Pathogenic Vibrio Sp Research Articles

COMPARATIVE STUDY ON THE INVITRO ANTIBACTERIAL ACTIVITY OF SELECTED MEDICINAL PLANTS AGAINST PATHOGENIC VIBRIO SPECIES FROM DISEASED PENAEUS MONODON (FAB)

The pathogenic Vibrio sp. were isolated from diseased shrimp of Penaeus monodon (FAB) using TCBS agar medium by streak plate method. Acalipha indica (L), Vinca rosea (L), Embilica officinalis (G), Ocimum sanctum(L) and Lucas aspera (L) medicinal plants are one of the most commonly used natural antimicrobial agents used traditionally. Hence an attempt has been made to screen the antivibrio activity of some selected medicinal plants against pathogenic Vibrio sp by disk diffusion method. Among the five plants tested, water extract of Lucas aspera showed the maximum zone of inhibition against Vibrio cholerae (18±3.4 mm in diameter) and the alcohol extract showed the maximum zone of inhibition against Vibrio harveyi (NL) (16±3 mm in diameter).

Isolation and Characterization of Specific Phages to Prepare a Cocktail Preventing Vibrio sp. Va-F3 Infections in Shrimp (Litopenaeus vannamei).

Vibrio is one of the most detrimental agents of shrimp premature death syndrome. Phage therapy for prevention and treatment of Vibrio infections has attracted increasing attentions due to the emergence of antibiotic-resistant bacterial variants. Here, we describe a workflow of preparing a phage cocktail against Vibrio infections for practical applications. Twenty Vibrio strains were isolated from the gut of diseased shrimp and aquaculture wastewater, and five of them were identified as pathogens causing shrimp vibriosis. Twenty-two lytic phages were then isolated using the above five pathogens as hosts, and five of them showed broad host ranges and high lytic capability against the Vibrio strains. Whole genomic sequencing and phylogenetic analysis of the five phages indicated that they are novel and belong to the Siphoviridae family. The phage cocktail consisting of these five phages showed higher efficiency in inhibiting the growth of pathogenic Vibrio sp. Va-F3 than any single phage in vitro. We then evaluated the performance of the phage cocktail in protecting shrimp against Vibrio sp. Va-F3 infections in situ. The results showed that shrimp survival rates could reach 91.4 and 91.6% in 7 days, for the cocktail-treated and the antibiotic-treated groups, respectively. By contrast, the shrimp survival rate of the group without any treatment was only 20.0%. Overall, this study describes a general workflow of how to prepare a phage cocktail and apply it in controlling bacterial infections in the shrimp aquaculture. Knowledge gained from this study will not only help fight against the shrimp vibriosis in practical but also facilitate the design of phage cocktails with a satisfying performance in controlling other animal diseases in aquaculture.

Targeted metabolomics to investigate antimicrobial activity of itaconic acid in marine molluscs.

Itaconic acid (ITA) has recently been identified as an antimicrobial metabolite in mammalian immune cells. The presence of ITA was alsoreported in different tissues of marine molluscs, indicating its role as an endogenous metabolite of molluscs. In addition, the accumulation of ITA has been observed in different tissues of mussels following pathogen challenges. However, the concentration of ITA in mussel tissues and the possible role of this metabolite in the molluscan innate immune system remain unknown. This study aims to quantitatively measure ITA levels in different tissues of marine mussels following an experimental challenge with Vibrio sp. DO1 isolate, and to identify the antimicrobial role of ITA in the innate immune system through the measurement of metabolic and immune alterations in tissues following the challenge. In this study, adult Perna canaliculus mussels were experimentally challenged with a pathogenic Vibrio sp. DO1 isolate. The metabolite profiles of five different tissues, including mantle, gill, muscle, hepatopancreas and haemolymph were obtained, and levels of ITA in each tissue were characterized using a gas chromatography-mass spectrometry (GC-MS) metabolomics approach. Flow cytometry was also employed to measure cell health parameters, including oxidative stress via reactive oxygen species (ROS) production, apoptosis via changes in mitochondrial membrane potential (MMP) and haemocyte viability. The ITA levels in mantle, gill, muscle and hepatopancreas tissues at 18-h post infection (hpi) with Vibrio sp. were 40.31, 41.71, 11.61 and 41.66ngmg-1, respectively. In haemolymph, the level of ITA was significantly increased from 95.25ngml-1 at 6 hpi to 174.36ngml-1 at 18hpi and 572.12ngml-1 at 60hpi. In line with the accumulation of ITA, we observed increased levels of metabolites within the tricarboxylic acid (TCA) cycle, anti-inflammatory metabolites and alterations of other metabolites associated with immune responses of the host. The flow cytometry analyses revealed increases in ROS production, apoptotic cells and decreases in cell viability. We reported on the production of ITA in different tissues of P. canaliculus mussels challenged with a marine pathogen which confirmed ITA as an antimicrobial metabolite. The findings revealed insights into the biosynthesis of ITA and suggests its role in antimicrobial and anti-inflammatory activities in the innate immune system. This study also provided insights into the innate immune system of bivalves and highlighted the potential use of ITA as a biomarker for shellfish health assessment in aquaculture.

Recommendations for identifying pathogenic Vibrio spp. as part of disease surveillance programmes in recirculating aquaculture systems for Pacific white shrimps (Litopenaeus vannamei).

Due to their pathogenic potential, identifying Vibrio species from recirculating aquaculture systems (RAS) for Pacific white shrimp (Litopenaeus vannamei) is of great importance to determine the risk for animal's as well as for the consumer's health. The present study compared identification results for a total of 93 Vibrio isolates, including type strains and isolates from shrimp aquaculture. Results from biochemical identifications, 16S rRNA sequencing, sequencing of the uridylate kinase encoding gene pyrH and analysis of the protein spectra assessed by MALDI-TOF MS were compared. The results achieved by these different methods were highly divergent for many of the analysed isolates and for several Vibrio spp difficulties in reliably identifying occurred. These difficulties mainly resulted from missing entries in digital databases, a low number of comparable isolates analysed so far, and high interspecific similarities of biochemical traits and nucleotide sequences between the closely related Vibrio species. Due to the presented data, it can be concluded that for identifying Vibrio spp. from samples in routine diagnostics, it is recommended to use MALDI-TOF MS analysis for a quick and reliable identification of pathogenic Vibrio sp. Nevertheless, editing the database, containing the main spectra of Vibrio is recommended to achieve reliable identification results.

Metabolomics Study of Immune Responses of New Zealand Greenshell™ Mussels (Perna canaliculus) Infected with Pathogenic Vibrio sp.

Vibrio coralliilyticus is a bacterial pathogen which can affect a range of marine organisms, such as corals, fish and shellfish, with sometimes devastating consequences. However, little is known about the mechanisms involved in the host-pathogen interaction, especially within molluscan models. We applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics to characterize the physiological responses in haemolymph of New Zealand Greenshell™ mussels (Perna canaliculus) injected with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Univariate data analyses of metabolite profiles in Vibrio-exposed mussels revealed significant changes in 22 metabolites at 6h post-infection, compared to non-exposed mussels. Among them, 10 metabolites were up-regulated, while 12 metabolites were down-regulated in infected mussels. Multivariate analyses showed a clear distinction between infected and non-infected mussels. In addition, secondary pathway analyses indicated perturbations of the host innate immune system following infection, including oxidative stress, inflammation and disruption of the TCA cycle, change in amino acid metabolism and protein synthesis. These findings provide new insights into the pathogenic mechanisms of Vibrio infection of mussels and demonstrate our ability to detect detailed and rapid host responses from haemolymph samples using a metabolomics approach.

Antibacterial activity of immobilized silver nanoparticles on TEPA ‐Den‐SiO 2 against shrimp pathogen, Vibrio sp. Persian1

In this study, we prepared silver nanoparticles immobilized onto silica sand beads as an antibacterial material against pathogenic luminous Vibrio sp. Persian1. Silica beads were modified with 3-aminopropyltriethoxysilane (APTES), cyanuric chlorid and tetraethylene pentamine, and silver nanoparticles were generated in various concentrations (0.5, 1, and 2 mM g−1 of silica beads) of AgNO3 on the surface using chemical reduction. Ag/TEPA-Den-SiO2 samples were characterized by TEM, FE-SEM/EDS, FT-IR and ICP OES and their antibacterial activity assayed by zone of inhibition and test tube tests against pathogenic Vibrio sp. The results of the zone inhibitory test revealed that all the Ag/TEPA-Den-SiO2 samples had an antibacterial effect against luminous Vibrio sp. Persian1. In addition, the tube test results showed 100% killing of bacteria in 2 h contact period. Ag/TEPA-Den-SiO2 samples maintained their antibacterial activity after 14-day immersion in seawater by slow release of silver ions. These results suggest that Ag/TEPA-Den-SiO2 substrates could be effective antibacterial materials for disinfection of seawater used to culture Penaeid shrimp larvae.

Beneficial uses of probiotics in mass scale production of marine ornamental fish

Mass scale culture of marine ornamental fish is facing many problems caused by pathogenic microorganisms. The accumulation of unconsumed feed and bad water quality influence the growth of pathogenic forms of the Vibrio sp. To avoid these problems probiotics are used as an alternative agent in vitro for efficacy based on the inhibition of growth of pathogenic Vibrio sp and also it decreased the concentrations of ammonium, nitrite, nitrate and phosphate ions. The present study revealed that after the use of probiotics in experimental tank the beneficial microbial load is drastically increased but decreased the level of pathogenic Vibrios. The bacterial population changed during every sampling. THB in the experimental tank before the application of probiotics ranged from 2.39 x 103 to 4.82 x 104. After the application of probiotics, THB population varied between 4.21x 106 and 7.43 x 107. In the control tank, THB count was found to be between 5.28x 103 and 2.1 x 105, but Vibrio load was high. Vibrio sp counts ranged from 6.82 x102 to 9.21 x 103 CFU/ml in the experimental tank prior to application of probiotics. After the application of probiotics, Vibrio population varied between 1.62 X102 and 3.96 x 102 CFU/ml. In the control tank, Vibrio count was found to be between 8.56 x102 and 2.1 x 104 CFU/ml. Key words: Probiotics, ornamental fish, disease.

Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites

High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Rivière d'Auray (Brittany) and Ronce (Marennes-oléron Basin, Poitou Charentes). Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with Vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production. Among the six families analyzed, three have shown high survival during summer (named as “resistant”, mean mortality 5.2%) and three experienced high mortality during summer (named as “susceptible”, 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events.

Microbial profile of organically enriched freshwater ponds in south‐easthern Nigeria

Samples of chicken manure and water from fish ponds receiving the latter were found to contain a relatively high bioload of micro‐organisms. Aerobic heterotropic counts in chicken manure were in the range of 1.75 x 10∗ colony forming units CFU per ml. with coliforms constituting about 7.4% of this value. Salmonella and Shigella similarly occurred in relatively high proportions. Iron oxidizing bacteria were also detected. Homogenized tissue of fish growing in the ponds was found to contain on the average 0.25% of total bioloads as pathogenic Vibrio sp. The implication of the use of chicken manure in fish ponds is discussed.

Lactic acid bacteria increase the resistance of turbot larvae,Scophthalmus maximus,against pathogenic vibrio

Characteristics of Lactic Acid Bacteria (LAB) isolated from rotifers, Brachionus plicutilis, resembled those of Lactobacillus plantarum or Carnobacterium sp. It was investigated whether the artificial maintenance of a high concentration of LAB in rotifers might increase their dietary value for turbot larvae, particularly when the fish were infected with pathogenic Vibrio sp. One LAB strain was cultivated, then introduced daily into the enrichment medium of rotifers. In this way, LAB were retrieved in large amounts in turbot, and a significant limitation of larval mortality rate was observed when turbot were challenged with pathogenic vibrio at day 9. The inoculum concentration of LAB had a decisive effect on survival rate, and the optimum was between 107 and 2 × 107 Colony Forming Units (CFU) daily added per ml of the enrichment medium (53% survival rate after 72 h of challenge, versus 8% for the infected control group without LAB).

Effect of sodium nifurstyrenate on the reduction of bacterial contamination of rotifers ( Brachionus plicatilis)

Although sodium nifurstyrenate (NFS-Na) has been shown effective in suppressing bacterial contamination of live-food rotifers ( Brachionus plicatilis), the earlier experiment dealt with a density of live food lower than used in a commercial marine hatchery. This study attempted to assess the effectiveness of NFS-Na with rotifers at greater densities. The drug was also tested with rotifers artificially contaminated with a bacterial pathogen ( Vibrio sp. INFL) of Japanese flounder larvae ( Paralichthys olivaceus). The viable bacterial counts on ZoBell's 2216e agar inoculated with samples drawn from the treated rotifers at densities of 1610, 8060 and 16 100 individuals/ml were slightly lower than the counts of untreated rotifers. There was no difference between samples treated with 5 or 10 μg/ml NFS-Na. On the other hand, the counts on TCBS agar at the three densities of rotifers showed a marked downward trend from 2.6 × 10 6 CFU/g to 2.9 × 10 4, 2.0 × 10 4 and 4.0 × 10 3 CFU/g after exposure to 5 μg/ml, and to 4.0 × 10 3, 4.0 × 10 3 and 7.0 × 10 3 CFU/g after exposure to 10 μg/ml of the drug for 5 h, respectively. A lower number of Vibrio isolates was observed to correspond with greater numbers of Moraxella and/or Pseudomonas in the NFS-Na-treated rotifers at all densities tested. For the Vibrio sp. INFL-loaded rotifers, the total and Vibrio sp. INFL counts at the beginning were 1.2 × 10 8 and 1.1 × 10 8 CFU/g on ZoBell agar but 1.6 × 10 7 and 1.3 × 10 7 CFU/g on TCBS agar, respectively. The number of bacteria in the control on both ZoBell and TCBS agar media was slightly lower during a 5-h period. In the NFS-Na-treated groups, the number of bacteria on the two media, particularly on the TCBS agar, was much lowered. The TCBS counts of rotifers at densities of 970 and 9700 individuals/ml decreased from 1.6 × 10 7 CFU/g to 9.1 × 10 3 and 2.9 × 10 3 CFU/g after exposure to 5 μg/ml, and to 3.2 × 10 3 and 8.2 × 10 2 CFU/g after exposure to 10 μg/ml of the drug for 5 h, respectively. No pathogenic Vibrio sp. INFL was detected from any treated rotifers after 3 h exposure.