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Post-Acute Sequelae of SARS-CoV-2 infection (PASC or "Long COVID"), includes numerous chronic conditions associated with widespread morbidity and rising healthcare costs. PASC has highly variable clinical presentations, and likely includes multiple molecular subtypes, but it remains poorly understood from a molecular and mechanistic standpoint. This hampers the development of rationally targeted therapeutic strategies. The NIH-sponsored "Researching COVID to Enhance Recovery" (RECOVER) initiative includes several retrospective/prospective observational cohort studies enrolling adult, pregnant adult and pediatric patients respectively. RECOVER formed an "OMICS" multidisciplinary task force, including clinicians, pathologists, laboratory scientists and data scientists, charged with developing recommendations to apply cutting-edge system biology technologies to achieve the goals of RECOVER. The task force met biweekly over 14 months, to evaluate published evidence, examine the possible contribution of each "omics" technique to the study of PASC and develop study design recommendations. The OMICS task force recommended an integrated, longitudinal, simultaneous systems biology study of participant biospecimens on the entire RECOVER cohorts through centralized laboratories, as opposed to multiple smaller studies using one or few analytical techniques. The resulting multi-dimensional molecular dataset should be correlated with the deep clinical phenotyping performed through RECOVER, as well as with information on demographics, comorbidities, social determinants of health, the exposome and lifestyle factors that may contribute to the clinical presentations of PASC. This approach will minimize lab-to-lab technical variability, maximize sample size for class discovery, and enable the incorporation of as many relevant variables as possible into statistical models. Many of our recommendations have already been considered by the NIH through the peer-review process, resulting in the creation of a systems biology panel that is currently designing the studies we proposed. This system biology strategy, coupled with modern data science approaches, will dramatically improve our prospects for accurate disease subtype identification, biomarker discovery and therapeutic target identification for precision treatment. The resulting dataset should be made available to the scientific community for secondary analyses. Analogous system biology approaches should be built into the study designs of large observational studies whenever possible. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei, causative agent of melioidosis, is a One Health concern as it is acquired directly from soil and water and causes disease in humans and agricultural and wild animals. We examined B. pseudomallei in soil and goats at a single farm in the Northern Territory of Australia where >30 goats acquired melioidosis over nine years. Methodology/Principal findings: We cultured 45 B. pseudomallei isolates from 35 goats and sampled soil in and around goat enclosures to isolate and detect B. pseudomallei and evaluate characteristics associated with its occurrence; 33 soil isolates were obtained from 1993–1994 and 116 in 2006. Ninety-two goat and soil isolates were sequenced; mice were challenged with six soil isolates to evaluate virulence. Sampling depth and total N/organic C correlated with B. pseudomallei presence. Twelve sequence types (STs) were identified. Most goat infections (74%) were ST617, some with high similarity to 2006 soil isolates, suggesting ST617 was successful at persisting in soil and infecting goats. ST260 and ST266 isolates were highly virulent in mice but other isolates produced low/intermediate virulence; three of these were ST326 isolates, the most common soil ST in 2006. Thus, virulent and non-virulent lineages can co-occur locally. Three genes associated with virulence were present in ST260 and ST266, absent in most ST326 isolates, and present or variably present in ST617. Conclusions/Significance: Agricultural animals can influence B. pseudomallei abundance and diversity in local environments. This effect may persist, as B. pseudomallei was detected more often from soil collected inside and adjacent to goat enclosures years after most goats were removed. Following goat removal, the low virulence ST326, which was not isolated from soil when goats were present, became the predominant ST in soil by 2006. Although multiple diverse lineages of B. pseudomallei may exist in a given location, some may infect mammals more efficiently than others. Author summary: We studied genomic diversity in the environmental bacterial pathogen, Burkholderia pseudomallei, which caused the disease melioidosis in goats at a small farm in northern Australia. By comparing genomes from 92 B. pseudomallei isolates from goats and soil, we discovered 12 diverse lineages at this location, three of which infected the majority of goats and were cultured from the soil of goat enclosures. We also conducted a mouse challenge experiment using six isolates from this farm and determined that two soil isolates displayed much greater virulence than the others. Finally, we investigated soil factors associated with B. pseudomallei occurrence and found 1) most B. pseudomallei isolates were sampled from irrigated soils, and 2) B. pseudomallei was much more likely to be encountered at soil depths of 30 cm than 10 cm. The presence of this pathogen in the soil severely impacted maintenance of the goat herd due to high mortality and emphasizes its One Health implications for other livestock operations in tropical countries where B. pseudomallei is endemic. Our findings provide insight into the importance of more fully understanding soil habitats that promote B. pseudomallei presence, thereby increasing the risk of melioidosis to humans and agricultural animals. [ABSTRACT FROM AUTHOR]

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic. [ABSTRACT FROM AUTHOR]

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles. [ABSTRACT FROM AUTHOR]

Background: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. Methodology/Principle findings: We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric (p^), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. Conclusion/Significance: We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection. Author summary: Melioidosis is a disease caused by the soil-dwelling bacterium Burkholderia pseudomallei, a Tier 1 Select Agent by the US Centers for Disease Control and Prevention (CDC), with the ability to naturally infect humans and non-human primates (NHP). Although melioidosis diagnosis in humans has been formally outlined, there is no standardized test available for B. pseudomallei exposure and melioidosis in NHP. NHP are important animal models in melioidosis research in addition to other scientific research. The goal of this study was to formally evaluate the first extensive serodiagnostic test for detection of B. pseudomallei exposure in NHP. The BurkPx assay comprised 21 B. pseudomallei specific antigens and was previously standardized in humans. Early IgM antibody responses were noted to carbohydrates whilst IgG antibody responses were mounted to multiple proteins during the second week of infection. The assay elicited low background reactivity to B. pseudomallei-naive NHP. Data collected from the BurkPx assay was fed into a multiple antigen model, which produced a p^ score used to classify the assay via thresholding. The final model produced a 100% specificity and 63.48% sensitivity among all samples; however, removal of early infection samples increased sensitivity to 95%. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative soil bacterium well recognized in Southeast Asia and northern Australia. However, wider and expanding global distribution of B. pseudomallei has been elucidated. Early diagnosis is critical for commencing the specific therapy required to optimize outcome. Serological testing using the indirect hemagglutination (IHA) antibody assay has long been used to augment diagnosis of melioidosis and to monitor progress. However, cross reactivity and prior exposure may complicate the diagnosis of current clinical disease (melioidosis). The goal of our study was to develop and initially evaluate a serology assay (BurkPx) that capitalized upon host response to multiple antigens. Antigens were selected from previous studies for expression/purification and conjugation to microspheres for multiantigen analysis. Selected serum samples from non-melioidosis controls and serial samples from culture-confirmed melioidosis patients were used to characterize the diagnostic power of individual and combined antigens at two times post admission. Multiple variable models were developed to evaluate multivariate antigen reactivity, identify important antigens, and determine sensitivity and specificity for the diagnosis of melioidosis. The final multiplex assay had a diagnostic sensitivity of 90% and specificity of 93%, which was superior to any single antigen in side-by-side comparisons. The sensitivity of the assay started at >85% for the initial serum sample after admission and increased to 94% 21 days later. Weighting antigen contribution to each model indicated that certain antigen contributed to diagnosis more than others, which suggests that the number of antigens in the assay can be decreased. In summation, the BurkPx assay can facilitate the diagnosis of melioidosis and potentially improve on currently available serology assays. Further evaluation is now required in both melioidosis-endemic and non-endemic settings. Author summary: Melioidosis has a wide diversity of symptoms, making laboratory diagnosis essential. The diagnosis of melioidosis is primarily by culture of the causative bacterium, Burkholderia pseudomallei, which is problematic for locations with limited laboratory resources and for laboratories inexperienced in handling B. pseudomallei. Early diagnosis of melioidosis facilitates the specific antimicrobial therapy that optimizes outcomes and substantially lowers mortality. We have developed and evaluated a multiple antigen-antibody serology assay using serial samples from culture confirmed melioidosis patients including the first sample after patient admission, together with non-melioidosis serum controls. Multiantigen reactivity was compared to single antigens and to the indirect hemagglutination (IHA) antibody assay data collected from routine patient testing. The results from testing patients with culture-confirmed melioidosis suggest that using multiple individual antigen assays coupled with models can improve the sensitivity and specificity of serological diagnosis using the same antigens evaluated individually. In addition, all evaluated antigens may not be needed to improve assay performance. Further antigen down selection and evaluation of the utility of this assay is now required in both melioidosis-endemic and non-endemic settings. [ABSTRACT FROM AUTHOR]

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wild type (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 μM, 8.5 μM, 24.1 μM, 8.2 μM and 13.6 μM, respectively. A separate experiment focusing on a combination of 10 μM stenoparib and 0.5 μM remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir. [ABSTRACT FROM AUTHOR]

Engineered natural killer (NK) cells represent a promising option for immune therapy option due to their immediate availability in allogeneic settings. Severe acute diseases, such as COVID-19, require targeted and immediate intervention. Here we show engineering of NK cells to express (1) soluble interleukin-15 (sIL15) for enhancing their survival and (2) a chimeric antigen receptor (CAR) consisting of an extracellular domain of ACE2, targeting the spike protein of SARS-CoV-2. These CAR NK cells (mACE2-CAR_sIL15 NK cells) bind to VSV-SARS-CoV-2 chimeric viral particles as well as the recombinant SARS-CoV-2 spike protein subunit S1 leading to enhanced NK cell production of TNF-α and IFN-γ and increased in vitro and in vivo cytotoxicity against cells expressing the spike protein. Administration of mACE2-CAR_sIL15 NK cells maintains body weight, reduces viral load, and prolongs survival of transgenic mice expressing human ACE2 upon infection with live SARS-CoV-2. These experiments, and the capacity of mACE2-CAR_sIL15 NK cells to retain their activity following cryopreservation, demonstrate their potential as an allogeneic off-the-shelf therapy for COVID-19 patients who are faced with limited treatment options. Severe COVID-19 requires immediate and targeted intervention that is efficient against SARS-CoV-2 and its variants. Authors show here the therapeutic potential of engineered natural killer cells that simultaneously express a chimeric antigen receptor targeting the spike protein of SARS-CoV-2, and IL-15, a cytokine that enhances the function and survival of their own. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796–1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1–2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei. Author summary: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. The evaluation of diversity within B. pseudomallei has largely been conducted utilizing clinical isolates despite almost all infections emerging from environmental exposure. In this study, we surveyed the genomic diversity of 169 isolates collected from a single soil sample in Ubon Ratchathani Province, Thailand. Seven different sequence types were identified, and substantial within-sequence-type gene diversity was observed. To test for differential virulence, 30 isolates were challenged in a mouse melioidosis model. A small number of isolates killed all mice, but most killed none, demonstrating the variable virulence potential of different B. pseudomallei isolates present in the single sample. A comparative genomics analysis identified multiple genes associated with virulence, including Hcp1, a component of the type VI secretion system and a known virulence factor. The results of this study have implications for the comprehensive environmental surveillance, environmental exposure, and differential virulence of B. pseudomallei. [ABSTRACT FROM AUTHOR]

