Investigation of the antimicrobial, antibiofilm, and immunomodulatory activities of cranberry (Vaccinium macrocarpon L.) proanthocyanidins against multidrug-resistant pathogens

Abstract

The increasing emergence of multidrug-resistant (MDR) pathogens poses a significant challenge to global healthcare, necessitating the development of alternative antimicrobial strategies. In the present study, cranberry extract was evaluated for its antimicrobial, antibiofilm, antioxidant, and anti-inflammatory activities against a panel of clinically relevant microorganisms, including Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). The extract demonstrated notable antimicrobial activity, with minimum inhibitory concentration (MIC) values ranging from 32 to 512 µg/mL, exhibiting relatively higher efficacy against Gram-positive bacteria. Significant antibiofilm activity was observed at sub-inhibitory concentrations, where cranberry extract reduced biofilm formation by up to 75–80% at 1/2 MIC and 50–65% at 1/4 MIC (p < 0.05) across tested isolates. Scanning electron microscopy (SEM) further supported these findings by revealing alterations in biofilm architecture following treatment. In addition, the extract exhibited strong antioxidant activity, achieving more than 85% free radical scavenging activity at higher concentrations. Cytotoxicity assessment revealed an IC₅₀ of approximately 450–500 µg/mL, with selectivity indices indicating a favorable therapeutic window, particularly against Gram-positive bacteria. Furthermore, in vivo evaluation using an infection model demonstrated improved survival rates (> 90%) and significant reductions in pro-inflammatory markers, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), nuclear factor kappa B (NF-κB), and cyclooxygenase-2 (COX-2), compared with untreated infected controls (p < 0.05). Collectively, these findings highlight the potential of cranberry extract as a multifunctional therapeutic agent with antimicrobial, antibiofilm, antioxidant, and anti-inflammatory properties. The observed efficacy against MDR pathogens suggests its potential application as a complementary or alternative strategy for the management of resistant infections.

Introduction

The global escalation of antimicrobial resistance (AMR) represents one of the most serious threats to modern healthcare systems. Multidrug-resistant (MDR) bacterial pathogens increasingly compromise the efficacy of conventional antibiotics, resulting in prolonged infections, higher mortality rates, and substantial economic burden on healthcare infrastructures (Patra et al. 2025). Recent global estimates indicate that bacterial AMR was directly responsible for approximately 1.27 million deaths in 2019, with projections suggesting that the burden may increase dramatically in the coming decades if effective countermeasures are not developed (Murray et al. 2022). Particularly concerning are MDR pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and resistant strains of Escherichia coli, which collectively contribute to a large proportion of hospital-acquired infections worldwide (Garlasco et al. 2026). The limited pipeline of new antibiotics, coupled with the rapid emergence of resistance, has intensified the search for alternative or adjunct therapeutic strategies capable of enhancing antimicrobial efficacy while minimizing selective pressure for resistance development (Schmidt et al. 2026).

Natural products derived from plants have attracted growing attention as promising sources of bioactive compounds with antimicrobial, antibiofilm, and immunomodulatory properties. Among these compounds, plant polyphenols have emerged as particularly attractive candidates due to their diverse chemical structures and multitarget biological activities (Tang et al. 2025). Polyphenolic compounds can interfere with microbial physiology through several mechanisms, including disruption of cell membranes, inhibition of essential metabolic enzymes, chelation of metal ions, and modulation of quorum-sensing pathways involved in bacterial communication and virulence regulation (De Rossi et al. 2025). Importantly, many polyphenols also demonstrate the ability to inhibit biofilm formation, a key virulence factor that enhances microbial persistence and confers up to 1000-fold increased tolerance to antimicrobial agents (Sharma et al. 2025). These properties make plant-derived polyphenols attractive candidates for the development of novel anti-infective or resistance-modifying therapies.

Cranberry (Vaccinium macrocarpon L.) is a well-known functional fruit rich in polyphenolic constituents, particularly proanthocyanidins (PACs), anthocyanins, flavonols, and phenolic acids. Among these compounds, cranberry-derived A-type proanthocyanidins have been extensively studied for their unique anti-adhesive activity against pathogenic bacteria, especially uropathogenic E. coli, where they inhibit bacterial attachment to epithelial cells and reduce colonization (Howell et al. 2022; Konesan et al. 2024). Beyond anti-adhesion activity, increasing evidence suggests that cranberry polyphenols also exhibit broader antimicrobial and antibiofilm properties against a variety of Gram-positive and Gram-negative pathogens (Ivanov et al. 2022; Zai et al. 2025). These compounds may interfere with bacterial membrane integrity, inhibit efflux pump systems, disrupt quorum-sensing signaling pathways, and reduce the production of virulence factors that contribute to infection persistence (Bhardwaj et al. 2021; Santos et al. 2021).

In addition to their direct antimicrobial effects, cranberry polyphenols possess significant antioxidant and anti-inflammatory activities that may contribute to improved host responses during infection. Polyphenolic compounds have been reported to regulate inflammatory signaling pathways, particularly the nuclear factor kappa B (NF-κB) pathway, which plays a central role in the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) during bacterial infections (Navaei-Alipour et al. 2021). Modulation of these inflammatory mediators may reduce tissue damage associated with severe infections and enhance host immune defense mechanisms.

Despite the growing interest in cranberry-derived phytochemicals, the biological efficacy of cranberry extracts depends strongly on the efficiency of extraction procedures used to recover bioactive compounds. Extraction conditions such as solvent composition, acidity, temperature, and extraction time significantly influence the yield and stability of proanthocyanidins and other phenolic compounds. Response surface methodology (RSM) has therefore emerged as a valuable statistical approach for optimizing extraction processes by simultaneously evaluating multiple experimental variables and their interactions (Klavins et al. 2022). Such optimization strategies are essential for maximizing the recovery of bioactive compounds and ensuring reproducible biological activity. Although several studies have investigated cranberry polyphenols in the context of urinary tract infections, their broader antimicrobial potential against clinically relevant MDR pathogens, as well as their combined antibiofilm and immunomodulatory effects, remains insufficiently explored (Jangid et al. 2025). Furthermore, relatively few studies have integrated extraction optimization with comprehensive in vitro antimicrobial testing and in vivo infection models to evaluate the therapeutic effect of cranberry-derived bioactive compounds (Manso et al. 2025).

Therefore, the present study aimed to optimize the extraction of proanthocyanidin-rich cranberry (Vaccinium macrocarpon L.) extract using response surface methodology and to evaluate its antioxidant, antimicrobial, and antibiofilm activities against clinically isolated MDR pathogens. In addition, the study investigated the immunomodulatory and protective effects of the optimized cranberry extract in an in vivo rat model of MDR Escherichia coli infection. By integrating extraction optimization, phytochemical characterization, microbiological assays, and in vivo validation, this work provides a comprehensive assessment of cranberry-derived polyphenols as potential adjunct strategies for combating multidrug-resistant infections.

Materials and methods

Extraction, optimization, and phytochemical characterization of cranberry fruit (Vaccinium macrocarpon L.)

Fully ripened cranberry fruits (Vaccinium macrocarpon L.) were obtained from a local retail market in September 2023. The cranberry fruits were kindly authenticated by Mrs. Therese Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture and Former Director of El-Orman Botanical Garden. The fruits were rinsed, stems removed, and homogenized to ensure uniformity. Cranberry press residues were selected as the extraction matrix due to their enrichment in proanthocyanidins. Preliminary trials were conducted using acetone-based solvents, and acidified systems were evaluated to enhance extraction efficiency by improving cell wall disruption and tannin solubilization.

For each extraction, 5.0 g of dried press residue was mixed with 50 mL of the selected acetone–acid solvent in a 100 mL container. Ultrasonic-assisted extraction was performed using a 300 W, 100 kHz water-cooled ultrasonic bath (Cole-Parmer, Chicago, IL, USA) for 15 min under controlled conditions. The mixture was filtered, and the residue was re-extracted twice under identical conditions. Filtrates were combined and adjusted to 200 mL, then stored at 4 °C and processed within 24 h. Portions were freeze-dried at − 85 °C under 0.01 mbar (Zirbus Technology GmbH, Bad Grund, Germany) for subsequent analyses (Klavins et al. 2022). Key variables, including acetone concentration, formic acid percentage, extraction time, and temperature, were optimized using a central composite design (CCD) with three factors at three levels and six center points. Experiments were conducted in randomized order to minimize bias, and the selected ranges are summarized in Table 1.

Table 1 Independent and dependent variables of CCD design with their respective levels and goals

Determination of total phenolic content (TPC)

Total phenolic compounds in cranberry extracts were measured using the Folin–Ciocalteu spectrophotometric method. Quantification of total phenolic content was first performed to provide a global estimation of bioactive compounds prior to detailed chromatographic profiling. Before analysis, each extract was diluted 1:20, and five concentration levels (12.5–200 mg/mL) were prepared using various solvents, including water, methanol, ethanol, acetonitrile, and acetone. For the assay procedure, 1 mL of the diluted extract was mixed with 1 mL of distilled water, followed by the addition of 500 µL of Folin–Ciocalteu reagent. The mixture was gently vortexed for approximately 1 min to ensure uniform reaction. Subsequently, 4 mL of 7.5% (w/v) sodium carbonate solution was added to promote color formation, and the reaction mixtures were incubated at 25 °C for 2 h in the absence of light to allow complete development of the chromophore. After incubation, absorbance readings were obtained at 765 nm using a UV–visible spectrophotometer. Quantitative determination was achieved using a gallic acid standard calibration curve prepared within a concentration range of 0–100 mg/L.

The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of fresh cranberry weight (Klavins et al. 2022).

Determination of total flavonoid content (TFC)

The total flavonoid content of cranberry extracts was determined using the aluminum chloride colorimetric assay. In brief, 1 mL of cranberry extract or catechin standard solution (5–200 mg/L) was combined with 4 mL of distilled water. Subsequently, 0.3 mL of 5% (w/v) sodium nitrite (NaNO₂) and 0.3 mL of 10% (w/v) aluminum chloride (AlCl₃) were added sequentially to the mixture. After allowing the reaction to proceed for 6 min, 2 mL of 1 M sodium hydroxide (NaOH) was introduced, and the final volume was adjusted to 10 mL with distilled water.

The absorbance of the resulting solution was recorded at 510 nm using a UV–visible spectrophotometer. Flavonoid concentration was calculated based on a catechin standard calibration curve and expressed as milligrams of catechin equivalents (CE) per 100 g of dry weight. All analyses were conducted in triplicate to ensure reproducibility (Bobinaitė et al. 2012).

Qualitative and quantitative profiling of cranberry extract constituents

Compositional profiling of proanthocyanidins, anthocyanins, flavonols, total phenolics, triterpenoids, and phytosterols was performed using an ACQUITY ultra-performance liquid chromatography (UHPLC) system coupled with a photodiode array (PDA) detector (Waters, Milford, MA, USA). Chromatographic separation was achieved on an ACE C18 reverse-phase column (100 × 2.1 mm, 1.7 μm), enabling efficient resolution of phenolic and terpenoid compounds.

Qualitative identification of compounds was carried out based on retention times, UV–visible spectral characteristics (λmax), and comparison with available reference standards and published literature data. Where authentic standards were available (e.g., gallic acid, catechin, quercetin), identification was confirmed by matching both retention times and UV spectra. For compounds lacking commercial standards, tentative identification was based on characteristic UV spectra and previously reported chromatographic behavior. Quantitative analysis was performed using peak area measurements obtained from the UHPLC chromatograms. External calibration curves were constructed using available reference standards at multiple concentrations, and results were expressed as mg/g of dry extract. For compounds without corresponding standards, semi-quantitative estimation was conducted using calibration curves of structurally related compounds. Anthocyanins and anthocyanidins were analyzed following Vilkickytė et al. using gradient elution with 10% aqueous formic acid (A) and acetonitrile (B), at 30 °C, a flow rate of 0.5 mL/min, an injection volume of 1 µL, and detection at 520 nm (Vilkickyte et al. 2021). Flavonols were quantified according to Urbštaitė et al. using acetonitrile and 0.1% aqueous formic acid, with detection at 360 nm (Urbstaite et al. 2022). Triterpenoids and phytosterols were determined following Šedbarė et al. using a methanol and 0.1% formic acid gradient system, with a flow rate of 0.2 mL/min, an injection volume of 1 µL, column temperature of 25 °C, and detection at 205 nm (Sedbare et al. 2022). All analyses were performed in triplicate to ensure reproducibility.

DPPH free radical scavenging assay

The antioxidant activity of cranberry extract was evaluated using a modified 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The extract obtained under optimized conditions (acetone/formic acid system) was evaporated to dryness and subsequently reconstituted in different solvents (water, methanol, ethanol, acetonitrile, and acetone) to assess the influence of solvent on antioxidant activity. Each extract solution was prepared at five concentration levels (12.5, 25, 50, 100, and 200 mg/mL). For each assay, 4 mL of the extract solution was mixed with 1 mL of freshly prepared DPPH solution and vortexed to ensure homogeneity. The reaction mixtures were incubated at room temperature in the dark for 45 min to allow sufficient interaction between antioxidant compounds and the DPPH radical. A control sample was prepared under identical conditions without the addition of extract. Following incubation, the decrease in absorbance was measured at 520 nm using a GENESYS 10 S^TM UV–visible spectrophotometer, with methanol used as the blank. Antioxidant activity was expressed as percentage (%) inhibition of DPPH radicals. All measurements were performed in triplicate.

The percentage of DPPH radical scavenging activity was calculated using the following equation:

$$ \% {\text{ Inhibition }} = {\text{ }}\left[ {\left( {{\text{A}}_{{{\text{control}}}} {-}{\text{ A}}_{{{\text{sample}}}} } \right)/{\text{A}}_{{{\text{control}}}} } \right]{\text{ }} \times {\text{ 1}}00 $$

(1)

where A_control represents the absorbance of the control reaction (without extract), and A_sample represents the absorbance in the presence of the extract. The half-maximal inhibitory concentration (IC₅₀) was defined as the concentration of extract required to inhibit 50% of the DPPH radical activity and was determined from the dose–response curve. Trolox was used as a reference antioxidant under identical experimental conditions, and a calibration curve was constructed. Antioxidant activity was additionally expressed as Trolox equivalent antioxidant capacity (TEAC), calculated from the Trolox standard curve (Yan et al. 2002).

Ferric reducing antioxidant power (FRAP) assay

The antioxidant reducing capacity of cranberry fruit extracts was assessed by measuring their ability to reduce ferric (Fe³⁺) ions to ferrous (Fe²⁺) ions. Extract solutions were prepared in distilled water at concentrations between 60 and 220 mg/mL. For each determination, 1.0 mL of the diluted extract was mixed with 3.5 mL of phosphate buffer (pH 6.6) and 3.5 mL of 1% (w/w) potassium ferricyanide solution. The mixtures were incubated in a water bath at 50 °C for 30 min to promote ferric ion reduction. After incubation, 2.5 mL of 10% (w/v) trichloroacetic acid was added to stop the reaction, followed by centrifugation for 10 min to obtain clear supernatants. Subsequently, 3.5 mL of the supernatant was combined with an equal volume of distilled water, and 1.0 mL of freshly prepared 0.1% (w/w) ferric chloride solution was added. The final mixtures were thoroughly vortexed, and absorbance was measured at 700 nm using a GENESYS 10 S™ UV–visible spectrophotometer. Increased absorbance values were interpreted as indicative of greater ferric reducing power and, consequently, enhanced antioxidant potential of the cranberry extract (Klavins et al. 2022).

Antimicrobial testing

The antimicrobial activity was evaluated against a panel of clinically characterized bacterial pathogens previously identified using both phenotypic and genotypic approaches. Initial phenotypic identification was performed using conventional microbiological techniques, including colony morphology, Gram staining, and standard biochemical assays, and was subsequently confirmed using the VITEK^® 2 automated identification system (bioMérieux, France). Antimicrobial susceptibility testing was conducted using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2023). All antimicrobial disks used in this study were commercially obtained from Oxoid™ Ltd. (Basingstoke, Hampshire, UK). Species-specific antimicrobial panels were selected according to CLSI recommendations and the clinical relevance of the investigated pathogens. Detailed information regarding the antimicrobial agents, disk potencies, and susceptibility testing panels is provided in Supplementary Methods S1. Antimicrobial susceptibility results were interpreted according to CLSI breakpoint criteria and categorized as susceptible (S), intermediate (I), or resistant (R). Multidrug resistance (MDR) was defined as resistance to at least one antimicrobial agent in three or more antimicrobial classes. In addition to the clinical isolates, reference strains including Salmonella enterica serovar Typhimurium ATCC 14,028, Bacillus spizizenii ATCC 6633, Aspergillus brasiliensis ATCC 16,404, and Bacillus subtilis ATCC 6633 were included as reference microorganisms for comparative evaluation of the antimicrobial activity of the cranberry extract. They were analyzed separately from the clinical isolates.

Agar well diffusion assay

The antimicrobial activity of cranberry extract was evaluated using an agar well diffusion assay adapted from previously published methodologies for the assessment of plant-derived antimicrobial agents (Purkait et al. 2020; Wayne 2008). A stock solution of cranberry extract was prepared by dissolving 0.5 g of dried extract in 1 mL of 3% (v/v) dimethyl sulfoxide (DMSO) (Sigma, Hamburg, Germany), resulting in a final concentration of 500 mg/mL. The solution was sterilized by filtration through a 0.22 μm membrane filter (Indiamart, Vadodara, India). Microbial inocula were prepared according to CLSI recommendations and adjusted to a 0.5 McFarland standard (approximately 1–2 × 10⁸ CFU/mL). Subsequently, 100 µL of each standardized microbial suspension was uniformly spread onto the surface of the appropriate agar medium. Mueller–Hinton agar (MHA) was used for non-fastidious bacterial isolates, whereas MHA supplemented with 5% defibrinated sheep blood was used for fastidious organisms, including Streptococcus viridans. Sabouraud dextrose agar (SDA) was used for fungal isolates, including Candida albicans, Aspergillus niger, and Aspergillus brasiliensis ATCC 16,404.

Wells of 10 mm diameter were aseptically prepared in the agar plates, and 100 µL of the cranberry extract was dispensed into each well. Gentamicin (10 µg/mL), penicillin (10 µg/mL), and fluconazole (25 µg/mL) were included as positive controls for Gram-negative bacteria, Gram-positive bacteria, and Candida albicans, respectively. These antimicrobial agents were prepared as standard solutions and applied directly into the wells. Wells containing 3% (v/v) DMSO served as negative controls.

Bacterial plates were incubated at 35 ± 1 °C for 16–20 h, while fungal plates were incubated at 28 °C for 48–72 h, according to CLSI-recommended growth conditions for the respective microorganisms. Following incubation, inhibition zone diameters were measured in millimeters (mm). All experiments were performed in triplicate, and results were expressed as mean inhibition zone diameters ± standard deviation (SD).

Determination of MICs

The minimum inhibitory concentrations (MICs) of cranberry extract against bacterial isolates were determined using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) M07 standard for dilution antimicrobial susceptibility testing of aerobic bacteria (CLSI, 2023). Serial two-fold dilutions of the extract were prepared in sterile 96-well microtiter plates using cation-adjusted Mueller–Hinton broth (CAMHB; Oxoid, UK). An initial stock solution of 2048 µg/mL was prepared, followed by serial two-fold dilutions to obtain final concentrations ranging from 1024 to 1 µg/mL. The stock solution was prepared in 3% (v/v) dimethyl sulfoxide (DMSO); however, dilution in broth ensured that the final DMSO concentration in each well did not exceed 1% (v/v).

Microbial suspensions were adjusted to a 0.5 McFarland standard and subsequently diluted in broth to achieve a final inoculum concentration of approximately 5 × 10⁵ CFU/mL in each well, in accordance with CLSI M07 recommendations. Following inoculation, the microtiter plates were incubated at 35 ± 1 °C for 16–20 h. MIC values were determined as the lowest concentration of cranberry extract that completely inhibited visible microbial growth. To support visual endpoint determination, optical density was measured at 620 nm using a microplate reader (BioTek, Quant) following incubation (Özkalp et al. 2010). Optical density measurements were used as a supplementary assessment of microbial growth and were not employed as the primary criterion for MIC determination. To minimize potential interference arising from the intrinsic color of the cranberry extract, extract-containing wells without microbial inoculum were included as blanks, and background absorbance values were subtracted where appropriate. Penicillin and gentamicin were included as reference antimicrobial agents for comparative purposes. For fungal isolates, MICs were determined using a broth microdilution procedure adapted for yeasts and filamentous fungi according to CLSI M27 and CLSI M38 guidelines, respectively. Yeast isolates (e.g., Candida albicans) were incubated at 35 ± 1 °C for 24–48 h, whereas filamentous fungi (e.g., Aspergillus niger and Aspergillus brasiliensis) were incubated at 28 °C for 48–72 h depending on their growth characteristics. Fluconazole was included as a reference antifungal agent for comparative purposes (Brito-Junior et al. 2024).

Determination of the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)

To assess the MBCs and MFCs, 10 µL of broth from the wells corresponding to MIC, 2 × MIC, and 4 × MIC values were transferred to the Muller-Hinton agar plate for bacteria and SDA for Candida albicans. Following subculturing, bacterial plates were incubated at 35 ± 1 °C for 18–24 h, while fungal plates were incubated at 28 °C for 48–72 h, depending on the growth characteristics of the organism. After incubation, the MBC was identified as the concentration at which no colony growth was observed (Kosakowska et al. 2024).

Antibiofilm activity

Microtiter plate biofilm assay

Biofilm-forming ability was evaluated using the microtiter plate crystal violet staining method with a certain focus on Gram-negative isolates, due to their strong biofilm-forming capacity and their established role in persistent and device-associated infections. Sub-inhibitory concentrations corresponding to 1/2 MIC and 1/4 MIC (ranging from 8 to 256 µg/mL depending on the isolate) were used to evaluate the antibiofilm activity. Overnight cultures grown in trypticase soy broth (TSB; HiMedia, Mumbai, India) were adjusted to 0.5 McFarland (OD₆₀₀) and diluted 1:20 in fresh TSB. A volume of 200 µL of each suspension was inoculated in triplicate into sterile 96-well polystyrene plates (Corning, NY, USA) and incubated at 37 °C for 24 h to allow biofilm formation. After incubation, planktonic cells were removed, and wells were gently washed once with phosphate-buffered saline (PBS). Plates were air-dried, and adherent biofilms were stained with 0.1% crystal violet for 15 min at room temperature, followed by washing to remove excess stain. The retained dye was solubilized using 33% acetic acid, and absorbance was measured at 630 nm using a microplate reader (ELx800, BioTek, USA). Wells containing sterile broth served as negative controls, while Acinetobacter baumannii ATCC 19,606 was used as a positive control. Results were expressed as mean absorbance values of triplicates. The cut-off optical density (ODc) was calculated as the mean OD of the negative control plus three standard deviations. Based on ODc values, isolates were classified as non-, weak, moderate, or strong biofilm producers (Haney et al. 2021).

Antibiofilm activities of cranberry extract at sub-inhibitory concentrations

The inhibitory effect of cranberry extracts on biofilm formation was assessed using a microtiter plate-based assay. In brief, 200 µL of bacterial suspensions standardized to a 0.5 McFarland turbidity were dispensed into sterile 96-well polystyrene microplates. Subsequently, 20 µL of cranberry extract was added to each well to obtain final concentrations equivalent to one-half (1/2 MIC) and one-quarter (1/4 MIC) of the previously determined minimum inhibitory concentration. The plates were incubated at 37 °C for 24 h to allow biofilm development in the presence of sub-inhibitory extract concentrations.

After incubation, wells were gently rinsed twice with phosphate-buffered saline (PBS) to remove planktonic and loosely attached cells. The remaining adherent biofilms were stained with 0.1% (v/v) crystal violet for 15 min. Excess stain was removed by washing, and the retained dye was dissolved using 33% (v/v) acetic acid. Biofilm biomass was quantified by measuring absorbance at 630 nm with a microplate reader (ELx800, BioTek, Winooski, VT, USA). All experiments were performed in triplicate to ensure reproducibility. Wells containing bacterial suspension without cranberry extract were used as positive controls for biofilm formation. The percentage of biofilm inhibition was calculated using the following equation:

$$ {\text{Biofilm inhibition }}\left( \% \right){\text{ }} = [({\text{ A}}_{{\text{c}}} - {\text{A}}_{{\text{s}}} /{\text{A}}_{{\text{c}}} ){\text{ }} \times {\text{1}}00] $$

(2)

where $\:{A}_{c}$represents the optical density at 630 nm of the positive control wells and $\:{A}_{s}$corresponds to the optical density of wells treated with cranberry extract (Sherif et al. 2021).

Scanning electron microscopy analysis of the antibiofilm activity of cranberry extract

To investigate the structural effects of cranberry extract on biofilm formation, a strongly biofilm-producing Acinetobacter baumannii isolate was selected for scanning electron microscopy (SEM) analysis. Briefly, 500 µL of a bacterial suspension adjusted to approximately 1 × 10⁸ CFU/mL was transferred into wells of a sterile 24-well tissue culture plate containing circular glass coverslips (13 mm diameter; Menzel Gläser, Braunschweig, Germany). For treatment groups, 500 µL of cranberry extract at sub-inhibitory concentration was added to each well, whereas control wells received no extract. Plates were incubated at 37 °C for 24 h to allow biofilm development on the coverslip surfaces. After incubation, the coverslips with adherent biofilms were carefully removed and prepared for SEM examination. Biofilms were first fixed in 2.5% (v/v) glutaraldehyde prepared in 0.1 M cacodylate buffer (pH 7.2) for 30 min at room temperature. Post-fixation was performed using osmium tetroxide. Samples were then dehydrated sequentially through a graded ethanol series (30, 50, 70, 90, and 100% v/v), with each step lasting 15 min to ensure complete dehydration. Following dehydration, specimens were sputter-coated with gold and examined using a scanning electron microscope (JEOL JSM-6390LV, Tokyo, Japan). Structural and morphological differences between treated and untreated biofilms were evaluated as previously described (Mlynek et al. 2022).

Growth rate analysis

The growth dynamics of bacterial isolates classified as strong biofilm formers were evaluated by monitoring optical density over time using a microplate-based growth curve assay, as described in recent studies on bacterial growth kinetics and biofilm-associated behavior (Azeredo et al. 2017). For each strain, 20 µL of an overnight culture adjusted to approximately 0.5 McFarland turbidity was inoculated into 180 µL of tryptic soy broth (TSB) in sterile 96-well microplates. The plates were incubated at 35 ± 1 °C under aerobic conditions, and bacterial growth was monitored by measuring optical density at 600 nm (OD₆₀₀) using a microplate reader (ELx800, BioTek, Winooski, VT, USA) at 4-h intervals over a total duration of 48 h.

Growth curves were constructed from mean absorbance values obtained from triplicate wells for each isolate. To evaluate the impact of sub-inhibitory concentrations on bacterial proliferation, cranberry extract was added at 1/2 MIC and 1/4 MIC to the respective wells. Untreated bacterial suspensions served as growth controls, while wells containing sterile medium were included as blanks for background correction. All experiments were performed in triplicate to ensure reproducibility.

Cytotoxicity activity

Vero cells (kidney epithelial cells of African green monkey (Cercopithecus aethiops) were seeded in 96-well plates (Thermo Scientific™ PCR Plate, 96-well, Waltham, MA, USA) at a density of 5000 cells/well. After 1-day incubation, cells were treated with varying concentrations of cranberry extract (10–1000 µg/mL) for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay was done after 37 °C incubation in a humidified 5% CO₂ environment. Cell viability was calculated as a percentage relative to untreated controls, and IC₅₀ values were determined from dose–response curves as previously determined (El-Sayed et al. 2025) .

In vivo studies

All rats received standard care according to the standard guidelines during all stages of the experiment.

Animals

Twenty adult male Wistar albino rats weighing 200–220 g were used. They were purchased from the animal house facility of the Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt. Prior to the start of the study, the animals were acclimatized for 15 days. Animals were handled and were housed in accordance with the Care and Use of Laboratory Animals recommendations and ARRIVE guidelines (https://arriveguidelines.org) (accessed on 20 April 2025). The whole study was reviewed and approved by the research ethical committee of the Faculty of Pharmacy, Ahram Canadian University, Egypt (Protocol approval number: Approval No. ACUC-FP-ACU-REC#1525).

Experimental design and oral infection model

A previously described oral infection model of MDR E.coli was implemented (El-Shiekh et al. 2023). All groups were fed orally by stomach gavage. Briefly, the rats were divided into four groups (Groups I-IV; 5 rats each) as follows:

The rats were assigned to four groups on a random basis (n = 5 per group) as follows:

• Group (I): Negative control, fed with 100 µL of normal saline with no bacterial cells.

• Group (II): Infected with 100 µL of 10⁸ CFU/ml of MDR E.coli not treated.

• Group (III): Infected with 100 µL of 10⁸ CFU/ml of MDR E.coli, then treated with cranberry extract.

• Group (IV): Positive control, infected with 100 µL of 10⁸ CFU/ml of MDR E.coli, then treated with Gentamycin.

Survival rate study

Survival of experimental animals was monitored for three days following bacterial challenge, and mortality rates were determined by documenting deaths and excluding deceased animals from further analysis. At the end of the observation period, surviving rats were anesthetized via intraperitoneal administration of a ketamine–xylazine combination (60 mg/kg ketamine and 10 mg/kg xylazine). Animals were then humanely euthanized by cervical dislocation. Spleens were aseptically harvested from all surviving animals and immediately fixed in 10% neutral-buffered formalin for subsequent histological evaluation. Tissue sections were later processed and stained with hematoxylin and eosin (H&E) for microscopic examination (Abdellatif et al. 2021).

Histopathological examination

As previously reported, all conventional methods for sample fixation and staining were adhered to in accordance with standard practices (Al-Sabaawy et al. 2021).

Immunohistochemistry

Formalin-fixed spleen specimens were processed overnight using an automated tissue processor (Sakura, Japan) and subsequently embedded in paraffin using an embedding station (Sakura, Japan). Paraffin blocks were sectioned at a thickness of 4 μm using a rotary microtome (Leica RM2245, Germany), and sections were mounted on super frost glass slides.

Deparaffinization was achieved through sequential immersion in xylene, followed by rehydration in descending concentrations of ethanol and finally distilled water. Antigen retrieval was conducted by heating tissue sections in citrate buffer using a microwave oven for 5 min. Immunohistochemical staining was performed employing an HRP/DAB detection kit (Abcam) according to the manufacturer’s instructions. Sections were incubated with a primary antibody targeting the pro-inflammatory cytokine interleukin-6 (IL-6) at a dilution of 1:400. After primary antibody incubation, sections were exposed to a horseradish peroxidase-conjugated secondary reagent, and immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as the chromogenic substrate. Slides were counterstained with Mayer’s hematoxylin and examined under a light microscope. The intensity and distribution of IL-6 expression were evaluated semi-quantitatively using a scoring system ranging from 0 (no detectable staining) to 5 (intense staining with widespread distribution). The immunohistochemical procedure was conducted in accordance with previously published methodology (Khan et al. 2020).

Collection of blood samples and estimation of biomarker levels

Blood samples were collected from the retro-orbital plexus under 4% lidocaine local anesthesia from all experimental groups three days after study initiation and before euthanasia (Moustafa et al. 2018). Serum was separated and used for the quantification of inflammatory and immunological biomarkers, including tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB p105), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and the inflammatory enzyme cyclooxygenase-2 (COX-2), also known as prostaglandin-endoperoxide synthase-2 (PTGS-2) (Nonoguchi et al. 2022). Serum concentrations of these mediators were determined using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits (Elabscience^®), following the manufacturer’s instructions. The assays were performed using pre-coated microplates containing capture antibodies specific to rat TNF-α, NF-κB p105, IL-6, IL-1β, and COX-2. Samples or standards were added to the antibody-coated wells, followed by incubation with a biotinylated detection antibody specific to each analyte. Subsequently, an avidin–horseradish peroxidase (HRP) conjugate was applied and incubated at 37 °C for 30 min. After the washing steps, a chromogenic substrate was added, resulting in a blue color reaction proportional to analyte binding.

Optical density (OD) was measured at 450 nm ± 2 nm using a microplate reader (FLUOstar Omega, BMG Labtech, Germany). Cytokine concentrations were calculated according to the respective standard curves. As previously reported, the OD signal is inversely proportional to the concentration of the measured parameter in rat samples (Alvarez et al. 2020; Navaei-Alipour et al. 2021). Unless otherwise stated, all biological assays (antimicrobial, antibiofilm, MIC, MBC/MFC, and in vivo studies) were performed using the optimized cranberry extract obtained from the acetone/formic acid extraction method. For antioxidant assays, the dried extract was reconstituted in different solvents (water, methanol, ethanol, acetonitrile, and acetone) to evaluate solvent-dependent activity.

Statistical analysis

All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA) and RStudio version 4.4.2 (R Foundation for Statistical Computing, Vienna, Austria) utilizing the fmsb and corrplot packages. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test was used to evaluate differences among groups when the data met the assumptions of normality and homogeneity of variance. This approach was selected to allow comparison across multiple experimental groups while controlling for type I error. A p-value < 0.05 was considered statistically significant.

For optimization studies, response surface methodology (RSM), model development, and visualization were performed using Design-Expert^® software (version 11.0). The adequacy and statistical significance of the developed models were assessed using analysis of variance (ANOVA), which was applied to evaluate the contribution and interaction effects of the independent variables within the experimental design framework.

Results

Extraction, optimization, and phytochemical characterization of cranberry fruit (Vaccinium macrocarpon L.)

Extraction of proanthocyanidins has been conducted using a variety of solvents and solvent additives depending on the material to be extracted, the types of proanthocyanidins, or the degree of proanthocyanidin polymerization in the specific biomass. Cranberry proanthocyanidins have been extracted using mainly acetone at different concentrations, occasionally adding different acids to lower the pH of the extraction medium, and at different times and temperatures. The variety of the used solvents suggests the need for extraction solvent standardization for cranberries and, more specifically, cranberry press residues, as the latter is the type of biomass with the greatest potential for the extraction of the valuable proanthocyanidins. The combined model effects of the previously chosen extraction variables were examined using RSM. CCD of 30 runs with 6 central points was performed in a randomized order. The actual values of the investigated factors, design, and observed results are displayed in Table 2. The summary of the ANOVA analysis of the used design can be found in Table S1. The obtained F and p values show model significance; therefore, the model can be used to predict total proanthocyanidin contents under any combination of the optimized variables, indicating adequate accuracy and general availability of the designed polynomial models.

Table 2 CCD design runs for the 4 different factors tested, showing the observed responses

The resulting quadratic CCD model equation obtained from the software is as follows:

$$ \begin{aligned} {\text{PAC content }} = & {\text{ 22}}.{\text{815 }} + {\text{ 1}}.{\text{321A }} - {\text{ }}0.{\text{339B }} + {\text{ }}0.{\text{335C }} \\ & + {\text{ }}0.{\text{983D }} + {\text{ }}0.{\text{185AB }} + {\text{ }}0.0{\text{6875AC }} \\ & + {\text{ }}0.{\text{3325AD }} - {\text{ }}0.0{\text{8125BC }} - {\text{ }}0.{\text{1725BD }} \\ & - {\text{ }}0.{\text{13125CD }} - {\text{ }}0.{\text{56833A}}^{2} {\text{ }} - {\text{ }}0.{\text{11833B}}^{2} {\text{ }} \\ & - {\text{ }}0.{\text{29333C}}^{2} {\text{ }} - {\text{ }}0.{\text{51833D}}^{2} \\ \end{aligned} $$

(3)

The Model F-value of 5336.26 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise, which proves the model is significant. P-values less than 0.0500 indicate model terms are significant. In this case, A, B, C, D, AB, AC, AD, BC, BD, CD, A², B², C², D² are significant model terms. Also, a low coefficient of variation of 0.1389 was obtained, indicating the good reliability of the experimental values. The coefficients of determination R² value, which was 0.9998, suggested that 99.98% of the variability in the response. The Predicted R² of 0.996 was in agreement with the Adjusted R² of 0.9996. Finally, the signal-to-noise ratio, or adequate precision, was equal to 276.7578. A ratio greater than 4 is desirable. Ratio of 276.758 indicates an adequate signal. This model can be used to navigate the design space. Figure 1 shows the three-dimensional response surface plots (3D plots), which revealed that the optimum conditions for PAC content were an acetone: formic acid ratio of 80:0.5, extraction time 12 min, and a temperature of 38 °C.

Fig. 1

Response surface plots illustrating the effects of extraction variables on proanthocyanidin (PAC) yield from cranberry press residues

The normal probability plots of residuals for PAC content (Fig. S1a) suggested that the residuals follow a normal plot as shown by the linear pattern.

The Box-Cox plots are useful tools that determine the utmost applicable power transformation. As shown by our results, the current lambda (λ = 1) was adequate (Fig. S1b). No transformation was recommended.

The predicted versus actual values plot displayed an acceptable agreement between the projected and the actual data (Fig. S1c).

The residuals versus Run number plot showed that the points were arbitrarily scattered around zero (Fig. S1d), which indicates that the model fits the data.

Determination of total phenolic content (TPC)

The calibration curve constructed using gallic acid standards exhibited excellent linearity over the concentration range of 50–500 µg/mL, as indicated by the regression equation (y = 0.003x − 0.014) and a high correlation coefficient (R² = 0.999), confirming the reliability of the Folin–Ciocalteu method for determining total phenolic content (TPC) expressed as gallic acid equivalents (Fig. S2).

The TPC values of Vaccinium macrocarpon fruit extracts obtained using different extraction solvents are presented in Table 3. Considerable variation in phenolic recovery was observed among the tested solvents, indicating a strong influence of solvent polarity on extraction efficiency. Methanol yielded the highest TPC value (28.6 ± 1.2 mg GAE/g fresh cranberry weight), followed by acetone (25.4 ± 1.0 mg GAE/g), ethanol (21.7 ± 0.9 mg GAE/g), and water (18.3 ± 0.8 mg GAE/g). Acetonitrile was the least effective extraction solvent, producing the lowest TPC value (14.9 ± 0.7 mg GAE/g fresh cranberry weight). These findings demonstrate that methanol was the most efficient solvent for extracting phenolic compounds from V. macrocarpon fruits under the tested conditions and support the importance of solvent selection for maximizing phenolic recovery.

Table 3 Total phenolic content (TPC) of Vaccinium macrocarpon fruit extracts was determined using the Folin–Ciocalteu method. Results are expressed as milligrams of gallic acid equivalents (GAE) per gram of fresh cranberry weight. Values represent mean ± standard deviation (SD) of triplicate experiments

Determination of total flavonoid content (TFC)

The total flavonoid content (TFC) of V. macrocarpon fruit extract was determined using the aluminum chloride colorimetric method and expressed as milligrams of catechin equivalents (CE) per 100 g of dry weight. The extract exhibited a high flavonoid content of 1580 ± 42 mg CE/100 g dry weight, indicating a substantial abundance of flavonoid compounds.

Qualitative and quantitative profiling of cranberry extract constituents

Chromatographic profiling of the optimized Vaccinium macrocarpon fruit extract by UHPLC-PDA revealed a complex phytochemical composition characterized by numerous peaks distributed across a broad retention time range (Fig. 2), indicating the presence of compounds with diverse polarities and chromatographic behaviors.

Fig. 2

UHPLC chromatogram of Vaccinium macrocarpon fruit extract

In the early elution region (0–15 min), several peaks corresponding to relatively polar constituents were detected. A distinct peak at a retention time (RT) of 7.04 min was assigned to gallic acid based on comparison with reference standards and UV–visible spectral characteristics. Additional peaks observed between RT 4.07 and 8.00 min were tentatively identified as anthocyanins and anthocyanidins, consistent with their known chromatographic behavior under reversed-phase conditions. Within the intermediate retention region (14–23 min), prominent peaks corresponding to flavan-3-ols and proanthocyanidin-related compounds were observed. A major peak at RT 14.03 min was assigned to proanthocyanidins, while another peak at RT 23.68 min was attributed to anthocyanin-related constituents. A prominent peak detected at RT 33.02 min was assigned to chlorogenic acid based on retention time matching and characteristic UV absorption spectra. This compound represented a substantial proportion of the total chromatographic area, suggesting that chlorogenic acid is an important phenolic constituent of the extract. At longer retention times (36–45 min), additional peaks corresponding to less polar compounds were observed and tentatively assigned to triterpenoid-related constituents. Furthermore, a peak at RT 52.06 min was assigned to rutin, while a late-eluting peak at RT 58.61 min was attributed to quercetin based on comparison with authenticated standards and UV spectral profiles. Quantitative analysis demonstrated that anthocyanins constituted the most abundant phytochemical class in the extract (6274 ± 76 µg/g), followed by triterpenoids (4194 ± 71 µg/g) and proanthocyanidin aglycones (3977 ± 44 µg/g). Flavonols were also present at appreciable concentrations (2967 ± 36 µg/g), whereas anthocyanidins accounted for 1406 ± 109 µg EE/g. Among the quantified phenolic acids, chlorogenic acid (469 ± 7 µg/g) and gallic acid (327 ± 4 µg/g) were detected at moderate levels. Overall, the UHPLC-PDA analysis demonstrated that V. macrocarpon extract possesses a chemically diverse phytochemical profile dominated by polyphenolic and triterpenoid constituents. These bioactive compounds have been widely associated with antioxidant, antimicrobial, and antibiofilm activities and may contribute to the biological effects observed in the present study. Compound assignments were based on retention times, UV–visible spectral characteristics, comparison with available reference standards, and published literature data.

