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Optimization and Testing of a Commercial Viability PCR Protocol to Detect Escherichia coli in Whole Blood
Kristi L Jones1, Federico Cunha1, Segundo Casaro1
1Department of Large Animal Clinical Sciences, University of Florida College of Veterinary Medicine, Gainesville, FL 32608, USA.
Abstract:
Bacteremia, specifically if progressed to sepsis, poses a time-sensitive threat to human and animal health. Escherichia coli is a main causative agent of sepsis in humans. The objective was to evaluate a propidium monoazide (PMA)-based viability PCR (vPCR) protocol to detect and quantify live E. coli from whole blood. We optimized the protocol by adding a eukaryotic-specific lysis step prior to PMA exposure, then used spiking experiments to determine the lower limit of detection (LOD) and linear range of quantification. We also compared the vPCR quantification method to standard colony count of spiked inoculum. Lastly, we calculated percent viability in spiked samples containing 50% live cells or 0% live cells. The LOD was 102 CFU/mL for samples containing live cells only and samples with mixed live and heat-killed cells. The linear range of quantification was 102 CFU/mL to 108 CFU/mL (R2 of 0.997) in samples containing only live cells and 103 CFU/mL to 108 CFU/mL (R2 of 0.998) in samples containing live plus heat-killed cells. A Bland-Altman analysis showed that vPCR quantification overestimates compared to standard plate count of the spiked inoculum, with an average bias of 1.85 Log10 CFU/mL across the linear range when only live cells were present in the sample and 1.98 Log10 CFU/mL when live plus heat-killed cells were present. Lastly, percent viability calculations showed an average 89.5% viable cells for samples containing 50% live cells and an average 19.3% for samples containing 0% live cells. In summary, this optimized protocol can detect and quantify viable E. coli in blood in the presence of heat-killed cells. Additionally, the data presented here provide the groundwork for further development of vPCR to detect and quantify live bacteria in blood in clinical settings.
Insights
This study optimized a viability PCR (vPCR) protocol to detect live Escherichia coli in blood, crucial for diagnosing sepsis. The method accurately quantifies viable bacteria, aiding rapid clinical detection of bloodstream infections.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Bacteremia and sepsis, often caused by Escherichia coli, are life-threatening conditions requiring rapid diagnosis.
- Accurate detection of viable bacteria in blood is critical for timely and effective treatment.
- Current methods may not always distinguish between live and dead bacteria, impacting treatment decisions.
Purpose of the Study:
- To evaluate and optimize a propidium monoazide (PMA)-based viability PCR (vPCR) protocol for detecting and quantifying live E. coli in whole blood.
- To determine the limit of detection (LOD) and linear range of quantification for the optimized vPCR protocol.
- To compare vPCR quantification with standard colony counts and assess its accuracy in determining bacterial viability.
Main Methods:
- Optimization of a PMA-based vPCR protocol by incorporating a eukaryotic-specific lysis step.
- Spiking experiments using known concentrations of live and heat-killed E. coli in whole blood samples.
- Comparison of vPCR results with standard plate counts (colony counts) and calculation of percent viability.
Main Results:
- The optimized vPCR protocol demonstrated a lower limit of detection (LOD) of 102 CFU/mL.
- A wide linear range of quantification (102–108 CFU/mL) was established for viable E. coli detection.
- vPCR generally overestimated bacterial load compared to standard plate counts, with an average bias of ~1.9 Log10 CFU/mL, and showed varying accuracy in calculating percent viability.
Conclusions:
- The optimized PMA-based vPCR protocol effectively detects and quantifies viable E. coli in blood, even in the presence of dead cells.
- The protocol shows promise for clinical applications in diagnosing bacteremia and sepsis.
- Further refinement is needed to address quantification biases and improve accuracy in viability assessments for clinical settings.
