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Hazan, Ronen N.

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Hazan

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Ronen N.

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Hazan, Ronen N.
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    Production of Pseudomonas aeruginosa Intercellular Small Signaling Molecules in Human Burn Wounds
    (SAGE-Hindawi Access to Research, 2011) Que, Yok-Ai; Hazan, Ronen N.; Ryan, Colleen; Milot, Sylvain; Lépine, François; Lydon, Martha; Rahme, Laurence
    Pseudomonas aeruginosa has developed a complex cell-to-cell communication system that relies on low-molecular weight excreted molecules to control the production of its virulence factors. We previously characterized the transcriptional regulator MvfR, that controls a major network of acute virulence functions in P. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (HAQs)—4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Though HHQ and PQS are produced in infected animals, their ratios differ from those in bacterial cultures. Because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human P. aeruginosa acute wound infection and whether their ratio is similar to that observed in P. aeruginosa-infected mice. We found that a clinically relevant P. aeruginosa isolate produced detectable levels of HAQs with ratios of HHQ and PQS that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. These molecules could be isolated from wound tissue as well as from drainage liquid. These results demonstrate for the first time that HAQs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection.
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    A Method for High Throughput Determination of Viable Bacteria Cell Counts in 96-Well Plates
    (BioMed Central, 2012) Hazan, Ronen N.; Que, Yok-Ai; Maura, Damien; Rahme, Laurence
    Background: There are several methods for quantitating bacterial cells, each with advantages and disadvantages. The most common method is bacterial plating, which has the advantage of allowing live cell assessment through colony forming unit (CFU) counts but is not well suited for high throughput screening (HTS). On the other hand, spectrophotometry is adaptable to HTS applications but does not differentiate between dead and living bacteria and has low sensitivity. Results: Here, we report a bacterial cell counting method termed Start Growth Time (SGT) that allows rapid and serial quantification of the absolute or relative number of live cells in a bacterial culture in a high throughput manner. We combined the methodology of quantitative polymerase chain reaction (qPCR) calculations with a previously described qualitative method of bacterial growth determination to develop an improved quantitative method. We show that SGT detects only live bacteria and is sensitive enough to differentiate between 40 and 400 cells/mL. SGT is based on the re-growth time required by a growing cell culture to reach a threshold, and the notion that this time is proportional to the number of cells in the initial inoculum. We show several applications of SGT, including assessment of antibiotic effects on cell viability and determination of an antibiotic tolerant subpopulation fraction within a cell population. SGT results do not differ significantly from results obtained by CFU counts. Conclusion: SGT is a relatively quick, highly sensitive, reproducible and non-laborious method that can be used in HTS settings to longitudinally assess live cells in bacterial cell cultures.
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    Honey’s Ability to Counter Bacterial Infections Arises from Both Bactericidal Compounds and QS Inhibition
    (Frontiers Research Foundation, 2012) Wang, Rui; Starkey, Melissa; Hazan, Ronen N.; Rahme, Laurence
    The ability of honey to kill bacterial pathogens in vitro and quickly clear even chronic or drug-resistant infections has been demonstrated by several studies. Most current research is focused on identifying the bactericidal compounds in honey, but the action of the compounds discovered is not sufficient to explain honey’s activity. By diluting honey to sub-inhibitory levels, we were able to study its impact on bacterial coordinated behavior, and discovered that honey inhibits bacterial quorum sensing (QS). Experiments to characterize and quantify honey’s effect on the QS networks of Pseudomonas aeruginosa revealed that low concentrations of honey inhibited the expression of MvfR, las, and rhl regulons, including the associated virulence factors. This research also establishes that inhibition of QS is associated with honey’s sugar content. Therefore, honey combats infections by two independent mechanisms acting in tandem: bactericidal components, which actively kill cells, and disruption of QS, which weakens bacterial coordination and virulence.
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    Homeostatic Interplay between Bacterial Cell-Cell Signaling and Iron in Virulence
    (Public Library of Science, 2010) Hazan, Ronen N.; He, Jianxin; Xiao, Gaoping; Dekimpe, Valérie; Apidianakis, Yiorgos; Lesic, Biliana; Astrakas, Christos; Déziel, Eric; Lépine, François; Rahme, Laurence
    Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.