A Simple Microbiome in the European Common Cuttlefish, Sepia officinalis.

The European common cuttlefish, Sepia officinalis, is used extensively in biological and biomedical research, yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16S rRNA amplicon sequencing, quantitative PCR (qPCR), and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae The esophagus was dominated by a single ASV of the genus Vibrio Imaging revealed bacteria in the family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at lower abundance, as determined by qPCR, and was found only scattered in the lumen rather than in a discrete layer via imaging analysis. Treatment of animals with the commonly used antibiotic enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did not significantly alter the relative abundance of bacteria overall between treated versus control animals. Data from the gills were dominated by a single ASV in the family Piscirickettsiaceae, which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity and that the esophagus and gills are major colonization sites.IMPORTANCE Microbes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio (Aliivibrio) fischeri in the light organ of the bobtail squid and the role of Alpha- and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio, as well as colonization of gills by Piscirickettsiaceae This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations, as well as for understanding and maintaining the health of cephalopods in mariculture

Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family

Aims: The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny. Methods and Results: We used a high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio, Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25–5000 nmol l-1 . Conclusions: This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles. Significance and Impact of the Study: The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains

AHL-mediated quorum sensing regulation: Role in controlling cytotoxicity, T6SSs and CRISPR-Cas systems in Aliivibrio wodanis

Aliivibrio wodanis has been associated with winter ulcer disease outbreaks. It has been reported that A. wodanis may act as a secondary pathogen in disease outbreaks and pathogenesis. The culture supernatant of A. wodanis causes a cytopathogenic effect (CPE) in various salmon cell lines. Moreover, in an Atlantic salmon bath challenge experiment, A. wodanis alone produces clinical symptoms in the fish. However, the contribution of A. wodanis to winter ulcer disease is not clear. Despite the knowledge to date and the research done in the field, there are still unanswered questions and a knowledge gap. Several Vibrio and Aliivibrio species use quorum sensing (QS) to regulate genes connected to host-pathogen interaction, virulence, survival, and adaptation mechanisms. In other aliivibrios, several QS systems, including the ainS/AinR and a transcriptional regulator litR (LuxR homologs), have been reported to regulate various phenotypic traits. Moreover, temperature is an essential factor that governs the prevalence of bacteria in the environment and host and is a potent regulator of pathogenesis in many bacteria. Hence the thesis aimed to understand better the QS`s role and the effect of temperature in A. wodanis. Our study reveals that ainS autoinducer synthase is required to produce the Acyl-Homoserine Lactone (AHL) 3OHC10-HSL in A. wodanis. Furthermore, we found that the 3OHC10-HSL production is cell density and temperature-dependent. The 3OHC10-HSL concentration was higher at 6°C, the temperature below the threshold temperature at which winter ulcer occurs, compared to 12°C. The results also showed that QS and temperature regulate various functions such as AHL production, motility, and production of proteases, hemolysin and siderophores. The cell culture study further revealed that cell density, QS and temperature influence the cytotoxicity in CHSE salmon cell lines. This suggests that A. wodanis produces cytolysins and cytotoxins that are implicated in cytotoxicity. Bacteria use the Type VI secretion system (T6SS) for multiple functions like iron transport, interspecies competition, virulence, and niche adaptations. A. wodanis co-exists with the main pathogen M. viscosa in the infected fish during the winter ulcer disease outbreaks. In addition, the bacterium has been shown to hinder the growth and virulence of M. viscosa. The thesis further investigated the mechanisms by which A. wodanis may survive together with M. viscosa in skin ulcers and during winter ulcer outbreaks. We found that the A. wodanis genome encodes three T6SSs (T6SS1-T6SS3) and auxiliary clusters (Aux1-4); and several potential Type VI secretion system effectors (T6SEs). In addition, the A. wodanis genome is found to contain a type IF CRISPR-Cas system. This suggests that these two mechanisms may play a role during the survival, adaptation, and immune function of A. wodanis in its natural environment, inside the host or during its co-existence with M. viscosa. Next, we analyzed the genome-wide transcriptomics of A. wodanis and QS mutants litR and ainS mutants grown at different temperatures and cell densities. We found that the genes involved in T6SSs, and CRISPR-Cas systems are regulated by cell density, temperature, and QS. The transcriptome analysis showed that the complete T6SS2 apparatus was less expressed in the litR/WT, suggesting litR regulates T6SS2 in A. wodanis. In this study, the transcriptome analysis demonstrated that deletion of litR decreased the hcp1 expression, a gene involved in bacterial competition and virulence in other bacteria. In addition to litR dependent expression, expression value of hcp decreased three times in litR/WT, HCD at 12°C when compared to 6°C and HCD. Our observation suggests that temperature 6°C and litR are crucial for expressing the genes related to virulence. Understanding the strain diversity of the same species is important in exploring strategies to survive in a changing environment. Finally, we wanted to study the genomic similarity and variation between A. wodanis isolates by performing a pan-genome analysis of twenty-two A. wodanis isolates collected from various locations across Norway. The analysis revealed an open pan-genome with a wide inter-species diversity in A. wodanis genomes. We examined the phylogenetic relatedness between the isolates using single nucleotide variants (SNVs) and core genes. The phylogenetic trees were distributed into five groups, where Group 2, 4 and 5 encompassed conserved isolates. The accessory genomes (shell and cloud) accounted for about 73% of the total pan-genome suggesting the genomes have acquired most of the genes through horizontal gene transfer (HGT). Whole and cloud genome functional annotation revealed a larger number of genes related to functional families such as metabolism, signaling and cellular processes and genetic information processing, suggesting they are involved in energy metabolism and environmental interactions. By further analyzing the twenty-two A. wodanis genomes, we identified diverse CRISPR-Cas systems, spacers, and prophages. About 60% of isolates encoded a different CRISPR-Cas system compared to the reference strain. Like the reference strain, the other twenty-one A. wodanis isolates also encode multiple T6SSs where the T6SS2 and Aux-2 are either absent or showed differences in about 80% (18 out of 22) of isolates. In addition to the T6SSs and CRISPR-Cas, other elements relevant for adaptation, virulence, and survival potentials, such as virulence factors (VFs) and biosynthetic gene clusters (BGCs), were explored. We have found that the predicted VFs and BGCs were conserved between the A. wodanis isolates. The results presented in this work yield knowledge about QS regulation of virulence, survival, and adaptation mechanisms in A. wodanis. This aids in understanding the mechanisms connected to co-existence of A. wodanis with M. viscosa and winter ulcer disease development and treatment

