Resistance and Adaptation of Bacteria to Non-Antibiotic Antibacterial Agents: Physical Stressors, Nanoparticles, and Bacteriophages.

Antimicrobial resistance is a significant threat to human health worldwide, forcing scientists to explore non-traditional antibacterial agents to support rapid interventions and combat the emergence and spread of drug resistant bacteria. Many new antibiotic-free approaches are being developed while the old ones are being revised, resulting in creating unique solutions that arise at the interface of physics, nanotechnology, and microbiology. Specifically, physical factors (e.g., pressure, temperature, UV light) are increasingly used for industrial sterilization. Nanoparticles (unmodified or in combination with toxic compounds) are also applied to circumvent in vivo drug resistance mechanisms in bacteria. Recently, bacteriophage-based treatments are also gaining momentum due to their high bactericidal activity and specificity. Although the number of novel approaches for tackling the antimicrobial resistance crisis is snowballing, it is still unclear if any proposed solutions would provide a long-term remedy. This review aims to provide a detailed overview of how bacteria acquire resistance against these non-antibiotic factors. We also discuss innate bacterial defense systems and how bacteriophages have evolved to tackle them.

Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics.

To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

Probing single-cell metabolism reveals prognostic value of highly metabolically active circulating stromal cells in prostate cancer.

Despite their important role in metastatic disease, no general method to detect circulating stromal cells (CStCs) exists. Here, we present the Metabolic Assay-Chip (MA-Chip) as a label-free, droplet-based microfluidic approach allowing single-cell extracellular pH measurement for the detection and isolation of highly metabolically active cells (hm-cells) from the tumor microenvironment. Single-cell mRNA-sequencing analysis of the hm-cells from metastatic prostate cancer patients revealed that approximately 10% were canonical EpCAM+ hm-CTCs, 3% were EpCAM- hm-CTCs with up-regulation of prostate-related genes, and 87% were hm-CStCs with profiles characteristic for cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells. Kaplan-Meier analysis shows that metastatic prostate cancer patients with more than five hm-cells have a significantly poorer survival probability than those with zero to five hm-cells. Thus, prevalence of hm-cells is a prognosticator of poor outcome in prostate cancer, and a potentially predictive and therapy response biomarker for agents cotargeting stromal components and preventing epithelial-to-mesenchymal transition.

Single-Cell Analysis Using Droplet Microfluidics.

Droplet microfluidics has revolutionized the study of single cells. The ability to compartmentalize cells within picoliter droplets in microfluidic devices has opened up a wide range of strategies to extract information at the genomic, transcriptomic, proteomic, or metabolomic level from large numbers of individual cells. Studying the different molecular landscapes at single-cell resolution has provided the authors with a detailed picture of intracellular heterogeneity and the resulting changes in cellular phenotypes. In addition, these technologies have aided in the discovery of rare cells in tumors or in the immune system, and left the authors with a deeper understanding of the fundamental biological processes that determine cell fate. This review aims to provide a detailed overview of the various droplet microfluidic strategies reported in the literature, taking into account the sometimes subtle differences in workflow or reagents that enable or improve certain protocols. Specifically, approaches to targeted- and whole-genome analysis, as well as whole-transcriptome profiling techniques, are reviewed. In addition, an up-to-date overview of new methods to characterize and quantify single-cell protein levels, and of developments to screen secreted molecules such as antibodies, cytokines, or metabolites at the single-cell level, is provided.

Phenotypic plasticity of Escherichia coli upon exposure to physical stress induced by ZnO nanorods.

Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed E. coli to a large number of collisions of around 105 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria's shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such E. coli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in ß-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Photoactive Langmuir-Blodgett, Freely Suspended and Free Standing Films of Carboxylate Ligand-Coated ZnO Nanocrystals.

A new possibility for the formation of macroscopic and photoactive structures from zinc oxide nanocrystals is described. Photoactive freely suspended and free-standing films of macroscopic area (up to few square millimeters) and submicrometer thickness (up to several hundreds of nanometers) composed of carboxylate ligand-coated zinc oxide nanocrystallites (RCO2-ZnO NCs) of diameter less than 5 nm are prepared according to a modified Langmuir-Schaefer method. First, the suspension of RCO2-ZnO NCs is applied onto the air/water interface. Upon compression, the films become turbid and elastic. The integrity of such structures is ensured by interdigitation of ligands stabilizing ZnO NCs. Great elasticity allows transfer of the films onto a metal frame as a freely suspended film. Such membranes are afterward extracted from the supporting frame to form free-standing films of macroscopic area. Because the integrity of the films is maintained by ligands, no abolishment of quantum confinement occurs, and films retain spectroscopic properties of initial RCO2-ZnO NCs. The mechanism of formation of thin films of RCO2-ZnO NCs at the air/water interface is discussed in detail.

Influence of nanomechanical stress induced by ZnO nanoparticles of different shapes on the viability of cells.

There is growing interest in nanostructures interacting with living organisms. However, there are still no general rules for the design of biocompatible nanodevices. Here, we present a step towards understanding the interactions between nanostructures and living cells. We study the influence of nanomechanical stress induced by zinc oxide (ZnO) nanostructures of different shapes on the viability of both prokaryotic (Gram-negative bacteria: Escherichia coli and Enterobacter aerogenes, and Gram-positive bacteria: Staphylococcus epidermidis and Corynebacterium glutamicum) and eukaryotic cells (yeast Saccharomyces cerevisiae and liver cancer cell line HepG2). Nanoparticles (NPs) and nanorods (NRs) of matching crystallographic structure (P63mc) and active surface area (in the order of 5 × 10(-2)µm(2)) are almost non-toxic for cells under static conditions. However, under conditions that enable collisions between ZnO nanostructures and cells, NRs appear to be more damaging compared to NPs. This is due to the increased probability of mechanical damage caused by nanorods upon puncturing of the cell wall and membranes. Gram-positive bacteria, which have thicker cell walls, are more resistant to nanomechanical stress induced by NRs compared to Gram-negative strains and eukaryotic cells. The presented results may be exploited to improve the properties of nanotechnology based products such as implants, drug delivery systems, antibacterial emulsions and cosmetics.

Towards Organized Hybrid Nanomaterials at the Air/Water Interface Based on Liquid-Crystal/ZnO Nanocrystals.

The ability to self-assemble nanosized ligand-stabilized metal oxide or semiconductor materials offers an intriguing route to engineer nanomaterials with new tailored properties from the disparate components. We describe a novel one-pot two-step organometallic approach to prepare ZnO nanocrystals (NCs) coated with deprotonated 4-(dodecyloxy)benzoic acid (i.e., an X-type liquid-crystalline ligand) as a model LC system (termed ZnO-LC1 NCs). Langmuir and Langmuir-Blodgett films of the resulting hybrids are investigated. The observed behavior of the ZnO NCs at the air/water interface is rationalized by invoking a ZnO-interdigitation process mediated by the anchored liquid-crystalline shell. The ordered superstructures form according to mechanism based on a ZnO-interdigitation process mediated by liquid crystals (termed ZIP-LC). The external and directed force applied upon compression at the air/water interface and the packing of the ligands that stabilize the ZnO cores drives the formation of nanorods of ordered internal structure. To study the process in detail, we follow a nontraditional protocol of thin-film investigation. We collect the films from the air/water interface in powder form (ZnO-LC1 LB), resuspend the powder in organic solvents and utilize otherwise unavailable experimental techniques. The structural and physical properties of the resulting superlattices were studied by using electron microscopy, atomic force microscopy, X-ray studies, dynamic light scattering, thermogravimetric analysis, UV/Vis absorption, and photoluminescence spectroscopy.