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Bacterial infection is rising as a threatening health issue. Because of the present delay in early diagnosis of bacterial diseases as well as the abuse of antibiotics, it has become a vital issue in the development of in-time detection and therapy of bacterial infections. Herein, we designed a multifunctional nanotheranostics platform based on the unique micro-environment of bacterial infections to achieve specific bioimaging and simultaneous inactivation of the target bacteria. We showed that in bacterial infections, the metal precursors (i.e., HAuCl 4 , FeCl 2 , and herring sperm DNA) could be readily bio-self-assembled to multifunctional nanoclusters (NCs) that exhibit luminescence, in which AuCl 4 − was biosynthesized via reductive biomolecules such as NADPH to the fluorescent AuNCs. The DNA may assist as an encapsulation and delivery vector, and Fe 2+ served as a fluorescence intensifier and reduced reactive oxygen species (ROS) to produce the iron oxides. While the bacteria were being visualized, the microenvironment-responsive NCs were enabled to sterilize bacteria efficiently due to electrostatic effect, cell membrane destruction, inhibition of biofilm formation, and ROS accumulation. Besides, the bio-responsive self-assembled NCs complexes contributed to accelerating bacteria-infected wound healing and showed negligible side effects in long-term toxicity tests in vivo. Also, intracellular molecules involved in microenvironmental response were investigated. The work may become an effective strategy for the detection and real-time sterilization of intractable bacterial infections.

High bacterial loads within chronic wounds increase the risk of infection and complication. Detection and localization of bacterial loads through point‐of‐care fluorescence (FL) imaging can objectively inform and support bacterial treatment decisions. This single time‐point, retrospective analysis describes the treatment decisions made on 1000 chronic wounds (DFUs, VLUs, PIs, surgical wounds, burns, and others) at 211 wound‐care facilities across 36 US states. Clinical assessment findings and treatment plans derived from them, as well as subsequent FL‐imaging (MolecuLight®) findings and any associated treatment plan changes, were recorded for analysis. FL signals indicating elevated bacterial loads were observed in 701 wounds (70.8%), while only 293 (29.6%) showed signs/symptoms of infection. After FL‐imaging, treatment plans changed in 528 wounds as follows: more extensive debridement (18.7%), more extensive hygiene (17.2%), FL‐targeted debridement (17.2%), new topical therapies (10.1%), new systemic antibiotic prescriptions (9.0%), FL‐guided sampling for microbiological analysis (6.2%), and changes in dressing selection (3.2%). These real‐world findings of asymptomatic bacterial load/biofilm incidence, and of the frequent treatment plan changes post‐imaging, are in accordance with clinical trial findings using this technology. These data, from a range of wound types, facilities, and clinician skill sets, suggest that point‐of‐care FL‐imaging information improves bacterial infection management.

Introduction: Biofilm is linked through a variety of mechanisms to the pathogenesis of chronic wounds. However, accurate biofilm detection is challenging, demanding highly specialized and technically complex methods rendering it unapplicable for most clinical settings. This study evaluated promising methods of bedside biofilm localization, fluorescence imaging of wound bacterial loads, and biofilm blotting by comparing their performance against validation scanning electron microscopy (SEM). Methods: In this clinical trial, 40 chronic hard-to-heal wounds underwent the following assessments: (1) clinical signs of biofilm (CSB), (2) biofilm blotting, (3) fluorescence imaging for localizing bacterial loads, wound scraping taken for (4) SEM to confirm matrix encased bacteria (biofilm), and (5) PCR (Polymerase Chain Reaction) and NGS (Next Generation Sequencing) to determine absolute bacterial load and species present. We used a combination of SEM and PCR microbiology to calculate the diagnostic accuracy measures of the CSB, biofilm blotting assay, and fluorescence imaging. Results: Study data demonstrate that 62.5% of wounds were identified as biofilm-positive based on SEM and microbiological assessment. By employing this method to determine the gold truth, and thus calculate accuracy measures for all methods, fluorescence imaging demonstrated superior sensitivity (84%) and accuracy (63%) compared to CSB (sensitivity 44% and accuracy 43%) and biofilm blotting (sensitivity 24% and accuracy 40%). Biofilm blotting exhibited the highest specificity (64%), albeit with lower sensitivity and accuracy. Using SEM alone as the validation method slightly altered the results, but all trends held constant. Discussion: This trial provides the first comparative assessment of bedside methods for wound biofilm detection. We report the diagnostic accuracy measures of these more feasibly implementable methods versus laboratory-based SEM. Fluorescence imaging showed the greatest number of true positives (highest sensitivity), which is clinically relevant and provides assurance that no pathogenic bacteria will be missed. It effectively alerted regions of biofilm at the point-of-care with greater accuracy than standard clinical assessment (CSB) or biofilm blotting paper, providing actionable information that will likely translate into enhanced therapeutic approaches and better patient outcomes.

Rapid antimicrobial susceptibility tests (ASTs) are essential tool for proper treatment of patients infected by Yersinia pestis (Y. pestis), the causative agent of plague, or for post-exposure prophylaxis of a population exposed to a naturally acquired or deliberately prepared resistant variant. The standard AST of Y. pestis is based on bacterial growth and requires 24–48 h of incubation in addition to the time required for prior isolation of a bacterial culture from the clinical or environmental sample, which may take an additional 24–48 h. In this study, we present a new and rapid AST method based on a fluorescence determination of the minimum inhibitory concentration (MIC). Our method includes the incubation of bacteria with an antibiotic, followed by staining of the bacteria with oxonol dye (SynaptoGreen C4/FM1–43), which enables the rapid detection of an antibiotic’s effect on bacterial viability. We show that stained, non-viable bacteria exhibit a spectral redshift and an increase in fluorescence intensity compared to intact control bacteria. Based on these criteria, we developed a rapid flow cytometer measurement procedure and a unique spectral intensity ratio (SIR) analysis that enables determination of antibiotic susceptibility for Y. pestis within 6 h instead of the 24 to 48 h required for the standard AST. This new rapid determination of antibiotic susceptibility could be crucial for reducing mortality and preventing the spread of disease.

Experiments were conducted to investigate the feasibility of applying constructed wetlands (CW) to treat a sanitary landfill leachate containing high nitrogen (TN) and bacterial contents. Under the tropical conditions (temperature of about 30 °C), the CW units operating at a hydraulic retention time (HRT) of 8 days yielded the best treatment efficiencies with BOD5 removal of 91%, TN removal of 96%, total and fecal coliforms (TC and FC) removal of more than 99%. Cadmium removal in the in the SFCW bed was found to be 99.7%. Mass balance analysis, based on TN contents of the plant biomass and dissolved oxygen (DO) and oxidation - reduction potential (ORP) values, suggested that 88% of the input TN were uptaken by the plant biomass. Fluorescence in situ hybridization (FISH) results revealed the predominance of bacteria including the heterotrophic and autotrophic bacteria responsible for BOD5 removal. Nitrifying bacteria was not found to be present in the SSFCW beds.

Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF₁-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF₁-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Key Points Single-molecule imaging of fluorescent fusion proteins can be used to track individual biomolecules that are moving in bacterial cells and to probe their diffusion or their directed or confined motion, which provides insights into various dynamic cellular processes. Single-molecule imaging, when combined with a method that actively controls the concentration of emitting fluorescent proteins, can be used to achieve super-resolution. Thus, it is now possible to visualize previously hidden structural details of protein localization patterns with a resolution that is no longer limited by the diffraction of light. Single-molecule and single-particle tracking have shown that the actin homologue MreB moves in a circumferential pattern around the bacterial cell, driven by cell wall synthesis. Other cytoskeletal protein structures, such as the FtsZ ring and PopZ nanodomains, have been characterized at subdiffraction spatial resolution. Super-resolution imaging has shown that some nucleoid-associated proteins bind DNA and form well-defined organizational regions, whereas others localize more randomly throughout the nucleoid. These data provide information about the spatial organization of the chromosome. The activities of DNA repair enzymes in live cells have been revealed by a combination of single-molecule tracking and single-molecule super-resolution imaging, which show a clear increase in repair rates under conditions of increased DNA damage. The spatial organization of transcription and translation has been investigated using single-particle tracking and super-resolution imaging of ribosomes and RNA polymerase, revealing clear differences between model organisms. Single-molecule imaging has shown that individual transcription factors search for their target DNA sequence by a combination of one-dimensional sliding along DNA and three-dimensional diffusive hopping between DNA stands. Here, Gahlmann and Moerner describe single-molecule imaging in live bacterial cells, which has transformed the study of bacterial cell biology. They discuss the insights that have been gained about the bacterial cytoskeleton, nucleoid organization and chromosome segregation and partitioning, as well as transcription and translation. The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Key Points The Bacillus subtilis spore coat is a multilayered protective structure composed of more than 70 different proteins. In addition to its protective role, the spore coat influences the process of spore germination and defines the type of interactions that spores can establish with various surfaces in the environment. Fluorescence microscopy in combination with high-resolution image analysis has produced a spatially scaled coat protein interaction network indicating that the coat is organized into four distinct layers. These studies led to the discovery of the outermost layer of the coat in B. subtilis , referred to as the spore crust. Time course analyses of spore coat assembly have revealed that two main steps can be distinguished in coat morphogenesis: the initial recruitment of proteins to the spore surface as a scaffold cap, followed by spore encasement in a series of successive waves. Coat assembly is regulated at the transcriptional level by the sequential expression of individual coat genes and at the protein level by a small group of coat morphogenetic proteins that coordinate both the recruitment of coat proteins to specific coat layers and spore encasement. Sporulation in Bacillus subtilis results in the formation of an endospore surrounded by a multilayered protective structure, known as the coat. In this Review, Patrick Eichenberger and colleagues describe recent studies that have illuminated the architecture of the coat and the dynamics of coat assembly. Sporulation in Bacillus subtilis involves an asymmetric cell division followed by differentiation into two cell types, the endospore and the mother cell. The endospore coat is a multilayered shell that protects the bacterial genome during stress conditions and is composed of dozens of proteins. Recently, fluorescence microscopy coupled with high-resolution image analysis has been applied to the dynamic process of coat assembly and has shown that the coat is organized into at least four distinct layers. In this Review, we provide a brief summary of B. subtilis sporulation, describe the function of the spore surface layers and discuss the recent progress that has improved our understanding of the structure of the endospore coat and the mechanisms of coat assembly.

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue–green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs. Near-infrared (NIR) fluorescent proteins (FPs) offer advantages for spectral multiplexing and deep-tissue imaging. Here the authors engineer a smaller NIR FP based on the unexplored cyanobacteriochrome photoreceptor and demonstrate its use in various applications in cell culture as well as whole-body imaging in vivo in mice.