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. 2016 Aug 8;55(33):9557-61.
doi: 10.1002/anie.201602763. Epub 2016 Jun 30.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

Nathan G Schoepp  1 , Eugenia M Khorosheva  1 , Travis S Schlappi  1 , Matthew S Curtis  1 , Romney M Humphries  2 , Janet A Hindler  2 , Rustem F Ismagilov  3
Affiliations

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

Nathan G Schoepp et al. Angew Chem Int Ed Engl. .

Erratum in

Abstract

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

Keywords: DNA replication; analytical methods; antibiotics; polymerase chain reaction; susceptibility.

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Figures

Figure 1
Figure 1
Quantitative PCR (qPCR) time course for exposure of A) susceptible (S) and B) resistant (R) UTI E. coli isolates to ciprofloxacin. Fold change values represent changes from t=0 min; error bars represent the upper and lower bounds of the 98% confidence interval (C.I.; see the SI). N=3 where N is the number of technical qPCR replicates. Significant differences (p value ≤ 0.02) are marked with a green check mark.
Figure 2
Figure 2
Comparison of susceptible and resistant isolates from UTI samples after a 15 min exposure with each of four antibiotics, analyzed by quantitative PCR. Fold change values represent change from t = 0 min; error bars are 98% C.I. (see SI), N=3. Significant (p-value ≤ 0.02) and nonsignificant differences detected using the susceptible isolate are marked with a green check and red × respectively.
Figure 3
Figure 3
AST results using dPCR. (A,B) Time course results for exposure of susceptible (A) and resistant (B) UTI E. coli isolates to ciprofloxacin. (C,D) Fold changes after treatment with all four antibiotics tested. Significant (p-value ≤ 0.02) and nonsignificant p-values for susceptible isolates are denoted with a green check and red × respectively. Samples treated with amoxicillin/clavulanic acid (D) were extracted using a non-denaturing protocol. Concentrations are calculated using Poisson statistics. Fold change values represent change from t = 0 min; all error bars are 98% C.I. (see SI), N=3 for qPCR, N=2 for dPCR.
Figure 4
Figure 4
A mechanistic investigation of AST by digital PCR (dPCR) after beta lactam exposure and non-denaturing DNA extraction using shearing to disrupt macromolecular assemblies; error bars for qPCR are 2.8 S.D. (see SI), N=3; error bars are 98%C.I. for dPCR (see SI), N=2. Significant (p-value ≤ 0.02) and nonsignificant p-values for susceptible isolates quantified using dPCR are denoted with a green check and red × respectively (see SI).
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