Monitoring Urban Beach Quality on a Summer Day: Determination of the Origin of Fecal Indicator Bacteria and Antimicrobial Resistance at Prophète Beach, Marseille (France).

A highly frequented beach in Marseille, France, was monitored on an hourly basis during a summer day in July 2018, to determine possible water and sand fecal pollution, in parallel with influx of beach users from 8 a.m. to 8 p.m. Fecal indicator bacteria were enumerated, together with four host-associated fecal molecular markers selected to discriminate human, dog, horse, or gull/seagull origins of the contamination. The antimicrobial resistance of bacteria in water and sand was evaluated by quantifying (i) the class 1, 2, and 3 integron integrase genes intI, and (ii) bla TEM, bla CTX-M, and bla SHV genes encoding endemic beta-lactamase enzymes. The number of beach users entering and leaving per hour during the observation period was manually counted. Photographs of the beach and the bathing area were taken every hour and used to count the number of persons in the water and on the sand, using a photo-interpretation method. The number of beach users increased from early morning to a peak by mid-afternoon, totaling more than 1,800, a very large number of users for such a small beach (less than 1 ha). An increase in fecal contamination in the water corresponded to the increase in beach attendance and number of bathers, with maximum numbers observed in the mid-afternoon. The human-specific fecal molecular marker HF183 indicated the contamination was of human origin. In the water, the load of Intl2 and 3 genes was lower than Intl1 but these genes were detected only during peak attendance and highest fecal contamination. The dynamics of the genes encoding B-lactamases involved in B-lactams resistance notably was linked to beach attendance and human fecal contamination. Fecal indicator bacteria, integron integrase genes intI, and genes encoding B-lactamases were detected in the sand. This study shows that bathers and beach users can be significant contributors to contamination of seawater and beach sand with bacteria of fecal origin and with bacteria carrying integron-integrase genes and beta lactamase encoding genes. High influx of users to beaches is a significant factor to be considered in order to reduce contamination and manage public health risk.

Responses of marine mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) after infection with the pathogen Vibrio splendidus.

Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosis, while clearance was observed within 24 h p.i. A recruitment of hemocytes at the injection site was found in mussel PAM, together with marked morphological changes in the volume of muscular fibers, with a recovery of muscle tissue organization after 48 h p.i. A concomitant impairment in the osmoregulatory processes were observed in PAM by an initial inhibition of aquaporins and increased immunopositivity of Na+/K+ ATPase ionic pump, strictly related to the histological alterations and hemocyte infiltration detected in PAM. Accordingly, an intense cell turnover activity was also recorded following the infection event. Overall, results indicated the hemolymph as the system responsible for the physiological adaptations in mussels to stressful factors, such as pathogenicity, for the maintenance of homeostasis and immune defence. Also, the osmotic balance and cell turnover can be used as objective diagnostic criteria to evaluate the physiological state of mussels following bacterial infection, which may be relevant in aquaculture and biomonitoring studies.

Occurrence of Vibrio cholerae in water reservoirs of Burkina Faso.

Africa is currently an important region in which cholera epidemics occur. Little is known about the presence of Vibrio cholerae in freshwater bodies in Africa. There are ca. 1700 lakes and reservoirs in Burkina Faso, most of which have been built within recent decades to secure water resources. The purpose of this study was to investigate the presence of V. cholerae in the water of reservoirs, using the most-probable-number polymerase chain reaction. Results showed that V. cholerae could be detected in water samples collected from 14 of 39 sampled reservoirs. The concentrations varied from 0 MPN/l to more than 1100 MPN/l. Fifty strains of V. cholerae isolated on CHROMagar™ vibrio were identified as V. cholerae non-O1/non-O139, none of which carried the ctxA gene. A significant positive correlation was found between the presence of V. cholerae in the reservoirs and both alkaline pH and phytoplankton biomass. V. cholerae was present in significantly higher numbers in reservoirs of urban areas than in rural areas. Since V. cholerae non-O1/non-O139 has been shown to be a causative agent of endemic diarrheal outbreaks, their presence in Burkina Faso reservoirs suggests they may play a role in gastroenteritis in that country.

