Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry

ABSTRACT The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

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Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.539-545.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 539-545 ◽

Cited By ~ 162

Author(s):

Feng Chen ◽

Jing-rang Lu ◽

Brian J. Binder ◽

Ying-chun Liu ◽

Robert E. Hodson

Keyword(s):

Flow Cytometry ◽

Image Analysis ◽

Digital Image ◽

Digital Image Analysis ◽

Flow Cytometric Analysis ◽

Epifluorescence Microscopy ◽

Flow Cytometric ◽

Viral Particles ◽

Sybr Gold ◽

Nucleic Acid Stain

ABSTRACT A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

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Polyamine Effects on Antibiotic Susceptibility in Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01472-06 ◽

2007 ◽

Vol 51 (6) ◽

pp. 2070-2077 ◽

Cited By ~ 76

Author(s):

Dong-Hyeon Kwon ◽

Chung-Dar Lu

Keyword(s):

Synergistic Effect ◽

Outer Membrane ◽

Antibiotic Susceptibility ◽

Efflux Pump ◽

Population Analysis ◽

Enteric Bacteria ◽

Dependent Manner ◽

E Coli ◽

Serovar Typhimurium ◽

Exogenous Spermine

ABSTRACT Biogenic polyamines (e.g., spermidine and spermine) are a group of essential polycationic compounds found in all living cells. The effects of spermine and spermidine on antibiotic susceptibility were examined with gram-negative Escherichia coli and Salmonella enterica serovar Typhimurium bacteria and clinical isolates of Pseudomonas aeruginosa and with gram-positive Staphylococcus aureus bacteria, including methicillin-resistant S. aureus (MRSA). Exogenous spermine exerted a dose-dependent inhibition effect on the growth of E. coli, S. enterica serovar Typhimurium, and S. aureus but not P. aeruginosa, as depicted by MIC and growth curve measurements. While the MICs of polymyxin and ciprofloxacin were in general increased by exogenous spermine and spermidine in P. aeruginosa, this adverse effect was not observed in enteric bacteria and S. aureus. It was found that spermine and spermidine can decrease the MICs of β-lactam antibiotics in all strains as well as other types of antibiotics in a strain-dependent manner. Significantly, the MICs of oxacillin for MRSA Mu50 and N315 were decreased more than 200-fold in the presence of spermine, and this effect of spermine was retained when assessed in the presence of divalent ions (magnesium or calcium; 3 mM) or sodium chloride (150 mM). The effect of spermine on the sensitization of P. aeruginosa and MRSA to antibiotics was further demonstrated by population analysis and time-killing assays. The results of checkerboard assays with E. coli and S. aureus indicated a strong synergistic effect of spermine in combination with β-lactams and chloramphenicol. The decreased MICs of β-lactams implied that the possible blockage of outer membrane porins by exogenous spermine or spermidine did not play a crucial role in most cases. In contrast, only the MIC of imipenem against P. aeruginosa was increased by exogenous spermine and spermidine, and this resistance effect was abolished in a mutant strain devoid of the outer membrane porin OprD. In E. coli, the MICs of carbenicillin, chloramphenicol, and tetracycline were decreased in two acrA mutants devoid of a major efflux pump, AcrAB. However, retention of the spermine effect on antibiotic susceptibility in two acrA mutants of E. coli suggested that the AcrAB efflux pump was not the target for a synergistic effect by spermine and antibiotics and ruled out the hypothesis of spermine serving as an efflux pump inhibitor in this organism. In summary, this interesting finding of the effect of spermine on antibiotic susceptibility provides the basis for a new potential approach against drug-resistant pathogens by use of existing β-lactam antibiotics.

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Molecular Analysis of Sucrose Metabolism ofErwinia amylovora and Influence on Bacterial Virulence

Journal of Bacteriology ◽

10.1128/jb.182.19.5351-5358.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5351-5358 ◽

Cited By ~ 42

Author(s):

Jochen Bogs ◽

Klaus Geider

Keyword(s):

Flow Cytometry ◽

Fire Blight ◽

Fluorescent Protein ◽

Sucrose Concentration ◽

Sucrose Metabolism ◽

Open Reading Frames ◽

Site Directed Mutagenesis ◽

Green Fluorescent Protein Gene ◽

E Coli ◽

Green Fluorescent

ABSTRACT Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scrregulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genesscrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scrregulons from Klebsiella pneumoniae and plasmid pUR400.scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) ofE. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a Km of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYABpromoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora.

