Paschalis Paranos, Spyros Pournaras, Joseph Meletiadis
{"title":"利用高通量微培养液生长抑制法检测噬菌体对产碳青霉烯酶肺炎克雷伯菌分离株的裂解活性。","authors":"Paschalis Paranos, Spyros Pournaras, Joseph Meletiadis","doi":"10.1007/s40121-024-01092-0","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>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.</p><p><strong>Methods: </strong>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<sup>9</sup>-5 × 10<sup>3</sup> PFU/ml) and two bacterial inocula (5 × 10<sup>6</sup> and 5 × 10<sup>8</sup> 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.</p><p><strong>Results: </strong>Statistically significant differences of growth inhibition at 6 and 24 h for phage concentrations ≥ 5 × 10<sup>8</sup> 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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":13592,"journal":{"name":"Infectious Diseases and Therapy","volume":" ","pages":"217-228"},"PeriodicalIF":4.7000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11782791/pdf/","citationCount":"0","resultStr":"{\"title\":\"Detection of Phage's Lytic Activity Against Carbapenemase-Producing Klebsiella pneumoniae Isolates Using a High-Throughput Microbroth Growth Inhibition Assay.\",\"authors\":\"Paschalis Paranos, Spyros Pournaras, Joseph Meletiadis\",\"doi\":\"10.1007/s40121-024-01092-0\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>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.</p><p><strong>Methods: </strong>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<sup>9</sup>-5 × 10<sup>3</sup> PFU/ml) and two bacterial inocula (5 × 10<sup>6</sup> and 5 × 10<sup>8</sup> 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.</p><p><strong>Results: </strong>Statistically significant differences of growth inhibition at 6 and 24 h for phage concentrations ≥ 5 × 10<sup>8</sup> 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.</p><p><strong>Conclusions: </strong>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.</p>\",\"PeriodicalId\":13592,\"journal\":{\"name\":\"Infectious Diseases and Therapy\",\"volume\":\" \",\"pages\":\"217-228\"},\"PeriodicalIF\":4.7000,\"publicationDate\":\"2025-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11782791/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Infectious Diseases and Therapy\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s40121-024-01092-0\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/12/22 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q1\",\"JCRName\":\"INFECTIOUS DISEASES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Infectious Diseases and Therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s40121-024-01092-0","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/12/22 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"INFECTIOUS DISEASES","Score":null,"Total":0}
Detection of Phage's Lytic Activity Against Carbapenemase-Producing Klebsiella pneumoniae Isolates Using a High-Throughput Microbroth Growth Inhibition Assay.
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.
期刊介绍：
Infectious Diseases and Therapy is an international, open access, peer-reviewed, rapid publication journal dedicated to the publication of high-quality clinical (all phases), observational, real-world, and health outcomes research around the discovery, development, and use of infectious disease therapies and interventions, including vaccines and devices. Studies relating to diagnostic products and diagnosis, pharmacoeconomics, public health, epidemiology, quality of life, and patient care, management, and education are also encouraged.
Areas of focus include, but are not limited to, bacterial and fungal infections, viral infections (including HIV/AIDS and hepatitis), parasitological diseases, tuberculosis and other mycobacterial diseases, vaccinations and other interventions, and drug-resistance, chronic infections, epidemiology and tropical, emergent, pediatric, dermal and sexually-transmitted diseases.
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