Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority.

Pediatric screen exposure and school related headache disability.

BACKGROUND AND OBJECTIVES: Prolonged screen exposure is often cited as a trigger for pediatric headache. We present initial findings evaluating the association between adolescent screen use type, duration, and school disability. METHODS: New patients aged 12-17 years presenting to a headache clinic were screened and surveyed regarding headache characteristics, behavioral habits, school attendance, and screen utilization. RESULTS: 99 adolescents (29 M, 70 F) with average age 14.8 years and average headache frequency of 17 days per month completed the survey. Patients missed an average of five full days and three partial days of school due to headaches over the 90 days prior to survey completion.No statistically significant correlation was found between type or duration of screen exposure and monthly headache frequency, school attendance, or school functioning. A small positive association was seen between increasing duration of computer use, total hours screen use, and school absenteeism. While most adolescents reported prolonged screen use (58.6%) and luminosity (64.6%) worsened headaches, no statistical difference was seen in average number of headache days per month. CONCLUSIONS: Average monthly headache frequency in an adolescent population was not significantly correlated with type or duration of screen exposure. Further studies are needed to elucidate how screen utilization impacts school related headache disability.

Natural History of Abortive Medication Withdrawal in the Management of Pediatric Medication Overuse Headache.

OBJECTIVE: The objective of this study is to document pain scores during withdrawal of abortive medication in patients diagnosed with medication overuse headache. DESIGN: Cross-sectional study. SETTING: Children's National Hospital's Headache Program. SUBJECTS: Patients 6-18 years of age who presented to the Headache Clinic at Children's National Hospital with presumed medication overuse headache between March 2017 and March 2019 were invited to participate. METHODS: Patients were instructed to abruptly discontinue overused medications and record their headache characteristics daily in a diary for 8 weeks. RESULTS: Fourteen diaries were returned and analyzed at a 4-week follow-up visit. Ninety-three percent of the patients were females, with a median age of 14.9 years (standard deviation [SD] = 2.0). The average headache intensity upon study entry was 4.7 out of 10 (SD = 2.5), and the average headache intensity upon study completion was 3.1 (SD = 2.5). Of the patients, 57% had daily headaches upon study entry, 71% had improved pain intensity from the first diary entry to the last diary entry, and 57% had complete headache resolution at an average of 7.6 days from medication discontinuation (SD = 5.1). Ibuprofen was the most overused medication (71%). CONCLUSIONS: Our findings suggest that medication overuse headache will improve in the majority of pediatric patients who abruptly stop the offending medication(s) in an average of 8 days from withdrawal. Average pain intensity was reduced by more than one point among all patients who stopped taking abortive medications. Further larger-scale studies on medication withdrawal in pediatric patients with medication overuse headache could help us better understand whether this management strategy is effective.

Criterion Validity and Reliability of Knee Frontal Plane Projection Angles Measured Using the Technique Application.

CONTEXT: Abnormal knee frontal plane projection angles (FPPA) during movement have been associated with patellofemoral pain. As such, clinicians are interested in valid and reliable instruments suitable for broad-based clinical use that allow them to objectively measure such variables. Therefore, the purpose of the current study was to examine the criterion validity and reliability of knee FPPA measures obtained by clinicians using a free tablet application called Technique. DESIGN: Validity/reliability study. METHODS: To examine validity, the same raters measured 10, two-dimensional criterion reference angles at the first testing session. To examine reliability, the knee FPPA of 16 subjects was measured by 6 raters (3 physical therapists and 3 student physical therapists) on 2 separate occasions while performing a single-limb step-down task. Validity was investigated by calculating the 95% limits of agreement, mean absolute differences, and Bland-Altman plots. Reliability was examined by calculating intraclass correlation coefficients and the SE of measure. RESULTS: For validity, the mean absolute difference between rater and criterion reference angle measures ranged from 0.20° to 0.90°. Ninety-five percent of expected errors between rater and criterion reference angle measures were 2.04° or less. For reliability, the intraclass correlation coefficient values for interrater and intrarater reliability were excellent ranging from .994 to .998 with SE of measure ranging from 0.44° to 0.84°. CONCLUSIONS: These findings indicate that knee FPPA measures obtained during a single-limb step-down task using the Technique tablet application are valid and reliable, and suitable for clinical use.

High-throughput interaction screens illuminate the role of c-di-AMP in cyanobacterial nighttime survival.

The broadly conserved signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) is essential for viability in most bacteria where it has been studied. However, characterization of the cellular functions and metabolism of c-di-AMP has largely been confined to the class Bacilli, limiting our functional understanding of the molecule among diverse phyla. We identified the cyclase responsible for c-di-AMP synthesis and characterized the molecule's role in survival of darkness in the model photosynthetic cyanobacterium Synechococcus elongatus PCC 7942. In addition to the use of traditional genetic, biochemical, and proteomic approaches, we developed a high-throughput genetic interaction screen (IRB-Seq) to determine pathways where the signaling nucleotide is active. We found that in S. elongatus c-di-AMP is produced by an enzyme of the diadenylate cyclase family, CdaA, which was previously unexplored experimentally. A cdaA-null mutant experiences increased oxidative stress and death during the nighttime portion of day-night cycles, in which potassium transport is implicated. These findings suggest that c-di-AMP is biologically active in cyanobacteria and has non-canonical roles in the phylum including oxidative stress management and day-night survival. The pipeline and analysis tools for IRB-Seq developed for this study constitute a quantitative high-throughput approach for studying genetic interactions.

Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome.

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemopreventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the "bare-bones", Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.

Variation of the aryl substituent on the piperazine ring within the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold unveils potent, non-competitive inhibitors of the inflammatory caspases.

The inflammatory caspases (caspase-1, -4 and -5) are potential therapeutic targets for autoimmune and inflammatory diseases due to their involvement in the immune response upon inflammasome formation. A series of small molecules based on the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold were synthesized with varying substituents on the piperazine ring. Several compounds were pan-selective inhibitors of the inflammatory caspases, caspase-1, -4 and -5, with the ethylbenzene derivative CK-1-41 displaying low nanomolar Ki values across this family of caspases. Three analogs were nearly 10 fold selective for caspase-5 over caspase-1 and -4. The compounds display non-competitive, time dependent inhibition profiles. To our knowledge, this series is the first example of small molecule inhibitors of all three inflammatory caspases.

Phage resistance mutations affecting the bacterial cell surface increase susceptibility to fungi in a model cheese community.

Diverse populations of bacteriophages infect and coevolve with their bacterial hosts. Although host recognition and infection occur within microbiomes, the molecular mechanisms underlying host-phage interactions within a community context remain poorly studied. The biofilms (rinds) of aged cheeses contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. In this study, we use cheese as a model for studying phage-microbe interactions by identifying and characterizing a tractable host-phage pair co-occurring within a model Brie-like community. We isolated a novel bacteriophage, TS33, that kills Hafnia sp. JB232, a member of the model community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese community, rendering it a prime candidate for the study of host-phage interactions. We performed growth assays of the Hafnia, TS33, and the fungal community members, Geotrichum candidum and Penicillium camemberti. Employing Random Barcode Transposon Sequencing experiments, we identified candidate host factors that contribute to TS33 infectivity, many of which are homologs of bacterial O-antigen genes. Hafnia mutants in these genes exhibit decreased susceptibility to phage infection, but experience negative fitness effects in the presence of the fungi. Therefore, mutations in O-antigen biosynthesis homologs may have antagonistic pleiotropic effects in Hafnia that have major consequences for its interactions with the rest of the community. Ongoing and future studies aim to unearth the molecular mechanisms by which the O-antigen of Hafnia mediates its interactions with its viral and fungal partners.

Microcystin Concentrations, Partitioning, and Structural Composition during Active Growth and Decline: A Laboratory Study.

Microcystin can be present in variable concentrations, phases (dissolved and particulate), and structural forms (congeners), all which impact the toxicity and persistence of the algal metabolite. Conducting incubation experiments with six bloom assemblages collected from the Chowan River, North Carolina, we assessed microcystin dynamics during active growth and biomass degradation. Upon collection, average particulate and dissolved microcystin ranged between 0.2 and 993 µg L-1 and 0.5 and 3.6 µg L-1, respectively. The presence of congeners MC-LA, -LR, -RR, and -YR was confirmed with MC-RR and MC-LR being the most prevalent. Congener composition shifted over time and varied between dissolved and particulate phases. Particulate microcystin exponentially declined in five of six incubations with an average half-life of 10.2 ± 3.7 days, while dissolved microcystin remained detectable until the end of the incubation trials (up to 100 days). Our findings suggest that concerns about food-web transfer via intracellular toxins seem most warranted within the first few weeks of the bloom peak, while dissolved toxins linger for several months in the aftermath of the event. Also, it was indicated there were differences in congener profiles linked to the sampling method. We believe this study can inform monitoring strategies and aid microcystin-exposure risk assessments for cyanobacterial blooms.

Longitudinal, Multi-Platform Metagenomics Yields a High-Quality Genomic Catalog and Guides an In Vitro Model for Cheese Communities.

Microbiomes are intricately intertwined with human health, geochemical cycles, and food production. While many microbiomes of interest are highly complex and experimentally intractable, cheese rind microbiomes have proven to be powerful model systems for the study of microbial interactions. To provide a more comprehensive view of the genomic potential and temporal dynamics of cheese rind communities, we combined longitudinal, multi-platform metagenomics of three ripening washed-rind cheeses with whole-genome sequencing of community isolates. Sequencing-based approaches revealed a highly reproducible microbial succession in each cheese and the coexistence of closely related Psychrobacter species and enabled the prediction of plasmid and phage diversity and their host associations. In combination with culture-based approaches, we established a genomic catalog and a paired 16-member in vitro washed-rind cheese system. The combination of multi-platform metagenomic time-series data and an in vitro model provides a rich resource for further investigation of cheese rind microbiomes both computationally and experimentally. IMPORTANCE Metagenome sequencing can provide great insights into microbiome composition and function and help researchers develop testable hypotheses. Model microbiomes, such as those composed of cheese rind bacteria and fungi, allow the testing of these hypotheses in a controlled manner. Here, we first generated an extensive longitudinal metagenomic data set. This data set reveals successional dynamics, yields a phyla-spanning bacterial genomic catalog, associates mobile genetic elements with their hosts, and provides insights into functional enrichment of Psychrobacter in the cheese environment. Next, we show that members of the washed-rind cheese microbiome lend themselves to in vitro community reconstruction. This paired metagenomic data and in vitro system can thus be used as a platform for generating and testing hypotheses related to the dynamics within, and the functions associated with, cheese rind microbiomes.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecule mediated BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis spp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and their role in complex communities should continue to be a priority.

Putting microbial interactions back into community contexts.

Microbial interactions are key aspects of the biology of microbiomes. Recently, there has been a shift in the field towards studying interactions in more representative contexts, whether using multispecies model microbial communities or by looking at interactions in situ. Across diverse microbial systems, these studies have begun to identify common interaction mechanisms. These mechanisms include interactions related to toxic molecules, nutrient competition and cross-feeding, access to metals, signaling pathways, pH changes, and interactions within biofilms. Leveraging technological innovations, many of these studies have used an interdisciplinary approach combining genetic, metabolomic, imaging, and/or microfluidic techniques to gain insight into mechanisms of microbial interactions and into the impact of these interactions on microbiomes.

Pediatric Headache Experience During the COVID-19 Pandemic.

OBJECTIVE: Headache disorders are exceedingly common in children and adolescents. The association between headaches, emotional stress, and disruptions in daily routines are well established. The goal of this study is to compare the experiences of patients with a preexisting diagnosis of a primary headache disorder in terms of headache frequency and severity, lifestyle techniques for headache prevention, screen use, and mood from before and after the onset of the COVID-19 pandemic. METHODS: Patients evaluated by the Headache Clinic at Children's National Hospital between Summer 2020 and Winter 2021 were enrolled in a patient registry. Patients completed a questionnaire examining changes in headache characteristics and lifestyle factors since the onset of the COVID-19 pandemic. RESULTS: A total of 107 patients completed the survey. Since the pandemic's onset, patients reported decreased physical activity (n = 59, 55%), increased frequency of chronic headaches from 40% (N = 42) to 50% (N = 54), and increased constant daily headaches from 22% (n = 24) to 36% (n = 38). Patients reported worsened anxiety (n = 58, 54%), mood (n = 50, 47%), and workload (n = 49, 46%). Sixty-one percent (n = 65) of patients reported using screens for school for more than 6 hours per day. The majority (n = 67, 63%) of patients indicated that they would prefer attending in-person school, with 14% (n = 15) responding that they preferred online school. CONCLUSION: Since the COVID-19 pandemic's onset, pediatric headache patients have experienced increasing headache frequency, worsening anxiety and mood, decreased physical activity, and increased screen usage. Although this study is limited by sample size and observational design, future population-based studies will further elucidate the impact of this pandemic on pediatric headache.

