Ultrasonography or Radiography for Suspected Pediatric Distal Forearm Fractures.

BACKGROUND: Data on whether ultrasonography for the initial diagnostic imaging of forearm fractures in children and adolescents is noninferior to radiography for subsequent physical function of the arm are limited. METHODS: In this open-label, multicenter, noninferiority, randomized trial in Australia, we recruited participants 5 to 15 years of age who presented to the emergency department with an isolated distal forearm injury, without a clinically visible deformity, in whom further evaluation with imaging was indicated. Participants were randomly assigned to initially undergo point-of-care ultrasonography or radiography, and were then followed for 8 weeks. The primary outcome was physical function of the affected arm at 4 weeks as assessed with the use of the validated Pediatric Upper Extremity Short Patient-Reported Outcomes Measurement Information System (PROMIS) score (range, 8 to 40, with higher scores indicating better function); the noninferiority margin was 5 points. RESULTS: A total of 270 participants were enrolled, with outcomes for 262 participants (97%) available at 4 weeks (with a window of ±3 days) as prespecified. PROMIS scores at 4 weeks in the ultrasonography group were noninferior to those in the radiography group (mean, 36.4 and 36.3 points, respectively; mean difference, 0.1 point; 95% confidence interval [CI], -1.3 to 1.4). Intention-to-treat analyses (in 266 participants with primary outcome data recorded at any time) produced similar results (mean difference, 0.1 point; 95% CI, -1.3 to 1.4). No clinically important fractures were missed, and there were no between-group differences in the occurrence of adverse events. CONCLUSIONS: In children and adolescents with a distal forearm injury, the use of ultrasonography as the initial diagnostic imaging method was noninferior to radiography with regard to the outcome of physical function of the arm at 4 weeks. (Funded by the Emergency Medicine Foundation and others; BUCKLED Australian New Zealand Clinical Trials Registry number, ACTRN12620000637943).

Diagnostic Accuracy of Point-of-Care Ultrasound Versus Radiographic Imaging for Pediatric Distal Forearm Fractures: A Randomized Controlled Trial.

STUDY OBJECTIVE: In patients aged 5 to 15 years with a clinically nondeformed distal forearm injury presenting to the emergency department (ED), we examined whether point-of-care ultrasound or radiographic imaging had better diagnostic accuracy, with the reference diagnosis determined by an expert panel review. METHODS: This multicenter, open-label, diagnostic randomized controlled trial was conducted in South East Queensland, Australia. Eligible patients were randomized to receive initial imaging through point-of-care ultrasound performed by an ED clinician or radiograph. Images were defined as "no," "buckle," or "other" fracture by the treating clinician. The primary outcome was the diagnostic accuracy of the treating clinician's interpretation compared against the reference standard diagnosis, which was determined retrospectively by an expert panel consisting of an emergency physician, pediatric radiologist, and pediatric orthopedic surgeon, who reviewed all imaging and follow-up. RESULTS: Two-hundred and seventy participants were enrolled, with 135 randomized to each initial imaging modality. There were 132 (97.8%) and 112 (83.0%) correctly diagnosed participants by ED clinicians in the point-of-care ultrasound and radiograph groups, respectively (absolute difference [AD]=14.8%; 95% confidence interval [CI] 8.0% to 21.6%; P<.001). Point-of-care ultrasound had better accuracy for participants with "buckle" fractures (AD=18.5%; 95% CI 7.1% to 29.8%) and "other" fractures (AD=17.1%; 95% CI 2.7% to 31.6%). No clinically important fractures were missed in either group. CONCLUSION: In children and adolescents presenting to the ED with a clinically nondeformed distal forearm injury, clinician-performed (acquired and interpreted) point-of-care ultrasound more accurately identified the correct diagnosis than clinician-interpreted radiographic imaging.

Shaping Nanobodies and Intrabodies against Proteoforms.

