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Aims: In case of biological hazards and pandemics, personal protective equipment of rescue forces is currently manually decontaminated with harmful disinfectants, primarily peracetic acid. To overcome current drawbacks regarding supply, handling and disposal of chemicals, the use of plasma processed air (PPA) represents a promising alternative for surface decontamination on site. In this study, the sporicidal efficiency of a portable plasma system, designed for field applications, was evaluated., Methods and Results: The developed plasma device is based on a dielectric barrier discharge (DBD) and operated with ambient air as process gas. PPA from the plasma nozzle was flushed into a treatment chamber (volume: 300 l) and bacterial endospores (Bacillus subtilis and Bacillus atrophaeus) dried on different surfaces were treated under variable conditions. Reductions in spores by more than 4 log 10 were found within 3 min of PPA exposure. However, the presence of endospores in agglomerates or in an organic matrix as well as the complexity of the respective surface microstructure negatively affected the inactivation efficiency. When endospores were embedded in a dried protein matrix, mechanical wiping with swabs during exposure to PPA increased the inactivation effect significantly. Gaseous ozone alone did not provide a sporicidal effect. Significant spore inactivation was only obtained when water vapour was injected into the PPA stream., Conclusion: The results show that endospores dried on surfaces can be reduced by several orders of magnitude within few minutes in a treatment chamber which is flushed with PPA from of a DBD plasma nozzle., Significance and Impact of the Study: Plasma processed air generated on site by DBD plasma nozzles could be a suitable alternative for the disinfection of various surfaces in closed rooms., (© 2019 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: The objective of this study was a comprehensive characterization of physiological changes of Salmonella enterica induced by intense broad spectrum pulsed light (PL). After exposing the bacteria to this nonthermal decontamination technology on a gel surface, multiple viability parameters beyond culturability were assessed., Methods and Results: By applying flow cytometry, a luciferin-luciferase bioluminescence assay and a microplate assay to measure the current redox activity, the impact of pulsed light on the membrane potential, membrane integrity, esterase activity, efflux pump activity, expression of the green fluorescent protein (GFP), respiration activity and ATP-content of Salm. enterica ATCC BAA-1045 was determined. These culture-independent methods for assessing the bacterial activity were compared to the ability to grow on tryptic soy agar. It is shown that this strain is rather sensitive to PL considering colony count reductions, while on the other hand unculturable bacteria still exhibit significant cellular energetic functions. However, this residual activity after PL exposure significantly decreases during sample storage in buffer for 24 h. This study also shows that the GFP expression of PL-treated cells which have rendered unculturable is severely reduced., Conclusions: This study reveals that although not all cellular functions of Salm. enterica are immediately shut down after PL exposure, the synthesis of new GFP is strongly reduced and affected to a similar extent as the culturability., Significance and Impact of the Study: It is shown for the first time, that even there is significant bacterial activity measurable after PL exposure, it is likely that nongrowing pathogenic bacteria like Salm. enterica are unable to express proteins, which is of great importance regarding their pathogenicity., (© 2016 The Society for Applied Microbiology.)

Aims: Pulsed light (PL) is an upcoming nonthermal decontamination technology mainly used for surface sterilization. The objective of this study was to investigate the extent of cellular damage caused by PL treatments of Listeria innocua and Escherichia coli on a polysaccharide surface in order to gain knowledge about the main inactivation pathways., Methods and Results: The impact of PL on the cellular ATP level was investigated as well as the bacterial ability to take up fluorescently labelled glucose (2-NBDG). Furthermore, the extent of DNA damages was assessed by qPCR. The ability of L. innocua and E. coli to photorepair under artificial daylight exposure was quantified. Finally, the induction of reactive oxygen species (ROS) and lipid peroxidation were studied by fluorometric detection of ROS and thiobarbituric acid reactive substances (TBARS). It is shown that intracellular ATP levels and glucose uptake ability do not correlate with the immediate loss of bacterial reproducibility, which indicates that cellular activity and energy may remain on a relatively high level, although growth on tryptic soy agar is not observable. Sequence specific investigation of PL induced DNA damages by qPCR revealed distinct differences between L. innocua and E. coli although the observed inactivation efficacy of PL by the culture based method was similar. Photoreactivation has been observed for both bacteria, a higher recovery rate of up to 2 log was seen in case of E. coli. Intracellular ROS and lipid peroxides were both detectable at relatively high fluencies with E. coli so the contribution of oxidative damage to microbial inactivation of PL cannot be excluded., Conclusions: Escherichia coli as well as L. innocua cells have proven to maintain residual cellular activity after having been exposed to PL even when they are not able to reproduce any more. High proportions of sublethal damages were also obvious with regard to occurring photoreactivation. The destruction of bacterial DNA seems to be the primary mechanism of inactivation of PL but the involvement of other factors like oxidative stress cannot be excluded., Significance and Impact of the Study: The observed data underline that bacteria are not immediately inactivated after exposure to PL as different indicators of cellular energy are still detectable even when cells do not reproduce on solid media any more. DNA is the primary target of PL, but as the extent of damage among different bacteria may not reveal their actual sensitivity, other destructive effects should also be considered., (© 2015 The Society for Applied Microbiology.)

The objective of this study was the determination of the efficiency of pulsed light (PL) treatments for the decontamination of endive salad and mung bean sprouts, as well as the assessment of quality changes in relation to discoloration and alteration of respiration activity. Produce samples were artificially inoculated with two bacterial test strains Escherichia coli (DSM 498) and Listeria innocua (DSM 20649) and exposed to PL at different energy doses. The inactivation efficiency with regard to the naturally occurring microbiota was also investigated. Besides microbiological investigations, color changes were determined as well as the produce respiration during chilled storage. The results indicated that inactivation of more than 2 log was possible with one flash in the case of fresh-cut salad, while the reduction on mung bean sprouts was limited to approximately 1.6 log with one flash, irrespective if the natural flora or inoculated E. coli or L. innocua were considered. The UV part of the PL proved to be exclusively responsible for the inactivation of microorganisms. Significant lower levels of microbial counts of treated compared with untreated samples were maintained for up to 6 days. In the case of endive salad, a dose-dependent progressive discoloration and increase in respiration was diminished by applying optical bandpass filters, which only slightly affected the inactivation efficiency. In contrast, PL treatments showed a positive effect on color and general appearance of mung bean sprouts, while the respiration was almost unaffected. However, care must be taken with regard to efficiency-limiting matrix effects and impact on food quality. These aspects have to be assessed for each treated product. The integration of PL in industrial food processing plants could be an alternative way to improve the microbial quality of fresh produce, and therefore have a positive impact on public health by reducing the risk of contaminations with pathogenic bacteria.

Aims: 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., Conclusions: 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., (© 2013 The Society for Applied Microbiology.)