The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps.

Chalcidoidea are mostly parasitoid wasps that include as many as 500 000 estimated species. Capturing phylogenetic signal from such a massive radiation can be daunting. Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 ultra-conserved elements (UCEs) for 433 taxa including all extant families, >95% of all subfamilies, and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between the molecular results and our collective knowledge of morphology and biology, we detected bias in the analyses that was driven by the saturation of nucleotide data. Our final results are based on a concatenated analysis of the least saturated exons and UCE datasets (2054 loci, 284 106 sites). Our analyses support an expected sister relationship with Mymarommatoidea. Seven previously recognized families were not monophyletic, so support for a new classification is discussed. Natural history in some cases would appear to be more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a transition from smaller soft-bodied wasps to larger and more heavily sclerotized wasps, with egg parasitism as potentially ancestral for the entire superfamily. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the "Angiosperm Terrestrial Revolution". Our dating analyses suggest a middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9-169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea may have undergone a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about the host taxa of chalcid wasps, their fossil record and Earth's palaeogeographic history.

What Whole Genome Sequencing Has Told Us About Pathogenic Vibrios.

When the first microbial genome sequences were published just 20 years ago, our understanding regarding the microbial world changed dramatically. The genomes of the first pathogenic vibrios sequenced, including Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus revealed a functional and phylogenetic diversity previously unimagined as well as a genome structure indelibly shaped by horizontal gene transfer. The initial glimpses into these organisms also revealed a genomic plasticity that allowed these bacteria to thrive in challenging and varied aquatic and marine environments, but critically also a suite of pathogenicity attributes. In this review we outline how our understanding of vibrios has changed over the last two decades with the advent of genomics and advances in bioinformatic and data analysis techniques, it has become possible to provide a more cohesive understanding regarding these bacteria: how these pathogens have evolved and emerged from environmental sources, their evolutionary routes through time and space, how they interact with other bacteria and the human host, as well as initiate disease. We outline novel approaches to the use of whole genome sequencing for this important group of bacteria and how new sequencing technologies may be applied to study these organisms in future studies.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

Global Expansion of Pacific Northwest Vibrio parahaemolyticus Sequence Type 36.

We report transcontinental expansion of Vibrio parahaemolyticus sequence type 36 into Lima, Peru. From national collections, we identified 7 isolates from 2 different Pacific Northwest complex lineages that surfaced during 2011-2016. Sequence type 36 is likely established in environmental reservoirs. Systematic surveillance enabled detection of these epidemic isolates.

The new tools revolutionizing Vibrio science.

As microbiologists we live in exciting times. A variety of technical and conceptual developments, particularly in the last decade have revolutionized the field of microbiology, redrawing the landscape, and entirely redefining what is possible. Perhaps this paradigm shift is no more apparent than in the study of vibrios. The family Vibrionaceae are almost unique as a group of bacteria to study in microbiology: they are genomically, phylogenetically and functionally diverse yet a distinct group of environmental bacteria encompassing important human and animal pathogens as well as non-pathogenic species such as ecologically critical symbionts. Sensitive to physiochemical stimuli, they are among the fasting replicating bacteria studied, capable of responding almost immediately to favourable environmental conditions such as those afforded by climate warming. Characterized by an unusual double chromosome and frequently carrying numerous cryptic plasmids - their genomes are often pockmarked with insertion elements, transposons, prophages and integrases - paying testament to past genomic promiscuity. With a strong affinity for environmental niches in freshwater and marine systems, they are among the most numerous bacteria present in our oceans, coasts and freshwater environments. As such they offer something for almost anyone interested in microbiology and represent an excellent example of field of microbiology that has benefitted hugely by advances across a gamut of disciplines - not just microbiological - but encompassing genomics, genetics, oceanography, ecological, earth observations sciences and data visualization, among others. We will briefly outline some of the most exciting, innovative and translational scientific advances that are currently being applied to these ecologically, environmentally and clinically important bacteria.

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3-5 million infections worldwide and 28.800-130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.

Epidemic Dynamics of Vibrio parahaemolyticus Illness in a Hotspot of Disease Emergence, Galicia, Spain.

Galicia in northwestern Spain has been considered a hotspot for Vibrio parahaemolyticus infections. Infections abruptly emerged in 1998 and, over the next 15 years, were associated with large outbreaks caused by strains belonging to a single clone. We report a recent transition in the epidemiologic pattern in which cases throughout the region have been linked to different and unrelated strains. Global genome-wide phylogenetic analysis revealed that most of the pathogenic strains isolated from infections were associated with globally diverse isolates, indicating frequent episodic introductions from disparate and remote sources. Moreover, we identified that the 2 major switches in the epidemic dynamics of V. parahaemolyticus in the regions, the emergence of cases and an epidemiologic shift in 2015-2016, were associated with the rise of sea surface temperature in coastal areas of Galicia. This association may represent a fundamental contributing factor in the emergence of illness linked to these introduced pathogenic strains.

Vibrio vulnificus: new insights into a deadly opportunistic pathogen.

Vibrio vulnificus is a Gram-negative aquatic bacterium first isolated by the United States (US) Centers for Disease Control and Prevention (CDC) in 1964. This bacterium is part of the normal microbiota of estuarine waters and occurs in high numbers in molluscan shellfish around the world, particularly in warmer months. Infections in humans are derived from consumption of seafood produce and from water exposure. Vibrio vulnificus is a striking and enigmatic human pathogen, yet many aspects related to its biology, genomics, virulence capabilities and epidemiology remain elusive and poorly understood. This pathogen is responsible for over 95% of seafood-related deaths in the United States, and carries the highest fatality rate of any food-borne pathogen. Indeed, infections associated with this pathogen that progress to primary septicaemia have a similar case fatality rate to category BSL 3 and 4 pathogens, such as anthrax, bubonic plague, Ebola and Marburg fever. Interestingly, V. vulnificus infections disproportionately affect males (∼85% of cases) and older patients (> 40 years), especially those with underlying conditions such as liver diseases, diabetes and immune disorders. New insights from molecular studies and comparative genomic approaches have offered tantalising insights into this pathogen. A recent increase and geographical spread in reported infections, in particular wound cases, underlines the growing international importance of V. vulnificus, particularly in the context of coastal warming. We outline and explore here a range of current data gaps regarding this important pathogen, and provide some current thoughts on approaches to elucidate key aspects associated with this bacterium.

Escherichia coli testing and enumeration in live bivalve shellfish - Present methods and future directions.

A wide variety of pathogenic agents such as bacteria, viruses and parasites can be greatly concentrated in filter feeding bivalve molluscan shellfish (BMS), that are grown in faecally contaminated waters. Human health risks associated with the consumption of BMS are also compounded by the traditional pattern of consuming them raw or lightly cooked. Because of these well-established food safety risks, food legislation such as that in Europe stipulates that BMS production areas are monitored for faecal contamination and classified accordingly. In this review we provide information regarding the background and use of methods for determining and quantifying Escherichia coli (E. coli) in shellfish matrices, focussing on the Most Probable Number (MPN) based approach. This review also discusses other techniques for determining E. coli in food matrices, as well as specific tests across a range of other food microbiology applications. This information draws on several sources: published peer-reviewed reports, data derived from proficiency testing/ring trials, depuration and challenge studies, as well as specific examples from BMS classification and long-term monitoring studies. We also provide a discussion on possible avenues for future direction regarding testing methods in this food microbiology sector.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Evaluation of the protection against norovirus afforded by E. coli monitoring of shellfish production areas under EU regulations.

