Renzo Masiclat

he/him | age 18 | Calgary, AB

Bronze Medal, CWSF 2023 | Defence Research and Development Canada Award

INTRODUCTION

Globally, it is estimated that around 150 million cases of urinary tract infections (UTI) occur every year (Flores-Mireles et al., 2015). More than one in ten men and more than half of women will experience a UTI in their lifetime, with many of them having recurring UTIs. Women are more prone to the infection because the urethra in a female is shorter and in closer proximity to the anus, where Escherichia coli is commonly found. Concerningly, a study by Ahmed et al., (2019) found that greater than 92% of bacteria that cause UTIs are resistant to at least one common antibiotic, and almost 80% are resistant to at least two. According to a report by the World Health Organization (2021), drug-resistant UTIs are a consequence of the larger problem of antimicrobial resistance (AMR), which happens when bacteria, viruses, fungi, and parasites no longer respond to medicines used to treat them. Therefore, an alternative therapeutic for UTIs is needed to provide patients with symptom relief and to improve their quality of life.

Cranberries are native to Atlantic Canada, where they grow in wetlands and marsh areas. Canada’s abundance of cranberries has made the country the second-largest supplier in the world, with a yearly production of around 160,000 tons. Cranberries are commonly referred to as a ‘superfood’ because of their therapeutic properties; they were first used by the Indigenous people, who also discovered their versatility. Over the years, cranberries have shown their potential in natural medicine; studies have shown the efficacy of cranberries and their potency in the prevention of certain types of cancers, such as the study by Rupasinghe et al. (2019) that found complete inhibition of HepG2 cell proliferation, a gene commonly associated with liver cancer. In addition, cranberries have been correlated with improved immune function and decreased blood pressure (Dohadwala et al., 2011). Intriguingly, the most significant area of research using cranberries is their effectiveness in lowering the risk of UTI. In addition to cranberries, some other kinds of berries, such as blueberries and blackberries, have also shown promise for acting as a therapeutic for UTIs. A study by Puupponen-Pimia et al., (2008) states that, “Phenolic compounds present in berries selectively inhibit the growth of human gastrointestinal pathogens. Complex phenolic polymers, such as ellagitannins, are strong antibacterial agents present in cloudberry, raspberry, and strawberry.”Aside from berries, there are also naturopathics that have been correlated with their antibacterial and antimicrobial activities. A literature review by Navabi et al., (2018) found that several Cinnamon zeylanicum bark extracts were tested in vitro against Klebsiella pneumonia 13883, Bacillus megaterium NRS, Pseudomonas aeruginosa ATCC 27859, Staphylococcus aureus 6538 P, Escherichia coli ATCC 8739, Enterobacter cloacae ATCC 13047, Corynebacterium xerosis UC 9165, Streptococcus faecalis DC 74, by the disk-diffusion method and showed high antibacterial activity. Additionally, Labrador Tea has been actively used in folklore medicine. Indigenous peoples were the first people to make use of this plant because of its medicinal properties. It has been associated with antibacterial, anti-cancer, and anti-diabetic properties. Korpinen et al. (2021) found that the phenolic extracts of Labrador tea inhibited the growth of some gram-negative and gram-positive bacteria. Therefore, the literature suggests the ability of berries, extracts, and Labrador Tea to act as therapeutics for UTIs, but this requires further elucidation.

Opportunistic bacteria are the primary cause of UTIs. The urinary tract can be infected by these bacteria, which are exceedingly prevalent in the human gastrointestinal system, by rising from the perineum to the anus. More than 90% of UTI cases are accounted for by the occurrence of E. coli in the bladder (Ernaes, 2021). Other causes of UTIs include poor hygiene, pregnancy, and structural problems in the urinary tract. Antibiotics are often prescribed to patients with UTI. At the same time, cranberry juice, vitamin supplements, and other food or drinks containing cranberries are commonly recommended for individuals with UTIs because of the A-type proanthocyanin (PAC). This is a bioactive compound of berries that could prevent opportunistic bacteria from sticking and growing on the bladder wall. PAC contains double A-type linkages, which are associated with strong interactions with surface virulence factors found on UTI-causing bacteria such as extra-intestinal pathogenic E. coli Urena- Saborio et al., (2021). Given the positive correlation between cranberries and their ability to minimize the symptoms of diseases, the first aim of this study is to investigate the effect of berry extracts on the growth of bacteria that are often associated with UTI. Aside from the berries mentioned, this study will also investigate the ability of Labrador Tea and cinnamon and their ability to prevent the growth of UTI-associated bacteria. We hypothesize that the berries, Labrador Tea, and cinnamon would have growth inhibition toward UTI-causing bacteria. This research is vital as it could identify if berries and other natural foods are possible sources of antibacterial agents to help treat and prevent UTIs.

MATERIALS & METHODS

Agar Preparation
Mueller-Hinton Agar (MHA) was used as it is commonly used for antibiotic susceptibility testing for the Kirby-Bauer disc diffusion method or standard antibiogram. Acid Hydrolysate of Casein, Agar, Beef Extract, and starch were dissolved in deionized water, as shown in Table 1.

**Table 1**: Composition of the Mueller-Hinton Agar with their corresponding measurements.

**Figure 1**: MHA plates on a fume hood.

The MHA medium was then heated on a hot plate with a magnetic stirrer until all contents were dissolved and the liquid temperature reached 100°C. The flask containing the mixture was then sterilized by autoclaving at 121°C for 15 minutes and left to cool until it reached 25°C. The medium was then poured into 50 Petri dishes in a fume hood to limit bacterial contamination and was left to rest for 30 minutes until the medium solidified, as shown in Figure 1. The MHA plates were then placed in the refrigerator for 2 hours.

**Figure 2**: Tryptic Soy Broth in test tubes before inoculation.

10 mL of Tryptic Soy broth was pipetted into 15 glass test tubes labelled with the names of the respective bacteria (Alcaligenes faecalis, Bacillus circulans, Escherichia coli, Escherichia coli B, Escherichia coli C, Escherichia coli K, Escherichia coli NW, Escherichia coli WT-K12, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhimurium, Staphylococcus aureus, and Streptococcus faecalis), as shown in Figure 2. One ceramic bead of the respective cryopreserved bacteria was then transferred to the respective test tubes using a tweezer. The test tubes were then incubated at 35°C for 24 hours, except for Pseudomonas aeruginosa and Pseudomonas fluorescens which are best cultured at 24°c, to let the bacteria grow.

Naturopathic
Blueberries, cranberries, blackberries, and strawberries were purchased from a Walmart Supercentre. The respective fruit juice extracts were manually collected by crushing the fruits using a pestle and extracting their juice extracts using a cheesecloth over a beaker. Commercial cranberry juice and jellied cranberry sauce, both from the brand Ocean Spray, were purchased from a Walmart Supercentre. Labrador Tea packages from the brand Burreal Heartland were purchased from Nutters Everyday Naturals. 50g of dried Labrador Tea was suspended in a beaker containing 100 ml of deionized water. The beaker was then placed in the microwave with the occasional manual stirring to let the contents of the tea dissolve with the water until the temperature reached 100°C. The mixture was then left to cool down to 25°C. Cinnamon powder was purchased from a Walmart Supercentre from the brand Great Value. 1g of cinnamon powder was then transferred to a weighing dish. It was then dissolved in a liquid MHA solution and gently mixed through manual stirring.

Preparation of test organisms in-vitro
The following organisms from the Hyperion Research Laboratory Stock Cultures were tested:

**Table 2**: A list of the bacteria used in this study with the acronym and associated strain designation.

Pharmaceutical antibiotics
Pharmaceutical antibiotics from Must Diagnostics (Merseyside, United Kingdom) were used as a positive control:

Erythromycin
Kanamycin
Neomycin
Streptomycin
Penicillin
Chloramphenicol

Tetracycline
Ciprofloxacin
Ampicillin
Ceftriaxone
Vancomycin
Ceftazidime

**Table 3**: Interpretive chart of the known zones of inhibition of pharmaceutical antibiotics used in this study.

The data tabulated in the above table are used as comparative data to compare the antibacterial activities of cranberry extracts with various antibiotics.

