Engineering Synthetic Antibiotics in Non-Traditional Pathways to Counter Antibiotic Resistance

Antibiotic resistance is a growing threat to global healthcare that requires immediate action to avoid the post-antibiotic era. The inherent ability of bacteria to obtain resistance and the lack of new antibiotics has led to the current antibiotic crisis. Current antibiotics are typically found through soil compound screens and only target proteins within three cellular pathways: cellular replication, cell wall biosynthesis, and protein biosynthesis. In the last decade, strains have been isolated which have resistance to nearly all available antibiotics highlighting the urgent need for intervention. In this work we investigated the rational design of non-naturally derived antibiotics which target bacterial processes outside of the three traditional antibiotic target pathways. Antisense therapeutics are nucleic acid oligomers that bind sequence-specifically via Watson-Crick base pairing with native nucleic acids and inhibit translation of the targeted gene. For this work, we use non-natural nucleic acid analog oligomers, peptide nucleic acids (PNA), for their demonstrated intracellular stability and high binding affinity for native nucleic acids. In our initial study, we show PNA oligomers targeted to TEM-1 β-lactamase re-sensitized drug-resistant Escherichia coli to a β-lactam antibiotic. We further adapted E. coli to low levels of PNA and β-lactam antibiotic and observed high variability in expression of stress response genes possibly suggesting a bet-hedging type adaptive resistance. In our next study, we designed PNA to target essential bacterial genes in non-traditional antibiotic target pathways. We designed the PNA against the genome sequences of non-pathogenic, drug sensitive E. coli, Klebsiella pneumoniae, and Salmonella enterica and subsequently tested their antibacterial action in multidrug-resistant (MDR) clinical isolates of the same three species. We found that 54% of predicted targets were effective at inhibiting the MDR pathogens demonstrating the ability to design sequence-specific yet still broad-pathogen antisense therapeutics. We further demonstrated that combinations of these essential gene antisense PNA and small molecule antibiotics function synergistically to enhance bacterial inhibition despite the clinical strains high antibiotic resistance. We next focused our efforts on designing an antimicrobial agent for perturbing bacterial redox homeostasis. Reactive oxygen species (ROS) have been studied for their effect on antibiotic efficacy and the emergence of drug resistance. In this work, we studied one ROS in particular, superoxide, for its role as an oxidative stress catalyst and its demonstrated disruption of metal homeostasis in bacteria. To controllably produce superoxide, we investigated the design of quantum dot nanoparticles. When quantum dots are excited over their nominal bandgap, excited electrons and holes are available for redox half reactions in the biological environment. In this work, we demonstrated the tuning of quantum dots for superoxide production from molecular oxygen and further showed the tuned nanoparticles inhibition of clinical isolates. Further, we established that E. coli could be eradicated from co-culture with mammalian cells; leaving the mammalian cells intact. Given the role that ROS have been shown to have in bactericidal antibiotic efficacy, we hypothesized that our superoxide-producing nanoparticles would function synergistically with small molecule antibiotics. Indeed, our designed superoxide generating nanoparticles potentiated the activity of antibiotics in clinical MDR isolates in spite of their antibiotic resistance. In this study, superoxide potentiated the activity of bactericidal antibiotics as well as bacteriostatic antibiotics, which had not been shown previously. To better understand the effect of superoxide generation on bacteria, we performed analysis of E. coli’s transcriptome during treatment. We removed mat

Engineering Synthetic Antibiotics in Non-Traditional Pathways to Counter Antibiotic Resistance

Antibiotic resistance is a growing threat to global healthcare that requires immediate action to avoid the post-antibiotic era. The inherent ability of bacteria to obtain resistance and the lack of new antibiotics has led to the current antibiotic crisis. Current antibiotics are typically found through soil compound screens and only target proteins within three cellular pathways: cellular replication, cell wall biosynthesis, and protein biosynthesis. In the last decade, strains have been isolated which have resistance to nearly all available antibiotics highlighting the urgent need for intervention. In this work we investigated the rational design of non-naturally derived antibiotics which target bacterial processes outside of the three traditional antibiotic target pathways. Antisense therapeutics are nucleic acid oligomers that bind sequence-specifically via Watson-Crick base pairing with native nucleic acids and inhibit translation of the targeted gene. For this work, we use non-natural nucleic acid analog oligomers, peptide nucleic acids (PNA), for their demonstrated intracellular stability and high binding affinity for native nucleic acids. In our initial study, we show PNA oligomers targeted to TEM-1 β-lactamase re-sensitized drug-resistant Escherichia coli to a β-lactam antibiotic. We further adapted E. coli to low levels of PNA and β-lactam antibiotic and observed high variability in expression of stress response genes possibly suggesting a bet-hedging type adaptive resistance. In our next study, we designed PNA to target essential bacterial genes in non-traditional antibiotic target pathways. We designed the PNA against the genome sequences of non-pathogenic, drug sensitive E. coli, Klebsiella pneumoniae, and Salmonella enterica and subsequently tested their antibacterial action in multidrug-resistant (MDR) clinical isolates of the same three species. We found that 54% of predicted targets were effective at inhibiting the MDR pathogens demonstrating the ability to design sequence-specific yet still broad-pathogen antisense therapeutics. We further demonstrated that combinations of these essential gene antisense PNA and small molecule antibiotics function synergistically to enhance bacterial inhibition despite the clinical strains high antibiotic resistance. We next focused our efforts on designing an antimicrobial agent for perturbing bacterial redox homeostasis. Reactive oxygen species (ROS) have been studied for their effect on antibiotic efficacy and the emergence of drug resistance. In this work, we studied one ROS in particular, superoxide, for its role as an oxidative stress catalyst and its demonstrated disruption of metal homeostasis in bacteria. To controllably produce superoxide, we investigated the design of quantum dot nanoparticles. When quantum dots are excited over their nominal bandgap, excited electrons and holes are available for redox half reactions in the biological environment. In this work, we demonstrated the tuning of quantum dots for superoxide production from molecular oxygen and further showed the tuned nanoparticles inhibition of clinical isolates. Further, we established that E. coli could be eradicated from co-culture with mammalian cells; leaving the mammalian cells intact. Given the role that ROS have been shown to have in bactericidal antibiotic efficacy, we hypothesized that our superoxide-producing nanoparticles would function synergistically with small molecule antibiotics. Indeed, our designed superoxide generating nanoparticles potentiated the activity of antibiotics in clinical MDR isolates in spite of their antibiotic resistance. In this study, superoxide potentiated the activity of bactericidal antibiotics as well as bacteriostatic antibiotics, which had not been shown previously. To better understand the effect of superoxide generation on bacteria, we performed analysis of E. coli’s transcripto

“Dear Diary, I’m Feeling...”: Within- Country Diversity in Guatemalan Adolescents’ Gratitude and Hassles

Gratitude is associated with subjective well-being in adolescents while hassles or everyday irritants likely detract from well-being. In Guatemala, a country with one of the highest rates of economic inequality in Latin America, adolescents’ experiences with gratitude and hassles may differ depending on socioeconomic status (World Bank, 2015). In the current qualitative study, we analyzed the within-country diversity of Guatemalan adolescents’ gratitude and hassles. The sample (N = 80) included data from two Guatemalan schools as part of a larger study. The first school served underprivileged youth (n = 37, Mage = 14.35, SD = 1.11, 48.6% girls), and the second served a middle-class population (n = 43, Mage = 15.77, SD = 1.15, 51.2% girls). Participants were randomly assigned to write about either their daily “gratitude” or “hassles” for 10 days. Journal responses were coded using thematic analysis (based on Braun & Clarke, 2006). Regarding gratitude, participants from both samples displayed appreciation for relationships with others and the support linked with those friendships, family members, and teachers, among others. However, hassles results showed distinct patterns between the samples. Students from the middle class school were often hassled by less severe hassles (e.g., waking up early) or by enduring the betrayal of false friends. Adolescents with few resources frequently mentioned feeling left out, ignored, and disrespected. Furthermore, distinct from the higher resource group, students with fewer resources mentioned feeling upset that close others experienced unkindness. Both groups shared negative experiences with peers, but the adolescents with more resources frequently reported indirect or relation aggression (e.g., gossiping, fakeness), versus the direct forms of unkindness (e.g., teasing) experienced by the adolescents with fewer resources. Such implications should be considered by school administrators to assess the needs of their students, and construct sample-specific interventions to foster gratitude and reduce hassles, thus promoting well-being

