Deactivation of Ascaris suum eggs using electroporation and sequential inactivation with chemical disinfection

Electroporation has been evaluated as a potential backend wastewater treatment for deactivation of Ascaris suum eggs in buffer solution. Initial results indicate that eggshell permeability is affected by the pulse train electric field strength and duration. Coupling electroporation with chemical exposure, using low concentrations of commercially available disinfectants, allows oxidizing agents to pass through the complex strata of the A. suum eggshell, specifically reaching the innermost embryonic environment, which leads to successful deactivation compared to either method used separately. The aim of this work is to identify and develop an alternative technique that efficiently inactivates helminth eggs present in wastewater.


INTRODUCTION
The health of approximately 2.5 billion people is negatively impacted by the prevalence of polluted water due to inadequate sanitation facilities and the consequent inability for individuals to practice safe personal hygiene methods ( Jiménez et al. ; Naidoo et al. ; Gyawali ). The global health issue associated with unpotable water sources suggests an increased risk of infectious disease not only caused by direct consumption or contact with wastewater but also for its use in 7% of the world's total irrigated land ( Jiménez et al. ).

Soil-transmitted helminthiasis (STH) is a global health
threat that affects 1 in 5 individuals, primarily in developing countries lacking safe Water, Sanitation, and Hygiene Current techniques used for helminth inactivation through affecting the ova strata include exposure to high temperature and pH, UV radiation, soaking in chemical agents (i.e., chlorination/oxidation), and anaerobic/aerobic digestion (Bandala et al. ; Maya et al. ; Oh et al. ; Gyawali ). Though these methods have proven successful, there is a lack of consistency regarding inactivation rates among the many studies on this topic (Gyawali ). The present research evaluates the efficacy of electroporation to prompt Ascaris suum deactivation by increasing eggshell permeability. Through penetration of the eggshell strata, the embryo's vulnerability to its external environment will be intensified due to significant structural damage or necrosis. Four hypotheses are thus investigated in regard to electroporation prompting pore formation in the eggshell strata by decreasing A. suum viability. These hypotheses include (i) prompting pore formation using electroporation, (ii) using short treatment durations with high pulsed electric fields, (iii) using longer treatment durations across mid-range electric fields, and (iv) implementing sequential inactivation with chemical exposure.   Figure 1(b)).

Incubation of samples
Each treated and untreated sample was dispensed onto a sterile Petri dish, and approximately 1 mL of 0.1 N H 2 SO 4 was pipetted into either the cuvette or conical tube to wash off any eggs that potentially remained on the interior surfaces. 10 mL of 0.1 N H 2 SO 4 was added to each Petri dish before they were gently swirled. The Petri dishes were covered with plastic Petri dish lids prior to incubation at 25-28 C for 28 days. The samples were aerated, by gently swirling the base of the Petri dish without the lid, and more 0.1 N H 2 SO 4 was added when necessary to prevent desiccation due to evaporation (Nelson & Darby ).

Preparation of samples for observation
After 28 days of incubation, the samples were removed from the incubator, pipetted into 15-mL plastic conical tubes, and left overnight at room temperature to promote sample sedimentation. The conical tubes were then centrifuged at 3,000 rpm for 10 min before the supernatant was aspirated, resulting in a 3 mL sample. The conical tubes were centrifuged again, at 3,000 rpm for 10 min, and aspirated to 1 mL to obtain the final, egg-containing deposit.
An aliquot of each sample, approximately 20 μL of the pellet-containing solution, was dispensed onto glass microscope slides. A glass coverslip was placed on top of each sample before the edges were sealed with clear nail polish to prevent leakage. The prepared slides were air dried and wiped with both saturated iodine and ethanol to prevent contamination prior to microscope observation. This process was also utilized for sample observation prior to incubation (see Process 2 in Figure 1(b)).

Enumeration and assessment of egg viability
The A. suum eggs were enumerated and assessed, using  Figure 1(b)). Internal fluorescence was observed across all tested voltage parameters. Therefore, electroporation successfully causes pore formation thereby permeabilizing the formerly impenetrable lipid layer (see Figure 2).
The examination of eggs that were treated with electroporation for less than 6 min, across various pulsed electric

). This would thereby explain a decrease in viability due
to the membrane's inability to recover from increased mechanical stress. Results from these experiments indicated that 6 min of electroporation using three different pulsed electric fields led to lower viability percentages but did not lead to 100% inactivation of A. suum (see Lots 3 and 5 in Figure 3).
It was concluded that electroporation, used alone in these experimental conditions, does not meet the requirements for a safe helminth treatment option, which supported the use of chemical co-exposure.
Due to the unsuccessful inactivation of all A. suum eggs, a modified approach to achieve deactivation was implemented  Table S3 in Supplemental Information). It is to be noted that   (Neu & Neu ; Rems & Mik-lavcǐč). The difference in influx of ROS will be impacted by the size of the pores formed considering larger pores contribute 86% of the fractional pore area, but smaller pores make up a greater percentage of the total pore area (Neu & Neu ). Ultimately, larger pores will take longer to seal, which may allow chemicals to pass more easily, and smaller pores may permit the passage of more fluid (Deipolyi et al. ). Both increase the amount of stress experienced by the membrane, the former being chemical and the latter being osmotic, therefore the significant contribution of both will be imperative to the overall destruction of helminths.
The aforementioned interpretation of pore depth was further tested by experiments evaluating pore transience after undergoing electroporation at 2,000 V/cm for 6 min.
Samples that were exposed to 10 mg/L of free chlorine 10 s, 30 s, 1 min, and 2 min post-electroporation all led to 100% inactivation (see Table S5 in Supplemental Information). This finding indicates that 2,000 V/cm for 6 min prompts the formation of pores that stay open long enough, meaning the cells cannot completely heal, thereby compromising the lipid membrane.
It was found that sequential inactivation using electroporation with 2,000 V/cm and exposure to 10 mg/L Cl 2 leads to 100% inactivation. Therefore, successful permeabilization of the A. suum eggshell requires a minimum electric field of 2,000 V/cm (see Lot 6 in Figure 3). Experimentation evaluating the use of another commercially available disinfectant, 3% hydrogen peroxide, for use in sequential inactivation, produced results that aligned with this finding (see Table S4 in Supplemental Information). Samples treated with 1,750 V/ cm for 6 min consistently have high viability rates, which could be the result of rapid pore sealing or insufficient pore formation. Therefore, electroporation at 2,000 V/cm prompts successful inactivation, regardless of the type of disinfectant.
Ultimately, it is hypothesized that 2,000 V/cm is the electric field strength required to permit the transmembrane potential to surpass the electroporation threshold of the innermost lipid layer (Tarek ). Despite the ability to generate free chlorine in PBS during electroporation, the concentration was negligible, 0.4 mg/L which did not significantly contribute to the inactivation results. Furthermore, chlorination of the samples occurred more than 1 min post-electroporation which indicates that despite the amount of time needed for pores to reseal, inactivation occurred because the A. suum eggs were successfully, and potentially irreversibly, porated.
Two aspects of this research paper are important to note due to their impact on the experimental method. First, the effects of electrolysis during electroporation must be considered to impartially evaluate the results presented here.
Although electrolysis occurs, as observed by bubbles forming on the electrode surface, the overall impact of the products formed on A. suum inactivation is considered to be negligible and has a second-order effect (Sale & Hamilton ; Neu & Neu ). This is supported by the pore creation concept, which indicates that the current passing through the pores varies based on the size of their radii, which is the result of the transmembrane potential responding to the pulsed electric field. Therefore, the effects of the applied electric field are of more importance in determining the exact causes of inactivation. Next, the amount of energy required for the transmembrane potential to surpass the electroporation threshold differs based on the composition of the lipid bilayer. Existing bonds between lipids and membrane-bound proteins must be broken to decrease membrane stability before pores can form (Tarek ).