Volume 25, Issue 6, Pages 1353-1362 (June 2017) Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity  Katie S. Flenker, Elliot L. Burghardt, Nirmal Dutta, William J. Burns, Julia M. Grover, Elizabeth J. Kenkel, Tyler M. Weaver, James Mills, Hyeon Kim, Lingyan Huang, Richard Owczarzy, Catherine A. Musselman, Mark A. Behlke, Bradley Ford, James O. McNamara  Molecular Therapy  Volume 25, Issue 6, Pages 1353-1362 (June 2017) DOI: 10.1016/j.ymthe.2017.03.015 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Endonuclease I Expression in E. coli and Measurement of Its DNase Activity (A) Cartoon of the DNA-SH quenched fluorescent probe. “F” indicates a fluorophore at the 5′ end and “Q” indicates a quencher at the 3′ end. (B) Lysates of the indicated Keio collection E. coli strains were incubated with the DNA-SH probe. A negative control (sample labeled as “Buffer”) consisted of the probe incubated with buffer only. The average fluorescence of reactions is shown, and the error bars indicate SD of the measurements. Note the minimal activity of the endonuclease I-knockout (endA KO) strains. (C) Anti-endonuclease I western blot confirms absence of endonuclease I from the endA knockout strains. (D) A DNA zymogram demonstrates that endonuclease I is a robust DNase of E. coli lysates as the predominant 25 kDa dark band seen in the Seattle 1946 and K12 Keio parental strains is absent in the endA knockout strain. (E) Anti-endonuclease I western blot of lysates of the indicated E. coli strains demonstrates endonuclease I expression in the CFT073 and J96 uropathogenic strains. Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 Evaluation of Probes with Chemically Modified Nucleotides (A–C) E. coli lysate (strain Seattle 1946) (A) or purified recombinant endonuclease I (B and C) was incubated with the indicated probes. The average fluorescence of each reaction is shown. The values in (A) and (B) are normalized to controls in which each probe was incubated in reaction buffer only. Note the robust activation of the 2′-Fl-SH probe in (A) and (B). The limit of detection of endonuclease I with the 2′-Fl-SH probe was found to be 25 fM endonuclease I (C). The values shown in (C) were background subtracted, and the background was determined by incubating probe in reaction buffer only. The error bars indicate SD of the measurements. Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Limit of Detection of the 2′-Fl-SH Probe for E. coli Serially diluted lysates of the indicated E. coli strains were incubated for 1 hr with the 2′-Fl-SH probe. The colony counts of the cultures were used to determine the effective number of CFUs per well of the fluorescence measurements. The values shown were background subtracted; and the background was determined by incubating probe in reaction buffer only. The error bars indicate SD of the measurements. Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Nuclease Assay Signals Are Elevated in E. coli-Positive Patient Urine Samples All samples with over 10,000 cfu/mL E. coli yielded higher nuclease signals than the no growth samples. The background subtracted nuclease assay signal averages of all samples that were positive for E. coli with the culture-based assays are shown. The labels on the x axis indicate results of culture-based assays, and the numbers indicate cfu/mL of indicated bacteria. Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 Urine Samples that Were Culture-Positive for Several Species of Enterobacteriaceae Produce Nuclease Signals Background subtracted nuclease assay signal averages of patient urine samples that yielded a single species or no growth with culture-based assays are shown. The labels on the x axis indicate results of culture-based assays, and the numbers indicate colony forming unit/milliliter of indicated bacteria (or yeast). Note that E. coli positive and no growth samples shown here are also included in the previous figure. Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 ROC Curves Depicting the Ability of the Nuclease Assay to Identify E. coli UTI and any UTI (A) For identification of E. coli UTI (A), the area under the curve is 0.893 (95% CI: 0.831–0.955), and, for identification of any UTI (B), the area under the curve is 0.829 (95% CI: 0.750–0.909). Molecular Therapy 2017 25, 1353-1362DOI: (10.1016/j.ymthe.2017.03.015) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions