{"id":1863,"date":"2015-03-28T07:10:15","date_gmt":"2015-03-28T07:10:15","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=1863"},"modified":"2020-04-26T07:25:41","modified_gmt":"2020-04-26T07:25:41","slug":"assessment-of-virulence-and-antimicrobial-susceptibility-of-vibrio-vulnificus-isolated-from-etroplus-suratensis-bloch","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol4no1\/assessment-of-virulence-and-antimicrobial-susceptibility-of-vibrio-vulnificus-isolated-from-etroplus-suratensis-bloch\/","title":{"rendered":"Assessment of Virulence and Antimicrobial Susceptibility of Vibrio vulnificus Isolated from Etroplus suratensis (Bloch)"},"content":{"rendered":"

Introduction <\/strong><\/p>\n

Fishes are one of the most primitive man\u2019s main foods in the earlier days as a hunter food gatherer. \u00a0It is the only sector which offers animal protein to a broad-cross section of the society thereby is in an advantageous position to ensure nutritional value. \u00a0The fish meal is highly proteinaceous in nature, when compared to other animal foods.\u00a0 Pickering and Steward, 1984 reported that overcrowding acts as an aquaculture related chronic stress or which reduces growth besides affecting the immune system in several species of fish like Atlantic salmon, Carp and Sea bream. \u00a0Due to over exploitation by human population our natural water resources have also become highly polluted through indiscriminate discharge of industrial, sewage and agricultural wastes. \u00a0Microbial pathogens are associated with the marine and fresh water aquatic animals.<\/p>\n

Vibriosis is an economically important disease of \u00a0fish, marine invertebrates and large marine mammals and is responsible for the high mortality rates in aquaculture worldwide. \u00a0The genus Vibrio<\/em> spp. cause fatal diseases commonly in Epinephelus<\/em> spp. Auguilla <\/em>spp. Lates calcarifer, Liza macrolepis, Orechromis <\/em>spp. Among Vibrio <\/em>spp., Vibrio \u00a0vulnificus<\/em> is one of the fish pathogen in marine and brackish waters (Thampuran et al<\/em>., 1998). Vibrionaceae members distributed in estuarine, marine environment and heterotrophic bacteria. They are halophilic, gram-negative, comma-shaped bacteria\u00a0 and V. vulnificus<\/em> cause diseases in human as well as terrestrial and aquatic animals. The common names for Vibrio <\/em>infections of fishes include \u201cred-pest\u201d of eels and \u201csalt- water furunculosis\u201d, \u201cred-boil\u201d and \u201cpike-pest\u201d Peggy et al<\/em>. (1996). \u00a0Oliver (1986) reported the production of extracellular enzymes and cytotoxicity by V. vulnificus. \u00a0Vibrio vulnificus <\/em>produce toxin such as cytotoxin, cytolysine also produce lipopolysaccharide (Tison et al<\/em>., 1986 and Yoshid, 1985).<\/p>\n

The present study mainly focused on isolation and identification of V. vulnificus<\/em> from the diseased fishes. A standard procedure was followed for identification of V. vulnificus <\/em>(Bergey\u2019s Determinative bacteriology, 1990). Virulence of V. vulnificus<\/em> was assessed by Lethal Dosage (LD50<\/sub>) method.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Isolation of the bacterium <\/strong><\/p>\n

Blood samples and tissue homogenates from the injected fishes were used to isolate the bacterium. \u00a0The blood samples were removed aseptically and then the clean supernatant of tissue homogenates were plated on to Zobell\u2019s Marine agar. \u00a0The colonies were stained and then identified as Vibrio<\/em> spp. based on morphology and motility. \u00a0The identified colonies were subsequently maintained in Alkaline peptone water (Larsen et al<\/em>., 1998) or Brain Heart Infusion Broth (BHIB). Furtherly it was plated on a selective medium, such as, Thiosulfate Citrate Bile salt Sucrose Agar (TCBS-Hi-media), Vibrio vulnificus<\/em> selective medium (VVM), and Sodium dodecyl sulfate – Polymixin B- Sucrose medium, (Arias et al<\/em>., 1999; Jofre et al<\/em>., 2000 and Bryant, 1987). Identification of V. vulnificus <\/em>done by Bergey\u2019s Manual Bacteriology (1990).<\/p>\n

Antibiotic Sensitivity Test<\/strong><\/p>\n

Sensitivity \/ Resistant pattern of isolate of \u00a0Vibrio vulnificus <\/em>was done by Kirby \u2013 Bauer\u00a0 method (Bauer and Kirby, 1966). All the test cultures were inoculated into tryptone soy broth TSB and incubated at 37\u00baC for 5 hours. Each strain of V. vulnificus<\/em> spreaded over the Muller Hinton Agar (MHA) plates, the panel of antibiotics were selected and placed aseptically over the MHA plates.\u00a0The plates were incubated at 37\u00b0C for 18- 24 hours. \u00a0Then the plates were examined for the presence of zone of inhibitions and results were interpreted according to the standard chart (Hi-media, India).<\/p>\n

Determination of Lethal Dosage<\/strong><\/p>\n

Five different dose regimes (10-4<\/sup><\/strong> -10–<\/sup><\/strong>8 <\/sup><\/strong>CFU \/ fish) were used to kill fifty percent of the challenged fishes, \u00a0Etroplus suratensis <\/em>that had been collected from backwater of Muttukadu and acclimatized at 37\u00b0C for 30 days prior to experimentation. Twelve fish per dose, six in each replicate were challenged through intramuscular injection, at the base of the dorsal fin with 0.1 ml suspension of the bacterium (Vibrio vulnificus<\/em>) were inoculated. Injected fishes were kept under observation for 7-10 days. Cumulative mortalities were used in calculating the LD50<\/sub> (Reed and Muench, 1938).<\/p>\n

Results and Discussion<\/strong><\/p>\n

In the present study, the injected fishes were sluggish, necrotic, hemorrhagic spot, boils and blackening of body surface, large sores and granulating lesions with feeble pale gills were identified. \u00a0The diseased fishes were collected and the samples from kidney and blood were cultured on Zobell\u2019s Marine agar, the non-swarming colonies with serrated margins were observed. \u00a0Typical green colour colonies were observed on TCBS agar. \u00a0In normal fish Etroplus suratensis<\/em>, the bacterium Vibrio vulnificus<\/em>, isolated from infected fishes were injected intramuscularly and the lesions were observed.<\/p>\n

The various biochemical characters of Vibrio vulnificus<\/em> isolated from infected fishes were tabulated in table 1. The characters of \u00a0V. vulnificus<\/em> it includes, gram negative rod, motile, growth at 0%, 0.5%, 3%, 5%, 6% and 8% NaCl, growth at 4\u00baC, 30\u00baC and 35\u00baC, gelatin and starch hydrolysis positive, oxidase and catalase positive, IMViC \u2013 Indole negative, MR negative, VP negative, citrate positive, growth on TCBS Agar, gas and acid from glucose, gas from fructose, maltose, \u00a0sucrose, galactose, mannose, arabinose and xylose. Stephenie et al<\/em>. (2010) studied the correlation of\u00a0\u00a0 D- mannitol fermentation with virulence – associated genotypic character in V. vulnificus<\/em>, which was isolated from oysters and water samples. Indole-negative V. vulnificus<\/em> strains isolated from a septicemic patient (Amaro, 1995) and indole-positive V. vulnificus<\/em> isolated from Danish eel farm (Dalsgaard et al<\/em>., 1998). Feifei Han et al<\/em>. (2009) reported the clinical and environmental types of V. vulnificus<\/em> isolated from Louisiana oysters. A culture – free method for the detection of Vibrio vulnificus<\/em> from costal seawater based on loop – mediated isothermal amplification targeting vcgC<\/em> gene (Yongjun Li et al<\/em>., 2010).\u00a0 Based on the present investigation, it is clearly indicated that the isolates from Etroplus suratensis<\/em> were V. vulnificus.<\/em><\/p>\n

Table 1: <\/strong>Biochemical characters of vibrio vulnificus.<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
S.No<\/strong><\/td>\nReactions for the species<\/strong><\/td>\nV. vulnificus <\/em><\/strong>from <\/strong><\/p>\n

E. suratensis<\/em><\/strong><\/td>\n<\/tr>\n

1<\/td>\nGram\u2019s Staining<\/td>\n–<\/td>\n<\/tr>\n
2<\/td>\nMotility<\/td>\n+<\/td>\n<\/tr>\n
3<\/td>\nGas from Glucose<\/td>\n–<\/td>\n<\/tr>\n
4<\/td>\nAcid from Sucrose<\/td>\n–<\/td>\n<\/tr>\n
5<\/td>\nAcid from Fructose<\/td>\n+<\/td>\n<\/tr>\n
6<\/td>\nAcid from Maltose<\/td>\n+<\/td>\n<\/tr>\n
7<\/td>\nAcid from D-Mannose<\/td>\n+<\/td>\n<\/tr>\n
8<\/td>\nAcids from D-Galactose<\/td>\n+<\/td>\n<\/tr>\n
9<\/td>\nAcid from D -Xylose<\/td>\n–<\/td>\n<\/tr>\n
10<\/td>\nAcid from L-Arabinose<\/td>\n–<\/td>\n<\/tr>\n
11<\/td>\nIndole Production<\/td>\n–<\/td>\n<\/tr>\n
12<\/td>\nMethyl Red<\/td>\n–<\/td>\n<\/tr>\n
13<\/td>\nVoges-Proskauer(VP)<\/td>\n–<\/td>\n<\/tr>\n
14<\/td>\nCitrate utilization<\/td>\n+<\/td>\n<\/tr>\n
15<\/td>\nUrease activity<\/td>\n–<\/td>\n<\/tr>\n
16<\/td>\nNitrate reduction<\/td>\n–<\/td>\n<\/tr>\n
17<\/td>\nGelatin hydrolysis<\/td>\n+<\/td>\n<\/tr>\n
18<\/td>\nStarch hydrolysis<\/td>\n+<\/td>\n<\/tr>\n
19<\/td>\nPhospholipids<\/td>\n+<\/td>\n<\/tr>\n
20<\/td>\nCatalase<\/td>\n+<\/td>\n<\/tr>\n
21<\/td>\nOxidase<\/td>\n+<\/td>\n<\/tr>\n
22<\/td>\nGrowth on TCBS agar<\/td>\n+<\/td>\n<\/tr>\n
23<\/td>\nGrowth at 0% Nacl<\/td>\n–<\/td>\n<\/tr>\n
24<\/td>\nGrowth at 0.5% Nacl<\/td>\n–<\/td>\n<\/tr>\n
25<\/td>\nGrowth at 3% Nacl<\/td>\n+<\/td>\n<\/tr>\n
26<\/td>\nGrowth at 5% Nacl<\/td>\n+<\/td>\n<\/tr>\n
27<\/td>\nGrowth at 6% Nacl<\/td>\n+<\/td>\n<\/tr>\n
28<\/td>\nGrowth at 8% Nacl<\/td>\n–<\/td>\n<\/tr>\n
29<\/td>\nGrowth at 4o <\/sup>C<\/td>\n–<\/td>\n<\/tr>\n
30<\/td>\nGrowth at 30o <\/sup>C<\/td>\n+<\/td>\n<\/tr>\n
31<\/td>\nGrowth at 35 o <\/sup>C<\/td>\n+<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

This study also showed the optimum temperature and salinity for the growth of organism. Temperature and salinity form a major controlling factor in aquaculture system. The optimum temperature lies between 30\u00b0-35\u00b0C supports normal growth (Beena, 2000). Fishes were much susceptible to temperature changes, which lead to temperature shock, followed by stress and thus are prone to infections. Salinity also forms important limiting factors in brackish water culture systems. The result of the present study, the sodium chloride concentration lies between 3% – 6% respectively.<\/p>\n

Sensitivity and Resistant pattern of Vibrio vulnificus<\/em> (54 – isolates) were studied and tabulated in table 2. A panel of antibiotics were employed, among these Gentamycin (25.5mm), Norfloxacin (25.0mm), Lomefloxacin (25.0mm), Nalidixic acid (23.5mm), Levofloxacin (22.5mm) and Co-trimoxazole (24.0mm) mean values were found to be sensitive and \u00a0Tetracycline and Cephotaxime \u00a0fail to inhibit V. vulnificus<\/em> \u00a0isolates. Antibiotics are frequently used to cure diseases but there is always a risk of bacteria developing resistance and residues in the product (Fjalestad et al.,<\/em> 1993). Vibrio vulnificus<\/em> isolates sensitive with many antibiotics and resistant with Tetracycline and Cephotaxime. \u00a0Li et al.<\/em> (1999) reported that Cefriaxone, Nalidixic acid, Chloramphenicol and Sulphamethoxazole were sensitive with isolates of V. vulnificus<\/em> isolated from moribund silver sea bream.<\/p>\n

Table 2: Sensitive and Resistance pattern of Vibrio vulnificus<\/em>\u00a0 <\/strong>(54 isolates) to Etroplus suratensis.<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
S. No<\/strong><\/td>\nAntibiotics<\/strong><\/td>\nSymbol<\/strong><\/td>\nDisc content (mcg)<\/strong><\/td>\nZone of inhibition(mm)<\/strong><\/td>\nResult<\/strong><\/td>\n<\/tr>\n
Minimum<\/strong><\/td>\nMaximum<\/strong><\/td>\nMean \u00b1 SEM*<\/strong><\/td>\n<\/tr>\n
1<\/td>\nGentamycin<\/td>\nG<\/strong><\/td>\n10<\/td>\n21<\/td>\n30<\/td>\n25.5 \u00b1 4.5<\/td>\nS<\/strong><\/td>\n<\/tr>\n
2<\/td>\nTetracycllin<\/td>\nT<\/strong><\/td>\n30<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\nR<\/strong><\/td>\n<\/tr>\n
3<\/td>\nNorflaxacin<\/td>\nNX<\/strong><\/td>\n10<\/td>\n22<\/td>\n28<\/td>\n25 \u00b1 3 .0<\/td>\nS<\/strong><\/td>\n<\/tr>\n
4<\/td>\nNalidixic acid<\/td>\nNA<\/strong><\/td>\n30<\/td>\n21<\/td>\n26<\/td>\n23.5 \u00b1 3.5<\/td>\nS<\/strong><\/td>\n<\/tr>\n
5<\/td>\nCephotaxime<\/td>\nCE<\/strong><\/td>\n30<\/td>\n18<\/td>\n22<\/td>\n20.0 \u00b1 2.0<\/td>\nR<\/strong><\/td>\n<\/tr>\n
6<\/td>\nLomefloxacin<\/td>\nLO<\/strong><\/td>\n10<\/td>\n22<\/td>\n28<\/td>\n25.0 \u00b1 3.0<\/td>\nS<\/strong><\/td>\n<\/tr>\n
7<\/td>\nLevofloxacin<\/td>\nLE<\/strong><\/td>\n5<\/td>\n19<\/td>\n26<\/td>\n22.5 \u00b1 3.5<\/td>\nS<\/strong><\/td>\n<\/tr>\n
8<\/td>\nCo-trimoxazole<\/td>\nCO<\/strong><\/td>\n1.25\/23.75 mcg<\/td>\n20<\/td>\n28<\/td>\n24.0 \u00b1 4.0<\/td>\nS<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*- Standard\u00a0 Error of the mean<\/strong><\/p>\n