EC Regulation 854/2004 requires the classification of bivalve mollusc harvesting areas according to the faecal pollution status of sites. It has been reported that determination of Escherichia coli in bivalve shellfish is a poor predictor of norovirus (NoV) contamination in individual samples. We explore the correlation of shellfish E. coli data with norovirus presence using data from studies across 88 UK sites (1,184 paired samples). We investigate whether current E. coli legislative standards could be refined to reduce NoV infection risk. A significant relationship between E. coli and NoV was found in the winter months (October to February) using data from sites with at least 10 data pairs (51 sites). We found that the ratio of arithmetic means (log10 E. coli to log10 NoV) at these sites ranged from 0.6 to 1.4. The lower ratios (towards 0.6) might typically indicate situations where the contribution from UV disinfected sewage discharges was more significant. Conversely, higher ratios (towards 1.4) might indicate a prevalence of animal sources of pollution; however, this relationship did not always hold true and so further work is required to fully elucidate the factors of relevance. Reducing the current class B maximum (allowed in 10% of samples) from 46,000 E. coli per 100 g (corresponding NoV value of 75750 ± 103) to 18,000 E. coli per 100 g (corresponding NoV value of 29365 ± 69) reduces maximum levels of NoV by a factor of 2.6 to 1; reducing the upper class B limit to 100% compliance with 4,600 E. coli per 100 g (corresponding NoV value of 7403 ± 39) reduces maximum levels of NoV by a factor of 10.2 to 1. We found using the UK filtered winter dataset that a maximum of 200 NoV corresponded to a maximum of 128 ± 7 E. coli per 100 g. A maximum of 1,000 NoV corresponded to a maximum of 631 ± 14 E. coli per 100 g.

Overexpression of antibiotic resistance genes in hospital effluents over time.

Objectives: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. Methods: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. Results: We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital ( ρ = 0.9, two-tailed P <0.0001) and farm ( ρ = 0.5, two-tailed P <0.0001) effluents and that two ß-lactam resistance genes ( bla GES and bla OXA ) were overexpressed in all hospital effluent samples. High ß-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. Conclusions: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Heat Wave-Associated Vibriosis, Sweden and Finland, 2014.

During summer 2014, a total of 89 Vibrio infections were reported in Sweden and Finland, substantially more yearly infections than previously have been reported in northern Europe. Infections were spread across most coastal counties of Sweden and Finland, but unusually, numerous infections were reported in subarctic regions; cases were reported as far north as 65°N, ≈100 miles (160 km) from the Arctic Circle. Most infections were caused by non-O1/O139 V. cholerae (70 cases, corresponding to 77% of the total, all strains were negative for the cholera toxin gene). An extreme heat wave in northern Scandinavia during summer 2014 led to unprecedented high sea surface temperatures, which appear to have been responsible for the emergence of Vibrio bacteria at these latitudes. The emergence of vibriosis in high-latitude regions requires improved diagnostic detection and clinical awareness of these emerging pathogens.

Patterns of Multi-Antibiotic-Resistant Escherichia Coli from Streams with No History of Antimicrobial Inputs.

A growing body of evidence suggests that contaminated environments may harbor a greater proportion of antibiotic-resistant microorganisms than unpolluted reference sites. Here, we report the screening of 427 Escherichia coli strains isolated from 11 locations on nine streams draining the US Department of Energy's Savannah River Site against a panel of five antibiotics. Streams were chosen to capture a wide range of watersheds from minimally disturbed to highly impacted. Overall, higher levels of resistance were found in waterborne E. coli that also generally exhibited low spatial variability. However, 3 of 11 locations also demonstrated elevated resistance levels in sediments. Two of these occurred in highly disturbed tributaries with no obvious sources of antimicrobials. To further investigate these patterns, we screened a subset of isolates obtained from three streams against 23 antibiotics or antibiotic combinations. A large proportion of these isolates (>40 %) demonstrated resistance to 10 or more antimicrobials, suggesting that environmental multi-antibiotic resistance may be prevalent in this bacterial commensal. Only 4 of 87 viable isolates were tested susceptible to all 23 antibiotics and combinations. Among these multi-antibiotic-resistant isolates, several demonstrated resistance to all structural classes of antimicrobial agents tested, including frontline antibiotics such as gatifloxacin and ciprofloxacin.

Comparative metagenomics reveals a diverse range of antimicrobial resistance genes in effluents entering a river catchment.

The aquatic environment has been implicated as a reservoir for antimicrobial resistance genes (ARGs). In order to identify sources that are contributing to these gene reservoirs, it is crucial to assess effluents that are entering the aquatic environment. Here we describe a metagenomic assessment for two types of effluent entering a river catchment. We investigated the diversity and abundance of resistance genes, mobile genetic elements (MGEs) and pathogenic bacteria. Findings were normalised to a background sample of river source water. Our results show that effluent contributed an array of genes to the river catchment, the most abundant being tetracycline resistance genes tetC and tetW from farm effluents and the sulfonamide resistance gene sul2 from wastewater treatment plant (WWTP) effluents. In nine separate samples taken across 3 years, we found 53 different genes conferring resistance to seven classes of antimicrobial. Compared to the background sample taken up river from effluent entry, the average abundance of genes was three times greater in the farm effluent and two times greater in the WWTP effluent. We conclude that effluents disperse ARGs, MGEs and pathogenic bacteria within a river catchment, thereby contributing to environmental reservoirs of ARGs.

Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014.

We report the first detection of tetrodotoxins (TTX) in European bivalve shellfish. We demonstrate that TTX is present within the temperate waters of the United Kingdom, along the English Channel, and can accumulate in filter-feeding molluscs. The toxin is heat-stable and thus it cannot be eliminated during cooking. While quantified concentrations were low in comparison to published minimum lethal doses for humans, the results demonstrate that the risk to shellfish consumers should not be discarded.

Impact of analytic provenance in genome analysis.

BACKGROUND: Many computational methods are available for assembly and annotation of newly sequenced microbial genomes. However, when new genomes are reported in the literature, there is frequently very little critical analysis of choices made during the sequence assembly and gene annotation stages. These choices have a direct impact on the biologically relevant products of a genomic analysis--for instance identification of common and differentiating regions among genomes in a comparison, or identification of enriched gene functional categories in a specific strain. Here, we examine the outcomes of different assembly and analysis steps in typical workflows in a comparison among strains of Vibrio vulnificus. RESULTS: Using six recently sequenced strains of V. vulnificus, we demonstrate the "alternate realities" of comparative genomics, and how they depend on the choice of a robust assembly method and accurate ab initio annotation. We apply several popular assemblers for paired-end Illumina data, and three well-regarded ab initio genefinders. We demonstrate significant differences in detected gene overlap among comparative genomics workflows that depend on these two steps. The divergence between workflows, even those using widely adopted methods, is obvious both at the single genome level and when a comparison is performed. In a typical example where multiple workflows are applied to the strain V. vulnificus CECT 4606, a workflow that uses the Velvet assembler and Glimmer gene finder identifies 3275 gene features, while a workflow that uses the Velvet assembler and the RAST annotation system identifies 5011 gene features. Only 3171 genes are identical between both workflows. When we examine 9 assembly/annotation workflow scenarios as input to a three-way genome comparison, differentiating genes and even differentially represented functional categories change significantly from scenario to scenario. CONCLUSIONS: Inconsistencies in genomic analysis can arise depending on the choices that are made during the assembly and annotation stages. These inconsistencies can have a significant impact on the interpretation of an individual genome's content. The impact is multiplied when comparison of content and function among multiple genomes is the goal. Tracking the analysis history of the data--its analytic provenance--is critical for reproducible analysis of genome data.

Viewing marine bacteria, their activity and response to environmental drivers from orbit: satellite remote sensing of bacteria.

Satellite-based remote sensing of marine microorganisms has become a useful tool in predicting human health risks associated with these microscopic targets. Early applications were focused on harmful algal blooms, but more recently methods have been developed to interrogate the ocean for bacteria. As satellite-based sensors have become more sophisticated and our ability to interpret information derived from these sensors has advanced, we have progressed from merely making fascinating pictures from space to developing process models with predictive capability. Our understanding of the role of marine microorganisms in primary production and global elemental cycles has been vastly improved as has our ability to use the combination of remote sensing data and models to provide early warning systems for disease outbreaks. This manuscript will discuss current approaches to monitoring cyanobacteria and vibrios, their activity and response to environmental drivers, and will also suggest future directions.