Shower dehumidification to reduce nontuberculous mycobacteria aerosolization.

OBJECTIVE: Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that can cause recalcitrant lung disease. Prior reports have demonstrated links between shower use and infections, yet the aerosolization of NTM from showerheads, as well as the humidity levels that may modulate NTM aerosolization from showerheads is less studied. The objective of the current study was to investigate the role of humidity in NTM aerosolization among showers in homes located in a geographic area with high lung disease incidence, Hawai'i, and test whether deployment of a dehumidifier in well-ventilated bathrooms reduce NTM exposure. RESULTS: Across two sampling events and five showers, existing NTM showerhead biofilms along with shower air were sampled at three points: pre-shower, post-shower, and post-dehumidification. In each of the sampling events, respiratory relevant NTM species were identified from shower biofilms, which were also detected in aerosolized shower air after showering events, but not after the shower was dehumidified and bathrooms vented. While sample size was small, these data suggest running a shower is a possible source of NTM aerosolization and using a commercial household dehumidifier in conjunction with opening bathroom doors and windows may be simple, cost-effective interventions to reduce environmental NTM exposures.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Bridging Place-Based Astrobiology Education with Genomics, Including Descriptions of Three Novel Bacterial Species Isolated from Mars Analog Sites of Cultural Relevance.

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T).

Genomic and microbiological analyses of iron acquisition pathways among respiratory and environmental nontuberculous mycobacteria from Hawai'i.

As environmental opportunistic pathogens, nontuberculous mycobacteria (NTM) can cause severe and difficult to treat pulmonary disease. In the United States, Hawai'i has the highest prevalence of infection. Rapid growing mycobacteria (RGM) such as Mycobacterium abscessus and M. porcinum and the slow growing mycobacteria (SGM) including M. intracellulare subspecies chimaera are common environmental NTM species and subspecies in Hawai'i. Although iron acquisition is an essential process of many microorganisms, iron acquisition via siderophores among the NTM is not well-characterized. In this study, we apply genomic and microbiological methodologies to better understand iron acquisition via siderophores for environmental and respiratory isolates of M. abscessus, M. porcinum, and M. intracellulare subspecies chimaera from Hawai'i. Siderophore synthesis and transport genes, including mycobactin (mbt), mmpL/S, and esx-3 were compared among 47 reference isolates, 29 respiratory isolates, and 23 environmental Hawai'i isolates. Among all reference isolates examined, respiratory isolates showed significantly more siderophore pertinent genes compared to environmental isolates. Among the Hawai'i isolates, RGM M. abscessus and M. porcinum had significantly less esx-3 and mbt genes compared to SGM M. chimaera when stratified by growth classification. However, no significant differences were observed between the species when grown on low iron culture agar or siderophore production by the chrome azurol S (CAS) assay in vitro. These results indicate the complex mechanisms involved in iron sequestration and siderophore activity among diverse NTM species.

Complete genome assembly of Hawai'i environmental nontuberculous mycobacteria reveals unexpected co-isolation with methylobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai'i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai'i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai'i, community scientists of Hawai'i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O'ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai'i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai'i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.

Genomic insights into the conservation status of the world's last remaining Sumatran rhinoceros populations.

Small populations are often exposed to high inbreeding and mutational load that can increase the risk of extinction. The Sumatran rhinoceros was widespread in Southeast Asia, but is now restricted to small and isolated populations on Sumatra and Borneo, and most likely extinct on the Malay Peninsula. Here, we analyse 5 historical and 16 modern genomes from these populations to investigate the genomic consequences of the recent decline, such as increased inbreeding and mutational load. We find that the Malay Peninsula population experienced increased inbreeding shortly before extirpation, which possibly was accompanied by purging. The populations on Sumatra and Borneo instead show low inbreeding, but high mutational load. The currently small population sizes may thus in the near future lead to inbreeding depression. Moreover, we find little evidence for differences in local adaptation among populations, suggesting that future inbreeding depression could potentially be mitigated by assisted gene flow among populations.

Genomic differentiation and local adaptation on a microgeographic scale in a resident songbird.

