Fig 1.
Overview of the pol-aAST shown for susceptible and resistant samples exposed to β-lactams.
(a) Treated aliquots are exposed to a β-lactam. In susceptible samples, β-lactams compromise cell wall integrity. (b) NAs are released from compromised cells, increasing NA accessibility to polymerase. (c) Released NAs in the susceptible treated aliquot amplify faster than NAs from intact cells in the control aliquot, resulting in a difference in TTP. No difference in amplification between control and treated aliquots is observed in resistant samples. (d) TTPD between control and treated aliquots is used to assess susceptibility. ABX, antibiotic; AST, antibiotic susceptibility testing; gDNA, genomic DNA; NA, nucleic acid; pol-aAST, polymerase-accessibility AST; R, resistant; RFU, relative fluorescent units; S, susceptible; TTP, time-to-positive; TTPD, time-to-positive difference.
Fig 2.
The pol-aAST requires non-lytic amplification conditions.
(a–b) Thermal profiles of LAMP and PCR. (c–d) LAMP and PCR amplification curves for a susceptible E. coli isolate exposed to ETP for 15 min. Blue and black lines are the average of triplicate samples. Grey lines represent standard deviation of triplicates. A difference in TTP for control and treated aliquots is observed for susceptible isolates when quantifying NAs using LAMP, but not PCR. Raw data are provided in S5 Table. AST, antibiotic susceptibility test/testing; Cq, quantitation cycle; ETP, ertapenem; LAMP, loop-mediated isothermal amplification; NA, nucleic acid; PCR, polymerase chain reaction; pol-aAST, polymerase-accessibility AST; RFU, relative fluorescent units; TTP, time-to-positive.
Fig 3.
Percentage of DNA released following antibiotic exposure.
Two susceptible and two resistant E. coli isolates were exposed to no antibiotic (control), CRO, ETP, or MEM for 15 min before filtering to separate intact cells from extracellular DNA. Experiments were performed in triplicate for all isolate/antibiotic combinations. Each point represents a single experiment; lines represent the average and standard deviation of replicate experiments. Raw data are provided in S6 Table. ABX, antibiotic; CRO, ceftriaxone; ETP, ertapenem; MEM, meropenem; R, resistant; S, susceptible.
Fig 4.
Validation of the pol-aAST method using control and antibiotic-treated aliquots.
(a) Example calculation of TTPD between control and treated aliquots (TTPDCT). The TTP (in minutes) of the control and treated aliquots are used to calculate TTPDCT. (b–d) The pol-aAST results using E. coli (b), K. pneumoniae (c), and Enterobacter spp. (d) isolates exposed to CRO, ETP, and MEM for 15 min. Red points represent isolates with either no detectable carbapenemase genes (Ec and Kp isolates) according to a published genotypic assay [67] and commercial assay [68] or no predictive genotype (Ebs isolates) according to the whole genome sequencing by the CDC [66]. S/R thresholds (dashed lines) were set halfway between the lowest susceptible and the highest resistant TTPDCT values. Raw data are provided in S3 Table. +ABX, antibiotic-treated; AST, antibiotic susceptibility testing; CDC, Centers for Disease Control and Prevention; CRO, ceftriaxone; CT, control to treated; ctrl, control; Ebs, E. aerogenes and E. cloacae collectively; Ec, E. coli; ETP, ertapenem; Kp, K. pneumoniae; MEM, meropenem; pol-aAST, polymerase-accessibility AST; R, resistant; RFU, relative fluorescent units; S, susceptible; TTP, time-to-positive; TTPD, time-to-positive difference; TTPDCT, TTPD control to treated.
Fig 5.
Validation of the pol-aAST method using lysed control and antibiotic-treated aliquots.
(a) Example calculation of TTPD between the lysed control and antibiotic-treated aliquots (TTPDLT). The TTP (in minutes) in the lysed control and antibiotic-treated aliquots are used to calculate TTPDLT. (b–d) The pol-aAST results using E. coli (b), K. pneumoniae (c), and Enterobacter spp. (d) isolates exposed to CRO, ETP, and MEM for 15 min. Red points represent isolates with either no detectable carbapenemase genes (Ec and Kp isolates) according to a published genotypic assay [67] and commercial assay [68], or no predictive genotype (Ebs isolates) according to the CDC [66]. S/R thresholds (dashed lines) were set halfway between the lowest susceptible and the highest resistant TTPDLT values except in the case of Enterobacter spp. treated with CRO (see text). Raw data are provided in S3 Table. +ABX, antibiotic-treated; AST, antibiotic susceptibility testing; CDC, Centers for Disease Control and Prevention; ctrl, control; CRO, ceftriaxone; Ebs, E. aerogenes and E. cloacae collectively; Ec, E. coli; ETP, ertapenem; Kp, K. pneumoniae; lc, lysed control; MEM, meropenem; pol-aAST, polymerase-accessibility AST; R, resistant; RFU, relative fluorescent units; S, susceptible; TTP, time-to-positive; TTPD, time-to-positive difference; TTPDLT, TTPD lysed-control to treated.
Fig 6.
Timed sample-to-answer pol-aAST using contrived urine samples spiked with either Ec or Kp.
(a) Because minimal sample handling is required for pol-aAST, all 4 contrived urine samples were run in parallel. (b) Urine samples were split into control and antibiotic-treated aliquots and incubated at 37°C for 13 min. A timer was started immediately after sample splitting. (c) All samples were added to pre-made LAMP mix and run in technical triplicate. (d) Samples were amplified using LAMP, and the fluorescence of reactions was monitored in real time. Once total fluorescence passed a predetermined threshold (indicating successful amplification), reactions were stopped and TTP values ported into an automated data-analysis spreadsheet. The timer was stopped as soon as the spreadsheet gave susceptibility calls. (e) Comparison of susceptibility calls with gold-standard AST categorization. Total assay time was 29.5 min. Raw data are provided in S3 Table. ABX, antibiotic; AST, antibiotic susceptibility test/testing; cntrl, control; Ec, E. coli; ETP, ertapenem; Kp, K. pneumoniae; LAMP, loop-mediated isothermal amplification; pol-aAST, polymerase-accessibility AST; R, resistant; RFU, relative fluorescent units; rt, real-time; S, susceptible; TTP, time-to-positive; TTPD, time-to-positive difference.
Fig 7.
Pilot testing of pol-aAST with clinical UTI samples with a modified protocol (see Methods and Discussion).
TTPDCT values for AMP and ETP susceptibility obtained by pol-aAST, with clinical UTI samples containing E. coli. Each point represents the TTPDCT value for one clinical sample tested once by pol-aAST (S2 and S4 Tables). LAMP was performed in technical triplicate, see S4 Table for values and statistical details. AMP, ampicillin; AST, antibiotic susceptibility testing; ETP, ertapenem; LAMP, loop-mediated isothermal amplification; pol-aAST, polymerase-accessibility AST; R, resistant; S, susceptible; TTPD, time-to-positive difference; TTPDCT, TTPD control to treated; UTI, urinary tract infection.