Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

Temperature-bounded development of Dirofilaria immitis larvae restricts the geographical distribution and seasonality of its transmission: case study and decision support system for canine heartworm management in Australia.

Dirofilaria immitis is the causative agent of canine heartworm disease. We used the established heartworm development unit (HDU) principle to map the extrinsic incubation period (EIP) of D. immitis in Australia using historical weather data from 2013-2022. We found weather conditions suitable for EIP completion showed substantial seasonality and geographical variability. Whilst a considerable percentage of the Australian territory showed suitable weather conditions to always support EIP completion (17%), only 2.7% of the 2021 Australian human population lived in this region. Therefore, 97% of the population lived in an area that changed its EIP suitability within the study period. EIP completion is required prior to D. immitis transmission, meaning that infection risk of D. immitis is seasonal and location-dependent, being disrupted each year for most of the human population's dogs. We developed an online, open access tool allowing us to visualise EIP completion across Australia historically and in near real-time. We aim to support veterinarians to make risk-based recommendations for dirofilariosis prevention by using the tool, available at https://heartworm-mapping.adelaide.edu.au/shiny/.

The causal effect of early retirement on medication use across sex and occupation: evidence from Danish administrative data.

We examine the causal effect of early retirement on medication use using Danish registry data. A reform in early retirement schemes in 2006 gradually increased eligibility ages from 60 to 64 differentially across birth cohorts. This enables an instrumental variable design that was applied using novel g-estimation methods that alleviate bias in binary outcome IV models. Our data allow studying patterns in the short run (ages 59½-60½) and in the long run (ages 57-63). For those who were eligible already at age 60, retirement did not change overall medication use. However, when investigating medication and population subgroups, we see that painkiller use decreases and hypertension medication as well as mental health medication use increase after retirement in almost all population subgroups. Moreover, males as well as the blue-collar occupation subgroups do show decreases in overall medication use after early retirement. In conclusion, our analyses reveal that retirement can have important heterogeneous health effects across population groups and are potentially informative about the welfare benefits of social insurance more broadly.

Outcomes of Biopsy Grade Group 1 Prostate Cancer Diagnosis in the Danish Population.

BACKGROUND: Debate regarding a nomenclature change for grade group 1 (GG 1) prostate cancer to noncancer has been revived, as this could be a powerful tool in reducing the overtreatment of indolent disease. OBJECTIVE: To describe outcomes for all men diagnosed with GG 1 prostate cancer in the Danish population, with a focus on men followed conservatively. DESIGN, SETTING, AND PARTICIPANTS: This was a population-based observational study using data from the Danish Prostate Registry. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We measured the cumulative incidence of curative treatment, endocrine treatment, and cause-specific mortality. RESULTS AND LIMITATIONS: The cumulative incidence of endocrine therapy at 10 yr was 5.3% (95% confidence interval [CI] 4.3-6.3%) for men with initial active surveillance and 21% (95% CI 19-23%) for men with initial watchful waiting for localized GG 1. In the GG1 cohort, the prostate cancer-specific mortality rate at 15 yr was 14% (95 CI% 11-16%) for men on watchful waiting, 10% (95 CI% 6.7-14%) for men with prostate-specific antigen <10 ng/ml on watchful waiting, and 16% (95 CI% 13-19%) for men who did not receive curative-intent treatment or histological assessment. The study is limited by the historic nature of the observations over a period during which diagnostic procedures and treatments have evolved. CONCLUSIONS: GG 1 cancer can lead to disease-specific mortality in men with localized prostate cancer, and changing the nomenclature for all men may lead to undertreatment. PATIENT SUMMARY: Key opinion leaders have suggested that prostate cancers of Gleason grade group 1 (GG 1) should be renamed as noncancer to reduce overtreatment. The argument is that low-grade cancer does not metastasize. However, our nationwide population-based study showed that death from prostate cancer can occur in some men diagnosed with GG 1 disease. These men should be considered in discussions on changing the name for GG 1 prostate cancer.

Health behavioral responses to parental myocardial infarction and impact on own risk of disease in the general population.

Aims: A family history of coronary heart disease increases one's own risk of experiencing cardiovascular disease and death. An implication of the hereditary nature of the disease is that individuals are provided information about their own risk when a parent is affected, potentially leading them to engage in behaviors that reduce their own risk. In this study, we assessed how a 10-year risk of a cardiovascular event, measured by SCORE, changes for the offspring in response to a parent experiencing a myocardial infarction. Methods: We analyzed 19,995 individuals from the general population in the Copenhagen City Heart Study of whom 2,071 had a parent, who suffered from a myocardial infarction during four decades of observation using fixed-effects regressions. Results: Following a parental myocardial infarction, individuals reduced their 10-year risk by 0.16 percentage points constituting a 7.1% reduction of baseline risk. Male participants had the largest change in the risk SCORE following an event of the mother, with a 12.4% reduction from the baseline risk. The degree of response contingent on their own level of risk was found to be the largest for individuals with a 10-year risk between 5% and 10%, who also showed a reduction in systolic blood pressure following paternal myocardial infarction. Parental myocardial infarction was associated with an increased smoking rate in individuals with a baseline risk above 10%, while reductions in risk were seen for individuals with a lower baseline risk. Conclusion: Following a parental event, individuals reduced their 10-year risk with the largest reductions in their own risk, as observed in men and individuals experiencing a maternal event. The response was the largest for individuals with a 10-year risk for myocardial infarction between 5 and 10%.

Relative diagnostic accuracy of point-of-care tests to rule-in Dirofilaria immitis infection in clinically suspect dogs: A systematic review and meta-analysis.

Canine heartworm, Dirofilaria immitis, can cause severe disease and sometimes death of the host. Associated clinical signs, lack of preventative usage and regional endemicity are unlikely sufficient by themselves to reach a definitive diagnosis. Several point-of-care (POC) diagnostic tests are commercially available to aid in-clinic diagnosis, however, there is variable diagnostic accuracy reported and no synthesis of published evidence. This systematic review aims at meta-analysing the likelihood ratio of a positive result (LR+) to inform the selection and interpretation of POC tests in practice to rule-in heartworm infection when there is clinical suspicion. Three literature index interfaces (Web of Science, PubMed, Scopus) were searched on November 11th, 2022, for diagnostic test evaluation (DTE) articles assessing at least one currently commercialised POC test. Risk of bias was assessed adapting the QUADAS-2 protocol and articles with no evidence of high risk of bias were meta-analysed if deemed applicable to our review objective. Substantial between DTE heterogeneity was investigated including potential threshold or covariate effects. A total of 324 primary articles were sourced and 18 were retained for full text review of which only three had low risk of bias in all four QUADAS-2 domains. Of the nine heartworm POC tests evaluated, only three, IDEXX SNAP (n DTEs = 6), Zoetis WITNESS (n DTEs = 3) and Zoetis VETSCAN (n DTEs = 5) could be analysed. Both WITNESS and VETSCAN DTEs showed substantial heterogeneity due to a putative threshold effect and no summary point estimates could be reported. SNAP DTEs showed acceptable heterogeneity, and a summary LR+ was estimated at 559.0 (95%CI: 24.3-12,847.4). The quality and heterogeneity of heartworm POC test DTEs was highly variable which restricted our summary of the diagnostic accuracy to only the SNAP test. A positive result from the SNAP test provides strong evidence of the presence of an infection with adult heartworm(s) in a dog patient and this test is warranted to rule-in clinical suspicion(s) in clinics. However, our review did not appraise the literature to assess the fitness of SNAP test, or any other POC tests, to rule-out heartworm infection in dogs without clinical suspicion or following heartworm therapy.

Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone.

Microbial assembly and metabolic potential in the subsurface critical zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study. Our results showed that subsurface microbes were highly localized and that communities were rarely interconnected. Microbial community composition as well as metabolic potential in carbon and nitrogen cycling varied even over short vertical distances. Further analyses indicated a strong depth-related covariation of community composition with a subset of 12 environmental variables. An analysis of dissolved organic carbon (DOC) quality via ultrahigh resolution mass spectrometry suggested that the SCZ was generally a low-carbon environment, with the relative portion of labile DOC decreasing and that of recalcitrant DOC increasing along the depth, selecting microbes from copiotrophs to oligotrophs and also impacting the microbial metabolic potential in the carbon cycle. Our study demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolism in the SCZ. IMPORTANCE In this study, we explored the links between geochemical parameters, microbial community structure and metabolic potential across the depth of sediment, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone. Our results revealed that microbes in the terrestrial subsurface can be highly localized, with communities rarely being interconnected along the depth. Overall, our research demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolic potential in different depths of subsurface terrestrial sediment. Such studies correlating microbial community analyses and geochemistry analyses, including high resolution mass spectrometry analyses of natural organic carbon, will further the fundamental understanding of microbial ecology and biogeochemistry in subsurface terrestrial ecosystems and will benefit the future development of predictive models on nutrient turnover in these environments.

Validation of a QTL associated with resistance to Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Vibriosis is a bacterial disease in fish caused by the Gram negative bacterium Vibrio anguillarum with severe impact on rainbow trout (Oncorhynchus mykiss) farming. Sustainable control methods should be developed and we here show that marker assisted selective breeding of fish naturally resistant to the disease is feasible. We have validated the use of a single nucleotide polymorphism (SNP) marker SNP AX-89,945,921 (QTL on chromosome 21). The QTL was previously found associated with resistance to vibriosis and described following a genome wide association analysis (GWAS) of trout exposed to the bacterium. For this validation spawners were genotyped by use of the 57 K Axiom®Trout Microarray (Affymetrix) and homozygous male fish carrying the allele with the SNP AX-89,945,921 were then selected and used to fertilize eggs from outbred female trout resulting in fish all carrying the SNP (QTL-fish). Control fish (non-QTL fish) were produced by fertilizing the same batch of eggs by use of male parents negative for the SNP. The fish were exposed in freshwater to V. anguillarum (water bath infection) at 19 C°. A total of 900 fish were challenged in a common garden set-up in triplicate. A bacterial solution of V. anguillarum (serotype O1) was added to each of three freshwater fish tanks, each with 150 QTL and 150 non-QTL fish. Fish were tagged by tail fin cut (upper/lower) to discern the two groups, whereafter fish were monitored around the clock to detect disease signs and remove moribund fish. Clinical vibriosis developed within two days in non-QTL-fish (overall morbidity of 70%). QTL fish developed clinical signs later and the morbidity was significantly lower and did not reach 50%. Rainbow trout farming may benefit from using the QTL associated with higher resistance towards vibriosis. The effect may be optimized in the future by use of both male and female parents homozygous for the marker allele.

Definitions and incidence rates of self-harm and suicide attempts in Europe: A scoping review.

INTRODUCTION: European countries use various terminologies for self-harm and attempted suicide, which are sometimes used interchangeably. This complicates cross-country comparisons of incidence rates. This scoping review aimed to examine the definitions used and the possibilities to identify and compare incidence rates of self-harm and attempted suicide in Europe. METHODS: A literature search was conducted in Embase, Medline and PsycINFO for studies published from 1990 to 2021, followed by grey literature searches. Data were collected for total populations originating from health care institutions or registries. Results were presented in tabular form supplemented by a qualitative summary by area. RESULTS: A total of 3160 articles were screened, resulting in 43 studies included from databases and further 29 studies from other sources. Most studies used the term 'suicide attempt' rather than 'self-harm' and reported person-based rates with annual incidence rates from age 15+. None of the rates were considered comparable due to different reporting traditions related to classification codes and statistical approaches. CONCLUSION: The present extensive literature on self-harm and attempted suicide cannot be used to compare findings between countries because of the high degree of heterogeneity among studies. International agreement on definitions and registration practices is needed to improve knowledge and understanding of suicidal behaviour.

Cats just want to have fun: Associations between play and welfare in domestic cats.

Play is often considered an indicator and promotor of animal welfare and may facilitate closer cat-human relationships. However, few studies have empirically investigated these associations. The current study aimed to investigate play-related factors associated with four welfare outcome measures in cats (Felis catus) including: cat quality of life; cat-guardian relationship quality; problem behaviour prevalence; and behavioural changes. An online survey was developed using demographic information, questions related to play and resources, free text sections and the following validated measures: cat quality of life (QOL), the cat owner relationship scale, and the adult playfulness trait scale. Responses were completed by 1,591 cat guardians from 55 countries. Higher cat playfulness scores and a greater number of games played were significantly associated with higher cat QOL scores while longer amounts of daily play, greater number of games, both cat and guardian initiating play and higher guardian playfulness scores were all significantly associated with higher cat-guardian relationship scores. Exclusively indoor housing was significantly associated with both higher cat QOL and higher cat-guardian relationships scores compared to cats with outdoor access. Behavioural changes associated with distress in cats were reported when play was absent. Play may be an important factor in assessing and maintaining cat welfare. Further research into the mechanisms of how play impacts welfare and cat-guardian relationships is needed.

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments.

Bacillus cereus strain CPT56D-587-MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF-matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%-40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species-largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.

Complete Genome Sequence of Bacillus cereus Strain CPT56D-587-MTF, Isolated from a Nitrate- and Metal-Contaminated Subsurface Environment.

Bacillus cereus strain CPT56D-587-MTF was isolated from nitrate- and toxic metal-contaminated subsurface sediment at the Oak Ridge Reservation (ORR) (Oak Ridge, TN, USA). Here, we report the complete genome sequence of this strain to provide genomic insight into its strategies for survival at this mixed-waste site.

Characteristics and outcomes of dog attacks to dogs and cats in Melbourne, Australia: A retrospective study of 459 cases (2018).

Dog-dog and dog-cat attacks can result in severe medical, financial, and emotional injury to pets and owners. The characteristics of dog-dog and dog-cat attack victims, the circumstances surrounding these attacks and the financial burden from veterinary visits is not reported in Australia. Medical records from 459 animals that were presented to the emergency service of four specialty hospitals in Melbourne, Australia in 2018 following a dog attack were assessed via univariate and multivariate methodologies with a retrospective case-control study design. Animals who had been attacked by a dog comprised 2.4% of the overall caseload at these four hospitals. Risk factors identified in dog-dog attack victims for presenting to a veterinary emergency hospital after being attacked were being cross-bred (OR = 1.4, p = 0.014, 95% CI = 1.07-1.84) and neutered (OR = 1.4, p = 0.035, 95% CI = 1.03-2.00). Being aged > 2-7years was protective (OR = 0.70, p = 0.010, CI = 0.48-0.88). Dogs from houses with a lower Socio-economic Indices for Areas score (SIEFA) were more likely to be attacked at home by a known attacker, compared to those from houses with a higher SIEFA score who were more likely to be attacked in public by a dog unknown to them (p = <0.001). Cats who presented following a dog attack had a 46.3% survival to discharge, compared to 91.8% in dogs (p < 0.001). Final cost of treatment for dogs and cats was similar (median AU $380 vs AU $360, respectively). Further research is needed to evaluate the population of dogs and cats attacked by dogs, to inform and direct public education campaigns aimed at reducing their incidence and overall burdens.

Genomic Features and Pervasive Negative Selection in Rhodanobacter Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer.

Rhodanobacter species dominate in the Oak Ridge Reservation (ORR) subsurface environments contaminated with acids, nitrate, metal radionuclides, and other heavy metals. To uncover the genomic features underlying adaptations to these mixed-waste environments and to guide genetic tool development, we sequenced the whole genomes of eight Rhodanobacter strains isolated from the ORR site. The genome sizes ranged from 3.9 to 4.2 Mb harboring 3,695 to 4,035 protein-coding genes and GC contents approximately 67%. Seven strains were classified as R. denitrificans and one strain, FW510-R12, as R. thiooxydans based on full length 16S rRNA sequences. According to gene annotation, the top two Cluster of Orthologous Groups (COGs) with high pan-genome expansion rates (Pan/Core gene ratio) were "replication, recombination and repair" and "defense mechanisms." The denitrifying genes had high DNA homologies except the predicted protein structure variances in NosZ. In contrast, heavy metal resistance genes were diverse with between 7 to 34% of them were located in genomic islands, and these results suggested origins from horizontal gene transfer. Analysis of the methylation patterns in four strains revealed the unique 5mC methylation motifs. Most orthologs (78%) had ratios of nonsynonymous to synonymous substitutions (dN/dS) less than one when compared to the type strain 2APBS1, suggesting the prevalence of negative selection. Overall, the results provide evidence for the important roles of horizontal gene transfer and negative selection in genomic adaptation at the contaminated field site. The complex restriction-modification system genes and the unique methylation motifs in Rhodanobacter strains suggest the potential recalcitrance to genetic manipulation. IMPORTANCE Despite the dominance of Rhodanobacter species in the subsurface of the contaminated Oak Ridge Reservation (ORR) site, very little is known about the mechanisms underlying their adaptions to the various stressors present at ORR. Recently, multiple Rhodanobacter strains have been isolated from the ORR groundwater samples from several wells with varying geochemical properties. Using Illumina, PacBio, and Oxford Nanopore sequencing platforms, we obtained the whole genome sequences of eight Rhodanobacter strains. Comparison of the whole genomes demonstrated the genetic diversity, and analysis of the long nanopore reads revealed the heterogeneity of methylation patterns in strains isolated from the same well. Although all strains contained a complete set of denitrifying genes, the predicted tertiary structures of NosZ differed. The sequence comparison results demonstrate the important roles of horizontal gene transfer and negative selection in adaptation. In addition, these strains may be recalcitrant to genetic manipulation due to the complex restriction-modification systems and methylations.

Genotype to ecotype in niche environments: adaptation of Arthrobacter to carbon availability and environmental conditions.

Niche environmental conditions influence both the structure and function of microbial communities and the cellular function of individual strains. The terrestrial subsurface is a dynamic and diverse environment that exhibits specific biogeochemical conditions associated with depth, resulting in distinct environmental niches. Here, we present the characterization of seven distinct strains belonging to the genus Arthrobacter isolated from varying depths of a single sediment core and associated groundwater from an adjacent well. We characterized genotype and phenotype of each isolate to connect specific cellular functions and metabolisms to ecotype. Arthrobacter isolates from each ecotype demonstrated functional and genomic capacities specific to their biogeochemical conditions of origin, including laboratory-demonstrated characterization of salinity tolerance and optimal pH, and genes for utilization of carbohydrates and other carbon substrates. Analysis of the Arthrobacter pangenome revealed that it is notably open with a volatile accessory genome compared to previous pangenome studies on other genera, suggesting a high potential for adaptability to environmental niches.

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella.

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

Whole-genome association study searching for QTL for Aeromonas salmonicida resistance in rainbow trout.

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, has extensive negative effects on wild and farmed salmonids worldwide. Vaccination induces some protection under certain conditions but disease outbreaks occur even in vaccinated fish. Therefore, alternative disease control approaches are required to ensure the sustainable expansion of rainbow trout aquaculture. Selective breeding can be applied to enhance host resistance to pathogens. The present work used genome-wide association study (GWAS) to identify quantitative trait loci (QTL) associated with A. salmonicida resistance in rainbow trout. A total 798 rainbow trout exposed to A. salmonicida by bath challenge revealed 614 susceptible and 138 resistant fish. Genotyping was conducted using the 57 K single nucleotide polymorphism (SNP) array and the GWAS was performed for survival and time to death phenotypes. We identified a QTL on chromosome 16 and located positional candidate genes in the proximity of the most significant SNPs. In addition, samples from exposed fish were examined for expression of 24 immune-relevant genes indicating a systematic immune response to the infection. The present work demonstrated that resistance to A. salmonicida is moderately heritable with oligogenic architecture. These result will be useful for the future breeding programs for improving the natural resistance of rainbow trout against furunculosis.

Deciphering Microbial Metal Toxicity Responses via Random Bar Code Transposon Site Sequencing and Activity-Based Metabolomics.

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al3+, the oxyanion CrO42-, and the oxycation UO22+. Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al3+ binds intracellular arginine, CrO42- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO22+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. Here, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment. IMPORTANCE Studying microbial interactions with their environment can lead to a deeper understanding of biological molecular mechanisms. In this study, two global techniques, RB-TnSeq and activity metabolomics, were successfully used to probe the interactions between a metal-resistant microorganism, Pantoea sp. strain MT58, and metals contaminating a site where the organism can be located. A number of novel metal-microbe interactions were uncovered, including Al3+ toxicity targeting arginine synthesis, which could lead to a deeper understanding of the impact Al3+ contamination has on microbial communities as well as its impact on higher-level organisms, including plants for whom Al3+ contamination is an issue. Using multiomic approaches like the one described here is a way to further our understanding of microbial interactions and their impacts on the environment overall.