Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

Rapid species identification of pathogenic bacteria from a minute quantity exploiting three-dimensional quantitative phase imaging and artificial neural network.

The healthcare industry is in dire need of rapid microbial identification techniques for treating microbial infections. Microbial infections are a major healthcare issue worldwide, as these widespread diseases often develop into deadly symptoms. While studies have shown that an early appropriate antibiotic treatment significantly reduces the mortality of an infection, this effective treatment is difficult to practice. The main obstacle to early appropriate antibiotic treatments is the long turnaround time of the routine microbial identification, which includes time-consuming sample growth. Here, we propose a microscopy-based framework that identifies the pathogen from single to few cells. Our framework obtains and exploits the morphology of the limited sample by incorporating three-dimensional quantitative phase imaging and an artificial neural network. We demonstrate the identification of 19 bacterial species that cause bloodstream infections, achieving an accuracy of 82.5% from an individual bacterial cell or cluster. This performance, comparable to that of the gold standard mass spectroscopy under a sufficient amount of sample, underpins the effectiveness of our framework in clinical applications. Furthermore, our accuracy increases with multiple measurements, reaching 99.9% with seven different measurements of cells or clusters. We believe that our framework can serve as a beneficial advisory tool for clinicians during the initial treatment of infections.

Delivery of antisense oligonucleotides using multi-layer coated gold nanoparticles to methicillin-resistant S. aureus for combinatorial treatment.

The spread of multidrug-resistant (MDR) bacterial infections has become a serious global threat. We introduce multi-layer coated gold nanoparticles (MLGNPs) delivering antisense oligonucleotides (ASOs) targeting the resistance gene of methicillin-resistant Staphylococcus aureus (MRSA), as a selective antimicrobial by restoring susceptibility. MLGNPs were prepared by multi-step surface immobilization of gold nanoparticles (GNPs) with polyethylenimine (PEI) and loaded with ASO targeting the mecA gene. The MLGNPs were shown to be efficiently internalized into various types of Gram-positive bacteria, including MRSA, Staphylococcus epidermidis, and Bacillus subtilis, which was superior to single-layer coated GNPs and free PEI polymer. The delivery of MLGNPs into MRSA resulted in up to 74% silencing of the mecA gene with high selectivity, in a dose-dependent manner. The treatment of MLGNPs to MRSA in the presence of oxacillin, a beta-lactam antibiotic, showed major suppression (~71%) of bacterial growth, due to the recovery of antibacterial sensitivity. Furthermore, the treatment of MLGNPs in a complex system showed preferential uptake into bacteria over mammalian cells, demonstrating the suitable characteristics of MLGNPs for selective delivery into bacteria. The current approach can be potentially applied for targeting various types of MDR bacterial infections by specific silencing of a resistance gene, as a combinatorial therapeutic used with conventional antibiotics.

Simple visualized readout of suppressed coffee ring patterns for rapid and isothermal genetic testing of antibacterial resistance.

We present a facile method based on the coffee ring effect that can rapidly detect antibiotic-resistant bacteria, as an affordable genetic testing platform. When a colloidal solution of particles is dropped onto a substrate surface, an outward capillary flow upon evaporation induces the migration of the particles to the periphery of the droplet, forming a characteristic ring pattern. Herein, we utilize capture DNA microbeads which in the presence of target nucleic acid, form suppressed ring patterns by hybridization-induced crosslinking of the microbeads. The coffee ring-based assay is integrated with isothermal amplification based on rolling circle amplification (RCA), to produce long, single-stranded target DNA and induce hybridization, via a one-step procedure (i-CoRi assay). The resultant ring patterns can be simply observed with the naked eye or recorded with a standard mobile device for readout. The i-CoRi assay was validated for the rapid and specific detection of the antibiotic resistance gene mecA for MRSA, showing that detection was possible at the sub-zeptomolar range (~0.2 zM) with the specificity of distinguishing 2 mismatched bases. The spatial patterns of the microbeads were characterized, showing the dense packing of the microbeads at the center of the droplet and thinning of the ring pattern for the MRSA target, which were distinct from the negative controls MSSA, E. coli, and P. aeruginosa. The images of the microbead patterns were also processed by a simple readout algorithm to discriminate the presence or absence of the coffee ring, to enable diagnostic decision making. The current method provides a rapid and versatile platform for the specific identification of bacterial pathogens and multidrug resistance, especially for diagnosis in resource-limited settings.

Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography.

Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug discovery, clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial resolution.

Ultra-fast and universal detection of Gram-negative bacteria in complex samples based on colistin derivatives.

Gram-negative bacteria are a significant cause of infections acquired in both hospital and community settings, resulting in a high mortality rate worldwide. Currently, a Gram-negative infection is diagnosed by symptom evaluation and is treated with empiric antibiotics which target both Gram-negative and Gram-positive bacteria. A rapid and simple diagnostic method would enable immediate and targeted treatment, while dramatically reducing antibiotic overuse. Herein, we introduce a method utilizing a fluorescent derivative of colistin (COL-FL), that can directly label the Gram-negative cell wall of live bacteria and universally detect the targets within 10 min. By using the COL-FL assay, we achieved the differential labeling of various Gram-negative pathogens related to hospital-acquired infections, which could be subsequently detected via spectrofluorometry and microscopy. Further, we determined that our method can be used for complex samples, such as combinations of multiple bacterial types; bacteria in the presence of mammalian cells; and bacteria with serum components. This assay can be integrated into a simple diagnostic platform for rapid screening tests and the stratification of Gram-negative bacterial infections in the clinic.

Rapid naked-eye detection of Gram-positive bacteria by vancomycin-based nano-aggregation.

Development of a rapid, point-of-care assay for diagnosing bacterial infections is crucial for subsequent treatment of the patient and preventing the overuse of antibiotics. Herein, we describe a rapid, one-step colorimetric assay based on the formation of nano-aggregates using nanobeads targeting Gram-positive bacteria. Vancomycin was immobilized onto blue-colored polymeric nanobeads to induce specific and multivalent binding with the Gram-positive bacterial cell wall and subsequent agglomeration. Without any pre-processing steps, the addition of various types of Gram-positive pathogens to the nanobeads resulted in the formation of blue precipitates, which could be observed with the naked eye in ∼30 min. We also utilized a porous filter system for the assay, which allowed discrimination of Gram-positive targets with higher selectivity, and demonstrated feasibility as a simple diagnostic assay with minimal technical components. We anticipate that the nanobead aggregation assay can be potentially applied as a rapid and simple sensing platform, which can be easily miniaturized and enable point-of-care diagnosis of Gram-positive infections.

A Carbon-Dot-Based Fluorescent Nanosensor for Simple Visualization of Bacterial Nucleic Acids.

A simple and facile method for sensing of nucleic acids is in great need for disease biomarker detection and diagnosis. Herein, a fluorescent nanosensor utilizing carbon dot nanoparticles is introduced that form visible precipitates in the presence of target DNA. Carbon dot nanoparticles are fabricated by microwave pyrolysis of polyethylenimine, which emits strong photoluminescence and can form precipitates when added to target DNA oligonucleotides. The precipitates can be easily visualized by UV illumination, and data can be acquired as images using a smartphone, which are analyzed for quantification. This carbon-dot-based assay allowed fluorescent sensing of target oligonucleotides with various sizes and visualization even with minimal amount of DNA (≈100 pmol). Finally, the assay can be applied as a nanosensor platform for detecting bacterial DNA for the antibiotic-resistance gene KPC-2 from Klebsiella pneumoniae. This method provides a simple technique for detecting molecular targets, showing wide applicability for diagnostics on the bedside or point-of-care testing.