Molecular characterization, expression and immune functions of two C-type lectin from Venerupis philippinarum.

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro, whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi, Vibrio splendidus and V. anguillarum, while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum.

Saline Environments as a Source of Potential Quorum Sensing Disruptors to Control Bacterial Infections: A Review.

Saline environments, such as marine and hypersaline habitats, are widely distributed around the world. They include sea waters, saline lakes, solar salterns, or hypersaline soils. The bacteria that live in these habitats produce and develop unique bioactive molecules and physiological pathways to cope with the stress conditions generated by these environments. They have been described to produce compounds with properties that differ from those found in non-saline habitats. In the last decades, the ability to disrupt quorum-sensing (QS) intercellular communication systems has been identified in many marine organisms, including bacteria. The two main mechanisms of QS interference, i.e., quorum sensing inhibition (QSI) and quorum quenching (QQ), appear to be a more frequent phenomenon in marine aquatic environments than in soils. However, data concerning bacteria from hypersaline habitats is scarce. Salt-tolerant QSI compounds and QQ enzymes may be of interest to interfere with QS-regulated bacterial functions, including virulence, in sectors such as aquaculture or agriculture where salinity is a serious environmental issue. This review provides a global overview of the main works related to QS interruption in saline environments as well as the derived biotechnological applications.

Antimicrobial effect of ethanol extract of leaf neem (Azadirachta indica A. Juss) on Listeria monocytogenes

Listeria monocytogenes es un patógeno causante de enfermedades alimentarias. En la búsqueda de controlar su propagación utilizando sustancias naturales se planteó el objetivo de mostrar si el extracto etanólico foliar de neem (Azadirachta Indica A. Juss.) tiene efecto antimicrobiano sobre L. monocytogenes ICTA-12446. El extracto se obtuvo a partir de hojas de neem sometidas a secado por 8 días, se redujeron de tamaño mecánicamente, se sometieron a maceración en frío por 3 días usando etanol 96% en recipientes ámbar, se filtró y concentró en rota evaporador. Se estandarizó el concentrado con dimetilsulfóxido (DMSO) a una concentración de 60 mg/L. Listeria monocytogenes ICTA-12446, fue inoculado en caldo nutriente junto con soluciones del extracto a diferentes concentraciones (20, 30, 40, 50 y 60 mg/L), se emplearon tiempos de contacto (2.5, 5, 10 y 15 minutos). Cumplido cada tiempo se realizaron diluciones seriadas e inocularon en agar nutritivo por extensión durante 24 h a 37ºC. Se efectuó el recuento en Unidades Formadoras de Colonias UFC. Al comparar las concentraciones del extracto se evidencia entre 20 y 60 mg/mL diferencia significativa, mientras que en 30, 40 y 50 mg/mL un comportamiento similar. Al contrastar tiempos de contacto, se observa que entre el tiempo 2.5 min y los restantes un p=0,03. El tiempo mínimo donde existió inhibición fue 2.5 minutos, y concentración mínima inhibitoria de 20 mg/mL. Los cuatro tiempos de contacto arrojan porcentajes de inhibición microbiana de 100% al emplear 60mg/mL. Se concluye que el extracto etanólico foliar de neem posee un efecto inhibitorio sobre Listeria monocytogenes(AU)

Listeria monocytogenes is a pathogen causing foodborne illness. In seeking to control its spread using natural substances in order to show if the leaf ethanol extract of neem (Azadirachta indica A. Juss) has antimicrobial effect on L. monocytogenes ICTA-12446, was proposed. The extract was obtained from neem leaves, which was subjected to drying for 8 days. It was reduced in size mechanically, and subjected to cold soak for 3 days, using 96% ethanol in amber vessels, filtered and concentrated in rot evaporator. Concentrated was solubilized with dimethylsulfoxide (DMSO) and standarized to achieve a concentration of 60 mg/mL Listeria monocytogenes was inoculated in nutrient broth with extract solutions at different concentrations (20, 30, 40, 50 and 60mg/mL), four contact times (2.5, 5, 10 and 15 minutes) were used. Completed each time it was diluted and inoculated on nutrient agar by extension for 24h at 37ºC. The count of Colony Forming Units UFC was taking. Comparing the concentrations of the extract between 20 and 60mg /mL significant difference was appreciate, while 30, 40 and 50 mg/mL show a similar behavior. Contrasting contact times observed between time 2.5 min and the remaining p = 0.03. The minimum time where there was some kind of inhibition was 2.5 minutes, and minima inhibitory concentration of 20mg/mL. The four contact times yield microbial inhibition percentages of 100% by using 60mg/L. It is concluded that ethanol extract of neem leaf has an inhibitory effect on L. monocytogenes(AU)

The in vivo extracellular life of facultative intracellular bacterial parasites: role in pathogenesis.

Classically labeled facultative intracellular pathogens are characterized by the ability to have an intracellular phase in the host, which is required for pathogenicity, while capable of extracellular growth in vitro. The ability of these bacteria to replicate in cell-free conditions is usually assessed by culture in artificial bacteriological media. However, the extracellular growth ability of these pathogens may also be expressed by a phase of extracellular infection in the natural setting of the host with pathologic consequences, an ability that adds to the pathogenic potential of the infectious agent. This infective capability to grow in the extracellular sites of the host represents an additional virulence attribute of those pathogens which may lead to severe outcomes. Here we discuss examples of infectious diseases where the in vivo infective extracellular life is well documented, including infections by Francisella tularensis, Yersinia pestis, Burkholderia pseudomallei, Burkholderia cenocepacia, Salmonella enterica serovar Typhimurium and Edwardsiella tarda. The occurrence of a phase of systemic dissemination with extracellular multiplication during progressive infections by facultative intracellular bacterial pathogens has been underappreciated, with most studies exclusively centered on the intracellular phase of the infections. The investigation of the occurrence of a dual lifestyle in the host among bacterial pathogens in general should be extended and likely will reveal more cases of infectious diseases with a dual infective intracellular/extracellular pattern.

Caldimicrobium rimae gen. nov., sp. nov., an extremely thermophilic, facultatively lithoautotrophic, anaerobic bacterium from the Uzon Caldera, Kamchatka.

An extremely thermophilic, strictly anaerobic, facultatively chemolithoautotrophic bacterium designated strain DS(T) was isolated from Treshchinnyi Spring, one of the hottest springs of the Uzon Caldera (Kamchatka, Russia). Cells of the novel organism were Gram-negative rods, about 1.0-1.2 microm long and 0.5 microm wide. The temperature range for growth was 52-82 degrees C, with an optimum at 75 degrees C. Growth was observed at pH 6.8-7.4, and the optimum pH was 7.0-7.2. Strain DS(T) was able to grow lithoautotrophically with hydrogen in the presence of CO(2) as a carbon source and thiosulfate or elemental sulfur as an electron acceptor. It also grew well with ethanol, fumarate, succinate or malate in the presence of thiosulfate. Yeast extract was not required for growth and did not stimulate growth. The genomic DNA G+C content was 35.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that the novel organism was a member of the family Thermodesulfobacteriaceae. On the basis of phylogenetic and physiological considerations, it is proposed that strain DS(T) represents a new genus and species, Caldimicrobium rimae gen. nov., sp. nov. The type strain of Caldimicrobium rimae is DS(T) (=DSM 19393(T) =VKM B-2460(T)).

Paenibacillus wynnii sp. nov., a novel species harbouring the nifH gene, isolated from Alexander Island, Antarctica.

Soil taken from 12 different locations at Mars Oasis on Alexander Island, Antarctica, yielded unidentified isolates of endospore-forming bacteria. Soil from four of the locations contained Gram-negative, facultatively anaerobic, motile rods that were able to grow at 4 degrees C and which formed ellipsoidal spores that lay paracentrally or subterminally in swollen or slightly swollen sporangia. All of the strains harboured the nitrogenase gene nifH. Phenotypic tests, amplified rDNA restriction analysis (ARDRA), fatty acid analysis and SDS-PAGE analysis suggested that the isolates represented a novel taxon of Paenibacillus. 16S rRNA gene sequence comparison supported the proposal of a novel species, Paenibacillus wynnii sp. nov. (type strain, LMG 22176(T)=CIP 108306(T)).

Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov.

The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

[The immuno-microbiological characteristics of the small intestine and the translocation of the enteral microflora in acute intestinal obstruction]. / Immuno-mikrobiologicheskaia kharakteristika tonkoi kishki i translokatsiia énteral'noi mikroflory pri ostroi neprokhodimosti kishechnika.

The complex examination of 72 patients with acute ileus (AI) of nontumor nature with different severity of endotoxicosis was carried out. The study revealed that AI was accompanied by deep suppression of the immunosecretory and motor evacuatory function of the small intestine, as well as by its pronounced bacterial contamination, mainly due to the significant quantitative prevalence of Gram-negative microflora. The combination of these factors played the key role in the increase of the permeability of the enteric barrier for symbiotic microflora and its massive translocation from the intestinal tract to the internal organs of the body (peritoneal exudate, portal bed), which directly correlated with the severity of endotoxicosis in AI patients. The deficiency of the barrier function of the liver was accompanied by the penetration of infective agents into the general blood stream, thus causing the development of endotoxic shock in AI patients. The analysis of the results thus obtained made it possible to determine the main ways for the elimination of intestinogenic intoxication in AI; they should be aimed at the bacterial decontamination of the small intestine, the restoration of its motor evacuatory and protective barrier functions, the liquidation of portal and systemic bacteremia, the correction of the functional deficiency of the liver.

Supplemental dietary arginine accelerates intestinal mucosal regeneration and enhances bacterial clearance following radiation enteritis in rats.

BACKGROUND: Arginine is a dibasic amino acid with significant metabolic and immunologic, effects especially in trauma and stress situations. Arginine supplementation has been shown to promote wound healing and improve immune system. We designed a study to evaluate the effects of supplemental dietary arginine on intestinal mucosal recovery and bacterial translocation and bacterial clearance after induction of radiation injury in rats. METHODS: Twenty-one male Sprague-Dawley rats were subjected to a single dose of 1100 rads of abdominal X radiation. Rats were divided into three groups; the first group received diet enriched with 2% arginine, the second group with 4% arginine, and the third group with isonitrogenous 4% glycine. Rats were sacrificed 7 days after the radiation. Blood was drawn for arginine levels and mesenteric lymph nodes were harvested for quantitative aerobic and anaerobic cultures. Segments of ileum and jejunum were evaluated for villous height, number of villi per centimeter of intestine, and the number of mucous cells per villous. RESULTS AND CONCLUSIONS: Arginine is absorbed reliably from the gut following oral administration. Dietary 4% arginine supplementation enhanced bacterial clearance from mesenteric lymph nodes compared to 2% arginine and 4% glycine supplemented diet following radiation enteritis in rats. Four percent arginine resulted in clear improvement in intestinal mucosal recovery when compared to 2% arginine and 4% glycine after abdominal irradiation in rats.

Shewanella woodyi sp. nov., an exclusively respiratory luminous bacterium isolated from the Alboran Sea.

Thirty-four strains of nonfermentative, respiratory, luminous bacteria were isolated from samples of squid ink and seawater from depths of 200 to 300 m in the Alboran Sea. Although these strains had a few properties similar to properties of Shewanella (Alteromonas) hanedai, they did not cluster phenotypically with any previously described bacterium. The nucleotide sequence of a 740-bp segment of luxA was not homologous with other known luxA sequences but clustered with the luxA sequences of Shewanella hanedai, Vibrio logei, Vibrio fischeri, and Photobacterium species. The 16S RNA gene from two strains was sequenced and was found to be most closely related to the S. hanedai 16S RNA gene. Based on the differences observed, we describe the new isolates as members of new species, Shewanella woodyi sp. nov. Strain ATCC 51908 (= MS32) is the type strain of this new species.

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue.

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens, was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Psudomonas was above 10(8) cfu ml-1. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas, not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1-2 log units from 10(9) to 10(10) cfu g-1 when the strains were grown on fish muscle blocks at 0 degrees C but the growth rate of S. putrefaciens was not affected.

Structure and properties of novel inclusions in Shewanella putrefaciens.

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.

Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass. Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration. Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration. During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier. As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased. Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2. Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2. Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1. We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates.