scholarly journals A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Atheesha Singh ◽  
Tobias George Barnard
Keyword(s):  
Flow Cytometry ◽  
Gastric Acid ◽  
Vibrio Cholerae ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Gastric Fluid ◽  
Simulated Gastric Fluid ◽  
Assessment Protocol ◽  
Pathogenic Vibrio ◽  
Vibrio Cholerae O1

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a “stressed state” due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

Surviving the acid barrier: responses of pathogenic Vibrio cholerae to simulated gastric fluid

2015 ◽  
Vol 100 (2) ◽  
pp. 815-824 ◽  
Author(s):  
Atheesha Singh ◽  
Tobias G. Barnard
Keyword(s):  
Vibrio Cholerae ◽  
Gastric Fluid ◽  
Simulated Gastric Fluid ◽  
Pathogenic Vibrio

scholarly journals The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

2006 ◽  
Vol 31 (4) ◽  
pp. 349-356 ◽  
Author(s):  
Luiz G. Chitarra ◽  
Peter Breeuwer ◽  
Tjakko Abee ◽  
Ruud W. Bulk
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Pathogenic Bacterium ◽  
Plant Pathogenic Bacterium ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Alternatives Assessment ◽  
Heat Treated ◽  
Calcein Am

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

scholarly journals Flow Cytometric Assessment of the Postantibiotic Effect of Methicillin on Staphylococcus aureus

1998 ◽  
Vol 42 (5) ◽  
pp. 1195-1199 ◽  
Author(s):  
M. T. E. Suller ◽  
D. Lloyd
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Respiratory Activity ◽  
Membrane Integrity ◽  
Postantibiotic Effect ◽  
Tetrazolium Chloride ◽  
Fluorescence Measurements ◽  
Flow Cytometric ◽  
Solid Agar

ABSTRACT The postantibiotic effect (PAE) following a 2-h exposure ofStaphylococcus aureus NCTC 6571 to methicillin (5× the MIC) was investigated with fluorescent probes, 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC), an indicator of respiratory activity, and the membrane potential-sensitive compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Counts of the numbers of CFU on solid agar correlated well with information gained from the CTC and DiBAC4(3) fluorescence intensity distributions obtained by flow cytometry and revealed that the postantibiotic effect was 3.1 h. Due to the capacity of flow cytometry to provide information on the heterogeneity of a bacterial population, both fluorescent probes identified the emergence of an active subpopulation 4 h after removal of the methicillin, indicating the recovery of a small percentage of the population. After removal of the methicillin and resuspension of the cells in methicillin-free medium, a further decrease in the respiratory activity and the membrane integrity of the population was observed, although the CFU counts hardly varied, indicating continued antibiotic-induced damage. Also, CTC fluorescence measurements identified numerous subpopulations during the PAE period; this suggests that the PAE is complex, with individual organisms exhibiting various degrees of recovery. Flow cytometry thus provides a rapid and sensitive alternative to traditional techniques that have been used to study PAE, with the added advantage that physiological changes can be detected as they arise.

scholarly journals Genome Sequence of a Pathogenic Vibrio cholerae O1 El Tor Strain Defective for the Entire Vibrio Pathogenicity Island 1, Isolated in Eastern Democratic Republic of the Congo

2020 ◽  
Vol 9 (26) ◽  
Author(s):  
Leonid M. Irenge ◽  
Jean-François Durant ◽  
Jérôme Ambroise ◽  
Prudence N. Mitangala ◽  
Bertrand Bearzatto ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Genome Sequence ◽  
Democratic Republic ◽  
Pathogenicity Island ◽  
Content Type ◽  
Pathogenic Vibrio ◽  
Republic Of The Congo ◽  
Vibrio Cholerae O1 ◽  
El Tor ◽  
North Kivu

ABSTRACT We report here a complete genome sequence of a Vibrio cholerae O1 El Tor (Inaba; sequence type 515 [ST515]) strain isolated from a cholera patient in North Kivu Province, Democratic Republic of the Congo (DRC), which showed a complete deletion (∼80 kb) of the Vibrio pathogenicity island 1.

262 THE EFFECT OF TRIS AND Botu-Bov® FOR BOVINE SEXED SPERM CRYOPRESERVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 211
Author(s):  
C. P. Freitas ◽  
J. A. Dell'Aqua-Junior ◽  
F. O. Papa ◽  
M. A. Alvarenga ◽  
A. M. Crespilho ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Semen Analysis ◽  
Bos Indicus ◽  
Membrane Integrity ◽  
Reproductive Age ◽  
Freezing Process ◽  
Control Group ◽  
Sperm Cryopreservation ◽  
High Concentration

Sexing of sperm by flow cytometry has been applied worldwide. However, the sorting as well as the cryopreservation process causes damage to the sperm cells. Therefore, the sexed sperm need enhanced protection during the freezing process. Thus, the aim of the present study was to compare the effect of two different extenders for sperm cryopreservation. Two ejaculates from 10 bulls (Bos taurus and Bos indicus) of reproductive age and used in commercial AI programs were used. The ejaculates were collected with an artificial vagina and the semen was analyzed and prepared for separation by flow cytometry. After sorting, the samples were split into two groups for freezing in TRIS-egg yolk or Botu-Bov� (Biotech Botucatu, Ltda., Sao Paulo, Brazil). Sperm were evaluated by computer-aided semen analysis (CASA) and fluorescent probes to determine plasma and acrosomal membrane integrity. The fertility of the spermwas also tested in an IVF program. Statistical analysis of sperm parameters was achieved using theTukey test with a significant level of P ≤ 0.05. Embryo production data after IVF were analyzed by chi-square test. There was a significant improvement in progressive motility with the use of Botu-Bov, compared with TRIS (67.4% v. 56.7%, respectively). There was also a statistical difference for curvilinear velocity (VCL) and lateral head displacement (ALH), with TRIS showing higher values than Botu-Bov (VCL = 206.6 v. 157.3 µm s–1, and ALH = 8.0 v. 5.9 µm, respectively). According to the literature, an ALH higher than 7 µm associated with a high VCL characterizes a vigorous and disordered movement indicating hyperactivity. The sperm frozen with the Botu-Bov had a higher straight movement (STR; 85.1%) and linearity (57.4%) than the TRIS group (79.8% and 45.6%, respectively). These features indicate a straighter and more uniform movement when Botu-Bov was used. The morphological analyses using fluorescent probes showed higher proportions of intact plasma and acrosomal membranes for Botu-Bov than for TRIS (50.1% v. 39.4% and 85.3% v. 71.2% for Botu-Bov and TRIS, respectively). Blastocyst formation after IVF was 21% (68/327; blastocysts/matured oocytes) for the TRIS group. This result was statistically different from the 30% (75/252) of blastocysts obtained with Botu-Bov and from the 34% (43/128) obtained in the control group. These results suggest that Botu-Bov provides better conditions for the maintenance of the viability of frozen sexed semen than does TRIS, probably due to the high concentration of essential and nonessential amino acids present in the Botu-Bov extender. This work was supported by Sexing Technologies – Brazil and HG Lagoa da Serra.

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample

2015 ◽  
Vol 63 ◽  
pp. 347-353 ◽  
Author(s):  
Numfon Khemthongcharoen ◽  
Wijit Wonglumsom ◽  
Assawapong Suppat ◽  
Kata Jaruwongrungsee ◽  
Adisorn Tuantranont ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Food Sample ◽  
Sensitive Detection ◽  
Dna Sensor ◽  
Pathogenic Vibrio ◽  
Vibrio Cholerae O1

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Вариабельность генома островов патогенности нетоксигенных штаммов Vibrio cholerae O1 биовара Эль Тор

2020 ◽  
Vol 56 (9) ◽  
pp. 1018-1033
Author(s):  
Н. И. Смирнова ◽  
А. А. Крицкий ◽  
Ж. В. Альхова ◽  
Е. Ю. Агафонова ◽  
Е. Ю. Щелканова ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Cholerae O1

Genomic Variability of Pathogenicity Islands in Nontoxigenic Strains of Vibrio cholerae O1 Biotype El Tor

2020 ◽  
Vol 56 (9) ◽  
pp. 1055-1069
Author(s):  
N. I. Smirnova ◽  
A. A. Kritsky ◽  
J. V. Alkhova ◽  
E. Yu. Agafonova ◽  
E. Yu. Shchelkanova ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Pathogenicity Islands ◽  
Genomic Variability ◽  
Vibrio Cholerae O1 ◽  
El Tor

scholarly journals Formulation of Quaternized Aminated Chitosan Nanoparticles for Efficient Encapsulation and Slow Release of Curcumin

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 449
Author(s):  
Ahmed M. Omer ◽  
Zyta M. Ziora ◽  
Tamer M. Tamer ◽  
Randa E. Khalifa ◽  
Mohamed A. Hassan ◽  
...  
Keyword(s):  
Slow Release ◽  
Sodium Tripolyphosphate ◽  
Chitosan Nanoparticles ◽  
Gastric Fluid ◽  
Release Profile ◽  
Simulated Gastric Fluid ◽  
Maximum Value ◽  
Structure Surface ◽  
Drug Encapsulation Efficiency

An effective drug nanocarrier was developed on the basis of a quaternized aminated chitosan (Q-AmCs) derivative for the efficient encapsulation and slow release of the curcumin (Cur)-drug. A simple ionic gelation method was conducted to formulate Q-AmCs nanoparticles (NPs), using different ratios of sodium tripolyphosphate (TPP) as an ionic crosslinker. Various characterization tools were employed to investigate the structure, surface morphology, and thermal properties of the formulated nanoparticles. The formulated Q-AmCs NPs displayed a smaller particle size of 162 ± 9.10 nm, and higher surface positive charges, with a maximum potential of +48.3 mV, compared to native aminated chitosan (AmCs) NPs (231 ± 7.14 nm, +32.8 mV). The Cur-drug encapsulation efficiency was greatly improved and reached a maximum value of 94.4 ± 0.91%, compared to 75.0 ± 1.13% for AmCs NPs. Moreover, the in vitro Cur-release profile was investigated under the conditions of simulated gastric fluid [SGF; pH 1.2] and simulated colon fluid [SCF; pH 7.4]. For Q-AmCs NPs, the Cur-release rate was meaningfully decreased, and recorded a cumulative release value of 54.0% at pH 7.4, compared to 73.0% for AmCs NPs. The formulated nanoparticles exhibited acceptable biocompatibility and biodegradability. These findings emphasize that Q-AmCs NPs have an outstanding potential for the delivery and slow release of anticancer drugs.

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