Eradicating Aquatic Biocontaminant Blooms With a Novel, Stabilized Ozonated Glycerol Antimicrobial

Problem

Water biocontamination is a global health crisis caused by microbial blooms.  Over 106 million people worldwide rely completely on biocontaminated water sources for consumption.  Microbial blooms in biocontaminated water cause many infectious diseases and are responsible for over 1.7 million deaths worldwide, annually.  In Canada, 40 Indigenous communities are currently under drinking water advisories due to biocontamination.  Current treatment modalities for the removal of aquatic biocontaminant blooms, such as ozone gas and chlorinated tablets, are highly unstable, toxic, economically burdensome, or produce harmful byproducts.  Thus, there is a need for new, safe, economical and chemically viable means of water bio-decontamination for communities without access to clean drinking water.  A treatment that addresses these limitations could have substantial implications for global health.

Solution

The biocontaminant eradication modality developed in this research was built upon two critical principles. First, given the limitations of ozone gas, an ideal biodecontaminant would be highly stable and preservable, non-toxic to humans, and economically viable, while still holding the advantages of traditional ozone gas modalities.  Second, eradicating microbial blooms by way of apoptosis (controlled, internally-induced cell-death) holds mechanistic advantages over necrosis (uncontrolled, externally-induced cell-death) in water bio-decontamination.  Therefore, a modality that could employ apoptosis as its mechanism of eradication was sought.  These criteria lead to the conceptualization of water bio-decontamination with ozonide: a matrix of ozone gas dissolved in glycerol gel.

Research Question

To what efficiency can ozonide eradicate aquatic microbial biocontaminant blooms of varying treatment-resistant capacities (vigors), both in unimicrobial and polymicrobial bloom cultures?

Phase 1: Unimicrobial Blooms

Bloom Cell Line Engineering with CRISPR-Cas9

Euglena gracilis blooms were grown and then bifurcated.  Low-vigor blooms were engineered using CRISPR-Cas 9, through homozygous deletion of the growth-signaling gene, IPT (IPT -/-). High-vigour blooms were of the wild type (IPT +/+).

Ozonated Glycerol Preparation

The ozonated glycerol treatment was pre-prepared.  O3 gas from an ozone generator was bubbled through anhydrous glycerol with a diffuser at 4oC.

Ozonide was applied in concentrations of 0, 10, 100 and 1000 µg/mL to samples of each bloom.  Sampling was done 24h and 48h post-treatment.

Quantification

Cell Viability

On Day 2, centrifuged pellets from each treated bloom sample were stained with Tryptan Blue, resuspended, then imaged with an inverted brightfield microscope. Viability was calculated as the percent of non-stained, viable cells.

DNA and RNA Electrophoresis

DNA and RNA extractions were performed on Days 1 and 2 for each treated bloom sample.  Electrophoresis gels were run and imaged.  Densitometric analysis of the intensity of electrophoresis lanes was performed using ImageJ software, and intensity data was normalized.

RT-qPCR

RNA isolated from Day 1 and Day 2 samples was converted into cDNA with reverse transcriptase.  The efficiency of pre-designed metacaspase primers was assessed, then the primers were added to the cDNA samples.  Samples were loaded into the RT-qPCR machine and run for 40 cycles.

Phase 2: Polymicrobial Blooms

Local Aquatic Bloom Extraction and Profiling

Polymicrobial co-culture bloom samples from a local waterway were cultured, then treated with 0, 10, 100 and 1000 µg/mL of ozonide.  Gram stain and morphological analysis were employed to profile the microbial class of the bloom.

Quantification

Colony Formation

A series dilution of the ozonide treated bloom samples was performed.  Samples were plated on agar and grown for ten days.  Colony Forming Units (CFU) were counted and colony concentration was calculated.

Data Analysis

Data were analyzed in GraphPad Prism software.  Significance between treatment conditions was tested by Two-Way ANOVA and a Post-HOC Tukey Multiple Comparisons test.  For the colony formation data, a non-linear regression curve and function to model the decay were generated.

Phase 1: Unimicrobial Blooms

Ozonide Significantly Reduces Viability (p<0.01)

In both high-vigor wildtype blooms (IPT +/+) and low-vigor, engineered knockout blooms (IPT -/-), the application of ozonide in concentrations of 10, 100 and 1000 µg/mL significantly decreased bloom viability.  In cell viability assays, apoptosis is hallmarked by an increase in non-viable, blue-stained, contracted cells, and a decrease in green, viable cells.  Results showed a high degree of tryptan blue stain of the treated bloom, indicating cell death; moreover, these cells also substantially decreased in size, consistent with apoptosis.  Notably, after applying 1000 µg/mL of ozonide to the low-vigor bloom, 0% of cells were viable.

Ozonide Significantly Fragments Microbial DNA (p<0.01)

In DNA electrophoresis, apoptosis is hallmarked by ladder-patterning fragmentation in fluorescent bands. In both high-vigor IPT +/+ and low-vigor IPT -/- blooms, the application of ozonide in concentrations of 10, 100 and 1000 µg/mL yielded significantly more fragmented, ladder-pattern DNA, relative to the untreated samples, as indicated by higher densitometric intensities of the ladder-pattern lanes.  Across all concentrations, the ozonide induced significant apoptosis.

Ozonide Significantly Degrades Microbial RNA (p<0.01)

In RNA electrophoresis, apoptosis is hallmarked by an increase in RNA degradation.  In both the high-vigor and low-vigor blooms, the application of ozonide in all concentrations yielded significantly higher concentration of degraded RNA relative to the untreated samples, as indicated by the ratio of band densitometric intensities.  This further supports the capacity of ozonide to eradicate high-vigor and low-vigor blooms.

Ozonide Significantly Upregulates the Metacaspase Gene (p<0.05)

Apoptotic E. gracilis cells are hallmarked by significant upregulation of the metacaspase genes.  RT-qPCR revealed that, in the high-vigor blooms on Day 1 and the low-vigor blooms on Day 2, the application of ozonide in concentration of 1000 µg/mL yielded significant upregulation of the metacaspase gene, relative to the untreated samples.  This metacaspase upregulation further indicates that ozonide eradicates blooms by the mechanism of apoptosis.

Phase 2: Polymicrobial Blooms

Identification of Gram-Negative, Rod-Shaped Coliform Bacterial Colonies

Gram stain and morphological analysis identified the microbial bloom co-culture colonies as gram-negative, non-spore forming, rod-shaped bacteria.  These distinguishing traits are consistent with the Coliform bacterial class, comprising species such as E. coli.

Ozonide Eradicates Polymicrobial Coliform Bacterial Colonies (p<0.01)

In the polymicrobial bloom colonies of coliform bacteria, the application of ozonide in all concentrations resulted in significant eradication across all dilution factors.  Notably, in the x1000 and x10000 dilutions, the observable CFUs were insignificant across all samples, meriting the designation of Too Few To Count.  Additionally, the non-linear regression successfully modeled the decay in bloom colonies as a function of ozonide concentration.  Based on the collected data, this function can be employed to calculate the concentration-dependent capacity of ozonide to eradicate microbial blooms.

Ozonide Eradicates High-Vigor and Low-Vigor Unimicrobial Blooms, and Coliform Polymicrobial Blooms by Apoptosis

Across all assays, results demonstrated that ozonide induced significant apoptosis in both high-vigor (IPT +/+) and low-vigor (IPT -/-) blooms.  The colony-formation results demonstrate ozonide’s eradication of coliform bacteria, a class comprising E. coli, among other species.  This has significant implications to global health in communities with limited access to clean drinking-water.  In Canada, there are currently 40 active drinking-water advisories in Indigenous communities, with many due to contamination with E. Coli.  The treatment developed in the present research can eradicate these pathogens, and therefore has the potential to improve access to clean drinking water.

Ozonide Offers Advantages Over Current Water-Treatment Modalities in Eradication Mechanism, Stability, Non-Toxicity and Cost-Effectiveness

Current water bio-decontamination modalities pose several limitations.  First, ozone gas kills by necrosis, and can cause intracellular contents to leak into the treated water.  Second, ozone gas is highly unstable; it must be generated on-site, seconds before application using highly-expensive machinery.  Additionally, before dissolution in water, ozone gas is toxic to humans.  Furthermore, chlorinated water-treatment tablets can produce carcinogenic byproducts in the treated water when at high concentrations.

The ozonated glycerol antimicrobial treatment developed in this research overcomes these limitations.  First, it eradicates by the mechanism of apoptosis.  Second, it is highly stable with a half-life of 90 days.  Third, it is non-toxic to humans; the ozone is dissolved into glycerol, an FDA approved sweetener.  Finally, the byproducts of dissolved ozonated glycerol in water are not harmful to humans.

Future Research

Future research will further explore the microbial species which this ozonide treatment modality can eradicate, as well as verification of its non-toxicity in human cell lines.

Additional microbial classes will be grown in co-culture and tested against the ozonated glycerol treatment to verify its capacity to induce apoptosis in a variety of aquatic bloom subtypes.

Ozonide resistance capacity of these microbial blooms will also be assessed, employing an antibiotic sensitivity test.

Finally, the non-toxicity of the ozonide treatment will be confirmed by assessing cytotoxicity in human mucosal digestive-tract cell lines, such as Caco-2 small-intestine and large-intestine enterocytes.

The author extends sincere gratitude to Dr. Neil Emery of Trent University for overseeing the project in his lab, and to Dr. Zhiyong Zhang of Trent University for his instruction and consistent support in the experimentation.  Through the understanding, welcoming and willing spirits of these mentors, the project was made possible.  Additionally, the author extends thanks to Zsofia Hatvani for her help in preliminary logistical project planning.

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