Phenotypic characterization and enterobacterial repetitive intergenic consensus polymerase chain reaction ( eric-pcr ) of Aeromonas spp . and Vibrio spp . strains isolated from Sparus aurata fish farm ( Khenis , Tunisia )

This study characterises the Aeromonas and Vibrio strains isolated from seawater and sediments out of Sparus aurata marine farm. The bacterial strains were isolated on Thiosulfate-Citrate-Bile-Sucrose (TCBS) agar plates and described using different bacteriological tests and using standardised micromethods “API 20 NE strips”. The exoenzymes production and antibiotics susceptibility were also investigated. The enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to evaluate the genetic diversity of the isolated strains. Two dominant genera of bacteria were found (Aeromonas and Vibrio). Aeromonas hydrophila strains were the dominant bacteria (56.5% of total isolates) followed by V. alginolyticus strains (26.1%). Most of the studied strains were -haemolytic, hydrolyze the DNA and produce many exoenzymes such as lecithinase, caseinase, amylase and lipase. All tested strains were resistant to at least three antimicrobial agents. The antibiotic resistance index was 0.683 for V. alginolyticus and 0.711 for A. hydrophila strains. The ERIC-PCR profiles among the isolated bacteria were generally heterogeneous, showing a high polymorphism between Aeromonas strains.

Gilthead sea bream (S. aurata L.) is one of the most important species reared in marine fish farms installed along Tunisian coastal areas.Members of the Vibrionaceae and Aeromonadaceae families are responsible for fish and aquatic animal pathologies (Martin and Phelepp, 1983;De Kinkelin et al., 1985).These bacteria cause several economic losses and high mortality in larvae of many species during the first days following hatching: S. aurata and D. labrax (Bakhrouf et al., 1992;Snoussi et al., 2006;Ben Kahla-Nakbi et al., 2007b).
Vibrio and Aeromonas genera are widespread of tropical and temperate waters around the word and they colonise the costal waters of the Mediterranean Sea (Caruso et al., 1998;Zanetti et al., 2000;Robert-Pillot et al., 2002;Snoussi at al., 2008b).Vibrio spp.strains are very normal inhabitants in estuarine and marine environments and are frequently isolated from bathing area as free-living bacteria and associated to different surfaces (Tamplin et al., 1990;Barbieri et al., 1999;Baffone et al., 2005).These strains were always associated with gastrointestinal and extra-intestinal troubles related to the consumption of insufficiently cooked seafood or deep contact with contaminated seawater (Ottaviani et al., 1995;Croci et al., 2001;Thompson et al., 2004).
Aeromonas hydrophila is a Gram-negative bacterium that infects a wide range of hosts including amphibians, reptiles, avians and mammals such as cows and man (Popoff, 1984), but it is most well known as a fish pathogen.It causes motile aeromonad septicemia (MAS), which results in great economic losses in fresh water fish farming worldwide.The pathogenesis of A. hydrophila is multifactorial.A variety of virulence factors may contribute to the overall virulence of this bacterium.These include extracellular products (Ljungh and Wadstrom, 1982;Leung and Stevenson, 1988;Anglka et al., 1995), S-layer (Dooley and Trust, 1988) and adhesins.It is suggested that A. hydrophila is entero-pathogenic, using disease by adhesive and entero-toxic mechanisms (Quinn et al., 1993).
In this study, we reported the isolation, characterisation, and ERIC 2-PCR typing of 23 Aeromonas spp.and Vibrio spp.strains isolated from seawater and sediments near a S. aurata rearing Farm (Monastir).The presence of Vibrio cholerae virulence genes toxR, toxS, toxRS and toxT genes were sought in the genome of all tested strains using PCR technique.Exoenzymes production and antimicrobial resistance have also been investigated.

Sampling site
The area of study (Figure 1) is localized on the eastern littoral of Tunisia (Monastir).This bay is localised between the latitudes 35°40 and 35°40 and the longitudes 10°48 and 10°54.The Monastir-Sayada seacoast is characterized by an algal bloom distribution during all the year especially during the three months of the summer related to an increase in the temperature with a rarefaction of fish capture.Seawater and sediment from the aquaculture farm installed along the seacoasts of Khenis (Monastir, Tunisia) were collected during the summer 2006.In the laboratory, Aeromonas spp.and Vibrio spp.research was carried out by filtering the water samples (500 ml) through 0.45 µm pore size filter membranes.These membranes were incubated in alkaline peptone water (1% NaCl, pH 8.6) for 24 h.The enrichments were then streaked onto Thiosulfate-citrate-bile salt-sucrose agar (TCBS agar, Biorad) and incubated for 18 to 24 and 30°C.Twenty five grams of sediment were diluted in Stomacher packet each containing 225 ml of alkaline peptone water supplemented with 1% NaCl.The homogenate was incubated at 30°C for 18 to 24 h.