Ecotoxicity of basil (Ocimum Basilicum) extract in aquaculture feeds: Is it really eco-safe for the aquatic environment?

Plant extract and essential oils are gaining application in aquaculture, but data about their environmental impact are limited and their potential effects on aquatic organisms are largely unknown. For this study, ecotoxicity tests were performed under standardized conditions on fish feed supplemented with 3 % w/w of a basil supercritical extract (F1-BEO; substance A), F1-BEO extract (substance B), and fish feed without F1-BEO extract (substance C) on three model species of different trophic levels (bacteria, primary producer, primary consumer) considered representative for freshwater (Aliivibrio fischeri, Raphidocelis subcapitata, Daphnia magna) and marine (A. fischeri, Phaeodactylum tricornutum, Paracentrotus lividus) ecosystems. Ecotoxicological response was largely comparable within the same trophic level (whichever the ecosystem). EC50 was not calculable in the concentration range here tested (3.9–500 mg/L) for freshwater and marine microalgae, suggesting that none of the substances were toxic for primary producers. Reduction of A. fischeri bioluminescence at the tested concentration (0.5–10 mg/L) was observed only for substance A (EC50 9.53 mg/L and 9 mg/L for freshwater and marine ecosystems, respectively). Notably, in P. lividus embryotoxicity was higher for substances A (EC50 1.80 mg/L) and C (EC50 4.6 mg/L) than for substance B (EC50 7.10 mg/L), suggesting a toxic effect due to feed dissolution. In contrast, substance B was more toxic (EC50 0.34 mg/L) in D. magna than substances A (EC50 3.98 mg/L) and C (EC50 5.50 mg/L). Based on the Globally Harmonized System of Classification and Labelling of Chemicals, all substances were categorized Acute 2, except for substance A which was categorized Acute 1 for D. magna. Overall, the substances were found to be potentially toxic for an aquatic ecosystem, especially for primary consumer. Further study of plant extract and essential oils is needed to better understand their effects and fate on the aquatic environment

Quorum sensing in Aliivibrio wodanis 06/09/139 and its role in controlling various phenotypic traits

Background Quorum Sensing (QS) is a cell-to-cell communication system that bacteria utilize to adapt to the external environment by synthesizing and responding to signalling molecules called autoinducers. The psychrotrophic bacterium Aliivibrio wodanis 06/09/139, originally isolated from a winter ulcer of a reared Atlantic salmon, produces the autoinducer N-3-hydroxy-decanoyl-homoserine-lactone (3OHC10-HSL) and encodes the QS systems AinS/R and LuxS/PQ, and the master regulator LitR. However, the role of QS in this bacterium has not been investigated yet. Results In the present work we show that 3OHC10-HSL production is cell density and temperature-dependent in A. wodanis 06/09/139 with the highest production occurring at a low temperature (6 °C). Gene inactivation demonstrates that AinS is responsible for 3OHC10-HSL production and positively regulated by LitR. Inactivation of ainS and litR further show that QS is involved in the regulation of growth, motility, hemolysis, protease activity and siderophore production. Of these QS regulated activities, only the protease activity was found to be independent of LitR. Lastly, supernatants harvested from the wild type and the ΔainS and ΔlitR mutants at high cell densities show that inactivation of QS leads to a decreased cytopathogenic effect (CPE) in a cell culture assay, and strongest attenuation of the CPE was observed with supernatants harvested from the ΔlitR mutant. Conclusion A. wodanis 06/09/139 use QS to regulate a number of activities that may prove important for host colonization or interactions. The temperature of 6 °C that is in the temperature range at which winter ulcer occurs, plays a role in AHL production and development of CPE on a Chinook Salmon Embryo (CHSE) cell line

Toxicity Assessment of Energetic Materials by Using the Luminescent Bacteria Inhibition Test

The luminescent bacteria inhibition test usingAliivibrio fischeriis a well-established method to determine the aquatic toxicity of soluble chemicals. More precisely, the effective concentration (EC50) after 15 and 30 min is determined in this test. The inhibition of natural bioluminescence of these bacteria gives a first idea of the toxicity of compounds towards some aquatic organisms. It is a cost and time efficient experimental method, which does not involve animals. In this contribution, the experimental set up, comparability with other measurements, and results of recently described compounds is presented. Different types of energetic materials such as coordination (e. g. [Cu(dtp)(3)](ClO4)(2)and [Fe(MTZ)(6)](ClO4)(2)), neutral (e. g. azidoethanol, 1- and 2-aminotetrazole) and ionic (e. g. polynitropyrazolates and PETNC salts) compounds were investigated and compared to commonly used materials, like RDX, ammonium perchlorate (AP) and azide salts. Furthermore, different substitution patterns and energetic functionalities such as azido-, nitro- and nitramino-groups were investigated

Production of cyathane type secondary metabolites by submerged cultures of Hericium erinaceus and evaluation of their antibacterial activity by direct bioautography

