Estimation of Microbial Viability Using Flow Cytometry

Davey, Hazel M.; Guyot, Stéphane

doi:10.1002/cpcy.72

Cited by 30 publications

(18 citation statements)

References 42 publications

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“…Among the different treatment groups, 4X MIC 99 showed a sharp increase in fluorescence, suggesting that CABI- 6 can disrupt the bacterial cell membrane potential ( Figure 3 C). To validate the membrane-disruptive property of CABI- 6 , we performed a propidium iodide (PI) uptake assay with where PI can stain nucleic acids of membrane raptured cells as it is a membrane impermeable dye [ 36 ]. We stained the bacterial cells after their treatment with different concentrations of CABI- 6 and measured the percentage of PI-positive cells using flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

N-methyl Benzimidazole Tethered Cholic Acid Amphiphiles Can Eradicate S. aureus-Mediated Biofilms and Wound Infections

Kakkar¹,

Chaudhary²,

Mehta³

et al. 2022

Molecules

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Infections associated with Gram-positive bacteria like S. aureus pose a major threat as these bacteria can develop resistance and thereby limit the applications of antibiotics. Therefore, there is a need for new antibacterials to mitigate these infections. Bacterial membranes present an attractive therapeutic target as these membranes are anionic in nature and have a low chance of developing modifications in their physicochemical features. Antimicrobial peptides (AMPs) can disrupt the microbial membranes via electrostatic interactions, but the poor stability of AMPs halts their clinical translation. Here, we present the synthesis of eight N-methyl benzimidazole substituted cholic acid amphiphiles as antibacterial agents. We screened these novel heterocyclic cholic acid amphiphiles against different pathogens. Among the series, CABI-6 outperformed the other amphiphiles in terms of bactericidal activity against S. aureus. The membrane disruptive property of CABI-6 using a fluorescence-based assay has also been investigated, and it was inferred that CABI-6 can enhance the production of reactive oxygen species. We further demonstrated that CABI-6 can clear the pre-formed biofilms and can mitigate wound infection in murine models.

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Section: Resultsmentioning

confidence: 99%

N-methyl Benzimidazole Tethered Cholic Acid Amphiphiles Can Eradicate S. aureus-Mediated Biofilms and Wound Infections

Kakkar¹,

Chaudhary²,

Mehta³

et al. 2022

Molecules

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“…This may not necessarily mean the cell is dead, but 'compromised' with decreased cellular functions, including biosynthesis of proteins and DNA, efflux pumping and respiration activity [35,36]. A membrane potential dye, DIBAC 4(3) is excluded from polarised cells that maintain membrane potential [37]. This means that well-energised (metabolically active) cells have low fluorescence DIBAC 4(3) staining.…”

Section: Resultsmentioning

confidence: 99%

Recombinant Protein Production with Escherichia coli in Glucose and Glycerol Limited Chemostats

Mitchell

Gogulancea

Smith

et al. 2021

Applied Microbiology

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Recently, there has been a resurgence of interest in continuous bioprocessing as a cost-optimised production strategy, driven by a rising global requirement for recombinant proteins used as biological drugs. This strategy could provide several benefits over traditional batch processing, including smaller bioreactors, smaller facilities, and overall reduced plant footprints and investment costs. Continuous processes may also offer improved product quality and minimise heterogeneity, both in the culture and in the product. In this paper, a model protein, green fluorescent protein (GFP) mut3*, was used to test the recombinant protein expression in an Escherichia coli strain with industrial relevance grown in chemostat. An important factor in enabling stable productivity in continuous cultures is the carbon source. We have studied the viability and heterogeneity of the chemostat cultures using a chemically defined medium based on glucose or glycerol as the single carbon source. As a by-product of biodiesel production, glycerol is expected to become a sustainable alternative substrate to glucose. We have found that although glycerol gives a higher cell density, it also generates higher heterogeneity in the culture and a less stable recombinant protein production. We suggest that manipulating the balance between different subpopulations to increase the proportion of productive cells may be a possible solution for making glycerol a successful alternative to glucose.

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“…This model attains excellent classification accuracy for all conditions in the PBS buffer and for HK and FP in the saline buffer. There remains a substantial number of SP classified as HK, which requires further analysis to understand the origin of the effect measurements because it can measure intensity from multiple fluorophores on the same microbe in a highthroughput manner, sampling thousands to tens of thousands of objects in minutes [47]. Thus, integrating fluorescence lifetime detection into flow cytometry would bring fluorescence lifetime techniques closer to routine diagnostic use by increasing confidence in conclusions drawn from SI-based measurements.…”

Section: Statistical Computationsmentioning

confidence: 99%

Toward absolute viability measurements for bacteria

Dunkers

Iyer

Jones

et al. 2021

Journal of Biophotonics

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We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ 1 , τ 2 and p 1 ), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach.

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Estimation of Microbial Viability Using Flow Cytometry

Cited by 30 publications

References 42 publications

N-methyl Benzimidazole Tethered Cholic Acid Amphiphiles Can Eradicate S. aureus-Mediated Biofilms and Wound Infections

N-methyl Benzimidazole Tethered Cholic Acid Amphiphiles Can Eradicate S. aureus-Mediated Biofilms and Wound Infections

Recombinant Protein Production with Escherichia coli in Glucose and Glycerol Limited Chemostats

Toward absolute viability measurements for bacteria

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