Vibrio harveyi phages: Isolation, characterization and evaluation of their potential as phage therapeutics on Vibrio harveyi in shrimp hatcheries

Aquaculture is a global industry providing food and employment thereby contributing to the economy. For the sustenance of aquaculture, disease management is a major requirement. Among the bacterial pathogens Vibrio harveyi remains to be the major one especially in shrimp culture systems. Rapid and mass mortality of shrimp larvae due to Vibrio harveyi infection is well known, and the pathogen causes serious economic losses in grow out systems as well. It suggests that a well defined management strategy has to be built up to protect the crop from Vibrio harveyi infection in aquaculture systems. Antibiotics have been the choice for quite some times which led to residues in meat and development of multidrug resistant bacteria which invited ban on their application. In this context several alternate options have been thought off such as probiotics, immunostimulants and vaccines. Phage therapy is yet another option. Phages being natural parasites of bacteria and are abundant in aquatic environments their application to control bacterial pathogens in aquaculture has commendable potential in lieu of antibiotics. For that matter the therapeutic effect of phages has been proven in several antibiotic resistant pathogens inclusive of Vibrio harveyi.

Molecular Approaches For Characterization And Determination Of Pathogenicity Of Vibrios With Special Reference To Vibrio Harveyi From Penaeus Monodon Larval Production Systems

The present study focuses on vibrios especially Vibrio harveyi isolated from shrimp (P. monodon) larval production systems from both east and west coasts during times of mortality. A comprehensive approach has been made to work out their systematics through numerical taxonomy and group them based on RAPD profiling and to segregate the virulent from non- virulent isolates based on the presence of virulent genes as well as their phenotypic expression. The information gathered has helped to develop a simple scheme of identification based on phenotypic characters and segregate the virulent from non virulent strains of V. harveyi.

Probiotic effect of Bacillus NL110 and Vibrio NE17 on the survival, growth performance and immune response of Macrobrachium rosenbergii (de Man)

Eight hundred and eighty-¢ve strains of bacterial isolates fromvarious samples associatedwith the natural habitat ofMacrobrachiumrosenbergii were screened for their probiotic potential. Two putative probionts namely Bacillus NL110 and Vibrio NE17 isolated from the larvae and egg samples, respectively, were selected for experimental studies and were introduced to the juveniles of M. rosenbergii (0.080 0.001g) through di¡erent modes such as through feed, water and both. The probiotic potential of the above bacteria in terms of improvements inwater quality, growth, survival, speci¢c growth rate (SGR), feed conversion ratio and immune parameters was evaluated. The treatment groups showed a signi¢cant improvement in SGR and weight gain (Po0.001). Survival among di¡erent treatment groups was better than that in the control group. There were also signi¢cant improvements in the water quality parameters such as the concentration of nitrate and ammonia in the treatment groups (Po0.05). Improvements in immune parameters such as the total haemocyte count (Po0.05), phenoloxidase activity and respiratory burst were also signi¢cant (Po0.001). It is concluded that screening of the natural micro£ora of cultured ¢sh and shell¢sh for putative probionts might yield probiotic strains of bacteria that could be utilized for an environment-friendly and organic mode of aquaculture.

Probiotic effect of Bacillus NL110 and Vibrio NE17 on the survival, growth performance and immune response of Macrobrachium rosenbergii (de Man)

Eight hundred and eighty-¢ve strains of bacterial isolates fromvarious samples associatedwith the natural habitat ofMacrobrachiumrosenbergii were screened for their probiotic potential. Two putative probionts namely Bacillus NL110 and Vibrio NE17 isolated from the larvae and egg samples, respectively, were selected for experimental studies and were introduced to the juveniles of M. rosenbergii (0.080 0.001g) through di¡erent modes such as through feed, water and both. The probiotic potential of the above bacteria in terms of improvements inwater quality, growth, survival, speci¢c growth rate (SGR), feed conversion ratio and immune parameters was evaluated. The treatment groups showed a signi¢cant improvement in SGR and weight gain (Po0.001). Survival among di¡erent treatment groups was better than that in the control group. There were also signi¢cant improvements in the water quality parameters such as the concentration of nitrate and ammonia in the treatment groups (Po0.05). Improvements in immune parameters such as the total haemocyte count (Po0.05), phenoloxidase activity and respiratory burst were also signi¢cant (Po0.001). It is concluded that screening of the natural micro£ora of cultured ¢sh and shell¢sh for putative probionts might yield probiotic strains of bacteria that could be utilized for an environment-friendly and organic mode of aquaculture

Detection and diversity of pathogenic Vibrio from Fiji

Here we investigate the diversity of pathogenic Vibrio species in marine environments close to Suva, Fiji. We use four distinct yet complementary analyses – biochemical testing, phylogenetic analyses, metagenomic analyses and molecular typing – to provide some preliminary insights into the diversity of vibrios in this region. Taken together our analyses confirmed the presence of nine Vibrio species, including three of the most important disease-causing vibrios (i.e. V. cholerae, V. parahaemolyticus and V. vulnificus), in Fijian marine environments. Furthermore, since toxigenic V. parahaemolyticus are present on fish for consumption we suggest these bacteria represent a potential public health risk. Our results from Illumina short read sequencing are encouraging in the context of microbial profiling and biomonitoring. They suggest this approach may offer an efficient and costeffective method for studying the dynamics of microbial diversity in marine environments over time.

Probiotic Effects of Lactic Acid Bacteria Against Vibrio Alginolyticus in Penaeus (Fenneropenaeus) Indicus (H. Milne Edwards)

Cell free extracts of four strains of Lactic acid bacteria (LAB) viz. Lactobacillus. acidophilus, Streptococcus.cremoris, Lactobacillus bulgaricus –56 and Lactobacillus bulgaricus –57 inhibited growth of Vibrio alginolyticus in nutrient broth. The antagonism of LAB to Vibrio alginolyticus was further confirmed by streak plating wherein suppression of growth of Vibrio was obtained. Juveniles of Penaeus indicus (average weight 0.985 ± 0.1 g) on administering orally a moist feed base containing 5 × 106 cells·g of the four LAB probionts for a period of four weeks showed better survival (56 to 72%) when challenged with V. alginolyticus by intra-muscular injection of 0.1 ml containing 3 × 109 cells·ml. Animals maintained on a diet devoid of bacterial biomass exhibited 80% mortality. No external or internal pathological changes were observed in shrimp fed with the LAB incorporated diets. Results showed inhibition of V. alginolyticus by LAB and stimulation of the non-specific immune response resulting in resistance to disease in the shrimp fed on LAB incorporated diets.

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi

Persistence of the antivibrio property of the potential antagonistic probiotics, Pseudomonas MCCB 102 and 103, at di¡erent temperatures, pH and in organic solvents was studied. The antivibrio compound was extracted, puri¢ed and characterized using thin-layer chromatography, high-pressure liquid chromatography, liquid chromatography-mass spectroscopy, UV^ Vis and nuclear magnetic resonance spectroscopy and identi¢ed as N-methyl-1-hydroxyphenazine, a phenazine antibiotic. The toxicity of the compound was tested in Penaeus monodon haemocyte culture and the IC50 valuewas found to be1.4 0.31mg L 1. The compound was found to be bacteriostatic at 0.5mg L 1. Its stability to varying temperature, pH, organic solvents, prolonged shelf-life and vibriostatic nature point to its suitability for prophylatic aquaculture application.

Reducing Vibrio load in Artemia nauplii usingantimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Isolation and characterization of broad spectrum bacteriophages lytic to Vibrio harveyi from shrimp farms of Kerala, India

Of 33 phages isolated from various shrimp farms in Kerala, India, six were segregated to have broad spectrum lytic efficiency towards 87 isolates of Vibrio harveyi with cross-infecting potential to a few other important aquaculture pathogens. They were further tested on beneficial aquaculture micro-organisms such as probiotics and nitrifying bacterial consortia and proved to be noninfective. Morphological characterization by transmission electron microscopy (TEM) and molecular characterization by RAPD and SDS-PAGE proved them distinct and positioned under Caudovirales belonging to Myoviridae and Siphoviridae

Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans

This is a valuable research work in which authors have demonstrated the antagonistic effects of pseudomonas on the growth of vibrio

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus.Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administeringAPV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.

Impact of process parameters on L-glutaminase production by marine Vibrio costicola in solid state fermentation using polystyrene as an inert support

Process parameters influencing e-glutaminase production by marine Vibrio costicola in solid state fermentation (SSF) using polystyrene as an inert support were optimised. Maximal enzyme yield (157 U/g dry substrate) was obtained at 2% (w/w) t:glutamine, 35°C and pH 7.0 after 24 h. Maltose and potassium dihydrogen phosphate at 1% (w/w) concentration enhanced enzyme yield by 23 and 18%, respectively, while nitrogen sources had an inhibitory effect. Leachate with high specific activity for glutaminase (4.2 U/mg protein) and low viscosity (0-966 Ns/m 2) was recovered from the polystyrene SSF system

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples from nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which are commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rifampicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimethoprirn, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Short Communication: Polystyrene-an inert carrier for L·glutaminase production by marine Vibrio costico/a under selld-state fermentation

Polystyrene beads, impregnated with mineral salts/glutamine medium as inert support, were used to produce L-glutaminase from Vibrio costicola by solid-state fermentation. Maximum enzyme yield, 88 U/g substrate, was after 36 h. Glucose at 10 g/kg enhanced the enzyme yield by 66%. The support system allowed glutaminase to be recovered with higher specific activity and lower viscosity than when a wheat-bran system was used