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SummaryOrdered collections of precise gene deletions inE. coliand other model microbes have proven to be invaluable resources to study the dispensability of the non-essential genome in different environmental and genetic contexts. The dispensability of canonically essential genes, however, has been largely unstudied in such contexts. Advances in gene editing, in particular CRISPR interference (CRISPRi), have enabled depletion of essential cellular machinery to study the downstream effects on bacterial physiology. Here, we describe the construction of an orderedE. coliCRISPRi collection, designed to knock down the expression of 356 essential genes with the induction of a catalytically inactive Cas9, harbored on the conjugative plasmid pFD152, that also encodes for expression of specific guide RNA scaffolds. This mobile CRISPRi library can be conjugated into other ordered genetic libraries, to assess combined effects of essential gene knockdowns with non-essential gene deletions. As proof of concept, we probed cell envelope synthesis with two crosses. First, we crossed a deletion in the genelppwith the entire CRISPRi library. Similarly, we crossed the CRISPRi knockdown oflolAwith ∼4,000 deletion strains of the Keio collection. These experiments revealed a number of notable genetic interactions for the essential phenotype probed and, in particular, showed supressing interactions for the loci in question.

Engineered surfaces that repel pathogens are of great interest due to their role in mitigating the spread of infectious diseases. A robust, universal, and scalable omniphobic spray coating with excellent repellency against water, oil, and pathogens is presented. The coating is substrate-independent and relies on hierarchically structured polydimethylsiloxane (PDMS) microparticles, decorated with gold nanoparticles (AuNPs). Wettability studies reveal the relationship between surface texturing of micro- and/or nano-hierarchical structures and the omniphobicity of the coating. Studies of pathogen transfer with bacteria and viruses reveal that an uncoated contaminated glove transfers pathogens to50 subsequent surfaces, while a coated glove picks up 10

The Comprehensive Antibiotic Resistance Database (CARD; card.mcmaster.ca) combines the Antibiotic Resistance Ontology (ARO) with curated AMR gene (ARG) sequences and resistance-conferring mutations to provide an informatics framework for annotation and interpretation of resistomes. As of version 3.2.4, CARD encompasses 6627 ontology terms, 5010 reference sequences, 1933 mutations, 3004 publications, and 5057 AMR detection models that can be used by the accompanying Resistance Gene Identifier (RGI) software to annotate genomic or metagenomic sequences. Focused curation enhancements since 2020 include expanded β-lactamase curation, incorporation of likelihood-based AMR mutations for Mycobacterium tuberculosis, addition of disinfectants and antiseptics plus their associated ARGs, and systematic curation of resistance-modifying agents. This expanded curation includes 180 new AMR gene families, 15 new drug classes, 1 new resistance mechanism, and two new ontological relationships: evolutionary_variant_of and is_small_molecule_inhibitor. In silico prediction of resistomes and prevalence statistics of ARGs has been expanded to 377 pathogens, 21,079 chromosomes, 2,662 genomic islands, 41,828 plasmids and 155,606 whole-genome shotgun assemblies, resulting in collation of 322,710 unique ARG allele sequences. New features include the CARD:Live collection of community submitted isolate resistome data and the introduction of standardized 15 character CARD Short Names for ARGs to support machine learning efforts.

The surface fouling of biomedical devices has been an ongoing issue in healthcare. Bacterial and blood adhesion in particular, severely impede the performance of such tools, leading to poor patient outcomes. Various structural and chemical modifications have been shown to reduce fouling, but all existing strategies lack the combination of physical, chemical, and economic traits necessary for widespread use. Herein, a lubricant infused, hierarchically micro- and nanostructured polydimethylsiloxane surface is presented. The surface is easy to produce and exhibits the high flexibility and optical transparency necessary for incorporation into various biomedical tools. Tests involving two clinically relevant, priority pathogens show up to a 98.5% reduction in the biofilm formation of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. With blood, the surface reduces staining by 95% and suppresses thrombin generation to background levels. Furthermore, the surface shows applicability within applications such as catheters, extracorporeal circuits, and microfluidic devices, through its effectiveness in dynamic conditions. The perfusion of bacterial media shows up to 96.5% reduction in bacterial adhesion. Similarly, a 95.8% reduction in fibrin networks is observed following whole blood perfusion. This substrate stands to hold high applicability within biomedical systems as a means to prevent fouling, thus improving performance.

Healthcare acquired infections are a major human health problem, and are becoming increasingly troublesome with the emergence of drug resistant bacteria. Engineered surfaces that reduce the adhesion, proliferation, and spread of bacteria have promise as a mean of preventing infections and reducing the use of antibiotics. To address this need, we created a flexible plastic wrap that combines a hierarchical wrinkled structure with chemical functionalization to reduce bacterial adhesion, biofilm formation, and the transfer of bacteria through an intermediate surface. These hierarchical wraps were effective for reducing biofilm formation of World Health Organization-designated priority pathogens Gram positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram negative Pseudomonas aeruginosa by 87 and 84%, respectively. In addition, these surfaces remain free of bacteria after being touched by a contaminated surface with Gram negative E. coli. We showed that these properties are the result of broad liquid repellency of the engineered surfaces and the presence of reduced anchor points for bacterial adhesion on the hierarchical structure. Such wraps are fabricated using scalable bottom-up techniques and form an effective cover on a variety of complex objects, making them superior to top-down and substrate-specific surface modification methods.