Core Genome Multilocus Sequence Typing and Antibiotic Susceptibility Prediction from Whole-Genome Sequence Data of Multidrug-Resistant Pseudomonas aeruginosa Isolates.

Over the past decade, whole-genome sequencing (WGS) has overtaken traditional bacterial typing methods for studies of genetic relatedness. Further, WGS data generated during epidemiologic studies can be used in other clinically relevant bioinformatic applications, such as antibiotic resistance prediction. Using commercially available software tools, the relatedness of 38 clinical isolates of multidrug-resistant Pseudomonas aeruginosa was defined by two core genome multilocus sequence typing (cgMLST) methods, and the WGS data of each isolate was analyzed to predict antibiotic susceptibility to nine antibacterial agents.

Acinetobacter baumannii Genomic Sequence-Based Core Genome Multilocus Sequence Typing Using Ridom SeqSphere+ and Antimicrobial Susceptibility Prediction in ARESdb.

Whole-genome sequencing (WGS) is rapidly replacing traditional typing methods for the investigation of infectious disease outbreaks. Additionally, WGS data are being used to predict phenotypic antimicrobial susceptibility. Acinetobacter baumannii, which is often multidrug-resistant, is a significant culprit in outbreaks in health care settings.

Defining Baseline Mechanisms of Cefiderocol Resistance in the Enterobacterales.

The objective of this study was to identify putative mechanisms contributing to baseline cefiderocol resistance among carbapenem-resistant Enterobacterales (CRE). We evaluated 56 clinical CRE isolates with no previous exposure to cefiderocol. Cefiderocol and comparator agent minimum inhibitory concentrations (MICs) were determined by broth microdilution.

Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing.

The increasing incidence of antimicrobial resistance (AMR) is a major global challenge. Routine techniques for molecular AMR marker detection are largely based on low-plex PCR and detect dozens to hundreds of AMR markers. To allow for comprehensive and sensitive profiling of AMR markers, we developed a capture-based next generation sequencing (NGS) workflow featuring a novel AMR marker panel based on the curated AMR database ARESdb.

Predictive Antibiotic Susceptibility Testing by Next-Generation Sequencing for Periprosthetic Joint Infections: Potential and Limitations.

Joint replacement surgeries are one of the most frequent medical interventions globally. Infections of prosthetic joints are a major health challenge and typically require prolonged or even indefinite antibiotic treatment. As multidrug-resistant pathogens continue to rise globally, novel diagnostics are critical to ensure appropriate treatment and help with prosthetic joint infections (PJI) management.

Core Genome Multilocus Sequence Typing and Prediction of Antimicrobial Susceptibility Using Whole-Genome Sequences of Escherichia coli Bloodstream Infection Isolates.

In total, 50 Escherichia coli bloodstream isolates from the clinical laboratory and 12 E. coli isolates referred for pulsed-field gel electrophoresis (PFGE) were sequenced, assessed for clonality using core genome multilocus sequence typing (cgMLST), and evaluated for genomic susceptibility predictions using ARESdb. Results of sequence typing using whole-genome sequencing (WGS)-based MLST and sequence type (ST)-specific PCR were identical.

Cefiderocol Activity Against Clinical Isolates Exhibiting Ceftolozane-Tazobactam Resistance.

Background: Mutations in the AmpC-AmpR region are associated with treatment-emergent ceftolozane-tazobactam (TOL-TAZ) and ceftazidime-avibactam (CAZ-AVI) resistance. We sought to determine if these mutations impact susceptibility to the novel cephalosporin-siderophore compound cefiderocol.

Methods: Thirty-two paired isolates from 16 patients with index isolates susceptible to TOL-TAZ and subsequent isolates available after TOL-TAZ exposure from January 2019 to December 2020 were included.

Genomic and Phenotypic Analysis of Linezolid-Resistant in a Tertiary Hospital in Innsbruck, Austria.

Whole genome sequencing is a useful tool to monitor the spread of resistance mechanisms in bacteria. In this retrospective study, we investigated genetic resistance mechanisms, sequence types (ST) and respective phenotypes of linezolid-resistant (LRSE, = 129) recovered from a cohort of patients receiving or not receiving linezolid within a tertiary hospital in Innsbruck, Austria. Hereby, the point mutation G2603U in the 23S rRNA ( = 91) was the major resistance mechanism followed by the presence of plasmid-derived ( = 30).

Learning From Limited Data: Towards Best Practice Techniques for Antimicrobial Resistance Prediction From Whole Genome Sequencing Data.

Antimicrobial resistance prediction from whole genome sequencing data (WGS) is an emerging application of machine learning, promising to improve antimicrobial resistance surveillance and outbreak monitoring. Despite significant reductions in sequencing cost, the availability and sampling diversity of WGS data with matched antimicrobial susceptibility testing (AST) profiles required for training of WGS-AST prediction models remains limited. Best practice machine learning techniques are required to ensure trained models generalize to independent data for optimal predictive performance.

A high leukocyte count and administration of hydrocortisone hamper PCR-based diagnostics for bloodstream infections.

Bloodstream infections (BSIs) require an accurate and fast identification of causative pathogens. Molecular diagnostics, in particular polymerase chain reaction (PCR)-based approaches for BSI diagnostics directly from whole blood, suffer from limitations such as inhibition leading to invalid results. In this retrospective study, we analyzed 23 parameters for their potential interference with LightCycler SeptiFast PCR tests (n = 2167) routinely performed at our institution.

Multicenter Evaluation of the Unyvero Platform for Testing Bronchoalveolar Lavage Fluid.

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, , in BAL fluid in ∼4.

Modifiable Risk Factors for the Emergence of Ceftolozane-tazobactam Resistance.

Background: Ceftolozane-tazobactam (TOL-TAZ) affords broad coverage against Pseudomonas aeruginosa. Regrettably, TOL-TAZ resistance has been reported. We sought to identify modifiable risk factors that may reduce the emergence of TOL-TAZ resistance.

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow.

Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking.

Large-scale assessment of antimicrobial resistance marker databases for genetic phenotype prediction: a systematic review.

Background: Antimicrobial resistance (AMR) is a rising health threat with 10 million annual casualties estimated by 2050. Appropriate treatment of infectious diseases with the right antibiotics reduces the spread of antibiotic resistance. Today, clinical practice relies on molecular and PCR techniques for pathogen identification and culture-based antibiotic susceptibility testing (AST).

Species Identification and Antibiotic Resistance Prediction by Analysis of Whole-Genome Sequence Data by Use of ARESdb: an Analysis of Isolates from the Unyvero Lower Respiratory Tract Infection Trial.

Whole-genome sequencing (WGS) is now routinely performed in clinical microbiology laboratories to assess isolate relatedness. With appropriately developed analytics, the same data can be used for prediction of antimicrobial susceptibility. We assessed WGS data for identification using open-source tools and antibiotic susceptibility testing (AST) prediction using ARESdb compared to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification and broth microdilution phenotypic susceptibility testing on clinical isolates from a multicenter clinical trial of the FDA-cleared Unyvero lower respiratory tract infection (LRTI) application (Curetis).

Clinical Resistome Screening of 1,110 Isolates Efficiently Recovers Diagnostically Relevant Antibiotic Resistance Biomarkers and Potential Novel Resistance Mechanisms.

Multidrug-resistant pathogens represent one of the biggest global healthcare challenges. Molecular diagnostics can guide effective antibiotics therapy but relies on validated, predictive biomarkers. Here we present a novel, universally applicable workflow for rapid identification of antimicrobial resistance (AMR) biomarkers from clinical isolates and quantitatively evaluate the potential to recover causal biomarkers for observed resistance phenotypes.

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates.

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years.

Assessing the heterogeneity of in silico plasmid predictions based on whole-genome-sequenced clinical isolates.

High-throughput next-generation shotgun sequencing of pathogenic bacteria is growing in clinical relevance, especially for chromosomal DNA-based taxonomic identification and for antibiotic resistance prediction. Genetic exchange is facilitated for extrachromosomal DNA, e.g.

Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy.

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart.

Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs.

Epigenome-Wide Association Study Identifies Cardiac Gene Patterning and a Novel Class of Biomarkers for Heart Failure.

Background: Biochemical DNA modification resembles a crucial regulatory layer among genetic information, environmental factors, and the transcriptome. To identify epigenetic susceptibility regions and novel biomarkers linked to myocardial dysfunction and heart failure, we performed the first multi-omics study in myocardial tissue and blood of patients with dilated cardiomyopathy and controls.

Methods: Infinium human methylation 450 was used for high-density epigenome-wide mapping of DNA methylation in left-ventricular biopsies and whole peripheral blood of living probands.

Comparing genome versus proteome-based identification of clinical bacterial isolates.

Whole-genome sequencing (WGS) is gaining importance in the analysis of bacterial cultures derived from patients with infectious diseases. Existing computational tools for WGS-based identification have, however, been evaluated on previously defined data relying thereby unwarily on the available taxonomic information.Here, we newly sequenced 846 clinical gram-negative bacterial isolates representing multiple distinct genera and compared the performance of five tools (CLARK, Kaiju, Kraken, DIAMOND/MEGAN and TUIT).

Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring.

The performance of a fiber optic and an optical conduit in-line attenuated total reflection mid-infrared (IR) probe during in situ monitoring of Penicillium chrysogenum fermentation were compared. The fiber optic probe was connected to a sealed, portable, Fourier transform infrared (FT-IR) process spectrometer via a plug-and-play interface. The optical conduit, on the other hand, was connected to a FT-IR process spectrometer via a knuckled probe with mirrors that had to be adjusted prior to each fermentation, which were purged with dry air.

Personalized Computer Simulation of Diastolic Function in Heart Failure.

The search for a parameter representing left ventricular relaxation from non-invasive and invasive diagnostic tools has been extensive, since heart failure (HF) with preserved ejection fraction (HF-pEF) is a global health problem. We explore here the feasibility using patient-specific cardiac computer modeling to capture diastolic parameters in patients suffering from different degrees of systolic HF. Fifty eight patients with idiopathic dilated cardiomyopathy have undergone thorough clinical evaluation, including cardiac magnetic resonance imaging (MRI), heart catheterization, echocardiography, and cardiac biomarker assessment.

Intact cell mass spectrometry as a progress tracking tool for batch and fed-batch fermentation processes.

Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth.