New methodology for viability testing in environmental samples

Biggerstaff, John; Puil, M. Le; Weidow, Brandy; Prater, Jason; Glass, Kathleen A.; Radosevich, Mark; White, David C.

doi:10.1016/j.mcp.2005.11.006

Cited by 65 publications

(49 citation statements)

References 10 publications

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“…Barbesti et al (6) devised a similar protocol, but using SG combined with PI (so that the researchers did not need to pay for the kit). Other authors have used other combinations of stains (e.g., Hoechst and PI [51] and Syto13 and Sytox Orange [11]). Using flow cytometry, the interpretation of the green-versus-red-fluorescence cytograms could provide information not only on "live" (green) and "dead" (red) cells but also on "green-plus-red" particles, which have been identified as "damaged" cells (30).…”

Section: Discussionmentioning

confidence: 99%

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

Falcioni

Papa

Gasol

2008

Appl Environ Microbiol

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Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 g ml ؊1 of propidium iodide, simultaneous to a 10؋ concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.Heterotrophic prokaryotes (Bacteria and Archaea) play a relevant role in planktonic marine microbial food webs (e.g., reference 5). As a relevant difference from other planktonic organisms, prokaryotes are known to sometimes be inactive or dead, but in the way that they are commonly enumerated (e.g., with DAPI [4Ј,6Ј-diamidino-2-phenylindole]), these cells are accounted for in budgets for prokaryote biomass since they are not distinguished from live and active cells.From an ecological point of view, at least three cell categories within microbial communities can be distinguished: the viable and active cells, which play a functional role and participate in biomass production at the time of sampling; the live but inactive cells (often called dormant cells), which do not participate in production at the time of sampling but have the potential for doing so; and the dead and therefore inactive cells, which do not have a role in the cycling of chemical elements, even though they might be retaining nutrients. The discrimination of these cellular categories is not without dispute: for example, the "active" term is often confounded with the "live" one and the "inactive" with the "dead" one (26). Moreover, different intrinsic levels of metabol...

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Section: Discussionmentioning

confidence: 99%

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

Falcioni

Papa

Gasol

2008

Appl Environ Microbiol

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“…Changes in IM integrity were determined using similarly prepared cultures by measuring ATP leakage with the CellTiter-Glo luminescent kit (Promega) as described previously (39). Membrane integrity was also assessed by using the Sytox orange (Molecular Probes) DNA stain (40,41). After growth to an OD 600 of 0.5 as described above, cells were resuspended in 25 ml of fresh TSB, and 2-ml samples were assayed.…”

Section: Methodsmentioning

confidence: 99%

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

et al. 2016

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Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-

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“…Detection limits for PCR and FISH are relatively high, being about 10 4 cells/ml and 10 6 cells/ ml, respectively. FISH is based on detection of 16s rRNA whose presence is not a direct proof of metabolic activity but rather an indication of potential viability (Biggerstaff, 2006). Real-time PCR and FISH have a limitation whereby counts of bacteria decreased but PCR and FISH results remained higher over the experimental period.…”

Section: Methods For Quantification Of Probiotic Culturesmentioning

confidence: 99%