Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity

Abstract

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli.

Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine

“…In soil, plasmid DNA degradation and the loss of transformation ability have been attributed to microorganisms . Bacteria, for example, can metabolize DNA for microbial growth and produce enzymes that disrupt DNA. , Here, removing material greater than 0.22 μm impacted the inactivation of the plasmid in the aged urine. Although most bacteria are larger than 0.22 μm, some are small enough to pass through 0.22 μm pores .…”

“…Although most bacteria are larger than 0.22 μm, some are small enough to pass through 0.22 μm pores . Extracellular nucleases are naturally excreted from bacteria and can remain active after the bacteria are removed . Nuclease enzymes can vary in size (∼20 up to 400 kDa), structure, and function and exhibit a range of sensitivities to elevated temperatures and pH values. − …”

Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity

“…However, while antigen tests use antibodies to specifically bind and detect their protein target, a NendoU test would use a fluorogenic enzymatic substrate to specifically detect the NendoU protein via its unique enzymatic properties. Rather than using assay reagents such as secondary antibodies coupled to reporter molecules for detection, this nuclease assay approach harnesses the enzymatic activity of the target protein to generate detectable signal directly (Burghardt et al, 2016; Flenker et al, 2017; Hernandez et al, 2014; Lopez-Alvarez et al, 2020). The approach thus requires fewer molecular components, with the primary component consisting of an oligonucleotide substrate that is produced with chemical synthesis.…”

Rapid and specific detection of Salmonella infections using chemically modified nucleic acid probes

“…Next, we attempted to calculate the detection limit for each probe by plotting the fluorescence signals (Y axis) versus the bacteria concentration in CFU mL -1 (X axis), using the same samples for both measurements and keeping the time constant for each acquisition point (Figure 5a and Figure 5b) [24,25]. Based on our screening method described herein (Figure S1), we are selecting our substrates by reducing from millions of possible substrates (12mer length, using 16 different nucleotides = 16 12 possibilities) to a few candidates, but expecting to reach high specificity and sensitivity for developing rapid system for Salmonella detection.…”