Comparative evaluation of viability assays and functional analysis of bbi29 in Borrelia burgdorferi

ABSTRACT In vitro viability assays are essential for studying the survival of Borrelia burgdorferi 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 B. burgdorferi 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 bbi29 , encoding a putative virulence-associated lipoprotein, in B. burgdorferi stress response. The contribution of bbi29 to B. burgdorferi mammalian infection was also examined using a murine model of Lyme disease. IMPORTANCE Lyme disease, caused by Borrelia burgdorferi , is the most common tick-borne disease in the United States. Assessment of B. burgdorferi 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 B. burgdorferi viability. We herein surprisingly found that the SYBR Green I/PI assay has significant caveats and does not provide accurate quantification of B. burgdorferi 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 bbi29 , we have refined our understanding of hypothetical proteins in B. burgdorferi pathogenesis. Together, this work highlights the importance of developing and validating experimental approaches and functionally characterizing B. burgdorferi genes with unknown functions.