Development of Recombinant Dihydrolipoamide Dehydrogenase Subunit Vaccine against Vibrio Infection in Large Yellow Croaker

Large yellow croaker (Larimichthys crocea), an economically important marine fish in China, has suffered from serious vibriosis, which has resulted in great economic losses for the large yellow croaker industry. Vaccination has been considered to be a safe and effective method to prevent and control vibriosis. However, due to the complex diversity and serotypes of the Vibrio genus, the progress of Vibrio vaccine development is still slow. In this study, we prepared recombinant Vibrio dihydrolipoamide dehydrogenase (rDLD) protein and investigated its potential as a candidate to be a subunit vaccine against Vibrio. The lysozyme activity and the rDLD-specific antibody level in sera of large yellow croakers immunized with rDLD were significantly higher than those in the control group, and the transcript levels of proinflammatory cytokines (IL-6, IL-8, IL-1β), MHC IIα/β, CD40, CD8α, IL-4/13A, and IL-4/13B were significantly up-regulated in the spleen and head kidney of large yellow croakers immunized with rDLD, suggesting that rDLD could induce both specific and nonspecific immune responses in this species. In addition, rDLD protein increased the survival rate of large yellow croakers against Vibrio alginolyticus and Vibrio parahaemolyticus, with the relative percent of survival (RPS) being 74.5% and 66.9%, respectively. These results will facilitate the development of a potential subunit vaccine against Vibrio in large yellow croaker aquaculture.

ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF Origanum vulgare AND Ocimum basilicum AGAINST Vibrio parahaemolyticus AND Vibrio vulnificus AND ADDITION OF THESE OILS ON Mugil platanus FILLETS

Species of the Vibrio genus are commonly reported as agents of food poisoning outbreaks associated with fish consumption. The objective of this study was to assess the antimicrobial activity of the essential oils of Origanum vulgare and Ocimum basilicum against Vibrio parahaemolyticus and Vibrio vulnificus, as well as to assess their addition to fillets of Mugil platanus and sensorial acceptance among consumers. The antimicrobial activity was analyzed by the disc diffusion test and minimal bactericidal concentration (MBC). M. platanus fillets, experimentally contaminated, were marinated for 24 hours in a solution containing 1.0% and 1.5% of essential oil of O. vulgare. For the sensory analysis, samples marinated in 1.5% of O. vulgare oil were prepared and offered to 100 testers. The oil of O. basilicum shows no antimicrobial activity, so its application would be ineffective; for such reason the subsequent tests were not performed. However, the essential oil of O. vulgare produced inhibition halo diameters ranging from 24.6 to 34.1 mm, on average, and the MBC ranging from 3.9% to 15.6%. When added to the product, the essential oil of O. vulgare eliminated the microorganisms that were experimentally inoculated. In the sensorial analysis, samples marinated in 1.5% of O. vulgare essential oil obtained an average of 6.82 of acceptance note among consumers.

Characterization of Potential Virulence Factors of Vibrio mimicus Isolated from Fishery Products and Water

Vibrio mimicus is a Gram-negative bacterium that is closely related to V. cholerae and causes gastroenteritis in humans due to contaminated fish consumption and seafood. This bacterium was isolated and identified from 238 analyzed samples of sea water, oysters, and fish. Twenty strains were identified as V. mimicus according to amplification of the vmhA gene, which is useful as a marker of identification of the species. The production of lipases, proteases, and nucleases was detected; 45% of the strains were able to produce thermonucleases and 40% were capable of producing hydroxamate-type siderophores, and the fragment of the iuT gene was amplified in all of the V. mimicus strains. Seventy-five percent of V. mimicus strains showed cytopathic effect on Chinese hamster ovary (CHO) cells and destruction of the monolayer, and 100% of the strains were adherent on the HEp-2 cell line with an aggregative adherence pattern. The presence of virulence factors in V. mimicus strains obtained from fishery products suggests that another member of the Vibrio genus could represent a risk to the consumer due to production of different metabolites that allows it to subsist in the host.

Insights into the chemistry of the amphibactin–metal (M3+) interaction and its role in antibiotic resistance

We have studied the diversity and specificity of interactions of amphibactin produced by Vibrio genus bacterium (Vibrio sp. HC0601C5) with iron and various metal ions in + 3 oxidation state in an octahedral (Oh) environment. To survive in the iron-deficient environment of their host, pathogenic bacteria have devised various efficient iron acquisition strategies. One such strategy involves the production of low molecular weight peptides called siderophores, which have a strong affinity and specificity to chelate Fe3+ and can thus facilitate uptake of this metal in order to ensure iron requirements. The Fe uptake by amphibactin and the release of iron inside the cell have been studied. Comparison of the interaction of different transition metal ions (M3+) with amphibactin has been studied and it reveals that Co and Ga form stable complexes with this siderophore. The competition of Co and Ga with Fe impedes iron uptake by bacteria, thereby preventing infection.

Isolation of agarase-producing bacteria from seawater and examination of the enzyme activity

This study aimed to isolate agarase-producing bacteria from seawater, and then determine activity of the agarase. Eight coastal surface seawater samples were collected from Ba Ria - Vung Tau province. Twenty-one bacterial strains that are capable of liquefying agar were isolated. These isolates produced disintegration zones around their colonies on agar plates with diameters ranging from 4.0 to 7.0 cm after an incubation period of 2 days at room temperature. Five bacterial strains (M1, M5, M7, M62B, and M71) that produced large halos on plates were identified belonging to Vibrio genus with identity > 96%. The crude enzyme activities of these strains ranged from 0.15 to 0.22 U/mL in reaction with agarose as substrate. Among isolated strains, the strain M71 showed the highest agarase activity, and was used to examine the degradation of seaweed. The hydrolysis of dried Gracilaria seaweed by the crude enzyme of M71 at concentration of 5% (v/v) released 915 μM/mL reducing sugar after a 24-h incubation period at 40oC.

Strategies of Controlling Vibriosis in Fish

The growth of the aquaculture industry is becoming more important in the food production sector. Presently, the biggest problem in aquaculture is bacterial diseases that infect the host thus causing outbreaks and economic losses. Vibriosis is a potentially lethal threat in commercial fish farming. Based on previous researches, various infections of the Vibrio genus have been reported hence their virulence is not fully understood. This review aims to provide enlightenment through research findings on Vibriosis-infected fish. Besides, some guidelines regarding control measures of Vibrio in aquaculture, such as the application of immunostimulant, antibiotics, probiotics and quorum sensing molecules, will be also reviewed.

CAPACIDAD DE ADHESIÓN A MOCO DE CORAL DE BACTERIAS AISLADAS DE Pocillopora sp. Y Porites panamensis DEL SUROESTE DEL GOLFO DE CALIFORNIA

El estudio de la microbiota en corales es de fundamental importancia para un mejor entendimiento de los procesos que determinan su asociación con el holobionte, sin embargo, aún se conoce poco acerca de los mecanismos básicos de dicha asociación. En esta investigación el objetivo fue aislar a partir de corales sin signos aparentes de enfermedad, las bacterias predominantes de la comunidad, determinar su capacidad de adhesión al moco producido por Pocillopora sp. e identificarlas molecularmente. Se recolectaron corales de los géneros Pocillopora sp. y P. panamensis, se cuantificó la población de cuatro grupos de microorganismos (expresados como Log de UFC g-1): bacterias mesófilas aerobias (4.7 - 6.4), bacterias ácido lácticas (˂1.0-5.8), bacterias del género Vibrio (˂1.0-4.5), así como hongos y levaduras (˂1.0-3.6). Se aislaron 156 cepas bacterianas del holobionte homogeneizado y se seleccionaron aquellas con mayor crecimiento a las 24 h para el ensayo de adhesión, que consistió en 25 cepas de Pocillopora sp. y 27 de Porites panamensis. El ensayo de adhesión al extracto crudo de moco de Pocillopora sp., marcado enzimáticamente con HRP mostró que el 82 % de las cepas se adhieren. Se extrajo ADN de todas las cepas, sin embargo, con los oligonucleótidos universales utilizados se obtuvieron productos de PCR solo de 32. Se identificaron molecularmente 14 de Pocillopora sp. y 18 de P. panamensis con base en la secuenciación y análisis del gen 16S DNAr. Las cepas identificadas correspondieron a 17 especies, donde predominó el género Bacillus, con 64 % en Pocillopora sp. y 44 % en P. panamensis. Las especies de bacterias que comparten estos corales son B. subtilis y Staphylococcus hominis. Se sugiere que las especies identificadas adherentes tienen la capacidad de colonizar el moco del coral, son comensales y potencialmente benéficas, debido a que fueron aisladas de corales aparentemente sanos. Adhesion ability to coral mucus of isolated bacteria from Pocillopora sp. and Porites panamensis of California Gulf southeast ABSTRACT. The study of coral microbiota is of fundamental importance for a better understanding of the processes that determine its association with the holobiont, however, little is known about the basic mechanisms of this association. In this research, the objective was to isolate from corals without apparent signs of disease, the predominant bacteria of the community, determine their ability to adhere to the mucus produced by Pocillopora sp. and identify them molecularly. Corals of the genera Pocillopora sp. and Porites panamensis were recollected, the population of four groups of microorganisms (expressed as Log of CFU g-1) was quantified. Results indicated aerobic mesophilic bacteria (4.7 - 6.4), lactic acid bacteria (˂1.0-5.8), bacteria of the Vibrio genus (˂ 1.0-4.5), as well as fungi and yeasts (˂1.0-3.6). 156 bacterial strains of the homogenized holobiont were isolated and those with the highest growth at 24 h were selected for the adhesion test, which consisted of 25 strains of Pocillopora sp. and 27 of P. panamensis. The adhesion test to the enzymatically labeled with HRP crude mucus extract of Pocillopora sp., showed that 82% of the strains adhere. DNA was extracted from all strains, however, with the universal oligonucleotides used, only 32 PCR products were obtained. 14 strains from Pocillopora sp. and 18 from P. panamensis were molecularly identified based on the sequencing and analysis of the 16S DNAr gene. The strains identified corresponded to 17 species, where the genus Bacillus predominated, with 64% in Pocillopora sp. and 44% in P. panamensis. The species of bacteria that share these corals are B. subtilis and Staphylococcus hominis. It is suggested that the identified adherent species have the ability to colonize coral mucus, are commensal and potentially beneficial, because they were isolated from apparently healthy corals.

IDENTIFIKASI BAKTERI PADA TUBUH LALAT RUMAH (Musca domestica Linn.) DI TEMPAT PEMBUANGAN AKHIR SAMPAH (TPA) DAN PASAR

ABSTRACT Several species of flies are the most important species in public health problems, especially as a disease transmission vector. One of them is Musca domestica. The role of flies in the spread of disease is as a mechanical vector, by bringing the seeds of disease through the limbs. Therefore it is necessary to understand what bacteria found in the body of M. domestica fly in Sukawinatan landfill, Palembang. This study aimed to find out the type of bacteria in the body of M. domestica in Sukawinatan landfill, Palembang and Jakabaring Main Market. This study was conducted from June to August 2017. The sampling site of home fly (M. domestica) was in Sukawinatan landfill and the Jakabaring main market of Palembang. Meanwhile, the research was conducted in Biology Laboratory, Faculty of Mathematics and Natural Sciences, Universitas PGRI Palembang and Microbiology Laboratory of Department of Biology, FMIPA UNSRI Indralaya. Bacteria found on the body of flies were 6 isolates, 4 bacterial isolates in the fly originating from Sukawinatan landfill and 2 bacterial isolates in the fly from Jakabaring main market. Four (4) bacteria were found in Sukawinatan landfill from Salmonella, Providencia, Escherichia and Vibrio genus. Meanwhile, bacteria found in fly species at Jakabaring main market were 2 bacteria from Salmonella and Proteus genus. Keywords: Bacteria; House fly (Musca domestica).

Characterization of prodiginine compounds produced by a Vibrio species isolated from salt flat sediment along the Florida Gulf Coast

Prodiginines are secondary metabolites produced by several known species of bacteria. These metabolites are known for their bright pigmentation and their potential medicinal uses. Biosynthesis of prodiginine compounds, including the well-studied prodigiosin, has been well characterized in Serratia marcescens and other bacterial species, including several marine bacteria. In an effort to isolate and identify natural products from marine organisms, an environmental sample was taken from a salt flat along the Florida Gulf Coast and cultured for bacterial growth. A bacterial species that produces a vibrant pink pigment was isolated and identified as a member of the Vibrio genus and was named MI-2. Whole genome sequencing identified a 13-gene operon with homology to the S. marcescens prodigiosin biosynthetic operon. The pigment produced by MI-2 was hypothesized to be composed of prodigiosin or related prodiginine compounds and was purified by flash column chromatography and identified by mass spectrometry.