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14 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Development of a high-throughput method for processing sponge-stick samples to detect viable Bacillus anthracis spores

Since the national validation of the sponge-stick based method for detection of Bacillus anthracis spores in environmental samples, there have not been focused efforts to address the low throughput nature of the method, which processes only one sample at one time. Sample processing remains a serious bottleneck for rapidly analyzing large numbers of samples expected from a biological warfare attack. Therefore, we developed a high-throughput method to simultaneously process multiple sponge-stick samples to be better prepared for rapid response and recovery after wide area anthrax incidents. In this method, sponges are placed in 50 mL tubes containing 25 mL extraction buffer and shaken to release spores, after which the suspension is recovered for analysis. Here, we determined that an additional extraction step, conducted in the same tubes with 10 mL buffer, further increased spore recovery from sponge-stick by approximately 10 %. We determined that orbital shaking and multi-tube vortexing were both more effective than reciprocating shaking for recovering spores. We conducted simultaneous processing of up to 12 sponge-stick samples and demonstrated comparable spore recovery efficiencies to the traditional low-throughput stomacher-based method (approximately 60 % recovery at 10 2 -spore level and 75 % recovery at 10 4 -spore level for both methods in three replicate experiments, P > 0.05 for two-tailed t-tests for each experiment and spore level). We also demonstrated that our high-throughput method could be integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis and could detect levels as low as 40 spores per sponge even when challenged by a PCR particulate contaminant.

Anthrax↗
DOE OSTI2025

Evaluation of a high-throughput method for processing sponge-stick samples to detect viable, non-spore-forming biothreat agents

After a bioterrorism incident, surface sampling is often used to determine the extent of contamination and exposure, guiding decontamination efforts and decisions for re-occupancy of affected sites. The sponge-stick (SS) is a preferred and commonly used device for sample collection to detect both spore-forming and non-spore-forming biothreat agents from non-porous surfaces. Here, in this study, a recently developed high-throughput method (HTM) for processing SS samples to detect viable Bacillus anthracis spores was adapted for detection of non-spore-forming biothreat agents, Yersinia pestis and Francisella tularensis. The scalable HTM was used to process up to 20 SS samples simultaneously, compared to the current stomacher-based method which processes one SS at a time. Comparisons of the HTM and the stomacher-based method were statistically indistinguishable for most experiments (P > 0.05) with HTM recoveries of 37–60 % for Y. pestis inoculated at 102–103 cells/SS and held 48 h at 4 °C to mimic sample transport/storage. The HTM was integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis to detect viable Y. pestis in the presence of particulate contamination (Arizona Test Dust, ATD). This approach detected Y. pestis inoculated at 20 cells/SS and ATD did not impact detection (P > 0.05). F. tularensis showed significantly lower recoveries between no-hold time and 48-h hold time (4 °C, P < 0.05) using the HTM, which further testing showed could be due to toxicity of the neutralizing buffer used for SS pre-wetting. With modifications, this method could enhance throughput capacity while maintaining similar recovery efficiencies to current methods for other non-spore-forming bacterial pathogens.

Biological and medical sciences↗
DOE OSTI2024

Fungal diversity and function in metagenomes sequenced from extreme environments

Fungi are increasingly recognized as key players in various extreme environments. Here we present an analysis of publicly-sourced metagenomes from global extreme environments, focusing on fungal taxonomy and function. The majority of 855 selected metagenomes contained scaffolds assigned to fungi. Relative abundance of fungi was as high as 10% of protein-coding genes with taxonomic annotation, with up to 289 fungal genera per sample. Despite taxonomic clustering by environment, fungal communities were more dissimilar than archaeal and bacterial communities, both for within- and between-environment comparisons. Relatively abundant fungal classes in extreme environments included Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, Saccharomycetes, and Sordariomycetes. Broad generalists and prolific aerial spore formers were the most relatively abundant fungal genera detected in most of the extreme environments, bringing up the question of whether they are actively growing in those environments or just surviving as spores. More specialized fungi were common in some environments, such as zoosporic taxa in cryosphere water and hot springs. Relative abundances of genes involved in adaptation to general, thermal, oxidative, and osmotic stress were greatest in soda lake, acid mine drainage, and cryosphere water samples.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Coccidioides undetected in soils from agricultural land and uncorrelated with time or the greater soil fungal community on undeveloped land

Coccidioidomycosis is a typically respiratory fungal disease that, in the United States, occurs primarily in Arizona and California. In California, most coccidioidomycosis cases occur in the San Joaquin Valley, a primarily agricultural region where the disease poses a risk for outdoor workers. We collected 710 soil samples and 265 settled dust samples from nine sites in the San Joaquin Valley and examined how Coccidioides detection varied by month, site, and the presence and abundance of other fungal species. We detected Coccidioides in 89 of 238 (37.4%) rodent burrow soil samples at five undeveloped sites and were unable to detect Coccidioides in any of 472 surface and subsurface soil samples at four agricultural sites. In what is the largest sampling effort undertaken on agricultural land, our results provide no evidence that agricultural soils in the San Joaquin Valley harbor Coccidioides . We found no clear association between Coccidioides and the greater soil fungal community, but we identified 19 fungal indicator species that were significantly associated with Coccidioides detection in burrows. We also did not find a seasonal pattern in Coccidioides detection in the rodent burrow soils we sampled. These findings suggest both the presence of a spore bank and that coccidioidomycosis incidence may be more strongly associated with Coccidioides dispersal than Coccidioides growth. Finally, we were able to detect Coccidioides in only five of our 265 near-surface settled dust samples, one from agricultural land, where Coccidioides was undetected in soils, and four from undeveloped land, where Coccidioides was common in the rodent burrow soils we sampled. Our ability to detect Coccidioides in few settled dust samples indicates that improved methods are likely needed moving forward, though raises questions regarding aerial dispersal in Coccidioides , whose key transmission event likely occurs over short distances in rodent burrows from soil to naïve rodent lungs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Estimating coccidioidomycosis endemicity while accounting for imperfect detection using spatio - temporal occupancy modeling

Coccidioidomycosis, or Valley fever, is an infectious disease caused by inhaling Coccidioides fungal spores. Incidence has risen in recent years, and it is believed the endemic region for Coccidioides is expanding in response to climate change. While Valley fever case data can help us understand trends in disease risk, using case data as a proxy for Coccidioides endemicity is not ideal because case data suffers from imperfect detection, including false positives (e.g., travel-related cases reported outside of endemic area) and false negatives (e.g., misdiagnosis or underreporting). Here we proposed a Bayesian, spatio-temporal occupancy model to relate monthly, county-level presence/absence data on Valley fever cases to latent endemicity of Coccidioides, accounting for imperfect detection. We used our model to estimate endemicity in the western United States. We estimated high probability of endemicity in southern California, Arizona, and New Mexico, but also in regions without mandated reporting, including western Texas, eastern Colorado, and southeastern Washington. We also quantified spatio-temporal variability in detectability of Valley fever, given an area is endemic to Coccidioides. We estimated an inverse relationship between lagged 3- and 9-month precipitation and case detection, and a positive association with agriculture. This work can help inform public health surveillance needs and identify areas that would benefit from mandatory case reporting.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Gene drive by Fusarium SKC1 is dependent on its competing allele

The Fusarium verticillioides SKC1 gene driver is transmitted to offspring in a biased manner through spore killing. The mechanism that allows SKC1 to kill non-SKC1 offspring while sparing others is poorly understood. Here we report that gene drive by SKC1 is dependent on SKC1's competing allele. We propose that SKC1's competing allele influences the ability of a genome defense process to detect SKC1, and we provide evidence that this genome defense process is meiotic silencing by unpaired DNA (MSUD). Furthermore, our findings suggest that the successful deployment of gene drivers to control pathogenic fungi will require researchers to consider how competing alleles influence the ability of gene drivers to be detected by genome defense processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Stepwise recombination suppression around the mating-type locus in an ascomycete fungus with self-fertile spores

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biochemical basis for the formation of organ-specific volatile blends in mint

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint ( Mentha ˣ piperita L.), spearmint ( Mentha spicata L.) and horsemint ( Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs – namely stems, rhizomes and roots – also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae -spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli , purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (–)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (–)-β-pinene and (–)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal

Abstract The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

Figueiredo, Ana M. (ORCID:0000000226236340)↗
DOE OSTI2023

Recalls of tattoo and permanent makeup inks in the United States and a follow-up microbiological survey of inks with a previous recall history

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA’s Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 10 3 and 1 × 10 5 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus -related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗
DOE OSTI2022

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de $D\hat{o}me$ (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Sulfurospirillum diekertiae sp. nov., a tetrachloroethene-respiring bacterium isolated from contaminated soil

Two anaerobic, tetrachloroethene- (PCE-) respiring bacterial isolates, designated strain ACS DCE T and strain ACS TCE , were characterized using a polyphasic approach. Cells were Gram-stain-negative, motile, non-spore-forming and shared a vibrioid- to spirillum-shaped morphology. Optimum growth occurred at 30°C and 0.1–0.4% salinity. The pH range for growth was pH 5.5–7.5, with an optimum at pH 7.2. Hydrogen, formate, pyruvate and lactate as electron donors supported respiratory reductive dechlorination of PCE to cis-1,2-dichloroethene (cDCE) in strain ACS DCE T and of PCE to trichloroethene (TCE) in strain ACS TCE . Both strains were able to grow with pyruvate under microaerobic conditions. Nitrate, elemental sulphur, and thiosulphate were alternative electron acceptors. Autotrophic growth was not observed and acetate served as carbon source for both strains. The major cellular fatty acids were C 16:1 ω7c, C 16:0 , C 14:0 and C 18:1 ω7c. Both genomes feature a circular plasmid. Strains ACS DCE T and ACS TCE were previously assigned to the candidate species 'Sulfurospirillum acididehalogenans'. Here, based on key genomic features and pairwise comparisons of whole-genome sequences, including average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity, strains ACS DCE T and ACS TCE , 'Ca. Sulfurospirillum diekertiae' strains SL2-1 and SL2-2, and the unclassified Sulfurospirillum sp. strain SPD-1 are grouped into one distinct species separate from previously described Sulfurospirillum species. Compared to Sulfurospirillum multivorans and Sulfurospirillum halorespirans, which dechlorinate PCE to cDCE without substantial TCE accumulation, these five strains produce TCE or cDCE as the end product. In addition, some cellular fatty acids (e.g., C 16:0 3OH, C 17:0 iso 3OH, C 17:0 2OH) were detected in strains ACS DCE T and ACS TCE but not in other Sulfurospirillum species. On the basis of phylogenetic, physiological and phenotypic characteristics, 'Ca. Sulfurospirillum acididehalogenans' and 'Ca. Sulfurospirillum diekertiae' are proposed to be merged into one novel species within the genus Sulfurospirillum, for which the name Sulfurospirillum diekertiae sp. nov. is proposed. Finally, the type strain is ACS DCE T (=JCM 33349 T = KCTC 15819 T =CGMCC 1.5292 T ).

59 BASIC BIOLOGICAL SCIENCES↗