Negative control: deionized water

Antimicrobial Assay

**Figure 3**: Example images of the petri dishes from the antibacterial assay.

This study quantitatively analyzed the growth of bacteria in the presence of various pure berry extracts, commercial cranberry juice, and jellied cranberry sauce using the agar disk-diffusion method. The selection of bacteria used (as shown in Table 2) serves as a representation of a UTI, as well as other bacteria, to investigate their effects on bacteria that do not cause UTI but other human diseases as well. The effect of pharmaceutical antibiotics with selected dosages towards the growth of the same bacteria was also investigated to act as a comparison for the antibacterial properties of berries, cinnamon, and Labrador Tea. 100µl of the bacterial strain of interest for the respective test tube, as listed in Table 2, were suspended on an agar plate using a pipette. The pipette tip was changed for every strain, and the test tubes were sterilized using the Bunsen burner to prevent microorganisms from entering the test tubes and potentially contaminating the cultures. The strains were then evenly spread on the plates using a cell spreader that was sterilized by dipping in a 95% ethyl alcohol and was then ignited by a flame.

Pharmaceutical antibiotic discs were evenly distributed on the plates to prevent the zones of inhibition from overlapping. The plates were then incubated at 35°C for 24 hours, except for the P. aeruginosa plate that was grown at 25°C for 24 hours. The zones of inhibition were then measured in millimetres using a ruler, with zero at the centre of the disk. Blank 6mm paper discs were impregnated with 25µl of each pure cranberry, blueberry, blackberry, and strawberry extracts or commercial cranberry juice. The jellied cranberry sauce was sliced into circular pieces about 6mm in diameter. To accurately analyze the antibacterial activities of berry extracts, the disks were then evenly placed onto agar plates inoculated with the various bacteria listed in Table 2. Blank 6mm paper discs were then impregnated with 25µl of the Labrador Tea. Labrador Tea was only tested against the first 5 strains of bacteria (first 5 strains: K. pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis) because of limited resources in the laboratory. Each plate of bacteria contained triplicates of the discs with the same volume and concentration of Labrador Tea Extract.

**Figure 4**: Preparation of the cinnamon gradient plate.

The gradient plate technique was used to determine the antibacterial activities of cinnamon. Due to the scarcity of square plates in the laboratory, a square glass container was used as a substitute. The square glass container was sterilized in the autoclave at 121ºC and 15 psi for 15 minutes. 15ml of the MHA was poured on the plate and was let to solidify on a slant position. 1g of the cinnamon powder was dissolved into 15 ml of liquid agar through gentle manual mixing. It was then poured on top of the first layer of plain agar and left to solidify for 30 minutes. Cinnamon was tested against the first 5 strains of bacteria (K. pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis) due to the scarcity laboratorial resources. Sterile cotton swabs were used to spread the strains of bacteria in a linear method that runs from the high-concentration through the low-concentration ends of the plate. The plate was then incubated at 35ºC for 24 hours. This was done to evaluate the antibacterial properties of cinnamon at different concentrations. The plate was observed for a line of growth of the bacteria from the high and low concentration ends. The growth inhibition was not quantified as it was only visually observed to see if the strains grew in areas with high concentration of cinnamon. The bacterial line was visible to the naked eye on both ends of the concentration, the cinnamon did not inhibit bacterial growth.

