Quorum sensing of Legionella pneumophila in biofilm : a longitudinal study

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Quorum sensing of Legionella pneumophila in biofilm : a longitudinal study


Author: Chu, Hei Man
Title: Quorum sensing of Legionella pneumophila in biofilm : a longitudinal study
Degree: M.Sc.
Year: 2014
Subject: Biofilms
Legionella pneumophila
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xii, 80 leaves : illustrations ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2759009
URI: http://theses.lib.polyu.edu.hk/handle/200/7590
Abstract: Introduction: Legionella are fastidious Gram-negative bacteria, found ubiquitously not only in natural aquatic environment, but also in man-made water supply systems, such as cooling towers, shower heads and hot water supply systems. Legionella pneumophila is the causative agent of Legionnaires’ disease, which is a severe and life-threatening form of pneumonia. L. pneumophila can reside in protozoa and biofilm for survival. Bacteria employ quorum sensing system for intraspecies or interspecies cell-cell communication in response to cell density. Upon activation of the system, different bacterial actions are coordinated, such as biofilm formation, virulence factors, bioluminescence and competence. In L. pneumophila, the lqs gene clusters (lqsA-lqsR-hdeD-lqsS) encode the quorum sensing system, consisting of the autoinducer synthase LqsA, sensor kinase LqsS and response regulator LqsR. Pseudomonas aeruginosa autoinducer 3-oxo-C12-HSL has been found to be able to suppress the biofilm formation and inhibit the growth of L. pneumophila at a high concentration of 50μM. However, the role of quorum sensing of L. pneumophila in regulation of the growth and biofilm development is still unclear. Aims: In this study, the role of quorum sensing of L. pneumophila in biofilms and the relationship between quorum sensing and cell density of L. pneumophila in biofilms were investigated. It was hypothesized that at high cell density, quorum sensing suppressed the growth of L. pneumophila in biofilm. Methods: Two biofilms were set up in bijou bottle containing AYE broth monospecies consisting of L. pneumophila only and multispecies consisting of L. pneumophila and bacteria collected in biofilms of water taps. The biofilms were incubated at 25℃ for 18 days. At every three days, medium was removed and rinsed to remove any planktonic cells. The biofilms were then resuspended in AYE broth. Bacteria count of viable and culturable L. pneumophila cells in biofilms was done on BCYE and GVPC agars respectively. L. pneumophila RNA was extracted and quantitative reverse transcription PCR was performed with primers of the lqsA, icmS and flaA genes for quorum sensing, virulence factor and flagella respectively.
Results: The growth patterns of L. pneumophila in both monospecies and multispecies biofilms were similar. The growth increased exponentially from day 3 to day 9. The bacterial cell counts dropped at day 12 due to nutrients depletion, but increased again at day 15 owing to renewal of nutrients. After that, the growth of L. pneumophila entered post-exponential phase and decreased. In monospecies biofilm, the lqsA gene was down-regulated with increasing cell density, while the icmS gene was expressed not related to the change in cell density. The flaA gene expression indicated the L. pneumophila was under replicative phase at most of the time, but transited to transmissive phase at high cell density to promote detachment of cells. In multispecies biofilm, the lqsA gene was up-regulated at nutrients rich days while down-regulated at nutrients poor days. The icmS gene was expressed at the level similar to that in planktonic cells for virulence. The flaA gene was down-regulated when the cell density was increasing, but up-regulated when the cells entered transmissive phase with decreasing cell density. Comparing the multispecies biofilm with monospecies biofilm, there were significant differences of the gene expression of the lqsA gene and the flaA gene, suggesting other bacteria were able to affect the behavior of L. pneumophila in biofilms. Conclusion: The results supported the hypothesis that at high cell density, the quorum sensing of L. pneumophila suppress its growth in biofilms. This may favor the survival and transmission of L. pneumophila in biofilms. The delicate molecular events remain unclear, more further studies are needed.

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