Molecular epidemiology of β-lactamases in ceftriaxone-resistant Enterobacterales bloodstream infections in the mid-Atlantic United States.

Ceftriaxone-resistant Enterobacterales remain a public health threat; contemporary data investigating their molecular epidemiology are limited. Five hundred consecutive ceftriaxone-resistant (MIC ≥ 4 µg/mL) Enterobacterales bloodstream isolates were collected between 2018 and 2022 from three Maryland hospitals. Broth microdilution confirmed antibiotic susceptibilities.

Three prolonged outbreaks of metallo-β-lactamase-producing in an Upper Austrian hospital, 2017-2023.

Unlabelled: In spring 2022, an increase in metallo-β-lactamase-producing (MBL-Pa) infections was detected in a hospital in Upper Austria. To identify the source of infection and to stop further transmissions, an epidemiological outbreak investigation including whole-genome sequencing (WGS)-based typing was conducted. The final case definition included cases admitted to the hospital between 2020 and 2023 with an MBL-Pa in one of the three genomic clusters identified.

A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens.

Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved.

Is Carbapenem Therapy Necessary for the Treatment of Non-CTX-M Extended-Spectrum β-Lactamase-Producing Enterobacterales Bloodstream Infections?

Background: Investigations into antibiotics for extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) bloodstream infections (BSIs) have focused on blaCTX-M genes. Patient outcomes from non-CTX-M-producing ESBL-E BSIs and optimal treatment are unknown.

Methods: A multicenter observational study investigating 500 consecutive patients with ceftriaxone-resistant Enterobacterales BSIs during 2018-2022 was conducted.

An NDM-Producing Clinical Isolate Exhibiting Resistance to Cefiderocol and the Combination of Ceftazidime-Avibactam and Aztreonam: Another Step Toward Pan-β-Lactam Resistance.

Background: Cefiderocol and ceftazidime-avibactam plus aztreonam (CZA-ATM) are preferred treatment regimens for New Delhi metallo-β-lactamase (NDM)-producing infections.

Methods: We report the case of a US patient who traveled to India to receive a renal transplant. He subsequently experienced pyelonephritis by an NDM-producing Broth microdilution and the broth disk elution method indicated resistance to all β-lactams, including cefiderocol and CZA-ATM.

Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing.

Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing.

Automated antimicrobial susceptibility testing and antimicrobial resistance genotyping using Illumina and Oxford Nanopore Technologies sequencing data among .

Whole-genome sequencing (WGS) enables the molecular characterization of bacterial pathogens. We compared the accuracy of the Illumina and Oxford Nanopore Technologies (ONT) sequencing platforms for the determination of AMR classes and antimicrobial susceptibility testing (AST) among 181 clinical isolates. Sequencing reads for each isolate were uploaded to AREScloud (Ares Genetics) to determine the presence of AMR markers and the predicted WGS-AST profile.

Padlock Probe-Based Generation of DNAzymes for the Colorimetric Detection of Antibiotic Resistance Genes.

The increasing emergence of multidrug- and pan-resistant pathogens requires rapid and cost-efficient diagnostic tools to contain their further spread in healthcare facilities and the environment. The currently established diagnostic technologies are of limited utility for efficient infection control measures because they are either cultivation-based and time-consuming or require sophisticated assays that are expensive. Furthermore, infectious diseases are unfortunately most problematic in countries with low-resource settings in their healthcare systems.

Monitoring of Rolling Circle Amplification on a Solid Support by Surface Plasmon Resonance and Optical Waveguide Spectroscopy.

The growth of surface-attached single-stranded deoxyribonucleic acid (ssDNA) chains is monitored using an evanescent wave optical biosensor that combines surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The "grafting-from" growth of ssDNA chains is facilitated by rolling circle amplification (RCA), and the gradual prolongation of ssDNA chains anchored to a gold sensor surface is optically tracked in time. At a sufficient density of the polymer chains, the ssDNA takes on a brush architecture with a thickness exceeding 10 μm, supporting a spectrum of guided optical waves traveling along the metallic sensor surface.

ResiDB: An automated database manager for sequence data.

The amount of publicly available DNA sequence data is drastically increasing, making it a tedious task to create sequence databases necessary for the design of diagnostic assays. The selection of appropriate sequences is especially challenging in genes affected by frequent point mutations such as antibiotic resistance genes. To overcome this issue, we have designed the webtool resiDB, a rapid and user-friendly sequence database manager for bacteria, fungi, viruses, protozoa, invertebrates, plants, archaea, environmental and whole genome shotgun sequence data.

Epidemiology of colistin-resistant, carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Croatia.

Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K.

Extended-spectrum beta-lactamases and plasmid diversity in urinary isolates of Escherichia coli in Croatia: a nation-wide, multicentric, retrospective study.

In recent years, a dramatic increase in the prevalence of Escherichia coli strains producing extended-spectrum β-lactamases (ESBLs) has been observed - both in the community and in healthcare settings. This multicentric study aimed to characterize ESBLs produced by E. coli isolates causing hospital-onset and community urinary tract infections, as well as to compare their antimicrobial sensitivity patterns, β-lactamase content and plasmid types.

PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

Purpose: A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia.

Methods: Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing.

Oli2go: an automated multiplex oligonucleotide design tool.

The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run.

Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes.

Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal species as well as 33 clinically relevant β-lactamases ( Barišić , 2016 ). By using 16S rRNA gene sequences as barcode elements in the padlock probes, and two different fluorescence channels for species and antibiotic resistance identification, we managed to cut the number of microarray probes needed by half.

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments.

Unlabelled: Human rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments.

Ion association by time-of-flight mass spectrometry: a study of V-P-O catalysts.

The oxidation catalysts (VO2)P207 and β-VOPO4 are studied by laser desorption time-of flight mass spectrometry. Mass spectra of both positive and negative ions are reported for these compounds. Individual scans are tested for ion association, that is, the consistent appearance of a given pair of ions in many scans.

An automatic electrometer ranging circuit for a spark-source mass-spectrometer.

An auto-ranging circuit which accommodates the erratic nature of the electrometer output signals from a spark-source mass-spectrometer is described briefly.

Simultaneous ion-beam collection for precise measurement of nuclidic ion-current ratios in spark-source mass spectrometry.

Simultaneous ion-beam collection techniques make significant improvement possible in the precision of measurements when electrical ion-detection is used with the spark ion-source. The advantage of reference of specific ion-current signals only to those from desired regions of the mass spectrum is that reproducible measurements may be made on heterogeneous samples such as oxide-graphite pellets. For example, isotopic ratio measurements from separate samplings of an oxide (pelleted with graphite) are reproducible to better than +/-0.

Relative sensitivity coefficients for rare earths in spark-source mass spectrometry.

Relative sensitivity coefficients have been computed for rare earth elements according to empirical models which have been proposed in the literature. Explanations concerning differences between the computed and observed values are based upon possible instrumental discrimination effects which negate some processes occurring in the ion source. Computations relating elemental physical constants to observed results in a semi-random manner are shown which support this contention.

Automatic control of the ion-illumination angle in a spark-source mass spectrometer.

Automatic adjustment of only the spark-gap width in a spark-source mass spectrometer does not ensure that optimum conditions of electrode geometry are maintained with respect to the ion-optics system. A device has been developed which simultaneously maintains a constant gap width and also a more constant z-axis ion-illumination angle. This is the first development to utilize ion-optics parameters to adjust the sparking electrodes automatically.