Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.

Diversity, equity, and inclusion (DEI) in medical education: DEI at the bedside.

BACKGROUND: The ability to recognize and address bias is an important communication skill not typically addressed during training. We describe the design of an educational curriculum that aims to identify and change behavior related to diversity, equity, and inclusion (DEI). "DEI at the Bedside" uses the existing infrastructure of bedside teaching and provides a tool to normalize DEI discussions and develop skills to address bias during a neurology inpatient rotation. METHODS: As part of traditional clinical rounds, team members on an inpatient service shared experiences with DEI topics, including bias. The team developed potential responses should they encounter a similar situation in the future. We report the results of our needs assessment and curriculum development to evaluate the feasibility of incorporating a DEI educational curriculum in the neurology inpatient setting. RESULTS: Forty-two DEI experiences were recorded. Medical students were the most frequent discussants (44%). Direction of bias occurred between healthcare team members (33%), against patients (31%), and patients against healthcare team members (28%). Experiences ranged from microaggressions to explicit comments of racism, sexism, and homophobia. CONCLUSIONS: Based on needs assessment data, we developed a DEI educational curriculum for the inpatient neurology setting aimed to improve knowledge and skills related to DEI topics as well as to normalize conversation of DEI in the clinical setting. Additional study will demonstrate whether this initiative translates into measurable and sustained improvement in knowledge of how bias and disparity show up in the clinical setting and behavioral intent to discuss and address them.

In-Hospital Methadone Enrollment: a Novel Program to Facilitate Linkage from the Hospital to the Opioid Treatment Program for Vulnerable Patients with Opioid Use Disorder.

INTRODUCTION: Methadone ameliorates opioid withdrawal among hospitalized patients with opioid use disorder (OUD). To continue methadone after hospital discharge, patients must enroll in an opioid treatment program (OTP) per federal regulations. Uncontrolled opioid withdrawal is a barrier to linkage from hospital to OTP. AIM: Describe a federally compliant In-Hospital Methadone Enrollment Team (IN-MEET) that enrolls hospitalized patients with OUD into an OTP with facilitated hospital to OTP linkage. SETTING: Seven hundred-bed university hospital in Aurora, CO. PROGRAM DESCRIPTION: A physician dually affiliated with a hospital's addiction consultation service and a community OTP completes an in-hospital, face-to-face medical assessment required by federal law and titrates methadone to comfort. An OTP-affiliated nurse with hospital privileges completes a psychosocial evaluation and provides case management by arranging transportation and providing weekly telephone check-ins. PROGRAM EVALUATION METRICS: IN-MEET enrollments completed, hospital to OTP linkage, and descriptive characteristics of patients who completed IN-MEET enrollments compared to patients who completed community OTP enrollments. RESULTS: Between April 2019 and April 2023, our team completed 165 IN-MEET enrollments. Among a subset of 73 IN-MEET patients, 56 (76.7%) presented to the OTP following hospital discharge. Compared to community OTP enrolled patients (n = 1687), a higher percentage of IN-MEET patients were older (39.7 years, standard deviation [SD] 11.2 years vs. 36.1 years, SD 10.6 years) and were unhoused (n = 43, 58.9% vs. n = 199, 11.8%). Compared to community OTP enrolled patients, a higher percentage of IN-MEET patients reported heroin or fentanyl as their primary substance (n = 53, 72.6% vs. n = 677, 40.1%), reported methamphetamine as their secondary substance (n = 27, 37.0% vs. n = 380, 22.5%), and reported they injected their primary substance (n = 46, 63.0% vs. n = 478, 28.3%). CONCLUSION: IN-MEET facilitates hospital to OTP linkage among a vulnerable population. This model has the potential to improve methadone access for hospitalized patients who may not otherwise seek out treatment.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Development of an Approach to Assessing Pediatric Fellows' Transport Medical Control Skills.

