Hybridation des acides déoxyribonucléiques de certaines bactéries marines vibrioïdes

The genotypic relationships established by DNA/DNA hybridization in vitro confirm the results obtained by earlier phenotypic analyses of certain vibrio-like marine bacteria. These strains are closely related to the species Photobacterium fischeri, and do not belong to the genus Vibrio. The group of marine, vibrio-like bacteria that require Na⁺ for growth is genotypically very heterogeneous.     

Vibrios Associated with Litopenaeus vannamei Larvae, Postlarvae, Broodstock, and Hatchery Probionts

Several bacteriological surveys were performed from 1994 to 1996 at different Litopenaeus vannamei hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased L. vannamei larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850 Vibrio type and reference strains. A selection of strains was further genotypically fine typed by AFLP. Vibrio alginolyticus is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity among V. alginolyticus strains, and the results suggest that putative probiotic and pathogenic strains each have specific genotypes. V. alginolyticus was found to be associated with larvae with the zoea 2 syndrome and the mysis mold syndrome, while different Vibrio species (V. alginolyticus and V. harveyi) are associated with the bolitas syndrome. V. harveyi is associated with diseased postlarvae, juveniles, and broodstock. The identities of the strains identified as V. harveyi by the Biolog system could not be unambiguously confirmed by AFLP genomic fingerprinting. Vibrio strain STD3-988 and one unidentified strain (STD3-959) are suspected pathogens of only juvenile and adult stages. V. parahaemolyticus, Photobacterium damselae, and V. mimicus are associated with juvenile and adult stages.     

Effects of mannan oligosaccharide dietary supplementation on performances of the tropical spiny lobsters juvenile (Panulirus ornatus,Fabricius 1798)

The effects of dietary mannan oligosaccharide (MOS) (Bio-Mos^®, Alltech, USA) on the growth, survival, physiology, bacteria and morphology of the gut and immune response to bacterial infection of tropical rock lobsters (Panulirus ornatus) juvenile were investigated. Dietary inclusion level of MOS at 0.4% was tested against the control diet (trash fish) without MOS inclusion. At the end of 56 days of rearing period, a challenged test was also conducted to evaluate the bacterial infection resistant ability of the lobsters fed the two diets. Lobster juvenile fed MOS diet attained 2.86 ± 0.07 g of total weigh and 66.67 ± 4.76% survival rate which were higher (P < 0.05) than the lobsters fed control diet (2.35 ± 0.14 g total weight and 54.76 ± 2.38% survival rate, respectively) thus providing the higher (P < 0.05) specific growth rate (SGR) and average weekly gain (AWG) of lobsters fed MOS diet. Physiological condition indicators such as wet tail muscle index (Tw/B), wet hepatosomatic index (Hiw) and dry tail muscle index (Td/B) of the lobsters fed MOS supplemented diet were higher (P < 0.05) than that of the lobsters fed the control diet. Bacteria in the gut (both total aerobic and Vibrio spp.) and gut's absorption surface indicated by the internal perimeter/external perimeter ratio were also higher (P < 0.05) when the lobsters were fed MOS diet. Lobsters fed MOS diet were in better immune condition showed by higher THC and GC, and lower bacteraemia. Survival, THC, GC were not different among the lobsters fed either MOS or control diet after 3 days of bacterial infection while bacteraemia was lower in the lobsters fed MOS diet. After 7 days of bacterial infection the lobsters fed MOS diet showed higher survival, THC, GC and lower bacteraemia than the lobsters fed the control diet. The experimental trial demonstrated the ability of MOS to improve the growth performance, survival, physiological condition, gut health and immune responses of tropical spiny lobsters juveniles.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Microbial Diversity within Early-Stage Cultured Panulirus ornatus Phyllosomas

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.     

Evaluation of bone marrow lesion volume as a knee osteoarthritis biomarker - longitudinal relationships with pain and structural changes: data from the Osteoarthritis Initiative

Introduction

Bone marrow lesion (BML) size may be an important imaging biomarker for osteoarthritis-related clinical trials and reducing BML size may be an important therapeutic goal. However, data on the interrelationships between BML size, pain, and structural progression are inconsistent and rarely examined in the same cohort. Therefore, we evaluated the cross-sectional and longitudinal associations of BML volume with knee pain and joint space narrowing (JSN).

Methods

A BML volume assessment was performed on magnetic resonance images of the knee collected at the 24- and 48-month Osteoarthritis Initiative visits from a convenience sample of 404 participants in the progression cohort. During the same visits, knee pain was assessed with WOMAC pain scores and knee radiographs were acquired and scored for JSN. BML volume was summed to generate a total knee volume and an index tibiofemoral compartment volume (compartment with greater baseline JSN). Primary analyses included multiple linear regressions (outcome = pain, predictor = total knee BML volume) and logistic regressions (outcome = JSN, predictor = index tibiofemoral compartment BML volume).

