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 .

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.

Ethanolamine metabolism through two genetically distinct loci enables Klebsiella pneumoniae to bypass nutritional competition in the gut.

Successful microbial colonization of the gastrointestinal (GI) tract hinges on an organism's ability to overcome the intense competition for nutrients in the gut between the host and the resident gut microbiome. Enteric pathogens can exploit ethanolamine (EA) in the gut to bypass nutrient competition. However, Klebsiella pneumoniae (K.

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.

Streptococcus ruminantium-associated sheep mastitis outbreak detected in Italy is distinct from bovine isolates.

Streptococcus ruminantium is the causative agent of several bovine and ovine diseases, however reports are uncommon and application of whole genome sequencing to identify is rare. We report for the first time, a severe ovine mastitis outbreak caused by S. ruminantium in Italy, 2022.

Bactabolize is a tool for high-throughput generation of bacterial strain-specific metabolic models.

Metabolic capacity can vary substantially within a bacterial species, leading to ecological niche separation, as well as differences in virulence and antimicrobial susceptibility. Genome-scale metabolic models are useful tools for studying the metabolic potential of individuals, and with the rapid expansion of genomic sequencing there is a wealth of data that can be leveraged for comparative analysis. However, there exist few tools to construct strain-specific metabolic models at scale.

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.

Genomic surveillance reveals antibiotic resistance gene transmission via phage recombinases within sheep mastitis-associated Streptococcus uberis.

Background: Streptococcus uberis is one of the main causative agents of ovine mastitis, however little is known about this global, environmental pathogen and its genomic mechanisms of disease. In this study, we performed genomic analysis on 46 S. uberis isolates collected from mastitis-infected sheep in Sardinia (Italy).

Transmission of Klebsiella strains and plasmids within and between grey-headed flying fox colonies.

The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood.

A curated collection of metabolic models reveals variable substrate usage and gene essentiality.

The species complex (KpSC) is a set of seven taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa.

Stable Recombinant-Gene Expression from a Live Bacterial Vector via Chromosomal Integration.

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing.

Sequence characterisation and novel insights into bovine mastitis-associated Streptococcus uberis in dairy herds.

Streptococcus uberis is one of the most frequent mastitis-causing pathogens isolated from dairy cows. Further understanding of S. uberis genetics may help elucidate the disease pathogenesis.

Survey and Sequence Characterization of Bovine Mastitis-Associated in Dairy Herds.

is frequently associated with mastitis in cattle. "Pathogenic" and "commensal" isolates appear to be genetically similar. With a few exceptions, no notable genotypic differences have been found between commensal and mastitis-associated .

Catalytically Active Bioseparation Resin Utilizing a Covalent Intermediate for Tagless Protein Purification.

Common protein purification processes rely on noncovalent affinity binding of the target protein to resin via fusion tags, which has limitations such as leakage of target during the removal of impurities, restricting stringency of wash conditions, and often requires postpurification removal of tags. Here, we developed the Catch, Hold, and Release (C-H-R) method by bioengineering a single-use resin composed of polyhydroxybutyrate beads codisplaying two SpyCatcher (SpyC) domains and Sortase A (SrtA). This core-shell resin was assembled by engineered .

Prediction of Burkholderia pseudomallei DsbA substrates identifies potential virulence factors and vaccine targets.

Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B.

Discovery and characterisation of circular bacteriocin plantacyclin B21AG from B21.

B21 isolated from Vietnamese sausage () has previously displayed broad antimicrobial activity against Gram-positive bacteria including foodborne pathogens and . This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach to identify and characterise plantacyclin B21AG from first principles.

Bioinformatic prospecting and phylogenetic analysis reveals 94 undescribed circular bacteriocins and key motifs.

Background: Circular bacteriocins are antimicrobial peptides produced by bacteria with a N and C termini ligation. They have desirable properties such as activity at low concentrations along with thermal, pH and proteolytic resistance. There are twenty experimentally confirmed circular bacteriocins as part of bacteriocin gene clusters, with transport, membrane and immunity proteins.

, the Cause of Spotty Liver Disease in Chickens: Transmission and Routes of Infection.

The epidemiology of Spotty Liver Disease (SLD) was investigated by assaying 1,840 samples collected from layer chickens and the environment in poultry farms across Australia for the presence of , the agent responsible SLD in chickens. A specific PCR and bacterial culture were used. Results showed that birds could be infected with up to 8 weeks before clinical SLD was manifested.

Survival Mechanisms of Identified by Genomic Analysis and Comparative Transcriptomic Analysis of and Derived Bacteria.

Chickens infected with or are largely asymptomatic, however, infection with the closely related species, , can result in Spotty Liver Disease (SLD). has been detected in the liver, bile, small intestine and caecum of SLD affected chickens. The survival and colonization mechanisms that uses to colonize chickens remain unknown.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

Background: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C.