Inactivation of bacterial endospores on surfaces by plasma processed air.

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 log10 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.

ATP-synthesis capacity of pulsed light-exposed bacteria.

The ability of four different bacteria to synthesize new ATP upon exposure to different doses of pulsed-light (PL) irradiation was investigated. The bacterial cells were PL treated on a gel surface, resuspended in phosphate buffered saline (PBS) and subsequently incubated in Tryptic Soy Broth (TSB) at 37°C. Cellular ATP levels were monitored during a 2h incubation period and compared to the respective colony count data. Although PL affected ATP production in a dose dependent manner, the results showed that bacteria, which had rendered unculturable after PL exposure, are still capable of generating significant quantities of ATP. Escherichia coli and Listeria innocua proved to be more resistant to PL than Salmonella enterica and Staphylococcus aureus, which was supported by the colony count data and the ATP synthesis capacity. These findings underline that bacteria undetectable by culture-based methods may still show cellular activity and synthesize new ATP.

Recent findings in pulsed light disinfection.

Nonthermal disinfection technologies are gaining increasing interest in the field of minimally processed food in order to improve the microbial safety or to extend the shelf life. Especially fresh-cut produce or meat and fish products are vulnerable to microbial spoilage, but, due to their sensitivity, they require gentle preservation measures. The application of intense light pulses of a broad spectral range comprising ultraviolet, visible and near infrared irradiation is currently investigated as a potentially suitable technology to reduce microbial loads on different food surfaces or in beverages. Considerable research has been performed within the last two decades, in which the impact of various process parameters or microbial responses as well as the suitability of pulsed light (PL) for food applications has been examined. This review summarizes the outcome of the latest studies dealing with the treatment of various foods including the impact of PL on food properties as well as recent findings about the microbicidal action and relevant process parameters.

Impact of pulsed light on cellular activity of Salmonella enterica.

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.

Pulsed light induced damages in Listeria innocua and Escherichia coli.

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.

Pulsed light decontamination of endive salad and mung bean sprouts and impact on color and respiration activity.

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.

Antimicrobial activity of hop extracts against foodborne pathogens for meat applications.

AIMS: The objective of this study was the fundamental investigation of the antimicrobial efficiency of various hop extracts against selected foodborne pathogens in vitro, as well as their activity against Listeria monocytogenes in a model meat marinade and on marinated pork tenderloins. METHODS AND RESULTS: In a first step, the minimum inhibitory concentrations (MIC) of three hop extracts containing either α- or ß-acids or xanthohumol were determined against test bacteria including L. monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli by a colorimetric method based on the measurement of bacterial metabolic activity. Moreover, the influence of either lactic or citric acid on the antimicrobial activity of the hop extracts was evaluated. The efficiency of hop extracts as a natural food preservative was then tested in a model meat marinade at 2 and 8°C, respectively, and finally on marinated pork. The experiments showed that Gram-positive bacteria were strongly inhibited by hop extracts containing ß-acids and xanthohumol (MIC values of 6.3 and 12.5 ppm, respectively), whereas the antimicrobial activity of the investigated α-acid extract was significantly lower (MIC values of 200 ppm). Gram-negative bacteria were highly resistant against all tested hop extracts. Acidification of the test media led to a decrease of the MIC values. The inhibitory activity of the hop extracts against L. monocytogenes was strongly reduced in a fat-containing model meat marinade, but the efficiency of ß-acids in this matrix could be increased by lowering pH and storage temperatures. By applying 0.5 % ß-acids at pH = 5 in a model marinade, the total aerobic count of pork tenderloins was reduced up to 0.9 log10 compared with marinated pork without hop extract after 2 weeks of storage at 5°C. CONCLUSIONS: ß-acid containing hop extracts have proven to possess a high antimicrobial activity against Gram-positive bacteria in vitro and in a practice-related application for food preservation. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial hop extracts could be used as natural preservatives in food applications to extend the shelf life and to increase the safety of fresh products.

Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli.

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.

Modification of bacterial structures by a low-temperature gas plasma and influence on packaging material.

