KATELYNN STANDELL

she/her | age 15 | Medicine Hat, AB

Sanofi Biogenius Canada Award, Canda Wide Science Fair (CWSF) 2023

In the United States, one person dies every fifteen minutes from an infection that common antibiotics cannot cure. With doctors consistently choosing antibiotics as a cure-all for infections, antibiotic resistance has become a major problem in our society. Due to the increase in antibiotic resistance, alternative treatments need to be implemented. Scientists and doctors have now turned back to an older method used to treat infections that predates antibiotics. In 1917, Felix d'Herelle discovered the bacteriophage; bacteriophages are viruses that have the ability to kill bacteria. He used that bacteriophage to treat antibiotic-resistant infections. This treatment is called phage therapy, which is capable of saving lives. In this experiment, environmental samples were tested on a variety of bacteria to look for plaques, which are evidence that the sample contains bacteriophages and the bacteria have been lysed. Environmental samples were filtered and eluted, streaked onto plates, and incubated overnight. The results demonstrate that bacteriophages can be isolated from raw sewage and water samples. The hope is that with the next steps of isolation, enrichment, and identification of bacteriophages, clinical trials could lead to future therapeutic uses to treat antibiotic-resistant infections.

INTRODUCTION

Once upon a time, a little bacteriophage infected a host cell. It then hijacked the host cell and replicated inside of it, causing it to lyse. This process is called the lytic cycle, something the bacteriophage has mastered. Some bacteriophages use the lysogenic cycle to replicate, while others use the lytic cycle. Lysogenic bacteriophages integrate their own genome into the host cell; as a result, bacteriophages that use this cycle take longer to lyse their host. Lysogenic bacteriophages have also been shown to contribute to the spread of antimicrobial resistance (AMR) (Kondo et al., 2021). On the other hand, lytic bacteriophages immediately kill their host after infecting them. This makes lytic bacteriophages the perfect predator for problematic bacteria.

In 1917, Felix d'Herelle was credited with the naming and discovery of the bacteriophage and their potential to kill bacteria (Clokie et al., 2011). He would use the feces of those suffering from bacterial dysentery to isolate bacteriophages which could be used to treat their infections (Brives & Pourraz, 2020). Thanks to d’Herelle, phage therapy later gained popularity throughout Europe and Asia (Dublanchet & Bourne, 2007). However, Alexander Fleming discovered penicillin in 1928, and this discovery caused the use of phage therapy to decline. The discovery of penicillin led to the overuse of penicillin as a cure-all for infections. By the 1950s, many bacteria were found to be resistant to antibiotics, not even twenty years after Fleming discovered penicillin (Crow et al., 1995). Now, nearly a century since Fleming’s discovery, AMR is a problem more than ever. The Global Burden of Disease Study (GBD) stated in 2019 the five most fatal pathogens were: Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumonia (Ikuta et al., 2022). In 2019, the Centers for Disease Control (CDC) reported that at least 35,900 people in the United States die every year from antibiotic-resistant infections, which is one person every fifteen minutes (Robert R. Redfield, 2019). Phage therapy has been used to treat antibiotic-resistant infections since 1917, but more bacteriophages must be found to treat the wide variety of antibiotic-resistant infections in patients.

Hypothesis
Bacteriophages can be isolated and enriched from environmental samples such as sewage samples and surface water samples through the detection of plaques.

MATERIALS & METHODS

Using the method described by Sobsey et al. (Sobsey et al., 1990), a double overlay agar method was originally used to isolate bacteriophages. This did not consistently produce plaques, and it was found that MgCl₂ and CaCl₂ were crucial for the development and growth of plaques. Modifying the method created by Van Twest and Kropinski (Van Twest & Kropinski, 2009), the following method was used: 100μL of host bacteria were inoculated, spread, and plated in a sterile environment onto coliphage agar plates. 500mL of coliphage broth was made and autoclaved using 2.5g tryptone, 0.5g glucose, 1.25g yeast extract, 4g NaCl, 1.5mL Tween 80, 0.12g CaCl₂, 5.1g MgCl₂, and 500mL deionized water. To make coliphage agar the same recipe was used as the coliphage broth, with the addition of 7.5g agar and a pinch of methylene blue.

