Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli
Article first published online: 5 DEC 2013
© 2013 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 116, Issue 3, pages 596–611, March 2014
How to Cite
Kramer, B. and Muranyi, P. (2014), Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli. Journal of Applied Microbiology, 116: 596–611. doi: 10.1111/jam.12394
- Issue published online: 14 FEB 2014
- Article first published online: 5 DEC 2013
- Accepted manuscript online: 17 NOV 2013 11:20PM EST
- Manuscript Accepted: 10 NOV 2013
- Manuscript Revised: 5 NOV 2013
- Manuscript Received: 5 AUG 2013
- Federal Ministry for Education and Research. Grant Number: 13N12427
- decontamination process;
- DNA damage;
- flow cytometry;
- pulsed light;
- reactive oxygen species;
- viability staining
The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli.
Methods and Results
Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L. innocua and E. coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable (VBNC) state after the pulsed light treatment. A high percentage of L. innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E. coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species (ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH-DA and RAPD-PCR, respectively.
A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample−1. Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes.
Significance and Impact of the Study
The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment.