Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

Abstract

Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters.

Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure

“…The use of cell impermeable dye, propidium iodine, where fluorescence increases when the membranes are disrupted, indicated that treatment with He and He-N 2 for 10 min with one hour post storage resulted in complete membrane disruption (TO + PI + ), which is not the case for He-O 2 plasma treatment which resulted in only injured cells with no membrane disruption but cell depolarization (TO + PI int ), less staining for thiazole orange. Our results are in line with other studies [ 29 , 42 – 44 ].…”

“…Flow cytometry analyzis confirmed these datas. Simultaneous staining with thiazole orange (TO) and propidium iodide (PI) allows the distinction of live (TO + PI − ), dead (TO + PI + ) with total membrane disruption, and injured (TO + PI int ) cell populations, a viable but non-culturable VBNC form with no total membrane disruption [ 42 ]. The results showed that the population within the grey circle (TO + PI − ) ( Fig 1E ), which refers to bacteria with high membrane potential disappeared regardless of the applied plasma treatment.…”

“…Some bacteria were no longer rod-shaped but rounded, becoming coccoid and losing their bacillus shape. Such damage has been described in the literature [ 29 , 42 – 44 ] and could be attributed to charged particles or electric field. To minimize the electrostatic forces exerted by charges, bacteria become round and if the charge effect or the electric field is too great, the membranes will break.…”

Contribution of Fluorescence Techniques in Determining the Efficiency of the Non-thermal Plasma Treatment

“…The inactivation mechanism of NTP is still unclear. However, three mechanisms are usually proposed: UV radiations of genetic material, photodesorption of cell by UV radiations and etching by ions and chemical reactive species ( Moisan et al, 2001 , 2002 ; De Geyter and Morent, 2012 ; Fröhling and Schlüter, 2015 ).…”

“…Non-thermal plasma is an ionized gas consisting of UV-VUV photons, charged particles, reactive species (e.g., reactive oxygen species – ROS, OH., O 2 , 1 O 2 - ; reactive nitrogen species – RNS, NO ∙ , ONOO ∙ , …), excited molecules (e.g., excited O 2 , N 2 , …) depending on the plasma process and the gas nature ( Moisan et al, 2001 ; Laroussi and Leipold, 2004 ; Gaunt et al, 2006 ; Liao et al, 2017 ). Nowadays there are numerous NTP techniques such as corona discharge, electric barrier discharge, microwave discharge or plasma jet that can inactivate various bacterial strains ( Lerouge et al, 2001 ; Moisan et al, 2001 ; Boscariol et al, 2008 ; Sureshkumar et al, 2010 ; Fröhling et al, 2012 ; Kvam et al, 2012 ; Alkawareek et al, 2014 ; Fröhling and Schlüter, 2015 ; Scholtz et al, 2015 ; Impe et al, 2018 ). These technologies differ from each other in terms of electrical current, discharge reactor designs, working pressures, and operating conditions (nature and flow rate of the gas).…”