Influence of Dielectric-barrier Discharge (DBD) Cold Plasma on Water Contaminated Bacteria Dielectric Barrier Discharge

after Results : It was found that using of different time of DBD plasma against different bacterial species give increase reduction rate until reaching lethal time of each species, this reduction time was species and isolates dependent, that the two L. pneumophila isolates have elimination time between 90 sec and 120 sec, while, S. paucimobilis two isolates need (75 -120) sec for total reduction and E. meningoseptica need only 45 sec for total destruction. The effect of sub-lethal dose of nonthermal DBD plasma on surviving bacterial cell on some virulence factors was studied for one isolate from each of L. pneumophila and S. paucimobilis . Results show that the ability of isolates for biofilm production was strongly reduced by these two isolates and the ability of tested isolates to produced protease, lipase and acid phosphatase enzymes were eliminated after exposed to sublethal dose of DBD plasma. Conclusion: Nonthermal DBD plasma is effective in treatment of tested isolates for relatively short exposure time. These activity is strain depending and survival cell after plasma treatment loss it is ability to exhibit some virulence property. From these investigations, it was proven that the DBD method was an effective and alternative application to conventional disinfectant methods of water treatment.


INTRODUCTION
Cooling water and water supply can contaminated with biofilms containing pathogen and opportunistic pathogens [1]. Among the most clinically relevant gram negative healthcare associated clinically important pathogens that identified in water samples were Legionella spp. and Sphingomonas paucimobilis.
The genus Legionella is primarily associated with Legionnaires' disease [2] L. pneumophila was first isolated by McDade et al. [3], and it was the only known species at that time, now there are more than 60 species and subspecies, about half of them are associated with Legionnaires' disease [4][5][6].
Sphingomonas paucimobilis was formally known as CDC Group IIk, biotype 1, then receiving its taxonomic place in 1977, when it named Pseudomonas paucimobilis [7].This bacterium placed in the genus Sphingomonas in 1990 [8]. S. paucimobilis can be widely distributed in natural environment. It has been isolated from soil, plant, water and other sources [9].
In medicine and biology plasma is known as the non-cellular fluid component of blood, while in physical sciences, ''plasma'' refers to the forth state of matter [10]. Plasma can be classified according to its temperature into: High temperature plasma and low temperature plasma (cold plasma). There are many schems for cold atmospheric plasma production, clinical applications of dielectric barrier discharge (DBD) plasma have been assessed in different areas, Joshi et al. [11] conclude that DBD plasma application can be a valuable in decontamination technique for removal of planktonic and biofilmembedded bacteria like methicillin-sensitive S. aureus and E. coli. In the environment, the contaminated surfaces play an important role in microorganisms transmission causing health associated infections [12]. Cahill et al. [13] highlights DBD cold plasma efficient antibacterial activity on bacterially contaminated surfaces at a short period of time and confirms its potential for routine used in clinical environment biodecontamination.
The goal of this study is to evaluated in vitro effectiveness of DBD cold plasma on Legionella and S. paucimobilis growth and survival.

Bacterial Isolates
Seven different bacterial isolates were isolated during this study, these isolates were as follow: two L. pneumophila isolates (Lp44, L77); Two S. paucimobilis isolates (S8, S148) and one E. meningoseptica isolate (E 11). All these isolates have the ability to form biofilm to produce acid phosphatase, protease and lipase enzymes.

DBD System
The DBD system used in this work, shown in Fig.  1 was designed and constricted locally by Dr. Hamid H. Murbat, in College of Science for women, Baghdad University, Department of Physics, its consist of two electrodes, each of them is made of copper rod with (50 mm) diameter surrounding by Teflon for isolated. One millimeter thickness quartz sheet was used as a dielectric material between two electrodes, the two electrodes were connected into high voltage transformer that Its voltages can vary between (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In our experiment the applied voltage was fixed at (6 KV). The samples were putted in the lower electrodes and the distance between the samples upper electrode and the samples was fixed at (1 mm).

Fig. 1. Locally design DBD plasma generator used in this study
Eleven different exposure time of DBD cold plasma were applied in vitro on different bacterial species which were E. meningoseptica, Legionella spp. and S. paucimobilis with bacterial concentration range between(1×10 2 -2×10 2 )cfu /ml . Bacterial suspension was adjusted spectrophotometrically. This experiment performed by place 100 microliter from each isolate on sterile Petri dish, this plates were exposed to DBD cold plasma generator shown in Fig. 1. Samples were exposed to plasma for (0,15, 30,45, 60,75, 90, 105,120,180, 240and 300) seconds, the exposure time was chosen according to previous study [14]. After that warm proper medium for each species (BCYEα for Legionella species , Muller-Hinton agar for other species was pour on Petri-dish over bacterial suspension, mixed well and leave to solidified at room temperature, three dishes leaved without exposure that served as control, all plates were incubated at 36±1 o C for 48-72 hours. All operations were done at sterile condition. Results were quantified by viable count. All experiments were performed with three replicate. The treatment effectiveness calculated by two values, the first one by the calculation of the percentage of cfu observed on agar for treated sample relative to cfu for control at each isolate where survival was observed. The second value is by estimation the percentage of reduction in standard time. This value is determined by comparing the reduction in bacterial concentration of the treated sample with that of control sample. Percentage of reduction (R) was calculated using the following formula [15]: Where: N o is initial number of bacteria before exposure to plasma. N t is number of bacteria recover after exposure to plasma at desired time(t).

Effect of Sublethal Dose of Cold Plasma Exposures on Surviving Bacteria
The effect of sublethal dose on survival bacteria ability to produce acid phosphatase, protease and lipase as described in [16] and to form biofilm as described in [17] were tested for two isolates (L77and S148) as describe above.

RESULTS AND DISCUSSION
It was found that using of different time of DBD plasma against different bacterial species give increase reduction rate until reaching lethal time of each species, this reduction time is species dependent, from These results were come in accordance with Joshi et al. [11] who demonstrate that, DBD killing responses were dependent on cell density and plasma exposure-time. These results clearly point out that sterilization by plasmas has very striking features in terms of practicality and effectiveness [18]. Kvam et al. [19] treat three antibiotic resistant microbial strains which exhibited rapid declines in intracellular ATP levels as a function of plasma duration, microscopically, observed that plasma rapidly inactivates bacterial cells and permeability of cell surface occur within few second after treatment, followed by loss of membrane integrity and leakage of intracellular components like ATP, protein and nucleic acid.
Statistic significant different was seen among reduction percentage by DBD plasma of different isolates at the same exposure time which sugust that bacterial killing by cold plasma is strain dipending process.
At time of writing this paper, there is no available local reported work concern cold plasma effectiveness on Legionella species, E. meningoseptica and S. paucimobilis. Other researcher work with another gram negative bacteria indicate cold plasma effectiveness like Laroussi [20] who reported cytoplasm leakage before total cell fragmentation of E. coli planktonic cells exposure to plasma discharges exceed 30 seconds. Park et al. [21] indicate bacterial distraction after 20 seconds of exposure to argon plasma discharge and Purevdorj et al. [22] observed similar results for Bacillus pumilus spore-forming bacteria. In another study found that cell walls of treated cells are damaged forming spheroplasts which are smaller in size and these spheroplasts are still alive but it nonculturable since they can retain an intact cell membrane [18]. Also Ziuzina et al. [23] observed that treatment of Salmonella, L. monocytogenes and E. coli by atmospheric cold plasma (ACP) for 30s reduced planktonic bacterial cells suspended in broth to undetectable levels, while biofilm attach bacteria need 300 seconds to reduced bacterial number to a maximum of 5 log 10 CFU/sample depending on conditions and bacterial type. While Yost and Joshi [24] suggested that plasma-treated solution contains superoxide and hydrogen peroxide like reactive species and their products which cause oxidative changes in cell components, and lead cell death.
In Iraq, Humud et al. [25] found that, Gramnegative species used in this experiment more susceptible to cold plasma needle treatment than Gram-positive bacteria. Hamad and Mahmood [26] used E. coli and S. aureus as a model system to optimize the conditions for bacterial treatment, they found that protraction of treatment time promote destruction effectiveness.
Khalaf et al. [27] treat E. coli and Staphylococcus spp bacteria for a period of time as sterilization process. They demonstrated that these two species were affected by operation conditions (increasing or decreasing) according to the produced plasma properties. Hussain et al. [28] indicated that when non-thermal argon needle plasma used against two species of Grampositive bacteria, plasma inactivated all bacterial population during 40 seconds. To our knowledge this is the first local study of the effect of cold plasma upon Legionella species, Sphingomonas paucimobilis and Elizabethkingia meningoseptica.

Effect of Sublethal Dose of Dbd on Survival Bacteria
Whilst the antibacterial action of non-thermal plasma is well characterized, relatively little attention has been paid to investigating the consequences of short plasma exposures on surviving microorganisms and their biomolecules [29]. The effect of sub-lethal dose of nonthermal DBD plasma on surviving bacterial cell was studied by test its ability to form biofilm on cover slid and glass tube and production of enzymes (acid phosphatase, protease and lipase). These tests were perform for two isolates Lp77 and S148 depending on DBD effectiveness results.

Coverslipe method
Results show that the ability of tested isolates for biofilm production was strongly reduced by these two isolates (Fig. 4) after exposure to sublethal dose of DB Dplasma.

Glass tube method
Result show strongly reduction in isolates ability to form biofilm in glass tube, that was confirm by the former result of coverslipe method (Table 2).

Enzyme production
Results show that the ability of tested isolates to produced protease, lipase and acid phosphatase was eliminated after exposed to sub-lethal dose of DBD plasma (Fig. 5).
Somolinos et al. [30] observed that sub-lethal injury cells are higher sensitive to stress conditions after treatment and my lose viability during subsequent storage under unfavorable conditions and they found that the occurrence of sub-lethal injury in plasma for treated bacteria is depending on treatment conditions, they also  Table 2. Detection of biofilm production by two method for L.pneumophila Lp77 isolate and S. paucimobilis S148 isolates after exposed to sub-lethal dose of DBD cold plasma

Bacterial Isolates code Biofilm Production by Coverslips method
Tube method Based on intensity of color by OD measured at 570 nm ± SD Control + 0.251 Lp77 -0.224 ± 0.0552 S148 -0.191± 0.0125 demonstrated that combined treatment is essential to completely eliminate microorganisms. During this process conditions must be fit to avoid repair of cell damage by prevent synthesis of necessary components and energy, revealing that sub-lethally injured cells are capable of repairing its damage.
Garcia et al. [31] observed that the present of E. coli sub-lethal injury cell maybe due to the ability of these cells to repair cell injury, also Garcia et al. [32] indicated that reduce the time of treatment low electric field strengths don't produce extensive sub-lethal injury cell, whereas, Vaze et al. [33] found significant changes in cell membrane and indicates that bacterial cells exposed to lethal dose completely inactivated while sublethal dose treated cells were re-grow. These researcher conclude that bacterial inactivation is not an-all or nothing-event case, and it may depend on the microorganism type and the treatment conditions [31,33,30]. Also Brelles-Marino [34] noted by scanning electron microscopic examination that these survival, treated-cells (injured cells), were also changed at the cellular level, therefore viability tests is needed before drawing conclusions, this may explain present study results, that bacterial ability to produce enzyme was effected by this treatment.
Flynn et al. [29] observed that nonthermal plasma treated Agrobacterium tumefaciens bacteria result in complete loss of pigment production gradually along with increase plasma exposure time. Pigment production is regulated by Quorum sensing (Qs) QS regulates many important cell functions in both Gram-negative and Gram-positive bacteria, including protein synthesis, metabolism, expression of virulence factors, biofilm maintenance and dispersal, biofilm formation, antibiotic resistance in addition to entry to stationary phase. Therefore, QS is a greatly attractive target for seek for new alternative antimicrobial [35]. Also Flynn et al. [29] found that non-thermal plasma can be used to attenuate bacterial Qs and interfere with Quorum sensing dependent virulence factor production in Gram-negative bacteria. This finding indicate the present of sub-lethal injured cell which exposed to low dose of cold plasma and lose their virulence factor gradually until they die, that was close to present study finding.

CONCLUSION
Nonthermal DBD plasma is effective on tested isolates treatment at relatively short exposure time. These activity is strain depending and survival bacterial cell after plasma treatment loss it is ability to exhibit some virulence property such as acidphosphatase, lipase and protease production in addition to loss its ability to form biofilm.