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|Mechanisms of the inhibition of biofilm formation by Sphingomonas spp.
|Sphingomonas;Biofilm Inhibition;Quorum Sensing;Drinking water;exopolymers;Chlorine resistance
|Colonization of bacteria from diverse ecological niches adopting a biofilm mode of sustenance on the drinking water framework endangers the quality of potable water. This necessitates an urgency to take up considerable safety measures. Ability of Sphingomonas, an oligotroph, to form biofilm play an important role in water borne infections by acting as a reservoir for pathogens and enhanced resistance to chlorine compounds. Growing body of evidences has suggested the role of cell to cell communication system called quorum sensing in formation of biofilm. Interference with bacterial communication system with suitable antagonists can offer a benign yet effective strategy for biofilm prevention. Microbial exopolymers, an attractable class of molecules, present an interesting proposition for exploitation. In view of this, the present study investigated the role of bacterial exopolymers modified chemically as quorum sensing inhibitors to prevent biofilm formation by Sphingomonas. A bacterial isolate with strong biofilm forming ability was isolated from drinking water samples and characterized as Sphingomonas paucimobilis strain MG6. Sphingomonas terrae MTCC 7766 was used as reference strain. A rapid technique for determining biofilm age by detecting changes in capacitance was developed. The ability of Sphingomonas to form a strong biofilm on various commonly used materials and evading chlorination in drinking water networks was studied. To understand the mechanism of biofilm formation, the signal molecule inducing quorum sensing in Sphingomonas was isolated and the identity of the later was characterized as a homolog of N-dodecanoyl-L-Homoserine lactone. The same was confirmed by a battery of qualitative and quantitative assays. In an attempt to develop quorum quenchers, we altered the surface properties of biopolymer extracted from Klebsiella terrigena to form quaternized biopolymer (QB) and N-methyl biopolymer (NMB). Their effect on quorum sensing and biofilm formation was investigated. It was observed that they had considerable potential for effectively reducing AHL molecule production, hence, leading to biofilm inhibition. AHL modulation by QB and NMB suggested that QB modulated both quorum sensing genes luxI and luxR activities particularly Lux R whereas NMB supposedly had no significant effect (p>0.05) on either of them. The role of AHL in biofilm formation was substantiated by the lack of biofilm formation of an AHL negative mutant obtained by transposon mutagenesis out of the library of 534 transconjugants. The effect of these compounds on the growth profile of Sphingomonas was also analyzed. QB was found to have major effect in reducing the growth of planktonic cells as compared to NMB. This was confirmed by analyzing leakage of potassium ions, UV –absorbing materials, proteins, glucose, Lactate dehydrogenase and ATP content of Sphingomonas after treatment with these compounds which inferred about QB in leading to cell damage while, NMB possessing negligible effect on the cell membrane. Based on the structure of the NMB, it was hypothesized to act through lactonolysis of lactone moiety of signal molecule of Sphingomonas resulting in ring opened product. Finally, the effect of QB and NMB on biofilm forming Sphingomonas under real time conditions was indicated. These were found to effectively reduce biofilm formation suggesting potential application of these compounds as a safe quorum sensing inhibitors for Sphingomonas.
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