AI Article Synopsis
- The study developed a microbroth assay to evaluate the host range and lytic activity of phages against carbapenemase-producing K. pneumoniae isolates, addressing limitations of the traditional agar overlay method.
- The host range was tested on 11 specific phages using various concentrations and bacterial inocula, with analysis of growth inhibition over 6 and 24 hours.
- Results showed significant differences in growth inhibition among phages and identified specific cutoffs for lysis detection, demonstrating the microbroth assay's effectiveness and reliability.
Article 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 × 10-5 × 10 PFU/ml) and two bacterial inocula (5 × 10 and 5 × 10 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 × 10 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.
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| http://dx.doi.org/10.1007/s40121-024-01092-0 | DOI Listing |
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