Ompetition, cooperation, and communication of microbes in complex systems such as biofilms

I have the following proposal, but I need to include the experiments that should be done in order to achieve the objectives of this proposal!


Title
Comparison of Horizontal Gene Transfer Among Bacteria in Biofilm and Established Rates of Resistance and Virulence Adaptations in the Bacteria s Natural Environment

Abstract
Research has shown that bacteria found in biofilms show an increased chance for undergoing horizontal gene transfer with neighboring organisms, both of the same species and in instances of two or more adjacent species. This research will explore the observed rate of genetic transfer and change exhibited by bacteria in biofilm conditions, and compare it to established data tracing the rate of genetic change in bacteria in their natural environments, including during human infection. Timelines detailing the growth of resistance and/or virulence in infectious and non-infectious naturally occurring and laboratory scenarios (non-biofilm) will be compared to the rates of genetic transfer and change observed in biofilm scenarios. Preliminary conclusions will be drawn as to a possible relationship between adaptations acquired in biofilm settings and increased (or decreased) risks of antibiotic resistance and/or virulence of certain strains and species of bacteria.

Proposal
Since its identification in 1959, horizontal gene transfer has greatly increased in prominence as a factor in genetic and evolutionary changes, especially amongst bacteria (Haagensen et al 2002; Sorensen et al 2005). Ongoing research has determined that especially for certain species and sub-species of bacteria, horizontal transfer is at least as important a method for effecting genetic change as conjugal or ancestral transfer (Maeda et al 2005). Despite this research, however, few studies have attempted to analyze horizontal gene transfer in a way that relates directly to practical concerns such as virulence and antibiotic resistance in infectious strains (Maeda et al 2005). While not engaged in studying horizontal gene transfer in its own right, this study will in part address this dearth of literature by making a preliminary establishment of such relationships through the comparison of established rates of adaptation leading to resistance and increased virulence, and findings concerning the rate of horizontal gene transfer that occur amongst specific bacterial strains situated in naturally occurring and laboratory biofilm.
The study of bacteria located in biofilm has formed an intriguing area of microbial study that has not always received the full attention of bacteriologists that the phenomenon warrants (Maeda et al 2005; Sorensen et al 2005). Horizontal gene transfer in certain bacteria has been found to occur more rapidly in biofilms than in other bacterial growth and colonization situations, including laboratory scenarios created to give optimum growth and transfer conditions (Haagensen et al 2002; Maeda et al 2005; Sorensen et al 2005). This leads to the conclusion that an understanding of horizontal gene transfer in biofilm is essential to an understanding of bacterial change and adaptation as a whole, especially as the viability of certain bacteria post-transfer seems to be completely unaffected by such transfers (Maeda et al 2005; Sorenson et al 2005). Reliable and effective techniques for marking, measuring, and modeling horizontal gene transfer in biofilm populations of bacteria have already been established and accepted by the scientific community, making further research in this area much easier to carry out (Haagensen et al 2002).
In the proposed research, an extensive literature review will be conducted to establish known patterns and rates of horizontal gene transfer amongst certain species and strains of abcteir when located in biofilm. A similar review will establish rates of horizontal gene transfer in situations other than biofilm adjacency, as well as the rate of bacterial adaptation leading to increased virulence and/or antibiotic resistance. If necessary, experimental research will be conducted to obtain more directly relevant information, though this is more appropriate for future research after preliminary results have been established by this study. The lack of directly applicable research does not reduce the interpretive values of certain other studies that relate at least tangentially to the research questions and objectives of the proposed study (Haagensen et al 2002; Maeda et al 2005; Sorensen et al 2005).

Objectives
The primary objective of the proposed research study will be to establish a preliminary relationship between the rate of horizontal gene transfer achieved by bacteria situated in biofilm and the observed rates of adaptation leading to increased virulence and/or antibiotic resistance in bacteria of the same species and strains. The majority of research that involves genetic gene transfer does not focus on such specific and practical results, though the degree of evidence achievable through established techniques of horizontal gene transfer mapping would certainly allow for such comparisons to be made (Haagensen et al 2002; Maeda et al 2005). This study will attempt to synthesize such diversified findings.
A secondary objective of this study will be to develop a more comprehensive understanding of the rate of horizontal gene transfer that occurs among bacteria in a biofilm, and possibly the mechanisms by which such transfers occur. This will occur as a natural outgrowth of the primary objective, which will necessarily lead to this understanding as various studies are analyzed and synthesized. General information regarding rates and mechanisms of horizontal gene transfer are abundant in the literature, and an explicit collation of the various findings in previously published research should yield valuable information both for the proposed study and for future research (Haagensen et al 2002; Sorensen et al 2005).

Methodology
The first method of obtaining the necessary data for this study will be through an extensive literature review. This will yield far more comprehensive results than would be feasible through experimentation, which is inherently and necessarily limited in scope and focus (Maeda et al 2005; Sorensen et al 2005). Mathematical modeling and statistical analysis of collected data will conducted using established methods (Maeda et al 2005; Sorensen et al 2005). A comparison of horizontal gene transfer rates in biofilm and rates of adaptation towards virulence and antibiotic resistance will be made to establish whether or not a true relationships exists, and in an attempt to determine the extent of that relationship.
Direct experimentation will be conducted using established methods of fluorescence introduction (Haagensen et al 2002)...............
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References

Haagensen, J.; Hansen, S. & Molin, S. (2002).  In situ detection of horizontal transfer of mobile genetic elements. FEMS microbiology ecology 42(2), pp. 261-8.

Maeda, S.; Ito, M.; Ando, T.; Ishimoto, Y.; Fuisawa, Y.; Takahashi, H.; Matsuda, A.; Sawamura, A. & Kato, S. (2005).  Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli. FEMS microbiology letters 255(1), pp. 115-20.

Sorensen, S.; Bailey, M.; Hansen, L;, Kroer, N. & Wuertz, S. (2005).  Studying plasmid horizontal transfer in situ: a critical review. Nature reviews: Microbiology 3(9), pp. 700-10.