Multicenter evaluation of the Selux Next-Generation Phenotyping antimicrobial susceptibility testing system.

The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.

Findings From the Step Up, Test Up Study of an Electronic Screening and Brief Intervention for Alcohol Misuse in Adolescents and Young Adults Presenting for HIV Testing: Randomized Controlled Efficacy Trial.

BACKGROUND: Substance use, particularly binge drinking of alcohol and noninjection substance use, is associated with increased risk for HIV infection among youth, but structured substance use screening and brief intervention are not often provided as part of HIV risk reduction. OBJECTIVE: The purpose of the study was to test the efficacy of a fully automated electronic screening and brief intervention, called Step Up, Test Up, to reduce alcohol misuse among adolescents and young adults presenting for HIV testing. Secondary objectives were reduction in sexual risk and uptake of pre-exposure prophylaxis (PrEP) for HIV prevention. METHODS: Youth aged 16 years to 25 years who presented for HIV testing at community-based locations were recruited for study participation. Those who screened at moderate to high risk on the Alcohol Use Disorders Identification Test were randomized (1:1) to either an electronic brief intervention or a time-attention control. The primary outcome was change in alcohol use at 1, 3, 6, and 12-month follow-ups. Negative binomial and log binomial regression analyses with generalized estimating equations were conducted to evaluate the intervention efficacy. RESULTS: Among a sample of 329 youth, there were no significant differences in alcohol use outcomes between conditions over time or at the 1, 3, 6, or 12-month time points. In terms of secondary outcomes, there was evidence of reduction in condomless insertive anal sex under the influence of alcohol and drugs at 12 months compared with 3 months in the intervention versus the attention control condition (incidence rate ratio=0.15, 95% CI 0.05-0.44); however, there were no other significant differences in sexual risk and no difference in PrEP engagement. CONCLUSIONS: We found no effect of electronic brief intervention to reduce alcohol use and some effect on sexual risk among youth aged 16 years to 25 years who present for HIV testing. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02703116; https://clinicaltrials.gov/ct2/show/NCT02703116. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s12889-020-8154-6.

A New Stage of the Caregiving Career: Informal Caregiving After Long-term Institutionalization.

BACKGROUND AND OBJECTIVES: The "unexpected career" of caregiving has previously been conceptualized in stages: community care through institutional placement/residence, ending with death of the care recipient. Transition programs such as Money Follows the Person (MFP) created a new stage of the caregiving career, caring for someone post-long-term institutionalization, about which little is known. Using Pearlin's Caregiver Stress Process Model, this study explores effects on caregivers from the return of their loved ones to the community after a long-term institutional stay. RESEARCH DESIGN AND METHODS: Cross-sectional surveys of 656 caregivers of persons transitioned through Connecticut's MFP program 2014-2018, completed 6 months posttransition. RESULTS: Regardless of the age/disability of the care recipient, and despite experiencing high caregiving intensity, caregivers experienced less burden, anxiety, and depression, and higher benefits of caregiving than demonstrated in literature for the general caregiving population. Most felt less stressed than before and during the participant's institutional stay. Factors associated with worse outcomes included worry about safety, strained finances, missing work, and desiring additional services. Black and Hispanic caregivers experienced lower burden and anxiety and higher benefits of caregiving than White caregivers. DISCUSSION AND IMPLICATIONS: By providing community supports to participants, transition programs can have broad ancillary benefits for caregivers and improve outcomes in the Pearlin model, lessening potentially deleterious effects of an unexpected return to intensive caregiving duties after institutional placement. Positive results for Black and Hispanic caregivers may reflect cultural expectations in caring for family that buffer the adverse effects of caregiving.

Predictors of Internet Health Information-Seeking Behaviors Among Young Adults Living With HIV Across the United States: Longitudinal Observational Study.

BACKGROUND: Consistent with young adults' penchant for digital communication, young adults living with HIV use digital communication media to seek out health information. Understanding the types of health information sought online and the characteristics of these information-seeking young adults is vital when designing digital health interventions for them. OBJECTIVE: This study aims to describe characteristics of young adults living with HIV who seek health information through the internet. Results will be relevant to digital health interventions and patient education. METHODS: Young adults with HIV (aged 18-34 years) self-reported internet use during an evaluation of digital HIV care interventions across 10 demonstration projects in the United States (N=716). Lasso (least absolute shrinkage and selection operator) models were used to select characteristics that predicted whether participants reported seeking general health and sexual and reproductive health (SRH) information on the internet during the past 6 months. RESULTS: Almost a third (211/716, 29.5%) and a fifth (155/716, 21.6%) of participants reported searching for general health and SRH information, respectively; 26.7% (36/135) of transgender young adults with HIV searched for gender-affirming care topics. Areas under the curve (>0.70) indicated success in building models to predict internet health information seeking. Consistent with prior studies, higher education and income predicted health information seeking. Higher self-reported antiretroviral therapy adherence, substance use, and not reporting transgender gender identity also predicted health information seeking. Reporting a sexual orientation other than gay, lesbian, bisexual, or straight predicted SRH information seeking. CONCLUSIONS: Young adults living with HIV commonly seek both general health and SRH information online, particularly those exploring their sexual identity. Providers should discuss the most commonly sought SRH topics and the use of digital technology and be open to discussing information found online to better assist young adults with HIV in finding accurate information. Characteristics associated with health information-seeking behavior may also be used to develop and tailor digital health interventions for these young adults.

A taxonomy of pragmatic measures of HIV preexposure prophylaxis use.

OBJECTIVES: As delivery of preexposure prophylaxis (PrEP) becomes an HIV prevention priority in the United States, standard, pragmatic measures of PrEP use are needed to compare and evaluate prevention implementation programs. By using readily available electronic health record data, we describe and compare measures of persistence and retention. DESIGN: Retrospective cohort. METHODS: Using electronic health record prescription data for patients at a large urban Federally Qualified Health Center from 2015 to 2019, we calculated measures of persistence and retention and compared them to pharmacy claims data, PrEP biomarkers, and HIV outcomes. RESULTS: Total PrEP time was 19.8 months on average. During this period, average adherence by medication prescription ratio (MRxR) was 89%; 77% of patients had an MRxR at least 85% and 90% have an MRxR at least 57%. Over the first 6 months, average proportion of days covered (PDC) at least 85% was 53% and PDC at least 57% was 57%. Prescription fill rates, based on claims data from a pharmacy partner, ranged from 45 to 60%. Using tenofovir-diphosphate as the gold standard, PDC had high sensitivity (97%) but low specificity (≤13%). As a measure of retention, over the first 6 months, 59% of patients had quarterly HIV tests. CONCLUSION: Total PrEP time is useful measure of overall persistence, while PDC can assess persistence and adherence at a specific time point. Adherence by PDC is more conservative compared with MRxR; both will overestimate true adherence. Retention in care can be measured by quarterly HIV tests. Using consistent terminology and reporting timepoints and adherence thresholds will help reporting and comparing PrEP delivery programs.

An Observational Study of Hypertension and Thromboembolism Among Transgender Patients Using Gender-Affirming Hormone Therapy.

Purpose: Given evidence from cisgender patients that sex hormones can impact risk for some forms of cardiovascular disease (CVD), there are concerns regarding CVD among transgender patients using gender-affirming hormone therapy (HT). Methods: Using a retrospective cohort at a U.S. urban federally qualified health center (FQHC) focused on sexual and gender minority health, we examined associations between HT in transgender patients and two specific CVD outcomes, hypertension (HTN) and thromboembolism (TE). We assessed outcomes by ICD-10 codes in electronic medical records (EMR) of 4402 transgender patients. Hormone use was assessed both by blood concentrations and by prescriptions, from EMR. Results: Nineteen transwomen (TW) (0.8%) had a TE and 49 (2.1%) developed HTN; among transmen (TM), 27 (1.5%) developed HTN and there were no significant associations between hormones and HTN. Among transwomen, there was no association between TE and HT as assessed by blood concentrations. However, recent progestin prescriptions were associated with an increased odds of TE (adjusted odds ratio [aOR] 2.95 [95% confidence interval; CI 1.02-8.57]), with possibly differential effects for medroxyprogesterone acetate versus micronized progesterone. Higher total testosterone blood concentrations were associated with greater odds of HTN in TW (aOR 1.16 [95% CI 1.01-1.33]), after controlling for body mass index. Among TW, ever having a progestin prescription was protective for HTN (aOR 0.36 [95% CI 0.15-0.87]). Conclusion: We found no associations between HT and HTN among TM, More research is needed to examine the effect of recent progestin, specifically medroxyprogesterone acetate, on TE among transwomen. The protective association between progestins and HTN among TW is reassuring.

Challenges to community transitions through Money Follows the Person.

OBJECTIVE: To examine the effects of transition challenges on the success and timeliness of transitions from institutions to community living for long-stay participants in the Money Follows the Person (MFP) Rebalancing Demonstration and determine whether outcomes vary by age and disability. DATA SOURCE: Secondary data on transition challenges for individuals enrolled in Connecticut's MFP program between December 2008 and December 2017. STUDY DESIGN: Challenges were analyzed for older adults, people with mental health disability, and people with physical disability. Bivariate and multivariate analyses investigated which transition challenges and selected demographic variables predict transition versus closure and length of transition period for each group. DATA EXTRACTION METHODS: The sample includes 3506 persons who attempted transition from institutions to community living and whose case concluded with transition or closure from 2015 to 2017. PRINCIPAL FINDINGS: The association between most transition challenges and the ability of long-stay institutional residents to return to the community, and to do so in a timely manner, varies significantly among older adults and younger persons with physical or mental health disabilities. For all groups, however, consumer engagement challenges predicted closure without transition (OR: 1.3-3.9) and housing challenges predicted longer transition periods (84-132 days). Length of institutional stay was associated with both outcomes for older adults and persons with physical disability. Other challenges, such as issues with services and supports, differed among the three groups on both outcomes. CONCLUSIONS: Knowledge of the effects of transition challenges on success and timeliness of transition for each group allows program managers and health and service providers to focus resources on addressing the most serious challenges. Particular emphasis should be placed on consumer engagement and housing challenges, and on targeting persons for transition early in their institutional stay. Federal and state transition programs can benefit by individualizing supports for residents to yield successful outcomes.

Correlations of HIV Preexposure Prophylaxis Indications and Uptake, Chicago, Illinois, 2015-2018.

Objectives. To examine gaps in identification of preexposure prophylaxis (PrEP) candidates, uptake, and use of PrEP by populations most likely to seroconvert.Methods. At a federally qualified health center in Chicago, Illinois, we used electronic medical records, prescription data, and our best approximation of Centers for Disease Control and Prevention PrEP guidelines to determine how many patients were indicated for PrEP relative to HIV diagnoses (indication:HIV), how many were on PrEP relative to indications (PrEP:indication), and how many were on PrEP relative to HIV diagnoses (PrEP:HIV). We compared these ratios across age, gender and orientation, race/ethnicity, and insurance.Results. Overall, there were 32 indications per incident diagnosis and 16 patients on PrEP per incident diagnosis. In adjusted models, Whites had higher indication:HIV and PrEP:HIV ratios compared with Blacks, men who have sex with men had higher indication:HIV and PrEP:HIV ratios compared with transwomen but lower PrEP:indication ratios, and uninsured patients had higher indication:HIV but lower PrEP:indication and PrEP:HIV ratios compared with those with insurance.Conclusions. PrEP use, relative to HIV diagnoses, differs by important patient characteristics. While improved guidelines will address some of the disparity, better approaches for determining PrEP candidates and more normalized patient-provider communication are needed to ensure better PrEP access to all individuals at high risk for HIV.

Efficacy of Computer-Based Telephone Counseling on Long-Term Adherence to Strength Training in Elderly Patients With Knee Osteoarthritis: A Randomized Trial.

OBJECTIVE: To determine whether the Boston Overcoming Osteoarthritis through Strength Training Telephone-Linked Communication (BOOST-TLC) program, a novel telephone-based, motivational, strength-training exercise-adherence counseling intervention, improved adherence to a strength-training exercise program over 2 years. METHODS: Participants were recruited for this 2-year, single-blind, parallel-arm randomized controlled trial from knee osteoarthritis patient registries, community newspapers, and online websites in Massachusetts. Eligibility criteria included age 50 years or older, painful knee osteoarthritis, and ability to use a telephone. Exclusion criteria included medical conditions precluding exercise, inflammatory arthritis, current regular strength training, planned knee replacement surgery, dementia, inability to follow exercise instructions, and inability to use the TLC system. After participating in a group exercise class, participants were randomized to receiving motivational telephone calls through the BOOST-TLC program for 24 months or the control. Both control and intervention participants received a monthly automated phone message reminder to continue the program. Exercise adherence was ascertained by a single self-report item scored 0-10, where 10 represented complete adherence. Outcomes were evaluated at 6, 12, 18, and 24 months. RESULTS: A total of 104 subjects were randomized, and 89 subjects (44 control, 45 TLC) completed the 24-month follow-up. There was no significant difference in adherence at 24 months between groups (mean for control group 4.01 [95% confidence interval (95% CI) 3.03, 4.99], mean for TLC subjects 3.63 [95% CI 2.70, 4.56]; P = 0.57). CONCLUSION: In those with knee osteoarthritis who had participated in an exercise program, frequent motivational telephone reminders did not increase adherence to strength-training exercise.

Exercise adherence: beliefs of adults with knee osteoarthritis over 2 years.

Exercise is an established treatment to alleviate pain and improve function among adults with knee osteoarthritis (KOA). However, long-term adherence to exercise is poor and effective approaches to support adherence are limited. Here, we report on an ancillary study to a randomized controlled trial (RCT) where the primary outcome was 2-year adherence to a home based strength-training program. The aims of this current study were to (i) explore experiences, feelings, and perspectives related to long-term adherence to exercise among adults with painful KOA participating in a 2-year RCT, and (ii) identify factors that influenced long-term adherence to exercise. Methods: We purposively recruited 25 subjects and conducted in-depth interviews at the 2-year RCT assessment. In the RCT participants completed a 6-week group exercise program followed by automated telephone calls. Findings: Three conceptual categories describing beliefs about exercise were identified: (1) monitoring; (2) knowledge of how to manage their exercise behaviors; and (3) benefits of exercise. Monitoring provided by peers and instructors during group exercise, and telephone technology were valued by participants. Participants who reported low adherence expressed ambivalence about the benefits of exercise and a desire for more social support. Those who reported high adherence exhibited self-determination and self-efficacy. Conclusions: A novel finding is the conceptual link of self-determination to high adherence to strength-training exercises over 2 years among adults with KOA. Implications for physical therapists include identifying patients' autonomy, competence, and relatedness needs to foster intrinsic control for exercise behavior.

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) Escherichia coli ST131 and Klebsiella pneumoniae ST258 led to identification of two subclasses of α-peptide/ß-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against E. coli, and clindamycin against K. pneumoniae. The study of six peptidomimetics was extended to Pseudomonas aeruginosa, confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-ßNPhe)8-NH2, exerted only a minor effect on the viability of mammalian cells (EC50 ≥ 124-210 µM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 µM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators.

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.

Quality Primary Care and Family Planning Services for LGBT Clients: A Comprehensive Review of Clinical Guidelines.

LGBT clients have unique healthcare needs but experience a wide range of quality in the care that they receive. This study provides a summary of clinical guideline recommendations related to the provision of primary care and family planning services for LGBT clients. In addition, we identify gaps in current guidelines, and inform future recommendations and guidance for clinical practice and research. PubMed, Cochrane, and Agency for Healthcare Research and Quality electronic bibliographic databases, and relevant professional organizations' websites, were searched to identify clinical guidelines related to the provision of primary care and family planning services for LGBT clients. Information obtained from a technical expert panel was used to inform the review. Clinical guidelines meeting the inclusion criteria were assessed to determine their alignment with Institute of Medicine (IOM) standards for the development of clinical practice guidelines and content relevant to the identified themes. The search parameters identified 2,006 clinical practice guidelines. Seventeen clinical guidelines met the inclusion criteria. Two of the guidelines met all eight IOM criteria. However, many recommendations were consistent regarding provision of services to LGBT clients within the following themes: clinic environment, provider cultural sensitivity and awareness, communication, confidentiality, coordination of care, general clinical principles, mental health considerations, and reproductive health. Guidelines for the primary and family planning care of LGBT clients are evolving. The themes identified in this review may guide professional organizations during guideline development, clinicians when providing care, and researchers conducting LGBT-related studies.

Providing quality family planning services to LGBTQIA individuals: a systematic review.

OBJECTIVE: Lesbian, gay, bisexual, transgender, queer/questioning, intersex and asexual (LGBTQIA) individuals have unique sexual and reproductive health needs; however, facilitators and barriers to optimal care are largely understudied. The objective of this study was to synthesize findings from a systematic review of the literature regarding the provision of quality family planning services to LGBTQIA clients to inform clinical and research strategies. STUDY DESIGN: Sixteen electronic bibliographic databases (e.g., PubMed, PSYCinfo) were searched to identify articles published from January 1985 to April 2016 relevant to the analytic framework. RESULTS: The search parameters identified 7193 abstracts; 19 descriptive studies met inclusion criteria. No studies assessed the impact of an intervention serving LGBTQIA clients on client experience, behavior or health outcomes. Two included studies focused on the perspectives of health care providers towards LGBTQIA clients. Of the 17 studies that documented client perspectives, 12 elucidated factors facilitating a client's ability to enter into care, and 13 examined client experience during care. Facilitators to care included access to a welcoming environment, clinicians knowledgeable about LGBTQIA needs and medical confidentiality. CONCLUSIONS: This systematic review found a paucity of evidence on provision of quality family planning services to LGBTQIA clients. However, multiple contextual facilitators and barriers to family planning service provision were identified. Further research is needed to assess interventions designed to assist LGBTQIA clients in clinical settings, and to gain a better understanding of effective education for providers, needs of specific subgroups (e.g., asexual individuals) and the role of the client's partner during receipt of care.

Detecting functional change in response to exercise in knee osteoarthritis: a comparison of two computerized adaptive tests.

BACKGROUND: The intent of this study was to examine and compare the ability to detect change of two patient reported outcome (PRO) instruments that use a computerized adaptive test (CAT) approach to measurement. The Patient Reported Outcomes Measurement Information System (PROMIS®) Physical Function scale is a generic PRO, while the Osteoarthritis Computerized Adaptive Test (OA-CAT) is an osteoarthritis-specific PRO. METHODS: This descriptive, longitudinal study was conducted in a community setting, involving individuals from the greater Boston area. INCLUSION CRITERIA: age > 50, self-reported doctor-diagnosed knee osteoarthritis (OA) and knee pain. The PROMIS® Physical Function CAT and OA-CAT Functional Difficulty scale were administered at baseline and at the conclusion of a 6-week exercise program. Effect sizes (ES) were calculated for both measures, and bootstrap methods were used to construct confidence intervals and to test for significant ES differences between the measures. RESULTS: The OA-CAT Functional Difficulty scale achieved an ES of 0.62 (0.43, 0.87) compared to the PROMIS® Physical Function CAT ES of 0.42 (0.24, 0.63). ES estimates for the two CAT measures were not statistically different. CONCLUSIONS: The condition-specific OA-CAT and generic PROMIS® Physical Function CAT both demonstrated the ability to detect change in function. While the OA-CAT scale showed larger effect size, no statistically significant difference was found in the effect size estimates for the generic and condition-specific CATs. Both CATs have potential for use in arthritis research. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov on 6/21/11 (Identifier NCT01394874 ).

Subgrouping and TargetEd Exercise pRogrammes for knee and hip OsteoArthritis (STEER OA): a systematic review update and individual participant data meta-analysis protocol.

INTRODUCTION: Knee and hip osteoarthritis (OA) is a leading cause of disability worldwide. Therapeutic exercise is a recommended core treatment for people with knee and hip OA, however, the observed effect sizes for reducing pain and improving physical function are small to moderate. This may be due to insufficient targeting of exercise to subgroups of people who are most likely to respond and/or suboptimal content of exercise programmes. This study aims to identify: (1) subgroups of people with knee and hip OA that do/do not respond to therapeutic exercise and to different types of exercise and (2) mediators of the effect of therapeutic exercise for reducing pain and improving physical function. This will enable optimal targeting and refining the content of future exercise interventions. METHODS AND ANALYSIS: Systematic review and individual participant data meta-analyses. A previous comprehensive systematic review will be updated to identify randomised controlled trials that compare the effects of therapeutic exercise for people with knee and hip OA on pain and physical function to a non-exercise control. Lead authors of eligible trials will be invited to share individual participant data. Trial-level and participant-level characteristics (for baseline variables and outcomes) of included studies will be summarised. Meta-analyses will use a two-stage approach, where effect estimates are obtained for each trial and then synthesised using a random effects model (to account for heterogeneity). All analyses will be on an intention-to-treat principle and all summary meta-analysis estimates will be reported as standardised mean differences with 95% CI. ETHICS AND DISSEMINATION: Research ethical or governance approval is exempt as no new data are being collected and no identifiable participant information will be shared. Findings will be disseminated via national and international conferences, publication in peer-reviewed journals and summaries posted on websites accessed by the public and clinicians. PROSPERO REGISTRATION NUMBER: CRD42017054049.

Macromolecule Biosynthesis Assay and Fluorescence Spectroscopy Methods to Explore Antimicrobial Peptide Mode(s) of Action.

Antimicrobial peptides (AMPs) are viable alternatives to the currently available antimicrobials, and numerous studies have investigated their possible use as therapeutic agents for specific clinical applications. AMPs are a diverse class of antimicrobials that often act upon the bacterial cell membrane but may exhibit additional modes of action. Identification of the multiple modes of action requires a comprehensive study at subinhibitory concentrations and careful data analysis since additional modes of action can be eclipsed by AMP action on the cell membrane.Techniques that measure the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods to identify AMP mode(s) of action. Detailed protocols designed to measure inhibition of DNA, RNA, protein, and cell wall synthesis or membrane de-energization are presented and discussed for optimal application of these two techniques as well as to enable accurate interpretation of the experimental findings.

Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of ß-Lactamase-Producing Escherichia coli.

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ß-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.