The marine biota is presently choaking and drowing in its own feces. All aquatic systems have become universal recipients of the human waste that have erupted from modernization. The advent of blue revolution, in marine and estuarine realms as important potential area of commercially important finfish and shellfish are now facing the danger of extensive use of near-shore marine areas as convenient means for the disposal of treated and untreated wastes in a most unscrupulous fashion. The genus Vibrio are gram-negative, curved, rod shaped, halophilic, non-spore forming bacteria, autochthonous inhabitants of the marine and estuarine environment. They occur in saline aquatic environments, both free in the water and bound to animate and inanimate surfaces (Huq et al., 1983; Montanaria et al., 1999). The members of the family Vibrionaceae are a signifigant component of the microflora which includes more than 64 species (Thompson et al., 2005). Few of these reports however, have looked at the likely risks from a microbiological food safety point of view (Edema et al., 2005; Okonko et al., 2009). In the last 20 years, many halophilic Vibrio species such as V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. hollisae, V. fluvialis V. mimicus, V. furnissii and V. damsella, have been implicated inhuman enteric infections, wound infections and septicemia due to the consumption of shellfish and exposureto seawater (Thompson et al., 2004; Merward et al., 2011). V.parahaemolyticus has been frequently involved in outbreaks of food-borne diseases in Worldwide (Dalsgaard et al., 2009). V.parahaemolyticus has often beenisolated from seawater, sediment and a variety of seafoods including shrimp, crab, oyster andClam due to its halophilic characteristics. Also, this microbial infection is characterized by diarrhoea, vomiting, snausea, abdominal cramps and low grade fever (Pinto et al., 2007). In addition, V. vulnificus is a potentially lethal food -borne pathogen that is capable of causing primary septicemia and necrotizing wound infections in susceptible individuals (Ballal et al., 2010).

In recent year, vibriosis has became one of the most important bacterial diseases in maricultured organisms, affecting a large number of fish and shellfish (Woo and Kelly, 1995; Wu and Pan, 1997).

V.vulnificus is a natural microflora of estuarine and coastal marine environments worldwide and have been isolated from sea and brackish water (Kirs et al., 2010), which made a significant health threat to humans who suffered from immune disorders, liver disease and hemochromotosis (Wafaa et al., 2011). V.alginolyticus are widely dispersed in coastal areas and have been associated with especially septicemia in human.

Marine fish’s contamination with Vibrios results in serious consequences relating to national productivity and development. Antibiotics and other chemotherapeutic agents are commonly used in fish farms either as feed additives or immersion baths to achieve either prophylaxis or therapy. It was observed that individual and multiple antibiotic resistances were associated with antimicrobial use. Acquired antibiotic resistance in bacteria is generally mediated by extra chromosomal plasmids and are transmitted to the next generation (vertical gene transfer) and also exchanged among different bacterial population (horizontal gene transfer). Extensive use of these antibiotics has resulted in an increase of drug-resistant bacteria as well as R-plasmids (Son et al., 1997).

At this backdrop, the present work was tailored in order to understand the status co of vibrios in marine fish and understand the resistant pattern of isolated vibrios against various antibiotics to unravel their resistance pattern in antibiogram and study the impact of ecological factors an its survivability.

1 Material and Methods

1.1 Sample collection

The sea fish samples Upeneus sp., Rastrelliger kanagarta, Sphyraena barracuda and Sardinella sp., (Locally called Nagarai, Ayalai, Ooli and Chalai) were collected from fish market at Thirunelveli and tuticorin in a pre sterilized polythene pack. All sea food samples were transported in individually labelled and sealed new plastics bags to avoid contamination. The samples were placed in sealed container containing ice pack and transported to the laboratory for further analysis within 6 hours of collection.

1.2 Enrichment procedure

The enrichment procedure improves the presence of Vibrios in seafish. 10 g of seafish muscles were taken and ground with 90 ml of 3% NaCl containing 1% alkaline peptone water (APW, pH: 8.6) (Adebayo – Tayo et al., 2011). The tubes for enrichment were incubated at 37℃ for 18 hours (Pinto et al., 2008).

1.3 Isolation procedure

Isolation of Vibrio sp was made on thiosulphate citrate bile salt sucrose agar media (TCBS, Himedia, India). From the enrichment tube, inoculum were streaked on TCBS agar plates and incubated at 37℃ for 18 to 24 hours. After incubation, the suspected colony types Vibrio like organisms-yellow and green) were picked out, and streaked again onto nutrient agar plus 2% NaCl to obtain pure cultures.

1.4 Phenotypic characteristics of the bacterial isolates and storage

Biochemical characterization of all the isolated bacterial colonies were subjected to the following tests with an addition of 2% NaCl in media at a final concentration except tests for growth in 0,1,3,6,8, and 10% NaCl (Castro et al., 2002). The isolated bacterial cultures were stored on nutrient agar slants with 2% sodium chloride and 20% glycerol were prepared and stored at 4℃ as stocks culture for further work (Manjusha and Sarita, 2011).

1.5 Antibiotic sensitivity test

Bacterial isolates were tested for anti microbial sensitivity through the disc diffusion method. The bacterial isolates were tested against the antibiotics impregnated discs of about 6mm diameter (Himedia Laboratories, Mumbai). Discs containing the following antibacterial agents were placed on spread plated Muller Hinton Agar plates (Himedia laboratories, Mumbai) supplemented with 2% NaCl and incubated overnight Amikacin (Ak, 30 μg), Ampicillin (Amp, 10 μg), Amoxyclave (Amc, 30 μg), Aztreonam (At, 30 μg), Ceftazidime (Ca, 30 μg), Cefotaxime (Ctx, 30 μg), Ciprofloxacin (Cf, 5 μg), Co-trimoxazole (cot, 25 μg), Erythromycin (E, 15 μg), Gentamycin (Gen, 10 μg), Imipenem (Ipm, 10 μg), Levofloxacin (Le, 5 μg), Methicillin (M, 10 μg), Nalidixic acid (Na, 30 μg), Netillin (Nt, 30 μg), Nitrofurantoin (Nf, 300 μg), Novobiocin (Nv, 5 μg), Tobramycin (Tob, 10 μg), Tetracyclin (T, 30 μg), Vancomycin (Va, 30 μg). After incubation, the diameter of the zone of inhibition, were measured in mm and compared with zone diameter interpretative chart to determine the sensitivity of the isolates to the antibiotics (Hi media, laboratory).

1.6 Effect of pH on Bacterial Growth

All the bacteria were inoculated into Nutrient broth containing different pH ranges (6, 7, 8, 9, 10) and incubated a 37℃. After incubation, the optical density was measured at 600 nm.

1.7 Effect of Temperature on Bacterial Growth

Bacterial isolates were inoculated into Nutrient broth (NB) broth and the tubes were incubated at different temperature range (20, 30, 40, 50 and 60℃) for 24 hours. After incubation, the optical density was measured at 600 nm.

1.8 Effect of Salinity on Bacterial Growth

All the bacterial isolates were inoculated into Nutrient broth preferred with different salinity ranges such as 5, 10, 15, 20, and 25% and incubated at 37℃. After incubation, the optical density was measured at 600 nm.

1.9 Effect of carbon and nitrogen sources on bacterial growth

All the bacterial strains were inoculated into Nutrient broth (NB) broth containing different carbon sources (Glucose, Sucrose, Lactose) and incubated 37℃. After incubation, the optical density was measured at 600 nm.

1.10 Effect of mineral sources on bacterial growth

All the bacterial strains were inoculated into Nutrient broth (NB) broth containing different mineral sources (Magnesium sulphate, Calcium chloride and Zinc sulphate) and incubated at 37℃. After incubation, the optical density was measured.

1.11 Isolation of plasmid DNA by rapid method (Kalaichelvan，2005)

The plasmid DNA was retrived using the following protocol as depicted by kalaiselven (2005).

2 Results

The prevalence and distribution of Vibrios were analyzed in marketed sea foods sample from Tuticorin and Thirunelveli market. Their antibiotic resistant pattern and molecular weight of the randomly selected Vibrio sp. were detected. In this work around 36 Vibrio sp., were isolated and identified through the biochemical tests were observed.

The amino acids (Arginine, Lysine, Ornithine) decarboxylase test were carried out in aerobic and anaerobic conditions to know the requirement of amino acids by the isolates and the results are tabulated in Table 1. Most of the isolates were positive to Lysine and ornithine, but exhibited negative result to Arginine. Sugar fermentation (Sucrose and Lactose) tests were carried out which showed most of the isolates to be negative to both the sugars and few were positive results.

The isolates were subjected salt tolerance character that was analyzed at different % of NaCl (1, 3, 6, 8, and 10). Apart from Vibrio cholerae other Vibrio sp could not grown in 0% NaCl, whereas all the isolates grew in 1% NaCl. V. alginolyticus alone grew at 10% NaCl.

Table 1 Decarboxylase testing for Vibrio sp. Isolated from marketed sea foods sample

36 Isolates of Vibrio sp. comprising of 5 genera were tested against 20 antibiotics (Table 2). The result of which are presented in the Table 3. 25 % of Vibrio sp., was totally resistance (100%) towards Ampicillin, Amoxyclave, imipenam, Methicillin and Nitrofurantoin while 100% sensitivity was shown by Levofloxacin.

Table 2 Distribution pattern of antibiogram among the 36 vibrio strain

Table 3 Antibiotics susceptibility test of Vibrio sp. isolated Vibrio sp. Isolated from Marketed sea foods sample

The isolates were subjected to various ecological factor such as various temperature (20℃, 30℃, 40℃, 50℃, 60℃), salinity range (5%, 10%, 15%, 20%, 25%), pH range (6,7,8,9,10), carbon (Glucose, Sucrose, Lactose) and mineral source (CaCl₂, MgSO₄, ZnSO₄), that were analyzed at marketed sea foods isolated vibrios. Apart from Vibrio cholerae other Vibrio sp., could not grown in 0% NaCl, whereas all the isolates grew in 1% NaCl (Table 4, Table 5).

Table 4 Effect of different pH on isolated Vibrio sp. growth from Marketed fish sample (The Values are measured in Optical Density)

Table 5 Effect of different carbon sources on isolated Vibrio spp. growth from marketed fish sample (The Values are measured in optical density)

For the isolated Vibrios sp, the highest (2.00 nm) and lowest growth (0.17 nm) range was observed from V.mimicus and V.paraheamolyticus based on various temperature (20℃^~60℃). V.cholera and V.vulnificus exhibited the lowest (0.00 nm) and highest (2.00) range in different salinity (5%~25%). Effect the different carbon source (Glucose, Sucrose and Lactose) on isolated vibrios sp. the lowest (0.02 nm) and highest (1.32 nm) value were observed in V.mimicus and v.alginolyticus. For the isolated Vibrio sp. at various pH levels (6~10), V.paraheamolyticus and V.mimicus are expressed to lowest (0.02) and hight range (1.09).

The plasmid profiling revealed that out of 11 isolates analysed for plasmid only 4 isolates V.parahaemolyticus (2), V.vulnificus (1), V. alginolyticus) and their molecular weight compared with 5000bp DNA ladder and their molecular weight identified in 4000bp and 3500bp, while the other 7 isolates were without plasmids that are represented in Figure 1, Figure 2 and Table 6.

Figure 1 Fishes in this study

Figure 2 Gel image of plasmid profiles of the randomly selected

Table 6 Plasmid formation of randamly selected the isolated Vibrios from marketed sea food samples

3 Discussion

Wafaa M.K.B., Walaa A.H., and Amani F.A., 2011, Detection of Salmonella and Vibrio species in some seafood in Alexandria, J. American Sci., 7(9): 663-668