Melissa Starkey

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms.

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation.

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.

Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Oral Pharmacologic Treatment of Type 2 Diabetes Mellitus: A Clinical Practice Guideline From the American College of Physicians

Description The American College of Physicians (ACP) developed this guideline to present the evidence and provide clinical recommendations on oral pharmacologic treatment of type 2 diabetes in adults. This guideline serves as an update to the 2012 ACP guideline on the same topic. This guideline is endorsed by the American Academy of Family Physicians. Methods This guideline is based on a systematic review of randomized, controlled trials and observational studies published through December 2015 on the comparative effectiveness of oral medications for type 2 diabetes. Evaluated interventions included metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase-4 (DPP-4) inhibitors, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors. Study quality was assessed, data were extracted, and results were summarized qualitatively on the basis of the totality of evidence identified by using several databases. Evaluated outcomes included intermediate outcomes of hemoglobin A1c, weight, systolic blood pressure, and heart rate; all-cause mortality; cardiovascular and cerebrovascular morbidity and mortality; retinopathy, nephropathy, and neuropathy; and harms. This guideline grades the recommendations by using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. Target Audience and Patient Population The target audience for this guideline includes all clinicians, and the target patient population includes adults with type 2 diabetes. Recommendation 1 ACP recommends that clinicians prescribe metformin to patients with type 2 diabetes when pharmacologic therapy is needed to improve glycemic control. (Grade: strong recommendation; moderate-quality evidence). Recommendation 2 ACP recommends that clinicians consider adding either a sulfonylurea, a thiazolidinedione, an SGLT-2 inhibitor, or a DPP-4 inhibitor to metformin to improve glycemic control when a second oral therapy is considered. (Grade: weak recommendation; moderate-quality evidence.) ACP recommends that clinicians and patients select among medications after discussing benefits, adverse effects, and costs.

Characterization of Colony Morphology Variants Isolated from Pseudomonas aeruginosa Biofilms

ABSTRACT In this study, we report the isolation of small, rough, strongly cohesive colony morphology variants from aging Pseudomonas aeruginosa PAO1 biofilms. Similar to many of the P. aeruginosa colony morphology variants previously described in the literature, these variants autoaggregate in liquid culture and hyperadhere to solid surfaces. They also exhibit increased hydrophobicity and reduced motility compared to the wild-type parent strain. Despite the similarities in appearance of our colony morphology variant isolates on solid medium, the isolates showed a range of responses in various phenotypic assays. These variants form biofilms with significant three-dimensional structure and more biomass than the wild-type parent. To further explore the nature of the variants, their transcriptional profiles were evaluated. The variants generally showed increased expression of the psl and pel loci, which have been previously implicated in the adherence of P. aeruginosa to solid surfaces. When a mutation in the psl locus was introduced into a colony morphology variant, the colony morphology was only partially affected, but hyperadherence and autoaggregation were lost. Finally, similar colony morphology variants were found in isolates from cystic fibrosis patients. These variants displayed many of the same characteristics as the laboratory variants, suggesting a link between laboratory and cystic fibrosis biofilms.

Diagnosis of Obstructive Sleep Apnea in Adults: A Clinical Practice Guideline From the American College of Physicians

DESCRIPTION The American College of Physicians (ACP) developed this guideline to present the evidence and provide clinical recommendations on the diagnosis of obstructive sleep apnea in adults. METHODS This guideline is based on published literature on this topic that was identified by using MEDLINE (1966 through May 2013), the Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews. Searches were limited to English-language publications. The clinical outcomes evaluated for this guideline included all-cause mortality, cardiovascular mortality, nonfatal cardiovascular disease, stroke, hypertension, type 2 diabetes, postsurgical outcomes, and quality of life. Sensitivities, specificities, and likelihood ratios were also assessed as outcomes of diagnostic tests. This guideline grades the evidence and recommendations by using ACPs clinical practice guidelines grading system. RECOMMENDATION 1 ACP recommends a sleep study for patients with unexplained daytime sleepiness. (Grade: weak recommendation, low-quality evidence). RECOMMENDATION 2 ACP recommends polysomnography for diagnostic testing in patients suspected of obstructive sleep apnea. ACP recommends portable sleep monitors in patients without serious comorbidities as an alternative to polysomnography when polysomnography is not available for diagnostic testing. (Grade: weak recommendation, moderate-quality evidence).

Venous Thromboembolism Prophylaxis in Hospitalized Patients: A Clinical Practice Guideline From the American College of

DESCRIPTION The American College of Physicians (ACP) developed this guideline to present the evidence and provide clinical recommendations on prophylaxis of venous thromboembolism for hospitalized nonsurgical patients (medical patients and patients with acute stroke). METHODS This guideline is based on published literature on the topic from 1950 through April 2011 that was identified by using MEDLINE, the Cochrane Library, and reference lists of pertinent randomized trials and systematic reviews to identify additional reports. Searches were limited to randomized trials and English-language publications. The primary outcome for this guideline was total mortality up to 120 days after randomization. Secondary outcomes included symptomatic deep venous thrombosis; all pulmonary embolisms; fatal pulmonary embolism; all bleeding events; major bleeding events; and, for mechanical prophylaxis, effects on skin. This guideline grades the evidence and recommendations by using the ACPs clinical practice guidelines grading system. RECOMMENDATION 1 ACP recommends assessment of the risk for thromboembolism and bleeding in medical (including stroke) patients prior to initiation of prophylaxis of venous thromboembolism (Grade: strong recommendation, moderate-quality evidence). RECOMMENDATION 2 ACP recommends pharmacologic prophylaxis with heparin or a related drug for venous thromboembolism in medical (including stroke) patients unless the assessed risk for bleeding outweighs the likely benefits (Grade: strong recommendation, moderate-quality evidence). RECOMMENDATION 3 ACP recommends against the use of mechanical prophylaxis with graduated compression stockings for prevention of venous thromboembolism (Grade: strong recommendation, moderate-quality evidence). POLICY IMPLICATION ACP does not support the application of performance measures in medical (including stroke) patients that promotes universal venous thromboembolism prophylaxis regardless of risk.

Treatment of Anemia in Patients With Heart Disease: A Clinical Practice Guideline From the American College of Physicians

DESCRIPTION The American College of Physicians (ACP) developed this guideline to present the evidence and provide clinical recommendations on the treatment of anemia and iron deficiency in adult patients with heart disease. METHODS This guideline is based on published literature in the English language on anemia and iron deficiency from 1947 to July 2012 that was identified using MEDLINE and the Cochrane Library. Literature was reassessed in April 2013, and additional studies were included. Outcomes evaluated for this guideline included mortality; hospitalization; exercise tolerance; quality of life; and cardiovascular events (defined as myocardial infarction, congestive heart failure exacerbation, arrhythmia, or cardiac death) and harms, including hypertension, venous thromboembolic events, and ischemic cerebrovascular events. The target audience for this guideline includes all clinicians, and the target patient population is anemic or iron-deficient adult patients with heart disease. This guideline grades the evidence and recommendations using the ACPs clinical practice guidelines grading system. RECOMMENDATION 1 ACP recommends using a restrictive red blood cell transfusion strategy (trigger hemoglobin threshold of 7 to 8 g/dL compared with higher hemoglobin levels) in hospitalized patients with coronary heart disease. (Grade: weak recommendation; low-quality evidence) RECOMMENDATION 2 ACP recommends against the use of erythropoiesis-stimulating agents in patients with mild to moderate anemia and congestive heart failure or coronary heart disease. (Grade: strong recommendation; moderate-quality evidence).

Oral Pharmacologic Treatment of Type 2 Diabetes Mellitus: A Clinical Practice Guideline Update From the American College of PhysiciansOral Pharmacologic Treatment of Type 2 Diabetes Mellitus

DESCRIPTION The American College of Physicians (ACP) developed this guideline to present the evidence and provide clinical recommendations on the comparative effectiveness and safety of type 2 diabetes medications. METHODS This guideline is based on a systematic evidence review evaluating literature published on this topic from 1966 through April 2010 that was identified by using MEDLINE (updated through December 2010), EMBASE, and the Cochrane Central Register of Controlled Trials. Searches were limited to English-language publications. The clinical outcomes evaluated for this guideline included all-cause mortality, cardiovascular morbidity and mortality, cerebrovascular morbidity, neuropathy, nephropathy, and retinopathy. This guideline grades the evidence and recommendations by using the American College of Physicians clinical practice guidelines grading system. RECOMMENDATION 1: ACP recommends that clinicians add oral pharmacologic therapy in patients diagnosed with type 2 diabetes when lifestyle modifications, including diet, exercise, and weight loss, have failed to adequately improve hyperglycemia (Grade: strong recommendation; high-quality evidence). RECOMMENDATION 2: ACP recommends that clinicians prescribe monotherapy with metformin for initial pharmacologic therapy to treat most patients with type 2 diabetes (Grade: strong recommendation; high-quality evidence). RECOMMENDATION 3: ACP recommends that clinicians add a second agent to metformin to treat patients with persistent hyperglycemia when lifestyle modifications and monotherapy with metformin fail to control hyperglycemia (Grade: strong recommendation; high-quality evidence).

Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post-transcriptionally by RsmA

Extracellular polysaccharides are important components of biofilms. In non‐mucoid Pseudomonas aeruginosa strains, the Pel and Psl polysaccharides are major structural components of the biofilm matrix. In this study, we demonstrate that the alternative σ‐factor RpoS is a positive transcriptional regulator of psl gene expression. Furthermore, we show that psl mRNA has an extensive 5′ untranslated region, to which the post‐transcriptional regulator RsmA binds and represses psl translation. Our observations suggest that upon binding RsmA, the region spanning the ribosome binding site of psl mRNA folds into a secondary stem‐loop structure that blocks the Shine–Dalgarno sequence, preventing ribosome access and protein translation. This constitutes a novel mechanism for translational repression by this family of regulators.