Targeted sequencing of bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies.

Unlabelled: Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen , by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene .

Ceftolozane/tazobactam disrupts Pseudomonas aeruginosa biofilms under static and dynamic conditions.

Background: Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P.

Wild-Type Domestication: Loss of Intrinsic Metabolic Traits Concealed by Culture in Rich Media.

Bacteria are typically isolated on rich media to maximise isolation success, removing them from their native evolutionary context. This eliminates selection pressures, enabling otherwise deleterious genomic events to accumulate. Here, we present a cautionary tale of these 'quiet mutations' which can persist unnoticed in bacterial culture lines.

A Finger-Actuated Sample-Dosing Capillary-Driven Microfluidic Device for Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) has attracted significant attention for rapid and accurate point-of-care diagnostics. However, integrating sample introduction, lysis, amplification, and detection steps into an easy-to-use, disposable system has so far been challenging. This has limited the uptake of the technique in practical applications.

Predicting drug resistance using artificial intelligence and clinical MALDI-TOF mass spectra.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used in clinical microbiology laboratories for bacterial identification but its use for detection of antimicrobial resistance (AMR) remains limited. Here, we used MALDI-TOF MS with artificial intelligence (AI) approaches to successfully predict AMR in , a priority pathogen with complex AMR mechanisms. The highest performance was achieved for modern β-lactam/β-lactamase inhibitor drugs, namely, ceftazidime/avibactam and ceftolozane/tazobactam.

Genomic investigation of multispecies and multivariant outbreak reveals key role of horizontal plasmid transmission.

Objectives: New Delhi metallo-β-lactamases (NDMs) are major contributors to the spread of carbapenem resistance globally. In Australia, NDMs were previously associated with international travel, but from 2019 we noted increasing incidence of NDM-positive clinical isolates. We investigated the clinical and genomic epidemiology of NDM carriage at a tertiary-care Australian hospital from 2016 to 2021.

Decisional Support Needed when Facing Tough Decisions: Survey of Parents with Children having Differences of Sex Development.

Introduction: Parents of infants and young children newly diagnosed with differences of sex development (DSD) commonly face medical and psychosocial management decisions at a time when they are first learning about the condition and cannot consult their child for input. The aim of this study was to identify areas of greatest need for parental decisional support.

Methods: 34 parents of children receiving care for DSD at one of three US children's hospitals participated in a survey to learn what clinical and psychosocial decisions needed to be made on behalf of their child.

Co-creating a suite of patient decision aids for parents of an infant or young child with differences of sex development: A methods roadmap.

Introduction: Parents and guardians of infants and young children with differences of sex development (DSD) often face numerous health and social decisions about their child's condition. While proxy health decisions can be stressful in any circumstance, they are further exacerbated in this clinical context by significant variations in clinical presentation, parental lack of knowledge about DSD, irreversibility of some options (e.g.

Trial of Vancomycin and Cefazolin as Surgical Prophylaxis in Arthroplasty.

Background: The addition of vancomycin to beta-lactam prophylaxis in arthroplasty may reduce surgical-site infections; however, the efficacy and safety are unclear.

Methods: In this multicenter, double-blind, superiority, placebo-controlled trial, we randomly assigned adult patients without known methicillin-resistant (MRSA) colonization who were undergoing arthroplasty to receive 1.5 g of vancomycin or normal saline placebo, in addition to cefazolin prophylaxis.

Genomic dissection of endemic carbapenem resistance reveals metallo-beta-lactamase dissemination through clonal, plasmid and integron transfer.

Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades of bla evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified the bla gene across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types.

Antimicrobial susceptibility of ceftolozane-tazobactam against multidrug-resistant Pseudomonas aeruginosa isolates from Melbourne, Australia.

We collected 163 clinical Pseudomonas aeruginosa isolates at a tertiary hospital specialising in adult cystic fibrosis (CF) and lung transplantation (LTx) in Melbourne, Australia, to explore the activity of ceftolozane-tazobactam (C/T) in populations at high-risk for antimicrobial resistance. Of these, 144 (88.3%) were collected from sputum, and 19 (11.

