Abstract
Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes—called bacterial ghosts (BGs)—that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.
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Acknowledgments
The authors would like to thank the companies BIRD-C GmbH & CoKG (Vienna, Austria) and Ardeypharm GmbH (Herdecke, Germany) for providing the strains and plasmids used in this work. All work presented here was initiated and partly financed by BIRD-C. Additional funding was received through the Austrian COMET Program by the Austrian Federal Ministry of Transport, Innovation and Technology (BMVIT); the Austrian Federal Ministry of Economy, Family and Youth (BMWFJ); and by the State of Styria (Styrian Funding Agency SFG).
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This study was funded through the Austrian COMET Program by the Austrian Federal Ministry of Transport, Innovation and Technology (BMVIT); the Austrian Federal Ministry of Economy, Family and Youth (BMWFJ); and by the State of Styria (Styrian Funding Agency SFG).
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Langemann, T., Mayr, U.B., Meitz, A. et al. Multi-parameter flow cytometry as a process analytical technology (PAT) approach for the assessment of bacterial ghost production. Appl Microbiol Biotechnol 100, 409–418 (2016). https://doi.org/10.1007/s00253-015-7089-9
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DOI: https://doi.org/10.1007/s00253-015-7089-9