A retrospective observational study of mNGS test utilization to examine the role of diagnostic stewardship at two academic medical centers.

Given the cost and unclear clinical impact of metagenomic next-generation sequencing (mNGS), laboratory stewardship may improve utilization. This retrospective observational study examines mNGS results from two academic medical centers employing different stewardship approaches. Eighty mNGS orders [54 cerebrospinal fluid (CSF) and 26 plasma] were identified from 2019 to 2021 at the University of Washington (UW), which requires director-level approval for mNGS orders, and the University of Utah (Utah), which does not restrict ordering. The impact of mNGS results and the relationship to traditional microbiology orders were evaluated. Nineteen percent (10/54) of CSF and 65% (17/26) of plasma studies detected at least one organism. Compared to CSF results, plasma results more frequently identified clinically significant organisms (31% vs 7%) and pathogens not detected by traditional methods (12% vs 0%). Antibiotic management was more frequently impacted by plasma versus CSF results (31% vs 4%). These outcome measures were not statistically different between study sites. The number and cumulative cost of traditional microbiology tests at UW were greater than Utah for CSF mNGS testing (UW: 46 tests, $6,237; Utah: 26 tests, $2,812; P < 0.05) but similar for plasma mNGS (UW: 31 tests, $3,975; Utah: 21 tests, $2,715; P = 0.14). mNGS testing accounted for 30%-50% of the total microbiology costs. Improving the diagnostic performance of mNGS by stewardship remains challenging due to low positivity rates and difficulties assessing clinical impact. From a fiscal perspective, stewardship efforts should focus on reducing testing in low-yield populations given the high costs of mNGS relative to overall microbiology testing expenditures. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) stewardship practices remain poorly standardized. This study aims to provide actionable insights for institutions that seek to reduce the unnecessary usage of mNGS. Importantly, we highlight that clinical impact remains challenging to measure without standardized guidelines, and we provide an actual cost estimate of microbiology expenditures on individuals undergoing mNGS.

A scoping review of health inequities in alcohol use disorder.

Background: Alcohol Use Disorder (AUD) poses a significant health burden on individuals. The burden occurs more frequently in the medically underserved, as well as racial and sexual minority populations. Ameliorating health inequities is vital to improving patient-centered care.Objectives: The objective of this scoping review is to chart the existing evidence on health inequities related to AUD and identify existing knowledge gaps to guide future equity-centered research.Methods: We performed a literature search using the Ovid (Embase) and MEDLINE (PubMed) databases for articles on AUD that were published in the 5-year period spanning from 2017 to 2021 and written in English. The frequencies of each health inequity examined were analyzed, and findings from each included study were summarized.Results: Our sample consisted of 55 studies for analysis. The most common inequity examined was by race/ethnicity followed by sex or gender. The least reported inequities examined were rural under-resourced areas and occupational status. Our findings indicate that significant research gaps exist in education, rural under-resourced populations, and LGBTQ+ communities with AUD.Conclusions: This scoping review highlights the gaps in research on inequities in AUD. To bridge the current gaps, we recommend research on the following: 1) triage screening tools and the use of telemedicine for rural, under-resourced populations; 2) interventions to increase treatment engagement and retention for women; and 3) community-based participatory methodologies for the LGBTQ+ communities.

Differences Between General Internal Medicine and Family Medicine Physicians' Initiation of Pre-Exposure Prophylaxis.

OBJECTIVE: Preexposure Prophylaxis (PrEP) is under-utilized in primary care. Given differences in treatment approaches for other conditions between family medicine (FM) and general internal medicine (GIM), this study compared PrEP-prescribing between FM and GIM physicians. METHODS: De-identified electronic health record data from a multi-state health care system was used in this retrospective observational study. The time period from 1/1/13 to 9/30/21 was used to identify PrEP eligible patients using measures of current sexually transmitted disease and condomless sex at the time of eligibility. Receipt of PrEP was measured in the 12 months after PrEP eligibility. The odds of receiving PrEP in GIM as compared to FM was computed before and after adjusting for demographics and physical and psychiatric comorbidities. RESULTS: The majority of eligible patients were 18 to 39 years of age, 60.9% were female and 71.6% were White race. Among PrEP eligible patients, 1.1% received PrEP in the first year after index date. Receiving PrEP was significantly more likely among patients treated in GIM versus FM (OR = 2.30; 95% CI:1.63-3.25). After adjusting for covariates, this association remained statistically significant (OR = 2.02; 95% CI:1.41-2.89). CONCLUSIONS: PrEP is grossly under-utilized in primary care. The majority of Americans enter the health care system through primary care and not through HIV providers or other specialties. Therefore, educational interventions are needed to increase confidence and knowledge and to encourage PrEP prescribing by FM and GIM physicians.

Anxiety disorders, benzodiazepine prescription, and incident dementia.

BACKGROUND: Prescribing benzodiazepines to older patients is controversial. Anxiety disorders and benzodiazepines have been associated with dementia, but literature is inconsistent. It is unknown if anxiety treated with a benzodiazepine, compared to anxiety disorder alone is associated with dementia risk. METHODS: A retrospective cohort study (n = 72,496) was conducted using electronic health data from 2014 to 2021. Entropy balancing controlled for bias by indication and other confounding factors. PARTICIPANTS: Eligible patients were ≥65 years old, had clinic encounters before and after index date and were free of dementia for 2 years prior to index date. Of the 72,496 eligible patients, 85.6% were White and 59.9% were female. Mean age was 74.1 (SD ± 7.1) years. EXPOSURE: Anxiety disorder was a composite of generalized anxiety disorder, anxiety not otherwise specified, panic disorder, and social phobia. Sustained benzodiazepine use was defined as at least two separate prescription orders in any 6-month period. MAIN OUTCOME AND MEASURES: ICD-9 or ICD-10 dementia diagnoses. RESULTS: Six percent of eligible patients had an anxiety diagnosis and 3.6% received sustained benzodiazepine prescriptions. There were 6640 (9.2%) incident dementia events. After controlling for confounders, both sustained benzodiazepine use (HR 1.28, 95% CI: 1.11-1.47) and a diagnosis of anxiety (HR 1.19, 95% CI: 1.06-1.33) were associated with incident dementia in patients aged 65-75. Anxiety disorder with sustained benzodiazepine, compared to anxiety disorder alone, was not associated with incident dementia (HR 1.18, 95% CI: 0.92-1.51) after controlling for confounding. Results were not significant when limiting the sample to those ≥75 years of age. CONCLUSIONS: Benzodiazepines and anxiety disorders are associated with increased risk for dementia. In patients with anxiety disorders, benzodiazepines were not associated with additional dementia risk. Further research is warranted to determine if benzodiazepines are associated with a reduced or increased risk for dementia compared to other anxiolytic medications in patients with anxiety disorders.

Trial Registry Searches in Plastic Surgery Systematic Reviews: A Meta-epidemiological Study.

INTRODUCTION: Clinical trial registry searches for unpublished clinical trial data are a means of mitigating publication bias within systematic reviews (SRs). The purpose of our study is to look at the rate of clinical trial registry searches conducted by SRs in the top five Plastic and Reconstructive Surgery journals. METHODS: We identified the top five plastic and reconstructive surgery journals using the Google h-5 index. We then searched Pubmed for SRs published in these journals and compared them to plastic surgery SRs published in the Cochrane Collaboration for SRs over the last 5 y. We included all SRs that were published within these top five journals and Cochrane between December 6, 2016 and December 6, 2021. We then conducted a secondary analysis on clinicaltrials.gov looking for unpublished clinical trials for 100 randomized SRs that did not conduct a clinical trial registry search. RESULTS: In SRs, 3.3% (17/512) from plastic surgery journals conducted trial registry searches. In comparison, 95.0% (38/40) of Cochrane Collaboration SRs conducted trial registry searches. Our secondary analysis found that 50% (50/100) of SRs could have included at least one unpublished clinical trial data set. CONCLUSIONS: We found that plastic surgery SRs rarely include searches for unpublished clinical trial data in clinical trial registries. To improve the data completeness of SRs in plastic surgery journals, we recommend journals alter their author guidelines to require a clinical trial registry search for unpublished literature.

Health inequities in mammography: A scoping review.

