Vagococcus jeotgali sp. nov., isolated from cutlassfish jeotgal, a traditional Korean fermented seafood.

A novel, Gram-positive, facultatively anaerobic, and non-motile bacterial strain, designated B2T-5T, was isolated from jeotgal, a traditional Korean fermented seafood. Colonies grown on gifu anaerobic medium agar plates were cream-coloured, irregular, and umbonate with curled margins. Optimal growth of strain B2T-5T occurred at 20 °C, pH 8.0, and in the presence of 1% (w/v) NaCl. Strain B2T-5T was negative for oxidase and catalase activity. Hippurate was not hydrolysed and acetoin was not produced. The major cellular fatty acids were C18 : 1 ω9c and C16 : 0. The cell-wall peptidoglycan was of the A4α type containing l-Lys-d-Asp. The predominant respiratory quinone was menaquinone 7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. According to the phylogenetic analysis based on 16S rRNA gene sequences, strain B2T-5T was most closely related to Vagococcus teuberi DSM 21459T, showing 98.2% sequence similarity. Genome sequencing of strain B2T-5T revealed a genome size of 2.0 Mbp and a G+C content of 33.8 mol%. The average nucleotide identities of strain B2T-5T with Vagococcus teuberi DSM 21459T, Vagococcus bubulae SS1994T, and Vagococcus martis D7T301T were 75.0, 74.7, and 75.1%, respectively. Based on the phenotypic, chemotaxonomic, and genotypic data, strain B2T-5T represents a novel species of the genus Vagococcus, for which the name Vagococcus jeotgali sp. nov. is proposed. The type strain is B2T-5T (=KCTC 21223T=JCM 35937T).

Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

Antibiotic susceptibility and resistance genes profiles of Vagococcus salmoninarum in a rainbow trout (Oncorhyncus mykiss, Walbaum) farm.

Disease outbreaks negatively affect fish production. Antimicrobial agents used in the treatment of diseases become ineffective over time because of antibiotic resistance developed by bacteria distributed in the aquaculture environment. This study was conducted for 4 months (cold period) in a fish farm to detect the fish disease, cold water streptococcosis. In the study, four brood stock showing disease signs were detected. Bacteria isolates were obtained and identified as Vagococcus salmoninarum. Antimicrobial susceptibility of V. salmoninarum was tested and antibiotic resistance gene profiles of V. salmoninarum isolates were screened. The phylogenetic relation of the isolates with the previously reported strains was evaluated. Antibiotic resistance developed by pathogenic bacteria is distributed in the aquaculture environment. The transfer of resistance genes from one bacterium to another is very common. This situation causes the antimicrobial agents used in the treatment of diseases to become ineffective over time. The disc diffusion test showed that all four isolates developed resistance to 13 (FFC30, AX25, C30, E15, CF30, L2, OX1, S10, T30, CRO30, CC2, PT15 and TY15) of the evaluated antibiotics and were about to develop resistance to six others (AM 10, FM 300, CFP75, SXT25, APR15 and TE30). Furthermore, antibiotic resistance genes tetA, sul1, sul2, sul3, dhfr1, ereB and floR were detected in the isolated strain. Moreover, the phylogenetic analysis showed that isolated V. salmoninarum strain (ESN1) was closely related to the bacterial strains isolated from USA and Jura.

The evolutionary mechanism and function analysis of two subgroups of histamine-producing and non-histamine-producing Tetragenococcus halophilus.

Tetragenococcus halophilus is a halophilic bacterium that existed in the fermentation of soy sauce and miso for flavor production and probiotic benefits. However, it is composed of two subgroups, histamine-producing and non-histamine-producing, with the former causing histamine accumulation and offering risks to food safety. Exploring the evolutionary mechanisms and physiological function of histamine-biosynthesis is of significance for understanding the formative mechanism of T. halophilus's strain-specificity and is helpful for microbial control. Using systematic genomic analysis, we found that plasmid acquisition and loss is the evolutionary form resulting in the two subgroups of T. halophilus. Two plasmids, plasmid α with 30 kb and plasmid ß with 4 kb existed in histamine-producing T. halophilus. We investigated the whole genetic information and proposed their genetic function in both two plasmids. The acquisition of histamine-producing plasmid enhanced the acid tolerance of histamine-producing T. halophilus but did not affect salt tolerance. More interestingly, we found that the existence of plasmid will promote the co-culture growth of T. halophilus. This study deepens our understanding of the formative mechanism of microbial species diversity, and provides our knowledge of the physiological function of histamine-producing plasmids.

