Open Veterinary Journal, (2025), Vol. 15(2): 813-819

Research Article

10.5455/OVJ.2025.v15.i2.30

Antimicrobial activity of cranberry juice (Vaccinium macrocarpon L.) ethanol extract against uropathogenic bacteria

Hayder Kamil Jabbar Al Kaabi^* and Baheeja A. Hmood

Basic and medical science department, College of Nursing, University of Al-Qadisiyah, Al-Diwaniyah City, Iraq

*Corresponding Author: Hayder Kamil Jabbar Al Kaabi. Basic and Medical Science Department, College of Nursing, University of Al-Qadisiyah, Al-Diwaniyah City, Iraq. Email: hayderalkaaby [at] qu.edu.iq

Submitted: 04/11/2024 Accepted: 03/01/2025 Published: 28/02/2025

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Abstract

Background: Cranberry fruit (Vaccinium macrocarpon L) contains a mixture of phytochemicals such as flavonoids which consist of flavonoids, anthocyanins, proanthocyanidins, catechins, phenolic acids, and triterpenoids, all of which have possible antimicrobial (antibacterial, antifungal, and antiviral) activity.

Aim: This study aimed to determine the antibacterial effect of the ethanol extract of cranberry juice (CJ) against uropathogen commonly found in urinary tract infections (UTIs) associated with pregnancy.

Methods: Cranberry fruits were purchased from local markets and juiced, filtered, and dried. The dried powder was extracted with 70% ethanol for 8–10 days. The bacterial isolates used in this study [Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Enterococcus sp., and coagulase-negative Staphylococci (CNS)] were collected from the Maternity and Children Hospital in Al-Diwaniyah City, Iraq. The antibacterial activity of the ethanol extract of CJ was measured using a standard Disc diffusion method. Sterile paper discs were soaked in 20 µl of different concentrations (12.5, 25, 50, and 100 mg/ml) of the extract, placed in Mueller–Hinton agar plates, and inoculated with bacterial cultures adjusted to 0.5 McFarland standards. Amoxicillin (30 µg) was used as a positive control, and 70% ethanol was used as a negative control.

Results: The ethanol solution of CJ displayed a significant (p < 0.05) inhibitory activity against all tested bacteria. The CNS showed the highest sensitivity with 100% inhibition, followed by S. aureus (90%), Enterococcus sp. (85%), P. vulgaris (75%), and E. coli (60%). The effect was dose dependent, as increasing the extract concentration resulted in broader inhibition zones.

Conclusion: The results show that the ethanol extract of CJ has potent antibacterial activity against uropathogenic bacteria, suggesting a possible role for CJ ethanol extract in treating UTIs when combined with recently emerging facts about increasing antibiotic resistance.

Keywords: Antibacterial activity, cranberry juice (Vaccinium macrocarpon l.), urinary tract infections (UTIs).

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Introduction

Natural foods with beneficial health effects, such as disease prevention, are referred to as functional foods. Cranberries, which contain simple sugars, sugar alcohols, organic acids, polyphenols, and vitamins, are widely recognized as one of the most popular functional foods. Numerous data support the notion that cranberry juice (CJ) can reduce the incidence of urinary tract infections (UTIs), the second most common infectious disease in humans after respiratory tract infections. UTIs are primarily caused by uropathogenic Escherichia coli strains that adhere to the epithelial cells of the urinary tract (González de Llano et al., 2020). Uropathogenic E. coli (UPEC) strains that harbor adhesions, such as fimbriae, that bind to the urothelium are responsible for UTI pathogenesis. CJ has been shown to inhibit the adherence of UPEC strains to uroepithelial cells (Mantzourani et al., 2019).

Cranberries are red, sour berries that are native to North America and can be eaten raw or sweetened. Juice concentration and CJ diet supplementation have been associated with several health benefits, such as UTIs and caries in children (O’Connor et al., 2019). However, the largest producers of cranberries grow the fruit specifically for acid juice production. The most common use of CJ is to prevent UTIs. UTIs are believed to occur through p-fimbriated UPEC bacteria that enter the bladder and cause infection (Das, 2020).

In the United States, there are 150 million cases of UTIs each year. Persistent infections are most associated with UPEC, Enterococcus spp., Klebsiella pneumoniae, and Proteus mirabilis. Some components of CJ inhibit bacteria adherence and biofilm formation and can be used as an alternative to antibiotics for the prevention and treatment of infections (O’Connor et al., 2019; González de Llano et al., 2020).

Many studies have proposed different mechanisms to explain the antimicrobial activity of CJ. Research attempts to understand the effects of CJ against bacteria, focusing on phytochemicals. Examples of phytochemicals found in CJ include organic acids and proanthocyanidins, the two types of cranberry metabolites most frequently studied (González de Llano et al., 2020).

This study aimed to determine the antibacterial effect of the ethanol extract of CJ against uropathogen commonly found in UTIs associated with pregnancy.

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Methods

Cranberry fruit collection and preparation

Cranberry fruits were purchased from local markets. They were juiced using an Omega 8,006 juicer. The juice was filtered through Whatman paper (No. 1. The filtered juice was dried at 30°C in an oven. The dried powder was stored at 4°C until needed.

Preparation of the ethanol plant extract

The dried cranberry powder was soaked in 2.5 l of 70% ethanol for 8–10 days as 1:5 (w/v) for 500 g of powder. It was stirred every 10 hours. After soaking, the mixture was filtered through Whatman filter paper No. 1. The ethanol filtrate was concentrated using a water bath at 40°C until a sticky semisolid mass formed. This product was stored at 4°C. A stock solution (100 mg/ml) was prepared by dissolving the dried extract in dimethyl sulfoxide.

Bacterial isolates and media

The UTI-bacterial isolates used were E. coli, Proteus vulgaris, Staphylococcus aureus, Enterococcus sp., and Coagulase-Negative Staphylococci (CNS). These data were obtained from the laboratory of Maternity and Children’s Hospital, Al-Diwaniyah, Saudi Arabia. Brian-Heart Infusion (BHI/Oxoid) media was used to grow the bacterial isolates, which were incubated at 37°C for 18 hours.

Antibacterial activity testing

The disc-diffusion method was used to test antibacterial activity in accordance with Ahmet et al. (2017). The culture turbidity was adjusted to 0.5 McFarlands. Bacterial suspensions were inoculated onto Mueller–Hinton agar plates as reported by Ahmet et al. (2017). Sterile paper discs (6 mm) were saturated with 20 µl of cranberry extract at concentrations of 12.5, 25, 50, and 100 mg/ml. Discs saturated with 20 µl of 70% ethanol were used as the negative control. Amoxicillin (30 µg) served as the positive control. The plates were incubated at 37°C for 24 hours. The inhibition zones were measured with a ruler.

Data analysis

Data are presented as mean and SD. The significance of the results was assessed using the ANOVA test with SPSS software. Differences were considered significant at p < 0.05.

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Results

Comparison of inhibition rates

Figure 1 presents a bar chart showing the level of inhibition of CJ extract against five uropathogenic bacteria: E. coli, P. vulgaris, S. aureus, Enterococcus sp, and CNS. The percentage of inhibition for each type of bacteria after treatment is displayed. The figure displays the susceptibility of CNS, S. aureus, and Enterococcus sp. to different concentrations of cranberry extract. On examination of the graphs, it can be easily concluded that the CNS was most sensitive to the cranberry extract, with a complete inhibition rate of 100%. This indicates that CJ extract completely suppressed the growth of the CNS in the tests conducted on it. Following this, S. aureus showed almost the same response to cranberry extract, with a very high inhibition rate of 90%. Enterococcus sp was also susceptible to the extract; however, with less inhibition rate was 85%.

Figure 1. Inhibition rates of CJ extract against bacterial species isolated from pregnant women with urinary tract infections.

Proteus vulgaris, in contrast, shows an inhibition percentage of only 75%. E. coli, the third type of bacteria, was inhibited at a percentage of 60%, the lowest inhibition rate of the three bacteria tested.

Concentration versus inhibition zone

Figure 2 depicts the effects of the CJ extract in terms of its concentration on the zone of inhibition of uropathogenic bacteria measured in millimeters. These bacteria include coli, P. vulgarus, S. aureus, Enterococcus sp, and CNS. For each bacterium, one line illustrates how this zone changed, and the concentration of the extract was increased.

Figure 2. Zone of inhibition at different concentrations of CJ extract against bacterial species isolated from pregnant women with urinary tract infections.

Therefore, by comparing the calculated mean zone of inhibition for. Here, 12.5 mg/ml to the inhibition properties of 100 mg/ml, a trend can be observed that increasing concentrations of CJ extract yielded larger zones of inhibition, and therefore stronger antibacterial properties. For example, E. coli exhibited an increase in clear inhibition zones from 12 mm in 12.5 mg/ml to 25 mm in 100 mg/ml. Similarly, for P. vulgaris, the ribbon chart revealed inhibition zones ranging from 13 to 26 mm.

Staphylococcus aureus showed the highest inhibition zones at 15 mm at the lowest concentration and up to 30 mm at the highest concentration, indicating that it was highly sensitive to cranberry extract. Enterococcus sp. also showed high inhibition zones at 14 mm at the lowest conc. and up to 28 mm at the highest conc. The CNS showed zone inhibition of 15 mm and up to 26 mm across the concentration band.

Comparison with antibiotic (Amoxicillin)

Figure 3. The effectiveness of CJ extract in a 100 mg/ml CJ extract with amoxicillin (30 µg) against five uropathogenic bacteria. The highest point in each bar indicates the zone of inhibition (in millimeters) for each treatment and species of bacteria.

Figure 3. Comparison of the zone of inhibition at 100 mg/ml of CJ extract and amoxicillin against bacterial species isolated from pregnant women with urinary tract infections.

The results indicated that, in all the bacteria tested, the concentration of 100% CJ extract generated significantly larger zones of inhibition than that of amoxicillin. The precise values are as follows: E. coli: 25 mm (Cranberry Extract) versus 9 mm (Amoxicillin) and P. vulgarus: 26 mm (Cranberry Extract) versus 11 mm (Amoxicillin)

Staphylococcus aureus was most sensitive to cranberry extract (30 mm zone of inhibition) followed by Amoxicillin (12 mm); for Enterococcus sp., the results showed that cranberry extract improved growth inhibition with a 28 mm inhibition zone; this was also noticed when Amoxicillin was used, which produced an 11 mm inhibition zone. The CNS showed results (26 mm), while amoxicillin revealed a 12-mm zone.

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Discussion

The prevalence of UTIs is steadily increasing, accounting for approximately 47 million patient visits to outpatient health clinics in the United States. Most of these visits (80%–90%) were for acute simple cystitis, an uncomplicated UTI with uncomplicated uropathogenic bacteria. Of these bacteria, UPEC is the most frequent, accounting for approximately 75%–95% of all cases. UPEC must first adhere to the surface of the urinary tract for the establishment of cystitis, capable of invading bladder epithelium cells, and overcoming host urinary factors for successful UTI. These attachment events are mainly mediated by kidney bean lectin-like adhesin (FimH) of type 1 fimbriae. The prevention of FimH-mediated attachment affects the pathogenicity of UPECs, leading to their excretion. CJ and its derivatives have been reported to have FimH-dependent anti-adhesion activity, leading to the investigation of the effects of CJ against UTI (Gbinigie et al., 2020).

CJ is a common fruit beverage made using Vaccinium macrocarpon fruit and is effective in preventing UTI infection in women. The effect of cranberry-containing products on UTI recurrence was first reported in 1994 using a clinical observational study. Since then, other clinical trials have been conducted using cranberry products, such as cranberry pills and capsules, to prevent a similar cumulative risk of UTIs in a meta-analysis. However, other clinical trials have challenged the efficacy of CJ. In 2011, the first such study was conducted, surprising researchers and largely affecting the perception of CJ within the medical environment. In response, other clinical trials were conducted to address this concern, expanding research beyond only CJ consumption, and involving factors such as age and indications for antibiotic prophylaxis. Further, meta-analyses of clinical trials have been performed, resulting in various conclusions, including evidence that CJ is beneficial, nonbeneficial, or only protective under bound conditions and limiting factors (Mantzourani et al., 2019).

The results suggest that CJ is a more effective and affordable method than the discovery of new antimicrobial compounds derived solely from natural sources or conducting extensive clinical trials using cranberry extract. Two scientific hypotheses have been formulated to explain the potential health benefits of CJ consumption: the antiadhesive effect of proanthocyanidin and its metabolite mechanism. The proanthocyanidin hypothesis states that PACs in cranberries prevent UTIs by altering the structure of bacterial fimbriae or the binding sites in uroepithelial cells infected with UPEC strains peptidic fimbrilin. These alterations impede the adhesion between bacteria and the epithelium, the first and crucial steps in the ascent of urinary pathogens, leading to inflammation and infection. Fluorescent in vitro and ex vivo studies have confirmed that PACs can prevent adhesion between type I-fimbriated UPEC strains and specific glycoproteins, i.e., uroplakins I and UTI89-adhesive p, which recognize the Fimbriae tip of the pathogenic strains (González de Llano et al., 2020). The second hypothesis states that PACs and other compounds are metabolized in the gut to form low-molecular phenolic acids, which are absorbed in the intestine and exert their effects on organs (Das, 2020).

These results agree with the well-documented antimicrobial action of CJ against uropathogenic bacteria, and several previous studies have shown the ability of cranberry extracts, especially high-proanthocyanidin ones, to inhibit the growth and adhesion of UTI-associated bacteria (Howell et al., 2005; Wojnicz et al., 2012; Vasileiou et al., 2013).

The dose-dependent inhibition of bacterial growth by CJ ethanol extract is consistent with the observations for the cranberry extracts tested by Ahmet et al. (2017) in their study on the antibacterial activity of various cranberry extracts against uropathogen. The observed dose-dependent inhibition is a result of increasing concentrations of bioactive compounds against bacteria, including anthocyanidins, proanthocyanidins, and flavanols, which are reported to impinge on microbial cell membranes, inhibit bacterial adhesion to host cell surfaces, and cause bacterial cell death.

The high sensitivity of the CNS and S. aureus to CJ extract observed in this study is consistent with previous reports. For example, Wojnicz et al. (2012) showed that the growth of S. aureus could be significantly inhibited by cranberry extracts, an activity that could be attributed to proanthocyanidins and other polyphenolic compounds. Similarly, Ahmet et al. (2017) reported the strong antibacterial effect of cranberry extracts against CNS strains isolated from UTIs.

The slight antagonist activity against gram-negative E. coli and P. vulgaris is consistent with the results obtained by Vasileiou et al. (2013), who reported that cranberry extracts showed greater activity against gram-positive bacteria than gram-negative strains. This difference in susceptibility can be explained by the structural differences in the cell walls between gram-positive and gram-negative bacteria; gram-negative bacteria are less permeable to antimicrobial compounds.

The findings of this study agree with previous research that proved that V. macrocarpon L is cytotoxic to bacteria. Similarly, regarding polyphenols, Lian et al. (2012) argued that β-methylated ellagitannin possesses more antimicrobial activities than ellagitannin. Furthermore, these were also reported by Lacombe et al. (2013). These compounds are found in the same cranberry used in many products. In this study, the term “American cranberry”, showed strong antimicrobial activity against E. coli O157:H7 and pathogenic S. aureus. Rauha et al. (2000) proved that the alcohol extracts of Finnish berries from the fruits of V. macrocarpon L exerted a positive impact on reducing the antimicrobial sensitivity of bacterial strains. Staphylococcus aureus showed moderate sensitivity, whereas E. coli showed high sensitivity to antimicrobials. The extract of this concentrate did not show an inhibitory effect on Staphylococcus epidermidis, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, and Candida albicans. Česonienė et al. (2009) investigated the antimicrobial features of the different wild varieties of European cranberry, based on a method, namely agar well diffusion, against these bacteria. European cranberry was shown to exhibit antimicrobial activity against various strains of bacteria that cause human disease. These include gram-negative (E. coli and Salmonella typhimurium) and gram-positive (Enterococcus faecalis, Listeria monocytogenes, S. aureus and B. subtilis) varieties.

The European cranberry inhibitory effect of antibacterial activity has been related to its higher polyphenol content and can be shown to be at least partly related to the activity of the polyphenolic subfraction in all tested concentrations and the proanthocyanidins at about 400 mg/100 g (Hellström et al., 2009). As a strength against infection, proanthocyanidins exhibit antimicrobial effects mainly by preventing bacterial adherence. The European cranberry has been found to have a proanthocyanidin content of A-type dimers and trimers, as confirmed by Loiseau et al. (2001) and Netto (2007), although it has a lower content than lingonberries and American cranberries because they contain substantial amounts of A-type oligomers and polymers with high molecular weight (Netto, 2007). Kylli et al. (2011) also reported the presence of procyanidin, prodelphinidin, and their catechin (C) and gallocatechin (G) derivatives as causative agents for the antimicrobial properties of V. microcarpon, as they can inhibit the growth of eight pathogenic strains. Most interestingly, the polymeric proanthocyanidin cranberry fraction showed significant antimicrobial activity against S. aureus, whereas no effect was seen on other bacterial strains, including S. enterica sv. Typhimurium, Lactobacillus rhamnosus, and E. coli.

Stobnicka and Gniewosz (2018) described the potential of European cranberry fruit extract as a natural preservative of minced pork meat. The antimicrobial activity of water, ethanol fruit, and pomace extracts, which were used for qualitative evaluation against the Gram-positive bacteria strains S. aureus and the Gram-negative strains L. monocytogenes, Salmonella enteritidis, and E. coli, was examined on fresh minced pork meat inoculated with 2.5% (v/w) extract. Extracts with antimicrobial activity were excluded from the analysis, and the percentage of inhibition was determined. In general, the water-ethanol fruit extract, as well as the pomace extract, exhibited pronounced inhibition, regardless of the units present. The pronounced antimicrobial activity was noted for Ethanol fruit and ethanol pomace extract against Gram-positive bacteria S. aureus. The pronounced inhibition was observed against all S. aureus strains present in the antimicrobial test, as well as the strains of L. monocytogenes, E. coli, and S. enteritidis. However, none of the tested extracts demonstrated antifungal activity against the selected strain of yeasts (C. albicans; an opportunistic fungus that becomes pathogenic when the host’s defenses are weakened). For the ethanol fruit and ethanol pomace extracts, the antibacterial activity against cell (%) growth was more pronounced. Stilbenes, which are predominantly resveratrol, but also the phenolic acids vanillic, caffeic, cinnamic, and syringic, are present at an approximately 10-fold higher concentration in the cranberry pomace extracts compared with fruit extracts, as well as the concentration of flavonols (quercetin as well as kaempferol as its total sum), especially quercetin, at approximately six-fold higher concentration. Besides the terpenes found in the cranberry fruit extract, which include the aromatic esters linalyl acetate and 3,5,5-trimethyl-2-cyclohexen-1-yl acetate, as well as beta-carotene, the ethanol-extracted fruit and pomace extracts contain, among others, the compound ursolic acid.

The ethanol extract of CJ was effective (p < 0.05) against all the bacteria studied, suggesting its potential use as a natural antimicrobial agent. The bacteria tested were CNS, which had the best resistance (100% inhibition), followed by S. aureus (90%), Enterococcus sp. (85%), P. vulgaris (75%), and E. coli (60%). The inhibitory effects were dose-dependent, and higher concentrations of the cranberry extract formed larger inhibition zones those of Tama et al. (2024) and Khatun et al. (2024). These studies indicate the emerging acceptance of natural remedies, including cranberry compounds, for fighting bacteria and infections, particularly in the urinary tract and in multidrug-resistant organisms.

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Conclusion

The results show that the ethanol extract of CJ has potent antibacterial activity against uropathogenic bacteria, suggesting a possible role for CJ ethanol extract in treating UTIs when combined with recently emerging facts about increasing antibiotic resistance. The findings support the medical utility of cranberry extract as a second or complementary strategy to address bacterial infections and challenges associated with antibiotic resistance.

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Acknowledgments

Not applicable.

Conflict of interest

There were no conflicts of interest in the present study, as indicated by the author.

Funding

No specific institutional funds were available for this study.

Data availability

The data will be provided upon request.

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