Yersinia pestis, causative agent of plague, occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species, including black‐tailed prairie dogs (Cynomys ludovicianus). How Y. pestis persists long‐term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed, including, among others, a separate enzootic transmission cycle that maintains Y. pestis without involvement of epizootic hosts and persistence of Y. pestis within epizootic host populations without causing high mortality within those populations. We live‐trapped and collected fleas from black‐tailed prairie dogs and other mammal species from sites with and without black‐tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y. pestis. Y. pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005, before and during a widespread epizootic that drastically reduced black‐tailed prairie dog populations in the affected colonies. Temporal and spatial patterns of Y. pestis occurrence in fleas and genotyping of Y. pestis present in some infected fleas suggest Y. pestis was introduced multiple times from sources outside the study area and once introduced, was dispersed between several sites. We conclude Y. pestis likely was not present in these black‐tailed prairie dog colonies prior to epizootic activity in these colonies. Although we did not identify likely enzootic hosts, we found evidence that deer mice (Peromyscus maniculatus) may serve as bridging hosts for Y. pestis between unknown enzootic hosts and black‐tailed prairie dogs. [ABSTRACT FROM AUTHOR]

Of the seven currently known botulinum neurotoxin-producing species of Clostridium , C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4+, and CD8+ T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis. [ABSTRACT FROM AUTHOR]

Background: Leptospira are shed into the environment via urine of infected animals. Rivers are thought to be an important risk factor for transmission to humans, though much is unknown about the types of environment or characteristics that favor survival. To address this, we screened for Leptospira DNA in two rivers in rural Ecuador where Leptospirosis is endemic. Results: We collected 112 longitudinal samples and recorded pH, temperature, river depth, precipitation, and dissolved oxygen. We also performed a series of three experiments designed to provide insight into Leptospira presence in the soil. In the first soil experiment, we characterized prevalence and co-occurrence of Leptospira with other bacterial taxa in the soil at dispersed sites along the rivers (n = 64). In the second soil experiment, we collected 24 river samples and 48 soil samples at three points along eight transects to compare the likelihood of finding Leptospira in the river and on the shore at different distances from the river. In a third experiment, we tested whether Leptospira presence is associated with soil moisture by collecting 25 soil samples from two different sites. In our river experiment, we found pathogenic Leptospira in only 4 (3.7%) of samples. In contrast, pathogenic Leptospira species were found in 22% of shore soil at dispersed sites, 16.7% of soil samples (compared to 4.2% of river samples) in the transects, and 40% of soil samples to test for associations with soil moisture. Conclusions: Our data are limited to two sites in a highly endemic area, but the scarcity of Leptospira DNA in the river is not consistent with the widespread contention of the importance of river water for leptospirosis transmission. While Leptospira may be shed directly into the river, onto the shores, or washed into the river from more remote sites, massive dilution and limited persistence in rivers may reduce the environmental load and therefore, the epidemiological significance of such sources. It is also possible that transmission may occur more frequently on shores where people are liable to be barefoot. Molecular studies that further explore the role of rivers and water bodies in the epidemiology of leptospirosis are needed. [ABSTRACT FROM AUTHOR]

Coccidioidomycosis, or Valley fever, is caused by two species of dimorphic fungi. Based on molecular phylogenetic evidence, the genus Coccidioides contains two reciprocally monophyletic species: C. immitis and C. posadasii. However, phenotypic variation between species has not been deeply investigated. We therefore explored differences in growth rate under various conditions. A collection of 39 C. posadasii and 46 C. immitis isolates, representing the full geographical range of the two species, was screened for mycelial growth rate at 37 °C and 28 °C on solid media. The radial growth rate was measured for 16 days on yeast extract agar. A linear mixed effect model was used to compare the growth rate of C. posadasii and C. immitis at 37 °C and 28 °C, respectively. C. posadasii grew significantly faster at 37 °C, when compared to C. immitis; whereas both species had similar growth rates at 28 °C. These results indicate thermotolerance differs between these two species. As the ecological niche has not been well-described for Coccidioides spp., and disease variability between species has not been shown, the evolutionary pressure underlying the adaptation is unclear. However, this research reveals the first significant phenotypic difference between the two species that directly applies to ecological research. [ABSTRACT FROM AUTHOR]

The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts. Herold et al. present an integrated meta-omics framework to investigate how mixed microbial communities, such as oleaginous bacterial populations in biological wastewater treatment plants, respond with distinct adaptation strategies to disturbances. They show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species. [ABSTRACT FROM AUTHOR]

Background: Binge drinking, an increasingly common form of alcohol use disorder, is associated with substantial morbidity and mortality; yet, its effects on the immune system's ability to defend against infectious agents are poorly understood. Burkholderia pseudomallei, the causative agent of melioidosis can occur in healthy humans, yet binge alcohol intoxication is increasingly being recognized as a major risk factor. Although our previous studies demonstrated that binge alcohol exposure increased B. pseudomallei near-neighbor virulence in vivo and increased paracellular diffusion and intracellular invasion, no experimental studies have examined the extent to which bacterial and alcohol dosage play a role in disease progression. In addition, the temporal effects of a single binge alcohol dose prior to infection has not been examined in vivo. Principal findings: In this study, we used B. thailandensis E264 a close genetic relative of B. pseudomallei, as useful BSL-2 model system. Eight-week-old female C57BL/6 mice were utilized in three distinct animal models to address the effects of 1) bacterial dosage, 2) alcohol dosage, and 3) the temporal effects, of a single binge alcohol episode. Alcohol was administered comparable to human binge drinking (≤ 4.4 g/kg) or PBS intraperitoneally before a non-lethal intranasal infection. Bacterial colonization of lung and spleen was increased in mice administered alcohol even after bacterial dose was decreased 10-fold. Lung and not spleen tissue were colonized even after alcohol dosage was decreased 20 times below the U.S legal limit. Temporally, a single binge alcohol episode affected lung bacterial colonization for more than 24 h after alcohol was no longer detected in the blood. Pulmonary and splenic cytokine expression (TNF-α, GM-CSF) remained suppressed, while IL-12/p40 increased in mice administered alcohol 6 or 24 h prior to infection. Increased lung and not intestinal bacterial invasion was observed in human and murine non-phagocytic epithelial cells exposed to 0.2% v/v alcohol in vitro. Conclusions: Our results indicate that the effects of a single binge alcohol episode are tissue specific. A single binge alcohol intoxication event increases bacterial colonization in mouse lung tissue even after very low BACs and decreases the dose required to colonize the lungs with less virulent B. thailandensis. Additionally, the temporal effects of binge alcohol alters lung and spleen cytokine expression for at least 24 h after alcohol is detected in the blood. Delayed recovery in lung and not spleen tissue may provide a means for B. pseudomallei and near-neighbors to successfully colonize lung tissue through increased intracellular invasion of non-phagocytic cells in patients with hazardous alcohol intake. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. Methodology/Principal findings: We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. Conclusions/Significance: B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7–20) and IgM (0–12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis. [ABSTRACT FROM AUTHOR]

Yersinia pestis was introduced to Brazil during the third plague pandemic and currently exists in several recognized foci. There is currently limited available phylogeographic data regarding Y. pestis in Brazil. We generated whole genome sequences for 411 Y. pestis strains from six Brazilian foci to investigate the phylogeography of Y. pestis in Brazil; these strains were isolated from 1966 to 1997. All 411 strains were assigned to a single monophyletic clade within the 1.ORI population, indicating a single Y. pestis introduction was responsible for the successful establishment of endemic foci in Brazil. There was a moderate level of genomic diversity but little population structure among the 411 Brazilian Y. pestis strains, consistent with a radial expansion wherein Y. pestis spread rapidly from the coast to the interior of Brazil and became ecologically established. Overall, there were no strong spatial or temporal patterns among the Brazilian strains. However, strains from the same focus tended to be more closely related and strains isolated from foci closer to the coast tended to fall in more basal positions in the whole genome phylogeny than strains from more interior foci. Overall, the patterns observed in Brazil are similar to other locations affected during the 3rd plague pandemic such as in North America and Madagascar. [ABSTRACT FROM AUTHOR]

Background: Binge drinking, an increasingly common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its effects on the immune system’s ability to defend against infectious agents are poorly understood. Burkholderia pseudomallei, the causative agent of melioidosis can occur in healthy humans, yet binge alcohol use is progressively being recognized as a major risk factor. Although our previous studies demonstrated that binge alcohol exposure results in reduced alveolar macrophage function and increased Burkholderia virulence in vitro, no experimental studies have investigated the outcomes of binge alcohol on Burkholderia spp. infection in vivo. Principal findings: In this study, we used the close genetic relatives of B. pseudomallei, B. thailandensis E264 and B. vietnamiensis, as useful BSL-2 model systems. Eight-week-old female C57BL/6 mice were administered alcohol comparable to human binge drinking episodes (4.4 g/kg) or PBS intraperitoneally 30 min before a non-lethal intranasal infection. In an initial B. thailandensis infection (3 x 105), bacteria accumulated in the lungs and disseminated to the spleen in alcohol administered mice only, compared with PBS treated mice at 24 h PI. The greatest bacterial load occurred with B. vietnamiensis (1 x 106) in lungs, spleen, and brain tissue by 72 h PI. Pulmonary cytokine expression (TNF-α, GM-CSF) decreased, while splenic cytokine (IL-10) increased in binge drunk mice. Increased lung and brain permeability was observed as early as 2 h post alcohol administration in vivo. Trans-epithelial electrical resistance (TEER) was significantly decreased, while intracellular invasion of non-phagocytic cells increased with 0.2% v/v alcohol exposure in vitro. Conclusions: Our results indicate that a single binge alcohol dose suppressed innate immune functions and increased the ability of less virulent Burkholderia strains to disseminate through increased barrier permeability and intracellular invasion of non-phagocytic cells. [ABSTRACT FROM AUTHOR]

West Nile Virus (WNV) has been detected annually in Maricopa County, Arizona, since 2003. With this in mind, we sought to determine if contemporary strains are endemic to the county or are annually imported. As part of this effort, we developed a new protocol for tiled amplicon sequencing of WNV to efficiently attain greater than 99% coverage of 14 WNV genomes collected directly from positive mosquito pools distributed throughout Maricopa County between 2014 and 2017. Bayesian phylogenetic analyses revealed that contemporary genomes fall within two major lineages; NA/WN02 and SW/WN03. We found that all of the Arizona strains possessed an amino acid substitution known to be under positive selection, which has arisen independently at least four times in Arizona. The SW/WN03 strains exhibited transient behavior, with at least 10 separate introductions into Arizona when considering both historical and contemporary strains. However, NA/WN02 strains are geographically differentiated and appear to be endemic in Arizona, with two clades that have been circulating for four and seven years. This establishment in Maricopa County provides the first evidence of local overwintering by a WNV strain over the course of several years in Arizona. Within a national context, the placement of eleven contemporary Arizona strains in the NA/WN02 lineage indicates while WNV first entered the northeastern United States in 1999, the most ancestral extant strains of WNV are now circulating in the American southwest. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the environmental bacillus that causes melioidosis; a disease clinically significant in Australia and Southeast Asia but emerging in tropical and sub-tropical regions around the globe. Previous studies have placed the ancestral population of the organism in Australia with a single lineage disseminated to Southeast Asia. We have previously characterized B. pseudomallei isolates from New Guinea and the Torres Strait archipelago; remote regions that share paleogeographic ties with Australia. These studies identified regional biogeographical boundaries. In this study, we utilize whole-genome sequencing to reconstruct ancient evolutionary relationships and ascertain correlations between paleogeography and present-day distribution of this bacterium in Australasia. Our results indicate that B. pseudomallei from New Guinea fall into a single clade within the Australian population. Furthermore, clades from New Guinea are region-specific; an observation possibly linked to limited recent anthropogenic influence in comparison to mainland Australia and Southeast Asia. Isolates from the Torres Strait archipelago were distinct yet scattered among those from mainland Australia. These results provide evidence that the New Guinean and Torres Strait lineages may be remnants of an ancient portion of the Australian population. Rising sea levels isolated New Guinea and the Torres Strait Islands from each other and the Australian mainland, and may have allowed long-term isolated evolution of these lineages, providing support for a theory of microbial biogeography congruent with that of macro flora and fauna. Moreover, these findings indicate that contemporary microbial biogeography theories should consider recent and ongoing impacts of globalisation and human activity. [ABSTRACT FROM AUTHOR]

Summary: Bacillus spores resist inactivation, but the extent of their persistence on common surfaces is unclear. This work addresses knowledge gaps regarding biothreat agents in the environment to reduce uncertainty in risk assessment models. Studies were conducted to investigate the long‐term inactivation of Bacillus anthracis and three commonly used surrogate organisms – B. cereus, B. atrophaeus and B. thuringiensis on three materials: laminate countertop, stainless steel and polystyrene Petri dishes. Viable spores were measured at 1, 30, 90, 196, 304 and 1038 days. Twelve different persistence models were fit to the data using maximum likelihood estimation and compared. The study found that (1) spore inactivation was not log‐linear, as commonly modelled; (2) B. thuringiensis counts increased at 24 h on all materials, followed by a subsequent decline; (3) several experiments showed evidence of a 'U' shape, with spore counts apparently decreasing and then increasing between 1 and 304 days; (4) spores on polystyrene showed little inactivation; and (5) the maximum inactivation of 56% was observed for B. atrophaeus spores on steel at 196 days. Over the range of surfaces, time durations and conditions (humidity controlled vs. uncontrolled) examined, B. thuringiensis most closely matched the behaviour of B. anthracis. This work is the first of its kind to jointly present an experimental and modelling study that characterizes the long‐term persistence of Bacillus spores on common surfaces by natural decay over a 3 year period. The implications of model selection and extrapolation beyond the final time point are elucidated for microorganisms that persist for long periods of time on environmental media. The study also informs the selection of suitable surrogates based on their behaviour under similar conditions over time. [ABSTRACT FROM AUTHOR]

Asymptomatic colonization with extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae has been described for humans, various mammal species, and birds. Here, antimicrobial resistant bacteria were recovered from dog feces originating in Germany, Kosovo, Afghanistan, Croatia, and Ukraine, with a subset of mostly E. coli isolates obtained from a longitudinal collection over twelve months. In vitro antimicrobial resistance testing revealed various patterns of resistance against single or all investigated beta-lactam antibiotics, with none of the 101 isolates resistant against two tested carbapenem antibiotics. Whole genome sequence analysis revealed bacteria species-specific patterns for 23 antimicrobial resistance coding DNA sequences (CDS) that were unapparent from the in vitro analysis alone. Phylogenetic analysis of single nucleotide polymorphisms (SNP) revealed clonal bacterial isolates originating from different dogs, suggesting transmission between dogs in the same community. However, individual resistant E. coli clones were not detected over a period longer than seven days. Multi locus sequence typing (MLST) of 85 E. coli isolates revealed 31 different sequence types (ST) with an accumulation of ST744 (n = 9), ST10 (n = 8), and ST648 (n = 6), although the world-wide hospital-associated CTX-M beta-lactamase producing ST131 was not detected. Neither the antimicrobial resistance CDSs patterns nor the phylogenetic analysis revealed an epidemiological correlation among the longitudinal isolates collected from a period longer than seven days. No genetic linkage could be associated with the geographic origin of isolates. In conclusion, healthy dogs frequently carry ESBL-producing bacteria, independent to prior treatment, which may be transmitted between individual dogs of the same community. Otherwise, these antimicrobial resistant bacteria share few commonalities, making their presence eerily unpredictable. [ABSTRACT FROM AUTHOR]

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples. [ABSTRACT FROM AUTHOR]

Abstract: Genomic studies have been used to identify genes underlying many important plant secondary metabolic pathways. However, genes for salicinoid phenolic glycosides (SPGs)—ecologically important compounds with significant commercial, cultural, and medicinal applications—remain largely undescribed. We used a linkage map derived from a full‐sib population of hybrid cottonwoods (Populus spp.) to search for quantitative trait loci (QTL) for the SPGs salicortin and HCH‐salicortin. SSR markers and primer sequences were used to anchor the map to the V3.0 P. trichocarpa genome. We discovered 21 QTL for the two traits, including a major QTL for HCH‐salicortin (R2 = .52) that colocated with a QTL for salicortin on chr12. Using the V3.0 Populus genome sequence, we identified 2,983 annotated genes and 1,480 genes of unknown function within our QTL intervals. We note ten candidate genes of interest, including a BAHD‐type acyltransferase that has been potentially linked to PopulusSPGs. Our results complement other recent studies in Populus with implications for gene discovery and the evolution of defensive chemistry in a model genus. To our knowledge, this is the first study to use a full‐sib mapping population to identify QTL intervals and gene lists associated with SPGs. [ABSTRACT FROM AUTHOR]

Brucellosis is a common livestock disease in the Middle East and North Africa, but remains poorly described in the region both genetically and epidemiologically. Traditionally found in goats and sheep, Brucella melitensis is increasingly recognized as infecting camels. Most studies of brucellosis in camels to date have focused on serological surveys, providing only limited understanding of the molecular epidemiology of circulating strains. We genotyped B. melitensis isolates from Omani camels using whole genome SNP assays and VNTRs to provide context for regional brucellosis cases. We identified a lineage of B. melitensis circulating in camels as well as in goats, sheep, and cattle in Oman. This lineage is genetically distinct from most genotypes from the Arabian Peninsula and from isolates from much of the rest of the Middle East. We then developed diagnostic assays that rapidly identify strains from this lineage. In analyses of genotypes from throughout the region, Omani isolates were genetically most closely related to strains from brucellosis cases in humans and livestock in North Africa. Our findings suggest an African origin for B. melitensis in Oman that has likely occurred through the trade of infected livestock. Moreover, African lineages of B. melitensis appear to be undersampled and consequently are underrepresented in genetic databases for Brucella. As we begin to more fully understand global genomic diversity of B. melitensis, finding and characterizing these unique but widespread lineages is essential. We predict that increased sampling of humans and livestock in Africa will reveal little known diversity in this important zoonotic pathogen. [ABSTRACT FROM AUTHOR]

Background: Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs. Methodology/Principal findings: To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort. Conclusions: The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for resource constraint laboratories. This is a practical formula that reduces resource-driven limitations to genetic research and promises to advance global collective knowledge of infectious diseases emanating from resource limited regions of the world. [ABSTRACT FROM AUTHOR]

The Gram-negative beta-proteobacterium Zoogloea sp. LCSB751 (LMG 29444) was newly isolated from foaming activated sludge of a municipal wastewater treatment plant. Here, we describe its draft genome sequence and annotation together with a general physiological and genomic analysis, as the first sequenced representative of the Zoogloea genus. Moreover, Zoogloea sp. gene expression in its environment is described using metatranscriptomic data obtained from the same treatment plant. The presented genomic and transcriptomic information demonstrate a pronounced capacity of this genus to synthesize poly-ß-hydroxyalkanoate within wastewater. [ABSTRACT FROM AUTHOR]