Identification of an operon and its regulator required for autoaggregation in Tetragenococcus halophilus.

IMPORTANCE: Tetragenococcus halophilus is a halophilic lactic acid bacterium generally used as a starter culture in fermenting soy and fish sauces. Aggregating strains can be useful in fermenting and obtaining clear soy sauce because cell clumps are trapped by the filter cake when the soy sauce mash is pressed. However, the genetic mechanisms of aggregation in T. halophilus are unknown. In this study, we identified genes encoding aggregation factor and its regulator. These findings may provide a foundation for developing improved T. halophilus starter cultures for soy sauce fermentation, leading to more efficient and consistent clear soy sauce production.

Temperature stress improved exopolysaccharide yield from Tetragenococcus halophilus: Structural differences and underlying mechanisms revealed by transcriptomic analysis.

This study aimed to enhance exopolysaccharide production by Tetragenococcus halophilus, and results showed that low temperature (20 °C) significantly improved exopolysaccharide production. Based on the analysis of batch fermentation kinetic parameters, a temperature-shift strategy was proposed, and the exopolysaccharide yield was increased by 28 %. Analysis of the structure of exopolysaccharide suggested that low temperature changed the molecular weight and monosaccharide composition. Transcriptomic analysis was performed to reveal mechanisms of low temperature improving exopolysaccharide production. Results suggested that T. halophilus regulated utilization of carbon sources through phosphotransferase system and increased the expression of key genes in exopolysaccharide biosynthesis to improve exopolysaccharide production. Meanwhile, metabolic pathways involved in glycolysis, amino acids synthesis, two-component system and ATP-binding cassette transporters were affected at low temperature. Results presented in this paper provided a theoretical basis for biosynthetic pathway of exopolysaccharide in T. halophilus and aided to strengthen its production and application in many areas.

Atypical Melissococcus plutonius strains: their characteristics, virulence, epidemiology, and mysteries.

Melissococcus plutonius is a Gram-positive lanceolate coccus that is the causative agent of European foulbrood, an important bacterial disease of honey bee brood. Although this bacterium was originally described in the early 20th century, a culture method for this bacterium was not established until more than 40 years after its discovery due to its fastidious characteristics, including the requirement for high potassium and anaerobic/microaerophilic conditions. These characteristics were considered to be common to the majority of M. plutonius strains isolated worldwide, and M. plutonius was also thought to be genetically homologous or clonal for years. However, non-fastidious variants of this species (designated as atypical M. plutonius) were very recently identified in Japan. Although the morphology of these unusual strains was similar to that of traditionally well-known M. plutonius strains, atypical strains were genetically very different from most of the M. plutonius strains previously isolated and were highly virulent to individual bee larva. These atypical variants were initially considered to be unique to Japan, but were subsequently found worldwide; however, the frequency of isolation varied from country to country. The background of the discovery of atypical M. plutonius in Japan and current knowledge on atypical strains, including their biochemical and culture characteristics, virulence, detection methods, and global distribution, are described in this review. Remaining mysteries related to atypical M. plutonius and directions for future research are also discussed.

Monitoring the growth dynamics of Tetragenococcus halophilus strains in lupine moromi fermentation using a multiplex-PCR system.

OBJECTIVE: The microbiota of a seasoning sauce fermentation process is usually complex and includes multiple species and even various strains of one species. Moreover, composition and cell numbers of individual strains vary over the course of the entire fermentation. This study demonstrates the applicability of a multiplex PCR system to monitor growth dynamics of Tetragenococcus (T.) halophilus strains in order to evaluate their performance and help to select the most competitive starter strain. RESULTS: In a previous study we isolated T. halophilus strains from multiple lupine moromi fermentation processes and characterized them. In this study we wanted to monitor the growth dynamics of these strains in a competitive lupine moromi model fermentation process using a multiplex PCR system. Therefore, pasteurized lupine koji was inoculated with eight different T. halophilus strains, six from lupine moromi, one from an experimental buckwheat moromi fermentation process and the type strain DSM 20,339T, to create the inoculated lupine moromi pilot scale fermentation process. With the multiplex PCR system, we could detect that all strains could grow in lupine moromi but, that TMW 2.2254 and TMW 2.2264 outperformed all other strains. Both strains dominated the fermentation after three weeks with cell counts between 4 × 106 to 4 × 107 CFU/mL for TMW 2.2254 and 1 × 107 to 5 × 107 CFU/mL for TMW 2.2264. The pH dropped to value below 5 within the first 7 days, the selection of these strains might be related to their acid tolerance.

