Comparative Whole-genome Sequencing Research Articles

Comparative whole-genome sequence analysis of Mycobacterium tuberculosis isolated from tuberculous meningitis and pulmonary tuberculosis patients

Tuberculous meningitis (TBM) is a severe form of tuberculosis with a high mortality rate. The factors associated with TBM pathogenesis are still unclear. Using comparative whole-genome sequence analysis we compared Mycobacterium tuberculosis (Mtb) isolates from cerebrospinal fluid of TBM cases (n = 73) with those from sputum of pulmonary tuberculosis (PulTB) patients (n = 220) from Thailand. The aim of this study was to seek genetic variants of Mtb associated with TBM. Regardless of Mtb lineage, we found 242 variants that were common to all TBM isolates. Among these variants, 28 were missense SNPs occurring mainly in the pks genes (involving polyketide synthesis) and the PE/PPE gene. Six lineage-independent SNPs were commonly found in TBM isolates, two of which were missense SNPs in Rv0532 (PE_PGRS6). Structural variant analysis revealed that PulTB isolates had 14 genomic regions containing 2–3-fold greater read depth, indicating higher copy number variants and half of these genes belonged to the PE/PPE gene family. Phylogenetic analysis revealed only two small clusters of TBM clonal isolates without support from epidemiological data. This study reported genetic variants of Mtb commonly found in TBM patients compared to PulTB patients. Variants associated with TBM disease warrant further investigation.

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa , causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca . In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

Clonal genome evolution and rapid invasive spread of the marbled crayfish

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarusfallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Chevrier's Field Mouse (Apodemus chevrieri) and Père David's Vole (Eothenomys melanogaster) in China Carry Orthohepeviruses that form Two Putative Novel Genotypes Within the Species Orthohepevirus C.

Hepatitis E virus (HEV) is the prototype of the family Hepeviridae and the causative agent of common acute viral hepatitis. Genetically diverse HEV-related viruses have been detected in a variety of mammals and some of them may have zoonotic potential. In this study, we tested 278 specimens collected from seven wild small mammal species in Yunnan province, China, for the presence and prevalence of orthohepevirus by broad-spectrum reverse transcription (RT)-PCR. HEV-related sequences were detected in two rodent species, including Chevrier's field mouse (Apodemus chevrieri, family Muridae) and Père David's vole (Eothenomys melanogaster, family Cricetidae), with the infection rates of 29.20% (59/202) and 7.27% (4/55), respectively. Further four representative full-length genomes were generated: two each from Chevrier's field mouse (named RdHEVAc14 and RdHEVAc86) and Père David's vole (RdHEVEm40 and RdHEVEm67). Phylogenetic analyses and pairwise distance comparisons of whole genome sequences and amino acid sequences of the gene coding regions showed that orthohepeviruses identified in Chinese Chevrier's field mouse and Père David's vole belonged to the species Orthohepevirus C but were highly divergent from the two assigned genotypes: HEV-C1 derived from rat and shrew, and HEV-C2 derived from ferret and possibly mink. Quantitative real-time RT-PCR demonstrated that these newly discovered orthohepeviruses had hepatic tropism. In summary, our work discovered two putative novel genotypes orthohepeviruses preliminarily named HEV-C3 and HEV-C4 within the species Orthohepevirus C, which expands our understanding of orthohepevirus infection in the order Rodentia and gives new insights into the origin, evolution, and host range of orthohepevirus.

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis.IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis.

Correction: Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.

[This corrects the article DOI: 10.1371/journal.pone.0183925.].

Optimal choice of k-mer in composition vector method for genome sequence comparison

Several proteins and genes are members of families that share a public evolutionary. In order to outline the evolutionary relationships and to recognize conserved patterns, sequence comparison becomes an emerging process. The current work investigates critically the k-mer role in composition vector method for comparing genome sequences. Generally, composition vector methods using k-mer are applied under choice of different value of k to compare genome sequences. For some values of k, results are satisfactory, but for other values of k, results are unsatisfactory. Standard composition vector method is carried out in the proposed work using 3-mer string length. In addition, special type of information based similarity index is used as a distance measure. It establishes that use of 3-mer and information based similarity index provide satisfactory results especially for comparison of whole genome sequences in all cases. These selections provide a sort of unified approach towards comparison of genome sequences.

Minimal standards for describing new species belonging to the families Campylobacteraceae and Helicobacteraceae: Campylobacter, Arcobacter, Helicobacter and Wolinella spp.

Ongoing changes in taxonomic methods, and in the rapid development of the taxonomic structure of species assigned to the Epsilonproteobacteria have lead the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Campylobacter and Related Bacteria to discuss significant updates to previous minimal standards for describing new species of Campylobacteraceae and Helicobacteraceae. This paper is the result of these discussions and proposes minimum requirements for the description of new species belonging to the families Campylobacteraceae and Helicobacteraceae, thus including species in Campylobacter, Arcobacter, Helicobacter, and Wolinella. The core underlying principle remains the use of appropriate phenotypic and genotypic methods to characterise strains sufficiently so as to effectively and unambiguously determine their taxonomic position in these families, and provide adequate means by which the new taxon can be distinguished from extant species and subspecies. This polyphasic taxonomic approach demands the use of appropriate reference data for comparison to ensure the novelty of proposed new taxa, and the recommended study of at least five strains to enable species diversity to be assessed. Methodological approaches for phenotypic and genotypic (including whole-genome sequence comparisons) characterisation are recommended.

A Comparison of Whole Genome Sequencing to Multigene Panel Testing in Hypertrophic Cardiomyopathy Patients

As DNA sequencing costs decline, genetic testing options have expanded. Whole exome sequencing and whole genome sequencing (WGS) are entering clinical use, posing questions about their incremental value compared with disease-specific multigene panels that have been the cornerstone of genetic testing. Forty-one patients with hypertrophic cardiomyopathy who had undergone targeted hypertrophic cardiomyopathy genetic testing (either multigene panel or familial variant test) were recruited into the MedSeq Project, a clinical trial of WGS. Results from panel genetic testing and WGS were compared. In 20 of 41 participants, panel genetic testing identified variants classified as pathogenic, likely pathogenic, or uncertain significance. WGS identified 19 of these 20 variants, but the variant detection algorithm missed a pathogenic 18 bp duplication in myosin binding protein C (MYBPC3) because of low coverage. In 3 individuals, WGS identified variants in genes implicated in cardiomyopathy but not included in prior panel testing: a pathogenic protein tyrosine phosphatase, non-receptor type 11 (PTPN11) variant and variants of uncertain significance in integrin-linked kinase (ILK) and filamin-C (FLNC). WGS also identified 84 secondary findings (mean=2 per person, range=0-6), which mostly defined carrier status for recessive conditions. WGS detected nearly all variants identified on panel testing, provided 1 new diagnostic finding, and allowed interrogation of posited disease genes. Several variants of uncertain clinical use and numerous secondary genetic findings were also identified. Whereas panel testing and WGS provided similar diagnostic yield, WGS offers the advantage of reanalysis over time to incorporate advances in knowledge, but requires expertise in genomic interpretation to appropriately incorporate WGS into clinical care. URL: https://clinicaltrials.gov. Unique identifier: NCT01736566.

Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300–500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

Outbreak and Cocirculation of Three Different Usutu Virus Strains in Eastern Germany.

Usutu virus (USUV) is a mosquito-borne flavivirus accounting for large-scale deaths in resident bird populations. In this study, we show the introduction of USUV to Eastern Germany resulting in massive death of birds, particularly blackbirds (Turdus merula). We found that three diverse USUV lineages ("Europe 3," "Africa 2," and "Africa 3-like") circulated simultaneously. Moreover, we detected USUV in Culex pipiens in a region where no dead birds were reported, strengthening the need for mosquito monitoring to uncover the spread of arboviruses. Furthermore, phylogenetic analyses revealed that mutations accumulated, in particular, in the NS3 region within short time periods. In addition, comparison of whole-genome sequences showed that diverse isolates of the cluster "Africa 3-like" are cocirculating in Germany due to independent introduction events.

Assessing genome-wide copy number variation in the Han Chinese population

BackgroundCopy number variation (CNV) is a valuable source of genetic diversity in the human genome and a well-recognised cause of various genetic diseases. However, CNVs have been considerably under-represented in...

Mangrovibacter phragmitis sp. nov., an endophyte isolated from the roots of Phragmites karka.

