Abstract:

APROACHES TO DEFINE THE PHYSIOLOGICAL STATE OF BACTERIA
Kirchen, S*; Brändle, M; Rieder, A; Kühl, B; Marten, S; Schwartz, T; Obst, U
Karlsruhe Institute of Technology, Institute fo Functional Interfaces, Department of Interface Microbiology, Karlsruhe, Germany

Upon environmental stress bacteria can enter different physiological stages including dormant states. In case of dormancies cultivation methods underestimate the amount of viable bacteria. Thus, new detection methods are needed to define the physiological state of bacteria. Therefore a number of different parameters were selected to study gene expression, membrane integrity and the cellular molecular pattern of stressed bacteria.
Using hygienically relevant bacteria the gene expression of the universal stress protein usp and other stress responsive targets were determined. The sigma factor rpoS, pbp5 involved in peptidoglycan synthesis and rcsA, a regulator in biofilm synthesis in Pseudomonas aeruginosa, enterococci and Escherichia coli respectively were investigated. Gene induction and repression of the above mentioned genes became obvious during stress application.
PCR based population analysis in combination with propidium monoazide (PMA) treatment was applied to determine the efficiency of innovative disinfection measures and to distinguish between killed, injured or living cells. Depending on the applied treatment, the investigated natural populations using PCR and subsequent denaturing gradient gel electrophoresis (DGGE) represented the living part of the communities exhibiting intact membrane integrities. Mass profiling with whole cell approaches using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and cluster analyses applied on enterococci resulted in distinct grouping of bacteria exhibiting different physiological behaviors.
The presented methods form a powerful analytical platform for the characterization of the physiological states of bacteria applicable to both pure cultures and natural mixed populations.

Abstract Category
31 Methodological Developments