Background Cataloguing the distribution of genes within natural bacterial populations is essential for understanding evolutionary processes and the genetic basis of adaptation. Advances in whole genome sequencing technologies have led to a vast expansion in the amount of bacterial genomes deposited in public databases. There is a pressing need for software solutions which are able to cluster, catalogue and characterise genes, or other features, in increasingly large genomic datasets. Results Here we present a pangenomics toolbox, PIRATE (Pangenome Iterative Refinement and Threshold Evaluation), which identifies and classifies orthologous gene families in bacterial pangenomes over a wide range of sequence similarity thresholds. PIRATE builds upon recent scalable software developments to allow for the rapid interrogation of thousands of isolates. PIRATE clusters genes (or other annotated features) over a wide range of amino acid or nucleotide identity thresholds and uses the clustering information to rapidly identify paralogous gene families and putative fission/fusion events. Furthermore, PIRATE orders the pangenome using a directed graph, provides a measure of allelic variation, and estimates sequence divergence for each gene family. Conclusions We demonstrate that PIRATE scales linearly with both number of samples and computation resources, allowing for analysis of large genomic datasets, and compares favorably to other popular tools. PIRATE provides a robust framework for analysing bacterial pangenomes, from largely clonal to panmictic species.

Cataloguing the distribution of genes within natural bacterial populations is essential for understanding evolutionary processes and the genetic basis of adaptation. Here we present a pangenomics toolbox, PIRATE (Pangenome Iterative Refinement And Threshold Evaluation), which identifies and classifies orthologous gene families in bacterial pangenomes over a wide range of sequence similarity thresholds. PIRATE builds upon recent scalable software developments to allow for the rapid interrogation of thousands of isolates. PIRATE clusters genes (or other annotated features) over a wide range of amino-acid or nucleotide identity thresholds and uses the clustering information to rapidly classify paralogous gene families into either putative fission/fusion events or gene duplications. Furthermore, PIRATE orders the pangenome using a directed graph, provides a measure of allelic variation and estimates sequence divergence for each gene family. We demonstrate that PIRATE scales linearly with both number of samples and computation resources, allowing for analysis of large genomic datasets, and compares favorably to other popular tools. PIRATE provides a robust framework for analysing bacterial pangenomes, from largely clonal to panmictic species.Availability: PIRATE is implemented in Perl and is freely available under an GNU GPL 3 open source license from https://github.com/SionBayliss/PIRATE. Contact: s.bayliss@bath.ac.uk

Climate change, changing farming practices, social and demographic changes and rising levels of antibiotic resistance are likely to lead to future increases in opportunistic bacterial infections that are more difficult to treat. Uncovering the prevalence and identity of pathogenic bacteria in the environment is key to assessing transmission risks. We describe the first use of the Wax moth larva Galleria mellonella, a well-established model for the mammalian innate immune system, to selectively enrich and characterize pathogens from coastal environments in the South West of the UK. Whole-genome sequencing of highly virulent isolates revealed amongst others a Proteus mirabilis strain carrying the Salmonella SGI1 genomic island not reported from the UK before and the recently described species Vibrio injenensis hitherto only reported from human patients in Korea. Our novel method has the power to detect bacterial pathogens in the environment that potentially pose a serious risk to public health.

Humans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps. Based on data from 1103 genomes of environmental and clinical isolates, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if long-range dispersal has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades. These results suggest that human activity, such as shipping, aquatic products trade and increased human migration between continents, are responsible for the change of distribution pattern of this species.

20Background Humans have profoundly affected the ocean environment but little is known 21 about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found 22 in warm coastal waters and causes gastroenteritis in humans and economically significant 23 disease in shrimps. 24Results Based on data from 1,103 genomes, we show that V. parahaemolyticus is divided 25 into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are 26 largely restricted to the US and Northern Europe, while the others are found worldwide, with 27 VppAsia making up the great majority of isolates in the seas around Asia. Patterns of 28 diversity within and between the populations are consistent with them having arisen by 29 progressive divergence via genetic drift during geographical isolation. However, we find that 30 there is substantial overlap in their current distribution. These observations can be reconciled 31 without requiring genetic barriers to exchange between populations if dispersal between 32 oceans has increased dramatically in the recent past. We found that VppAsia isolates from the 33 US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than 34 VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within 35 epidemic lineages, we estimate that recombination affects about 0.017% of the genome per 36year, implying that the genetic mixture has taken place within the last few decades. 37Conclusions These results suggest that human activity, such as shipping and aquatic products 38 trade, are responsible for the change of distribution pattern of this marine species. 39 40 Keywords 41 Vibrio parahaemolyticus, population structure, biogeography, anthropogenic change, ocean 42 dispersal 43 44 3 45 Background 46 Hospitable environments for particular marine microbes can be separated by large distances 47 but whether dispersal barriers substantially influence their distribution and evolution is 48 unknown. There are many studies of distribution of marine microbes e.g. [1-4], but these 49 typically survey patterns of macro-scale diversity. Differences in species level or genus level 50 composition between locations are as likely to reflect environmental heterogeneity as 51 dispersal, making the patterns difficult to interpret. Recent spread of microbes between 52 continents has been documented for lineages that cause pathogenic infection of humans, 53 including notorious clonal groups within Vibrio parahaemolyticus and Vibrio cholerae [5-8]. 54 However, these lineages are unusual in using humans as vectors, which might facilitate long-55 range dispersal as in the case of the Haitian cholera outbreak [9]. We currently have little 56 information on rates of spread of the great majority of environmental organisms that do not 57 colonize large-animal hosts. 58 59 V. parahaemolyticus prefers warm coastal waters and causes gastroenteritis in humans [10, 60 11]. Disease outbreaks became common from 1990s and became global, due to spread of 61 pa...

Vibrio anguillarum is the causative agent of vibriosis in many species important to aquaculture. We generated whole genome sequence (WGS) data on a diverse collection of 64 V. anguillarum strains, which we supplemented with 41 publicly available genomes to produce a combined dataset of 105 strains. These WGS data resolved six major lineages (L1-L6), and the additional use of multilocus sequence analysis (MLSA) clarified the association of L1 with serotype O1 and Salmonidae hosts (salmon/trout), and L2 with serotypes O2a/O2b/O2c and Gadidae hosts (cod). Our analysis also revealed a large-scale homologous replacement of 526-kb of core genome in an L2 strain from a con-specific donor. Although the strains affected by this recombination event are exclusively associated with Gadidae, we find no clear genetic evidence that it has played a causal role in host specialism. Whilst it is established that Vibrio species freely recombine, to our knowledge this is the first report of a contiguous recombinational replacement of this magnitude in any Vibrio genome. We also note a smaller accessory region of high single nucleotide polymorphism (SNP) density and gene content variation that contains lipopolysaccharide biosynthesis genes which may play a role in determining serotype.

Climate change, changing farming practices, social and demographic changes and rising levels of antibiotic resistance are likely to lead to future increases in opportunistic bacterial infections that are more difficult to treat. Uncovering the prevalence and identity of pathogenic bacteria in the environment is key to assessing transmission risks. We describe the first use of the Wax moth larva Galleria mellonella, a well-established model for the mammalian innate immune system, to selectively enrich and characterize pathogens from coastal environments in the South West of the U.K. Whole-genome sequencing of highly virulent isolates revealed amongst others a Proteus mirabilis strain carrying the Salmonella SGI1 genomic island not reported from the U.K. before and the recently described speciesVibrio injenensis hitherto only reported from human patients in Korea. Our novel method has the power to detect novel bacterial pathogens in the environment that potentially pose a serious risk to public health.

Bacteria from the family Vibrionaceae have been implicated in mass mortalities of farmed Pacific oysters (Magallana gigas) in multiple countries, leading to substantial impairment of growth in the sector. In Ireland there has been concern that Vibrio have been involved in serious summer outbreaks. There is evidence that Vibrio aestuarianus is increasingly becoming the main pathogen of concern for the Pacific oyster industry in Ireland. While bacteria belonging to the Vibrio splendidus clade are also detected frequently in mortality episodes, their role in the outbreaks of summer mortality is not well understood. To identify and characterize strains involved in these outbreaks, 43 Vibrio isolates were recovered from Pacific oyster summer mass mortality episodes in Ireland from 2008 to 2015 and these were whole-genome sequenced. Among these, 25 were found to be V. aestuarianus (implicated in disease) and 18 were members of the V. splendidus species complex (role in disease undetermined). Two distinct clades of V. aestuarianus – clade A and clade B – were found that had previously been described as circulating within French oyster culture. The high degree of similarity between the Irish and French V. aestuarianus isolates points to translocation of the pathogen between Europe’s two major oyster-producing countries, probably via trade in spat and other age classes. V. splendidus isolates were more diverse, but the data reveal a single clone of this species that has spread across oyster farms in Ireland. This underscores that Vibrio could be transmitted readily across oyster farms. The presence of V. aestuarianus clades A and B in not only France but also Ireland adds weight to growing concern that this pathogen is spreading and impacting Pacific oyster production within Europe.