Identification of Capsular Polysaccharide Synthesis Loci Determining Bacteriophage Susceptibility in Tetragenococcus halophilus.

Bacteriophages infecting Tetragenococcus halophilus, a halophilic lactic acid bacterium, have been a major industrial concern due to their detrimental effects on the quality of food products. Previously characterized tetragenococcal phages displayed narrow host ranges, but there is little information on these mechanisms. Here, we revealed the host's determinant factors for phage susceptibility using two virulent phages, phiYA5_2 and phiYG2_4, that infect T. halophilus YA5 and YG2, respectively. Phage-resistant derivatives were obtained from these host strains, and mutations were found at the capsular polysaccharide (CPS) synthesis (cps) loci. Quantification analysis verified that capsular polysaccharide production by the cps derivatives from YG2 was impaired. Transmission electron microscopy observation confirmed the presence of filamentous structures outside the cell walls of YG2 and their absence in the cps derivatives of YG2. Phage adsorption assays revealed that phiYG2_4 adsorbed to YG2 but not its cps derivatives, which suggests that the capsular polysaccharide of YG2 is the specific receptor for phiYG2_4. Interestingly, phiYA5_2 adsorbed and infected cps derivatives of YG2, although neither adsorption to nor infection of the parental strain YG2 by phiYA5_2 was observed. The plaque-surrounding halos formed by phiYA5_2 implied the presence of the virion-associated depolymerase that degrades the capsular polysaccharide of YA5. These results indicated that the capsular polysaccharide is a physical barrier rather than a binding receptor for phiYA5_2 and that phiYA5_2 specifically overcomes the capsular polysaccharide of YA5. Thus, it is suggested that tetragenococcal phages utilize CPSs as binding receptors and/or degrade CPSs to approach host cells. IMPORTANCE T. halophilus is a halophilic lactic acid bacterium that contributes to the fermentation processes for various salted foods. Bacteriophage infections of T. halophilus have been a major industrial problem causing fermentation failures. Here, we identified the cps loci in T. halophilus as genetic determinants of phage susceptibility. The structural diversity of the capsular polysaccharide is responsible for the narrow host ranges of tetragenococcal phages. The information provided here could facilitate future studies on tetragenococcal phages and the development of efficient methods to prevent bacteriophage infections.

Vagococcus luciliae sp. nov., isolated from the common green bottle fly Lucilia sericata.

The genus Vagococcus belongs to the family Enterococcaceae (order Lactobacillales) and is closely related to the genus Enterococcus. Currently, 19 species of the genus have been validly named. In this study, we isolated strain G314FT from the common green bottle fly Lucilia sericata collected in Germany. Sequencing of its almost-complete 16S rRNA gene revealed that the isolate belongs to the genus Vagococcus, being closely related to Vagococcus bubulae SS1994T with high sequence identity (99.50 %), followed by Vagococcus martis D7T301T (98.86 %), Vagococcus vulneris SS1995T (98.71 %), Vagococcus teuberi DSM 21459T (98.64 %), Vagococcus silagei 2B-2T (98.64 %) and Vagococcus penaei CD276T (98.64 %). Genome sequencing of strain G314FT was performed by a combination of Illumina and Oxford Nanopore technology, yielding a circular genome with a size of 2 139 468 bp and an 11 kb plasmid. Average nucleotide identity and digital DNA-DNA hybridization values were calculated between G314FT and its closest-related taxa, and found to be <91 % and <40 %, respectively, thus strongly supporting that strain G314FT represents a novel species of the genus Vagococcus. Phylogenetic and core protein-based phylogenomic trees revealed that G314FT was closely related to a group of three species, V. bubulae SS1994T, V. martis D7T301T and V. teuberi DSM 21459T. Comparatively, the genome of G314FT is the smallest in the group of the four related species, and the biochemical pathway comparison using BlastKOALA revealed that G314FT has lost some amino acid biosynthetic proteins; however, it has gained enzymes for carbohydrate metabolism. Phenotypically, G314FT was consistent with other species of the genus Vagococcus including a negative catalase reaction and non-motility. Using the polyphasic approach, our data supports that the isolate represents a new species, for which we propose the name Vagococcus luciliae G314FT (=DSM 112651T= CCM 9164T).

Targeted Screening for Spontaneous Insertion Mutations in a Lactic Acid Bacterium, Tetragenococcus halophilus.

Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for T. halophilus makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence ISTeha4, belonging to the IS4 family, is translocated at an extremely high frequency in T. halophilus and causes insertional mutations at various loci. We developed a method named targeting spontaneous insertional mutations in genomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. IMPORTANCE Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, ISTeha4, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of T. halophilus.

Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal.

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium.

BACKGROUND: Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. RESULTS: The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. CONCLUSIONS: Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

Construction of a Novel Shuttle Vector for Tetragenococcus species based on a Cryptic Plasmid from Tetragenococcus halophilus.

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Emr), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

Selective extracellular secretion of small double-stranded RNA by Tetragenococcus halophilus.

Small double-stranded RNAs (dsRNAs) abundantly produced by lactic acid bacteria demonstrate immunomodulatory activity and antiviral protective immunity. However, the extracellular secretion of dsRNA from lactic acid bacteria and their compositional and functional differences compared to the intracellular dsRNA is unknown. In this study, we compared the intracellular and secreted extracellular dsRNA of the lactic acid bacteria, Tetragenococcus halophilus, commonly present in fermented foods, by growing in RNA-free and RNase-free media. We used RNA deep sequencing and in-silico analysis to annotate potential regulatory functions for the comparison. A time series sampling of T. halophilus culture demonstrated growth phase-dependent dynamics in extracellular dsRNA secretion with no major change in the intracellular dsRNA profile. The RNA deep sequencing resulted in thousands of diverse dsRNA fragments with 14-21 nucleotides in size from T. halophilus culture. Over 70% of the secreted extracellular dsRNAs were unique in their sequences compared to the intracellular dsRNAs. Furthermore, the extracellular dsRNA abundantly contains sequences that are not T. halophilus genome encoded, not detected intracellularly and showed higher hits on human transcriptome during in-silico analysis, which suggests the presence of extrachromosomal mobile regulatory elements. Further analysis showed significant enrichment of dsRNA target genes of human transcriptome on cancer pathways and transcription process, indicating the extracellular dsRNA of T. halophilus is different not only at the sequence level but also in function. Studying the bacterial extracellular dsRNA is a promising area of future research, particularly for developing postbiotic fermented functional foods and understanding the impact of commensal gut bacteria on human health.

Investigation of Melissococcus plutonius isolates from 3 outbreaks of European foulbrood disease in commercial beekeeping operations in western Canada.

European foulbrood (EFB) disease is an economically important bacterial disease of honey bee larvae caused by enteric infection with Melissococcus plutonius. In this study, we investigated 3 clinical outbreaks of EFB disease in commercial beekeeping operations in western Canada in the summer of 2020 and characterized the Melissococcus plutonius isolates cultured from these outbreaks according to genetic multi-locus sequence type and i n vitro larval pathogenicity. We isolated M. plutonius sequence type 19 from EFB outbreaks in British Columbia and Alberta, and a novel M. plutonius sequence type 36 from an EFB outbreak in Saskatchewan. In vitro larval infection with each M. plutonius isolate was associated with decreased larval survival in vitro by 58.3 to 70.8% (P < 0.001) compared to non-infected controls. Further elucidation of mechanisms of virulence of M. plutonius, paired with epidemiologic investigation, is imperative to improve EFB management strategies and mitigate risks of EFB outbreaks in western Canada.

Enquête sur des isolats de Melissococcus plutonius provenant de trois éclosions de loque e uropéenne dans des exploitations apicoles commerciales de l'Ouest canadien. La loque européenne (EFB) est une maladie bactérienne économiquement importante des larves d'abeilles mellifères causée par une infection entérique par Melissococcus plutonius. Dans cette étude, nous avons enquêté sur trois éclosions cliniques de la maladie EFB dans des exploitations apicoles commerciales dans l'ouest du Canada à l'été 2020 et caractérisé les isolats de Melissococcus plutonius cultivés à partir de ces éclosions selon le typage génomique multilocus et la pathogénicité larvaire in vitro. Nous avons isolé le type de séquence 19 de M. plutonius des éclosions d'EFB en Colombie-Britannique et en Alberta, et une nouvelle séquence de type 36 de M. plutonius d'une éclosion d'EFB en Saskatchewan. L'infection larvaire in vitro avec chaque isolat de M. plutonius était associée à une diminution de la survie larvaire in vitro de 58,3 à 70,8 % (P < 0,001) par rapport aux témoins non infectés. Une élucidation plus poussée des mécanismes de virulence de M. plutonius, associée à une enquête épidémiologique, est impérative pour améliorer les stratégies de gestion de l'EFB et atténuer les risques d'épidémies d'EFB dans l'Ouest canadien.(Traduit par Dr Serge Messier).

Comparative genome analysis of Vagococcus fluvialis reveals abundance of mobile genetic elements in sponge-isolated strains.

BACKGROUND: Vagococcus fluvialis is a species of lactic acid bacteria found both free-living in river and seawater and associated to hosts, such as marine sponges. This species has been greatly understudied, with no complete genome assembly available to date, which is essential for the characterisation of the mobilome. RESULTS: We sequenced and assembled de novo the complete genome sequences of five V. fluvialis isolates recovered from marine sponges. Pangenome analysis of the V. fluvialis species (total of 17 genomes) showed a high intraspecific diversity, with 45.5% of orthologous genes found to be strain specific. Despite this diversity, analyses of gene functions clustered all V. fluvialis species together and separated them from other sequenced Vagococcus species. V. fluvialis strains from different habitats were highly similar in terms of functional diversity but the sponge-isolated strains were enriched in several functions related to the marine environment. Furthermore, sponge-isolated strains carried a significantly higher number of mobile genetic elements (MGEs) compared to previously sequenced V. fluvialis strains from other environments. Sponge-isolated strains carried up to 4 circular plasmids each, including a 48-kb conjugative plasmid. Three of the five strains carried an additional circular extrachromosomal sequence, assumed to be an excised prophage as it contained mainly viral genes and lacked plasmid replication genes. Insertion sequences (ISs) were up to five times more abundant in the genomes of sponge-isolated strains compared to the others, including several IS families found exclusively in these genomes. CONCLUSIONS: Our findings highlight the dynamics and plasticity of the V. fluvialis genome. The abundance of mobile genetic elements in the genomes of sponge-isolated V. fluvialis strains suggests that the mobilome might be key to understanding the genomic signatures of symbiosis in bacteria.

Microbial community changes induced by a newly isolated salt-tolerant Tetragenococcus muriaticus improve the volatile flavor formation in low-salt fish sauce.

Application of low-salt fish sauce is restricted by its easy spoilage and poor flavor formation. In this study, a salt-tolerant strain Tetragenococcus muriaticus with powerful salt tolerance was isolated as starter for the low-salt fish sauce fermentation. The 16S rRNA gene high-throughput sequencing showed that Staphylococcus was the main microbial genus at the beginning of fermentation, reaching 73.75%, followed by Tetragenococcus (16.36%) and Pseudomonas (6.68%), while Tetragenococcus quickly took over the dominant position with the relative abundance over 56.9% after 10 d fermentation and peaked at the end of fermentation. There were a total of 144 volatile compounds identified by HS-SPME-GC-MS, among which aldehydes, esters, alcohols, heterocycles, ketones, and acids were the main volatile flavor compounds and were mainly produced by Tetragenococcus, Synechococcus, Rhodococcus, Stenotrophomonas, Achromobacter, and Brucella based on the correlation network maps constructed by the Pearson's correlation coefficient after O2PLS evaluation. The Sobs, ACE, Chao, and Shannon indexes of microbial community were significantly reduced, while the Simpson index was markedly enhanced in the low-salt fish sauce fermented with T. muriaticus compared with them in the fish sauce without starter addition. The addition of T. muriaticus also obviously improved the types and concentrations of main volatile flavor compounds in the low-salt fish sauce. The correlation network map showed that Tetragenococcus was the only genus that played a crucial role in the spoilage microorganism inhibition and volatile flavor improvement in the fish sauce. T. muriaticus can be developed as a potential microbial starter for the industrial production of low-salt fish sauce.

Lack of evidence for trans-generational immune priming against the honey bee pathogen Melissococcus plutonius.

Trans-generational immune priming involves the transfer of immunological experience, acquired by the parents after exposure to pathogens, to protect their progeny against infections by these pathogens. Such natural mechanisms could be exploited to prevent disease expression in economically important insects, such as the honey bee. This mechanism occurs when honey bee queens are exposed to the pathogenic bacterium Paenibacillus larvae. Here, we tested whether natural or experimental exposure to Melissococcus plutonius-another bacterium triggering a disease in honey bee larvae-reduced the susceptibility of the queen's progeny to infection by this pathogen. Because the immunological response upon pathogen exposure can lead to fitness costs, we also determined whether experimental exposure of the queens affected them or their colony negatively. Neither natural nor experimental exposure induced protection in the honey bee larvae against the deleterious effects of M. plutonius. Our results provided no evidence for the occurrence of trans-generational immune priming upon exposure of the queen to M. plutonius. Whether this lack was due to confounding genetic resistance, to unsuitable exposure procedure or to the absence of trans-generational immune priming against this pathogen in honey bees remains to be determined.

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V. salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C. maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V. salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C. maltaromaticum and V. salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C. maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C. maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.