Potential of Marine Strains of Pseudoalteromonas to Improve Resistance of Juvenile Sea Bass to Pathogens and Limit Biofilm Development.

The European sea bass (Dicentrarchus labrax), one of the most produced marine fish species in Europe, is acutely vulnerable to multiple infectious hazards. In this study, we investigated the potential probiotic effect of some marine Pseudoalteromonas bacterial strains against two major pathogens of this species, Vibrio harveyi and the nervous necrosis virus (NNV), and examined their antibiofilm effect. Impregnation phase was done by repeated immersion of juvenile's sea bass during 8 to 12 weeks in seawater containing the probiotic candidates at a concentration of 106 CFU/mL. Four candidates were tested: (1) a combination of two strains producing antimicrobial compounds, hCg-42 and hOe-125; (2) strain 3J6, with known antibiofilm properties; (3) strain RA15, from the same genus, but with no identified probiotic effect; and (4) a control group without probiotics. At the end of the impregnation phase, fish underwent an infection challenge with V. harveyi or with a pathogenic strain of NNV and mortality was monitored. For the V. harveyi challenge, improved survival rates of 10 and 25% were obtained for the RA15 and the mix hCg-42 + hOe-125-impregnated groups, respectively. For the NNV challenge, no significant benefic effect of the probiotics on infection kinetics or cumulative mortality was observed. At the end of the impregnation phase, the maximal thickness of biofilm was significantly lower in the 3J6, double strain, and RA15 groups, compared with the non-impregnated control group. This study highlights the interesting probiotic potential of marine bacteria to limit mortalities induced by bacterial pathogens as well as biofilm development.

New insights into the haemo- and coelo-microbiota with antimicrobial activities from Echinodermata and Mollusca.

AIMS: The aim of this study was to investigate the diversity of bacteria with antimicrobial activity present in the coelomic fluid and haemolymph of wild and healthy echinodermata and mollusca. METHODS AND RESULTS: Collection expeditions of healthy marine molluscs and echinoderms were conducted in the Glenan archipelago in spring 2014. Members of the culturable microbiota present in the haemolymph, (haemo-microbiota) of Haliotis tuberculata (gastropoda, abalone) and Mytilus edulis (bivalvia, mussel), as well as in the coelomic fluid (coelo-microbiota) of Echinus esculentus (echinoidea, sea urchin) and Holothuria forskali (Holothuroidea, holothurian) were screened for antimicrobial activity, and further identified using 16S rRNA sequencing. Except for E. esculentus, culturable bacteria in the internal fluids of all studied organisms (mussel, abalone and holothurian) were more abundant than in seawater. The haemo- and coelo-microbiota with antimicrobial activity differed significantly between host species, in terms of abundance and diversity. Indeed, higher numbers were isolated from mussel than from abalone haemolymph. Moreover, in mussels and holothurians, bacteria with antimicrobial activities were predominantly Vibrio spp. (respectively 55 and 45%), while Pseudoalteromonas spp. were the most abundant (50%) in abalone haemolymph. Nevertheless, the activity spectra of these bacteria mainly included marine pathogens affiliated to the Vibrio genus. CONCLUSION: The haemo- and coelo-microbiota with antimicrobial activities were significantly related to their host species and differed in terms of abundance and diversity. These bacteria may play a key role in host homeostasis against pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study brings new knowledge on the diversity of bacteria present in the internal fluids of two marine molluscs and two echinoderms and their antimicrobial activities towards marine pathogens.

Antibacterial activity of lactophoricin, a synthetic 23-residues peptide derived from the sequence of bovine milk component-3 of proteose peptone.

A synthetic peptide of 23 residues corresponding to the carboxyterminal 113 to 135 region of component-3 of proteose peptone (PP3) has been investigated with regard to its antibacterial properties. This cationic amphipathic peptide that we refer to as lactophoricin, displayed a growth-inhibitory activity against both gram-positive and gram-negative bacteria. For most of the strains tested, bacterial growth was observed in the presence of lactophoricin except for Streptococcus thermophilus. In that case, lactophoricin exhibited a minimum inhibitory concentration of 10 microM and a minimum lethal concentration of 20 microM. No hemolysis of human red blood cells was detected for peptide concentrations between 2 to 200 microM, indicating that lactophoricin would be noncytotoxic when used in this concentration range.

