{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T19:06:10Z","timestamp":1780513570022,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,1]]},"group-title":"Microbiology","reference-count":23,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Phage therapy is increasingly considered a promising alternative for treating multidrug-resistant (MDR) infections. However, its clinical application remains limited by challenges in isolating effective phages against resistant clinical strains and by the limited ability of in vitro assays to predict performance in real biological environments. While biological matrices are known to influence phage activity, these effects are not well characterised.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    A phage-resistant\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    isolate from a patient with recurrent MDR urinary tract infection was used as the model organism. Conventional isolation methods failed to recover effective phages, leading to the development of TEASER-i (Transient EDTA- and Ion-Assisted Sequential Enrichment &amp; Recovery). Recovered phages were characterised using adsorption assays, one-step growth kinetics, and time-kill experiments. Their antibacterial activity was evaluated both\n                    <jats:italic>in vitro<\/jats:italic>\n                    and in\n                    <jats:italic>ex vivo<\/jats:italic>\n                    human matrices (whole blood, serum, plasma, and urine). Phage efficacy was quantified using maximum log reduction (\n                    <jats:italic>E<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>max<\/jats:italic>\n                    <\/jats:sub>\n                    ), area under the curve (AUC), and phage-to-bacteria ratio (PBR).\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    A novel TEASER-i method optimised for difficult-to-treat Gram-negative infections, enabled recovery of a functionally effective \u00d8sewage-derived\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    phage, which outperformed a \u00d8urine-derived\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    phage that showed slower replication and lower burst size. Phage activity varied significantly in blood, serum, and plasma. Urine supported the most sustained antibacterial effect. In many cases, early bacterial reduction was followed by regrowth. Sustained activity was associated with maintenance of favourable PBR values, while negative PBR corresponded to treatment failure. At 96 h, only two conditions maintained favourable phage load (log\n                    <jats:sub>10<\/jats:sub>\n                    <jats:italic>PBR<\/jats:italic>\n                    &gt; 0): the\n                    <jats:italic>S. aureus<\/jats:italic>\n                    phage in urine (+1.66) and the sewage-derived\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    phage in serum (+1.32).\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>\n                    Phage efficacy depends not only on intrinsic lytic capacity but also on the ability to persist and amplify within specific biological environments. Conventional isolation and\n                    <jats:italic>in vitro<\/jats:italic>\n                    screening may therefore overestimate therapeutic potential. Combining optimised isolation strategies with\n                    <jats:italic>ex vivo<\/jats:italic>\n                    evaluation provides a more realistic framework for phage selection and clinical translation.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.29.728643","type":"posted-content","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T13:05:16Z","timestamp":1780319116000},"source":"Crossref","is-referenced-by-count":0,"title":["Impairment of bacteriophage activity in blood: a case study revealing constraints in phage isolation and translation"],"prefix":"10.64898","author":[{"given":"Braira","family":"Wahid","sequence":"first","affiliation":[{"name":"Bacteriophage Research and Innovation Laboratory (BRIL), College for Science and Engineering, Flinders University, Bedford Park 5042, South Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teddy","family":"Teo","sequence":"additional","affiliation":[{"name":"College of Medicine and Public Health, Flinders Medical Complex, Adelaide, SA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinxin","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lawrence","family":"Zang","sequence":"additional","affiliation":[{"name":"Bacteriophage Research and Innovation Laboratory (BRIL), College for Science and Engineering, Flinders University, Bedford Park 5042, South Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anjali","family":"Bandara","sequence":"additional","affiliation":[{"name":"Bacteriophage Research and Innovation Laboratory (BRIL), College for Science and Engineering, Flinders University, Bedford Park 5042, South Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qurat-ul-ain","family":"Ashraf","sequence":"additional","affiliation":[{"name":"School of Sciences, University of Management and Technology, Lahore, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morgyn","family":"Warner","sequence":"additional","affiliation":[{"name":"Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9087-258X","authenticated-orcid":false,"given":"Peter","family":"Speck","sequence":"additional","affiliation":[{"name":"Bacteriophage Research and Innovation Laboratory (BRIL), College for Science and Engineering, Flinders University, Bedford Park 5042, South Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060311151126000_2026.05.29.728643v1.1","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.2261"},{"issue":"7","key":"2026060311151126000_2026.05.29.728643v1.2","doi-asserted-by":"crossref","first-page":"877","DOI":"10.3390\/antibiotics10070877","article-title":"Pseudomonas aeruginosa PAO 1 in vitro time\u2013kill kinetics using single phages and phage formulations\u2014modulating death, adaptation, and resistance","volume":"10","year":"2021","journal-title":"Antibiotics"},{"issue":"6","key":"2026060311151126000_2026.05.29.728643v1.3","doi-asserted-by":"crossref","first-page":"1220","DOI":"10.4269\/ajtmh.22-0303","article-title":"Isolation and characterization of Pseudomonas aeruginosa phages with a broad host spectrum from hospital sewage systems and their therapeutic effect in a mouse model","volume":"108","year":"2023","journal-title":"The American Journal of Tropical Medicine and Hygiene"},{"issue":"1","key":"2026060311151126000_2026.05.29.728643v1.4","doi-asserted-by":"crossref","first-page":"19393","DOI":"10.1038\/s41598-021-98457-z","article-title":"Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa","volume":"11","year":"2021","journal-title":"Scientific Reports"},{"key":"2026060311151126000_2026.05.29.728643v1.5","doi-asserted-by":"crossref","unstructured":"B. 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