Investigation of possible mutagenic changes induced to pseudomonas putida by pulsed electric field treatment

Summary form only given. During the last years the pulsed electric field (PEF) method entered several fields of application. A promising application is the inactivation of microorganism by inducing pores in cell membranes (electroporation) caused by electric field exposure. During our studies the Pseudomonas putida (DSM 291) strain was suspended in PBS buffer at a conductivity of 2 mS/cm and subjected to electric field pulses in a commercially available electroporation cuvette (BTX Ltd.). 10 square pulses of 100 kV/cm and 600 ns pulse duration delivered to the cell suspension led to an inactivation of 3.5 log rates. By increasing the pulse number up to 200, the maximum viability reduction saturates at 6 log rates. This saturation is already well described in the literature. However, there is a demand to understand the killing effect of pulsed electric field treatment. One possible explanation could be the existence of "electro-resistant" bacteria. To prove this hypothesis the bacteria which survived a PEF treatment were cultivated for 3 days and again treated with 10 pulses (100 kV/cm and 600 ns pulse duration). This procedure was repeated over 30 generations. The cell inactivation was determined by plating on agar for two sets of cells every third cycle. The inactivation rate was 3.5plusmn0.8 and remained constant over the 30 cycle. No "electro-resistance" could be observed during this time. To confirm this result we used the pulsed field gel electrophoresis (PFGE) for genetic characterization of bacterial strains. Restriction enzymes cutting specific DNA-cognition sites were applied to total genomic DNA of Pseudomonas putida cultures. This restriction digests delivered strain specific DNA fragments in number and size, which were separated by PFGE on an acrylamide gel. The resulting identical DNA band patterns revealed no indication for a genetic modification during the repeated disinfection treatment. Other possible agents for inducing genetic changes could be ch- oride (Cl₂ ) and hydrogen peroxide (H₂O₂) which are generated during PEF treatment in water and in buffers containing Cl^- ions. The experimental results from this investigation will also be presented