Analysis of Microbiomes from Ultra-Low Biomass Surfaces Using Novel Surface Sampling and Nanopore Sequencing.

The rapid assessment of microbiomes from ultra-low biomass environments such as cleanrooms or hospital operating rooms has a number of applications for human health and spacecraft manufacturing. Current techniques often employ lengthy protocols using short-read DNA sequencing technology to analyze amplified DNA and have the disadvantage of a longer analysis time and lack of portability. Here, we demonstrate a rapid (~24 hours) on-site nanopore-based sequencing approach to characterize the microbiome of a NASA Class 100K cleanroom where spacecraft components are assembled. This approach employs a modified protocol of Oxford Nanopore's Rapid PCR Barcoding Kit in combination with the recently developed Squeegee-Aspirator for Large Sampling Area (SALSA) surface sampling device. Results for these ultra-low biomass samples revealed DNA amplification ~1 to 2 orders of magnitude above process control samples and were dominated primarily by Paracoccus and Acinetobacter species. Negative control samples were collected to provide critical data on background contamination, including Cutibacerium acnes, which most likely originated from the sampling reagents-associated microbiome (kitome). Overall, these results provide data on a novel approach for rapid low-biomass DNA profiling using the SALSA sampler combined with modified nanopore sequencing. These data highlight the critical need for employing multiple negative controls, along with using DNA-free reagents and techniques, to enable a proper assessment of ultra-low biomass samples.

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Draft genome sequencing of Naganishia species isolated from the polar environments.

The draft genomes of five Naganishia strains were sequenced using MinION and annotated using Funannotate pipeline. Phylogenetic and genomic analyses were performed to provide their genetic relationships, diversity, and potential functional capabilities. This approach will aid in understanding their potential to survive under microgravity and their resilience to extreme environments.

Draft Genome Sequences of Fungi Isolated from Mars 2020 Spacecraft Assembly Facilities.

During the Mars 2020 mission, several fungal strains were isolated from surfaces where spacecraft components were assembled. Draft genome sequencing and characterization will help identify the genes responsible for radiation resistance, supporting the development of countermeasures to prevent fungal contamination of extraterrestrial environments.

Genome Insight and Comparative Pathogenomic Analysis of Nesterenkonia jeotgali Strain CD08_7 Isolated from Duodenal Mucosa of Celiac Disease Patient.

Species of the genus Nesterenkonia have been isolated from different ecological niches, especially from saline habitats and reported as weak human pathogens causing asymptomatic bacteraemia. Here, for the first time we are reporting the genome sequence and pathogenomic analysis of a strain designated as CD08_7 isolated from the duodenal mucosa of a celiac disease patient, identified as Nesterenkonia jeotgali. To date, only five strains of the genus Nesterenkonia (N. massiliensis strain NP1T, Nesterenkonia sp. strain JCM 19054, Nesterenkonia sp. strain F and Nesterenkonia sp. strain AN1) have been whole genome sequenced and annotated. In the present study we have mapped and compared the virulence profile of N. jeotgali strain CD08_7 along with other reference genomes which showed some characteristic features that could contribute to pathogenicity. The RAST (Rapid Annotation using Subsystem Technology) based genome mining revealed more genes responsible for pathogenicity in strain CD08_7 when compared with the other four sequenced strains. The studied categories were resistance to antibiotic and toxic compounds, invasion and intracellular resistance, membrane transport, stress response, osmotic stress, oxidative stress, phages and prophages and iron acquisition. A total of 1431 protein-encoding genes were identified in the genome of strain CD08_7 among which 163 were predicted to contribute for pathogenicity. Out of 163 genes only 59 were common to other genome, which shows the higher levels of genetic richness in strain CD08_7 that may contribute to its functional versatility. This study provides a comprehensive analysis on genome of N. jeotgali strain CD08_7 and possibly indicates its importance as a clinical pathogen.