AIMS: To investigate the effect of a cascaded dielectric barrier discharge (CDBD) treatment on the biological structure of a selected bacterium and on the properties of different polymer films. METHODS AND RESULTS: Inactivation kinetics were measured using air as the process gas and using Bacillus atrophaeus spores and vegetative cells, which had been homogeneously distributed on a surface. The changes to the outer coats and the DNA of the endospores and cells after plasma treatment were determined using biomolecular and chemical methods. The experiments showed that damage to the DNA molecules and changes in the cell walls can be observed as a consequence of the CDBD treatment. Furthermore, the influence of the plasma treatment on the properties of various polymer films was investigated using a variety of test methods. Except the sealing strength where a slight decrease was observed (max. 20%), no negative changes of the material properties have occurred. CONCLUSIONS: CDBD treatment can affect the DNA of spores and cells, depending on the treatment time. At the same time, practically relevant inactivation rates on packaging materials were observed, without any significant changes to the material properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about CDBD mechanisms was acquired from a biological point of view, and the suitability of the method for treating polymer films was demonstrated.

Antimicrobial efficiency of titanium dioxide-coated surfaces.

AIMS: Development and evaluation of an antimicrobially active titanium dioxide coating. METHODS AND RESULTS: For this purpose, titanium dioxide coatings were applied to glass slides by using a sol-gel method and then exposed to a light source. The antimicrobial efficiency was determined by a count reduction test for selected test strains (Aspergillus niger, Bacillus atrophaeus, Kocuria rhizophila), which were homogenously sprayed onto surface. The bacterial count of K. rhizophila was reduced by up to 3.3 log(10) on titanium dioxide samples within 4 h of UV-A light exposure. Experiments with spore formers did not lead to any significant log reduction. A further aspect of this work was to evaluate the effect of selected parameters (relative humidity, inoculation density, radiation intensity) on the antimicrobial efficiency to gain knowledge for further optimization procedures. At a high relative humidity (85% r.h.), increased inactivation was observed for K. rhizophila (up to 5.2 log(10)). Furthermore, a dependency of the antimicrobial effect on the radiation intensity and the inoculation density was identified. CONCLUSIONS: Antimicrobial surfaces and coatings based on titanium dioxide have the potential to effectively inactivate vegetative micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the antimicrobial efficiency of titanium dioxide was gained. This is a prerequisite for industrial applications to improve hygiene, food quality and safety.

Influence of relative gas humidity on the inactivation efficiency of a low temperature gas plasma.

AIMS: To investigate the effect of relative gas humidity on the inactivation efficiency of a cascaded dielectric barrier discharge (CDBD) in air against Aspergillus niger and Bacillus subtilis spores on PET foils. METHODS AND RESULTS: The inactivation kinetics as a function of treatment time were determined using synthetic air with different relative humidity as the process gas. Spores of A. niger and B. subtilis respectively were evenly sprayed on PET foils for use as bioindicators. In the case of A. niger, increased spore mortality was found at a high relative gas humidity of 70% (approx. 2 log(10)). This effect was more evident at prolonged treatment times. In contrast, B. subtilis showed slightly poorer inactivation at high gas humidity. CONCLUSIONS: Water molecules in the process gas significantly affect the inactivation efficiency of CDBD in air. SIGNIFICANCE AND IMPACT OF THE STUDY: Modifying simple process parameters such as the relative gas humidity can be used to optimize plasma treatment to improve inactivation of resistant micro-organisms such as conidiospores of A. niger.

Sterilization efficiency of a cascaded dielectric barrier discharge.

AIMS: To investigate the microbial inactivation efficiency of a newly developed cascaded dielectric barrier discharge (CDBD) set-up against various micro-organisms on polyethylene terephthalate (PET) foils. METHODS AND RESULTS: Inactivation kinetics in dependency of time were produced with air as process gas and test strains like Salmonella serotype Mons, Staphylococcus aureus and Escherichia coli and spores of Bacillus atrophaeus, Aspergillus niger and Clostridium botulinum, which were homogeneously distributed on the sample surface by a spray method. Highest count reduction was observed for the vegetative cells with at least 6.6 log(10) within 1 s. Aspergillus niger was the most resistant test strain with an inactivation rate of about 5 log(10) in 5 s. CONCLUSIONS: For industrial applications it is necessary to evaluate new sterilization methods against a broad range of different micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: CDBD plasma is a fast and effective technology for decontamination of heat sensitive materials in few seconds.