RESULTS

Various antibiotics were tested for their ability to inhibit the growth of K. pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis. From the data in Figure 5, S. aureus and K. pneumoniae were susceptible to ciprofloxacin with an inhibition zone of 29 mm and 35 mm, respectively. E. coli and S. aureus were susceptible to ampicillin with inhibition zones of 15mm and 35 mm, respectively. K. pneumoniae was tested as susceptible to ceftriaxone with an inhibition zone of 22 mm. S. aureus was susceptible to Vancomycin with a zone of inhibition of 15 mm. E. coli was susceptible to Ceftazidime with a ZOI of 23mm while K. pneumoniae tested as intermediate with a ZOI of 17mm. S. aureus tested as intermediate against erythromycin with a ZOI of 21 mm. S. aureus and K. pneumoniae were susceptible to Kanamycin with a ZOI of 20 mm and 22 mm, respectively. E. coli, S. aureus, and P. aeruginosa tested as intermediate against Neomycin with a ZOI of 15 mm. E. coli, S. aureus and K. pneumoniae tested as intermediate against Streptomycin with ZOI of 17 mm, 12 mm, and 20 mm, respectively. E. coli, S. aureus and K. pneumoniae were susceptible to Chloramphenicol with a ZOI of 25 mm, 22 mm, and 25 mm, respectively.

**Figure 5**: Antibacterial Activity of Pharmaceutical Antibiotics.

Table 4 depicts the antibacterial activities of berries, cinnamon, and Labrador tea. Interestingly, only cranberries revealed significant results. Labrador Tea was found to have no protection against any type of bacteria, which was unexpected due to the data produced by other relevant studies. Cinnamon also did not inhibit the growth of any of the other bacteria, which was also unexpected. Blueberries were found to be inhibiting against E. coli only, while strawberry extract was inhibiting only to S. aureus. While cinnamon and blackberry were resistant to all strains. Figure 6 demonstrates a visual representation of the antibacterial assay results using blueberry, blackberry, and strawberry extract. Figure 7 depicts the gradient plate of cinnamon.

**Figure 4**: Antibacterial activity of berries, cinnamon, and Labrador tea.

From the data in Figure 8, A. faecalis tested susceptible against cranberry extracts with the ZOI mean of 12.8 mm is compared to the susceptibility values of Vancomycin (≥ 12 mm). B. circulans and P. fluorescens tested intermediate against cranberry extracts with a ZOI mean of 11.6 mm when compared to the values of Vancomycin.

**Figure 8**: Antibacterial activity of cranberry extract against 10 strains of bacteria.

Figure 9 shows that Streptococcus had the largest ZOI at an average of 10mm. S. aureus had an average ZOI of 9.4mm, P. aeruginosa had an average ZOI of 9mm, while both K. pneumoniae and E. coli had the same average ZOI of 8.6mm.

**Figure 9**: The antibacterial activity of pure cranberry extracts against different strains of bacteria.

From the data in Figure 10, P. fluorescens, P. aeruginosa, and S. aureus were susceptible to jellied cranberry sauce when compared to the values of Vancomycin with inhibition zones of 12.2mm, 12mm, and 18mm, respectively. A, faecalis, E. coli NW, E. coli, and K. pneumoniae tested as intermediate against jellied cranberry sauce when compared to the values of Vancomycin with inhibition zones of 9.6mm, 10.2mm, 10mm, and 10mm, respectively.

**Figure 10**: The antibacterial activity of jellied cranberry sauce against species of pathogenic and non-pathogenic bacteria.

From the data in Figure 11, P. fluorescens and S. aureus showed susceptibility against commercial cranberry juice when compared to the values of Vancomycin in Table 1 with zones of inhibition mean of 13.6mm and 12mm, respectively. While P. aeruginosa, A, faecalis, and E. coli were intermediate against commercial cranberry juice with zones of inhibition mean of 9.3mm, 11mm, and 10.2mm, respectively.

**Figure 11**: Antibacterial activity of commercial cranberry juice against strains of bacteria.

DISCUSSION

Previous studies in the literature found that cranberry extracts and their bioactive compounds inhibited the growth of several other cells and bacteria (González de Llano et al., 2020). Based on this, it was initially hypothesized that cranberries would inhibit the growth of E. coli strains. Cranberries, in all forms (fruit, jellied sauce, and juice extract), effectively inhibited the growth of the following bacterial strains: P. fluorescens, S. aureus, and A. faecalis when compared to the susceptibility values of Vancomycin (≤ 12mm). Data also suggests that cranberries, in all forms, have a promising antimicrobial component as they inhibit E. coli growth. However, it was unexpected that cranberries in all forms would have the greatest effect on P. fluorescens, S. aureus, and A. faecalis instead of E. coli. Intriguingly, there was no significant effect on the growth of E. coli if it the ZOI were to be analyzed with respect to the susceptibility values of the pharmaceutical antibiotics present in this study. Commercial cranberry juice and jellied cranberry sauce showed intermediate inhibitions toward E. coli, which could be traced back to as to why people with UTI are often recommended to drink cranberry juice. It is worth investigating the combination of naturopathics and berries against UTI-associated bacteria as it is possible that the amalgamation of their bioactive compounds can inhibit bacterial growth.

Inhibition of these three bacterial strains was relative to the inhibition elicited by Vancomycin, as shown in Figure 5. P. fluorescens can cause bacteremia in humans, with most reported cases being attributable either to transfusion of contaminated blood products or to use of contaminated equipment associated with intravenous infusions (Scales et al., 2014). S. aureus is the leading cause of skin and soft tissue infections such as abscesses (boils), furuncles, and cellulitis. A. faecalis has been associated with endocarditis, bacteremia, meningitis, endophthalmitis, skin and soft tissue infections, urinary tract infections, otitis media, peritonitis, and pneumonia (Huang, 2020). Collectively, this data suggests that cranberries could act as a therapeutic for more than simply UTIs. It was expected that Labrador Tea and cinnamon would inhibit bacterial growth, however, both Labrador Tea and cinnamon did not inhibit the growth of any of the bacteria tested in this study. Table 4 results show that blueberries inhibited the growth of E. coli, and strawberries inhibited the growth of S. aureus. This was intriguing because, based on currently published studies about their therapeutic properties, they could inhibit pathogenic bacteria from growing. This would suggest that there is a vital need to investigate further their bioactive constituents and how their potential can contribute to medicinal use.

This study only looks at the antibacterial activities of the berries and other naturopathic through a single method, the Kirby-Bauer disc diffusion. Thus, this limits the ability to thoroughly investigate the antibacterial activities of the various berries tested, cinnamon and Labrador Tea, to the greatest degree. Another method that can be used to evaluate the antibacterial properties of berries, cinnamon, and Labrador Tea is through the flow cytofluorometric method, where cells are stained with DiOC5(3), carbocyanine dyes used for membrane potential measurements, and the decrease in stain intensity determines treatment effectivity (Balouiri et al., 2016).

Some discs impregnated with the same compound showed inhibition, while others showed no inhibition towards the same strain of bacteria. This would suggest that there could have been errors in the collection of test organisms and the antibacterial assay. In addition, cranberries were the only substance tested in different forms (juice and jellied sauce). Future studies should test the other berries in various forms for their ability to inhibit the growth of UTI-associated bacteria. Due to limited resources, processed forms of Labrador Tea and cinnamon were used. Therefore, their genuine constituents and compounds may have been exposed to contamination, which could explain the lack of antibacterial properties of these compounds.

CONCLUSION

A wide range of research is being conducted on the bioactive components of cranberries and how they could help in modern medicine. The objective of this research was to evaluate the inhibitory potential of cranberries in different forms, other berries, as well as other naturopathic compounds compared to various antibiotics against the growth of pathogenic bacteria typically linked to UTIs. Through the Kirby-Bauer disc diffusion method, the antibacterial properties of cranberries, blackberries, blueberries, strawberries, cinnamon, and Labrador Tea were quantitatively measured in the diameter of inhibition zones. The cranberries, in all forms (extract, jelly and juice), effectively inhibited the growth of E. coli, a bacterial species commonly associated with UTIs. Conversely, strawberries, blackberries, blueberries, cinnamon, and Labrador Tea did not yield promising results against E. coli, albeit more research is required to test these compounds and their antibacterial properties. Future research should focus on comparing the application of cranberries, in multiple forms, and antibiotics to clinical swab culture samples of UTIs or in vivo using mouse models of UTIs. This would provide greater translation and insight into the use of cranberries as a therapeutic and prophylaxis option for UTIs.

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In-Vitro Exploration of the Antimicrobial Activities of Berries, Cinnamon, and Tea Against Urinary Tract Infection- Associated BacteriA