Antibacterial Applications of Low-Pressure Plasma on Degradation of Multidrug Resistant V. cholera

Abstract

The existence of Vibrio cholera (V. cholera) is a major health problem in many parts of the world; therefore, the treatments of V. cholera have always remained necessary for public safety, health, and environmental protection. In the last few decades, plasma discharges have proven to be a novel technique of sterilization against infectious bacteria such as V. cholera. In this research, a low-pressure plasma (LPP) technique has been introduced for the degradation of multidrug resistant V. cholera. The V. cholera strains with 10⁷ CFUs (colony-forming units) were treated by low-pressure plasma, with and without ${\mathrm{H}}_2{\mathrm{O}}_2$ injection into the sterilization chamber, to investigate and report the adverse effects of plasma on V. cholera. The results demonstrated that plasma treatment has significant effects on the degradation of V. cholera in the presence of ${\mathrm{H}}_2{\mathrm{O}}_2$ vapors inside the plasma sterilization chamber. The time-course study of the bactericidal effects revealed that there is no regeneration or increase in the number of V. cholera colonies after plasma treatment.

1. Introduction

Low-temperature and low-pressure plasmas have shown significant applications in the biomedical field through bactericidal disinfection, primarily due to the reactive species generated by it [1,2]. Plasma-produced energetic electrons, intense electric fields, and UV radiation, along with some specific reactive oxidant species, such as reactive oxygen (${\mathrm{O}}^{.}$), ozone (O₃), and hydroxyl radicals (${\mathrm{OH}}^{.}$), play a vital role in bactericidal degradation, depending upon their redox potential and the reaction kinetics of each reactive species [3,4,5]. These reactive species and the UV radiation, accompanied by intense electric fields and plasma heating, interact with the bacterial cells, causing cell membrane damage as well as the breakage of the chemical bonds of the double-strand DNA structure and the oxidation of protein [6,7,8,9,10,11,12].

Vibrio Cholera (V. cholera) is a Gram-negative, halophile rod-like species which is considered to be the root cause of several cholera pandemics worldwide as well as local outbreaks. Owing to the halophile nature of the species, it thrives in water and spreads out through the vegetation and the animals that feed on it. The World Health Organization (WHO) has confirmed the outbreak of cholera in different parts of world due to water-source contamination [13]. This contamination is mostly caused by fecal pollution by animal or human sources [14]. Any object coming in contact with these contaminated sources also becomes affected. The sterilization of such contaminated objects infected by infectious bacteria is an active field of research [15]. Calfee et al. studied the effects of various disinfectants against different household materials, such as glass, wood, carpets, etc., and concluded that the complete inactivation is a challenge. Oseri-Asare et al. devised a disinfectant against V. cholera using akpeteshie, a local Ghanian beverage [16]. Several other techniques, such as sono-photolysis, photo-catalysis, photo-electro-catalysis, etc., are amongst the noteworthy UV-based sterilization techniques [17]. Marugán et al. demonstrated the effectiveness of solar disinfection of water with the use of photo-Fenton oxidative reaction. H₂O₂ generation within cells leads to the accumulation of hydroxyl radicals by photo-Fenton reaction, which eventually leads to cell damage [18,19]. All of the presented methods have shown some disadvantages and limitations with respect to the volume of the object to be treated, the environmental effects, and the ease of access. LPP is amongst the few techniques that can produce UV, energetic electrons, and various radicals which are considered to be V. cholera inhibitors [20]. Plasma sterilization has proven to be an effective mechanism for the degradation of V. cholera, as compared to the pure-chemical and traditional treatments.

In this research, V. cholera was treated by low-temperature and low-pressure plasma generated inside a stainless-steel sterilization chamber. It was observed that the inhibition of Gram-negative V. cholera increases with the increase in the plasma treatment time. The addition of ${\mathrm{H}}_2{\mathrm{O}}_2$ fumes in the plasma sterilization chamber resulted in the enhancement of the yield rate of ROS. To diagnose the effects of plasma treatment on V. cholera degradation, different characterization techniques were adopted, including spectral diagnostics, counting the colony-forming units (CFUs), inactivation efficiencies, kill rate, and retardant and bactericidal effects [21,22,23]. The results proved that H₂O₂-assisted plasma discharge caused a reduction in colony-forming units (CFUs) and disabled the process of the regeneration of V. cholera, as well as DNA wall distortion. Figure 1 represents the graphical representation of the reaction of the plasma-generated reactive species with V. cholera for its DNA distortion.

This research will provide baseline data for the advance of the biological, medical, and agricultural fields where quick and effective bactericidal disinfection is needed.

2. Materials and Methods

2.1. Bacterial Strains Used and Growth Conditions

The clinical samples of multidrug resistant V. cholera strains (NP6) (resistant to erythromycin, chloramphenicol, nalidixic acid, streptomycin and sulfamethoxazole-trimethoprim) were collected from Microbiology and Public Health Lab, COMSATS University, Pakistan. Vibrio cholera and were revived on Thiosulfate Citrate bile salts sucrose (TCBS) (Sigma-Aldrich, Ireland Ltd.) agar plates. For each set of experiments, overnight cultures (16 to 18 h) of bacterial grown in Lauria broth at 37 °C were used.

2.2. Bacterial Cell Survival Assay

Overnight cultures of each bacterial strain (~10⁷ CFU) were used to inoculate autoclaved distilled water to make a water suspension. Samples from this water suspension were then exposed to plasma for different times starting from 15 to 45 s, with a step of 15 s. Each sample was then serially diluted and spot plated on TCBS agar plates; and then, these plates were incubated at 37 °C for 24 h. The number of colonies on each plate was counted, and the colony-forming units per ml (CFU/mL) were calculated using the following formula.

$$\mathrm{Colony}\mathrm{forming}\mathrm{unit}\left(\mathrm{CFU}/\mathrm{mL}\right)=\frac{\mathrm{No}.\mathrm{of}\mathrm{colonies}\times \mathrm{Dilution}\mathrm{factor}}{\mathrm{Volume}\mathrm{of}\mathrm{culture}\mathrm{plated}}$$

(1)

The killing % overall inactivation efficiency of the plasma was calculated using the following formula:

$$\mathrm{Killing}\%=\left(1-\frac{\mathrm{No}.\mathrm{ofCFU}/\mathrm{mlin}\mathrm{treated}\mathrm{samples}}{\mathrm{No}.\mathrm{ofCFU}/\mathrm{mlin}\mathrm{untreated}\mathrm{control}\mathrm{samples}}\right)\times 100$$

(2)

An untreated water suspension and un-inoculated water were used as the positive and negative controls.

3. Experiment

The sterilization of V. cholera was achieved by generating an air plasma in a stainless steel chamber (30 cm long and 20 cm in diameter) by using a DC voltage of 5 kV. The chamber’s schematic is shown in Figure 2a, while Figure 2b represents a typical view of the discharge. The samples were treated for different time intervals, including 15, 30, and 45 s, at 0 mbar air pressure (base pressure of rotary vane pump inside chamber when there were no ${\mathrm{H}}_2{\mathrm{O}}_2$ vapors) and 0.1 mbar (due to 5.9 sccm of injected vaporized H₂O₂). The plasma-treated plates were visually diagnosed, and the colony-forming units (CFUs) were counted.

The CFUs were observed after 12 h of treatment and were monitored for up to 48 h, with an interval of 12 h to observe the physical and biological changes in the CFUs.

4. Results and Discussion

The degradation of V. cholera was characterized by visual analysis, the inactivation efficiency of the plasma, and the plasma-retardant effects on the bactericidal regrowth.

4.1. Spectral Analysis

Optical emission spectrum (OES) facilitates in the understanding of the types of reactive species generated inside a low-pressure plasma sterilization chamber, under different experimental conditions. The result of the emission line spectrum shows that as the high voltage is applied, the emission spectrum of${\mathrm{OH}}^{.}$ radicals, hydrogen, and oxidative species is obtained. The spectrum was recorded by a high-resolution spectrometer (HR-4000 CG-UV-NIR) through a fiber optic with a resolution of 0.5 nm; the wavelength range was from 200 nm to 900 nm. The ${\mathrm{OH}}^{.}$ radical’s peak is at 309 nm; the Balmer α peak of the hydrogen was observed at 656 nm, the Balmer β peak at 484 nm, and the reactive oxygen peaks at 777 nm and 844 nm, respectively. Figure 3 represents the OES of the reactive species generated inside the sterilization chamber with and without using ${\mathrm{H}}_2{\mathrm{O}}_2$ vaporized gas.

The emission spectrum reveals a higher concentration of ${\mathrm{OH}}^{.}$ radicals and reactive oxidant species with the addition of vaporized H₂O₂ gas. The spectrum represents the fact that the intensity of oxidant species generated by the low-pressure plasma discharge significantly depends upon the H₂O₂ vaporized gas injection. The ${\mathrm{OH}}^{.}$ radicals, hydrogen (H_α, H_β), and oxygen species O & O₂ with the redox potentials (2.80 V, 0, 2.07 V and 1.23 V), respectively, interact with the V. Cholera cells. The reaction of such ROS can damage its integrity as well as prevent the colony-forming units from regenerating.

4.2. Inhabitation of Vibrio Cholera

Figure 4 represents typical images of the V. cholera colonies used for the determination of CFUs under different plasma treatment times. The V. cholera colonies were calculated to evaluate the number of colonies that survived after plasma treatment. The electric field produced by applying high voltage in a low-pressure sterilization chamber has a potentially negative impact on microorganisms by reducing their metabolic activity and damaging their cell membranes.

The exposure of bacterial cells to the electric field has an intense effect on the degradation of V. cholera. The intense electric field causes a change in the potential gradient on both sides of the cell membrane, while the interaction of the electric field and the cell membrane causes cell stress. The field reacts with the outer surface of cell membrane, overcoming its tensile strength and causing its rupture [24].

An increase in plasma treatment time caused a larger potential gradient interaction with the cell membranes, resulting in cell death. Figure 5 graphically illustrates the reduction in CFUs for different plasma treatment times.

The low-pressure plasma generated in the sterilization chamber leads to the formation of UV radiation, ${\mathrm{OH}}^{.}$ radicals, energetic electrons, reactive oxygen, super oxide and reactive nitrogen species. These reactive species react with the cell membrane of V. cholera and cause cell death. The UV radiation inactivates the V. cholera by shattering its DNA structure. The injection of ${\mathrm{H}}_2{\mathrm{O}}_2$, results in the reaction with O₃, generated in the sterilization chamber and enhanced by the formation of ${\mathrm{OH}}^{.}$ radicals. The highly reactive ${\mathrm{OH}}^{.}$ radicals interact with the V. cholera DNA structure, resulting in its segregation from the other cell wall. ROS and RNS both act as intercellular and intracellular segregators. These reactive species react with both the inner and the outer membrane and increase the distance between the double strands of DNA structure and cause mutation of the DNA.

The results demonstrated that with the increasing treatment time and ${\mathrm{H}}_2{\mathrm{O}}_2$ injection the growth of V. cholera becomes precluded. The excess of ROS directly reacted with the DNA structure and damaged the integrity of V. cholera by increasing the oxidative stress. With the injection of ${\mathrm{H}}_2{\mathrm{O}}_2$ the high yield rate of reactive species was observed. ROS, RNS, and ${\mathrm{OH}}^{.}$ radicals are highly antibacterial for V. cholera and cause chromosomal aberration, which is due to the direct interaction of the ROS with the DNA. The reactive species damage all three main molecules (liquids, proteins, and nucleic acid) of the DNA cell, which leads to the cell death and prevents them from regenerating.

4.3. Inactivation Efficiency and Kill Rate

Figure 6a represents the inactivation efficiency of the plasma treatment for V. cholera, and Figure 6b represents the kill rate of the plasma treatment time. The inactivation efficiency demonstrates the percentage of colonies inactivated by the plasma treatment and its dependence on the hydrogen peroxide injection rate and plasma treatment time. The inactivation efficiency $\eta$ of V. cholera is calculated as [22]:

$$\eta =\left(1-N_0/N_T\right)\times 100\%$$

(3)

N₀ = initial concentration of colonies without any treatment;

N_T = concentration of colonies after plasma treatment.

The log reduction (kill rate) of V. cholera was calculated by the formula:

$$ln\left(\frac{N_t}{N_0}\right)$$

(4)

N = number of colonies after plasma treatment;

N₀ = number of colonies before plasma treatment.

It describes the relative number of living and dead cells.

The plasma-generated ROS reacts with the protein and amino acid of the V. cholera DNA structure and inactivates it. These reactive species accumulate, leading to vigorous V. cholera cell damage.

4.4. Bactericidal Retardant Effect

Low-pressure plasma in the sterilization chamber has proven to be effective for the degradation and inhabitation of V. cholera. The reactive species produced in the LPP sterilization chamber kept V. cholera in a stationary phase and prevented its growth. Figure 7a,b represents the bactericidal retardant effects of the plasma treatment. The plasma-treated numbers of colonies were counted after 12, 24, 36, and 48 h to observe any change in the number of colonies within the agar plates.

The results demonstrated that after plasma treatment the growth of V. cholera never regenerated. The reason is that ROS play a vital role in intercellular signaling and regulation. These ROS resulted in oxidative stress that caused cell damage and cell death. By increasing the plasma treatment time, the number of colonies of V. cholera was reduced. The addition of ${\mathrm{H}}_2{\mathrm{O}}_2$ vapors caused the enrichment of the production of more oxidant reactive species and the emanation of the cell stress. No change was observed in the cultural inhabitation of V. cholera after the LPP treatment.

5. Conclusions

Low-pressure plasma has the possibility to penetrate even into nonhomogeneous surfaces, cavities, and fissures down to the micrometer scale. Plasma sterilization is a promising sterilization method in the field of the protection and conservation of materials from microorganisms such as V. cholera. The low-pressure plasma generated within the sterilization chamber offered its effectiveness on V. cholera sterilization. The use of ${\mathrm{H}}_2{\mathrm{O}}_2$ and the increasing of the plasma treatment time resulted in a higher reduction in the V. cholera habitation. The highly reactive oxidant species, along with the presence of energetic electrons, the electric field, and the UV radiation generated by LPP, play a vital role in the castration of V. cholera. The plasma treatment not only affected cell culture media but also damaged the cell-growth viability and the colony-forming ability. The LPP sterilization of V. cholera is a rapid and efficient sterilization approach.