The influence of the environment on biofilms of oral bacteria

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Li, Yung-Hua
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The overall objective of this project as to study the influence of two major ecological factors, carbohydrate and pH, on the accumulation of oral bacterial biofilms and the survival of bacteria in biofilms. The study was first initiated to define some characteristics of mono-culture biofilms of selected oral bacteria under different nutrient conditions. The work in this part would provide baseline data to determine the relationship between nutritional carbohydrates and the development, structure and organization of biofilms. Biofilms of four oral organisms, S. mutans (BM71), S. sanguis (SK78) and A. naeslundii genospecies 1 (ATCC12104) and 2 (WVU627), were developed at dilution rate of 0.1 h$\sp{-1}$ at pH 7.0 under three different nutrient conditions. The results showed that the accumulation of biofilms in terms of increase of biomass, matrix or cell number was generally a function of the accumulation time. An increase in the concentration of sucrose enhanced the accumulation of biofilms and this was moresignificant when the medium was modified to contain a higher ratio of sucrose to other nutrients. The sucrose-enhanced accumulation of biofilms, particularly of S. mutans (BM71), was characterized by a dominant increase in the extracellular matrix, which contained up to 70.1% of carbohydrates under some conditions such as in Dx8/S/Excess medium. Scanning electron microscopy showed that under sucrose limitation, biofilms formed during 1 day were generally composed of a single layer of cells, whereas biofilms accumulating for 5 days usually consisted of multiple layers of cells. The data also showed that biofilms of different bacteria differed in their resistance to removal by shear force. The actinomyces were retained on the surface better than were the streptococci, but the retention of the streptococci was enhanced by increased production of matrix modulated by sucrose concentration. The second part of this project was carried out to examine the effects of carbohydrates and pH on population shifts in bi-culture biofilms grown in continuous culture. The results showed that shifts of bacterial populations in biofilms in response to fluctuating low pH did not necessarily follow the same pattern as that of cells grown in the planktonic phase. Bacterial cells in biofilms survived better under fluctuating pH stress, probably because of 'inevitable surface effects' such as cell retention and the development of heterogeneous environments in the biofilms. In the third part of this project, the acid tolerance responses of biofilm cells were examined to determine the pH survival mechanisms of biofilm cells in low pH environments. The study was first carried out by using S. mutans (BM71) and A. naeslundii (WVU627) as two models to determine whether oral bacteria showed the adaptive acid tolerance responses (ATR) which had been described in several enteric bacteria. The results showed that S. mutans (BM71) expressed at least two acid tolerance systems, a log-phase ATR system induced by low pH and a general acid resistance system in stationary-phase cells. The survival of A. naeslundii (WVU627) to lethal acid only involved a general acid tolerance system in stationary-phase cells and this strain lacked of the log-phase ATR system. (Abstract shortened by UMI.)