{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:56:07Z","timestamp":1790690167048,"version":"4.1.0"},"reference-count":70,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI181738, AI152983"],"award-info":[{"award-number":["AI181738, AI152983"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">In vitro<\/jats:italic>\n                      viability assays are essential for studying the survival of\n                      <jats:italic toggle=\"yes\">Borrelia burgdorferi<\/jats:italic>\n                      after exposure to stressful conditions. In contrast to the cumbersome and tedious manual microscopic counting method, fluorescence-based assays are more rapid and convenient and thus can be used in a high-throughput format. In the SYBR Green I\/propidium iodide (PI) assay, spirochetes are labeled with different fluorescent dyes, and fluorescence ratios are then compared to calculate the ratio between live and dead spirochetes. Stained spirochetes are also manually counted under a fluorescence microscope or analyzed via flow cytometry to obtain the live\/dead ratio. In this study, we compared these methods to determine the most accurate technique for quantifying spirochete viability. Our results showed that, compared with the SYBR Green I\/PI assay that carries inherent caveats, both the fluorescent microscopic counting method and the flow cytometry method provide more reliable and accurate results for\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      viability assays, and flow cytometry offers significant advantages in speed and reduced labor compared to manual microscopic counting. Leveraging the flow cytometry-based viability assay, we investigated the role of gene\n                      <jats:italic toggle=\"yes\">bbi29<\/jats:italic>\n                      , encoding a putative virulence-associated lipoprotein, in\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      stress response. The contribution of\n                      <jats:italic toggle=\"yes\">bbi29<\/jats:italic>\n                      to\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      mammalian infection was also examined using a murine model of Lyme disease.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Lyme disease, caused by\n                      <jats:italic toggle=\"yes\">Borrelia burgdorferi<\/jats:italic>\n                      , is the most common tick-borne disease in the United States. Assessment of\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      growth and viability has been traditionally challenging due to the slow growth and unique morphology of the spirochete. Recently, an optimized SYBR Green I\/PI assay was developed and has since been used by a number of laboratories to assess\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      viability. We herein surprisingly found that the SYBR Green I\/PI assay has significant caveats and does not provide accurate quantification of\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      viability. Rather, our data showed that flow cytometry is a superior alternative to quantify spirochete viability, providing a valuable methodological framework for future studies. Additionally, by investigating the role and requirement for gene\n                      <jats:italic toggle=\"yes\">bbi29<\/jats:italic>\n                      , we have refined our understanding of hypothetical proteins in\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      pathogenesis. Together, this work highlights the importance of developing and validating experimental approaches and functionally characterizing\n                      <jats:italic toggle=\"yes\">B. burgdorferi<\/jats:italic>\n                      genes with unknown functions.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01574-26","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:03:27Z","timestamp":1790687007000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Comparative evaluation of viability assays and functional analysis of\n                    <i>bbi29<\/i>\n                    in\n                    <i>Borrelia burgdorferi<\/i>"],"prefix":"10.1128","author":[{"given":"Connor","family":"Waldron","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/032db5x82","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Medicine, University of South Florida","place":["Tampa, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sierra","family":"George","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/032db5x82","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Medicine, University of South Florida","place":["Tampa, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7427-1856","authenticated-orcid":true,"given":"Zhiming","family":"Ouyang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/032db5x82","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Medicine, University of South Florida","place":["Tampa, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.15319"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.14460"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00951-19"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.00090-24"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.72.11.6433-6445.2004"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.182.10.2909-2918.2000"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2011.07691.x"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0111809"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.3390\/sci7030104"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/emi.2015.31"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0117207"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI39401"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.00743"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkp129"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0908236106"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics9090542"},{"key":"e_1_3_4_18_2","first-page":"125","article-title":"Stationary phase persister\/biofilm microcolony of Borrelia burgdorferi causes more severe disease in a mouse model of Lyme arthritis: implications for understanding persistence, Post-treatment Lyme Disease Syndrome (PTLDS), and treatment failure","volume":"27","author":"Feng J","year":"2019","unstructured":"Feng J, Li T, Yee R, Yuan Y, Bai C, Cai M, Shi W, Embers M, Brayton C, Saeki H, Gabrielson K, Zhang Y. 2019. 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