Harnessing the Diversity of Burkholderia spp. Prophages for Therapeutic Potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here, we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested phages were tested for lytic activity against the same 32 isolates. Temperate phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that Burkholdera cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Pituitary Adenomas as a Barometer for Health Care Access.

Introduction Expanding access to care has been shown to impact patient care and disease epidemiology for different disease states, but has not been studied in pituitary adenoma. We hypothesize that increasing access to care-which includes diagnostics-through the Affordable Care Act (ACA) and Medicaid expansion has increased identification of pituitary adenomas. Methods The National Cancer Institute's Surveillance, Epidemiology, and End Results database was utilized to identify patients with pituitary adenomas from 2007-to 2016 yielding 39,120 cases. Demographic, histologic, and insurance data were extracted. After stratification based on their insurance status, they were plotted to examine trends in insurance status after introduction of the ACA and Medicaid expansion. Magnetic resonance imaging (MRI) data was gathered from the Organization for Economic Co-operation and Development. A linear regression model was developed to describe the relationship between pituitary adenoma discovery and the number of MRI exams. Results Pituitary adenoma diagnoses (37.6%) and MRI examinations per 1,000 in the U.S. (32.3%) increased concurrently from 2007 to 2016. Linear regression analysis revealed a statistically significant relationship ( p = 0.0004). Those patients without insurance diagnosed with pituitary adenomas decreased 36.8% after Medicaid expansion ( p = 0.023). With respect to Medicaid utilization, significant increases of 28.5% ( p = 0.014) and 30.3% ( p = 0.00096) were noted after both the ACA enactment and Medicaid expansion, respectively. Conclusion The ACA has expanded health care access which has increased the ability to identify patients with pituitary adenomas. The present study also provides evidence that access to care is important for less prevalent diseases such as pituitary adenomas.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

Impact of Insurer on Papillary Thyroid Carcinoma Outcomes, Stage, and Treatment Modalities.

BACKGROUND: While the impact of insurance has been described for thyroid cancer as a whole, we sought to further characterize this relationship for the papillary sub-group (PTC). METHODS: Those patients with primary site thyroid tumors from 2007 to 2016 with histology-confirmed PTC were extracted from the SEER database. These parameters yielded 103 219 participants for demographic, extent of disease, and treatment parameter study and 103 025 for outcome studies. RESULTS: Compared to their counterparts, those with Medicaid were more likely to have stage T3 or greater (<.0001) disease at presentation. Those with Medicare/private insurance were more likely to have No staging at diagnosis (P < .0001). Similarly, those with Medicaid exhibited poorer overall (98.0%, 90.9%, 81.6% vs 98.9%, 95.0%, 90.0%; P < .0001) and cause-specific (99.3%, 98.0%, 95.8% vs 99.7%, 99.1%, 98.4%; P < .0001) survival after 1, 5, and 9 years respectively. CONCLUSION: Insurer has a significant impact on the stage at diagnosis of papillary thyroid carcinoma while having limited effect on the treatment modalities offered. Statistically significant overall and cause-specific mortality differences were appreciated but are likely clinical insignificant. Further work to elucidate the social factors likely affecting these patients is warranted.

Pituitary Carcinoma Diagnosis and Survival Improvement, with Affordable Care Act Correlation: A SEER Database Study.

Introduction Pituitary carcinomas are challenging tumors to diagnose and treat due to their rarity and limited data surrounding their etiology. Traditionally, these patients have exhibited poor survival. Over the last several decades, our understanding of pituitary carcinomas has dramatically increased, and there have been recent initiatives to improve patient access to health care, including the Affordable Care Act (ACA). This study investigates whether there were any changes in incidence and treatment outcomes of pituitary carcinoma that correlated with these advances. Methods A retrospective case review was conducted utilizing the Surveillance, Epidemiology, and End Results (SEER) database of the National Cancer Institute. Those with primary site pituitary tumors with noncontiguous metastases were identified from 1975 to 2016. Demographic data, overall, and cause-specific outcomes were obtained. The data were analyzed using SPSS to generate 5-year Kaplan-Meier curves. Results The incidence of pituitary carcinoma pre- and post-ACA was 0.31 and 2.14 diagnoses/year, respectively. This represents a significant increase (Chi-square, p < 0.00002). In addition, 1-, 2-, and 5-year overall survival of these patients was determined to be 88.2, 74.0, and 66.6% which was significantly improved compared with prior studies. Cause-specific survival of these patients follow similar trends exhibiting 94.1, 79.0, 71.1% after 1, 2, and 5 years, respectively. Conclusion The survival for pituitary carcinoma has improved significantly which signals a change in how practitioners should counsel their patients. There is a significant surge in the number of cases in the post-ACA timeline, which suggests that improving patient access has played a part in wider recognition and treatment initiation for this disease.

The Role of Vitamin D as a Prognostic Marker in Papillary Thyroid Cancer.

The role of vitamin D in modulating several cancer-related pathways has received an increasing amount of attention in the past several years. Previous literature has found an abundance of evidence of vitamin D exerting an anti-proliferative, anti-inflammatory, and pro-differentiation effect in various types of cancers including breast, colon, prostate, and pancreatic cancer. Although the link between vitamin D and thyroid cancer remains controversial, both biochemical evidence and clinical studies have attempted to establish a link between papillary thyroid carcinoma (PTC) and vitamin D status. Furthermore, the use of vitamin D as a prognostic marker has received increased attention, both in regards to clinical outcomes and cancer staging. In this review, we briefly discuss the metabolism and proposed mechanism of action of vitamin D in the context of PTC, and explore links between modulators in the vitamin D pathway and progression of PTC. We provide evidence from both clinical studies as well as molecular studies of metabolic targets, including vitamin D receptor and activating enzymes exerting an effect on PTC tissue, which indicate that vitamin D may play a significant prognostic role in PTC.

Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections.

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract, L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infection took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, to further investigate the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure, we hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. To test this hypothesis, respiratory activity in anaerobically grown L. monocytogenes was stimulated by exogenous fumarate and subsequent intracellular pathogenesis was assessed. The results showed that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of respiratory activity in L. monocytogenes in modulating the outcome of subsequent intracellular infections.

Listeria monocytogenes Response to Propionate Is Differentially Modulated by Anaerobicity.

Propionate is a common food preservative and one of the major fermentation acids in the intestines. Therefore, exposure to propionate is frequent for foodborne pathogens and likely takes place under suboxic conditions. However, it is not clear whether the absence of oxygen affects how pathogens respond to propionate. Here, we investigated how propionate exposure affects Listeria monocytogenes growth and virulence factor production under aerobic or anaerobic conditions and showed that oxygen indeed plays a key role in modulating L. monocytogenes response to propionate. Under aerobic conditions, propionate supplementations had no effect on planktonic growth but resulted in decreased adherent growth. Under anaerobic conditions, propionate supplementations resulted in a pH-dependent inhibition of planktonic growth and increased adherent growth. Cultures grown with propionate accumulated higher levels of acetoin under aerobic conditions but lower levels of ethanol under both aerobic and anaerobic conditions. Metabolic perturbations by propionate were also evident by the increase in straight chain fatty acids. Finally, propionate supplementations resulted in increased listeriolyin O (LLO) production under anaerobic conditions but decreased LLO production under aerobic conditions. These results demonstrate for the first time that the presence or absence of oxygen plays a critical role in shaping L. monocytogenes responses to propionate.

On the retrieval of crystallographic information from atom probe microscopy data via signal mapping from the detector coordinate space.

Atom probe tomography is a powerful microscopy technique capable of reconstructing the 3D position and chemical identity of millions of atoms within engineering materials, at the atomic level. Crystallographic information contained within the data is particularly valuable for the purposes of reconstruction calibration and grain boundary analysis. Typically, analysing this data is a manual, time-consuming and error prone process. In many cases, the crystallographic signal is so weak that it is difficult to detect at all. In this study, a new automated signal processing methodology is demonstrated. We use the affine properties of the detector coordinate space, or the 'detector stack', as the basis for our calculations. The methodological framework and the visualisation tools are shown to be superior to the standard method of crystallographic pole visualisation directly from field evaporation images and there is no requirement for iterations between a full real-space initial tomographic reconstruction and the detector stack. The mapping approaches are demonstrated for aluminium, tungsten, magnesium and molybdenum. Implications for reconstruction calibration, accuracy of crystallographic measurements, reliability and repeatability are discussed.

Metabolic determinants in Listeria monocytogenes anaerobic listeriolysin O production.

Listeria monocytogenes is a human pathogen and a facultative anaerobe. To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures-a phenotype observed in wild type and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA. To explore potential regulatory mechanisms, we established that the addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.

An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse.

BACKGROUND: We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). RESULTS: We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. CONCLUSIONS: Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET(+/P)) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET(+/+)) mice, whereas transport activity in mice carrying two A457P alleles (NET(P/P)) was nearly abolished. NET(+/P) and NET(P/P) mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET(+/P) mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET(+/P) mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

Investing in HIV services while building Kenya's health system: PEPFAR's support to prevent mother-to-child HIV transmission.

Trade-offs may exist between investments to promote health system strengthening, such as investments in facilities and training, and the rapid scale-up of HIV/AIDS services. We analyzed trends in expenditures to support the prevention of mother-to-child transmission of HIV in Kenya under the President's Emergency Plan for AIDS Relief (PEPFAR) from 2005 to 2010. We examined how expenditures changed over time, considering health system strengthening alongside direct treatment of patients. We focused on two organizations carrying out contracts under PEPFAR: the Elizabeth Glaser Pediatric AIDS Foundation and FHI360 (formerly Family Health International), a nonprofit health and development organization. We found that the average unit expenditure, or the spending on goods and services per mother living with HIV who was provided with antiretroviral drugs, declined by 52 percent, from $567 to $271, during this time period. The unit expenditure per mother-to-infant transmission averted declined by 66 percent, from $7,117 to $2,440. Meanwhile, the health system strengthening proportion of unit expenditure increased from 12 percent to 33 percent during the same time period. The analysis suggests that PEPFAR investments in prevention of mother-to-child transmission of HIV in Kenya became more efficient over time, and that there was no strong evidence of a trade-off between scaling up services and investing in health systems.