Prevalence of, and Resident and Facility Characteristics Associated With Antipsychotic Use in Assisted Living vs. Long-Term Care Facilities: A Cross-Sectional Analysis from Alberta, Canada

BACKGROUND: Potentially inappropriate antipsychotic use in long-term care (LTC) facilities has been the focus of significant policy and clinical attention over the past 20 years. However, most initiatives aimed at reducing the use of these medications have overlooked assisted living (AL) settings. OBJECTIVE: We sought to compare the prevalence of antipsychotic use (including potentially inappropriate use) among older AL and LTC residents and to explore the resident and facility-level factors associated with use in these two populations. METHODS: We performed cross-sectional analyses of 1089 residents (mean age 85 years; 77% female) from 59 AL facilities and 1000 residents (mean age 85 years; 66% female) from 54 LTC facilities, in Alberta, Canada. Research nurses completed comprehensive resident assessments at baseline (2006-2007). Facility-level factors were assessed using standardized administrator interviews. Generalized linear models were used to estimate odds ratios for associations, accounting for clustering by facility. RESULTS: Over a quarter of residents in AL (26.4%) and LTC (31.8%) were using antipsychotics (p = 0.006). Prevalence of potentially inappropriate use was similar in AL and LTC (23.4 vs. 26.8%, p = 0.09). However, among users, the proportion of antipsychotic use deemed potentially inappropriate was significantly higher in AL than LTC (AL: 231/287 = 80.5%; LTC: 224/318 = 70.4%; p = 0.004). In both settings, comparable findings regarding associations between resident characteristics (including dementia, psychiatric disorders, frailty, behavioral symptoms, and antidepressant use) and antipsychotic use were observed. Few facility characteristics were associated with overall antipsychotic use, but having a pharmacist on staff (AL), or an affiliated physician (LTC) was associated with a lower likelihood of potentially inappropriate antipsychotic use. CONCLUSION: Our findings illustrate the importance of including AL settings in clinical and policy initiatives aimed at reducing inappropriate antipsychotic use among older vulnerable residents

Cognitive Reserve and Mild Cognitive Impairment

Background and Objectives Little is known about the effect of education or other indicators of cognitive reserve on the rate of reversion from mild cognitive impairment (MCI) to normal cognition (NC) or the relative rate (RR) of reversion from MCI to NC vs progression from MCI to dementia. Our objectives were to (1) estimate transition rates from MCI to NC and dementia and (2) determine the effect of age, APOE, and indicators of cognitive reserve on the RR of reversion vs progression using multistate Markov modeling. Methods We estimated instantaneous transition rates between NC, MCI, and dementia after accounting for transition to death across up to 12 assessments in the Nun Study, a cohort study of religious sisters aged 75+ years. We estimated RRs of reversion vs progression for age, APOE, and potential cognitive reserve indicators: education, academic performance (high school grades), and written language skills (idea density, grammatical complexity). Results Of the 619 participants, 472 were assessed with MCI during the study period. Of these 472, 143 (30.3%) experienced at least one reverse transition to NC, and 120 of the 143 (83.9%) never developed dementia (mean follow-up = 8.6 years). In models adjusted for age group and APOE, higher levels of education more than doubled the RR ratio of reversion vs progression. Novel cognitive reserve indicators were significantly associated with a higher adjusted RR of reversion vs progression (higher vs lower levels for English grades: RR ratio = 1.83; idea density: RR ratio = 3.93; and grammatical complexity: RR ratio = 5.78). Discussion Knowledge of frequent reversion from MCI to NC may alleviate concerns of inevitable cognitive decline in those with MCI. Identification of characteristics predicting the rate of reversion from MCI to NC vs progression from MCI to dementia may guide population-level interventions targeting these characteristics to prevent or postpone MCI and dementia. Research on cognitive trajectories would benefit from incorporating predictors of reverse transitions and competing events, such as death, into statistical modeling. These results may inform the design and interpretation of MCI clinical trials, given that a substantial proportion of participants may experience improvement without intervention

Depression rating scales for detection of major depression in people with dementia

This is the protocol for a review and there is no abstract. The objectives are as follows: To identify the accuracy of depression rating scales as screening tools for detecting DpD and compare the diagnostic accuracy of different depression rating scales for detecting MDD among adults with Alzheimer's disease and related forms of dementia. To examine factors that may impact on the accuracy of depression rating scales that are used to diagnose depression. We will examine the reference standard used for verification of DpD, baseline prevalence of DpD in the study population, age of the underlying study population, gender of participants, type of dementia (any-cause dementia versus Alzheimer’s disease), study setting (community or primary care setting, long-term care, tertiary care setting), and study country as potential sources of heterogeneity. We will also evaluate the effects of using different cut-points of individual depression rating scales on the diagnostic accuracy of the scales

Utilizing population-based clinical and administrative data to explore the relevance of frailty to cholinesterase inhibitor use and discontinuation at nursing home transition.

Introduction Cholinesterase inhibitors (ChEIs) are medications used to treat cognitive symptoms associated with Alzheimer’s disease. Previous studies examining the determinants of continued use or withdrawal of ChEIs during the transition into a nursing home have lacked detailed clinical information needed to understand the range of factors associated with pharmacotherapeutic decision-making. Objectives and Approach Population-based clinical and administrative health databases were linked to examine patterns of ChEI use among 47,851 adults (aged 66+) with dementia newly admitted to nursing homes in Ontario between April 2011-March 2015. We examined whether resident frailty, among other factors, was associated with ChEI discontinuation in the following year. Frailty was calculated using a validated 72-item index derived from the Resident Assessment Instrument (RAI-MDS 2.0). Discontinuation was defined as a 30-day period when no dispensations occurred and no supply of ChEI was available. Subdistribution hazard models estimated the association between resident characteristics and discontinuation, accounting for competing risk of death. Results Over one-third (36.7%) of residents were receiving a ChEI at admission and this proportion was lower among those defined as frail (33.6%) vs. non-frail (40.7%) at admission. Among those on a ChEI at admission, 82.3% continued use and 17.7% discontinued during the following year. After accounting for resident characteristics, ChEI type and previous use, the incidence of discontinuation was 15% higher in frail residents vs. non-frail residents (hazard ratio (HR)= 1.15, 95\% confidence interval (CI) [1.01,1.30]). Residents with severe aggressive behaviours (HR=1.82, 95% CI [1.60, 2.07]), and higher levels of cognitive impairment (HR=1.29, 95% CI [1.10, 1.51]) were more likely to discontinue. Residents aged 85+ (HR=0.69, 95% CI [0.61, 0.77]) and those who were widowed (HR=0.84, 95% CI [0.77, 0.91]) were less likely to discontinue. Conclusion/Implications Most residents who entered on a ChEI continued treatment during follow-up. The availability of linked clinical and administrative data allowed for a novel exploration of predictors of ChEI discontinuation. Frailty, severity of cognitive impairment and aggressive behaviours were associated with ChEI discontinuation; whereas selected sociodemographic factors predicted continued use