Background Fungi of the phylum Basidiomycota are well-known to form a broad spectrum of biologically active secondary metabolites, especially low molecular weight compounds such as terpenoids. Hericium erinaceus produces various cyathane type diterpenoids including erinacines. However, no quantitative data and production kinetics have been reported on the biosynthesis of the erinacines C and P in submerged cultures. In the present study, the production of erinacine C was optimized, and the product formation kinetics as well as the antimicrobial activity were studied by high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and direct bioautography. Results Oatmeal and Edamin® K were identified to be crucial media components for an efficient production of erinacine C. The highest concentrations of erinacine C were obtained in the optimized culture medium on the 9th culture day (approximately 260 mg L-1). The production of erinacine P was strongly time dependent. The maximum concentration of erinacine P of 184 mg L-1 was observed on the third culture day. Afterwards, the concentrations of erinacine P decreased while the concentrations of erinacine C steadily increased. Comparable results were obtained by HPTLC with UV detection and HPLC with diode-array detection (DAD) analyses. Direct bioautography allowed for an additional analysis of the antimicrobial activity of the secondary metabolites. Conclusions The C and N sources oatmeal and Edamin® K induced the formation of erinacine C. Detailed product formation kinetics of the erinacines C and P have been reported for the first time. HPTLC combined with the Aliivibrio fischeri bioassay allowed for an instant detection of cyathane diterpenoids in crude extracts and for an evaluation of the antimicrobial activity of the secondary metabolites directly on the plate

Phylogenetic analysis reveals an ancient gene duplication as the origin of the MdtABC efflux pump.

The efflux pumps from the Resistance-Nodulation-Division family, RND, are main contributors to intrinsic antibiotic resistance in Gram-negative bacteria. Among this family, the MdtABC pump is unusual by having two inner membrane components. The two components, MdtB and MdtC are homologs, therefore it is evident that the two components arose by gene duplication. In this paper, we describe the results obtained from a phylogenetic analysis of the MdtBC pumps in the context of other RNDs. We show that the individual inner membrane components (MdtB and MdtC) are conserved throughout the Proteobacterial species and that their existence is a result of a single gene duplication. We argue that this gene duplication was an ancient event which occurred before the split of Proteobacteria into Alpha-, Beta- and Gamma- classes. Moreover, we find that the MdtABC pumps and the MexMN pump from Pseudomonas aeruginosa share a close common ancestor, suggesting the MexMN pump arose by another gene duplication event of the original Mdt ancestor. Taken together, these results shed light on the evolution of the RND efflux pumps and demonstrate the ancient origin of the Mdt pumps and suggest that the core bacterial efflux pump repertoires have been generally stable throughout the course of evolution

Developing the MAR databases – Augmenting Genomic Versatility of Sequenced Marine Microbiota

This thesis introduces the MAR databases as marine-specific resources in the genomic landscape. Paper 1 describes the curation effort and development leading to the MAR databases being created. It results in the highly valued reference database MarRef, the broader MarDB, and the marine gene catalog MarCat. Definition of a marine environment, the curation process, and the Marine Metagenomics Portal as a public web-service are described. It facilitates scientists to find marine sequence data for prokaryotes and to explore rich contextual information, secondary metabolites, updated taxonomy, and helps in evaluating genome quality. Many of these database advancements are covered in Paper 2. This includes new entries and development of specific databases on marine fungi (MarFun) and salmon related prokaryotes (SalDB). With the implementation of metagenome assembled and single amplified genomes it leads up to the database quality evaluation discussed in Paper 3. The lack of quality control in primary databases is here discussed based on estimated completeness and contamination in the genomes of the MAR databases. Paper 4 explores the microbiota of skin and gut mucosa of Atlantic salmon. By using a database dependent amplicon analysis, the full-length 16 rRNA gene proved accurate, but not a game-changer in taxonomic classification for this environmental niche. The proportion of dataset sequences lacking clear taxonomic classification suggests lack of diversity in current-day databases and inadequate phylogenetic resolution. Advancing phylogenetic resolution was the subject of Paper 5. Here the highly similar species of genus Aliivibrio became delineated using six genes in a multilocus sequence analysis. Five potentially novel species could in this way be delineated, which coincided with recent genome-wide taxonomy listings. Thus, Paper 4 and 5 parallel those of the MAR databases by providing insight into the inter-relational framework of bioinformatic analysis and marine database sources

Natural replacement of vertically inherited lux‐rib genes of Photobacterium aquimaris by horizontally acquired homologues

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92440/1/emi4355_sm_FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92440/2/emi4355_sm_TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92440/3/emi4355.pd