Spirostrain-Accelerated Chemiexcitation of Dioxetanes Yields Unprecedented Detection Sensitivity in Chemiluminescence Bioassays.

Chemiluminescence is a fascinating phenomenon that involves the generation of light through chemical reactions. The light emission from adamantyl-phenoxy-1,2-dioxetanes can glow from minutes to hours depending on the specific substituent present on the dioxetane molecule. In order to improve the light emission properties produced by these chemiluminescent luminophores, it is necessary to induce the chemiexcitation rate to a flash mode, wherein the bulk of light is emitted instantly rather than slowly over time. We report the realization of this goal through the incorporation of spirostrain release into the decomposition of 1,2-dioxetane luminophores. DFT computational simulations provided support for the hypothesis that the spiro-cyclobutyl substituent accelerates chemiexcitation as compared to the unstrained adamantyl substituent. Spiro-linking of cyclobutane and oxetane units led to greater than 100-fold and 1000-fold emission enhancement, respectively. This accelerated chemiexcitation rate increases the detection sensitivity for known chemiluminescent probes to the highest signal-to-noise ratio documented to date. A turn-ON probe, containing a spiro-cyclobutyl unit, for detecting the enzyme ß-galactosidase exhibited a limit of detection value that is 125-fold more sensitive than that for the previously described adamantyl analogue. This probe was also able to instantly detect and image ß-gal activity with enhanced sensitivity in E. coli bacterial assays.

Chromogenic culture media complements diagnostic cytology in the visual identification of pathogenic skin bacteria in dogs and cats.

In dogs and cats, bacterial skin infections (pyoderma and otitis externa) are a common cause for visiting the veterinary clinic. The most frequent skin pathogens are Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, often requiring different therapeutic antibiotic protocols. Unfavorably, existing diagnostics based on cytology cannot reveal bacterial species but only bacterial shapes such as cocci or rods. This microscopic limitation could be overcome by clinical translation of affordable chromogenic media, which enable species identification based on bacterial colonies growing in different colors and sizes. In this study, we determined how well inexperienced general veterinary clinicians identified bacterial pathogens from the skin and ears on two commercial (Chromatic™ MH and Flexicult® Vet) and one custom-made Mueller Hinton agar-based chromogenic medium. For this purpose, four veterinarians evaluated 100 unique samples representing 10 bacterial species. On average, clinicians correctly identified between 72.1 and 86.3% of bacterial species. Colony colors developed quickly on the Chromatic™ MH medium, leading to the highest 81.6% identification accuracy after 24 h incubation. However, Flexicult® Vet exhibited the highest accuracy of 86.3% after prolonged 48 h incubation. Evaluators easily recognized bacteria displaying uniquely colored colonies like green-brown Pseudomonas aeruginosa, blue Enterococcus faecalis, orange-brown Proteus spp., and red Escherichia coli. Oppositely, staphylococci shared uncharacteristically pale pink colonies causing misidentifications among the genus, deteriorating overall accuracy by around 10 percentage points (from 90.9%). Another reason for identification errors was the evaluators' inexperience, reflected in not recognizing colony size differences. For example, although Streptococcus canis exhibited the tiniest colonies, the species was frequently mistaken for other cocci. Finally, around 10% of errors were negligence-related slips due to unconsidered sample history. To conclude, the introduction of chromogenic media into veterinary clinics can significantly complement diagnostics in skin inflammations by identifying pathogen species in around 80% of cases. The extra information may help in therapeutic dilemmas on antibiotics and standard antimicrobial susceptibility testing. Additional personnel training and evaluation help by visuals, flowcharts, checklists, and, if necessary, microbiologists could further improve identification accuracy.

Fluorogenic in vitro activity assay for the main protease Mpro from SARS-CoV-2 and its adaptation to the identification of inhibitors.

This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET (Fluorescence Resonance Energy Transfer) substrates. The protocol is based on an aminomethyl coumarin substrate. High sensitivity, specificity, and an easily detectable fluorescent read-out are the advantages offered by this rapid assay, which allows high throughput screening of new Mpro inhibitors.

New Crystalline Salts of Nicotinamide Riboside as Food Additives.

NR+ is a highly effective vitamin B3 type supplement due to its unique ability to replenish NAD+ levels. While NR+ chloride is already on the market as a nutritional supplement, its synthesis is challenging, expensive, and low yielding, making it cumbersome for large-scale industrial production. Here we report the novel crystalline NR+ salts, d/l/dl-hydrogen tartrate and d/l/dl-hydrogen malate. Their high-yielding, one-pot manufacture does not require specific equipment and is suitable for multi-ton scale production. These new NR+ salts seem ideal for nutritional applications due to their bio-equivalence compared to the approved NR+ chloride. In addition, the crystal structures of all stereoisomers of NR+ hydrogen tartrate and NR+ hydrogen malate and a comparison to the known NR+ halogenides are presented.

Chemiluminescence Detection of Hydrogen Sulfide Release by ß-Lactamase-Catalyzed ß-Lactam Biodegradation: Unprecedented Pathway for Monitoring ß-Lactam Antibiotic Bacterial Resistance.