Toll signal transduction pathway in bivalves: complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis.

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C. gigas, suggests that the sequence data inferred from Mytilus cDNAs may not be exhaustive. Most of the Toll pathway genes were constitutively and ubiquitously expressed in M. galloprovincialis, although at different levels, and clearly induced after in vivo injection with bacteria. Such over-transcription was more rapid and intense with Gram-negative than with Gram-positive bacteria. Injection of a fungus modulated the transcription of few Toll pathway genes, with the induction levels of TLR/MyD88 complex being always less intense. Purified LPS and ß-glucans had marginal effect whereas peptidoglycans were ineffective. At the moment, we found no evidence of an IMD transcript in bivalves. In conclusion, mussels possess a complete Toll pathway which can be triggered either by Gram-positive or Gram-negative bacteria.

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signaling pathway in Lophotrochozoa.

Individual variability of mytimycin gene expression in mussel.

The antifungal peptide mytimycin (MytM) is synthesized by hemocytes of the Mediterranean mussel, Mytilus galloprovincialis. In addition to sequence and gene structure diversities previously reported from pooled hemocytes, the present report focused on the expression of mytm gene in individual M. galloprovincialis, before and after challenge. Within untreated mussel, MytM mRNA was observed by ISH in about 42% of circulating hemocytes, characterized by large, diffuse nucleus. Injection with Fusarium oxysporum increased such percentage, but in only some of the mussels. Similarly, MytM gene expression increased after injection in only some of the mussels, as measured by qPCR. Responders and not responders are common evidence in any given population of organisms. Nevertheless, even if the use of proper pool size selection has been practised to find out and evaluate the most common response trends, individual analyses must be regarded as optimal.

MIF from mussel: coding sequence, phylogeny, polymorphism, 3D model and regulation of expression.

Three macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5'-UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3'-UTR of 349 nucleotides. As for other MIFs, M. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter α-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but not to Chlamys farreri and Biomphalaria glabrata. Numerous mutation points were observed within the Mg-MIF ORF, defining 11 amino acid variants within the mussels from Palavas-France and 14 amino acid variants within the mussels from Palermo-Italy. The 2 major variants from Palavas were identical to 2 of the 4 major variants from Palermo. In all the 18 Mg-MIF variants, residues involved in tautomerase and in oxidoreductase activities were conserved. Generally, one mussel expressed 2 Mg-MIF amino acid sequences but with different frequencies of occurrence. Mg-MIF is constitutively expressed principally in hemocytes and in the mantle. In contrast to other animal models, Mg-MIF expression was always down regulated following challenge by bacteria and fungi, confirming previous data obtained with microarray. Down regulation started as soon as 1 h and Mg-MIF expression returned to background 9-48 h after the challenge. Exception was regarding the yeast, Candidaalbicans, down-regulation between 9 and 72 h, suggesting yeast and bacteria-filamentous fungi trigger different mechanisms of elimination.

Gene expression specificity of the mussel antifungal mytimycin (MytM).

We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 10(4) spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 ± 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.

Myostatin inactivation increases myotube size through regulation of translational initiation machinery.

Myostatin deficiency leads in skeletal muscle overgrowth but the precise molecular mechanisms underlying this hypertrophy are not well understood. In this study, to gain insight into the role of endogenous myostatin in the translational regulation, we used an in vitro model of cultured satellite cells derived from myostatin knock-out mice. Our results show that myostatin knock-out myotubes are larger than control myotubes and that this phenotype is associated with an increased activation of the Akt/mTOR signaling pathway, a known regulator of muscle hypertrophy. These results demonstrate that hypertrophy due to myostatin deficiency is preserved in vitro and suggest that myostatin deletion results in an increased protein synthesis. Accordingly, the rates of global RNA content, polysome formation and protein synthesis are all increased in myostatin-deficient myotubes while they are counteracted by the addition of recombinant myostatin. We furthermore demonstrated that genetic deletion of myostatin stimulates cap-dependent translation by positively regulating assembly of the translation preinitiation complex. Together the data indicate that myostatin controls muscle hypertrophy in part by regulating protein synthesis initiation rates, that is, translational efficiency.