OBJECTIVE: The objective of this scoping review is to chart the existing evidence on health inequities related to mammography and identify existing knowledge gaps to guide future research. METHODS: This scoping review followed guidelines from the Joanna Briggs Institute and the PRISMA extension for scoping reviews. In July 2022, we searched PubMed and Ovid Embase for published articles on mammography screening, published between 2011 and 2021, written in English, and examining at least one health inequity as defined by the NIH. Screening and charting were both performed in a masked, duplicate manner. Frequencies of each health inequity examined were analyzed and main findings from each included study were summarized. RESULTS: Following screening, our sample consisted of 128 studies. Our findings indicate that mammography screening was less likely in historically marginalized groups, patients who live in rural areas, and in women with low income status and education level. Significant research gaps were observed regarding the LGBTQ + community and sex and gender. No trends between inequities investigated over time were identified. DISCUSSION: This scoping review highlights the gaps in inequities research regarding mammography, as well as the limited consensus across findings. To bridge existing research gaps, we recommend research into the following: 1) assessments of physician knowledge on the LGBTQ + community guidelines, 2) tools for health literacy, and 3) culturally competent screening models.

The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.

Achievement of glycemic control and antidepressant medication use in comorbid depression and type 2 diabetes.

BACKGROUND: Existing studies designed to determine if depression treatment in patients with type 2 diabetes (T2D) is associated with improved glycemic control have produced inconsistent results. The present study investigated the link between acute phase antidepressant medication treatment and achievement of glycemic control in patients with T2D using nationally distributed electronic health record data. METHODS: A retrospective cohort study (n = 7332) was conducted using nationally distributed Optum® de-identified electronic health record data from 2010 to 2018. Eligible patients were 18-64 years old and had T2D, depression, and poor glycemic control. Antidepressant medication treatment was categorized into acute phase treatment (≥12 weeks), less than acute phase (<12 weeks) or no treatment. Glycemic control was defined as HbA1c < 7.0 % (53 mmol/mol). Propensity scores (PS) and inverse probability of treatment weighting (IPTW) controlled for confounding. Extended Cox models measured the association between duration of antidepressant medication treatment and glycemic control at 0 to 36 months, 36 to 72 months and ≥72 months. RESULTS: After controlling for confounding, compared to no treatment, acute phase treatment was significantly associated with achieving glycemic control within 36 months (HR 1.17, 95 % CI 1.02-1.34). No association was observed beyond 36 months. There was no association between acute vs. less than acute phase treatment and glycemic control. LIMITATIONS: We were unable to measure decreased depression severity which could contribute to glycemic control. CONCLUSIONS: For patients with T2D and hyperglycemia, acute phase antidepressant medication may enable glycemic control. Further research is needed to establish mechanisms for this association.

Reporting of patient-reported outcomes in trials on alcohol use disorder: a meta-epidemiological study.

OBJECTIVES: Currently, limited research exists to assess the extent of patient-reported outcome (PRO) reporting among randomised controlled trials (RCTs) evaluating alcohol use disorder (AUD). We sought to investigate the completeness of reporting of PROs using the Consolidated Standards of Reporting Trials-PRO (CONSORT-PRO) extension in AUD RCTs. DESIGN SETTING: Meta-epidemiological study. METHODS: We searched MEDLINE, Embase and the Cochrane Central Register of Controlled Trials (CENTRAL) on 29 June 2021 for published RCTs focused on AUD. Following these searches, title and abstract screening, and full-text screening were performed by two investigators. To be included, a study must have employed a randomised trial design, published in English, focused on treatment of AUD and included at least one PRO. Trials meeting inclusion criteria were evaluated for completeness of reporting using the CONSORT-PRO extension adaptation. These trials were also evaluated for risk of bias (RoB) using the Cochrane RoB V.2.0 tool. Additionally, an exploratory analysis of each RCT's therapeutic area was extracted using the Mapi Research Trust's ePROVIDE platform. Screening and data collection were all performed in masked, duplicate fashion. MAIN OUTCOME MEASURES: PRO completeness of reporting, identification of factors associated with completeness of reporting and PRO measures used in RCTs to evaluate patients with AUD. RESULTS: Nineteen RCTs were evaluated in our analysis. Our primary outcome, the mean completion score for CONSORT-PRO, was 40.8%. Our secondary outcome-the identification of factors associated with completeness of reporting-found that trials published after 2014 (ie, 1 year after the publication of the CONSORT-PRO extension) were 15.0% more complete than trials published before 2014. We found no additional associations with better reporting. CONCLUSIONS: We found that the completeness of PRO reporting in RCTs involving AUD was deficient. Complete reporting of PROs is instrumental in understanding the effects of interventions, encourages patient participation in their treatment and may increase clinician confidence in the value of PROs. High quality treatment strategies for AUD require properly reported PROs.

Demographic factors, psychiatric and physical comorbidities associated with starting preexposure prophylaxis in a nationally distributed cohort.

Due to a large number of small studies and limited control for confounding, existing evidence regarding patient characteristics associated with PrEP initiation is inconsistent. We used a large electronic health record cohort to determine which demographic, physical morbidity and psychiatric conditions are associated with PrEP initiation. Eligible adult (≥18 years) patients were selected from the Optum® de-identified Electronic Health Record dataset (2010-2018). Non-HIV sexually transmitted diseases and high risk sexual behavior was used to identify patients eligible for PrEP. A fully adjusted Poisson regression model estimated the association between age, gender, race, insurance status, comorbidity index, depression, anxiety, dysthymia, severe mental illness, substance use disorder and nicotine dependence/smoking and rate of PrEP initiation. The cohort (n = 30,909) was mostly under 40 years of age (64.3%), 67.6% were female and 58.2% were White. The cumulative incidence of PrEP initiation was 1.3% (n = 408). Patients ≥60 years of age, compared to 18-29 year olds and Black compared to White patients had significantly lower rates of PrEP initiation. Anxiety disorder was significantly associated with higher rate of PrEP initiation (RR = 1.67; 95%CI:1.20-2.33) and nicotine dependence/smoking with a lower rate (RR = 0.73; 95%CI:0.54-0.97). PrEP is underutilized, and a race disparity exists in PrEP initiation. In the context of existing research, nicotine dependence/smoking is the patient characteristic most consistently associated lower rates of starting PrEP. Given the high prevalence of smoking in PrEP eligible patients, physicians may want to integrate discussions of smoking cessation in patient-provider decisions to start PrEP.

Sickle Cell Disease and Quality of Life: An Evaluation of Reporting of Patient-Reported Outcomes in Randomized Controlled Trials.

Sickle cell disease significantly impacts one's quality of life (QOL); thus, randomized controlled trials (RCTs) have integrated patient-reported outcomes (PROs) to assess patients' health from their perspective. We aim to evaluate the completeness of reporting of PROs included in sickle cell disease RCTs. We searched MEDLINE, Embase and Cochrane Central Register of Controlled Trials (CENTRAL) for published sickle cell disease RCTs with at least one PRO measure from 2006 to 2021. In a masked, duplicate fashion, two investigators evaluated RCTs using the Consolidated Standards of Reporting in Trials (CONSORT)-PRO adaptation and Cochrane Collaboration Risk of Bias (RoB) 2.0 tool. The primary objective was mean percent completeness of the CONSORT-PRO adaptation. Additional relationships between trial characteristics and completeness of reporting were evaluated. Mean completeness of reporting of RCTs was 41.49% (SD = 20.90). Randomized controlled trials with primary outcomes were more complete (57.50%, SD = 8.33) than RCTs with secondary PROs (33.48%, SD = 20.91). We did not find a significant difference in completion between trials with primary PROs and secondary PROs (t1 = 2.07; p = 0.06). Our secondary objectives included factors that may be associated with completeness of PRO reporting. Of the 12 included studies, five were considered to be overall 'high' RoB (41.67%). In each of the five domains, the majority of studies received 'low' RoB evaluations. Incomplete PRO reporting was common within sickle cell RCTs. Therefore, we recommend future RCTs including PROs should take measures to increase completeness of reporting.

Diagnosis and Treatment of Bipolar Illness in the Primary Care Office.

Bipolar Affective Disorder (BPAD) is frequently encountered in the primary care office and must be considered in the differential diagnosis of all patients with mood dysregulation. Appreciation for the range of bipolar illness has evolved in recent years, and the overlap of bipolar illness with trauma-based diagnosis such as Post-Traumatic Stress Disorder (PTSD) and Borderline Personality Disorder must be considered. Treatment of BPAD is divided into manic, depressive, and maintenance phases, each with different pharmacologic considerations. First line agents for the acute manic phase include lithium, valproic acid, and second generation antipsychotics (SGAs). First line agents for depressive phase include lamotrigine, lithium, and the SGAs lurasidone and quetiapine. For bipolar maintenance therapy, lamotrigine, valproic acid, and lithium are first line options. Finally, nonpharmacologic interventions including psychoeducation can be extremely helpful for patients and their families to successfully participate in the management of their disease.

Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile.

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or 'pdcB switch', determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

Editorial conflicts of interest related to the management of stable ischemic heart disease.

OBJECTIVE: Our study examines the association between the favorability of percutaneous coronary intervention (PCI) and/or coronary artery bypass surgery (CABG) and the presence of conflicts of interest (COIs) among authors. METHODS: We used the "Citing Articles" tool on the New England Journal of Medicine website to identify editorials on the use of PCI/CABG for stable ischemic heart disease. Authors were rated as "supportive," "neutral," or "critical" of these interventions based on the content of their editorials. COIs for each author were identified using past publications found on Scopus, PubMed, or a general internet search. RESULTS: A total of 606 articles were identified, and data were extracted from 56 of them. Among the 149 authors, 64 (43.0%) had a COI. Of these 64 authors, 19 (29.7%) disclosed their COI, while 45 (70.3%) did not. Overall, among authors with a COI, there was no association between disclosed and undisclosed COIs and the authors' view of PCI/CABG [χ2 (2, N = 64) = 1.63, p = .44]. If an author was associated with Medtronic, Abbott, or Boston Scientific, they were more likely to favor PCI/CABG if they had an undisclosed COI relative to authors who disclosed COIs [χ2 (1, N = 31) = 5.04, p = .025]. Authors publishing in a cardiology journal were more likely to view PCI/CABG favorably relative to those publishing in a general medicine journal [χ2 (2, N = 62) = 7.17, p = .028]. CONCLUSION: Editors should adopt policies to counteract the unbalancing effects that COIs have on medical opinions and evidence.

Quality of reporting among systematic reviews underpinning the ESC/ACC guidelines on ventricular arrhythmias and sudden cardiac death.

OBJECTIVES: The main objective of this study was to assess the methodological and reporting quality of the systematic reviews (SRs) supporting the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) clinical practice guidelines (CPGs) recommendations for the management of patients with ventricular arrhythmias and sudden cardiac death (SCD). As a secondary objective, we sought to determine: (1) the proportion of Cochrane SRs were cited; and (2) whether Cochrane SRs scored higher on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR-2) appraisals. DESIGN: Cross-sectional analysis. MAIN OUTCOME MEASURES: We searched for CPGs published by the ESC and the ACC from 2010 to 2020. We selected the CPGs for ventricular arrhythmias and the prevention of SCD. The reference sections were searched for SRs. Two independent investigators evaluated eligible SR using the PRISMA checklist and the AMSTAR-2 assessment tool. RESULTS: Two CPGs for ventricular arrhythmia and SCD were included in this study. Fifty-five SRs were included in our analysis. Across all SRs, the mean PRISMA score was 0.70. The lowest scoring PRISMA item related to the presence of a pre-published protocol (item 5, score 0.17). Overall, 40% of included SRs were found to have 'critically low' AMSTAR-2 ratings. One of the lowest scoring items for AMSTAR-2 was reporting of sources of funding (item 10). The 4 Cochrane SRs that were included scored higher on both assessment tools than non-Cochrane studies, specifically in PRISMA overall completion (88.7% vs 69.7%). CONCLUSION: Our study suggests the methodological and reporting quality of SRs used within ESC and ACC CPGs is insufficient, as demonstrated by the lack of adherence to both AMSTAR-2 and PRISMA checklists. Given the importance of CPGs on clinical decision making, and ultimately patient care, the methodological rigour and quality reporting within SRs used in CPGs should be held to the highest standard within the field of cardiology.

Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis.

The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the United States. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hypervirulent isolate (strain [str.] R20291) and a historic strain (str. 630), validating both with in vitro and in vivo data sets. Growth simulations revealed significant correlations with measured carbon source usage (positive predictive value [PPV] ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the pentose phosphate pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in higher-order phenotypes like bacterial pathogenesis. IMPORTANCE Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curated C. difficile GENREs for both a well-studied laboratory strain (str. 630) and a more recently characterized hypervirulent isolate (str. R20291). In silico validation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization data sets. Subsequent exploration of context-specific metabolism during both in vitro growth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differential C. difficile virulence is associated with distinct metabolic programs related to use of carbon sources and provide a platform for identification of novel therapeutic targets.

Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile.

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the "cmr switch," and expression of cmrRST is also regulated by cyclic diguanylate (c-di-GMP) through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase-locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase-locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independently of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of the cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCE Clostridioides difficile is a leading cause of hospital-acquired intestinal infections in the United States. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here, we define the complex, multilevel regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.