Population genetic and evolution analysis of controversial genus Edwardsiella by multilocus sequence typing.

At present, the genus Edwardsiella compiles five species: E. tarda, E. hoshinae, E. ictaluri, E. piscicida and E. anguillarum. Some species of this genus such us E. ictaluri and E. piscicida are important pathogens of numerous fish species. With the description of the two latter species, the phylogeny of Edwardsiella became more complicated. With the aim to clarify the relationships among all species in the genus, a multilocus sequence typing (MLST) approach was developed and applied to characterize 56 isolates and 6 reference strains belonging to the five Edwardsiella species. Moreover, several analyses based on the MLST scheme were performed to investigate the evolution within the genus, as well as the influence of recombination and mutation in the speciation. Edwardsiella isolates presented a high genetic variability reflected in the fourteen sequence types (ST) represented by a single isolates out of eighteen total ST. Mutation events were considerably more frequent than recombination, although both approximately equal influenced the genetic diversification. However, the speciation among species occurred mostly by recombination. Edwardsiella genus displays a non-clonal population structure with some degree of geographical isolation followed by a population expansion of E. piscicida. A database from this study was created and hosted on pubmlst.org (http://pubmlst.org/edwardsiella/).

Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Streptococcus phocae strains isolated from Atlantic salmon (Salmo salar) cage-farmed in Chile. Four salmon and three seal isolates showed minor differences in the SDS-PAGE protein analysis. Thus, a major protein band present in the salmon isolates, of approximately 22.4 kDa, was absent in the pinniped strains, regardless of the growth media employed. In addition, the pinniped strains showed protein bands with molecular masses of 71.5 and 14.2 kDa, when grown on trypticase soy agar supplemented with 1% NaCl, or 25.6 kDa, when grown on Columbia blood agar, not present in the Atlantic salmon strains. A high similarity in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra of the strains was observed, although some minor peaks were absent in the fish isolates. Fatty acid methyl esters from isolates with different host origin significantly (P<0.05) differed in the content of C16:0, C17:0, C18:1ω9c, C20:4ω6,9,12,15c and summed features 3, 5 and 8. The salmon isolates formed a separate cluster in the phylogenetic analysis of housekeeping genes, separately or as concatenated sequences. Sequence divergences among salmon and seal strains were in the range of inter-subspecies differentiation for groEL (2.5%), gyrB (1.8%), recN (2.1%), rpoB (1.7%) and sodA (2.0%) genes. DNA-DNA hybridization results confirmed those of sequencing, showing reassociation values between seal and salmon strains close to the borderline of species definition. Differences in growth at low temperatures and in the haemolytic capacities were also observed between both groups of isolates. On the basis of all these results, the salmon isolates represent a novel subspecies of S. phocae, for which the name Streptococcus phocae subsp. salmonis subsp. nov. is proposed. The type strain is C-4T (=CECT 7921T=DSM 24768T). The subspecies Streptococcus phocae subsp. phocae subsp. nov. is automatically created. An emended description of S. phocae is also provided.

In vitro quenching of fish pathogen Edwardsiella tarda AHL production using marine bacterium Tenacibaculum sp. strain 20J cell extracts.

Quorum quenching (QQ) has become an interesting alternative for solving the problem of bacterial antibiotic resistance, especially in the aquaculture industry, since many species of fish-pathogenic bacteria control their virulence factors through quorum sensing (QS) systems mediated by N-acylhomoserine lactones (AHLs). In a screening for bacterial strains with QQ activity in different marine environments, Tenacibaculum sp. strain 20J was identified and selected for its high degradation activity against a wide range of AHLs. In this study, the QQ activity of live cells and crude cell extracts (CCEs) of strain 20J was characterized and the possibilities of the use of CCEs of this strain to quench the production of AHLs in cultures of the fish pathogen Edwardsiella tarda ACC35.1 was explored. E. tarda ACC35.1 produces N-hexanoyl-L-homoserine lactone (C6-HSL) and N-oxohexanoyl-L-homoserine lactone (OC6-HSL). This differs from profiles registered for other E. tarda strains and indicates an important intra-specific variability in AHL production in this species. The CCEs of strain 20J presented a wide-spectrum QQ activity and, unlike Bacillus thuringiensis serovar Berliner ATCC10792 CCEs, were effective in eliminating the AHLs produced in E. tarda ACC35.1 cultures. The fast and wide-spectrum AHL-degradation activity shown by this member of the Cytophaga-Flexibacter-Bacteroidetes group consolidates this strain as a promising candidate for the control of AHL-based QS pathogens, especially in the marine fish farming industry.

Nonleaching Biocidal N-Halamine-Functionalized Polyamine-, Guanidine-, and Hydantoin-Based Coatings.

Fibrous materials with inherent antimicrobial properties can help in real-time deactivation of microorganisms, enabling multiple uses while reducing secondary infections. Coatings with antiviral polymers enhance the surface functionality for existing and potential future pandemics. Herein, we demonstrated a straightforward route toward biocidal surface creation using polymers with nucleophilic biguanide, guanidine, and hydantoin groups that are covalently attached onto a solid support. Biocidal poly(N-vinylguanidine) (PVG) and poly(allylamine-co-4-aminopyridine-co-5-(4-hydroxybenzylidene)hydantoin) (PAH) were introduced for coating applications along with commercially available polyvinylamine (PVAm) and poly(hexamethylene biguanide) (PHMB). Nonleaching coatings were created by first fabricating bifunctional siloxane or isocyanate precursor coatings on the cotton, nylon-cotton, and glass fiber fabric, followed by the polymer attachment. The developed grafting methods ensured the stability of the coating and the reuse of the material while maintaining the biocidal properties. Halogenation of polymer-coated fabric was conducted by aqueous solutions of sodium hypochlorite or in situ generation of hypobromous acid (HOBr), resulting in surfaces coated by N-halamines with high contents of active > N-Cl or > N-Br groups. The polymer-coated fabrics were stable in multiple laundry cycles and maintained hydrophilic character after coating and halogenation. Halogenated polymer-coated fabrics completely inactivated human respiratory coronavirus based on a contact-killing mechanism and were shown to be reusable after recharging with bromine or chlorine.

Multifunctional polymeric guanidine and hydantoin halamines with broad biocidal activity.

Prolonged and excessive use of biocides during the coronavirus disease era calls for incorporating new antiviral polymers that enhance the surface design and functionality for existing and potential future pandemics. Herein, we investigated previously unexplored polyamines with nucleophilic biguanide, guanidine, and hydantoin groups that all can be halogenated leading to high contents of oxidizing halogen that enables enhancement of the biocidal activity. Primary amino groups can be used to attach poly(N-vinylguanidine) (PVG) and poly(allylamine-co-4-aminopyridine-co-5-(4-hydroxybenzylidene)hydantoin) (PAH) as well as a broad-spectrum commercial biocide poly(hexamethylene biguanide) (PHMB) onto a solid support. Halogenation of polymer suspensions was conducted through in situ generation of excess hypobromous acid (HBrO) from bromine and sodium hydroxide or by sodium hypochlorite in aqueous solutions, resulting in N-halamines with high contents of active > N-Br or > N-Cl groups. The virucidal activity of the polymers against human respiratory coronavirus HCoV-229E increased dramatically with their halogenation. Brominated PHMB-Br showed activation activity value > 5 even at 1 mg/L, and complete virus inhibition was observed with either PHMB-Br or PAH-Br at 10 mg/mL. Brominated PVG-Br and PAH-Br possessed fungicidal activity against C. albicans, while PHMB was fungistatic. PHMB, PHMB-Br and PAH polymers demonstrated excellent bactericidal activity against the methicillin-resistant S. aureus and vancomycin-resistant E. faecium. Brominated polymers (PHMB-Br, PVG-Br, PAH-Br) were not toxic to the HeLa monolayers, indicating acceptable biocompatibility to cultured human cells. With these features, the N-halamine polymers of the present study are a worthwhile addition to the arsenal of biocides and are promising candidates for development of non-leaching coatings.

The secretome of the fish pathogen Tenacibaculum maritimum includes soluble virulence-related proteins and outer membrane vesicles.

Tenacibaculum maritimum, the etiological agent of tenacibaculosis in marine fish, constitutively secretes extracellular products (ECPs) in which protein content has not been yet comprehensively studied. In this work, the prevalence of extracellular proteolytic and lipolytic activities related to virulence was analyzed in 64 T. maritimum strains belonging to the O1-O4 serotypes. The results showed the existence of a great intra-specific heterogeneity in the enzymatic capacity, particularly within serotype O4. Thus, the secretome of a strain belonging to this serotype was characterized by analyzing the protein content of ECPs and the possible production of outer membrane vesicles (OMVs). Notably, the ECPs of T. maritimum SP9.1 contain a large amount of OMVs that were characterized by electron microscopy and purified. Thus, ECPs were divided into soluble (S-ECPs) and insoluble fractions (OMVs), and their protein content was analyzed by a high-throughput proteomic approach. A total of 641 proteins were identified in ECPs including some virulence-related factors, which were mainly found in one of the fractions, either OMVs or S-ECPs. Outer membrane proteins such as TonB-dependent siderophore transporters and the type IX secretion system (T9SS)-related proteins PorP, PorT, and SprA appeared to be mainly associated with OMVs. By contrast, putative virulence factors such as sialidase SiaA, chondroitinase CslA, sphingomyelinase Sph, ceramidase Cer, and collagenase Col were found only in the S-ECPs. These findings clearly demonstrate that T. maritimum releases, through surface blebbing, OMVs specifically enriched in TonB-dependent transporters and T9SS proteins. Interestingly, in vitro and in vivo assays also showed that OMVs could play a key role in virulence by promoting surface adhesion and biofilm formation and maximizing the cytotoxic effects of the ECPs. The characterization of T. maritimum secretome provides insights into ECP function and can constitute the basis for future studies aimed to elucidate the full role of OMVs in the pathogenesis of fish tenacibaculosis.

Combined sterilization and fabrication of drug-loaded scaffolds using supercritical CO2 technology.

The access of biodegradable scaffolds to the clinical arena is constrained by the absence of a suitable sterilization technique for the processing of advanced polymeric materials. Sterilization with supercritical CO2 (scCO2) may circumvent some technological limitations (e.g., low temperature, no chemical residues on the material), although scCO2 can plasticize the polymer depending on the processing conditions used. In this latter case, the integration of the manufacturing and sterilization processes is of particular interest to obtain sterile and customized scaffolds in a single step. In this work, scCO2 was exploited as a concomitantly foaming and sterilizing agent for the first time, developing a one-step process for the production of vancomycin-loaded poly(ε-caprolactone) (PCL) bone scaffolds. The effect of the CO2 contact time on the sterility levels of the procedure was investigated, and the sterilization efficiency was evaluated against dry spores (Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus). Vancomycin-loaded PCL scaffolds had relevant sustained release profiles for the prophylaxis of infections at the grafted area, even those caused by methicillin-resistant Staphylococcus aureus (MRSA). The biological performance of the scaffolds was evaluated in vitro regarding human mesenchymal stem cells (hMSCs) attachment and growth. Finally, the biocompatibility and angiogenic response of the manufactured sterile scaffolds was assessed in ovo through chick chorioallantoic membrane (CAM) assays.

Genomic characterization of Tenacibaculum maritimum O-antigen gene cluster and development of a multiplex PCR-based serotyping scheme.

Tenacibaculum maritimum is a devastating bacterial pathogen affecting a large variety of marine fish species. It is responsible for significant economic losses in aquaculture farms worldwide. Different typing methods have been proposed to analyse bacterial diversity and population structure. Serological heterogeneity has been observed and up to four different serotypes have been described so far. However, the underlying molecular factors remain unknown. By combining conventional serotyping and genome-wide association study, we identified the genomic loci likely involved in the O-antigen biosynthesis. This finding allowed the development of a robust multiplex PCR-based serotyping scheme able to detect subgroups within each serotype and therefore performs better than conventional serotyping. This scheme was successfully applied to a large number of isolates from worldwide origin and retrieved from a large variety of fish species. No obvious correlations were observed between the mPCR-based serotype and the host species or the geographic origin of the isolates. Strikingly, the distribution of mPCR-based serotypes does not follow the core genome phylogeny. Nevertheless, this simple and cost-effective mPCR-based serotyping method could be useful for different applications such as population structure analysis, disease surveillance, vaccine formulation and efficacy follow-up.

Supercritical CO2 sterilization: An effective treatment to reprocess FFP3 face masks and to reduce waste during COVID-19 pandemic.

The outbreak of COVID-19 pandemic unveiled an unprecedented scarcity of personal protective equipment (PPE) available in sanitary premises and for the population worldwide. This situation fostered the development of new strategies to reuse PPE that would ensure sterility and, simultaneously, preserve the filtering properties of the materials. In addition, the reuse of PPEs by reprocessing could reduce the environmental impact of the massive single-use and disposal of these materials. Conventional sterilization techniques such as steam or dry heat, ethylene oxide, and gamma irradiation may alter the functional properties of the PPEs and/or leave toxic residues. Supercritical CO2 (scCO2)-based sterilization is herein proposed as a safe, sustainable, and rapid sterilization method for contaminated face masks while preserving their performance. The functional (bacterial filtration efficiency, breathability, splash resistance, straps elasticity) properties of the processed FFP3 face masks were evaluated after 1 and 10 cycles of sterilization. Log-6 sterilization reduction levels were obtained for face masks contaminated with Bacillus pumilus endospores at mild operating conditions (CO2 at 39 °C and 100 bar for 30 min) and with low contents of H2O2 (150 ppm). Physicochemical properties of the FFP3 face masks remained unchanged after reprocessing and differences in efficacy were not observed neither in the filtration tests, following UNE-EN 14683, nor in the integrity of FFP3 filtration after the sterilization process. The herein presented method based on scCO2 technology is the first reported protocol achieving the reprocessing of FFP3 masks up to 10 cycles while preserving their functional properties.

Efficacy of Ciprofloxacin, Metronidazole and Minocycline in Ordered Mesoporous Silica against Enterococcus faecalis for Dental Pulp Revascularization: An In-Vitro Study.

Pulp revascularization of teeth with necrotic pulp has become an alternative treatment in cases with immature apex. Microbial control is essential to achieve a successful outcome and continued root development. Enterococcus faecalis (E. faecalis) is the most frequently isolated bacterial species in root canals of endodontically failed teeth. Our main goal was to compare the in-vitro antimicrobial efficacy of different antibiotic formulations delivered by ordered mesoporous silica (OMS) against E. faecalis. To determine antibiotic susceptibility, we tested OMS and triple antibiotic paste (TAP; ciprofloxacin:metronidazole:minocycline) with different reagents in different concentrations, using the Kirby−Bauer disk diffusion method. OMS and metronidazole showed no antibacterial activity against E. faecalis. Mixtures of OMS and antibiotics in proportions of 2:2:14 and 4:1:7 (mg/L of ciprofloxacin:metronidazole:minocycline, respectively) showed the lowest antibacterial activity. The antibacterial activity of the combined solutions of ciprofloxacin and metronidazole was significantly higher (p < 0.005). Combinations in different concentrations of minocycline, ciprofloxacin, and metronidazole in OMS have shown activity against E. faecalis, although the combined use of ciprofloxacin and metronidazole has shown the most effective results. This study demonstrates the efficacy of intracanal antibiotic combination paste activity against E. faecalis, avoiding the use of minocycline, whose undesirable effect of teeth staining is a common problem for patients and professionals in dental clinic.

Bactericidal core-shell paramagnetic nanoparticles functionalized with poly(hexamethylene biguanide).

Bactericidal paramagnetic particles were obtained either through the attachment of a conjugate of poly(ethyleneimine) (PEI) and poly(hexamethylene biguanide) (PHMBG) to the surface of magnetite (Fe(3)O(4)) particles, or via the sol-gel encapsulation of magnetite particles with a functional silane (3-glycidoxypropyl trimethoxysilane) and subsequent binding of the polysiloxane shell by the amine/imine groups of PHMBG. The encapsulated core-shell particles possess a high saturation magnetization, which is preserved for more than 10 months while in contact with air in aqueous suspensions. The minimum inhibitory concentration (MIC) of the encapsulated particles for eight types of bacteria was size-dependent, with polydisperse submillimeter particles possessing a several-fold higher MIC than analogous particles sized below 250 nm. The encapsulated particles are biocompatible and nontoxic to mammalian cells such as mouse fibroblasts. The particles efficiently bind both glycopeptide components mimicking the gram-positive bacteria membranes and whole bacteria, and possess broad-range bactericidal activity. The cell-particle complexes can be captured, manipulated, and removed by means of a magnet.

Supercritical CO2 technology for one-pot foaming and sterilization of polymeric scaffolds for bone regeneration.

Sterilization is a quite challenging step in the development of novel polymeric scaffolds for regenerative medicine since conventional sterilization techniques may significantly alter their morphological and physicochemical properties. Supercritical (sc) sterilization, i.e. the use of scCO2 as a sterilizing agent, emerges as a promising sterilization method due to the mild operational conditions and excellent penetration capability. In this work, a scCO2 protocol was implemented for the one-pot preparation and sterilization of poly(ε-caprolactone) (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds. The sterilization conditions were established after screening against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) vegetative bacteria and spores of Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus. The transition from the sterilization conditions (140 bar, 39 °C) to the compressed foaming (60 bar, 26 °C) was performed through controlled depressurization (3.2 bar/min) and CO2 liquid flow. Controlled depressurization/pressurization cycles were subsequently applied. Using this scCO2 technology toolbox, sterile scaffolds of well-controlled pore architecture were obtained. This sterilization procedure successfully achieved not only SAL-6 against well-known resistant bacteria endospores but also improved the scaffold morphologies compared to standard gamma radiation sterilization procedures.

Binding of functionalized paramagnetic nanoparticles to bacterial lipopolysaccharides and DNA.

Magnetite and metallic cobalt-based nanoparticles with sizes ranging from 10 to 300 nm and surface-functionalized with poly(hexamethylene biguanide) (PHMBG) are introduced as capable lipopolysaccharide (LPS)-sequestering agents. The nanoparticles efficiently bind to whole E. coli cells and can be used to separate the cells effectively from suspension using a magnet. A fluorescence dye displacement assay shows strong affinities of the nanoparticles for lipid A, the glycolipid component of LPS responsible for septic shock. The particle-lipid A affinity is of the same order of magnitude or higher than that of polymyxin B. The affinity of smaller (< 50 nm) magnetite particles modified with PHMBG to lipid A is several-fold higher than that of their larger counterparts (> 100 nm) due to their higher surface area to volume ratio. The nanoparticles possess high saturation capacity for double-stranded lambdaDNA from E. coli, with which particle-polyelectrolyte complexes are formed. The PHMBG-modified nanoparticles are potent bactericides, inhibiting E. coli viability and growth at concentrations at < or = 10 microg/mL.

Contact lens hydrophobicity and roughness effects on bacterial adhesion.

PURPOSE: This study was designed to assess whether hydrogel contact lens (CL) surface hydrophobicity and roughness affect Staphylococcus epidermidis adhesion. METHODS: Bacterial adhesion experiments were performed on two unworn silicone hydrogel and three unworn conventional hydrogel CLs using the S.epidermidis strain CECT 4184. Microbial colonization was assessed by conducting counts expressed as colony-forming units. CL hydrophobicity was determined through water contact angle measurements and the roughness parameters such as mean surface roughness (Ra), kurtosis (Rku), and skewness (Rsk) were determined through atomic force microscopy in Tapping Mode. RESULTS: The conventional CLs showed similar water contact angles (p > 0.05) and were classified as hydrophilic. The silicone hydrogel CLs yielded hydrophobic contact angles with no significant differences between them (p > 0.05). The lenses with the highest (nelfilcon A and ocufilcon B) or lowest (comfilcon A and omafilcon A) Ra values displayed a lesser or greater extent of spikiness of their surfaces, respectively. All lenses showed a predominance of peaks (Rsk > 0) over troughs. S. epidermidis adhered more to the hydrophobic CLs (p < 0.05). Omafilcon A and comfilcon A, which showed the lowest Ra values among the hydrophilic and hydrophobic lenses, respectively, returned the lowest bacterial adhesion scores (p < 0.05). CONCLUSIONS: Our results suggest that more hydrophobic CLs are more prone to S. epidermidis adhesion. Although the Ra appears to be related to S. epidermidis adhesion, the influence of Rku and Rsk on this variable remains unclear.

Streptococcus phocae, an emerging pathogen for salmonid culture.

This work describes the characterization of the causal agent of disease outbreaks that, from 1999, occurred repeatedly during the summer months (temperatures higher than 15 degrees C) in Atlantic salmon (Salmo salar) cage-farmed in Chile affecting both smolts and adult fish cultured in estuary and marine waters, reaching in some occasions a cumulative mortality up to 25% of the affected population. Diseased fish showed exophthalmia with accumulation of purulent and haemorrhagic fluid around eyes, and ventral petechial haemorrhages. At necropsy, haemorrhage in the abdominal fat, pericarditis, and enlarged liver, spleen and kidney are common pathological changes. Gram-stained smears revealed the presence of Gram-positive cocci, beta-hemolytic, negative for oxidase and catalase tests. Although biochemical characterization of the isolates using the miniaturized system rapid ID 32 Strep suggested their assignation to genus Gemella, sequencing and RFLP analysis of the 16S rRNA revealed that bacteria associated with the mortalities belong to Streptococcus phocae. Serological studies demonstrated that all the salmon isolates are antigenically homogeneous, which can facilitate the development of preventive measures and, although sharing some antigenical determinants, they belong to a different Lancefield group than the type strain isolated from seals. On the basis of these facts, we conclude that the species S. phocae is an emerging pathogen for salmonid culture in Chile, and it should be included as a new member of the warm water streptococcosis.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile.

Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The characteristics of isolates were compared with the type strain V. ordalii ATCC 33509T. The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their antigenic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS electrophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demonstrated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area.

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species.

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum. The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA-DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.

Draft Genome Sequence of Edwardsiellapiscicida Strain ACC35.1 Isolated from Diseased Turbot (Scophthalmus maximus) in Europe.

Edwardsiella piscicida is a bacterial fish pathogen with a high degree of virulence. The strain ACC35.1 was isolated from diseased turbot in Europe. The draft genome sequence comprises 3.84 Mb with a G+C content of 59.8% and >3,450 protein-coding genes.

Draft Genome Sequence of the Fish Strain Edwardsiella tarda NCIMB 2034.

Edwardsiella tarda is an important pathogen for fish. The strain NCIMB 2034, obtained from the National Collection of Industrial Food and Marine Bacteria, was isolated from unknown diseased fish in the United States. The draft genome sequence has 3.79 Mb with a G+C content of 57.1% and >3,340 protein-coding genes.