Favipiravir in early symptomatic COVID-19, a randomised placebo-controlled trial.

Background: Well tolerated antivirals administered early in the course of COVID-19 infection when the viremia is highest could prevent progression to severe disease. Favipiravir inhibits SARS-CoV-2 viral replication with evidence of clinical benefit in open label trials. Placebo controlled studies of people with early symptomatic COVID-19 with regular assessments of SARS-CoV-2 viral load can determine if it has an antiviral effect and improves clinical outcomes.

A survey of healthcare professionals' perceptions of the decisional needs of parents with an infant born with a disorder/difference of sex development.

Introduction: Parents of infants born with differences in sex development (DSD) face many difficult decisions. As part of a larger project designed to develop educational interventions to promote shared decision making, this study assessed healthcare professionals' (HCPs) perceptions of parental decision-making needs when an infant is born with a DSD.

Methods: A cross-sectional web-based survey following the Ottawa Decision Support Framework was conducted in two waves, between October 2020 and June 2022.

Attributable mortality and excess length of stay associated with third-generation cephalosporin-resistant Enterobacterales bloodstream infections: a prospective cohort study in Suva, Fiji.

Objectives: There are scant primary clinical data on antimicrobial resistance (AMR) burden from low- and middle-income countries (LMICs). We adapted recent World Health Organization methodology to measure the effect of third-generation cephalosporin resistance (3GC-R) on mortality and excess length of hospital stay in Fiji.

Methods: We conducted a prospective cohort study of inpatients with Enterobacterales bloodstream infections (BSIs) at Colonial War Memorial Hospital, Suva.

SARS-CoV-2 infection results in immune responses in the respiratory tract and peripheral blood that suggest mechanisms of disease severity.

Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens.

Epidemiology, antimicrobial resistance and outcomes of bacteraemia in a tertiary hospital in Fiji: A prospective cohort study.

Background: bacteraemia (SAB) is one of the commonest bloodstream infections globally and is associated with a high mortality rate. Most published data comes from temperate, high-income countries. We describe the clinical epidemiology, microbiology, management and outcomes of patients with SAB treated in a tropical, middle-income setting at Fiji's largest hospital.

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in .

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of , mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins.

A Cost-effectiveness Analysis Comparing Pembrolizumab-Axitinib, Nivolumab-Ipilimumab, and Sunitinib for Treatment of Advanced Renal Cell Carcinoma.

Objectives: The US Food and Drug Administration (FDA) approved nivolumab-ipilimumab and pembrolizumab-axitinib as first-line treatments for metastatic, clear-cell, renal cell carcinoma (mRCC) based on results from CheckMate 214 and KEYNOTE-426. Our objective was to compare the adjusted, lifetime cost-effectiveness between nivolumab-ipilimumab, pembrolizumab-axitinib, and sunitinib for patients with mRCC.

Materials And Methods: A 3-state Markov model was developed comparing nivolumab-ipilimumab and pembrolizumab-axitinib to each other and sunitinib, over a 20-year lifetime horizon from a US medical center perspective.

Crystal structure of TcpK in complex with oriT DNA of the antibiotic resistance plasmid pCW3.

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution.

The Tcp conjugation system of Clostridium perfringens.

The Gram-positive pathogen Clostridium perfringens possesses a family of large conjugative plasmids that is typified by the tetracycline resistance plasmid pCW3. Since these plasmids may carry antibiotic resistance genes or genes encoding extracellular or sporulation-associated toxins, the conjugative transfer of these plasmids appears to be important for the epidemiology of C. perfringens-mediated diseases.

The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth.

Analysis of the virulence-associated RevSR two-component signal transduction system of Clostridium perfringens.

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence.

TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase.

Virulence Plasmids of Spore-Forming Bacteria.

Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C.

Two novel membrane proteins, TcpD and TcpE, are essential for conjugative transfer of pCW3 in Clostridium perfringens.

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus.