A New Era for Space Life Science: International Standards for Space Omics Processing.

Space agencies have announced plans for human missions to the Moon to prepare for Mars. However, the space environment presents stressors that include radiation, microgravity, and isolation. Understanding how these factors affect biology is crucial for safe and effective crewed space exploration. There is a need to develop countermeasures, to adapt plants and microbes for nutrient sources and bioregenerative life support, and to limit pathogen infection. Scientists across the world are conducting space omics experiments on model organisms and, more recently, on humans. Optimal extraction of actionable scientific discoveries from these precious datasets will only occur at the collective level with improved standardization. To address this shortcoming, we established ISSOP (International Standards for Space Omics Processing), an international consortium of scientists who aim to enhance standard guidelines between space biologists at a global level. Here we introduce our consortium and share past lessons learned and future challenges related to spaceflight omics.

Genomic characterization of Salmonella Cerro ST367, an emerging Salmonella subtype in cattle in the United States.

BACKGROUND: Within the last decade, Salmonella enterica subsp. enterica serovar Cerro (S. Cerro) has become one of the most common serovars isolated from cattle and dairy farm environments in the northeastern US. The fact that this serovar is commonly isolated from subclinically infected cattle and is rarely associated with human disease, despite its frequent isolation from cattle, has led to the hypothesis that this emerging serovar may be characterized by reduced virulence. We applied comparative and population genomic approaches to (i) characterize the evolution of this recently emerged serovar and to (ii) gain a better understanding of genomic features that could explain some of the unique epidemiological features associated with this serovar. RESULTS: In addition to generating a de novo draft genome for one Salmonella Cerro strain, we also generated whole genome sequence data for 26 additional S. Cerro isolates, including 16 from cattle operations in New York (NY) state, 2 from human clinical cases from NY in 2008, and 8 from diverse animal sources (7 from Washington state and 1 from Florida). All isolates sequenced in this study represent sequence type ST367. Population genomic analysis showed that isolates from the NY cattle operations form a well-supported clade within S. Cerro ST367 (designated here "NY bovine clade"), distinct from isolates from Washington state, Florida and the human clinical cases. A molecular clock analysis indicates that the most recent common ancestor of the NY bovine clade dates back to 1998, supporting the recent emergence of this clone.Comparative genomic analyses revealed several relevant genomic features of S. Cerro ST367, that may be responsible for reduced virulence of S. Cerro, including an insertion creating a premature stop codon in sopA. In addition, patterns of gene deletion in S. Cerro ST367 further support adaptation of this clone to a unique ecological or host related niche. CONCLUSIONS: Our results indicate that the increase in prevalence of S. Cerro ST367 is caused by a highly clonal subpopulation and that S. Cerro ST367 is characterized by unique genomic deletions that may indicate adaptation to specific ecological niches and possibly reduced virulence in some hosts.

Comparative analysis of genome sequences covering the seven cronobacter species.

BACKGROUND: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. CONCLUSIONS/SIGNIFICANCE: Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.

Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.

A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.

Genome sequencing reveals diversification of virulence factor content and possible host adaptation in distinct subpopulations of Salmonella enterica.

BACKGROUND: Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous. Here we used a comparative genomics approach to define the population structure of Salmonella enterica subsp. enterica, and identify clade-specific genes that may be the result of ecological specialization. RESULTS: Multi-locus sequence analysis (MLSA) and single nucleotide polymorphisms (SNPs) data for 16 newly sequenced and 30 publicly available genomes showed an unambiguous subdivision of S. enterica subsp. enterica into at least two subpopulations, which we refer to as clade A and clade B. Clade B strains contain several clade-specific genes or operons, including a ß-glucuronidase operon, a S-fimbrial operon, and cell surface related genes, which strongly suggests niche specialization of this subpopulation. An additional set of 123 isolates was assigned to clades A and B by using qPCR assays targeting subpopulation-specific SNPs and genes of interest. Among 98 serovars examined, approximately 20% belonged to clade B. All clade B isolates contained two pathogenicity related genomic islands, SPI-18 and a cytolethal distending toxin islet; a combination of these two islands was previously thought to be exclusive to serovars Typhi and Paratyphi A. Presence of ß-glucuronidase in clade B isolates specifically suggests an adaptation of this clade to the vertebrate gastrointestinal environment. CONCLUSIONS: S. enterica subsp. enterica consists of at least two subpopulations that differ specifically in genes involved in host and tissue tropism, utilization of host specific carbon and nitrogen sources and are therefore likely to differ in ecology and transmission characteristics.

Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss.

BACKGROUND: The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. RESULTS: To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. CONCLUSIONS: Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides an example of a group of bacteria that appears to evolve through a loss of virulence rather than acquisition of virulence characteristics. While Listeria includes a number of species-like clades, many of these putative species include clades or strains with atypical virulence associated characteristics. This information will allow for the development of genetic and genomic criteria for pathogenic strains, including development of assays that specifically detect pathogenic Listeria strains.

Hydrogels for osteochondral repair based on photocrosslinkable carbamate dendrimers.

First generation, photocrosslinkable dendrimers consisting of natural metabolites (i.e., succinic acid, glycerol, and beta-alanine) and nonimmunogenic poly(ethylene glycol) (PEG) were synthesized divergently in high yields using ester and carbamate forming reactions. Aqueous solutions of these dendrimers were photocrosslinked with an eosin-based photoinitiator to afford hydrogels. The hydrogels displayed a range of mechanical properties based on their structure, generation size, and concentration in solution. All of the hydrogels showed minimal swelling characteristics. The dendrimer solutions were then photocrosslinked in situ in an ex vivo rabbit osteochondral defect (3 mm diameter and 10 mm depth), and the resulting hydrogels were subjected to physiologically relevant dynamic loads. Magnetic resonance imaging (MRI) showed the hydrogels to be fixated in the defect site after the repetitive loading regimen. The ([G1]-PGLBA-MA) 2-PEG hydrogel was chosen for the 6 month pilot in vivo rabbit study because this hydrogel scaffold could be prepared at low polymer weight (10 wt %) and possessed the largest compressive modulus of the 10% formulations, a low swelling ratio, and contained carbamate linkages, which are more hydrolytically stable than the ester linkages. The hydrogel-treated osteochondral defects showed good attachment in the defect site and histological analysis showed the presence of collagen II and glycosaminoglycans (GAGs) in the treated defects. By contrast, the contralateral unfilled defects showed poor healing and negligible GAG or collagen II production. Good mechanical properties, low swelling, good attachment to the defect site, and positive in vivo results illustrate the potential of these dendrimer-based hydrogels as scaffolds for osteochondral defect repair.

Photo cross-linkable Biodendrimers as ophthalmic adhesives for central lacerations and penetrating keratoplasties.

PURPOSE: Biodendrimer-based hydrogel adhesives were derived from biocompatible building blocks and poly(ethylene glycol) of 3,400, 10,000 and 20,000 g/mole. The leaking pressures were determined for these adhesives when used to seal 4.1-mm central lacerations and penetrating keratoplasties (PKPs) in enucleated porcine eyes. METHODS: Three biodendrimers, ([G1]-PGLSA-MA)(2)-PEG(3,400), ([G1]-PGLSA-MA)(2)-PEG(10,000), and ([G1]-PGLSA-MA)(2)-PEG(20,000), at a range of weight percents were each photo cross-linked in the presence of a photo-initiator to form a hydrated network. These biodendrimer-based adhesives were applied directly to a 4.1-mm linear central laceration. In a PKP, the corneal button was initially secured with 8 or 16 sutures and then sealed with the adhesive. RESULTS: For the 4.1-mm central lacerations, the ([G1]-PGLSA-MA)(2)-PEG(3,400) at 20% and 40% wt/vol, the ([G1]-PGLSA-MA)(2)-PEG(10,000) at 10 and 20% wt/vol, and the ([G1]-PGLSA-MA)(2)-PEG(20,000) at 20% wt/vol held to leaking pressures above 200 mm Hg. In the autograft with 16 sutures, the 20% wt/vol of the ([G1]-PGLSA-MA)(2)-PEG(3,400), ([G1]-PGLSA-MA)(2)-PEG(10,000), and ([G1]-PGLSA-MA)(2)-PEG(20,000) held to a pressure at or above 100 mm Hg. In the autograft with eight sutures, the ([G1]-PGLSA-MA)(2)-PEG(10,000) and ([G1]-PGLSA-MA)(2)-PEG(20,000) formulations at 20% wt/vol held to leaking pressures of 85 +/- 22 and 80 +/- 30 mm Hg, respectively. CONCLUSIONS: The 10% wt/vol ([G1]-PGLSA-MA)(2)-PEG(10,000) formulation withheld leaking pressures above 200 mm Hg when used to secure a 4.1 mm central laceration. The 20% wt/vol ([G1]-PGLSA-MA)(2)-PEG(10,000) and ([G1]-PGLSA-MA)(2)-PEG(20,000) formulations, with 8 or 16 sutures, secured the PKP well above normal IOP. Biodendrimer-based adhesives are of potential use for repairing corneal wounds.

Dendritic supramolecular assemblies for drug delivery.

Dendritic supramolecular assemblies were formed in water with Reichardt's dye or the anticancer drug 10-hydroxycamptothecin and the dendritic macromolecule, ([G4]-PGLSA-OH)2-PEG3400.