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

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

Nicotinamide Riboside Derivatives: Single Crystal Growth and Determination of X-ray Structures

For the first time, the X-ray structure of nicotinamide riboside could be determined. Five nicotinamide riboside (NR) derivatives in their native, thioamide and triacetyl protected form could be crystallized as their chloride and bromide salts. The single crystals were obtained with the help of the vapor diffusion technique. The use of the much slower layering technique for crystallization led to the decomposition of the nicotinamide ribosides yielding the corresponding nicotinamide salts. The χ torsion angles of the five nicotinamide riboside derivatives were compared with those obtained from the two nicotinamide adenine dinucleotide crystal and the three protein nicotinamide riboside cocrystal structures

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

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

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

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

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 β-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 β-gal activity with enhanced sensitivity in E. coli bacterial assays

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

Chemiluminescence is a fascinating phenomenon involving 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 decomposition of 1,2-dioxetane luminophores. DFT computational simulations provided support for the hypothesis that the spiro-cyclobutyl 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 β-galactosidase, exhibited a Limit-of-Detection value that is 125-fold more sensitive than the previously described adamantyl analogue. This probe was also able to instantly detect and image β-gal activity with enhanced sensitivity in E. coli bacterial assays