Elucidating forces capable of driving species diversification in the face of gene flow remains a key goal in evolutionary biology. Song sparrows, Melospiza melodia, occur as 25 subspecies in diverse habitats across North America, are among the continent's most widespread vertebrate species, and are exemplary of many highly variable species for which the conservation of locally adapted populations may be critical to their range-wide persistence. We focus here on six morphologically distinct subspecies resident in the San Francisco Bay region, including three salt-marsh endemics and three residents in upland and riparian habitats adjacent to the Bay. We used reduced-representation sequencing to generate 2,773 SNPs to explore genetic differentiation, spatial population structure, and demographic history. Clustering separated individuals from each of the six subspecies, indicating subtle differentiation at microgeographic scales. Evidence of limited gene flow and low nucleotide diversity across all six subspecies further supports a hypothesis of isolation among locally adapted populations. We suggest that natural selection for genotypes adapted to salt marsh environments and changes in demography over the past century have acted in concert to drive the patterns of diversification reported here. Our results offer evidence of microgeographic specialization in a highly polytypic bird species long discussed as a model of sympatric speciation and rapid adaptation, and they support the hypothesis that conserving locally adapted populations may be critical to the range-wide persistence of similarly highly variable species.

Pre-extinction Demographic Stability and Genomic Signatures of Adaptation in the Woolly Rhinoceros.

Ancient DNA has significantly improved our understanding of the evolution and population history of extinct megafauna. However, few studies have used complete ancient genomes to examine species responses to climate change prior to extinction. The woolly rhinoceros (Coelodonta antiquitatis) was a cold-adapted megaherbivore widely distributed across northern Eurasia during the Late Pleistocene and became extinct approximately 14 thousand years before present (ka BP). While humans and climate change have been proposed as potential causes of extinction [1-3], knowledge is limited on how the woolly rhinoceros was impacted by human arrival and climatic fluctuations [2]. Here, we use one complete nuclear genome and 14 mitogenomes to investigate the demographic history of woolly rhinoceros leading up to its extinction. Unlike other northern megafauna, the effective population size of woolly rhinoceros likely increased at 29.7 ka BP and subsequently remained stable until close to the species' extinction. Analysis of the nuclear genome from a ∼18.5-ka-old specimen did not indicate any increased inbreeding or reduced genetic diversity, suggesting that the population size remained steady for more than 13 ka following the arrival of humans [4]. The population contraction leading to extinction of the woolly rhinoceros may have thus been sudden and mostly driven by rapid warming in the Bølling-Allerød interstadial. Furthermore, we identify woolly rhinoceros-specific adaptations to arctic climate, similar to those of the woolly mammoth. This study highlights how species respond differently to climatic fluctuations and further illustrates the potential of palaeogenomics to study the evolutionary history of extinct species.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Genomics of rapid ecological divergence and parallel adaptation in four tidal marsh sparrows.

Theory suggests that different taxa having colonized a similar, challenging environment will show parallel or lineage-specific adaptations to shared selection pressures, but empirical examples of parallel evolution in independent taxa are exceedingly rare. We employed comparative genomics to identify parallel and lineage-specific responses to selection within and among four species of North American sparrows that represent four independent, post-Pleistocene colonization events by an ancestral, upland subspecies and a derived salt marsh specialist. We identified multiple cases of parallel adaptation in these independent comparisons following salt marsh colonization, including selection of 12 candidate genes linked to osmoregulation. In addition to detecting shared genetic targets of selection across multiple comparisons, we found many novel, species-specific signatures of selection, including evidence of selection of loci associated with both physiological and behavioral mechanisms of osmoregulation. Demographic reconstructions of all four species highlighted their recent divergence and small effective population sizes, as expected given their rapid radiation into saline environments. Our results highlight the interplay of both shared and lineage-specific selection pressures in the colonization of a biotically and abiotically challenging habitat and confirm theoretical expectations that steep environmental clines can drive repeated and rapid evolutionary diversification in birds.

Are Rapid Influenza Antigen Tests Still Clinically Useful in Today's Molecular Diagnostics World?

Influenza virus infection and disease historically contribute to widespread cases of seasonal morbidity and in some cases mortality. Prompt and accurate diagnosis is crucial for optimal patient management. Rapid influenza direct antigen testing (RIDT) offers a faster turn-around-time for results but test performance (ie, sensitivity and specificity) varies widely. Nucleic acid amplification testing (NAAT) can offer a viable alternative. The objective of this retrospective study was to compare the test performance of RIDT with NAAT. RIDT testing included the Directigen EZ Flu A+B or the Veritor System for Rapid Detection of Flu A+B. NAAT employed the SimplexaTM Flu A/B™ RSV assay. A total of 5,795 specimens collected from October to March for the 2012/2013 (n=953), 2013/2014 (n=2060) and 2014/2015 (n=2783) seasons were co-tested by RIDT and NAAT. Using NAAT as the gold standard, RIDT tests had a sensitivity range of 0 to 15.7% and a specificity of 98.2 to 100% for influenza type A. For influenza type B, RIDT tests had a sensitivity of 0 to 33.3% and a specificity of 98.9 to 100%. These findings suggest that RIDT has unacceptably low sensitivity for both influenza A and influenza B, despite high specificity. The key advantage of RIDT in previous years (faster turnaround time) has been challenged by newer NAAT technology that provides results in a turn-around-time comparable to RIDT, but with superior test performance.