Diversity of coding sequences and gene structures of the antifungal peptide mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis.

Knowledge on antifungal biomolecules is limited compared to antibacterial peptides. A strictly antifungal peptide from the blue mussel, Mytilus edulis named mytimycin (MytM) was reported in 1996 as partial NH(2) 33 amino acid sequence. Using back-translations of the previous sequence, MytM-related nucleotide sequences were identified from a normalized Mytilus galloprovincialis expressed sequence tag library. Primers designed from a consensus sequence have been used to obtain a fragment of 560 nucleotides, including the complete coding sequence of 456 nucleotides. Precursor is constituted by a signal peptide of 23 amino acids, followed by MytM of 54 amino acids (6.2-6.3 kDa, 12 cysteines) and C-terminal extension of 75 amino acids. Only two major amino acid precursor sequences emerged, one shared by M. galloprovincialis from Venice and Vigo, the other belonging to M. galloprovincialis from Palavas, with nine amino acid differences between the two MytM. Predicted disulfide bonds suggested the presence of two constrained domains joined by amino acidic NIFG track. Intriguing was the presence of conserved canonical EF hand-motif located in the C-terminus extension of the precursor. The MytM gene was found interrupted by two introns. Intron 2 existed in two forms, a long (1,112 nucleotides) and a short (716 nucleotides) one resulting from the removal of the central part of the long one. Both the short (GenBank FJ804479) and the long (GenBank FJ804478) genes are simultaneously present in the mussel genome.

Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.

Mussels live in diverse coastal environments experience various physical, chemical and biological conditions, which they counteract with functional adjustments and heritable adaptive changes. In order to investigate possible differences in immune system capabilities, we analyzed by qPCR the expression levels of 4 immune genes (defensin, mytilin B, myticin B, lysozyme) and HSP70 in the Mediterranean mussel, Mytilus galloprovincialis collected in 3 European farming areas {Atlantic Ocean-Ría de Vigo-Spain (RV), French Mediterranean Gulf of Lion-Palavas-Prévost lagoon (PP) and Northern Adriatic Sea-Venice-Italy (VI)} in response to one injection of one of the 3 bacterial species (Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus), and to heat shock or cold stress. We confirmed that the 5 genes are constitutively expressed in hemocytes, defensin being the less expressed, myticin B the highest. As suspected, the same gene resulted differently expressed according to mussel group, with the biggest difference being for HSP70 and lysozyme and lowest expression of all the 5 genes in mussels from RV. In addition, gene expression levels varied according to the challenge. Most frequent effect of bacterial injections was down-regulation, especially for mytilin B and myticin B. Heat shock enhanced transcript levels, particularly in mussels from RV, whereas cold stress had no effect. In situ hybridization of labelled probes on mussel hemocytes indicated that bacterial injections did not change the mRNA patterns of defensin and myticin B whereas mytilin B mRNA almost disappeared. In conclusion, these results demonstrated that constitutive level, nature and intensity of immune gene expression regulations strongly depended from mussel group, and support the concept of gene-environment interactions.

Influence of temperature, salinity and E. coli tissue content on immune gene expression in mussel: results from a 2005-2008 survey.

Several bivalves, including mussels, suffered from mortalities particularly in summer. To look for the possible effect of environmental parameters on immune capacities, Mytilus galloprovincialis were collected monthly from August 2005 to July 2008 from the Palavas Laguna, French Mediterranean coast. Q-PCR was used to quantify the expression of three antimicrobial peptide genes (defensin, mytilin B and myticin B), in addition to lysozyme and HSP70. House keeping gene was 28S rRNA. Defensin, myticin B and lysozyme appeared more expressed in spring-summer than in winter. In contrast, HSP70 expression was higher in winter. Statistical studies using principal component analysis (PCA) and multiple regression models revealed positive influence of temperature on 28S rRNA, defensin, myticin B and lysozyme expressions, but not on mytilin B and HSP70. The positive influence was significant for defensin and lysozyme expression, but relationships cannot be quantified. Similarly, salinity appeared to influence defensin expression, but this relationship cannot be quantified neither. E. coli tissue content appeared without influence. Consequently, there was no clear relationship between environmental parameters and immune-related gene expressions, demonstrating anti-infectious capabilities cannot be evaluated using only the expression of such genes as markers.