 <\/p>\n

The occurrence of resistance to OXA by fish pathogen been reported to have increased over the recent years. \u00a0Development of drug resistance by fish pathogen has frequently been reported. Disease resistance genes have not been identified in fish. However, the production of\u00a0\u00a0 transgenic fish with enhanced resistance to specific diseases remains as a possibility for the future (Fjalestad et al<\/em>., 1993). \u00a0Although\u00a0\u00a0 the present study recommends the use of sensitive drugs in aquaculture, it is suggested that these antibiotics should be assessed against the set of standards as reported by Austin and Austin (1987) in order to ensure safety.<\/p>\n

The lethal dose (LD50<\/sub>) values of Vibrio vulnificus <\/em>were recorded in table 3.<\/em>\u00a0 The LD50<\/sub> value for the juvenile Etroplus suratensis<\/em> was found to be 104.93<\/sup>. Melanisation followed by reddening and swelling at the site of injection were observed as immediate changes, one to three hours post injection with 10-7<\/sup>and 10-8 <\/sup>CFU \/ fish of V. vulnificus. \u00a0<\/em>Mean lethal dose 50% LD50<\/sub> represent the number of bacteria needed to kill 50% of the inoculated fishes (Reed and Muench, 1938). This test has given a more quantitative assessment of the vaccine potency than the Relative Percent Survival (RPS) method (Ellis, 1988). Higher lethality due to injection challenge was by direct access of the bacterium to the circulatory system.<\/p>\n

Tables 3 (a) : <\/strong>Survival pattern of Etroplus suratensis<\/em> injected with Vibrio vulnificus <\/em>(Intramuscular Route).<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Dilution Factor<\/strong><\/td>\nReplicates<\/strong><\/td>\nDay Post Injection (Dpi)<\/strong><\/td>\nTotal Mortality<\/strong><\/td>\nTotal Survival<\/strong><\/td>\n<\/tr>\n
1<\/strong><\/td>\n2<\/strong><\/td>\n3<\/strong><\/td>\n4<\/strong><\/td>\n5<\/strong><\/td>\n6<\/strong><\/td>\n7<\/strong><\/td>\n<\/tr>\n
10-4<\/sup><\/td>\nR1<\/td>\n6<\/td>\n6<\/td>\n6<\/td>\n6<\/td>\n5<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n8<\/td>\n<\/tr>\n
R2<\/td>\n6<\/td>\n6<\/td>\n6<\/td>\n6<\/td>\n6<\/td>\n5<\/td>\n4<\/td>\n<\/tr>\n
10-5<\/sup><\/td>\nR1<\/td>\n6<\/td>\n6<\/td>\n5<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n3<\/td>\n7<\/td>\n5<\/td>\n<\/tr>\n
R2<\/td>\n6<\/td>\n5<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n3<\/td>\n2<\/td>\n<\/tr>\n
10-6<\/sup><\/td>\nR1<\/td>\n6<\/td>\n5<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n3<\/td>\n3<\/td>\n8<\/td>\n4<\/td>\n<\/tr>\n
R2<\/td>\n6<\/td>\n6<\/td>\n5<\/td>\n4<\/td>\n3<\/td>\n2<\/td>\n1<\/td>\n<\/tr>\n
10-7<\/sup><\/td>\nR1<\/td>\n5<\/td>\n5<\/td>\n4<\/td>\n3<\/td>\n3<\/td>\n2<\/td>\n1<\/td>\n11<\/td>\n1<\/td>\n<\/tr>\n
R2<\/td>\n6<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n3<\/td>\n2<\/td>\n0<\/td>\n<\/tr>\n
10-8<\/sup><\/td>\nR1<\/td>\n5<\/td>\n5<\/td>\n5<\/td>\n3<\/td>\n2<\/td>\n1<\/td>\n0<\/td>\n12<\/td>\n0<\/td>\n<\/tr>\n
R2<\/td>\n6<\/td>\n5<\/td>\n4<\/td>\n4<\/td>\n3<\/td>\n2<\/td>\n0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 3 (b) : <\/strong>Determination of LD50<\/sub> values of\u00a0 Vibrio vulnificus <\/em>isolated <\/strong>from Etroplus suratensis<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n
CFU 0.1ml\/fish<\/strong><\/td>\nMortality<\/strong><\/td>\nSurvival<\/strong><\/td>\nCumulative Mortality<\/strong><\/td>\nCumulative Survival<\/strong><\/td>\nMortality rate<\/strong><\/td>\nMortality Percentage<\/strong><\/td>\n<\/tr>\n
108<\/sup><\/td>\n12<\/td>\n–<\/td>\n42<\/td>\n–<\/td>\n42.42<\/td>\n100.00<\/td>\n<\/tr>\n
107<\/sup><\/td>\n11<\/td>\n01<\/td>\n30<\/td>\n01<\/td>\n30.31<\/td>\n96.80<\/td>\n<\/tr>\n
106<\/sup><\/td>\n08<\/td>\n04<\/td>\n19<\/td>\n05<\/td>\n19.24<\/td>\n79.10<\/td>\n<\/tr>\n
105<\/sup><\/td>\n07<\/td>\n05<\/td>\n11<\/td>\n10<\/td>\n11.21<\/td>\n52.38<\/td>\n<\/tr>\n
104<\/sup><\/td>\n04<\/td>\n08<\/td>\n04<\/td>\n18<\/td>\n4.22<\/td>\n18.18<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

\"Vol4No1_Ass_Vija_for1\"<\/p>\n

Biosca et al.<\/em> (1993) reported the presence of the capsule in Vibrio vulnificus<\/em> biotype 2 and its relationship to virulence for eels. Capsule expression, siderophore production, ability to use hemin and exotoxin production expresses the virulence factors for human (Amaro et al.,<\/em> 1995).\u00a0 Amaro et al.<\/em> (1997) reported that the lipopolysaccharide O-side chain of V. vulnificus<\/em> E is a virulence determinant for eels. Iron acquisition system is involved in the virulence mechanism of V. vulnificus<\/em>. Sung Young Goo et al.<\/em> (2006) reported the presence of OmpU, <\/em>a major outer membrane proteins is an important virulence factor involved in the adherence of V. vulnificus <\/em>to the host cell. Lee et al.<\/em> (2010) reported the presence of I1pA protein of V. vulnificus <\/em>is an important virulence factor involved in the adhesion. The utilization of N-Acetylneuraminic acid and a Sialic acid involved in V. vulnificus <\/em>pathogenesis (Jeong et al<\/em>., 2009). Fouz et al.<\/em> (2002) reported LD50<\/sub> value of V.<\/em> vulnificus<\/em> to be 1.0 X 10-6<\/sup> CFU \/ ml and 1.1 X 10-7<\/sup> CFU \/ ml for eel and tilapia group.<\/p>\n

The virulence of the bacterium depended on the type of species, its susceptibility and also the some extent and environmental conditions.\u00a0 The results of the present study revealed that Vibrio vulnificus<\/em> can become a potential threat to the culture of brackish water species.\u00a0 The present study fulfilled that this kind of study will certainly promote the identification and prevention of Vibriosis from the aquaculture industries.<\/p>\n

Acknowledgement<\/strong><\/p>\n

The author is thankful to Central Institute of Brackish water and Aquaculture (CIBA), Govt. of India, Muttukadu, Chennai, Tamil Nadu for providing Lab facilities and encouragement.<\/p>\n

References<\/strong><\/p>\n

    \n
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