Draft Genome Sequence of the Fluconazole-Resistant Yarrowia lipolytica Clinical Isolate CBS 18115.

Yarrowia lipolytica is a nonconventional yeast of industrial interest that can sometimes act as an opportunistic pathogen and is responsible for invasive fungal infections. We report the draft genome sequence of the fluconazole-resistant strain CBS 18115, which was isolated from a blood culture. The Y132F substitution in ERG11, which was previously described in fluconazole-resistant Candida isolates, was identified.

Draft Genome Sequence of the Fluconazole-Resistant Candida palmioleophila Clinical Isolate CBS 18098.

Candida palmioleophila belongs to the Saccharomycetales. This opportunistic yeast which has been associated with invasive infections in human and animals, warrants a specific attention as it is frequently misidentified and display reduced susceptibility to fluconazole. Here, we report the first draft genome of C. palmioleophila, obtained from a clinical isolate.

Type IX Secretion System Effectors and Virulence of the Model Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here, we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted 10 genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding 10 secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

Persistent BK Polyomavirus Viruria is Associated with Accumulation of VP1 Mutations and Neutralization Escape.

To investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific "holes" in the patient's humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies.

Colonization kinetics and implantation follow-up of the sewage microbiome in an urban wastewater treatment plant.

The Seine-Morée wastewater treatment plant (SM_WWTP), with a capacity of 100,000 population-equivalents, was fed with raw domestic wastewater during all of its start-up phase. Its microbiome resulted from the spontaneous evolution of wastewater-borne microorganisms. This rare opportunity allowed us to analyze the sequential microbiota colonization and implantation follow up during the start-up phase of this WWTP by means of regular sampling carried out over 8 months until the establishment of a stable and functional ecosystem. During the study, biological nitrification-denitrification and dephosphatation occurred 68 days after the start-up of the WWTP, followed by flocs decantation 91 days later. High throughput sequencing of 18S and 16S rRNA genes was performed using Illumina's MiSeq and PGM Ion Torrent platforms respectively, generating 584,647 16S and 521,031 18S high-quality sequence rDNA reads. Analyses of 16S and 18S rDNA datasets show three colonization phases occurring concomitantly with nitrification, dephosphatation and floc development processes. Thus, we could define three microbiota profiles that sequentially colonized the SM_WWTP: the early colonizers, the late colonizers and the continuous spectrum population. Shannon and inverse Simpson diversity indices indicate that the highest microbiota diversity was reached at days 133 and 82 for prokaryotes and eukaryotes respectively; after that, the structure and complexity of the wastewater microbiome reached its functional stability. This study demonstrates that physicochemical parameters and microbial metabolic interactions are the main forces shaping microbial community structure, gradually building up and maintaining a functionally stable microbial ecosystem.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare, requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN The ΔgldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the ΔgldN mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The ΔgldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease.IMPORTANCE Bacterial cold-water disease, caused by F. psychrophilum, is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.

Draft Genome Sequence of the Fish Pathogen Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare MS-FC-4 is a highly virulent genetic group 1 (formerly genomovar I) strain isolated from rainbow trout (Oncorhynchus mykiss). The draft genome consists of three contigs totaling 3,449,277 bp with 2,811 predicted open reading frames. F. columnare MS-FC-4 is a model strain for functional genomic analyses.

Genomic Diversity and Evolution of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout (Oncorhynchus mykiss) worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of F. psychrophilum, a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare.

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS.IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.

Complete Genome Sequence of Flavobacteriumpsychrophilum Strain OSU THCO2-90, Used for Functional Genetic Analysis.

We report here the complete annotated genome sequence of Flavobacterium psychrophilum OSU THCO2-90, isolated from Coho salmon (Oncorhynchus kisutch) in Oregon. The genome consists of a circular chromosome with 2,343 predicted open reading frames. This strain has proved to be a valuable tool for functional genomics.

From the Flavobacterium genus to the phylum Bacteroidetes: genomic analysis of dnd gene clusters.

Phosphorothioate modification of DNA and the corresponding DNA degradation (Dnd) phenotype that occurs during gel electrophoresis are caused by dnd genes. Although widely distributed among Bacteria and Archaea, dnd genes have been found in only very few, taxonomically unrelated, bacterial species so far. Here, we report the presence of dnd genes and their associated Dnd phenotype in two Flavobacterium species. Comparison with dnd gene clusters previously described led us to report a noncanonical genetic organization and to identify a gene likely encoding a hybrid DndE protein. Hence, we showed that dnd genes are also present in members of the family Flavobacteriaceae, a bacterial group occurring in a variety of habitats with an interesting diversity of lifestyle. Two main types of genomic organization of dnd loci were uncovered probably denoting their spreading in the phylum Bacteroidetes via distinct genetic transfer events.

Complete genome sequence of Flavobacterium indicum GPSTA100-9T, isolated from warm spring water.

We report here the complete annotated genome sequence of Flavobacterium indicum CIP 109464(T) (= GPTSA100-9(T)), isolated from warm spring water in Assam, India. The genome sequence of F. indicum revealed a number of interesting features and genes in relation to its environmental lifestyle.

Complete genome sequence of the fish pathogen Flavobacterium branchiophilum.

Members of the genus Flavobacterium occur in a variety of ecological niches and represent an interesting diversity of lifestyles. Flavobacterium branchiophilum is the main causative agent of bacterial gill disease, a severe condition affecting various cultured freshwater fish species worldwide, in particular salmonids in Canada and Japan. We report here the complete genome sequence of strain FL-15 isolated from a diseased sheatfish (Silurus glanis) in Hungary. The analysis of the F. branchiophilum genome revealed putative mechanisms of pathogenicity strikingly different from those of the other, closely related fish pathogen Flavobacterium psychrophilum, including the first cholera-like toxin in a non-Proteobacteria and a wealth of adhesins. The comparison with available genomes of other Flavobacterium species revealed a small genome size, large differences in chromosome organization, and fewer rRNA and tRNA genes, in line with its more fastidious growth. In addition, horizontal gene transfer shaped the evolution of F. branchiophilum, as evidenced by its virulence factors, genomic islands, and CRISPR (clustered regularly interspaced short palindromic repeats) systems. Further functional analysis should help in the understanding of host-pathogen interactions and in the development of rational diagnostic tools and control strategies in fish farms.