Combined Use of Fluorescent Reporters and Flow Cytometry for Simultaneous Monitoring of Bacterial Growth and Gene Expression on Plant Roots

Measuring the bacterial growth potential of seawater reverse osmosis (SWRO) feed water is an issue that is receiving growing attention. This study developed and demonstrated the applicability of the flow-cytometry (FCM)-based bacterial growth potential (BGP) method to assess the biofouling potential in SWRO systems using natural microbial consortium. This method is relatively fast (2–3 days) compared to conventional bioassays. The effect of the potential introduction of nutrients during measurement has been studied thoroughly to achieve the lowest measure value of about 45,000 cells/mL, which is equivalent to about (10 µg-C glucose/L). The BGP method was applied in two full-scale SWRO plants that included (i) dissolved air flotation (DAF) and ultra-filtration (UF); (ii) dual-media filtration (DMF) and cartridge filter (CF), which were compared with the cleaning frequency of the plants. A significant reduction (54%) in BGP was observed through DAF–UF as pre-treatment (with 0.5 mg Fe3+/L), while there was a 40% reduction by DMF–CF (with 0.8 mg Fe3+/L). In terms of the absolute number, the SWRO feed water after DAF–UF supports 1.5 × 106 cells/mL, which is 1.25 times higher than after DMF–CF. This corresponds to the higher cleaning-in-place (CIP) frequency of SWRO with DAF–UF compared to DMF–CF as pre-treatment, indicating that the BGP method has an added value in monitoring the biofouling potential in SWRO systems.

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Fructose Enhanced Reduction of Bacterial Growth on Nanorough Surfaces

MRS Proceedings ◽

10.1557/opl.2013.35 ◽

2013 ◽

Vol 1498 ◽

pp. 73-78 ◽

Cited By ~ 2

Author(s):

N. Gozde Durmus ◽

Erik N. Taylor ◽

Kim M. Kummer ◽

Thomas J. Webster

Keyword(s):

Bacterial Growth ◽

Biofilm Formation ◽

Antibacterial Agents ◽

Bacteria Growth ◽

Planktonic Bacteria ◽

Combined Use ◽

Wide Range ◽

Metabolic Stimulation ◽

Antibacterial Surfaces ◽

First Time

ABSTRACTBiofilms are a major source of medical device-associated infections, due to their persistent growth and antibiotic resistance. Recent studies have shown that engineering surface nanoroughness has great potential to create antibacterial surfaces. In addition, stimulation of bacterial metabolism increases the efficacy of antibacterial agents to eradicate biofilms. In this study, we combined the antibacterial effects of nanorough topographies with metabolic stimulation (i.e., fructose metabolites) to further decrease bacterial growth on polyvinyl chloride (PVC) surfaces, without using antibiotics. We showed for the first time that the presence of fructose on nanorough PVC surfaces decreased planktonic bacteria growth and biofilm formation after 24 hours. Most importantly, a 60% decrease was observed on nanorough PVC surfaces soaked in a 10 mM fructose solution compared to conventional PVC surfaces. In this manner, this study demonstrated that bacteria growth can be significantly decreased through the combined use of fructose and nanorough surfaces and thus should be further studied for a wide range of antibacterial applications.

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Glucocorticoid receptor expression on circulating leukocytes differs between healthy male and female adults

Journal of Clinical and Translational Science ◽

10.1017/cts.2016.20 ◽

2017 ◽

Vol 1 (2) ◽

pp. 108-114 ◽

Cited By ~ 13

Author(s):

Kim D. Lu ◽

Shlomit Radom-Aizik ◽

Fadia Haddad ◽

Frank Zaldivar ◽

Monica Kraft ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Sexual Dimorphism ◽

Natural Killer Cells ◽

Glucocorticoid Receptor ◽

Natural Killer ◽

Peripheral Blood ◽

Healthy Adults ◽

Peripheral Blood Mononuclear ◽

Killer Cells

IntroductionThe glucocorticoid receptor (GR) is a key receptor involved in inflammatory responses and is influenced by sex steroids. This study measured GR expression on circulating leukocyte subtypes in males and females.MethodsA total of 23 healthy adults (12 female) participated in this study. GR expression was measured in leukocyte subtypes using flow cytometry. Peripheral blood mononuclear cell (PBMC) gene expression of GR (NR3C1), GR β, TGF-β1 and 2, and glucocorticoid-induced leucine zipper (GILZ) were determined by real-time polymerase chain reaction.ResultsLeukocyte GR was lower in females, particularly in granulocytes, natural killer cells, and peripheral blood mononuclear cells (p≤0.01). GR protein expression was different across leukocyte subtypes, with higher expression in eosinophils compared with granulocytes, T lymphocytes, and natural killer cells (p<0.05). There was higher gene expression of GR β in males (p=0.03).ConclusionsThis is the first study to identify sexual dimorphism in GR expression in healthy adults using flow cytometry. These results may begin to explain the sexual dimorphism seen in many diseases and sex differences in glucocorticoid responsiveness.

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Quantitative assessment of bacterial growth phase utilizing flow cytometry

Journal of Microbiological Methods ◽

10.1016/j.mimet.2019.105760 ◽

2019 ◽

Vol 167 ◽

pp. 105760

Author(s):

K.M. Khomtchouk ◽

M. Weglarz ◽

L.A. Bekale ◽

I. Koliesnik ◽

P.L. Bollyky ◽

...

Keyword(s):

Flow Cytometry ◽

Bacterial Growth ◽

Growth Phase ◽

Quantitative Assessment

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Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy

Innate Immunity ◽

10.1177/1753425917709508 ◽

2017 ◽

Vol 23 (5) ◽

pp. 440-448 ◽

Cited By ~ 3

Author(s):

Reza Gholamnezhadjafari ◽

Nader Tajik ◽

Reza Falak ◽

Reza Aflatoonian ◽

Sanaz Dehghan ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Inflammatory Factors ◽

Control Group ◽

Organ Donors ◽

Complement Components ◽

Tnf Α ◽

Brain Dead

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

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Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells

mBio ◽

10.1128/mbio.01435-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 16

Author(s):

Shiou-Ling Lu ◽

Chih-Feng Kuo ◽

Hao-Wen Chen ◽

Yi-Shuan Yang ◽

Ching-Chuan Liu ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Epithelial Cells ◽

Bacterial Growth ◽

Group A Streptococcus ◽

Multiorgan Failure ◽

Specific Inhibitor ◽

Low Ph ◽

Bafilomycin A1 ◽

Group A

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells.IMPORTANCEPrevious reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.

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