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Multidrug-resistant TB is successfully treated in less than 60% of cases by long, expensive, and aggressive treatments. Mycobacteriophages, viruses that can infect bacteria such as\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      \u2014the species responsible for TB\u2014have the potential to redefine TB prevention and treatments. However, the development of phage-based products necessitates the assessment of numerous parameters, including virulence and adsorption, to ensure their performance and quality. In this work, we characterized the virulence of three different mycobacteriophages (Fionnbharth, Muddy, and D29), alone and as cocktails, against a TB model host (\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      ) under planktonic and early-stage biofilm growth conditions. Phage D29 and cocktails containing D29 had the highest virulence under all conditions. Interestingly, phages Fionnbharth and Muddy and their combination showed higher virulence against early-stage biofilm than against the planktonic phenotype. Adsorption assays indicated that all three phages had lower adsorption efficiencies on the early-stage biofilm phenotype than on the planktonic one, suggesting a reduced availability of receptors in the former. Given that, despite these lower adsorption efficiencies, the virulence of the phages and phage cocktails was either unchanged or higher against the early-stage biofilm, this phenotype must display properties that are favorable to other steps of the infection process. These results inform us on the dynamics of mycobacteriophage infections, both alone and in cocktail formulations, under different host growth conditions, serving as a basis for the development of phage products targeting mycobacteria biofilms.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      This study provides a systematic investigation of the virulence of three mycobacteriophages, Fionnbharth, Muddy, and D29, and their combinations as cocktails against\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      . We also included considerations on the hydrodynamic conditions (shaking and not shaking) and host phenotype (planktonic and early-onset biofilm cultures) during the infection process and adsorption of the phage to the host. We showed that virulence was strongly affected by phenotype and that higher virulence shown against the early-onset biofilm phenotype was not linked to faster adsorption to the host. We also showed that phage D29 and cocktails containing this phage had the highest virulence. These results are important as they provide a framework for a better evaluation and development of phage-based treatment against mycobacterial infections.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01920-24","type":"journal-article","created":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T09:02:06Z","timestamp":1747818126000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Comparative study on the virulence of mycobacteriophages"],"prefix":"10.1128","volume":"99","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1589-1566","authenticated-orcid":false,"given":"Ilaria","family":"Rubino","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0160cpw27","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemical and Materials Engineering, University of Alberta","place":["Edmonton, 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