The bacterium Burkholderia ubonensis is commonly co-isolated from environmental specimens harbouring the melioidosis pathogen, Burkholderia pseudomallei. B. ubonensis has been reported in northern Australia and Thailand but not North America, suggesting similar geographic distribution to B. pseudomallei. Unlike most other Burkholderia cepacia complex (Bcc) species, B. ubonensis is considered non-pathogenic, although its virulence potential has not been tested. Antibiotic resistance in B. ubonensis, particularly towards drugs used to treat the most severe B. pseudomallei infections, has also been poorly characterised. This study examined the population biology of B. ubonensis, and includes the first reported isolates from the Caribbean. Phylogenomic analysis of 264 B. ubonensis genomes identified distinct clades that corresponded with geographic origin, similar to B. pseudomallei. A small proportion (4%) of strains lacked the 920kb chromosome III replicon, with discordance of presence/absence amongst genetically highly related strains, demonstrating that the third chromosome of B. ubonensis, like other Bcc species, probably encodes for a nonessential pC3 megaplasmid. Multilocus sequence typing using the B. pseudomallei scheme revealed that one-third of strains lack the “housekeeping” narK locus. In comparison, all strains could be genotyped using the Bcc scheme. Several strains possessed high-level meropenem resistance (≥32 μg/mL), a concern due to potential transmission of this phenotype to B. pseudomallei. In silico analysis uncovered a high degree of heterogeneity among the lipopolysaccharide O-antigen cluster loci, with at least 35 different variants identified. Finally, we show that Asian B. ubonensis isolate RF23-BP41 is avirulent in the BALB/c mouse model via a subcutaneous route of infection. Our results provide several new insights into the biology of this understudied species. [ABSTRACT FROM AUTHOR]

Background: Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. Methodology/Principal findings: We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. Conclusions/Significance: Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location. [ABSTRACT FROM AUTHOR]

The rapid pace of bacterial evolution enables organisms to adapt to the laboratory environment with repeated passage and thus diverge from naturally-occurring environmental (“wild”) strains. Distinguishing wild and laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to convergent phenotypes, difficulty in detecting certain types of mutations, or perhaps because some adaptive modifications are epigenetic. Monitoring protein abundance, a molecular measure of phenotype, can overcome some of these difficulties. We have assembled a collection of Yersinia pestis proteomics datasets from our own published and unpublished work, and from a proteomics data archive, and demonstrated that protein abundance data can clearly distinguish laboratory-adapted from wild. We developed a lasso logistic regression classifier that uses binary (presence/absence) or quantitative protein abundance measures to predict whether a sample is laboratory-adapted or wild that proved to be ~98% accurate, as judged by replicated 10-fold cross-validation. Protein features selected by the classifier accord well with our previous study of laboratory adaptation in Y. pestis. The input data was derived from a variety of unrelated experiments and contained significant confounding variables. We show that the classifier is robust with respect to these variables. The methodology is able to discover signatures for laboratory facility and culture medium that are largely independent of the signature of laboratory adaptation. Going beyond our previous laboratory evolution study, this work suggests that proteomic differences between laboratory-adapted and wild Y. pestis are general, potentially pointing to a process that could apply to other species as well. Additionally, we show that proteomics datasets (even archived data collected for different purposes) contain the information necessary to distinguish wild and laboratory samples. This work has clear applications in biomarker detection as well as biodefense. [ABSTRACT FROM AUTHOR]

We examined 5 tularemia cases in Arizona, USA, during 2015-2017. All were caused by Francisella tularensis group A.II. Genetically similar isolates were found across large spatial and temporal distances, suggesting that group A.II strains are dispersed across long distances by wind and exhibit low replication rates in the environment. [ABSTRACT FROM AUTHOR]

Leptospirosis is a major zoonotic disease with widespread distribution and a large impact on human health. Carrier animals excrete pathogenic Leptospira primarily in their urine. Infection occurs when the pathogen enters a host through mucosa or small skin abrasions. Humans and other animals are exposed to the pathogen by direct contact with urine, contaminated soil or water. While many factors influence environmental cycling and the transmission of Leptospira to humans, the load of pathogenic Leptospira in the environment is likely to play a major role. Peridomestic rats are often implicated as a potential source of human disease; however exposure to other animals is a risk factor as well. The aim of this report is to highlight the importance of various carrier animals in terms of the quantity of Leptospira shed into the environment. For this, we performed a systematic literature review and a meta-analysis of the amount of pathogen that various animal species shed in their urine. Results: The quantity of pathogen has been reported for cows, deer, dogs, humans, mice, and rats, in a total of 14 research articles. We estimated the average Leptospira per unit volume shed by each animal species, and the daily environmental contribution by considering the total volume of urine excreted by each carrier animal. Rats excrete the highest quantity of Leptospira per millilitre of urine (median = 5.7 × 106 cells), but large mammals excrete much more urine and thus shed significantly more Leptospira per day (5.1 × 108 to 1.3 × 109 cells). Conclusions: Here we illustrate how, in a low-income rural Ecuadorian community, host population demographics, and prevalence of Leptospira infection can be integrated with estimates of shed Leptospira to suggest that peridomestic cattle may be more important than rats in environmental cycling and ultimately, transmission to humans. [ABSTRACT FROM AUTHOR]

Background: Prevention of nosocomial transmission of infections is a central responsibility in the healthcare environment, and accurate identification of transmission events presents the first challenge. Phylogenetic analysis based on whole genome sequencing provides a high-resolution approach for accurately relating isolates to one another, allowing precise identification or exclusion of transmission events and sources for nearly all cases. We sequenced 24 methicillin-resistant Staphylococcus aureus (MRSA) genomes to retrospectively investigate a suspected point source of three surgical site infections (SSIs) that occurred over a one-year period. The source of transmission was believed to be a surgical team member colonized with MRSA, involved in all surgeries preceding the SSI cases, who was subsequently decolonized. Genetic relatedness among isolates was determined using whole genome single nucleotide polymorphism (SNP) data. Results: Whole genome SNP typing (WGST) revealed 283 informative SNPs between the surgical team member's isolate and the closest SSI isolate. The second isolate was 286 and the third was thousands of SNPs different, indicating the nasal carriage strain from the surgical team member was not the source of the SSIs. Given the mutation rates estimated for S. aureus, none of the SSI isolates share a common ancestor within the past 16 years, further discounting any common point source for these infections. The decolonization procedures and resources spent on the point source infection control could have been prevented if WGST was performed at the time of the suspected transmission, instead of retrospectively. Conclusions: Whole genome sequence analysis is an ideal method to exclude isolates involved in transmission events and nosocomial outbreaks, and coupling this method with epidemiological data can determine if a transmission event occurred. These methods promise to direct infection control resources more appropriately. [ABSTRACT FROM AUTHOR]

Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis is a zoonotic disease responsible for high morbidity around the world, especially in tropical and low income countries. Rats are thought to be the main vector of human leptospirosis in urban settings. However, differences between urban and low-income rural communities provide additional insights into the epidemiology of the disease. Methodology/Principal findings: Our study was conducted in two low-income rural communities near the coast of Ecuador. We detected and characterized infectious leptospira DNA in a wide variety of samples using new real time quantitative PCR assays and amplicon sequencing. We detected infectious leptospira in a high percentage of febrile patients (14.7%). In contrast to previous studies on leptospirosis risk factors, higher positivity was not found in rats (3.0%) but rather in cows (35.8%) and pigs (21.1%). Six leptospira species were identified (L. borgpetersenii, L kirschnerii, L santarosai, L. interrogans, L noguchii, and an intermediate species within the L. licerasiae and L. wolffii clade) and no significant differences in the species of leptospira present in each animal species was detected (χ2 = 9.89, adj.p-value = 0.27). Conclusions/Significance: A large portion of the world’s human population lives in low-income, rural communities, however, there is limited information about leptospirosis transmission dynamics in these settings. In these areas, exposure to peridomestic livestock is particularly common and high prevalence of infectious leptospira in cows and pigs suggest that they may be the most important reservoir for human transmission. Genotyping clinical samples show that multiple species of leptospira are involved in human disease. As these genotypes were also detected in samples from a variety of animals, genotype data must be used in conjunction with epidemiological data to provide evidence of transmission and the importance of different potential leptospirosis reservoirs. [ABSTRACT FROM AUTHOR]

Coxiella burnetii is a gram-negative bacterium that is the etiologic agent of the zoonotic disease Q fever. Common reservoirs of C. burnetii include sheep, goats, and cattle. These animals shed C. burnetii into the environment, and humans are infected by inhalation of aerosols. A survey of 1622 environmental samples taken across the United States in 2006-2008 found that 23.8% of the samples contained C. burnetii DNA. To identify the strains circulating in the U.S. environment, DNA from these environmental samples was genotyped using an SNP-based approach to derive sequence types (ST) that are also compatible with multispacer sequence typing methods. Three different sequence types were observed in 31 samples taken from 19 locations. ST8 was associated with goats and ST20 with dairy cattle. ST16/26 was detected in locations with exposure to various animals and also in locations with no direct animal contact. Viable isolates were obtained for all three sequence types, but only the ST20 and ST16/26 isolates grew in acidified citrate cysteine medium (ACCM)-2 axenic media. Examination of a variety of isolates with different sequence types showed that ST8 and closely related isolates did not grow in ACCM-2. These results suggest that a limited number of C. burnetii sequence types are circulating in the U.S. environment and these strains have close associations with specific reservoir species. Growth in ACCM-2 may not be suitable for isolation of many C. burnetii strains. [ABSTRACT FROM AUTHOR]

The hyper-virulent emm59 genotype of invasive group A Streptococcus was identified in northern Arizona in 2015. Eighteen isolates belonging to a genomic cluster grouped most closely with recently identified isolates in New Mexico. The continued transmission of emm59 in the southwestern United States poses a public health concern. [ABSTRACT FROM AUTHOR]

Background: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/ Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. Results: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. Conclusions: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found. [ABSTRACT FROM AUTHOR]

Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey. [ABSTRACT FROM AUTHOR]