Bacterial samples were filtered with a filter tower using a 0.2μm filter membrane. The filter membrane was then placed into a small sterile plastic bag with 5mL of elution buffer and massaged before being placed into an Ultrasonic Cleaner for 5 minutes. Van Twest and Kropinski’s Elution Buffer was used with 43.2mM CaCl₂, and 20mM MgSO₄·7H₂O added. The liquid in the bag was then streaked onto the plates prepared previously. The plates were then incubated at 37 °C or room temperature overnight. The leftover samples in the bag were placed into a freezer to preserve them. Plates were observed the next day.

If a plaque was found, it was isolated and enriched. This was done by pipetting 10mL of coliphage broth into a sterile tube with 100μL of the host bacteria. The plaque was transferred with a loop and incubated overnight. The enriched plaque containing the bacteriophage was then tested against its host bacteria. A line of the host bacteria was streaked onto a coliphage agar plate. 5μL of the isolated bacteriophage was then pipetted onto the streak of bacteria previously prepared in two places. The plate was incubated overnight and observed the next day. In addition to testing against its host bacteria, the isolated bacteriophage was also tested against all other bacteria in the experiment.

Fresh stock cultures were made regularly by pipetting 10mL of coliphage broth into a sterile tube; the previous culture was then vortexed and transferred into the new tube with a loop. The fresh culture was then incubated overnight.

All infectious materials were autoclaved and properly disposed of after use. A variety of environmental samples were tested on Streptococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. All the bacteria used in this experiment were provided by Hyperion Research Ltd.

RESULTS

The most susceptible bacteria to lysis by phages were Escherichia coli and Klebsiella pneumoniae, but plaques were also found on all bacteria being tested (Figure 1). 93% of Escherichia coli plaques came from either sewage water or surface water samples, with the other 7% coming from well water samples. No Escherichia coli plaques were found in clam water samples (Figure 2a). 50% of Klebsiella pneumoniae plaques were found on sewage water samples, but plaques were found at least once on every sample type tested (Figure 2b). Streptococcus faecalis plaques were only found in surface water samples (Figure 2c). Surface water and well water samples were responsible for 75% and 25% of Staphylococcus aureus plaques, respectively. No Staphylococcus aureus was found in sewage water or clam water samples (Figure 2d). 75% of Pseudomonas aeruginosa plaques came from surface water samples with the other 25% coming from well water samples. No Pseudomonas aeruginosa was found in sewage water or clam water samples (Figure 2e).

The plaques discovered ranged in size across plates (Figures 3a-d). Successfully isolated plaques were tested on the other bacteria species to see if they would kill species that were not their host. The isolated phages consistently killed their host bacteria but struggled to kill the other bacteria they were tested on as shown in Figure 4a and b.

Methods were evaluated and refined to minimise opportunities for error and contamination. During the study, it was discovered that some of the stock cultures had become contaminated while making fresh cultures. New cultures were grown using the original stock cultures that were made at the beginning of the experiment. Any previous samples that could have been affected by the stock cultures were retested.

Pseudomonas aeruginosa was originally incubated at 37°C, but later incubated at room temperature which resulted in better growth.

During experimentation, two samples were thought to have been contaminated due to the presence of colonies that were not killed by phages. To make sure they were not contaminated, they were tested again using the liquid preserved in the freezer and the leftover original sample. Both the retested sample and the original sample showed the same kind of colonies suggesting they were phage-resistant bacteria, not contaminants.

**Figure 1:** Different types of plaques were found in a variety of environmental samples. The chart shows the types of plaques and the water samples they were found in.

**Figure 2:** Different water samples produced specific types of plaques. a) Chart comparing which type of samples produced the most *Escherichia coli* plaques. b) Chart comparing which type of samples produced the most *Klebsiella pneumoniae* plaques. c) Chart comparing which type of samples produced the most *Streptococcus faecalis* plaques. d) Chart comparing which type of samples produced the most *Staphylococcus aureus* plaques. e) Chart comparing which type of samples produced the most *Pseudomonas aeruginosa* plaques.