ß-Lactamase positive bacteria represent a growing threat to human health because of their resistance to commonly used antibiotics. Therefore, development of new diagnostic methods for identification of ß-lactamase positive bacteria is of high importance for monitoring the spread of antibiotic-resistant bacteria. Here, we report the discovery of a new biodegradation metabolite (H2S), generated through ß-lactamase-catalyzed hydrolysis of ß-lactam antibiotics. This discovery directed us to develop a distinct molecular technique for monitoring bacterial antibiotic resistance. The technique is based on a highly efficient chemiluminescence probe, designed for detection of the metabolite, hydrogen sulfide, that is released upon biodegradation of ß-lactam by ß-lactamases. Such an assay can directly indicate if antibiotic bacterial resistance exists for a certain examined ß-lactam. The assay was successfully demonstrated for five different ß-lactam antibiotics and eight ß-lactam resistant bacterial strains. Importantly, in a functional bacterial assay, our chemiluminescence probe was able to clearly distinguish between a ß-lactam resistant bacterial strain and a sensitive one. As far as we know, there is no previous documentation for such a biodegradation pathway of ß-lactam antibiotics. Bearing in mind the data obtained in this study, we propose that hydrogen sulfide should be considered as an emerging ß-lactam metabolite for detection of bacterial resistance.

Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase.

Adenosine triphosphate (ATP) is one of the most important indicators of cell viability. Extracellular ATP (eATP) is commonly detected in cultures of both eukaryotic and prokaryotic cells but is not the focus of current scientific research. Although ATP release has traditionally been considered to mainly occur as a consequence of cell destruction, current evidence indicates that ATP leakage also occurs during the growth phase of diverse bacterial species and may play an important role in bacterial physiology. ATP can be conveniently measured with high sensitivity in luciferase-based bioluminescence assays. However, wild-type luciferases suffer from low stability, which limit their use. Here we demonstrate that an engineered, thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces. Different bacterial species show distinct patterns of eATP accumulation and decline. Real-time monitoring of eATP allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. Furthermore, the method is able to rapidly detect the effect of antibiotics on bacterial cultures as Ampicillin sensitive strains challenged with beta lactam antibiotics showed strongly increased accumulation of eATP even in the absence of growth, as determined by optical density. Patterns of eATP determined by real-time luminescence measurement could be used to infer the minimal inhibitory concentration of Ampicillin. Compared to conventional antibiotic susceptibility testing, the method presented here is faster and more sensitive, which is essential for better treatment outcomes and reducing the risk of inducing antibiotic resistance. Real-time eATP bioluminescence assays are suitable for different cell types, either prokaryotic or eukaryotic, thus, permitting their application in diverse fields of research. It can be used for example in the study of the role of eATP in physiology and pathophysiology, for monitoring microbial contamination or for antimicrobial susceptibility testing in clinical diagnostics.

Modified Enzyme Substrates for the Detection of Bacteria: A Review.

The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.

Nicotinamide Riboside-The Current State of Research and Therapeutic Uses.

Nicotinamide riboside (NR) has recently become one of the most studied nicotinamide adenine dinucleotide (NAD+) precursors, due to its numerous potential health benefits mediated via elevated NAD+ content in the body. NAD+ is an essential coenzyme that plays important roles in various metabolic pathways and increasing its overall content has been confirmed as a valuable strategy for treating a wide variety of pathophysiological conditions. Accumulating evidence on NRs' health benefits has validated its efficiency across numerous animal and human studies for the treatment of a number of cardiovascular, neurodegenerative, and metabolic disorders. As the prevalence and morbidity of these conditions increases in modern society, the great necessity has arisen for a rapid translation of NR to therapeutic use and further establishment of its availability as a nutritional supplement. Here, we summarize currently available data on NR effects on metabolism, and several neurodegenerative and cardiovascular disorders, through to its application as a treatment for specific pathophysiological conditions. In addition, we have reviewed newly published research on the application of NR as a potential therapy against infections with several pathogens, including SARS-CoV-2. Additionally, to support rapid NR translation to therapeutics, the challenges related to its bioavailability and safety are addressed, together with the advantages of NR to other NAD+ precursors.

Chemiluminescent Carbapenem-Based Molecular Probe for Detection of Carbapenemase Activity in Live Bacteria.

Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatment due to the acquisition of antibiotic resistance. This resistance can be largely attributed to the antibiotic-hydrolyzing enzymes that the bacteria produce. Current carbapenem "wonder drugs", such as doripenem, ertapenem, meropenem, imipenem, and so on, are resistant to regular ß-lactamases, but susceptible to carbapenemases. Even worse, extended exposure of bacteria to these drugs accelerates the spread of resistance genes. In order to preserve the clinical efficacy of antibacterial treatment, carbapenem drugs should be carefully regulated and deployed only in cases of a CPO infection. Early diagnosis is therefore of paramount importance. Herein, we report the design, synthesis, and activity of the first carbapenemase-sensitive chemiluminescent probe, CPCL, which may be used to monitor CPO activity. The design of our probe enables enzymatic cleavage of the carbapenem core, which is followed by a facile 1,8-elimination process and the emission of green light through rapid chemical excitation. We have demonstrated the ability of the probe to detect a number of clinically relevant carbapenemases and the successful identification of CPO present in bacterial cultures, such as those used for clinical diagnosis. We believe that our use of "turn-on" chemiluminescence activation will find significant application in future diagnostic assays and improve antibacterial treatment.

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small-Molecule Chemiluminescence Probes.

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.