Application of Flow Cytometry to the Detection of Pathogenic Bacteria

Kennedy, Deirdre; Wilkinson, Martin G.

doi:10.21775/cimb.023.021

Cited by 45 publications

(27 citation statements)

References 81 publications

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“…Monitoring of changes in sub-populations of Lactic Acid Bacteria (LAB) starters and probiotic strains in fermented dairy products has been undertaken in a number of studies. The application of FCM assays using multiple stain combinations in the monitoring of pathogen survival in other food types (Cronin & Wilkinson, 2009;Kennedy & Wilkinson, 2017) has also provided fascinating insights into the degree of heterogeneity which develops in microbial populations during storage under various conditions. However, in many of the published studies few direct comparisons have been carried out between data obtained by FCM enumeration and that obtained by traditional plate counting.…”

Section: Immuno-fcm and Bacterial Enumerationmentioning

confidence: 99%

“…Data is collected from individual stained cells and the degree of uptake of a particular stain allows discrimination of cells into discrete sub-populations. Thus, FCM data may reflect differing properties such as the extent of cell membrane integrity, functionality of membrane potential, presence of intracellular enzyme activity and DNA base composition (Egli & Kotzsch, 2015;Kennedy & Wilkinson, 2017;Overton, 2015;Wilkinson, 2016). In this way, additional data can be obtained on individual cell physiology and structure by staining of cells with specific fluorescent dyes.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Wilkinson

2018

Trends in Food Science & Technology

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128

102

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Section: Immuno-fcm and Bacterial Enumerationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Wilkinson

2018

Trends in Food Science & Technology

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128

102

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“…specifically label target cells within a sample, rendering it one of the most rapid and potentially sensitive microbial detection and enumeration techniques (2)(3)(4). Despite this, little work has been undertaken to understand the factors affecting the antibody labeling of bacteria for subsequent immunofluorescence FCM analysis (compared to a growing interest in antibody performance in other fields) (5).…”

mentioning

confidence: 99%

Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Kennedy

Cronin

Piterina

et al. 2019

Appl Environ Microbiol

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The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sublethal heat; exposure to organic acids, salt, and sodium hydroxide; and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/propidium iodide (PI), DiOC2(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labeling by commercial anti-S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensities of stained cells to those of unstained cells [MFI/MFI(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, resulted in significantly lower fluorescence values compared to controls. IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting, should be undertaken. Researchers should be aware of physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

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“…FCM is a method based on the flow of single cells past one or more lasers ( Kennedy & Wilkinson, 2017 ). Scatter and fluorescence data are recorded from thousands of individual cells per second.…”

Section: Dairy Industry Need For Rapid Methods Organisms Of Interestmentioning

confidence: 99%

“…FCM has the potential to do much more in a dairy setting than perform total counts. Combined with antibody tagging and physiological staining the technology has the potential to become the basis of a multiplexed detection system (up to a dozen antibodies and stains can be simultaneously applied) which will detect live, dead or vital microbes down to strain level ( Kennedy & Wilkinson, 2017 ).…”

Section: Dairy Industry Need For Rapid Methods Organisms Of Interestmentioning

confidence: 99%

Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry

O’Grady

Cronin

Tierney

et al. 2020

Advances in Applied Microbiology

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This review presents the results of a study into the offering of rapid microbial detection assays to the Irish dairy industry. At the outset, a consultation process was undertaken whereby key stakeholders were asked to compile a list of the key microorganisms of interest to the sector. The resultant list comprises 19 organisms/groups of organisms divided into five categories: single pathogenic species ( Cronobacter sakazakii , Escherichia coli and Listeria monocytogenes ); genera containing pathogenic species ( Bacillus , Clostridium , Listeria , Salmonella ; Staphylococcus ); broad taxonomic groupings (Coliforms, Enterobacteriaceae, fecal Streptococci, sulfite reducing bacteria/sulfite reducing Clostridia [SRBs/SRCs], yeasts and molds); organisms displaying certain growth preferences or resistance as regards temperature (endospores, psychrotrophs, thermodurics, thermophiles); indicators of quality (total plate count, Pseudomonas spp.). A survey of the rapid assays commercially available for the 19 organisms/groups of organisms was conducted. A wide disparity between the number of rapid tests available was found. Four categories were used to summarize the availability of rapid assays per organism/group of organisms: high coverage (> 15 assays available); medium coverage (5–15 assays available); low coverage (< 5 assays available); no coverage (0 assays available). Generally, species or genera containing pathogens, whose presence is regulated-for, tend to have a good selection of commercially available rapid assays for their detection, whereas groups composed of heterogenous or even undefined genera of mainly spoilage organisms tend to be “low coverage” or “no coverage.” Organisms/groups of organisms with “low coverage” by rapid assays include: Clostridium spp.; fecal Streptococci; and Pseudomonas spp. Those with “no coverage” by rapid assays include: endospores; psychrotrophs; SRB/SRCs; thermodurics; and thermophiles. An important question is: why have manufacturers of rapid microbiological assays failed to respond to the necessity for rapid methods for these organisms/groups of organisms? The review offers explanations, ranging from the technical difficulty involved in detecting as broad a group as the thermodurics, which covers the spores of multiple sporeforming genera as well at least six genera of mesophilic nonsporeformers, to the taxonomically controversial issue as to what constitutes a fecal Streptococcus or SRBs/SRCs. We review two problematic areas for assay developers: validation/certification and the nature of dairy food matrices. Development and implementation of rapid alternative test methods for the dairy industry is influenced by regulations relating to both the microbiological quality standards and the criteria alterna...

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Application of Flow Cytometry to the Detection of Pathogenic Bacteria

Cited by 45 publications

References 81 publications

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry

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