Figure 3: Isolation of Escherichia coli plaques from sewage and water samples. a and b) Escherichia coli plaques found on sewage samples. c and d) Escherichia coli plaques found on surface water samples.

Figure 4: Enriched Escherichia coli plaques were tested on Escherichia coli and Klebsiella pneumoniae. a and b) The enriched bacteriophage killed most of the Escherichia coli (left side of plate) but did not kill the Klebsiella pneumoniae (right side of plate).

DISCUSSION

As mentioned above, Escherichia coli and Klebsiella pneumoniae plaques were often found in sewage water samples. These results make sense considering raw sewage water often contains both of those bacteria (Seguni et al., 2023). Streptococcus faecalis was only isolated from surface water samples, which would lead to the belief that these bacteriophages could only be isolated from surface water; however, Streptococcus faecalis plaques were particularly difficult to isolate during the course of this project, and more research would need to be done to confirm this. Previous studies have shown both Staphylococcus aureus and Pseudomonas aeruginosa can be found in water samples, which would support the results that 75% of plaques from both bacteria were found in surface water samples (LeChevallier & Seidler, 1980; Mena & Gerba, 2009).

In this experiment, the isolated and enriched bacteriophages were further tested against bacteria other than the bacteriophages' host species. This served two different purposes: it reinforced the hypothesis that bacteriophages are specific to their host, and it also proved that the isolated bacteriophages were able to kill their host. During this experiment, Escherichia coli plaques were isolated, enriched, and tested against other bacteria species. Time after time, the isolated Escherichia coli plaques were able to kill Escherichia coli but were unable to kill any of the other bacteria. Research done by Sundar et al. showed similar results. Their experiment tested isolated Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa bacteriophages. Their results concluded that the bacteriophages they isolated for Escherichia coli and Salmonella typhi were specific to their host, while Pseudomonas aeruginosa had a broad host range (M. Sundar et al., 2009). The results from that study support the results gathered in this experiment.

Many of these experiments were performed during the fall and winter seasons, making samples of interest harder to obtain due to freezing temperatures and weather hindering access to fresh samples. As a result, previously preserved samples had to be used in place of some fresh samples. In the future, fresh samples would be ideal for best results. The results that were gathered show that the bacteriophages can be isolated from a simple sewage sample, but a variety of diverse types of samples is crucial to get several types of isolated bacteriophages.

Clams were originally thought to harbor bacteriophages due to their filter feeding nature (Echeverría-Vega et al., 2019). Clam water was originally going to be tested, but unfortunately the clams did not survive long enough for bacteriophage isolation to be performed, and no bacteriophages were found in the river water they were living in. Future research could be done on isolating bacteriophages in clams and their environment, as well as other environmental samples such as soil or compost. Repetition of this experiment could help to eliminate sources of error, particularly those related to contamination. Further experimentation could help provide more data about bacteriophages and which types of samples are best for isolating bacteriophages. Supplementary research may also aid in the development of a better method for plaque enrichment. Future work to improve isolation, enrichment, and identification of bacteriophages would allow more bacteriophages to be used in a clinical setting (Cui et al., 2019).

CONCLUSION

Throughout this experiment it was demonstrated that bacteriophages can be isolated and enriched from environmental samples. After identifying the right method and exposing different environmental samples to a variety of different bacteria, plaques were found capable of killing their host bacteria species. These bacteriophages were isolated and enriched to gain an understanding of the bacteriophage, what the bacteriophage needs to survive, and the types of plaque that can be easily produced from specific environmental samples. Future work could be focused on using these isolated bacteriophages in further experimentation to identify the phage types as well as limitations on their bacteria-killing abilities. Overall, the results of this experiment move us further towards the goal of finding more bacteriophages to treat the wide variety of antibiotic-resistant infections in patients.

ACKNOWLEDGEMENTS

Thanks to Dr. Peter Wallis, Hyperion Research Ltd., and all the staff who helped and supported me throughout this entire process. Thanks to my family for their support and driver's licence. As well, thanks to the people who helped me edit this project.

REFERENCES

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Set Phages to Kill: The Next Step in AMR