity found in shellfi sh samples collected in Castellammare polyculture area refl ected that of the waters. Indeed, in oysters, V. parahaemolyticus has been found to concentrate by up to 10 4 times compared to the surrounding water [60], even if the mechanism of association (i.e. specifi c gut microbionts, resulting from selective enrichment, or aspecifi c uptake of transient microbiota from the aquatic environment) remains unclear [56]. Also in water the distribution of this species, together with V. alginolyticus, is closely related to temperature and salinity [71,85]. V.
parahaemolyticus prefers water at temperature higher than 20°C [86], while below 16°C V. parahaemolyticus disappears from waters, surviving in the sediments [87]. V. parahaemolyticus and V. alginolyticus growth is also favoured by organic matter, therefore these species usually prefer coastal and brackish eutrophic temperate environments [51].
Through a meta-analysis of environmental variables affecting vibrios distribution, temperature and salinity were found to be the main variables explaining the variance of total Vibrio abundance in the water, while other variables were only marginally predictive of vibrios proliferation in the environment [56].
Seasonal variability in the vibrios composition was found in Cape Peloro lakes by Zaccone, et al. [49], who reported high percentages (approaching 70% of the total) of V. vulnifi cus in winter, at temperatures of 16.11 o C, while V. parahaemolyticus was detected in autumn, winter and early spring seasons with percentages of 10-14% of the total. and aTDH related hemolysin (TRH) [88], while proteases and hemolysins are considered to be relevant for the pathogenesis of V. vulnifi cus [53] and V. fl uvialis [79]. In the vibrios isolated from Castellammare Gulf shellfi sh and water, proteases, hemolysins, lipases and lecithinases were widespread. In the bacteria isolated from shellfi sh, higher percentages of positive responses were found in spring at impact sites, while in autumn the polyculture practice was associated to a reduction in the expression of enzymes related to proteolysis and haemolysis.
The expression of enzymatic activities -as a proxy of virulencein spring was higher in the bacterial strains isolated from water than from shellfi sh. In Mexico, strains of V. vulnifi cus isolated from oysters were found to be 100% proteolytic, 97.8% were lecithinase-positive and 79.8% lipase positive [66].

Conclusions
To our knowledge, only a few papers have dealt with the microbiological quality of shellfi sh produced in polyculture experiments and knowledge of bacterial communities growing in these conditions is still scarce [89]. Therefore, our study aimed at contributing to this topic in marine environments.
The analysis of whole quantitative data obtained in Castellammare Gulf confi rmed that, due to their fi lterfeeding behaviour, shellfi sh were able to concentrate bacteria inside their body, compared to the surrounding waters. The simultaneous increase of faecal pollution indicators and marine heterotrophic bacteria close to polyculture activities suggested that the inputs of organic matter (such as from food residuals and faecal pellets) stimulated the bacterial growth.
In spite of this, no detrimental effects due to the polyculture experiment were observed in the bacteriological quality of both Citation: Caruso  In the light of these considerations, IMTA systems could represent an attractive, ecofriendly, practice to diversify local production, promoting the competitiveness of aquaculture and reducing the weight of importation from other Countries.