Sex-dependent variations and timing of thyroid growth during puberty.

Marked changes in thyroid function occur during puberty as an adaptation to body and sexual development. Characteristics of thyroid growth were investigated in 259 healthy adolescents (110 girls and 149 boys), aged 11--17 yr, in an urban area of Switzerland with sufficient iodine supply. The thyroid volume determined by ultrasonography was correlated with chronological age, body weight, body height, cervical circumference, body mass index, and body surface area (BSA). Iodide concentration was measured in urine. The increase in thyroid volume mainly occurred between 11--15 yr (age at maximum thyroid growth rate, 12.5 yr) and was best correlated with BSA in both genders (girls, r(2) = 0.38; boys, r(2) = 0.49). The BSA-related thyroid growth was almost constant throughout puberty in boys and similar in girls up to menarche, but 14.5% larger in girls after menarche (P < 0.01). Percentiles of thyroid volume were lower than WHO reference values despite low normal urinary iodide concentration (median, 0.75 micromol/L). These findings suggest that physiological thyroid growth during puberty is mainly influenced by growth factors involved in somatic development and further modulated by sex steroid secretion profiles. The thyroid growth spurt coinciding with menarche in girls may contribute to a higher incidence of goiter during mid- to late puberty.

[Iodine nutrition and prevalence of goiter in adolescents in the Canton of Vaud]. / Apport iodé et prévalence du goitre chez les adolescents dans le canton de Vaud.

Iodine deficiency disorders virtually disappeared in Switzerland after iodized salt was introduced in 1922 and the iodine content increased from 3.75 to 7.5 mg/kg in 1962 and to 15 mg/kg in 1980. However, a decreasing iodine intake has recently been reported again. The status of iodine nutrition in the Canton of Vaud was therefore assessed in 348 representative adolescents aged 11 to 17 years from the urban area of Lausanne in 1995. Thyroid size was estimated by inspection and palpation and thyroid volume measured by ultrasonography. Iodine concentration was determined in urine. Thyroid enlargement, as assessed by clinical examination, was found in 15% of the adolescents (stage 1a: 12.9%, 1b: 1.8%, 2: 0.3%), but its positive predictive value was low. The goitre prevalence determined by ultrasonography was only 1.4%. In addition, 4% of the adolescents had nodular or diffuse echo-structure abnormalities. Percentiles of thyroid volume as a function of sex, chronological age and body surface area were lower than WHO reference values. Median iodine concentration in urine was 92 micrograms/l (56.6% < 100 micrograms/l), slightly below WHO recommendations. Iodized cooking salt was consumed by 82% of the adolescents. In conclusion, the adolescents living in the Canton of Vaud had a low normal iodine intake with subclinical thyroid abnormalities. These findings support the recent decision to increase the salt iodine content to 20-30 mg/kg in order to prevent iodine deficiency during puberty and pregnancy. Percentiles of thyroid volume determined in the present study can be used as local reference.

Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family.

Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P. sauvagii. In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi. To date, 6 dermaseptin B mature peptides, 24-34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P. bicolor. To assess dermaseptin related genes further, we screened a P. bicolor genomic library with 32P-labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin). A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23-42% amino acids identities with other members of the family. Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene. Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 microM. Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that if efficiently depolarizes the plasma membrane.

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins.

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence of two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti-Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M(r) of Mesenterocin 52A appeared identical to the previously described Mesentericin Y105. In contrast, Mesenterocin 52B possessed a M(r) of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins: NH2-KGVLGWLSMASSALTGPQQPNSPWLAKIKNHK.

Covalent structure, synthesis, and structure-function studies of mesentericin Y 105(37), a defensive peptide from gram-positive bacteria Leuconostoc mesenteroides.

A 37-residue cationic antimicrobial peptide named mesentericin Y 105(37) was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 105(37) contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 105(37) was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 105(37) is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 105(37) in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 105(37) targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum alpha-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 105(37) are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative alpha-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 105(37) is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.

The putative immunity protein of the gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment.

Immunity proteins are though to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105(37) bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.