Characterization of bacterial strains
Bacterial strains growing on TCBS plates were subjected to standard morphological, physiological and biochemical plate and tube tests using the procedures described previously by Snoussi et al. (2006Snoussi et al. ( , 2008a)).A commercial miniaturized API 20 NE system (Bio Mérieux, Marcy l'Étoile, France) was used to identify the strains isolated.For exoenzymes productions, all strains were tested for  (Snoussi et al., 2009).
The antibiotic resistance index (ARI) of each bacterial population was determined using the following formula: ARI = y/nx, where y was the actual number of resistance determinants recorded in a population of a size n and x was the total number of antibacterial tested for in the sensitivity test.Based on the occurrence of resistance to more than three antibiotics, the isolates were grouped as multiple antibiotic resistant isolates (Manjusha et al., 2005).
The multiple antibiotic resistance (MAR) index of the isolates was done as referred to described by Krumperman (1983).The MAR index was defined as a/b where a represents the number of multiple antibiotics to which the particular isolate is resistant and b as the number of multiple antibiotics to which the particular isolates were exposed.
The gels were viewed under ultraviolet light and photographed by the Gel Doc (Bio-Rad, USA).The number and size of DNA fragments were evaluated by visual inspection and using Gel Pro Analyser 3.1 software.Dendrogram based on the unweighted pair group method of arithmetic averages (UPGMA) and Jaccard's correlation coefficient on the basis of ERIC-2 patterns was built using the MVSP 3.1 software (Snoussi et al., 2008a).

Distribution of toxR, toxT, toxS and toxRS genes
Primers for the amplification of the toxRS genes were designed to be complementary to the 5' and 3' ends of the toxR and the 5' and 3' ends of toxS.For toxT amplification, primers toxT1 and toxT2 were designed.All primers used in this study were summarized in Table 1.Amplification reactions were performed in a 25 l reaction mixture contained 3 l of genomic DNA, 5 l of green Go Taq buffer (5x), 200 M of each deoxynucleoside triphosphates (dNTP), 25 M of each primer and 1U of GO Taq DNA polymerase (Promega, USA).The mixtures were incubated for 5 mn at 94°C, followed by 35 cycles of amplifications.Each cycle consisted of a step of denaturation at 94°C for 40 s, annealing for 40 s, and primer extension for 1 mn at 72°C.The mixtures were kept at 72°C for 10 mn between two cycles.The annealing temperatures were: 58°C for toxR, toxS, toxT and toxRS.
All PCR-positive strains had the presence of the virulence genes confirmed by repeating the PCR three times independently.All bands produced were normalized by comparing molecular weight marker (Hyper Ladder II DNA molecular weight marquer, Bioline) between different gels.The amplification products were visualized by staining the gel with ethidium bromide after electrophoresis at 90 V for 90 min in 1% agarose gel (FMC, Bio products).All DNA amplifications were performed in a DNA thermal cycler (PTC-100,

