scholarly journals Flow Cytometric Monitoring of Antibiotic-Induced Injury in Escherichia coli Using Cell-Impermeant Fluorescent Probes

2000 ◽  
Vol 44 (3) ◽  
pp. 676-681 ◽  
Author(s):  
Fiona C. Mortimer ◽  
David J. Mason ◽  
Vanya A. Gant
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Antimicrobial Agents ◽  
Flow Cytometric Analysis ◽  
Bacterial Cells ◽  
Nucleic Acid Binding ◽  
Permeabilized Cells ◽  
Sytox Green ◽  
Flow Cytometric

ABSTRACT Three fluorescent nucleic acid binding dyes—propidium iodide, TO-PRO-1, and SYTOX green—were evaluated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells ofEscherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-1. Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent. Large changes in fluorescence intensity were observed for all the dyes subsequent to β-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis. Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts. Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.

scholarly journals Flow Cytometric Investigation of Filamentation, Membrane Patency, and Membrane Potential in Escherichia coli following Ciprofloxacin Exposure

2000 ◽  
Vol 44 (3) ◽  
pp. 682-687 ◽  
Author(s):  
H. J. Wickens ◽  
R. J. Pinney ◽  
D. J. Mason ◽  
V. A. Gant
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Division ◽  
Membrane Potential ◽  
Nucleic Acid ◽  
Nutrient Broth ◽  
Nucleic Acid Binding ◽  
Dye Uptake ◽  
Flow Cytometric ◽  
Drug Removal

ABSTRACT Ninety-eight percent of the cells in a population ofEscherichia coli in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial morphology, cell membrane potential, and ability to exclude nucleic acid-binding dye. These changes persisted beyond the end of the classically defined PAE and were not a definite indicator of cell death as defined by loss of colony formation, which related at least in part to filamentation.

scholarly journals Application of Bacteriophages on Shiga Toxin-Producing Escherichia coli (STEC) Biofilm

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1423
Author(s):  
Nicola Mangieri ◽  
Roberto Foschino ◽  
Claudia Picozzi
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Bacterial Cells ◽  
Abiotic Surfaces ◽  
Continuous Presence ◽  
Different Strains ◽  
Better Than

Shiga toxin-producing Escherichia coli are pathogenic bacteria able to form biofilms both on abiotic surfaces and on food, thus increasing risks for food consumers. Moreover, biofilms are difficult to remove and more resistant to antimicrobial agents compared to planktonic cells. Bacteriophages, natural predators of bacteria, can be used as an alternative to prevent biofilm formation or to remove pre-formed biofilm. In this work, four STEC able to produce biofilm were selected among 31 different strains and tested against single bacteriophages and two-phage cocktails. Results showed that our phages were able to reduce biofilm formation by 43.46% both when used as single phage preparation and as a cocktail formulation. Since one of the two cocktails had a slightly better performance, it was used to remove pre-existing biofilms. In this case, the phages were unable to destroy the biofilms and reduce the number of bacterial cells. Our data confirm that preventing biofilm formation in a food plant is better than trying to remove a preformed biofilm and the continuous presence of bacteriophages in the process environment could reduce the number of bacteria able to form biofilms and therefore improve the food safety.

scholarly journals Transformation of Rickettsia prowazekii to Erythromycin Resistance Encoded by the Escherichia coli ereB Gene

2000 ◽  
Vol 182 (11) ◽  
pp. 3289-3291 ◽  
Author(s):  
Lyudmila I. Rachek ◽  
Andria Hines ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Flow Cytometric Analysis ◽  
Etiologic Agent ◽  
Erythromycin Resistance ◽  
Rickettsia Prowazekii ◽  
Flow Cytometric ◽  
Epidemic Typhus ◽  
Green Fluorescent

ABSTRACT Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression inR. prowazekii of the Escherichia coli ereBgene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene inR. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.

scholarly journals Flow Cytometric Analysis of Adherence of Porphyromonas gingivalis to Oral Epithelial Cells

2007 ◽  
Vol 75 (5) ◽  
pp. 2484-2492 ◽  
Author(s):  
Rishi D. Pathirana ◽  
Neil M. O'Brien-Simpson ◽  
Kumar Visvanathan ◽  
John A. Hamilton ◽  
Eric C. Reynolds
Keyword(s):  
Porphyromonas Gingivalis ◽  
Competitive Inhibition ◽  
Flow Cytometric Analysis ◽  
Large Cell ◽  
Cell Aggregates ◽  
Bacterial Cells ◽  
Kb Cells ◽  
Flow Cytometric ◽  
Logarithmic Relationship ◽  
Oral Epithelial

