Detection of Phage's Lytic Activity Against Carbapenemase-Producing Klebsiella pneumoniae Isolates Using a High-Throughput Microbroth Growth Inhibition Assay.

Abstract

Introduction: The host range of phages is usually assessed with the agar overlay method. However, this method is both cumbersome and subjective. Therefore, a microbroth assay was developed to assess host range and lytic activity patterns of phages in the agar overlay method against a collection of carbapenemase-producing Klebsiella pneumoniae (CRKP) isolates.

Methods: The host range of 11 K. pneumoniae-specific phages against 8 non-repetitive well-characterized CRKP isolates was assessed with the agar overlay method and a microbroth assay by monitoring optical density (OD) at 630 nm for 24 h at different phage concentrations (5 × 10⁹-5 × 10³ PFU/ml) and two bacterial inocula (5 × 10⁶ and 5 × 10⁸ CFU/ml). The lytic activity of phage-bacteria pairs with transparent/semi-transparent (N = 7), turbid (N = 6), and no (N = 6) lysis in overlay agar method was compared statistically with the growth inhibition at 6 and 24 h in the microbroth assay with analysis of variance (ANOVA), receiver operating characteristic curves (ROC) curves and Fisher's exact test. Optimal cutoffs were determined, and sensitivity and specificity were calculated.

Results: Statistically significant differences of growth inhibition at 6 and 24 h for phage concentrations ≥ 5 × 10⁸ PFU/ml for both inocula were found between phages with transparent/semi-transparent, turbid, and no lysis. ROC curve analysis indicated an optimal growth inhibition cutoff of ≥ 31% at high phage and bacteria concentrations for detecting phages with lysis and ≥ 61% at high-phage and low-bacteria concentrations for detecting phages with transparent/semi-transparent lysis with sensitivity/specificity 100%/100% and 100%/86%, respectively.

Conclusions: The microbroth growth inhibition assay provided fast, reliable, and objective results for K. pneumoniae phage host-range lytic activity differentiating different patterns of lysis in a high-throughput format.