Results

This sample was 49% female with a mean age of 63 (9.2 standard deviation (SD)) years, and 71% had radiographic osteoarthritis in the study knee. Larger baseline BMLs were associated with greater baseline knee pain (P = 0.01), the presence of JSN at baseline (odds ratio (OR) = 1.50, 95% confidence interval (CI) = 1.23 to 1.83), and JSN progression (OR = 1.27, 95%CI = 1.11 to 1.46). Changes in total knee BML volume had a positive association with changes in knee pain severity (P = 0.004) and this association may be driven by knees that were progressing from no or small baseline BMLs to larger BMLs. In contrast, we found no linear positive relationship between BML volume change and JSN progression. Instead, regression of medial tibiofemoral BML volume was associated with JSN progression compared to knees with no or minimal changes in BML volume (OR = 3.36, 95%CI = 1.55 to 7.28). However, follow-up analyses indicated that the association between JSN progression and BML volume change may primarily be influenced by baseline BML volume.

Conclusion

Large baseline BMLs are associated with greater baseline knee pain, the presence of JSN at baseline, and disease progression. Additionally, BML regression is associated with decreased knee pain but not a reduced risk of concurrent JSN progression.     

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 administering APV26 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.     

Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae

Observations of a spontaneous infection in Crassostrea virginica larvae at Milford Laboratory indicate that the causative microorganism, Vibrio sp., was present in the filtered, uv light-treated sea water in very low concentrations. The disease was usually noted within 6–10 days after fertilization of eggs and mortality was usually complete about 6–8 days later. The disease could be induced by adding either washed cells of the pathogen or the broth filtrate. Observations made when the filtrate was employed indicate that the bacterium produced a toxin capable of breaking down the tissue structure of the oyster larvae. Mortalities were prevented with 100 mg of neomycin/liter of oyster egg culture.     

Detection of quorum sensing systems of bacteria isolated from fouled marine organisms

N-Acyl homoserine lactones (AHLs or N-AHLs) are a class of signaling molecules involved in bacterial quorum sensing (qs) that have recently been proposed as mediators of the fouling process. In this study, we determined the presence of AHLs in the following marine bacteria strains, which were collected in Santa Marta Bay (Colombia) from heavily fouled surfaces: Ochrobactrum sp., Vibrio sp. (23-6PIN), Vibrio campbellii, Vibrio sp. (11-6DEP), Ochrobactrum pseudogringnonense, Shewanella sp., Vibrio harveyi and Alteromonas sp. The detection and identification of AHLs was conducted using the microbial biosensor Escherichia coli (pSB401) and GC–MS and HPLC-MS analyses. We found that all isolated marine strains had quorum sensing systems mediated by either N-butanoyl homoserine lactone or N-hexanoyl homoserine lactone and in some cases by both. These results are in agreement with the theory that qs is involved in the fouling process. It is noteworthy to mention that we identified qs systems for the first time in bacteria of the genera Ochrobactrum and Alteromonas.     

Pectic Enzyme Activities of Bacteria Associated with Rotted Onions (Allium cepa)

The aerobic bacteria associated with soft rot in onions (Allium cepa) were isolated and identified as a Vibrio sp., Micrococcus epidermidis, Pseudomonas cepacia, an Acinetobacter sp., a Xanthomonas sp., Bacillus polymyxa, and Bacillus megaterium. With the cup-plate assay method, no pectin hydrolase could be detected from any of these isolates when they were cultured in pectin medium, but lyase and pectinesterases were detectable. Onion tissue cultures showed pectin hydrolase activity for P. cepacia and B. polymyxa and lyase and pectinesterase activities for all of the isolates, usually at higher levels of activity than those of the pectin medium culture filtrates. In both culture media, Vibrio sp. showed the highest lyase and pectinesterase activities. In the viscometric test, all of the isolates achieved at least a 50% decrease in viscosity for lyase enzyme, with M. epidermidis and Vibrio sp. recording viscosity decreases as high as 83%. The ability to cause soft rot in onion bulbs was demonstrated by P. cepacia and Xanthomonas sp. Benzoic acid at a concentration of 0.8 mg/ml caused total suppression of enzyme production, whereas sodium benzoate at this concentration reduced pectinesterase production by 71% and lyase production by 72%. The possible use of these preservatives in the control of soft rot in onions is noted.     

A Novel Agarolytic β-Galactosidase Acts on Agarooligosaccharides for Complete Hydrolysis of Agarose into Monomers

Marine red macroalgae have emerged to be renewable biomass for the production of chemicals and biofuels, because carbohydrates that form the major component of red macroalgae can be hydrolyzed into fermentable sugars. The main carbohydrate in red algae is agarose, and it is composed of d-galactose and 3,6-anhydro-l-galactose (AHG), which are alternately bonded by β1-4 and α1-3 linkages. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium (Vibrio sp. strain EJY3); the enzyme is annotated as Vibrio sp. EJY3 agarolytic β-galactosidase (VejABG). Unlike the lacZ-encoded β-galactosidase from Escherichia coli, VejABG does not hydrolyze common substrates like lactose and can act only on the galactose moiety at the nonreducing end of AOS. The optimum pH and temperature of VejABG on an agarotriose substrate were 7 and 35°C, respectively. Its catalytic efficiency with agarotriose was also similar to that with agaropentaose or agaroheptaose. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of this novel β-galactosidase offers an enormous advantage in the saccharification of agarose or agar in red macroalgae for its use as a biomass feedstock for fermentable sugar production.     

Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.     

Pectinase Activity of Anaerobic and Facultatively Anaerobic Bacteria Associated with Soft Rot of Yam (Diascorea rotundata)

Anaerobic and facultatively anaerobic bacteria associated with soft rot of yam (Diascorea rotundata) were isolated by the looping-out method and found to consist of Clostridium (three isolates), Corynebacterium (three isolates), Vibrio (one isolate), and Bacillus lentus (one isolate). Enzyme assay for hydrolase, lyase, and pectinesterase activities by the cup-plate method showed that except for Vibrio sp., B. lentus, and two isolates of Corynebacterium no pectinase activity could be detected for organisms cultured on pectin medium. Most of the cultures on yam tissue, however, showed activities for the three enzymes. The viscometric assay for hydrolase and lyase enzymes indicated a significant level of hydrolase activity (a 40.90% decrease in viscosity for Vibrio sp. and Corynebacterium spp.), but no lyase activity for most of the isolates. Two isolates of Corynebacterium and B. lentus caused changes in fresh yams suggestive of soft rot.     

Extracellular Signal Molecule(s) Involved in the Carbon Starvation Response of Marine Vibrio sp. Strain S14

The role of exogenous metabolites as putative signal molecules mediating and/or regulating the carbon starvation adaptation program in Vibrio sp. strain S14 was investigated. Addition of the stationary-phase supernatant extract (SSE) of Vibrio sp. strain S14 to logarithmic-phase cells resulted in a significant number of carbon starvation-induced proteins being up-regulated. Halogenated furanones, putative antagonists of acylated homoserine lactones (AHLs), inhibited the synthesis of proteins specifically induced upon carbon starvation. The effect of the furanone was the opposite of that caused by SSE with respect to the up- and down-regulation of protein expression, indicating that both the furanone and the putative signalling molecules were acting on the same regulatory pathway. Culturability was rapidly lost when Vibrio sp. strain S14 was starved in the presence of the furanone at a low concentration. The furanone also had a negative effect on the ability of carbon-starved cells to mount resistance against UV irradiation and hydrogen peroxide exposure. The SSE of Vibrio sp. strain S14 had the ability to provide cross-protection against the loss in viability caused by the furanone. We have further demonstrated that the SSE taken from low- as well as high-cell-density cultures of Vibrio sp. strain S14 induced luminescence in Vibrio harveyi. Taken together, the results in this report provide evidence that Vibrio sp. strain S14 produces extracellular signalling metabolites during carbon and energy starvation and that these molecules play an important role in the expression of proteins crucial to the development of starvation- and stress-resistant phenotypes.     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

A comparison of Fe bioavailability and binding of a catecholate siderophore with virus-mediated lysates from the marine bacterium Vibrio alginolyticus PWH3a

We have examined the bioavailability of Fe complexed to a siderophore produced by the heterotrophic marine bacterium Vibrio alginolyticus isolate PWH3a and Fe from ligand-complexes present in virus-mediated lysates (using phage PWH3a-P1) of this same bacterium. Fe-binding functional groups, stability constants and the bioavailability of Fe associated with these two separate ligand pools were determined and contrasted to previous work. Under low-Fe growth conditions, axenic cultures of V. alginolyticus PWH3a were shown to produce catecholate siderophores, while neither catecholate nor hydroxamate-type Fe-binding moieties were detected in virus-generated cell lysates. Analysis of the overall binding strength using electrochemical techniques revealed that the siderophore-containing organic extract and the organics in the virus-mediated lysates had Fe-binding constants comparable to the weaker L₂-type ligands found throughout the water column in seawater. A further purification of the siderophore-containing extract revealed a ligand with a stability constant of logK′_FeL,Fe3+ = 22.3, typical for siderophores and L₁-type of ligands found in marine surface waters. In assimilation studies, the Fe in the lysate was found to be more bioavailable to our model heterotrophic bacterium, autotrophic cyanobacterium and eukaryotic diatom cultures than the catecholate siderophore produced by the Vibrio sp. This work demonstrates that the Fe-containing components of virus-mediated cell lysates are different than siderophores secreted by these same cells, and as such continues to build the argument supporting the importance of virus-mediated Fe regeneration in marine surface waters.     

Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.

Τhe β-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes β-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V₁₀₉VTS₁₁₂ as playing a key role in the enzyme reaction.     

Microbial Transformation of Squalene: Terminal Methyl Group Oxidation by Corynebacterium sp

Corynebacterium sp. strain SY-79 was isolated from soil, using squalene as a carbon source. Microbiological properties of this bacterium are described. The metabolic product of this bacterium from squalene was identified as 2,6,10,15,19, 23-hexamethyl-2,6,10,14,18,22-tetracosahexaenedioic acid (squalenedioic acid).