ABSTRACT By using fluorescence microscopy, fluorescently labeled Porphyromonas gingivalis W50 was shown to adhere to oral epithelial (KB) cells as discrete cells or small cell aggregates, whereas P. gingivalis ATCC 33277 bound as large cell aggregates. Flow cytometric analysis showed that for P. gingivalis W50 there was a logarithmic relationship between the bacterial cell ratio (BCR), that is the number of bacterial cells to KB cells, and the percentage of KB cells with W50 cells attached. This percentage of KB cells with W50 attached reached a plateau of ∼84% cells at a BCR of 500:1. In contrast, a quadratic relationship was observed between BCR and the percentage of KB cells with P. gingivalis ATCC 33277 attached, reaching a maximum of 47% at a BCR of 100:1 but decreasing to 7% at a BCR of 1,000:1. The lower binding of ATCC 33277 at high cell concentrations was attributed to autoaggregation. P. gingivalis W50 cells treated with an inhibitor (Nα-p-tosyl-l-lysine chloromethyl ketone [TLCK]) of its RgpA-Kgp proteinase-adhesin complex exhibited significantly reduced binding to KB cells than to untreated cells, suggesting a role for proteinase activity in binding to KB cells. Competitive inhibition with purified proteinase-active and TLCK-inactivated RgpA-Kgp complex significantly decreased the adherence of P. gingivalis W50 cells to KB cells. Furthermore, isogenic mutants of P. gingivalis W50 lacking the kgp gene product, but not the rgpA or rgpB gene products, exhibited significantly decreased adherence to KB cells compared to the wild type.

scholarly journals Molecular Fossils from Microorganisms Preserved in Glacial Ice

2015 ◽  
Author(s):  
P. Buford Price ◽  
J. Jeffrey Morris ◽  
Ryan C. Bay ◽  
Ajeeth Adhikari ◽  
Stephen J. Giovannoni ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Vertical Motion ◽  
Ice Cores ◽  
Microbial Evolution ◽  
Bacterial Cells ◽  
Triple Junctions ◽  
Genetic Studies ◽  
Glacial Ice ◽  
Flow Cytometric ◽  
Glacial Environments

The study of microbial evolution is hindered by the fact that microbial populations leave few fossils. We hypothesized that bacterial cells preserved in ancient ice could be used as a molecular fossil record if their DNA could be extracted and sequenced. Channels formed along triple junctions of ice crystals contain liquid “veins” in which microbial cells may be preserved intact. Since vertical motion through the ice matrix is impossible, microbes found in ice cores are representative of microbes present at the time the ice was formed. We detected chlorophyll fluorescence in intact ice cores taken from Greenland and Antarctica. Flow cytometric analysis localized at least some of this fluorescence to particles < 1 μm in diameter. Metagenomic analysis of meltwater indeed revealed sequences similar to modern strains of the picocyanobacterial genera Synechococcus and Prochlorococcus, and some of these sequences were distinct from any sequences known from modern oceans or glacial environments. Our study is a first proof-of-concept of the use of ice cores as records of microbial evolution, and we suggest that future genetic studies with higher vertical resolution in the cores might shed light on the pace and character of evolution of these ecologically important cells.

scholarly journals Relationships between cytometric characteristics of high and low nucleic-acid bacterioplankton cells, bacterial production and environmental parameters along a longitudinal gradient across the Mediterranean Sea

2010 ◽  
Vol 7 (6) ◽  
pp. 8245-8279 ◽  
Author(s):  
F. Van Wambeke ◽  
P. Catala ◽  
P. Lebaron
Keyword(s):  
Nucleic Acid ◽  
Mediterranean Sea ◽  
Bacterial Production ◽  
Environmental Parameters ◽  
Early Summer ◽  
Bacterial Cells ◽  
Green Fluorescence ◽  
Flow Cytometric ◽  
Chlorophyll Distribution ◽  
The Mediterranean

Abstract. Heterotrophic bacterioplankton abundance and production were determined along vertical (down to bathypelagic layers) and latitudinal (from 4.9° E to 32.7° E) gradients across the Mediterranean Sea in early summer 2008. Abundance and flow cytometric characteristics (green fluorescence and side scatter signals) of high nucleic acid (HNA) and low nucleic acid (LNA) bacterial cells were investigated using flow cytometry. Contrarily to what is generally observed, the percentage of total bacteria represented by HNA cells (%HNA, range 30–69%) decreased with increased bacterial production (range 0.15–44 ng C l−1 h−1) although this negative relation was poorly explained (log-log regression r2=0.19). The %HNA as well as the mean side scatter of this group increased significantly with depth in the meso and bathypelagic layers. Our results demonstrated that vertical stratification with regard to chlorophyll distribution above, within or below the deep chlorophyll maximum plays an important role in influencing the distribution of cells, and in the relationships between the flow cytometric parameters and environmental variables such as chlorophyll a or bacterial production. Relationships between green fluorescence and side scatter of both HNA and LNA cells depended largely on chlorophyll distribution over the water column, suggesting that the dynamic link between HNA and LNA cells differs vertically.

scholarly journals Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

1998 ◽  
Vol 64 (7) ◽  
pp. 2697-2700 ◽  
Author(s):  
P. Lebaron ◽  
P. Catala ◽  
N. Parthuisot
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Salmonella Typhimurium ◽  
Viability Assessment ◽  
Bacterial Viability ◽  
Sytox Green ◽  
Nucleic Acid Stain

ABSTRACT The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations ofEscherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.

Sign in / Sign up

Export Citation Format

Share Document