Profiling chromatin accessibility responses in human neutrophils with sensitive pathogen detection.

Sepsis, sequela of bloodstream infections and dysregulated host responses, is a leading cause of death globally. Neutrophils tightly regulate responses to pathogens to prevent organ damage. Profiling early host epigenetic responses in neutrophils may aid in disease recognition. We performed assay for transposase-accessible chromatin (ATAC)-seq of human neutrophils challenged with six toll-like receptor ligands and two organisms; and RNA-seq after Escherichia coli exposure for 1 and 4 h along with ATAC-seq. ATAC-seq of neutrophils facilitates detection of pathogen DNA. In addition, despite similarities in genomic distribution of differential chromatin changes across challenges, only a fraction overlaps between the challenges. Ligands depict shared signatures, but majority are unique in position, function, and challenge. Epigenomic changes are plastic, only ∼120 are shared by E coli challenges over time, resulting in varied differential genes and associated processes. We identify three classes of gene regulation, chromatin access changes in the promoter; changes in the promoter and distal enhancers; and controlling expression through changes solely in distal enhancers. These and transcription factor footprinting reveal timely and challenge specific mechanisms of transcriptional regulation in neutrophils.

A Novel Platform Using RNA Signatures To Accelerate Antimicrobial Susceptibility Testing in Neisseria gonorrhoeae.

The rise of antimicrobial-resistant pathogens can be attributed to the lack of a rapid pathogen identification (ID) or antimicrobial susceptibility testing (AST), resulting in delayed therapeutic decisions at the point of care. Gonorrhea is usually empirically treated, with no AST results available before treatment, thus contributing to the rapid rise in drug resistance. Here, we present a rapid AST platform using RNA signatures for Neisseria gonorrhoeae Transcriptome sequencing (RNA-seq) followed by bioinformatic tools was applied to explore potential markers in the transcriptome profile of N. gonorrhoeae upon minutes of azithromycin exposure. Validation of candidate markers using quantitative real-time PCR (qRT-PCR) showed that two markers (arsR [NGO1562] and rpsO) can deliver accurate AST results across 14 tested isolates. Further validation of our susceptibility threshold in comparison to MIC across 64 more isolates confirmed the reliability of our platform. Our RNA markers combined with emerging molecular point-of-care systems has the potential to greatly accelerate both ID and AST to inform treatment.

RNA markers for ultra-rapid molecular antimicrobial susceptibility testing in fluoroquinolone-treated Klebsiella pneumoniae.

OBJECTIVES: Traditional antimicrobial susceptibility testing (AST) is growth dependent and time-consuming. With rising rates of drug-resistant infections, a novel diagnostic method is critically needed that can rapidly reveal a pathogen's antimicrobial susceptibility to guide appropriate treatment. Recently, RNA sequencing has been identified as a powerful diagnostic tool to explore transcriptional gene expression and improve AST. METHODS: RNA sequencing was used to investigate the potential of RNA markers for rapid molecular AST using Klebsiella pneumoniae and ciprofloxacin as a model. Downstream bioinformatic analysis was applied for optimal marker selection. Further validation on 11 more isolates of K. pneumoniae was performed using quantitative real-time PCR. RESULTS: From RNA sequencing, we identified RNA signatures that were induced or suppressed following exposure to ciprofloxacin. Significant shifts at the transcript level were observed as early as 10 min after antibiotic exposure. Lastly, we confirmed marker expression profiles with concordant MIC results from traditional culture-based AST and validated across 11 K. pneumoniae isolates. recA, coaA and metN transcripts harbour the most sensitive susceptibility information and were selected as our top markers. CONCLUSIONS: Our results suggest that RNA signature is a promising approach to AST development, resulting in faster clinical diagnosis and treatment of infectious disease. This approach is potentially applicable in other models including other pathogens exposed to different classes of antibiotics.

Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing.

In the face of looming threats from multi-drug resistant microorganisms, there is a growing need for technologies that will enable rapid identification and drug susceptibility profiling of these pathogens in health care settings. In particular, recent progress in microfluidics and nucleic acid amplification is pushing the boundaries of timescale for diagnosing bacterial infections. With a diverse range of techniques and parallel developments in the field of analytical chemistry, an integrative perspective is needed to understand the significance of these developments. This review examines the scope of new developments in assay technologies grouped by key enabling domains of research. First, we examine recent development in nucleic acid amplification assays for rapid identification and drug susceptibility testing in bacterial infections. Next, we examine advances in microfluidics that facilitate acceleration of diagnostic assays via integration and scale. Lastly, recentdevelopments in biosensor technologies are reviewed. We conclude this review with perspectives on the use of emerging concepts to develop paradigm-changing assays.

A 'culture' shift: Application of molecular techniques for diagnosing polymicrobial infections.

With the advancement of microbiological discovery, it is evident that many infections, particularly bloodstream infections, are polymicrobial in nature. Consequently, new challenges have emerged in identifying the numerous etiologic organisms in an accurate and timely manner using the current diagnostic standard. Various molecular diagnostic methods have been utilized as an effort to provide a fast and reliable identification in lieu or parallel to the conventional culture-based methods. These technologies are mostly based on nucleic acid, proteins, or physical properties of the pathogens with differing advantages and limitations. This review evaluates the different molecular methods and technologies currently available to diagnose polymicrobial infections, which will help determine the most appropriate option for future diagnosis.

A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood.

BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.

Microbial Typing by Machine Learned DNA Melt Signatures.

There is still an ongoing demand for a simple broad-spectrum molecular diagnostic assay for pathogenic bacteria. For this purpose, we developed a single-plex High Resolution Melt (HRM) assay that generates complex melt curves for bacterial identification. Using internal transcribed spacer (ITS) region as the phylogenetic marker for HRM, we observed complex melt curve signatures as compared to 16S rDNA amplicons with enhanced interspecies discrimination. We also developed a novel Naïve Bayes curve classification algorithm with statistical interpretation and achieved 95% accuracy in differentiating 89 bacterial species in our library using leave-one-out cross-validation. Pilot clinical validation of our method correctly identified the etiologic organisms at the species-level in 59 culture-positive mono-bacterial blood culture samples with 90% accuracy. Our findings suggest that broad bacterial sequences may be simply, reliably and automatically profiled by ITS HRM assay for clinical adoption.

Expression of tmRNA in mycobacteria is increased by antimicrobial agents that target the ribosome.

The specialized RNA, tmRNA, is a central component of prokaryote trans-translation; a process that salvages stalled translational complexes. Evidence from other bacteria suggested that exposure to ribosome inhibitors elevated tmRNA levels, although it was unclear whether such changes resulted from increased tmRNA synthesis. Consequently, this study was initiated to determine the effect of ribosome inhibitors on the expression of tmRNA in mycobacteria. Exposure of Mycobacterium smegmatis to ribosome-targeting antimicrobial agents was associated with increased levels of the tmRNA precursor, pre-tmRNA, and mature tmRNA. For example, exposure to 16 µg mL⁻¹ erythromycin for 3 h increased pre-tmRNA and tmRNA by 18- and 6-fold, respectively. Equivalent results were found following exposure of Mycobacterium bovis BCG to streptomycin. Exposure to antimicrobial agents with nonribosome targets did not affect tmRNA levels. The increased tmRNA levels were associated with increased output from the ssrA promoter, which controls tmRNA transcription, without evidence of a change in tmRNA degradation. These results suggest that the upregulation of tmRNA expression was an important response of bacteria to exposure to ribosome-inhibiting antimicrobial agents.

Intrinsic macrolide resistance in rapidly growing mycobacteria.

This study reports the discovery of erm genes in seven species of rapidly growing mycobacteria (RGM): Mycobacterium boenickei, M. goodii, M. houstonense, M. mageritense, M. neworleansense, M. porcinum, and M. wolinskyi. This study further substantiates the role of erm genes in intrinsic macrolide resistance in RGM.

Intrinsic macrolide resistance of the Mycobacterium tuberculosis complex is inducible.

Mycobacterium tuberculosis is intrinsically resistant to macrolides, a characteristic associated with expression of the erm(37) gene. This intrinsic resistance was found to be inducible with clarithromycin and the ketolide HMR3004. Furthermore, underlying the phenotypic induction was an increase in erm(37) mRNA levels.