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Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

ABSTRACT: Vibriosis, a hemorrhagic septicemic disease caused by the bacterium Vibrio anguillarum, is an important bacterial infection in Danish sea-reared rainbow trout. Despite of vaccination, outbreaks still occur, likely because the vaccine is based on V. anguillarum strains from abroad/other hosts than rainbow trout. Information about the genetic diversity of V. anguillarum specifically in Danish rainbow trout, is required to investigate this claim. Consequently, the aim of the present investigation was to sequence and to characterize a collection of 44 V. anguillarum strains obtained primarily from vibriosis outbreaks in Danish rainbow trout. The strains were sequenced, de novo assembled, and the genomes examined for the presence of plasmids, virulence, and acquired antibiotic resistance genes. To investigate the phylogeny, single nucleotide polymorphisms were identified, and the pan-genome was calculated. All strains carried tet(34) encoding tetracycline resistance, and 36 strains also contained qnrVC6 for increased fluoroquinolone/quinolone resistance. But interestingly, all strains were phenotypic sensitive to both oxytetracycline and oxolinic acid. Almost all serotype O1 strains contained a pJM1-like plasmid and nine serotype O2A strains carried the plasmid p15. The distribution of virulence genes was rather similar across the strains, although evident variance among serotypes was observed. Most significant, almost all serotype O2 and O3 strains, as well as the serotype O1 strain without a pJM1-like plasmid, carried genes encoding piscibactin biosynthesis. Hence supporting the hypothesis, that piscibactin plays a crucial role in virulence for pathogenic strains lacking the anguibactin system. The phylogenetic analysis and pan-genome calculations revealed great diversity within V. anguillarum. Serotype O1 strains were in general very similar, whereas considerable variation was found among serotype O2A strains. The great diversity within the V. anguillarum serotype O2A genomes is most likely the reason why vaccines provide good protection from some strains, but not from others. Hopefully, the new genomic data and knowledge provided in this study might help develop an optimized vaccine against V. anguillarum in the future to reduce the use of antibiotics, minimize economic losses and improve the welfare of the fish.

SUBMITTER: Hansen MJ

PROVIDER: S-EPMC7719292 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

Hansen Mie Johanne MJ Kudirkiene Egle E Dalsgaard Inger I

PeerJ 20201203

Vibriosis, a hemorrhagic septicemic disease caused by the bacterium Vibrio anguillarum, is an important bacterial infection in Danish sea-reared rainbow trout. Despite of vaccination, outbreaks still occur, likely because the vaccine is based on V. anguillarum strains from abroad/other hosts than rainbow trout. Information about the genetic diversity of V. anguillarum specifically in Danish rainbow trout, is required to investigate this claim. Consequently, the aim of the pr ...[more]

PMID: 33344086

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Project description:UnlabelledThe genetic heterogeneity of the close relatives Vibrio anguillarum and Vibrio ordalii, both serious pathogens of fish causing extensive losses in aquaculture, was studied. Eight housekeeping genes, i.e., atpA, ftsZ, gapA, gyrB, mreB, rpoA, topA, and pyrH, were partially sequenced in 116 isolates from diverse fish species and geographical areas. The eight genes appear to be under purifying selection, and the genetic diversity in the total data set was estimated to be 0.767 ± 0.026. Our multilocus sequence analysis (MLSA) scheme identified several widespread clonal complexes and resolved the isolates, for the most part, according to serotype. Serotype O2b isolates from diseased cod in Norway, Ireland, and Scotland were found to be extremely homogeneous. Horizontal gene transfer appears to be fairly common within and between clonal complexes. Taken together, MLSA and in silico DNA-DNA hybridization (DDH) calculations suggest that some isolates previously characterized as V ordalii, i.e., 12B09, FF93, FS144, and FS238, are in fact V. anguillarum isolates. The precise taxonomic situation for two isolates from Atlantic cod that display several traits consistent with V. ordalii, i.e., NVI 5286 and NVI 5918, and a single environmental strain that was previously considered to represent V. ordalii, i.e., FF167, is less clear.ImportanceIt is still being debated whether V. anguillarum and V ordalii represent separate bacterial species. Our study addresses this issue and elucidates the degree of genetic variability within this group of closely related bacteria, based on a substantial number of isolates. Our results clearly illustrate the existence of different populations among putative V ordalii isolates. On the basis of additional full-length genomic analysis, we conclude that most environmental isolates previously identified as V ordalii lie firmly within the species V. anguillarum While bona fide fish-pathogenic V ordalii isolates display a very close genetic relationship with V. anguillarum, they combine a clearly divergent evolutionary pattern with clear phenotypic differences. The study also highlights the need for further characterization of fish-pathogenic isolates from the northern Atlantic region that share phenotypic characteristics with V. ordalii but are genetically closer to V. anguillarum The retention of taxonomic distinctions between the phenotypically different groups of bacteria is of practical advantage to microbial ecologists and veterinarians.

Project description:BackgroundVibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number.ResultsTo generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified.ConclusionsOur study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains.

Project description:BACKGROUND: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements. RESULTS: Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 microg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer. CONCLUSIONS: The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.

Project description:Chlamydia (C.) gallinacea is a recently identified bacterium that mainly infects domestic chickens. Demonstration of C. gallinacea in human atypical pneumonia suggests its zoonotic potential. Its prevalence in chickens exceeds that of C. psittaci, but genetic and genomic research on C. gallinacea is still at the beginning. In this study, we conducted whole-genome sequencing of C. gallinacea strain JX-1 isolated from an asymptomatic chicken, and comparative genomic analysis between C. gallinacea strains and related chlamydial species.The genome of C. gallinacea JX-1 was sequenced by single-molecule, real-time technology and is comprised of a 1,059,522-bp circular chromosome with an overall G?+?C content of 37.93% and sequence similarity of 99.4% to type strain 08-1274/3. In addition, a plasmid designated pJX-1, almost identical to p1274 of the type strain, except for two point mutations, was only found in field strains from chicken, but not in other hosts. In contrast to chlamydial species with notably variable polymorphic membrane protein (pmp) genes and plasticity zone (PZ), these regions were conserved in both C. gallinacea strains. There were 15 predicted pmp genes, but only B, A, E1, H, G1 and G2 were apparently intact in both strains. In comparison to chlamydial species where the PZ may be up to 50 kbp, C. gallinacea strains displayed gene content reduction in the PZ (14 kbp), with strain JX-1 having a premature STOP codon in the cytotoxin (tox) gene, while tox gene is intact in the type strain. In multilocus sequence typing (MLST), 15 C. gallinacea STs were identified among 25 strains based on cognate MLST allelic profiles of the concatenated sequences. The type strain and all Chinese strains belong to two distinct phylogenetic clades. Clade of the Chinese strains separated into 14 genetically distinct lineages, thus revealing considerable genetic diversity of C. gallinacea strains in China.In this first detailed comparative genomic analysis of C. gallinacea, we have provided evidence for substantial genetic diversity among C. gallinacea strains. How these genetic polymorphisms affect C. gallinacea biology and pathogenicity should be addressed in future studies that focus on phylogenetics and host adaption of this enigmatic bacterial agent.

Project description:Since first identified in December of 2019, COVID-19 has been quickly spreading to the world in few months and COVID-19 cases are still undergoing rapid surge in most countries worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adapts and evolves rapidly in nature. With the availability of 16,092 SARS-CoV-2 full genomes in GISAID as of 13 May, we removed the poor-quality genomes and performed mutational profiling analysis for the remaining 11,183 viral genomes. Global analysis of all sequences identified all single nucleotide polymorphisms (SNPs) across the whole genome and critical SNPs with high mutation frequency that contributes to five-clade classification of global strains. A total of 119 SNPs were found with 74 non-synonymous mutations, 43 synonymous mutations and two mutations in intergenic regions. Analysis of geographic pattern of mutational profiling for the whole genome reveals differences between each continent. A transition mutation from C to T represents the most mutation types across the genome, suggesting rapid evolution and adaptation of the virus in host. Amino acid (AA) deletions and insertions found across the genome results in changes in viral protein length and potential function alteration. Mutational profiling for each gene was analysed, and results show that nucleocapsid gene demonstrates the highest mutational frequency, followed by Nsp2, Nsp3 and Spike gene. We further focused on non-synonymous mutational distributions on four key viral proteins, spike with 75 mutations, RNA-dependent-RNA-polymerase with 41 mutations, 3C-like protease with 22 mutations and Papain-like protease with 10 mutations. Results show that non-synonymous mutations on critical sites of these four proteins pose great challenge for development of anti-viral drugs and other countering measures. Overall, this study provides more understanding of genetic diversity/variability of SARS-CoV-2 and insights for development of anti-viral therapeutics.

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Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

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Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

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