RESULTS AND DISCUSSION
A total of 13 A. hydrophila strains, two A. salmonicida spp.salmonicida, two V. vulnificus and six V. alginolyticus strains were isolated from all samples tested in this work (Table 2).In fact, eight strains (34.78 %) were isolated from seawater and 15 strains from the sediment (65.2 %).
The biochemical activities of the Aeromonas spp.(15  2).In fact, out of 13 hydrophila strains tested, eleven biotypes were identified and five biotypes were identified out of six strains tested.All A. hydrophila strains were identified as Gram-negative motile fermentative bacilli, producing enzymes like catalase and oxidase, susceptible to Vibriostatic compound O/129.In this study, the identifi cation procedure of environmental Vibrio and strains using the API 20 NE strips was modified in order to inoculate a loopful of a bacterial suspension prepar on sterile saline water (2.5% NaCl).All strains tested with this modification and using the API Web software were successfully identified with a high level of acceptance previous studies, we reported the isolation and characterization of large populations of strains isolated from the same area associated with a high mortality affecting S. aurata larvae using A and RapID TM NF plus strips (Snoussi et al., 2008a(Snoussi et al., 2008b)).
. (8 strains) tested on API 20 NE strips demonstrated the heterogeneity of the two populations studied (Table 2).In fact, out of 13 A.
strains tested, eleven biotypes were identified and five biotypes were identified out of six V. alginolyticus strains were identified as negative motile fermentative bacilli, producing enzymes like catalase and oxidase, susceptible to In this study, the identifiand Aeromonas 20 NE strips was modified in order to inoculate a loopful of a bacterial suspension prepared % NaCl).All strains tested with Web software were ssfully identified with a high level of acceptance.In previous studies, we reported the isolation and characterization of large populations of V. alginolyticus strains isolated from the same area associated with a larvae using API 20 E NF plus strips (Snoussi et al., 2008a; 20 NE strips, all strains were positive for the following tests: reduction of nitrates, hydrolysis of esculin glucosamine.All strains grew respectively with 3 and 8% of salmonicida strains reduction of nitrates, glucose fermentation, hydrolysis of esculin and gelatine, -galactosidase and assimilation of the following substrates: D-glucose, galactosidase, potassium glucosamine.All V. alginolyticus strains (n = 2) grew in ly with 3, 8 and 10% of nitrate reductase, indole production, glucose fermentation, hydrolysis of esculin and gelatine, and assimilation of malic acid.For exoenzymes production, all 15) and Vibrio spp.(n = 8) strains produced several hydrolytic enzymes including: protease, gelatinase, lipase, DNase and lecithinase.All these strains were haemolytic on Human Blood agar.All these data were in accordance with our previous findings regarding alginolyticus and A. hydrophila different Tunisian marine biotopes (Ben Kahla al., 2006, Ben Kahla-Nakbi et al., Nakbi et al., 2009;Snoussi et al., 2006;2008a, 2008c).In fact, several virulence including adhesins, enterotoxins, hemolysins, proteases, DNases, lipases and other extracellular enzymes, have been identified in Aeromonas bacterial products cause tissue damage and aid in establishing an infection by overcoming host defenses and by providing nutrients for bacterial proliferation (Agarwal et al., 1998).
Figure 2 represents the percentage of resistance to t selected antibiotics tested in the present study.strains of Aeromonas and chloramphenicol, erythromycin, nitrofurantoin and gentamycin while 95.65% of strains were resistant to ampicillin and cefotaxime.The lowest perc resistance were noted when acid nalidixic (21.73 ciprofloxacin (26.08 %) were tested.
All, the isolates were multi resistance to at least three different antibiotics with multiple resistance index ranging from alginolyticus, from 0.583 to 0.833 for hydrophila strains.The antibiotic resistance index was 0.683 for V. alginolyticus and indole production, glucose fermentation, hydrolysis of esculin and gelatine, and assimilation of malic acid.For exoenzymes production, all Aeromonas spp.(n = 8) strains produced several hydrolytic enzymes including: protease, gelatinase, lipase, DNase and lecithinase.All these strains werehaemolytic on Human Blood agar.All these data were in accordance with our previous findings regarding V.
A. hydrophila strains isolated from different Tunisian marine biotopes (Ben Kahla-Nakbi et Nakbi et al., 2007b, Ben Kahla-2009;Snoussi et al., 2006;2008a, 2008b, several virulence-associated factors including adhesins, enterotoxins, hemolysins, proteases, DNases, lipases and other extracellular enzymes, have Aeromonas spp.Some of these bacterial products cause tissue damage and aid in establishing an infection by overcoming host defenses and by providing nutrients for bacterial proliferation Figure 2 represents the percentage of resistance to the selected antibiotics tested in the present study.The 23 and Vibrio were resistant to chloramphenicol, erythromycin, nitrofurantoin and gentamycin while 95.65% of strains were resistant to ampicillin and cefotaxime.The lowest percentages of oted when acid nalidixic (21.73%) and ciprofloxacin (26.08 %) were tested.
All, the isolates were multi-resistants, defined as resistance to at least three different antibiotics with multiple resistance index ranging from 0.5 to 0.75 to V.
, from 0.583 to 0.833 for Aeromonas strains.The antibiotic resistance index was and 0.711 for A. hydrophila strains.The high value of AR index was recorded for the two A. salmonicida sp.salmonicida strains (0.791).All these data are summarized in Table 3.In a previous study, we studied the resistance of 28 strains isolated from the seawater of the seacoast of Khenis (Monastir) to 18 antibiotics including those tested in the present work (Snoussi et al., 2008a), and we founded different values of MAR indexes for V. alginolyticus strains ranging from 0.3 to 1 and a high value of antibiotic resistance index (ARI = 0.856).While, in another marine biotope (Lac of Bizerte), the multiple antibiotic resistance index of the 31 V. alginolyticus strains was ranging from 0.25 to 0,833 and with a low value of ARI = 0,449 (Snoussi et al., 2010).
In a recent study, Devi et al. (2009) founded that the multiple antibiotic resistance (MAR) index was found to be lower (0.2) indicating the meager resistance acquired by the isolates.Parveen et al. (1997) was the first to propose the MAR index for differentiating the sources of antibiotic pollution.The occurrence of MAR among the bacterial species could be a problem associated with transfer or resistance to other organisms of human or veterinary significance.As evident in the present study, V. parahaemolyticus isolates have not so far developed resistance to life saving drugs like tetracycline, nalidixic acid followed by chloramphenicol, trimethoprim and nitrofurantoin, although in a very few number of isolates, resistance was noticed towards gentamycin indicating the safety of seafood products.The significance of MAR in different ecosystem was studied by Kaspar et al. (1990) and the results of the present study are in agreement with their study.