Polymorphism of mytilin B mRNA is not translated into mature peptide.

Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding sequences (cds) segregating in two different clusters. Most of the base mutations were silent, and the number of pro-peptide variants was restricted to four. Finally, as the two amino acid replacements occurred within COOH-terminus, mature peptide from mytilin B appeared unique. Multiple sequencing of partial mytilin B gene from one mussel revealed that one to four randomly distributed mutation points occurred within intron-3. Only one sequence out of the 91 analyzed contained 16 mutation points. In addition, this sequence was the only one containing four out of the six mutation points occurring within exon-4, that code for most of the COOH-terminus domain, including the unique amino acid replacement. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure for COOH-terminus domain.

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses.

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.

NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides.

Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II. Hydrophilic and hydrophobic areas of mytilin account for 63% and 37%, respectively, a ratio very close to that of MGD-1 (64% and 36%). One linear and three cyclic fragments were designed from the interstrand loop sequence known to retain the biological activities in MGD-1. Only the fragment of 10 amino acids (C10C) constrained by two disulfide bonds in a stable beta-hairpin structure was able to inhibit the mortality of Palaemon serratus shrimp injected with white spot syndrome virus (WSSV). Fifty percent inhibition was obtained by in vitro pre-incubation of WSSV with 45 microM of C10C compared with 7 microM for mytilin. Interaction between the fragment and the virus occurred very rapidly as 40% survival was recorded after only 1 min of pre-incubation. In addition, C10C was capable of inhibiting in vitro growth of Vibrio splendidus LGP32 (MIC 125 microM), Vibrio anguillarum (MIC 2mM), Micrococcus lysodeikticus and Escherichia coli (MIC 1mM). Destroying the cysteine-stabilized alphabeta structure or shortening the C10C fragment to the C6C fragment with only one disulfide bond resulted in loss of both antiviral and antibacterial activities. Increasing the positive net charge did not enforce the antibacterial activity and completely suppressed the antiviral one. The C10C-designed peptide from mytilin appeared comparable in composition and structure with protegrin, tachyplesin and polyphemusin.

Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels.

Defensin, mytilin and myticin are antimicrobial peptides (AMP) involved in mussel innate immunity. Their in vitro antibacterial activity is different according to the targeted bacterial species. To determine if this specificity is correlated to different regulations of gene expressions, adult mussels were challenged in vivo with either Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus or by heat shock. RNAs were isolated from circulating hemocytes and AMP mRNAs were quantified by Q-PCR using 28S rRNA as housekeeping gene. In addition, HSP70 gene expression was also quantified as representing non-specific response to stress. In naïve mussels, the three AMP mRNAs were present in dramatically different quantities. Compared to defensin, myticin was expressed 300-fold more and mytilin 30-fold more. HSP70 was found expressed 80-fold more than defensin. AMP genes were differentially regulated according to the challenging bacteria, M. lysodeikticus being the only one inducing down-regulation. Such variations in mRNA quantities were observed immediately after challenging, lasting less than 24h. Only V. anguillarum effect was observed later, between 12h and 3 days post-challenge. Compared to their background expression in naïve mussels, the major effect of V. splendidus was the decrease of mytilin and myticin mRNAs, V. anguillarum mainly increased both mytilin and HSP70 mRNAs, whereas M. lysodeikticus almost suppressed defensin mRNA. As expected, heat shock increased HSP70 mRNA, but also myticin mRNA. Consequently, AMP genes responded specifically to the challenges, confirming that at least some of the innate immune mechanisms are specifically orientated.

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.