# P-1302. Klebsiella pneumoniae Phage Susceptibility Testing (PST): Comparison Between a Spot Plaque Assay and an Optical Density-Based Liquid Assay

## TL;DR

This study compares two methods for testing phage susceptibility in antibiotic-resistant Klebsiella pneumoniae to help standardize phage therapy approaches.

## Contribution

The study evaluates the agreement between a plaque assay and a liquid assay for phage susceptibility testing using specific cutoff thresholds.

## Key findings

- Using a 4-hour hold time cutoff in the liquid assay showed complete agreement with the plaque assay for three phages.
- A 8-hour hold time cutoff in the liquid assay resulted in lower agreement with the plaque assay results.
- The plaque assay classified more phage-bacteria combinations as susceptible compared to the liquid assay at the 8-hour cutoff.

## Abstract

Phage therapy has gained increasing interest as an approach to treat antimicrobial- resistant bacterial infections. Standardized, reproducible PST methods are needed to rapidly identify active phages for potential clinical use. Here, PST of 24 clinical isolates of carbapenem-resistant K. pneumoniae (CR-Kp) to 6 K. pneumoniae phages (3 from KlebPhaCol and 3 from phage hunting) was assessed using a plaque assay (PA) and a liquid assay (LA), and results compared.

Phage Susceptibility Testing: Agreement Between Plaque Assay and Liquid Assay

For the PA, 2µL of 10−1-10−8 phage dilutions were spotted on bacterial laws of CR-Kp (double agar overlay), and plaques scored upon overnight incubation at 37°C [0 (no activity), 1+ (lysis from without), 2+ (concentration-dependent lysis), 3+ (clear plaques, small or medium size), 4+ (clear plaques of large size); scores ≥ 2+ were considered susceptible]. Testing was performed on 3 different days. If ≥ 2 replicates scored ≥ 2+, the result was classified as susceptible.

For the LA, phages and bacteria were combined (MOI=10) in 96-well plates [phage-bacteria combinations (3 replicates), phage controls (106 PFU/mL), bacterial controls (BC, 105 CFU/mL), media sterility controls] and plates incubated at 37°C for 24h, with OD600 measured every 15 min. Hold time (HT) — delay in achieving inflection at the beginning of exponential growth phase — was calculated for phage-treated compared to BC wells. Testing was performed on 2 different days. Mean HT was computed for each phage-bacteria combination and results assessed as susceptible or not using cutoff HTs of 4h and 8h.

Using a LA HT cutoff of 4h, there was complete agreement with results of the PA for 3 phages, and 96, 79 and 67% overall agreement for 3 other phages (Table). Using a LA HT cutoff of 8h, there was complete negative agreement for 4 phages, but lower overall agreement between the LA and PA (58-96%). Increased classification as susceptible by PA compared to LA was the main source of disagreement when using a LA HT cutoff of 8h.

Here, two PST assays were compared, and greater agreement was found when using a 4h compared to an 8h HT LA cutoff. These results represent a step forward in the standardization of PST methods, bearing in mind that correlation of results with pre-clinical and clinical outcomes is needed.

Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication, a patent on PET imaging of bacterial infection: a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication, a patent on PET imaging of bacterial infection|MicuRx Pharmaceuticals and bioMérieux: Grant/Research Support|PhAST, Day Zero Diagnostics, DEEPULL DIAGNOSTICS, S.L., Nostics, HealthTrackRx, bioMérieux and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course: Honoraria