When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining

Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered ‘dead’. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant

Brain metastasis

Brain metastasis, which commonly arises in patients with lung cancer, breast cancer and melanoma, is associated with poor survival outcomes and poses distinct clinical challenges. The brain microenvironment, with its unique cell types, anatomical structures, metabolic constraints and immune environment, differs drastically from microenvironments of extracranial lesions, imposing a distinct and profound selective pressure on tumour cells that, in turn, shapes the metastatic process and therapeutic responses. Accordingly, the study of brain metastasis could uncover new therapeutic targets and identify novel treatment approaches to address the unmet clinical need. Moreover, such efforts could provide insight into the biology of primary brain tumours, which face similar challenges to brain metastases of extracranial origin, and vice versa. However, the paucity of robust preclinical models of brain metastasis has severely limited such investigations, underscoring the importance of developing improved experimental models that holistically encompass the metastatic cascade and/or brain microenvironment. In this Viewpoint, we asked four leading experts to provide their opinions on these important aspects of brain metastasis biology and management.M.V. acknowledges support from the Ministry of Economy and Competitiveness (MINECO) grant MINECO-Retos SAF2017-89643-R, the Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award 2017 (498103), the Beug Foundation's Prize for Metastasis Research 2017, Fundacion Ramon Areces (CIVP19S8163), Worldwide Cancer Research (19-0177), Horizon 2020 Funding and Emerging Technologies (FET) Open (828972), the Clinic and Laboratory Integration Program Cancer Research Institute (CRI) Award 2018 (54545) and Spanish Association Against Cancer (AECC) Coordinated Translational Groups 2017 (GCTRA16015SEOA). M.V. is a Ramon y Cajal Investigator (RYC-2013-13365) and a member of the European Molecular Biology Organization Young Investigator Programme (EMBO YIP; 4053). L.G. is supported by CIHR Operating Grant PJT-162234, Terry Fox Research Institute grant TFRI1087, Canadian Cancer Society Research Institute CCS#705799, the Cancer Research Society and the C17 Research Network.S