Asynchronous demographic responses to Pleistocene climate change in Eastern Nearctic vertebrates.

Pleistocene climatic cycles altered species distributions in the Eastern Nearctic of North America, yet the degree of congruent demographic response to the Pleistocene among codistributed taxa remains unknown. We use a hierarchical approximate Bayesian computational approach to test if population sizes across lineages of snakes, lizards, turtles, mammals, birds, salamanders and frogs in this region expanded synchronously to Late Pleistocene climate changes. Expansion occurred in 75% of 74 lineages, and of these, population size trajectories across the community were partially synchronous, with coexpansion found in at least 50% of lineages in each taxonomic group. For those taxa expanding outside of these synchronous pulses, factors related to when they entered the community, ecological thresholds or biotic interactions likely condition their timing of response to Pleistocene climate change. Unified timing of population size change across communities in response to Pleistocene climate cycles is likely rare in North America.

Comparing the temporal colonization and microbial diversity of showerhead biofilms in Hawai'i and Colorado.

The household is a potential source of opportunistic pathogens to humans, a particularly critical issue for immunodeficient individuals. An important human-microbe interface is the biofilm that develops on showerhead surfaces. Once microbe-laden biofilms become aerosolized, they can potentially be inhaled into the lungs. Understanding how quickly a new showerhead becomes colonized would provide useful information to minimize exposure to potentially pathogenic environmental microbes. High school scientists sampled the inner surfaces of pre-existing and newly fitted showerheads monthly over a nine-month period and applied standard microbiologic culture techniques to qualitatively assess microbial growth. Water chemistry was also monitored using commercial test strips. Sampling was performed in households on Oahu, Hawai'i and Denver, Colorado, representing warm/humid and cold/arid environments, respectively. Pre-existing showerheads in Hawai'i showed more diverse microbial growth and significantly greater microbial numbers than a comparable showerhead from Colorado. New, chrome-plated or plastic showerheads in Hawai'i showed diverse and abundant growth one month after installment compared to new showerheads from Colorado. The pH, total chlorine and water hardness levels varied significantly between the Hawai'i and Colorado samples. Enthusiastic student and teacher participation allowed us to answer long-standing questions regarding the temporal colonization of microbial biofilms on pre-existing and new showerhead surfaces.

Detecting concerted demographic response across community assemblages using hierarchical approximate Bayesian computation.

Methods that integrate population-level sampling from multiple taxa into a single community-level analysis are an essential addition to the comparative phylogeographic toolkit. Detecting how species within communities have demographically tracked each other in space and time is important for understanding the effects of future climate and landscape changes and the resulting acceleration of extinctions, biological invasions, and potential surges in adaptive evolution. Here, we present a statistical framework for such an analysis based on hierarchical approximate Bayesian computation (hABC) with the goal of detecting concerted demographic histories across an ecological assemblage. Our method combines population genetic data sets from multiple taxa into a single analysis to estimate: 1) the proportion of a community sample that demographically expanded in a temporally clustered pulse and 2) when the pulse occurred. To validate the accuracy and utility of this new approach, we use simulation cross-validation experiments and subsequently analyze an empirical data set of 32 avian populations from Australia that are hypothesized to have expanded from smaller refugia populations in the late Pleistocene. The method can accommodate data set heterogeneity such as variability in effective population size, mutation rates, and sample sizes across species and exploits the statistical strength from the simultaneous analysis of multiple species. This hABC framework used in a multitaxa demographic context can increase our understanding of the impact of historical climate change by determining what proportion of the community responded in concert or independently and can be used with a wide variety of comparative phylogeographic data sets as biota-wide DNA barcoding data sets accumulate.

Archaeological evidence of validity of fish populations on unexploited reefs as proxy targets for modern populations.

Reef-fish management and conservation is hindered by a lack of information on fish populations prior to large-scale contemporary human impacts. As a result, relatively pristine sites are often used as conservation baselines for populations near sites affected by humans. This space-for-time approach can only be validated by sampling assemblages through time. We used archaeological remains to evaluate whether the remote, uninhabited Northwestern Hawaiian Islands (NWHI) might provide a reasonable proxy for a lightly exploited baseline in the Main Hawaiian Islands (MHI). We used molecular and morphological techniques to describe the taxonomic and size composition of the scarine parrotfish catches present in 2 archaeological assemblages from the MHI, compared metrics of these catches with modern estimates of reproductive parameters to evaluate whether catches represented by the archaeological material were consistent with sustainable fishing, and evaluated overlap between size structures represented by the archaeological material and modern survey data from the MHI and the NWHI to assess whether a space-for-time substitution is reasonable. The parrotfish catches represented by archaeological remains were consistent with sustainable fishing because they were dominated by large, mature individuals whose average size remained stable from prehistoric (AD approximately 1400-1700) through historic (AD 1700-1960) periods. The ancient catches were unlike populations in the MHI today. Overlap between the size structure of ancient MHI catches and modern survey data from the NWHI or the MHI was an order of magnitude greater for the NWHI comparison, a result that supports the validity of using the NWHI parrotfish data as a proxy for the MHI before accelerated, heavy human impacts in modern times.

Genetic variation over 10,000 years in Ctenomys: comparative phylochronology provides a temporal perspective on rarity, environmental change and demography.

An understanding of how ecological traits influence past species response to environmental change can aid our future predictions of species persistence. We used ancient DNA and serial coalescent modelling in a hypothesis-testing framework to reveal differences in temporal genetic variation over 10,000 years for two species of subterranean rodents that currently differ in rarity (abundance, range size and habitat specificity) and mating system, but that reside in the same volcanically active region. Comparative phylochronologic analyses indicated little genetic change and suggest genetic stability in the solitary widespread Ctenomys haigi over thousands of years. In contrast, we found a pattern of haplotypic turnover in the rare and currently endangered Ctenomys sociabilis. Serial coalescent modelling indicated that the best-fit models of microevolutionary change included gene flow between isolated populations for this species. Although C. haigi and C. sociabilis are congeners that share many life history traits, they have behavioural, habitat-preference and population-size differences that may have resulted in contrasting patterns of temporal variation during periods of environmental change.

Generalist dinoflagellate endosymbionts and host genotype diversity detected from mesophotic (67-100 m depths) coral Leptoseris.

BACKGROUND: Mesophotic corals (light-dependent corals in the deepest half of the photic zone at depths of 30-150 m) provide a unique opportunity to study the limits of the interactions between corals and endosymbiotic dinoflagellates in the genus Symbiodinium. We sampled Leptoseris spp. in Hawaii via manned submersibles across a depth range of 67-100 m. Both the host and Symbiodinium communities were genotyped, using a non-coding region of the mitochondrial ND5 intron (NAD5) and the nuclear ribosomal internal transcribed spacer region 2 (ITS2), respectively. RESULTS: Coral colonies harbored endosymbiotic communities dominated by previously identified shallow water Symbiodinium ITS2 types (C1_ AF333515, C1c_ AY239364, C27_ AY239379, and C1b_ AY239363) and exhibited genetic variability at mitochondrial NAD5. CONCLUSION: This is one of the first studies to examine genetic diversity in corals and their endosymbiotic dinoflagellates sampled at the limits of the depth and light gradients for hermatypic corals. The results reveal that these corals associate with generalist endosymbiont types commonly found in shallow water corals and implies that the composition of the Symbiodinium community (based on ITS2) alone is not responsible for the dominance and broad depth distribution of Leptoseris spp. The level of genetic diversity detected in the coral NAD5 suggests that there is undescribed taxonomic diversity in the genus Leptoseris from Hawaii.

Application of universally amplifying plastid primers to environmental sampling of a stream periphyton community.

To demonstrate the utility of universal plastid primers for probing of environmental samples, we extracted DNA from a tropical stream periphyton community and created two environmental clone libraries. We demonstrate the recovery of DNA sequences corresponding to the major groups of algae observed microscopically in the sample, illustrating the utility of these primers for analysis of environmental samples. Using a touchdown polymerase chain reaction technique, almost 99% of recovered sequences correspond to plastid-containing or cyanobacterial taxa, which allows algae to be targeted to the almost complete exclusion of noncyanobacterial prokaryotes and nonplastid-containing eukaryotes.