BACKGROUND AND OBJECTIVE: Pediatric interfacility transport teams facilitate access to subspecialty care, and physicians often guide management remotely as transport medical control (TMC). Pediatric subspecialty fellows frequently perform TMC duties, but tools assessing competency are lacking. Our objective was to develop content validity for the items required to assess pediatric subspecialty fellows' TMC skills. METHODS: We conducted a modified Delphi process among transport and fellow education experts in pediatric critical care medicine, pediatric emergency medicine, neonatal-perinatal medicine, and pediatric hospital medicine. The study team generated an initial list of items on the basis of a literature review and personal experience. A modified Delphi panel of transport experts was recruited to participate in 3 rounds of anonymous, online voting on the importance of the items using a 3-point Likert scale (marginal, important, essential). We defined consensus for inclusion as ≥80% agreement that an item was important/essential and consensus for exclusion as ≥80% agreement that an item was marginal. RESULTS: The study team of 20 faculty drafted an initial list of items. Ten additional experts in each subspecialty served on the modified Delphi panel. Thirty-six items met the criteria for inclusion, with widespread agreement across subspecialties. Only 1 item, "discussed bed availability," met the criteria for inclusion among some subspecialties but not others. The study team consolidated the final list into 26 items for ease of use. CONCLUSIONS: Through a consensus-based process among transport experts, we generated content validity for the items required to assess pediatric subspecialty fellows' TMC skills.

Staphylococcus Aureus Swabbing and Decolonization Before Neuromodulation Procedures: A Systematic Review and Meta-Analysis.

INTRODUCTION: Staphylococcus aureus (S aureus) is the foremost bacterial cause of surgical-site infection (SSI) and is a common source of neuromodulation SSI. Endogenous colonization is an independent risk factor for SSI; however, this risk has been shown to diminish with screening and decolonization. MATERIALS AND METHODS: A systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines using the PubMed, Cochrane Library, and Embase data bases from inception to January 1, 2022, for the purposes of identifying all studies reporting on the use of S aureus swabbing and/or decolonization before neuromodulation procedures. A random-effects meta-analysis was performed using the metaphor package in R to calculate odds ratios (OR). RESULTS: Five observational cohort studies were included after applying the inclusion and exclusion criteria. The average study duration was 6.6 ± 3.8 years. Three studies included nasal screening as a prerequisite for subsequent decolonization. Type of neuromodulation included spinal cord stimulation in two studies, deep brain stimulation in two studies, intrathecal baclofen in one study, and sacral neuromodulation in one study. Overall, 860 and 1054 patients were included in a control or intervention (ie, screening and/or decolonization) group, respectively. A combination of nasal mupirocin ointment and a body wash, most commonly chlorhexidine gluconate soap, was used to decolonize throughout. Overall infection rates were observed at 59 of 860 (6.86%) and ten of 1054 (0.95%) in the control and intervention groups, respectively. Four studies reported a significant difference. The OR for intervention (screen and/or decolonization) vs no intervention was 0.19 (95% CI, 0.09-0.37; p < 0.001). Heterogeneity between studies was nonsignificant (I2 = 0.43%, τ2 = 0.00). CONCLUSIONS: Preoperative S aureus swabbing and decolonization resulted in significantly decreased odds of infection in neuromodulation procedures. This measure may represent a worthwhile tool to reduce neuromodulation SSI, warranting further investigation.

Potential risk modifiers for severe intraventricular hemorrhage in very low birthweight infants requiring transport.

BACKGROUND: Very low birthweight (VLBW) infants must undergo transport when born at a facility unequipped for their care. Previous research suggests an increased risk for intraventricular hemorrhage (IVH) associated with transport. It is unknown whether logistical aspects of transport, particularly mode and distance, or skill level of the resuscitation team are drivers of risk. OBJECTIVE: To determine if the transport vehicle, distance traveled, or absence of advanced resuscitation team increased risk for severe IVH in outborn VLBW infants. DESIGN/METHODS: Outborn VLBW infants, transported by specialized team via helicopter or ambulance to a Level IV NICU, were included; inborn VLBW infants served as controls. Infants transported >24 h after birth, by referring center's team, or without head ultrasound were excluded. Baseline clinical data were collected along with IVH grade, transport vehicle, distance traveled, and skill of resuscitation team. RESULTS: Two hundred and ninety-three outborn were matched to 293 inborn infants. Outborn infants had increased incidence of severe IVH even when controlling for antenatal steroids, race, delivery method, and surfactant use (17% vs. 11%, OR = 1.6, 95% CI = 1.1-2.7). Despite this increased incidence, severe IVH was not associated with transport vehicle (p = .90; OR = 0.76, 95% CI = 0.34-1.7), distance traveled (p = .13; OR 0.84, 95% CI = 0.60-1.2), or skill of resuscitation team (p = .18; OR = 0.49, 95% CI = 0.21-1.1). CONCLUSION: Compared to inborn, outborn infants had increased risk of severe IVH. Transport vehicle, distance traveled, and the skill of resuscitation team did not significantly impact risk.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Draft Genome Sequence of the Redox-Active Enteric Bacterium Citrobacter portucalensis Strain MBL.

We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid.

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

Implementation of Telemedicine in Pediatric and Neonatal Transport.

OBJECTIVE: Telemedicine uses video technology to communicate visual clinical information. This study aimed to implement telemedicine in pediatric and neonatal transport, assess its value, and identify barriers. METHODS: This prospective study implemented telemedicine before transport to a tertiary care children's hospital. A preimplementation survey assessed attitudes toward telemedicine and perceived barriers. During the 12-week pilot, a video connection was initiated between transport and medical control. We collected survey results measuring telemedicine usefulness and hindrance after each use. A postimplementation survey assessed opinions about when telemedicine was useful. RESULTS: Initially, 82% of users had no direct experience with telemedicine. Perceived utility and burden of telemedicine varied significantly by department. During the study, telemedicine was offered 65% of the time, initiated in 47% of cases, and successful in 30% of cases. The greatest barrier was connectivity. Over time, transport members and physicians found telemedicine to be significantly more useful. In 14 cases, telemedicine changed patient outcome or management. Providers who reported a change in management rated telemedicine as significantly more useful. CONCLUSION: This prospective pilot successfully implemented telemedicine before pediatric transport. Telemedicine was more useful in patients with visual findings on examination and, in some cases, changed the clinical outcome.

Addressing medically underserved populations through maternal-fetal transport: a geographic analysis.

Objective: Despite regionalization of maternal care in the USA, there is little contemporary information on characteristics and utilization of maternal-fetal transport. We used geographic analysis to investigate referral and transportation patterns of the maternal-fetal transport service at our institution.Methods: This is a retrospective cohort study of all calls taken by our maternal-fetal transfer service. Call logs were abstracted, and diagnoses, gestational ages, referring hospital, and mode of transportation were analyzed. The United States Health Resources and Services Administration's Medically Underserved Area (MUA) designations were used to identify hospitals in these areas. Geographic information system software was used to analyze and map geographic variables, including frequency of transfers from each hospital, distance traveled, mode of transfer, and MUAs.Results: From November 2012 to March 2017, there were 835 telephone consults without transfer and 1682 patients transferred from 103 hospitals. Preterm labor was the most common diagnosis (n = 338, 20%), followed by hypertensive disorders (n = 231, 14%). There were 738 transfers (44%) from MUAs, and 20 (19%) of hospitals were critical access hospitals, accounting for 121 (7%) transfers. One-way trips from a referring hospital accounted for 659 patients, and the transport team was dispatched in 1023 cases. The median distance traveled was 24 mi. For hospitals within 50 mi, the mean difference in transport time between air and ground was <1 h, yet there were 73 air round trips for hospitals <50 mi from our hospital.Conclusion: The transfer system is robust and supports underserved hospitals; however, the service could be deployed more efficiently.

Beyond "Read More": An Intervention to Improve Faculty Written Feedback to Learners.

BACKGROUND: High-quality feedback is necessary for learners' development. It is most effective when focused on behavior and should also provide learners with specific next steps and desired outcomes. Many faculty struggle to provide this high-quality feedback. OBJECTIVE: To improve the quality of written feedback by faculty in a department of medicine, we conducted a 1-hour session using a novel framework based on education literature, individual review of previously written feedback, and deliberate practice in writing comments. METHODS: Sessions were conducted between August 2015 and June 2018. Participants were faculty members who teach medical students, residents, and/or fellows. To measure the effects of our intervention, we surveyed participants and used an a priori coding scheme to determine how feedback comments changed after the session. RESULTS: Faculty from 7 divisions participated (n = 157). We surveyed 139 participants postsession and 55 (40%) responded. Fifty-three participants (96%) reported learning new information. To more thoroughly assess behavioral changes, we analyzed 5976 feedback comments for students, residents, and fellows written by 22 randomly selected participants before the session and compared these to 5653 comments written by the same participants 1 to 12 months postsession. Analysis demonstrated improved feedback content; comments providing nonspecific next steps decreased, and comments providing specific next steps, reasons why, and outcomes increased. CONCLUSIONS: Combining the learning of a simple feedback framework with an immediate review of written comments that individual faculty members previously provided learners led to measured improvement in written comments.

Extracellular Electron Transfer Transcends Microbe-Mineral Interactions.

Extracellular electron transfer (EET) allows microbes to drive their metabolism through interactions with minerals or electrodes. In recent work, Light et al. (2018) discover a specialized EET pathway in Listeria monocytogenes with homologs in pathogens and gut commensals, suggesting that EET plays important roles in diverse environments.

A Systematic, Intensive Statistical Investigation of Data from the Comprehensive Analysis of Reported Drugs (CARD) for Compliance and Illicit Opioid Abstinence in Substance Addiction Treatment with Buprenorphine/naloxone.

BACKGROUND: Buprenorphine and naloxone (bup/nal), a combination partial mu receptor agonist and low-dose delta mu antagonist, is presently recommended and used to treat opioid-use disorder. However, a literature review revealed a paucity of research involving data from urine drug tests that looked at compliance and abstinence in one sample. METHOD: Statistical analysis of data from the Comprehensive Analysis of Reported Drugs (CARD) was used to assess compliance and abstinence during treatment in a large cohort of bup/nal patients attending chemical-dependency programs from eastern USA in 2010 and 2011. RESULTS: Part 1: Bup/nal was present in 93.4% of first (n = 1,282; p <.0001) and 92.4% of last (n = 1,268; p <.0001) urine samples. Concomitantly, unreported illicit drugs were present in 47.7% (n = 655, p =.0261) of samples. Patients who were compliant to the bup/nal prescription were more likely than noncompliant patients to be abstinent during treatment (p =.0012; odds ratio = 1.69 with 95% confidence interval (1.210, 2.354). Part 2: An analysis of all samples collected in 2011 revealed a significant improvement in both compliance (p < 2.2 × 10-16) and abstinence (p < 2.2 × 10-16) during treatment. Conclusion/Importance: While significant use of illicit opioids during treatment with bup/nal is present, improvements in abstinence and high compliance during maintenance-assisted therapy programs may ameliorate fears of diversion in comprehensive programs. Expanded clinical datasets, the treatment modality, location, and year of sampling are important covariates, for further studies. The potential for long-term antireward effects from bup/nal use requires consideration in future investigations.

The Colorful World of Extracellular Electron Shuttles.

Descriptions of the changeable, striking colors associated with secreted natural products date back well over a century. These molecules can serve as extracellular electron shuttles (EESs) that permit microbes to access substrates at a distance. In this review, we argue that the colorful world of EESs has been too long neglected. Rather than simply serving as a diagnostic attribute of a particular microbial strain, redox-active natural products likely play fundamental, underappreciated roles in the biology of their producers, particularly those that inhabit biofilms. Here, we describe the chemical diversity and potential distribution of EES producers and users, discuss the costs associated with their biosynthesis, and critically evaluate strategies for their economical usage. We hope this review will inspire efforts to identify and explore the importance of EES cycling by a wide range of microorganisms so that their contributions to shaping microbial communities can be better assessed and exploited.

Polar Intermetallics Pr5 Co2 Ge3 and Pr7 Co2 Ge4 with Planar Hydrocarbon-Like Metal Clusters.

Planar hydrocarbon-like metal clusters may foster new insights linking organic molecules with conjugated π-π bonding interactions and inorganic structures in terms of their bonding characteristics. However, such clusters are uncommon in polar intermetallics. Herein, we report two polar intermetallic phases, Pr5 Co2 Ge3 and Pr7 Co2 Ge4 , both of which feature such planar metal clusters, namely, ethylene-like [Co2 Ge4 ] clusters plus the concatenated forms and polyacene-like [Co2 Ge2 ]n ribbons in Pr5 Co2 Ge3 , and 1,2,4,5-tetramethylbenzene-like [Co4 Ge6 ] cluster in Pr7 Co2 Ge4 . Just as in the related planar organic structures, these metal-metalloid species are dominated by covalent bonding interactions. Both compounds magnetically order at low temperature with net ferromagnetic components: Pr5 Co2 Ge3 through a series of transitions below 150 K and Pr7 Co2 Ge4 through a single ferromagnetic transition at 19 K. Spin-polarized electronic structure calculations for Pr7 Co2 Ge4 reveal strong spin-orbit coupling within Pr and considerable magnetic contributions from Co atoms. This work suggests that similar structural chemistry can emerge for other rare-earth/late-transition-metal/main-group systems.

Enzymatic Degradation of Phenazines Can Generate Energy and Protect Sensitive Organisms from Toxicity.

UNLABELLED: Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed. IMPORTANCE: Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines.

Systematic evaluation of "compliance" to prescribed treatment medications and "abstinence" from psychoactive drug abuse in chemical dependence programs: data from the comprehensive analysis of reported drugs.

This is the first quantitative analysis of data from urine drug tests for compliance to treatment medications and abstinence from drug abuse across "levels of care" in six eastern states of America. Comprehensive Analysis of Reported Drugs (CARD) data was used in this post-hoc retrospective observational study from 10,570 patients, filtered to include a total of 2,919 patients prescribed at least one treatment medication during 2010 and 2011. The first and last urine samples (5,838 specimens) were analyzed; compliance to treatment medications and abstinence from drugs of abuse supported treatment effectiveness for many. Compared to non-compliant patients, compliant patients were marginally less likely to abuse opioids, cannabinoids, and ethanol during treatment although more likely to abuse benzodiazepines. Almost 17% of the non-abstinent patients used benzodiazepines, 15% used opiates, and 10% used cocaine during treatment. Compliance was significantly higher in residential than in the non-residential treatment facilities. Independent of level of care, 67.2% of the patients (n = 1963; P<.001) had every treatment medication found in both first and last urine specimens (compliance). In addition, 39.2% of the patients (n = 1143; P<.001) had no substance of abuse detected in either the first or last urine samples (abstinence). Moreover, in 2010, 16.9% of the patients (n = 57) were abstinent at first but not at last urine (deteriorating abstinence), the percentage dropped to 13.3% (n = 174) in 2011; this improvement over years was statistically significant. A longitudinal analysis for abstinence and compliance was studied in a randomized subset from 2011, (n = 511) representing 17.5% of the total cohort. A statistically significant upward trend (p = 2.353×10-8) of abstinence rates as well as a similar but stronger trend for compliance ((p = 2.200×10-16) was found. Being cognizant of the trend toward drug urine testing being linked to medical necessity eliminating abusive screening, the interpretation of these valuable results require further intensive investigation.