New Daily Persistent Headache in a Pediatric Population.

INTRODUCTION: New daily persistent headache (NDPH) is a primary headache disorder characterized by an intractable, daily, and unremitting headache lasting for at least 3 months. Currently, there are limited studies in the pediatric population describing the characteristics of NDPH. OBJECTIVE: The objective of the current study is to describe the characteristics of NDPH in pediatric patients presenting to a headache program at a tertiary referral center. METHODS: The participants in the current study were pediatric patients who attended the Headache Clinic at Children's National Hospital between 2016 and 2018. All patients seen in the Headache Clinic were enrolled in an institutional review board-approved patient registry. RESULTS: Between 2016 and 2018, NDPH was diagnosed in 245 patients, representing 14% of the total headache population. NDPH patients were predominantly female (78%) and white (72%). The median age was 14.8 years. The median pain intensity was 6 of 10 (standard deviation = 1.52). Most patients reported experiencing migrainous features, namely, photophobia (85%), phonophobia (85%), and a reduced activity level (88%). Overall, 33% of patients had failed at least 1 preventive medication, and 56% had failed at least 1 abortive medication. Furthermore, 36% of patients were additionally diagnosed with medication overuse headache. CONCLUSION: NDPH is a relatively frequent disorder among pediatric chronic headache patients. The vast majority of these patients experience migrainous headache characteristics and associated symptoms and are highly refractory to treatment-as evidenced by a strong predisposition to medication overuse headache and high rates of failed preventive management.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Interaction with the justice system and other factors associated with pregnant women's self-report and continuation of use of marijuana.

BACKGROUND: Marijuana use in pregnancy is common and self-report of use to healthcare providers is variable. This study evaluates the relationship between prior interaction with the justice system as well as other factors associated with self-report and continuation of use of marijuana in pregnancy. METHODS: This retrospective cohort study included women with a positive urine toxicology test for marijuana at their initial prenatal visit. Prior interaction with the judicial system was determined by querying a publicly available database. Those who did and did not self-report use were compared using chi square and Fisher's exact test for analysis of categorical variables and t test for comparison of means. A logistic regression was performed to determine factors related to remaining positive at the time of birth. RESULTS: Of 597 women included in analysis, 308 (51.6%) self-reported use. African American women were more likely to self-report use (p = 0.02). Interaction with the justice system was not statistically predictive of self-report. Of 483 (80.9%) who had birth outcome data available, 117 (24.2%) had positive marijuana tests at the time of delivery. Women who tested positive at birth were older (27.3 v 25, p < 0.01), more likely to present later for care (p = 0.02) and more likely to have self-reported their use at their initial visit (p = 0.03). DISCUSSION: Discrepancies exist between pregnant women's self-report of marijuana use and biologic testing results. Women with positive toxicology testing who self-report use may do so due to more problematic use and may benefit from enhanced interventions.

Interaction of oxidative stress and neurotrauma in ALDH2-/- mice causes significant and persistent behavioral and pro-inflammatory effects in a tractable model of mild traumatic brain injury.

Oxidative stress induced by lipid peroxidation products (LPP) accompanies aging and has been hypothesized to exacerbate the secondary cascade in traumatic brain injury (TBI). Increased oxidative stress is a contributor to loss of neural reserve that defines the ability to maintain healthy cognitive function despite the accumulation of neuropathology. ALDH2-/- mice are unable to clear aldehyde LPP by mitochondrial aldehyde dehydrogenase-2 (Aldh2) detoxification and provide a model to study mild TBI (mTBI), therapeutic interventions, and underlying mechanisms. The ALDH2-/- mouse model presents with elevated LPP-mediated protein modification, lowered levels of PSD-95, PGC1-α, and SOD-1, and mild cognitive deficits from 4 months of age. LPP scavengers are neuroprotective in vitro and in ALDH2-/- mice restore cognitive performance. A single-hit, closed skull mTBI failed to elicit significant effects in WT mice; however, ALDH2-/- mice showed a significant inflammatory cytokine surge in the ipsilateral hemisphere 24 h post-mTBI, and increased GFAP cleavage, a biomarker for TBI. Known neuroprotective agents, were able to reverse the effects of mTBI. This new preclinical model of mTBI, incorporating significant perturbations in behavior, inflammation, and clinically relevant biomarkers, allows mechanistic study of the interaction of LPP and neurotrauma in loss of neural reserve.

BLANKA: an Algorithm for Blank Subtraction in Mass Spectrometry of Complex Biological Samples.

Multispecies microbiome systems are known to be closely linked to human, animal, and plant life processes. The growing field of metabolomics presents the opportunity to detect changes in overall metabolomic profiles of microbial species interactions. These metabolomic changes provide insight into function of metabolites as they correlate to different species presence and the observed phenotypic changes, but detection of subtle changes is often difficult in samples with complex backgrounds. Natural environments such as soil and food contain many molecules that convolute mass spectrometry-based analyses, and identification of microbial metabolites amongst environmental metabolites is an informatics problem we begin to address here. Our microbes are grown on solid or liquid cheese curd media. This medium, which is necessary for microbial growth, contains high amounts of salts, lipids, and casein breakdown products which make statistical analyses using LC-MS/MS data difficult due to the high background from the media. We have developed a simple algorithm to carry out background subtraction from microbes grown on solid or liquid cheese curd media to aid in our ability to conduct statistical analyses so that we may prioritize metabolites for further structure elucidation. Graphical Abstract .

Accuracy of tobramycin levels obtained from central venous access devices in patients with cystic fibrosis is technique dependent.

OBJECTIVE: Patients with cystic fibrosis (CF) frequently require prolonged aminoglycoside antibiotic therapy administered via central venous access devices (CVADs). Therefore, the effect of the volume of saline flush used prior to drawing blood on tobramycin levels obtained from CVADs was investigated to determine the potential for inappropriate drug dosing decisions. PATIENTS AND METHODS: This was a prospective study comparing tobramycin levels in blood samples obtained simultaneously from CVADs and from venipuncture. Two saline flushing protocols were tested. During Phase I, CVADs were flushed with 3 mL of normal saline. The saline flush volume was increased to 10 to 20 mL during Phase II. All patients had CF and ranged in age from 10 months to 20 years. RESULTS: Twenty-eight paired peak levels were collected from 19 patients during Phase I of the study, and 30 paired samples were obtained from 20 patients using the revised collection protocol during Phase II. The median (range) peak tobramycin level obtained from CVADs using the original flushing protocol was 13.1 (3.4 to 52.0) compared to 9.5 (3.1 to 46.4) microg/mL for those drawn peripherally. Use of CVAD-obtained levels would have caused inappropriate dosing changes in 43% of cases. Increasing the saline flush volume improved the accuracy of the tobramycin levels obtained from CVADs. Median peak tobramycin levels using the modified flushing protocol were 17.3 (3.6 to 47.0) and 19.0 (3.3 to 42.1) microg/mL obtained from a CVAD or venipuncture, respectively. The intraclass correlation coefficient for this protocol was 0.90 (lower 95% confidence limit = 0.80). Additionally, 87% of these CVAD-obtained levels provided accurate information for clinical decision making. CONCLUSIONS: Peak tobramycin levels drawn from CVADs flushed with a small flush volume are falsely elevated compared to those obtained by peripheral venipuncture. The use of a larger saline flush greatly improves the accuracy of these levels. Therefore, care should be taken to base clinical decisions on tobramycin levels obtained by peripheral venipuncture or following an appropriate large volume saline flush.

Changes in the genetic requirements for microbial interactions with increasing community complexity.

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. coli's genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. Genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher-order interactions. Our work provides a framework for using the model organism E. coli as a readout to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem.