Optimization of the antimicrobial peptide Bac7 by deep mutational scanning.

BACKGROUND: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiotics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacterial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. RESULTS: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline-rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. CONCLUSION: We demonstrated that the presented platform can be used to establish the sequence-activity relationship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicrobial drug candidates.

High-Throughput Optimization of Recombinant Protein Production in Microfluidic Gel Beads.

Efficient production hosts are a key requirement for bringing biopharmaceutical and biotechnological innovations to the market. In this work, a truly universal high-throughput platform for optimization of microbial protein production is described. Using droplet microfluidics, large genetic libraries of strains are encapsulated into biocompatible gel beads that are engineered to selectively retain any protein of interest. Bead-retained products are then fluorescently labeled and strains with superior production titers are isolated using flow cytometry. The broad applicability of the platform is demonstrated by successfully culturing several industrially relevant bacterial and yeast strains and detecting peptides or proteins of interest that are secreted or released from the cell via autolysis. Lastly, the platform is applied to optimize cutinase secretion in Komagataella phaffii (Pichia pastoris) and a strain with 5.7-fold improvement is isolated. The platform permits the analysis of >106 genotypes per day and is readily applicable to any protein that can be equipped with a His6 -tag. It is envisioned that the platform will be useful for large screening campaigns that aim to identify improved hosts for large-scale production of biotechnologically relevant proteins, thereby accelerating the costly and time-consuming process of strain engineering.

Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale.

The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.

Parallel Sampling of Nanoliter Droplet Arrays for Noninvasive Protein Analysis in Discrete Yeast Cultivations by MALDI-MS.

Miniaturization of cell-based assays enables the analysis of secreted compounds from low cell numbers down to a single cell. Droplet microfluidics is a well-established tool for high-throughput single-cell analysis. Nevertheless, the integration of label-free bioanalytical techniques like mass spectrometry is still ongoing. For example, without additional separation steps, droplet-enclosed cells do not survive the analysis. Cell separation techniques for droplets have been reported, but could not yet be coupled to mass spectrometric analysis. Here, we present a simple approach for high-throughput cell separation in parallel in nanoliter droplets and demonstrate that it can be used for qualitative analysis of protein secretion by the yeast Komagataella phaffii. Using a custom-made droplet spotter, we generated an array of 200 droplets of nanoliter volumes on a glass plate, each containing approximately 500 cells. After cultivation for 24 h, a second plate was placed above the droplet array and brought in contact with the droplets. All droplets were sampled in parallel by plate-based droplet splitting. The nanoliter samples of the supernatant could be interfaced with mass spectrometry and we were able to detect the protein brazzein (his-tagged, 7445 Da) in all but two droplets. Additionally, we show that the cells were viable after the cell separation and a sample from one spot could be transferred to a cultivation tube. An advantage of our protocol is that each cell suspension is directly linked to the analysis result by its position. Furthermore, we demonstrate that our method is capable of splitting around 6000 droplets in a few seconds. In the future, additional processing steps on a small scale, such as desalting and protein digestion, could be developed and will enable structural proteomics in nanoliter volumes.

Early Infectious Diseases Specialty Intervention Is Associated With Shorter Hospital Stays and Lower Readmission Rates: A Retrospective Cohort Study.

Background: Intervention by infectious diseases (ID) physicians improves outcomes for inpatients in Medicare, but patients with other insurance types could fare differently. We assessed whether ID involvement leads to better outcomes among privately insured patients under age 65 years hospitalized with common infections. Methods: We performed a retrospective analysis of administrative claims data from community hospital and postdischarge ambulatory care. Patients were privately insured individuals less than 65 years old with an acute-care stay in 2014 for selected infections, classed as having early (by day 3) or late (after day 3) ID intervention, or none. Key outcomes were mortality, cost, length of the index stay, readmission rate, mortality, and total cost of care over the first 30 days after discharge. Results: Patients managed with early ID involvement had shorter length of stay, lower spending, and lower mortality in the index stay than those patients managed without ID involvement. Relative to late, early ID involvement was associated with shorter length of stay and lower cost. Individuals with early ID intervention during hospitalization had fewer readmissions and lower healthcare payments after discharge. Relative to late, those with early ID intervention experienced lower readmission, lower spending, and lower mortality. Conclusions: Among privately insured patients less than 65 years old, treated in a hospital, early intervention with an ID physician was associated with lower mortality rate and shorter length of stay. Patients who received early ID intervention during their hospital stay were less likely to be readmitted after discharge and had lower total healthcare spending.

Executive Summary: 2015 Infectious Diseases Society of America (IDSA) Clinical Practice Guidelines for the Diagnosis and Treatment of Native Vertebral Osteomyelitis in Adults.

These guidelines are intended for use by infectious disease specialists, orthopedic surgeons, neurosurgeons, radiologists, and other healthcare professionals who care for patients with native vertebral osteomyelitis (NVO). They include evidence and opinion-based recommendations for the diagnosis and management of patients with NVO treated with antimicrobial therapy, with or without surgical intervention.

2015 Infectious Diseases Society of America (IDSA) Clinical Practice Guidelines for the Diagnosis and Treatment of Native Vertebral Osteomyelitis in Adults.

These guidelines are intended for use by infectious disease specialists, orthopedic surgeons, neurosurgeons, radiologists, and other healthcare professionals who care for patients with native vertebral osteomyelitis (NVO). They include evidence and opinion-based recommendations for the diagnosis and management of patients with NVO treated with antimicrobial therapy, with or without surgical intervention.

Infectious diseases specialty intervention is associated with decreased mortality and lower healthcare costs.

BACKGROUND: Previous studies, largely based on chart reviews with small sample sizes, have demonstrated that infectious diseases (ID) specialists positively impact patient outcomes. We investigated how ID specialists impact mortality, utilization, and costs using a large claims dataset. METHODS: We used administrative fee-for-service Medicare claims to identify beneficiaries hospitalized from 2008 to 2009 with at least 1 of 11 infections. There were 101 991 stays with and 170 336 stays without ID interventions. Cohorts were propensity score matched for patient demographics, comorbidities, and hospital characteristics. Regression models compared ID versus non-ID intervention and early versus late ID intervention. Risk-adjusted outcomes included hospital and intensive care unit (ICU) length of stay (LOS), mortality, readmissions, hospital charges, and Medicare payments. RESULTS: The ID intervention cohort demonstrated significantly lower mortality (odds ratio [OR], 0.87; 95% confidence interval [CI], .83 to .91) and readmissions (OR, 0.96; 95% CI, .93 to .99) than the non-ID intervention cohort. Medicare charges and payments were not significantly different; the ID intervention cohort ICU LOS was 3.7% shorter (95% CI, -5.5% to -1.9%). Patients receiving ID intervention within 2 days of admission had significantly lower 30-day mortality and readmission, hospital and ICU length of stay, and Medicare charges and payments compared with patients receiving later ID interventions. CONCLUSIONS: ID interventions are associated with improved patient outcomes. Early ID interventions are also associated with reduced costs for Medicare beneficiaries with select infections.

A Thermoplastic Microsystem to Perform Antibiotic Susceptibility Testing by Monitoring Oxygen Consumption.

Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 µL of a diluted bacterial suspension (OD600 = 0.02) and provides resistance profiles about 2-3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains.

Contrasting patterns of viral load response in transplant recipients with BK polyomavirus DNAemia on leflunomide therapy.

BACKGROUND: Reduction in immunosuppression is considered the therapy of proven benefit for BKV infection in renal transplantation, but the use of leflunomide has also been reported. It was observed at this center that the patterns of viral load response while on leflunomide appear to fall into two distinct types. METHODS: Medical records of 22 kidney and kidney-pancreas recipients at a single center who received leflunomide therapy for BKV DNAemia were reviewed. Information was collected on demographics, BKV viral loads, other antiviral therapy, immunosuppressive drug levels and doses, adverse effects, and graft and patient outcomes. RESULTS: Eighteen of 22 cleared BKV viremia, and 12 of 22 had preserved allograft function; only two graft losses occurred in the screening era among leflunomide-treated patients. Two patterns of viral load reduction were observed, termed the "smooth" and the "zigzag" pattern, which differed in mean time to clear of BKV DNA (2.9 vs. 19.5 months, p = 0.0073). Graft preservation was correlated with lower serum creatinine (SCr) at the start of leflunomide therapy. CONCLUSIONS: Long courses and "zigzag" fluctuations in viral load can occur in patients who eventually clear BKV on leflunomide with preserved allograft function. Intermittent increases in viral load do not necessarily portend therapeutic failure. Although the utility of leflunomide is still debated in the transplant community, this information may be useful to clinicians who choose to use it in selected patients.

Discovery of antimicrobials by massively parallelized growth assays (Mex).

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. Here we present Mex, a method for the high-throughput discovery of novel antimicrobials, that relies on E. coli self-screening to determine the bioactivity of more than ten thousand naturally occurring peptides. Analysis of thousands of E. coli growth curves using next-generation sequencing enables the identification of more than 1000 previously unknown antimicrobial peptides. Additionally, by incorporating the kinetics of growth inhibition, a first indication of the mode of action is obtained, which has implications for the ultimate usefulness of the peptides in question. The most promising peptides of the screen are chemically synthesized and their activity is determined in standardized susceptibility assays. Ten out of 15 investigated peptides efficiently eradicate bacteria at a minimal inhibitory concentration in the lower µM or upper nM range. This work represents a step-change in the high-throughput discovery of functionally diverse antimicrobials.

Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression.

Microfluidic methods for the formation of single and double emulsion (DE) droplets allow for the encapsulation and isolation of reactants inside nanoliter compartments. Such methods have greatly enhanced the toolbox for high-throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed compartments limit the applicability of droplet microfluidics. Here, a strategy is introduced for on-demand delivery of reactants in DEs. Lipid vesicles are used as reactant carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant sodium dodecyl sulfate (SDS). The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the vesicles' lipid bilayer. The versatility of the method is demonstrated with two critical applications chosen as representative assays for high-throughput screening: the induction of gene expression in bacteria and the initiation of an enzymatic reaction. This method not only allows for the release of the lipid vesicle content inside DEs to be synchronized for all DEs but also for the release to be triggered at any desired time.