A facultatively anaerobic, Gram-stain-negative, rod-shaped, nitrogen-fixing, endophytic bacterial strain designated MP23T was isolated from the roots of Phragmites karka growing in Chilika Lagoon, Odisha, India. Strain MP23T was slightly halophilic, and the optimal NaCl concentration and temperature for growth were 1 % and 30 °C, respectively. On the basis of 16S rRNA gene sequence similarities, strain MP23T was affiliated to the family Enterobacteriaceae and most closely related to Mangrovibacter yixingensis KCTC 42181T and Mangrovibacter plantisponsor DSM 19579T with 99.71 % similarity, followed by Salmonella enterica subsp. salamae DSM 9220T (97.22 %), Cronobacter condimenti LMG 26250T (97.14 %) and Salmonella enterica subsp. diarizonae DSM 14847T (97 %). Sequence analysis of 16S rRNA, hsp60, gyrB and rpoB genes showed that strain MP23T formed a phylogenetic cluster with M. yixingensis KCTC 42181T and M. plantisponsor DSM 19579T indicating that it belongs to the genus Mangrovibacter. The major cellular fatty acids were C16 : 0, C18 : 1ω6c and/or C18 : 1ω7c, C16 : 1ω6c and/or C16 : 1ω7c, C14 : 0, C14 : 0 3-OH and/or iso-C16 : 1 I and C17 : 0 cyclo. Polar lipids of strain MP23T consisted of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 50.3 mol%. Based on experimental DNA-DNA hybridization values and average nucleotide identity derived from in silico comparison of whole-genome sequences, strain MP23T could be distinguished from its closest neighbours. We therefore conclude that strain MP23T represents a novel species of the genus Mangrovibacter for which the name Mangrovibacter phragmitis sp. nov. is proposed. The type strain is MP23T (=DSM 100250T=KCTC 42580T).

Epidemiological and Molecular Characterization of an Invasive Group A Streptococcus emm32.2 Outbreak.

ABSTRACTAn emm32.2 invasive group A streptococcus (iGAS) outbreak occurred in Liverpool from January 2010 to September 2012. This genotype had not previously been identified in Liverpool, but was responsible for 32% (14/44) of all iGAS cases reported during this time period. We performed a case-case comparison of emm32.2 iGAS cases with non-emm32.2 control iGAS cases identified in the Liverpool population over the same time period to assess patient risk factors for emm32.2 iGAS infection. The emm32.2 iGAS cases were confined to the adult population. We show that homelessness, intravenous drug use, and alcohol abuse predisposed patients to emm32.2 iGAS disease; however, no obvious epidemiological linkage between the patients with emm32.2 iGAS could be identified. Comparative whole-genome sequencing analysis of emm32.2 iGAS and non-emm32.2 control isolates was also performed to identify pathogen factors which might have driven the outbreak. We identified 19 genes, five of which had previously been implicated in virulence, which were present in all of the emm32.2 iGAS isolates but not present in any of the non-emm32.2 control isolates. We report that a novel emm32.2 genotype emerged in Liverpool in 2010 and identified a specific subset of genes, which could have allowed this novel emm32.2 genotype to persist in a disadvantaged population in the region over a 3-year period.

BMPR1B mutation causes Pierre Robin sequence.

BackgroundWe investigated a large family with Pierre Robin sequence (PRS).Aim of the studyThis study aims to determine the genetic cause of PRS.ResultsThe reciprocal translocation t(4;6)(q22;p21) was identified to be segregated with PRS in a three-generation family. Whole-genome sequencing and Sanger sequencing successfully detected breakpoints in the intragenic regions of BMRP1B and GRM4. We hypothesized that PRS in this family was caused by (i) haploinsufficiency for BMPR1B or (ii) a gain of function mechanism mediated by the BMPR1B-GRM4 fusion gene. In an unrelated family, we identified another BMPR1B-splicing mutation that co-segregated with PRS.ConclusionWe detected two BMPR1B mutations in two unrelated PRS families, suggesting that BMPR1B disruption is probably a cause of human PRS.MethodsGTG banding, comparative genomic hybridization, whole-genome sequencing, and Sanger sequencing were performed to identify the gene causing PRS.

Polynucleobacter wuianus sp. nov., a free-living freshwater bacterium affiliated with the cryptic species complex PnecC.

Strain QLW-P1FAT50C-4T, isolated from a shallow, acidic freshwater pond located in the Austrian Alps at an altitude of 1300 m, was characterized by investigation of phenotypic, chemotaxonomic and genomic traits. As shown previously, phylogenetic analyses based on 16S rRNA gene sequences placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The major fatty acids of the strain were C16 : 1ω7c and C18 : 1ω7c. The strain has a genome of 2.23 Mbp with a DNA G+C content of 44.9 mol%. The strain encodes a seemingly complete gene cluster for anoxygenic photosynthesis but lacks typical genes for CO2 assimilation. In order to resolve the phylogenetic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were analysed. The phylogenetic reconstruction obtained did not place strain QLW-P1FAT50C-4T close to any of the five previously described species within subcluster PnecC. Pairwise average nucleotide identity (ANI) comparisons of whole-genome sequences suggested that strain QLW-P1FAT50C-4T (=DSM 24008T=CIP 111100T) represents a novel species, for which we propose the name Polynucleobacter wuianus sp. nov.

Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains.

Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

Evolutionary Dynamics of Pandemic Methicillin-Sensitive Staphylococcus aureus ST398 and Its International Spread via Routes of Human Migration.

ABSTRACTMethicillin-susceptible Staphylococcus aureus (MSSA) accounts for the majority of S. aureus infections globally, and yet surprisingly little is known about its clonal evolution. We applied comparative whole-genome sequencing (WGS) analyses to epidemiologically and geographically diverse ST398-MSSA, a pandemic lineage affecting both humans and livestock. Bayesian phylogenetic analysis predicted divergence of human-associated ST398-MSSA ~40 years ago. Isolates from Midwestern pigs and veterinarians differed substantially from those in New York City (NYC). Pig ST398 strains contained a large region of recombination representing imports from multiple sequence types (STs). Phylogeographic analyses supported the spread of ST398-MSSA along local cultural and migratory links between parts of the Caribbean, North America, and France, respectively. Applying pairwise single-nucleotide polymorphism (SNP) distances as a measure of genetic relatedness between isolates, we observed that ST398 not only clustered in households but also frequently extended across local social networks. Isolates collected from environmental surfaces reflected the full diversity of colonizing individuals, highlighting their potentially critical role as reservoirs for transmission and diversification. Strikingly, we observed high within-host SNP variability compared to our previous studies on the dominant methicillin-resistant Staphylococcus aureus (MRSA) clone USA300. Our data indicate that the dynamics of colonization, persistence, and transmission differ substantially between USA300-MRSA and ST398-MSSA. Taken together, our study reveals local and international routes of transmission for a major MSSA clone, indicating key impacts of recombination and mutation on genetic diversification and highlighting important ecological differences from epidemic USA300. Our study demonstrates extensive local and international routes of transmission for a major MSSA clone despite the lack of substantial antibiotic resistance.

Brevibacterium massiliense (Roux and Raoult 2009) is a later heterotypic synonym of Brevibacterium ravenspurgense (Mages, Frodl, Bernard and Funke 2009), using whole-genome sequence analysis as a comparative tool.

A patient strain derived from urine was found by 16S rRNA gene sequencing to be closely related (99.6 % identity) to sequences derived from both Brevibacterium ravenspurgense CCUG 56047T and Brevibacterium massilienseCCUG 53855T. Those species had been described during the same 11 month period in 2008-2009. Further characterization revealed that those isolates could not be readily distinguished from each other biochemically, by cellular fatty acids, antimicrobial susceptibility, MALDI-TOF MS, 16S rRNA gene sequencing or by whole-genome sequence (WGS) analyses. By WGS comparison, these isolates had an aerage nucleotide identity using blastn (ANIb) scores of 95.7 % or higher to each other, DNA G+C content in the range of 62.3 mol%-62.4 mol%, with genome sizes ranging from 2.28×106 to 2.41×106 bases. Based on these data, we propose that the name B. massiliense is a later heterotypic synonym of B. ravenspurgense and provide an emended description of B. ravenspurgense.

Characterization of clinical and environmental isolates of Vibrio cidicii sp. nov., a close relative of Vibrio navarrensis.

Four Vibrio spp. isolates from the historical culture collection at the Centers for Disease Control and Prevention, obtained from human blood specimens (n=3) and river water (n=1), show characteristics distinct from those of isolates of the most closely related species, Vibrio navarrensis and Vibrio vulnificus, based on phenotypic and genotypic tests. They are specifically adapted to survival in both freshwater and seawater, being able to grow in rich media without added salts as well as salinities above that of seawater. Phenotypically, these isolates resemble V. navarrensis, their closest known relative with a validly published name, but the group of isolates is distinguished from V. navarrensis by the ability to utilize l-rhamnose. Average nucleotide identity and percent DNA-DNA hybridization values obtained from the pairwise comparisons of whole-genome sequences of these isolates to V. navarrensis range from 95.4-95.8 % and 61.9-64.3 %, respectively, suggesting that the group represents a different species. Phylogenetic analysis of the core genome, including four protein-coding housekeeping genes (pyrH, recA, rpoA and rpoB), places these four isolates into their own monophyletic clade, distinct from V. navarrensis and V. vulnificus. Based on these differences, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio cidicii sp. nov. is proposed; strain LMG 29267T (=CIP 111013T=2756-81T), isolated from river water, is the type strain.