ERIC-2 finger printing
Figure 3 represents the patterns obtained with ERIC 2 primer tested with A. hydrophila and Vibrio spp.strains isolated from seawater and sediment of the fish farm in Khenis (Monastir).This technique applied to the 23  isolates showed a reproducible patterns consisting of 4 to 9 bands ranging from 200 pb to 971 pb for Aeromonas spp.strains and from 181 pb to 1480 pb for Vibrio spp.strains estimated using the Gel Pro Analyzer 3.1 software.The phylogenetic analysis of patterns generated by ERIC 2-PCR (Figure 4) based on UPGMA method (Jaccard's coefficient) has shown that both Vibrio and Aeromonas strains exhibit high amount of heterogeneity not only according to the origin (seawater, sediment) but also within the same samples.We found in this study that strains isolated from the same origin were genetically different as they were grouped in different clusters.Moreover, strains originated from the same sample were genetically heterogeneous as they fell into different clusters.Eleven genotypes of Aeromonas spp.were identified and only tree A. hydrophila strains originated from two different samples (seawater and sediment) were grouped into the same cluster and were considered as closely related strains (100% of similarity).The six V. alginolyticus strains originated from seawater and sediment were genetically heterogeneous as they were grouped in different clusters.
In a previous studies, we used the same technique (ERIC 2-PCR) to type different populations of V. alginolyticus strains isolated from different Tunisian marine biotopes (Snoussi et al., 2006;2008a;2010).In a previous study, Ben Kahla-Nakbi et al. (2006) used ERIC2-PCR fingerprinting method to type V. alginolyticus strains isolated from internal organs and external lesions of diseased gilthead sea bream and sea bass and their growing area harvested from a Tunisian farm and reported a high level of genomic diversity.In fact, 19 genotypes out 34 strains studied were reported by Ben Kahla-Nakbi et al. (2006) with patterns including 2 to 7 bands and there is no correlation between genotypes and season of recovery was signaled.

Distribution of virulence genes
The virulence genes toxR, toxS, toxT and toxRS were sought in the genome of all isolates by PCR (Table 3).The expected toxRS fragment was amplified from the chromosome of only two strains of V. alginolyticus out of six tested and interestingly from one A. hydrophila strain (isolate 5).All strains were negative for the amplification of toxR, toxS, and toxT virulence genes.In a previous study, we reported a large distribution of V. cholerae genes among the Vibrio spp.isolated from mussels and their growing area from the lac of Bizerte (Snoussi et al., 2010) and from Monastir-Sayada lagoon (Snoussi et al., 2008b).It is now clear that in aquatic biotopes, V. alginolyticus might be continually undergoing genetic change by the acquisition of DNA originated from Vibrio cholerae strains (Sechi et al. 2000;Deriu et al., 2002;Baffone et al., 2005;Xie et al., 2005;Snoussi et al., 2008b;2010;Ben Kahla-Nakbi et al., 2009;).

Conclusion
In this study, we found a large distribution of Aeromonas and Vibrio strains in the marine fish farm installed in Khenis (Lagoon of Monastir-Sayada) with several virulence factors (lipolytic, proteolytic and haemolytic properties) and we confirm the use of the ERIC 2-PCR as an easy and rapid tool for the genetic typing of Aeromonas and Vibrio strains.Mejdi et al. 2927

Figure 1 .
Figure 1.Localization of the site of study.

Figure 3 .
Figure 3.DNA fingerprints generated on 1% agarose gel by ERIC 2-PCR analysis of A. hydrophila and Vibrio spp.strains isolated from seawater and sediment.(M): Hyper Ladder II DNA molecular weight marquer (Bioline) and (T) negative control.

Figure 4 .
Figure 4. based on the unweighted pair group method of arithmetic averages and Jaccard's correlation coefficient on the basis of ERIC-2 patterns for the 23 strains of Aeromonas spp.and Vibrio spp.isolated from seawater and sediment from the aquaculture installation in Khenis.Numbers on the horizontal axis indicate the percentage of similarity.

Table 1 .
Selected primers of four Vibrio cholerae virulence genes investigated in this study.

Table 2 .
Biochemical of Aeromonas spp.and Vibrio spp.strains isolated from seawater and sediment tested using the API 20 NE strips.

Table 3 .
MAR index and distribution of four V. cholerae virulence genes in genome of Aeromonas spp.and Vibrio spp.strains.