Paranos Paschalis, Pournaras Spyros, Meletiadis Joseph
Clinical Microbiology Laboratory, Medical School, Attikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece.
Infect Dis Ther. 2025 Jan;14(1):217-228. doi: 10.1007/s40121-024-01092-0. Epub 2024 Dec 22.
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.
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.
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.
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.
噬菌体的宿主范围通常采用琼脂覆盖法进行评估。然而，该方法既繁琐又主观。因此，开发了一种微量肉汤测定法，以评估在琼脂覆盖法中噬菌体对一组产碳青霉烯酶肺炎克雷伯菌（CRKP）分离株的宿主范围和裂解活性模式。
采用琼脂覆盖法和微量肉汤测定法，通过在不同噬菌体浓度（5×10 - 5×10 PFU/ml）和两种细菌接种量（5×10和5×10 CFU/ml）下监测630 nm处的光密度（OD）24小时，评估11种肺炎克雷伯菌特异性噬菌体对8株特征明确的非重复性CRKP分离株的宿主范围。对在覆盖琼脂法中呈现透明/半透明裂解（N = 7）、浑浊裂解（N = 6）和无裂解（N = 6）的噬菌体-细菌对的裂解活性，与微量肉汤测定法中6小时和24小时的生长抑制情况进行方差分析（ANOVA）、受试者工作特征曲线（ROC）分析和Fisher精确检验。确定最佳临界值，并计算敏感性和特异性。
对于两种接种量，当噬菌体浓度≥5×10 PFU/ml时，在6小时和24小时的生长抑制方面，具有透明/半透明裂解、浑浊裂解和无裂解的噬菌体之间存在统计学显著差异。ROC曲线分析表明，在高噬菌体和细菌浓度下，检测有裂解的噬菌体的最佳生长抑制临界值≥31%，在高噬菌体和低细菌浓度下，检测有透明/半透明裂解的噬菌体的最佳生长抑制临界值≥61%，敏感性/特异性分别为100%/100%和100%/86%。
微量肉汤生长抑制测定法为肺炎克雷伯菌噬菌体宿主范围裂解活性提供了快速、可靠且客观的数据，能够以高通量形式区分不同的裂解模式。