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Using Colistin as a Trojan Horse: Inactivation of Gram-Negative Bacteria with Chlorophyllin

Antibiotics ◽

10.3390/antibiotics8040158 ◽

2019 ◽

Vol 8 (4) ◽

pp. 158 ◽

Cited By ~ 3

Author(s):

Richter ◽

Krüger ◽

Prasad ◽

Gastiger ◽

Bodenschatz ◽

...

Keyword(s):

Outer Membrane ◽

Combination Treatment ◽

Salmonella Enterica Serovar Typhimurium ◽

Gram Negative Bacteria ◽

Photodynamic Inactivation ◽

Gram Negative ◽

E Coli ◽

Serovar Typhimurium ◽

Polymyxin E ◽

Toxic Concentrations

Colistin (polymyxin E) is a membrane-destabilizing antibiotic used against Gram-negative bacteria. We have recently reported that the outer membrane prevents the uptake of antibacterial chlorophyllin into Gram-negative cells. In this study, we used sub-toxic concentrations of colistin to weaken this barrier for a combination treatment of Escherichia coli and Salmonella enterica serovar Typhimurium with chlorophyllin. In the presence of 0.25 µg/mL colistin, chlorophyllin was able to inactivate both bacteria strains at concentrations of 5–10 mg/L for E. coli and 0.5–1 mg/L for S. Typhimurium, which showed a higher overall susceptibility to chlorophyllin treatment. In accordance with a previous study, chlorophyllin has proven antibacterial activity both as a photosensitizer, illuminated with 12 mW/cm2, and in darkness. Our data clearly confirmed the relevance of the outer membrane in protection against xenobiotics. Combination treatment with colistin broadens chlorophyllin’s application spectrum against Gram-negatives and gives rise to the assumption that chlorophyllin together with cell membrane-destabilizing substances may become a promising approach in bacteria control. Furthermore, we demonstrated that colistin acts as a door opener even for the photodynamic inactivation of colistin-resistant (mcr-1-positive) E. coli cells by chlorophyllin, which could help us to overcome this antimicrobial resistance.

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Automation of human sperm cell analysis by flow cytometry

Clinical Chemistry ◽

10.1093/clinchem/43.5.801 ◽

1997 ◽

Vol 43 (5) ◽

pp. 801-807 ◽

Cited By ~ 15

Author(s):

Fulvio Ferrara ◽

Rita Daverio ◽

Giuliano Mazzini ◽

Pierangelo Bonini ◽

Giuseppe Banfi

Keyword(s):

Flow Cytometry ◽

Flow Cytometric Analysis ◽

Human Sperm ◽

Sperm Analysis ◽

Cell Counts ◽

Flow Cytometric ◽

Sperm Counts ◽

The Mean ◽

Nucleic Acid Stain ◽

Automated Methods

Abstract Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 × 106 sperms/mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.

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An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry

Viruses ◽

10.3390/v13010102 ◽

2021 ◽

Vol 13 (1) ◽

pp. 102

Author(s):

Mara Elena Heinrichs ◽

Daniele De Corte ◽

Bert Engelen ◽

Donald Pan

Keyword(s):

Flow Cytometry ◽

Marine Sediment ◽

Biogeochemical Cycles ◽

Organic Content ◽

Epifluorescence Microscopy ◽

Active Components ◽

Flow Cytometric ◽

Sediment Particles ◽

High Organic Content ◽

Accurate Quantification

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R2 = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.

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Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

10.1101/480392 ◽

2018 ◽

Author(s):

Srijan Jindal ◽

Harish Thampy ◽

Philip J. Day ◽

Douglas B. Kell

Keyword(s):

Flow Cytometry ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Lag Phase ◽

E Coli ◽

Flow Cytometric ◽

Size Number ◽

Antibiotic Susceptibility Test ◽

Carbocyanine Dye ◽

Rapid Flow

AbstractCells ofE. coliwere grown in LB medium, taken from a stationary phase of 2-4h, and reinoculated into fresh media at a concentration (105.mL-1or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energisation (using a carbocyanine dye) and DNA distribution. It turned out that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of reinoculation, providing the possibility of a very rapid antibiotic susceptibility test, on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential Urinary Tract Infection (UTI) samples taken from a doctor’s surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

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A Gene Encoding l-Methionine γ-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundiil-Methionine γ-Lyase

Journal of Bacteriology ◽

10.1128/jb.187.11.3889-3893.2005 ◽

2005 ◽

Vol 187 (11) ◽

pp. 3889-3893 ◽

Cited By ~ 48

Author(s):

Ilya V. Manukhov ◽

Daria V. Mamaeva ◽

Sergei M. Rastorguev ◽

Nicolai G. Faleev ◽

Elena A. Morozova ◽

...

Keyword(s):

Shigella Flexneri ◽

Open Reading Frames ◽

Gene Encoding ◽

High Sequence Identity ◽

E Coli ◽

The Family ◽

Serovar Typhimurium ◽

Identification And Characterization ◽

Reading Frames

ABSTRACT Citrobacter freundii cells produce l-methionine γ-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine γ-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.

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Rapid assessment of antibiotic effects on Escherichia coli by bis-(1,3-dibutylbarbituric acid) trimethine oxonol and flow cytometry.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.9.2001 ◽

1997 ◽

Vol 41 (9) ◽

pp. 2001-2005 ◽

Cited By ~ 51

Author(s):

R I Jepras ◽

F E Paul ◽

S C Pearson ◽

M J Wilkinson

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Membrane Potential ◽

Rapid Assessment ◽

Physiological Changes ◽

Analytical Technique ◽

E Coli ◽

Flow Cytometric ◽

Bacterial Cell Viability ◽

Sensitive Analytical Technique

The effects of selected antibiotics on Escherichia coli were studied by flow cytometry with the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The actions of azithromycin, cefuroxime, and ciprofloxacin at five times the MIC on E. coli were compared by the traditional CFU assay and flow cytometry. Changes in viable counts of bacteria determined with DiBAC4(3) and by flow cytometry following treatment with the antibiotics showed trends similar to those found by the CFU assays. However, viable counts determined by flow cytometry following antibiotic treatment were 1 to 2 logs higher than those determined by the corresponding CFU assays. All the results obtained by flow cytometry were provided within 10 min after sampling, whereas the conventional CFU assay results took at least 18 h. The results indicated that flow cytometry is a sensitive analytical technique that can rapidly monitor the physiological changes of individual microorganisms following antibiotic action and can provide information on the mode of action of a drug. The membrane potential probe DiBAC4(3) provides a robust flow cytometric indicator for bacterial cell viability.

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Curli Loci of Shigella spp

Infection and Immunity ◽

10.1128/iai.68.6.3780-3783.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3780-3783 ◽

Cited By ~ 55

Author(s):

Harry Sakellaris ◽

Nerissa K. Hannink ◽

Kumar Rajakumar ◽

Dieter Bulach ◽

Meredith Hunt ◽

...

Keyword(s):

Selective Pressure ◽

Shigella Flexneri ◽

Pcr Analysis ◽

Environmental Isolates ◽

E Coli ◽

Wide Range ◽

Serovar Typhimurium ◽

Genes Encoding ◽

Shigella Spp ◽

Pathoadaptive Mutations

ABSTRACT An unstable chromosomal element encoding multiple antibiotic resistance in Shigella flexneri serotype 2a was found to include sequences homologous to the csg genes encoding curli in Escherichia coli and Salmonella enterica serovar Typhimurium. As curli have been implicated in the virulence of serovar Typhimurium, we investigated thecsg loci in all four species of Shigella. DNA sequencing and PCR analysis showed that the csg loci of a wide range of Shigella strains, of diverse serotypes and different geographical distributions, were almost universally disrupted by deletions or insertions, indicating the existence of a strong selective pressure against the expression of curli. Strains of enteroinvasive E. coli (EIEC), which share virulence traits with Shigella spp. and cause similar diseases in humans, also possessed insertions or deletions in the csg locus or were otherwise unable to produce curli. Since the production of curli is a widespread trait in environmental isolates of E. coli, our results suggest that genetic lesions that abolish curli production in the closely related genus Shigella and in EIEC are pathoadaptive mutations.

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