Proteoforms expand genomic diversity and direct developmental processes. While high-resolution mass spectrometry has accelerated characterization of proteoforms, molecular techniques working to bind and disrupt the function of specific proteoforms have lagged behind. In this study, we worked to develop intrabodies capable of binding specific proteoforms. We employed a synthetic camelid nanobody library expressed in yeast to identify nanobody binders of different SARS-CoV-2 receptor binding domain (RBD) proteoforms. Importantly, employment of the positive and negative selection mechanisms inherent to the synthetic system allowed for amplification of nanobody-expressing yeast that bind to the original (Wuhan strain RBD) but not the E484 K (Beta variant) mutation. Nanobodies raised against specific RBD proteoforms were validated by yeast-2-hybrid analysis and sequence comparisons. These results provide a framework for development of nanobodies and intrabodies that target proteoforms.

Yarning to reduce take own leave events in First Nations patients presenting to the Emergency Department-presenting the qualitative themes and co-design of the Deadly RED project.

ISSUE ADDRESSED: The Deadly RED redesign and implementation research aimed to improve take own leave (TOL) rates within a Queensland emergency department by providing a culturally competent care pathway. METHODOLOGY: A mixed methods pre/post evaluation of the feasibility, acceptability and usability of the Deadly RED pathway for First Nations patients presenting to ED was performed. This pathway combined early welcome and information sharing, introduction of screening and follow up for patients who TOL and enhanced access to alternative community healthcare. Yarning circles facilitated co-design of research protocols and tools while a purposefully designed research Yarn enhanced understanding of the 'story' of the people. Qualitative analysis of Yarns allowed deductive themes to be extracted. A Participatory Action Research (PAR) approach and Indigenous research methodology involving First Nations people in design, knowledge sharing and joint ownership of results was used. RESULTS: Common themes from the 85 yarns included the negative impact of long waiting times and positive impact from wholistic care. Unique themes identified included interpretation of waiting room placement and paracetamol prescription as a dismissal. Knowledge dissemination from yarning drove improvements in communications and processes to promote treatment completion resulting in elimination of these themes in post implementation yarns. Eighteen patients who had TOL were included in the post implementation yarns, however only eight of these believed that their treatment was incomplete. CONCLUSION: The use of yarning for consumer engagement has allowed deeper understanding of the reasons for TOL in First Nations emergency patients. The reciprocal knowledge sharing has guided targeted improvements in wholistic emergency care and communication resulting in First Nations patients feeling their care is complete even when the 'number' reports otherwise. SO WHAT?: Indigenous Research methodology including yarning with First Nations patients suggests alternative engagement methods to guide enhanced quality of care monitoring for ED presentations.

CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.

Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.

Challenges of metagenomics and single-cell genomics approaches for exploring cyanobacterial diversity.

Cyanobacteria have played a crucial role in the history of early earth and continue to be instrumental in shaping our planet, yet applications of cutting edge technology have not yet been widely used to explore cyanobacterial diversity. To provide adequate background, we briefly review current sequencing technologies and their innovative uses in genomics and metagenomics. Next, we focus on current cell capture technologies and the challenges of using them with cyanobacteria. We illustrate the utility in coupling breakthroughs in DNA amplification with cell capture platforms, with an example of microfluidic isolation and subsequent targeted amplicon sequencing from individual terrestrial thermophilic cyanobacteria. Single cells of thermophilic, unicellular Synechococcus sp. JA-2-3-B'a(2-13) (Syn OS-B') were sorted in a microfluidic device, lysed, and subjected to whole genome amplification by multiple displacement amplification. We amplified regions from specific CRISPR spacer arrays, which are known to be highly diverse, contain semi-palindromic repeats which form secondary structure, and can be difficult to amplify. Cell capture, lysis, and genome amplification on a microfluidic device have been optimized, setting a stage for further investigations of individual cyanobacterial cells isolated directly from natural populations.

Evaluation of a pathway to address take own leave events for First Nations peoples presenting for emergency care: The Deadly RED project.

OBJECTIVE: The 'Deadly RED' project primarily aimed to improve culturally competent care to reduce the number of First Nations patients presenting to a Queensland ED who 'Take own leave' (TOL). The secondary aim was to evaluate the implementation project. METHODS: A pre/post-test quasi experimental study design using mixed methods was co-designed with adherence to Indigenous research considerations. Quantitative analysis of First Nations presentations before and after Deadly RED implementation was performed using SPSS. Qualitative analysis of transcribed research yarns in NVIVO was coded and themed for analysis. Staff experiences and perspectives were collated using electronically distributed surveys and process audits were performed. RESULTS: A total of 1096 First Nations presentations June to August 2021 and 1167 in the matched 2022 post-implementation period were analysed. Significantly more patients were recorded as TOL post-implementation (13.0% pre vs 21.3% post) and representations rates were unchanged. Forty-six staff surveyed identified improvements in all parameters including cultural appropriateness and quality of care. Qualitative analysis of 85 research yarns revealed themes migrated to increasingly acceptable, accessible, and usable care. Notably, 45% of the First Nation's patients recorded as TOL self-reported that their treatment was complete. The study was feasible as 80% of packs distributed and 73% follow-up screening after TOL. CONCLUSIONS: The Deadly RED evaluation revealed significant discrepancies in the reported data points of TOL and the 'story' of the First Nations persons experience of appropriate and completed care. Staff awareness and cultural capability improved significantly, and yarning allowed knowledge translation and improvements in communication which contributed to a better healthcare experience for First Nations patients attending our ED.

Hi-C metagenome sequencing reveals soil phage-host interactions.

Bacteriophages are abundant in soils. However, the majority are uncharacterized, and their hosts are unknown. Here, we apply high-throughput chromosome conformation capture (Hi-C) to directly capture phage-host relationships. Some hosts have high centralities in bacterial community co-occurrence networks, suggesting phage infections have an important impact on the soil bacterial community interactions. We observe increased average viral copies per host (VPH) and decreased viral transcriptional activity following a two-week soil-drying incubation, indicating an increase in lysogenic infections. Soil drying also alters the observed phage host range. A significant negative correlation between VPH and host abundance prior to drying indicates more lytic infections result in more host death and inversely influence host abundance. This study provides empirical evidence of phage-mediated bacterial population dynamics in soil by directly capturing specific phage-host interactions.

RNA Viruses Linked to Eukaryotic Hosts in Thawed Permafrost.

Arctic permafrost is thawing due to global warming, with unknown consequences on the microbial inhabitants or associated viruses. DNA viruses have previously been shown to be abundant and active in thawing permafrost, but little is known about RNA viruses in these systems. To address this knowledge gap, we assessed the composition of RNA viruses in thawed permafrost samples that were incubated for 97 days at 4°C to simulate thaw conditions. A diverse RNA viral community was assembled from metatranscriptome data including double-stranded RNA viruses, dominated by Reoviridae and Hypoviridae, and negative and positive single-stranded RNA viruses, with relatively high representations of Rhabdoviridae and Leviviridae, respectively. Sequences corresponding to potential plant and human pathogens were also detected. The detected RNA viruses primarily targeted dominant eukaryotic taxa in the samples (e.g., fungi, Metazoa and Viridiplantae) and the viral community structures were significantly associated with predicted host populations. These results indicate that RNA viruses are linked to eukaryotic host dynamics. Several of the RNA viral sequences contained auxiliary metabolic genes encoding proteins involved in carbon utilization (e.g., polygalacturosase), implying their potential roles in carbon cycling in thawed permafrost. IMPORTANCE Permafrost is thawing at a rapid pace in the Arctic with largely unknown consequences on ecological processes that are fundamental to Arctic ecosystems. This is the first study to determine the composition of RNA viruses in thawed permafrost. Other recent studies have characterized DNA viruses in thawing permafrost, but the majority of DNA viruses are bacteriophages that target bacterial hosts. By contrast RNA viruses primarily target eukaryotic hosts and thus represent potential pathogenic threats to humans, animals, and plants. Here, we find that RNA viruses in permafrost are novel and distinct from those in other habitats studied to date. The COVID-19 pandemic has heightened awareness of the importance of potential environmental reservoirs of emerging RNA viral pathogens. We demonstrate that some potential pathogens were detected after an experimental thawing regime. These results are important for understanding critical viral-host interactions and provide a better understanding of the ecological roles that RNA viruses play as permafrost thaws.

Whole gene amplification and protein separation from a few cells.

Despite the growing interest to explore untapped microbial gene and protein diversity, no single platform has been able to acquire both gene and protein information from just a few cells. We present a microfluidic system that simultaneously performs on-chip capillary electrophoresis for protein analysis and whole genome amplification (WGA), and we demonstrate this by doing both for the same cohort of cyanobacterial cells. This technology opens avenues for studying protein profiles of precious environmental microbial samples and simultaneously accessing genomic information based on WGA.

Bedside Ultrasound Conducted in Kids with distal upper Limb fractures in the Emergency Department (BUCKLED): a protocol for an open-label non-inferiority diagnostic randomised controlled trial.

BACKGROUND: Children frequently present to the emergency department (ED) with forearm injuries and often have x-rays to determine if there is a fracture. Bedside ultrasound, also known as point-of-care ultrasound (POCUS), is an alternative diagnostic test used to rapidly diagnose a fracture at the time of examination, without exposing children to ionising radiation. Prospective studies have demonstrated high agreement between POCUS and x-ray findings. However, whether the initial imaging modality affects the patient's medium-term physical function is unknown. METHODS: This is a multicentre, open-label, non-inferiority randomised controlled trial conducted in Australian EDs. Recruitment will continue until 112 children with distal forearm injuries (including 48 buckle fractures) per trial arm have achieved the primary outcome measure. Patients aged 5-15 years presenting with an isolated, acute, clinically non-angulated, distal forearm injury with suspected fracture will have their initial diagnostic approach randomised to either POCUS, performed by a credentialled practitioner, or x-ray imaging. If a cortical breach fracture is identified on POCUS, the patient will receive x-rays and have usual care. If a buckle fracture is identified, the patient will have their forearm placed in a splint and be discharged home. Patients will be followed up at 1, 4 and 8 weeks. The primary outcome is upper limb physical function at 4 weeks, as determined by the Pediatric Upper Extremity Short Patient-Reported Outcomes Measurement Information System (PROMIS) tool. Secondary outcomes include healthcare costs, satisfaction, pain, complications, rates of imaging, ED length of stay and diagnostic accuracy. DISCUSSION: If POCUS is non-inferior to x-ray in terms of patient's medium-term physical function, it may have an effect on overall health care resource use, including the number of x-ray performed and earlier ED discharge. Although prospective studies have confirmed the accuracy of POCUS, this will be the first RCT to assess non-inferiority of functional outcomes of POCUS to diagnose non-angulated paediatric distal forearm injuries, compared to x-ray. POCUS may be of particular importance in settings where access to x-ray imaging can be limited either during or after-hours, as it can aid the triaging and management of patients. TRIAL REGISTRATION: Prospectively registered with the ANZCTR on 29 May 2020 ( ACTRN12620000637943 ).

DNA Viral Diversity, Abundance, and Functional Potential Vary across Grassland Soils with a Range of Historical Moisture Regimes.

Soil viruses are abundant, but the influence of the environment and climate on soil viruses remains poorly understood. Here, we addressed this gap by comparing the diversity, abundance, lifestyle, and metabolic potential of DNA viruses in three grassland soils with historical differences in average annual precipitation, low in eastern Washington (WA), high in Iowa (IA), and intermediate in Kansas (KS). Bioinformatics analyses were applied to identify a total of 2,631 viral contigs, including 14 complete viral genomes from three deep metagenomes (1 terabase [Tb] each) that were sequenced from bulk soil DNA. An additional three replicate metagenomes (∼0.5 Tb each) were obtained from each location for statistical comparisons. Identified viruses were primarily bacteriophages targeting dominant bacterial taxa. Both viral and host diversity were higher in soil with lower precipitation. Viral abundance was also significantly higher in the arid WA location than in IA and KS. More lysogenic markers and fewer clustered regularly interspaced short palindromic repeats (CRISPR) spacer hits were found in WA, reflecting more lysogeny in historically drier soil. More putative auxiliary metabolic genes (AMGs) were also detected in WA than in the historically wetter locations. The AMGs occurring in 18 pathways could potentially contribute to carbon metabolism and energy acquisition in their hosts. Structural equation modeling (SEM) suggested that historical precipitation influenced viral life cycle and selection of AMGs. The observed and predicted relationships between soil viruses and various biotic and abiotic variables have value for predicting viral responses to environmental change. IMPORTANCE Soil viruses are abundant but poorly understood. Because soil viruses regulate the dynamics of their hosts and potentially key processes in soil ecology, it is important to understand them better. Here, we leveraged massive DNA sequencing to unearth previously unknown soil viruses. We found that soil viruses differed across a historical gradient of precipitation. We compared soil viruses from Iowa, which is traditionally wetter, to those from Washington, which is traditionally drier, and from Kansas, which is intermediate. This study provides strong evidence that changes in historical precipitation impact not only the types of soil viruses but also their functional potential.

Moisture modulates soil reservoirs of active DNA and RNA viruses.

Soil is known to harbor viruses, but the majority are uncharacterized and their responses to environmental changes are unknown. Here, we used a multi-omics approach (metagenomics, metatranscriptomics and metaproteomics) to detect active DNA viruses and RNA viruses in a native prairie soil and to determine their responses to extremes in soil moisture. The majority of transcribed DNA viruses were bacteriophage, but some were assigned to eukaryotic hosts, mainly insects. We also demonstrated that higher soil moisture increased transcription of a subset of DNA viruses. Metaproteome data validated that the specific viral transcripts were translated into proteins, including chaperonins known to be essential for virion replication and assembly. The soil viral chaperonins were phylogenetically distinct from previously described marine viral chaperonins. The soil also had a high abundance of RNA viruses, with highest representation of Reoviridae. Leviviridae were the most diverse RNA viruses in the samples, with higher amounts in wet soil. This study demonstrates that extreme shifts in soil moisture have dramatic impacts on the composition, activity and potential functions of both DNA and RNA soil viruses.

Development and Analysis of a Stable, Reduced Complexity Model Soil Microbiome.

The soil microbiome is central to the cycling of carbon and other nutrients and to the promotion of plant growth. Despite its importance, analysis of the soil microbiome is difficult due to its sheer complexity, with thousands of interacting species. Here, we reduced this complexity by developing model soil microbial consortia that are simpler and more amenable to experimental analysis but still represent important microbial functions of the native soil ecosystem. Samples were collected from an arid grassland soil and microbial communities (consisting mainly of bacterial species) were enriched on agar plates containing chitin as the main carbon source. Chitin was chosen because it is an abundant carbon and nitrogen polymer in soil that often requires the coordinated action of several microorganisms for complete metabolic degradation. Several soil consortia were derived that had tractable richness (30-50 OTUs) with diverse phyla representative of the native soil, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. The resulting consortia could be stored as glycerol or lyophilized stocks at -80°C and revived while retaining community composition, greatly increasing their use as tools for the research community at large. One of the consortia that was particularly stable was chosen as a model soil consortium (MSC-1) for further analysis. MSC-1 species interactions were studied using both pairwise co-cultivation in liquid media and during growth in soil under several perturbations. Co-abundance analyses highlighted interspecies interactions and helped to define keystone species, including Mycobacterium, Rhodococcus, and Rhizobiales taxa. These experiments demonstrate the success of an approach based on naturally enriching a community of interacting species that can be stored, revived, and shared. The knowledge gained from querying these communities and their interactions will enable better understanding of the soil microbiome and the roles these interactions play in this environment.

Probing the ecological and evolutionary history of a thermophilic cyanobacterial population via statistical properties of its microdiversity.

Despite extensive DNA sequencing data derived from natural microbial communities, it remains a major challenge to identify the key evolutionary and ecological forces that shape microbial populations. We have focused on the extensive microdiversity of the cyanobacterium Synechococcus sp., which is a dominant member of the dense phototrophic biofilms in the hot springs of Yellowstone National Park. From deep amplicon sequencing of many loci and statistical analyses of these data, we showed previously that the population has undergone an unexpectedly high degree of homologous recombination, unlinking synonymous SNP-pair correlations even on intragenic length scales. Here, we analyze the genic amino acid diversity, which provides new evidence of selection and insights into the evolutionary history of the population. Surprisingly, some features of the data, including the spectrum of distances between genic-alleles, appear consistent with primarily asexual neutral drift. Yet the non-synonymous site frequency spectrum has too large an excess of low-frequency polymorphisms to result from negative selection on deleterious mutations given the distribution of coalescent times that we infer. And our previous analyses showed that the population is not asexual. Taken together, these apparently contradictory data suggest that selection, epistasis, and hitchhiking all play essential roles in generating and stabilizing the diversity. We discuss these as well as potential roles of ecological niches at genomic and genic levels. From quantitative properties of the diversity and comparative genomic data, we infer aspects of the history and inter-spring dispersal of the meta-population since it was established in the Yellowstone Caldera. Our investigations illustrate the need for combining multiple types of sequencing data and quantitative statistical analyses to develop an understanding of microdiversity in natural microbial populations.

Evaluation of a multiple-encounter in situ simulation for orientation of staff to a new paediatric emergency service: a single-group pretest/post-test study.

AIM: To assess the utility of a multiple-encounter in-situ (MEIS) simulation as an orientation tool for multidisciplinary staff prior to opening a new paediatric emergency service. METHODS: A single-group pretest/post-test study was conducted. During the MEIS simulation, multidisciplinary staff with participant or observer roles managed eight children (mannequins) who attended triage with their parent/guardians (clinical facilitators) for a range of emergency presentations (structured scenarios designed to represent the expected range of presentations plus test various clinical pathways/systems). Participants were debriefed to explore clinical, systems and crisis-resource management issues. Participants also completed a pre-intervention and post-intervention questionnaire comprising statements about role confidence and orientation adequacy. Pre-test and post-test results were analysed using t-test and Wilcoxon signed rank test. RESULTS: Eighty-nine staff participated in the MEIS simulation, with the majority completing the pre-simulation and post-simulation questionnaire. There was a significant improvement in post-intervention versus pre-intervention Likert scores for role confidence and orientation adequacy (p=0.001 and <0.001, respectively); effect sizes suggested the greatest impact was on orientation adequacy. Nearly all scenarios resulted in significant increases in participants' confidence levels. CONCLUSIONS: The MEIS simulation was of utility in orientation of staff, at least with respect to self-reported role confidence and orientation adequacy. Its effectiveness in practice or compared with other orientation techniques was not assessed, but it did identify several flaws in planned systems allowing remediation prior to opening.

Paediatric high-flow nasal cannula therapy in children with bronchiolitis: A retrospective safety and efficacy study in a non-tertiary environment.

OBJECTIVE: The objective was to examine the safety and efficacy of high-flow nasal cannula (HFNC) therapy for children with bronchiolitis in a non-tertiary paediatric setting. METHODS: This was a single-centre retrospective study conducted over 26 months (March 2013-April 2015) on children aged 1-23 months with suspected bronchiolitis, who commenced on HFNC therapy in either the ED or the ward. Changes with respect to baseline data were analysed for effect on work of breathing (WOB), heart rate (HR) and respiratory rate (RR). Data was analysed using a linear mixed effects model and adjusted for age (≤12 months and >12 months) and location (ED vs ward). Transfer to a tertiary environment, escalation of care and adverse event rates were also recorded. RESULTS: A total of 61 children commenced on HFNC therapy, with flow rates ranging from 0.6 to 3.3L/kg/min. The proportion of patients with higher WOB scores appeared to reduce within 60 min of initiation of therapy. There was also a progressive reduction in surrogate markers of respiratory distress (HR and RR), with significant reductions evident by 60 min (P < 0.05). There were no adverse events related to HFNC therapy. The transfer rate was 13%. It was predominantly due to lack of improvement of physiological parameters post initiation of HFNC therapy. None of the transferred patients required escalation of care. CONCLUSION: Within the limitations of this study it appears HFNC therapy may be safely commenced in both age groups in a non-tertiary ED or ward, with an appropriate level of observation and robust transfer criteria.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.

Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers.

The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2-13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics.

Creation and Analysis of a Virome: Using CRISPR Spacers.

Advances in sequencing technology have allowed for the study of complex and previously unexplored microbial and viral populations; however, linking host-phage partners using in silico techniques has been challenging. Here, we describe the flow-through for creation of a virome, and its subsequent analysis with the viral assembly and analysis module "Viritas," which we have recently developed. This module allows for binning of contigs based on tetranucleotide frequencies, putative phage-host partner identification by CRISPR spacer matching, and identification of ORFs.