DPPH free radical scavenging assay

The antioxidant activity of Vaccinium macrocarpon fruit extracts was evaluated using the DPPH radical scavenging assay, and the results demonstrated a clear concentration-dependent increase in radical inhibition for all tested solvent systems (Fig. S3). Among the extracts, the methanol extract exhibited the highest antioxidant activity, reaching approximately 98% inhibition at 200 mg/mL. The acetone extract showed the second-highest activity, achieving approximately 75% inhibition at the same concentration. Ethanol and water extracts displayed moderate antioxidant activity, with maximum inhibition values of approximately 65 and 52%, respectively, whereas the acetonitrile extract exhibited the lowest activity, reaching only about 38% inhibition at 200 mg/mL. Trolox, used as the reference antioxidant, demonstrated strong radical scavenging activity across all tested concentrations, with inhibition values of approximately 35, 55, 75, 90, and 98% at concentrations of 25, 50, 100, 150, and 200 mg/mL, respectively. At lower concentrations (25–50 mg/mL), Trolox exhibited substantially greater antioxidant activity than all cranberry extracts. However, at higher concentrations, particularly at 200 mg/mL, the antioxidant activity of the methanolic extract approached that of Trolox, while the acetone, ethanol, water, and acetonitrile extracts remained considerably less effective. Overall, the antioxidant activity of the extracts followed the order: methanol > acetone > ethanol > water > acetonitrile.

Reducing power assay FRAP

The ferric reducing antioxidant power (FRAP) assay revealed that the cranberry extract exhibited a clear concentration-dependent increase in reducing activity across the evaluated concentration range of 60–220 mg/mL. Progressive elevation of extract concentration resulted in a corresponding rise in absorbance measured at 700 nm, reflecting an enhanced capacity of the extract to mediate the reduction of ferric (Fe³⁺) ions to ferrous (Fe²⁺) ions (Table S2). At lower concentrations, the cranberry extract demonstrated a moderate reducing ability, whereas a marked amplification of reducing power was observed as the concentration increased. The highest concentration tested produced the maximum reducing response, indicating a strong electron-donating capability of the bioactive constituents present in the extract. This pattern highlights the involvement of cranberry-derived compounds in redox-mediated reactions that underpin antioxidant activity. The pronounced ferric-reducing capacity observed can be largely attributed to the abundance of phenolic compounds and flavonoids in cranberry extract, which are well recognized for their ability to donate electrons or hydrogen atoms, thereby stabilizing reactive species. Collectively, these findings confirm that cranberry extract possesses substantial ferric-reducing potential, reinforcing its suitability as a natural antioxidant source and supporting the results of complementary antioxidant assays.

Antimicrobial activity

Characterization and antimicrobial susceptibility profiles of tested isolates

Antimicrobial susceptibility testing revealed that all investigated clinical isolates exhibited multidrug-resistant (MDR) phenotypes, demonstrating resistance to at least one antimicrobial agent in three or more antimicrobial classes according to CLSI criteria. The tested Gram-negative isolates, including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, showed high resistance frequencies toward β-lactam antibiotics, particularly cephalosporins and carbapenems, in addition to marked resistance against fluoroquinolones and aminoglycosides. Resistance to cefepime (FEP), ceftriaxone (CRO), meropenem (MEM), levofloxacin (LEV), ciprofloxacin (CIP), gentamicin (GEN), and amikacin (AMK) was commonly observed among the tested isolates. In contrast, tigecycline (TGC) demonstrated comparatively lower resistance rates across most isolates.

Similarly, Gram-positive isolates, including methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative staphylococci (CONS), and Streptococcus viridans, demonstrated pronounced MDR profiles characterized by elevated resistance against β-lactams, macrolides, lincosamides, tetracyclines, and fluoroquinolones. MRSA isolates exhibited particularly high resistance to cefoxitin, penicillin, erythromycin, clindamycin, ciprofloxacin, and levofloxacin, whereas vancomycin and linezolid retained relatively preserved activity against most isolates.

The comparative antimicrobial resistance patterns among the investigated Gram-negative pathogens are illustrated in the radar plot (Fig. S4). As shown in Fig. S4, Acinetobacter baumannii exhibited the highest overall resistance burden, particularly against carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, indicating its highly resistant phenotype. Pseudomonas aeruginosa and Klebsiella pneumoniae also displayed elevated resistance profiles across most tested antibiotics, whereas Escherichia coli demonstrated comparatively lower resistance frequencies, especially toward aminoglycosides. Among the tested antimicrobial agents, tigecycline (TGC) showed the lowest resistance rates across all bacterial species, suggesting retained antimicrobial efficacy against several MDR isolates.

Overall, all tested isolates met the criteria for multidrug resistance, defined as resistance to at least one agent in three or more antimicrobial classes. These susceptibility profiles confirm the MDR status of the selected panel and support their suitability for evaluating the antimicrobial activity of cranberry extract.

Agar well diffusion assay

The antimicrobial activity of Vaccinium macrocarpon fruit extract was evaluated using the agar well diffusion assay under standardized conditions. The extract produced clear and reproducible zones of inhibition against all tested microorganisms, with inhibition zone diameters ranging from 10.8 ± 0.4 mm to 17.0 ± 2.2 mm. The overall activity pattern is illustrated in Fig. S5 , while detailed inhibition zone measurements are presented in Table S3 . No inhibition was observed for the negative control (3% DMSO, 0 mm).

Among Gram-positive bacteria, Bacillus subtilis ATCC 6633 exhibited the highest susceptibility to the cranberry extract, with an inhibition zone of 18.0 ± 0.6 mm, followed by Bacillus spizizenii ATCC 6633 (15.5 ± 1.5 mm), Streptococcus viridans (14.8 ± 0.5 mm), Enterococcus faecalis (13.5 ± 0.7 mm), methicillin-resistant Staphylococcus aureus (MRSA; 12.4 ± 0.6 mm), and coagulase-negative staphylococci (CoNS; 10.8 ± 0.4 mm). Penicillin, used as the positive control, produced inhibition zones ranging from 10.2 ± 0.3 mm to 12.0 ± 0.4 mm. These results demonstrate that the extract retained measurable inhibitory activity against isolates displaying resistance to conventional antimicrobial agents.

Among Gram-negative bacteria, Salmonella enterica serovar Typhimurium ATCC 14,028 demonstrated the greatest susceptibility, with an inhibition zone of 14.8 ± 1.2 mm. Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa exhibited inhibition zones of 12.2 ± 0.8 mm, 11.8 ± 0.8 mm, 11.5 ± 0.6 mm, and 10.8 ± 0.4 mm, respectively. Gentamicin, used as the positive control, produced inhibition zones within the range of 11.0 ± 0.4 mm. The cranberry extract also demonstrated antifungal activity against all tested fungal strains. Candida albicans exhibited the largest inhibition zone (13.5 ± 0.5 mm), followed by Aspergillus brasiliensis ATCC 16,404 (13.0 ± 0.8 mm) and Aspergillus niger (12.2 ± 0.6 mm). Fluconazole, used as the antifungal control, produced inhibition zones ranging from 11.2 ± 0.4 mm to 12.8 ± 0.5 mm. Overall, Gram-positive bacteria displayed greater susceptibility to the cranberry extract than Gram-negative bacteria, with Bacillus spp. showing the largest inhibition zones. In contrast, Pseudomonas aeruginosa exhibited the lowest susceptibility among the tested bacterial isolates. These findings indicate broad-spectrum antimicrobial activity of the cranberry extract against both bacterial and fungal pathogens.

Determination of MICs

The minimum inhibitory concentrations (MICs) of cranberry extract were determined against multidrug-resistant (MDR) bacterial and fungal isolates and compared with standard antimicrobials. The extract showed measurable inhibitory activity against all tested organisms, with MIC values ranging from 32 to 512 µg/mL. Statistical analysis using the Kruskal–Wallis test followed by Dunn’s post hoc test indicated significant differences compared with controls for several microorganisms (p < 0.05).

Greater susceptibility was observed among Gram-positive bacteria. Streptococcus viridans and Bacillus spizizenii ATCC 6633 exhibited the lowest MIC values (32 µg/mL), while Bacillus subtilis ATCC 6633 and CONS showed MICs of 64 µg/mL. Methicillin-resistant Staphylococcus aureus (MRSA) was inhibited at 128 µg/mL, lower than that observed for penicillin (512 µg/mL; p < 0.05), indicating measurable activity against resistant Gram-positive strains.

Gram-negative bacteria showed higher MIC values, including Escherichia coli (128 µg/mL), Salmonella enterica serovar Typhimurium ATCC 14,028 (64 µg/mL), and Klebsiella pneumoniae and Acinetobacter baumannii (256–512 µg/mL), while Pseudomonas aeruginosa exhibited the highest MIC (512 µg/mL). This reduced susceptibility may be related to intrinsic resistance mechanisms such as limited membrane permeability and efflux activity.

Antifungal activity was also observed, with MIC values of 64 µg/mL against Candida albicans and 128–256 µg/mL against Aspergillus spp. Lower MIC values were observed compared with fluconazole for C. albicans (64 vs. 256 µg/mL; p < 0.05), while comparable activity was noted against Aspergillus spp. No activity was detected for the DMSO control.

Overall, these results indicate antimicrobial activity of the cranberry extract against a range of MDR microorganisms under the tested conditions, with greater effects observed for Gram-positive bacteria and yeasts.

Determination of MBC/MFC

The bactericidal and fungicidal activity of cranberry extract was evaluated by determining minimum bactericidal and fungicidal concentrations (MBC/MFC) relative to MIC values. MBC/MFC values ranged from 64 to 1024 µg/mL and were consistently two-fold higher than MIC values, resulting in MBC/MIC and MFC/MIC ratios of 2 across all tested microorganisms.

Statistical analysis using the Kruskal–Wallis test followed by Dunn’s post hoc test indicated significant differences between MIC and MBC/MFC values (p < 0.05), as well as among microbial groups. Gram-positive bacteria and fungi exhibited lower MBC/MFC values compared with Gram-negative bacteria (p < 0.05). Among Gram-positive bacteria, Streptococcus viridans, Bacillus spizizenii ATCC 6633, and Bacillus subtilis ATCC 6633 showed low MIC and MBC values, with consistent MBC/MIC ratios of 2. Methicillin-resistant Staphylococcus aureus (MRSA) showed bactericidal activity at 256 µg/mL (p < 0.05 compared with penicillin).

Gram-negative bacteria required higher concentrations to achieve bactericidal effects, with Acinetobacter baumannii and Pseudomonas aeruginosa showing the highest MBC values (1024 µg/mL). However, the consistent MBC/MIC ratio of 2 suggests cidal activity under the tested conditions. Antifungal activity was also observed, with Candida albicans and Aspergillus spp. showing MFC/MIC ratios of 2 and lower MFC values than fluconazole (p < 0.05).

Overall, the results indicate that the extract exhibits bactericidal and fungicidal activity under experimental conditions, with variation among microbial groups.

Antibiofilm activity

Microtiter Plate biofilm formation assay, antibiofilm activities of cranberry extract at sub-inhibitory concentrations

Cranberry extract reduced biofilm formation across tested Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA). The strongest effects were observed at sub-inhibitory concentrations, particularly at 1/2 MIC, with a significant concentration-dependent reduction in biofilm biomass (one-way ANOVA, p < 0.05). Stratification of isolates based on baseline biofilm production showed that strong biofilm formers (A. baumannii and P. aeruginosa) exhibited significant reductions in biofilm formation at sub-MIC levels (p < 0.05). Moderate biofilm formers (E. coli and K. pneumoniae) also showed reduced biofilm formation at 1/2 and 1/4 MIC, with both concentration and species influencing the extent of inhibition (p < 0.05). In weak biofilm producers, reductions were observed, but were not always statistically significant (p > 0.05), likely due to low baseline biofilm formation. Overall analysis confirmed that both extract concentration and biofilm-forming capacity significantly affected biofilm inhibition (two-way ANOVA, p < 0.05), with greater reductions observed at 1/2 MIC. Importantly, sub-inhibitory concentrations did not markedly affect bacterial growth, indicating that biofilm inhibition was independent of antimicrobial effects (Fig. 3a, b). Biofilm inhibition ranged approximately from 50% to 80% across the tested microorganisms. The highest inhibition was observed in A. baumannii (≈ 75–80%), followed by MRSA (≈ 65–70%) and E. coli (≈ 60%). Moderate effects were observed for K. pneumoniae and P. aeruginosa (≈ 55–60%), while Candida albicans showed lower inhibition (≈ 50–55%). Variability among replicates was observed; however, the overall trend supports consistent antibiofilm activity across microbial groups. Overall, cranberry extract was associated with reduced biofilm formation across diverse microorganisms under the tested conditions.

Fig. 3

Growth kinetics and antibiofilm activity of cranberry extract against selected pathogens. a Growth curves of Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans monitored over 48 h. Bacterial growth was measured spectrophotometrically at 600 nm (OD₆₀₀) at 4 h intervals. Data represent mean values of triplicate experiments. b Dose-dependent antibiofilm activity of cranberry extract at 1/2 MIC and 1/4 MIC. Values represent mean ± SD (n = 3)

Scanning electron microscope examination (SEM)

SEM examination of the untreated control revealed the presence of a compact and continuous biofilm characterized by a dense aggregation of A. baumannii cells adherent to the surface (Fig. 4a). In contrast, biofilms exposed to cranberry extract showed a noticeable reduction in surface coverage and biofilm density, with less compact cell aggregation compared to the untreated control. Notably, treatment with cranberry extract resulted in a pronounced antibiofilm effect (Fig. 4b), demonstrating greater inhibition of biofilm formation compared with the lower-intensity treatment condition. These SEM observations are in agreement with the quantitative results obtained from the microtiter plate biofilm assay, confirming the strong antibiofilm activity of cranberry extract against A. baumannii.

Fig. 4

Scanning electron micrographs of Acinetobacter baumannii biofilms formed in the absence and presence of cranberry extract. a Untreated control showing dense biofilm architecture with extensive bacterial aggregation and extracellular matrix formation on the glass coverslip surface. b Biofilm formed in the presence of cranberry extract at sub-inhibitory concentration, demonstrating disrupted architecture, reduced bacterial clustering, and diminished extracellular matrix deposition

Cytotoxicity activity

The cytotoxic effects of Vaccinium macrocarpon fruit extract on Vero cells were evaluated using the MTT assay following 24 h of exposure to concentrations ranging from 10 to 1000 µg/mL. As shown in Fig. 5, the extract exhibited a concentration-dependent reduction in cell viability, demonstrating a typical sigmoidal dose–response relationship.

Fig. 5

Cytotoxicity of cranberry extract on Vero cells

At the lowest tested concentrations (10–50 µg/mL), the extract displayed minimal cytotoxicity, with cell viability remaining above 90% relative to the untreated control. Exposure to 100 µg/mL resulted in a slight reduction in viability to approximately 82%, while treatment with 200 µg/mL decreased viability to approximately 70%. A more pronounced cytotoxic effect was observed at higher concentrations, with cell viability declining to approximately 55% at 400 µg/mL. Further increases in extract concentration resulted in substantial reductions in cell viability, reaching approximately 30% at 800 µg/mL and approximately 18–20% at 1000 µg/mL.

The dose–response curve indicated that the half-maximal inhibitory concentration (IC₅₀) of the cranberry extract was approximately 450–500 µg/mL. These findings demonstrate that the extract exhibits relatively low cytotoxicity at concentrations below 200 µg/mL, whereas higher concentrations induce a marked reduction in Vero cell viability. The observed concentration-dependent cytotoxic profile suggests a reasonable safety margin between biologically active concentrations and concentrations associated with significant cytotoxic effects.

Experimental design and oral infection model

Survival rate

The survival percentage graph illustrates the effect of cranberry extract treatment on survival in rats experimentally infected with multidrug-resistant (MDR) Escherichia coli. Animals were allocated into four groups: a negative control (Group I), an untreated infected group (Group II), an infected group treated with cranberry extract (Group III), and a positive control group treated with gentamycin (Group IV).

Group I (negative control), which received normal saline without bacterial challenge, exhibited 100% survival, confirming the absence of procedure-related mortality and supporting the validity of the experimental conditions. In contrast, Group II (infected, untreated) showed a marked reduction in survival, decreasing to 60%, reflecting the virulence of the MDR E. coli strain in the absence of intervention.

Group III (infected and treated with cranberry extract) was associated with improved survival, with survival rates of 100% under the experimental conditions. This observation suggests a protective effect of the cranberry extract in this model; however, these findings should be interpreted cautiously, given the limited sample size and short experimental duration.

Group IV (infected and treated with gentamycin) also demonstrated high survival rates (approximately 90%), consistent with the expected activity of gentamycin. Both treatment groups showed improved survival compared to the untreated infected group (Fig. S6). Direct comparisons between treatments should be interpreted with caution within the constraints of the experimental design.

Overall, these results demonstrate that cranberry extract significantly improved survival outcomes in the experimental model. Although a beneficial effect was observed, direct comparisons with gentamycin should be interpreted with caution, given the experimental limitations. The findings support the potential of cranberry extract as an effective therapeutic or adjunct strategy in the management of MDR bacterial infections.

Histopathological examination

Histological examination of spleen sections from the negative control group (Group I), which received normal saline without bacterial challenge, revealed normal splenic architecture. The white and red pulp regions were clearly distinguishable and well organized, with preserved structural integrity. Lymphoid follicles appeared intact, with normal cellular density and morphology, and no evidence of lymphoid depletion or hyperplasia. The red pulp exhibited normal splenic cords and sinusoids, without signs of congestion, hemorrhage, or vascular dilation. No inflammatory cell infiltration or tissue disruption was observed, confirming the absence of pathological alterations and supporting its role as a baseline control (Fig. 6a).

Fig. 6

Histopathological examination of rat spleen tissue stained with hematoxylin and eosin (H&E). a Normal control group (I) normal splenic architecture showing intact white pulp with well-defined lymphoid follicles. b Group (II) shows severe depletion of white pulp (arrow), characterized by a mafollicles. ((ion in lymphoid cellularity. c Group (III) shows mild depletion of white pulp (arrow), with a slight reduction in lymphoid elements. d Group (IV) shows moderate depletion of white pulp (arrow), showing noticeable but incomplete loss of lymphoid tissue. Representative photomicrographs are shown for each experimental group

In contrast, spleen sections from the infected untreated group (Group II) showed marked pathological alterations, including disruption of normal architecture, lymphoid depletion, expansion and congestion of the red pulp, and extensive inflammatory cell infiltration. Areas of cellular degeneration and focal necrosis were also evident, consistent with infection-associated tissue injury (Fig. 6b).

In Group III (infected and treated with cranberry extract), histological features were associated with partial restoration of splenic architecture compared with the untreated infected group. Improved distinction between white and red pulp regions was observed, along with reduced lymphoid depletion and decreased inflammatory cell infiltration. Evidence of severe necrosis and cellular degeneration was less apparent, suggesting attenuation of infection-associated tissue damage under the experimental conditions (Fig. 6c).

In Group IV (infected and treated with gentamycin), a degree of structural improvement was also observed relative to the untreated group, with partial reorganization of splenic architecture and reduced inflammatory changes. However, residual lymphoid depletion and mild congestion persisted in some areas (Fig. 6d).

Overall, both treatment groups demonstrated histological improvements compared with the untreated infected group. However, these findings should be interpreted cautiously within the constraints of the experimental design, and direct comparisons between treatments remain limited.

Immunohistochemistry

Immunohistochemical staining for interleukin-6 (IL-6) in spleen sections from the negative control group (Group I), which received normal saline without bacterial challenge, demonstrated minimal to absent IL-6 immunoreactivity. The splenic tissue showed predominantly negative staining, with only occasional faint DAB signals, consistent with basal physiological expression levels. Both the white and red pulp regions exhibited no appreciable IL-6 overexpression, indicating the absence of inflammatory activation (Fig. 7a).

Fig. 7

Immunohistochemical detection of IL-6 expression in rat spleen white pulp (IHC–peroxidase–DAB). a Normal control group (I) showing basal IL-6 expression within the white pulp.b Group (II) shows severe positive IL-6 expression in the white pulp, indicated by intense brown DAB staining. c Group (III) shows mild positive IL-6 immunoreactivity in the white pulp. d Group (IV) shows moderate positive IL-6 expression within the splenic white pulp. Brown coloration represents positive immunostaining for IL-6, while nuclei are counterstained with hematoxylin

In contrast, spleen sections from the infected untreated group (Group II) revealed marked upregulation of IL-6 expression, with extensive DAB-positive staining observed throughout the tissue. IL-6–positive cells were widely distributed in both white and red pulp regions, reflecting an elevated inflammatory response associated with systemic infection (Fig. 7b).

In Group III (infected and treated with cranberry extract), IL-6 expression was reduced compared with the untreated infected group. The tissue exhibited weak to moderate staining, with fewer IL-6–positive cells distributed within the splenic parenchyma. This pattern suggests attenuation of the inflammatory response under the experimental conditions, although residual cytokine expression remained evident (Fig. 7c).

In Group IV (infected and treated with gentamycin), a reduction in IL-6 expression was also observed relative to the untreated group, with scattered DAB-positive cells indicating decreased inflammatory activity. However, focal IL-6 positivity persisted in certain regions, suggesting incomplete resolution of the inflammatory response (Fig. 7d).

Overall, both treatment groups were associated with a reduction in IL-6 expression compared with the untreated infected group. However, these findings should be interpreted cautiously within the limitations of the experimental model, and direct comparisons between treatments remain limited.

Collection of blood samples and estimation of biomarker levels

Figure 8 illustrates the effects of cranberry extract treatment on systemic inflammatory and immune markers in rats infected with multidrug-resistant (MDR) Escherichia coli, compared with untreated infected animals and gentamycin-treated controls.

Fig. 8

Effect of cranberry extract treatment on systemic inflammatory biomarkers in infected rats. a Tumor necrosis factor-α (TNF-α), b interleukin-6 (IL-6), c interleukin-1β (IL-1β), d nuclear factor kappa B p105 (NF-κB p105), and e cyclooxygenase-2 (COX-2/PTGS-2) levels

Across all panels (a–e), Group II (infected, untreated) exhibited a marked and statistically significant elevation in pro-inflammatory mediators, including TNF-α, IL-6, IL-1β, NF-κB p105, and COX-2, compared with the negative control (Group I) (p < 0.001–0.0001), indicating a pronounced inflammatory response following MDR E. coli infection.

In Group III (cranberry extract–treated), a reduction in all measured biomarkers was observed relative to Group II (p < 0.05–0.01). Levels of TNF-α, IL-6, and IL-1β were lower compared with the untreated infected group, suggesting attenuation of cytokine-associated inflammatory responses under the experimental conditions. Similarly, reduced levels of NF-κB p105 and COX-2 were observed, which may be indicative of modulation of inflammatory signaling pathways, although this was not directly investigated in the present study. Quantitatively, cranberry extract was associated with approximate reductions of 2.6-, 3.3-, 2.8-, 2.8-, and 2.7-fold in TNF-α, IL-6, IL-1β, NF-κB p105, and COX-2 levels, respectively, compared with Group II.

In Group IV (gentamycin-treated), inflammatory markers were also reduced relative to the untreated infected group, consistent with its antibacterial activity. While differences between treatment groups were observed, direct comparisons should be interpreted cautiously within the limitations of experimental design.

Overall, these findings indicate that cranberry extract was associated with a reduction in inflammatory markers and may have a modulatory effect on inflammatory responses in this experimental model. However, further studies are required to validate these observations and to clarify the underlying mechanisms.

Discussion

The accelerating global crisis of antimicrobial resistance (AMR) represents one of the most urgent threats facing contemporary medicine. Multidrug-resistant (MDR) pathogens are progressively eroding the clinical efficacy of established antibiotic classes, leading to prolonged hospitalizations, increased mortality, and a mounting economic burden on health systems worldwide (Murray et al. 2022; Patra et al. 2025). The World Health Organization has identified carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA) as critical and high-priority pathogens, for which new therapeutic options are urgently needed (Sati et al. 2025). Against this backdrop, plant-derived polyphenols have attracted growing research interest as potential adjunct or alternative antimicrobial strategies, owing to their multitarget mechanisms of action and comparatively lower propensity for inducing resistance (Tang et al. 2025). The present study integrates RSM-guided extraction optimization, comprehensive phytochemical profiling, and coordinated in vitro and in vivo evaluation to characterize the antimicrobial, antibiofilm, antioxidant, and immunomodulatory activities of Vaccinium macrocarpon (cranberry) proanthocyanidin-rich extract against clinically relevant MDR pathogens.

A central contribution of the present study is the systematic optimization of the proanthocyanidin extraction process from cranberry press residues using response surface methodology (RSM) based on a central composite design (CCD). The developed quadratic model demonstrated exceptional predictive accuracy (R² = 0.9998; adequate precision ratio = 276.76), confirming its suitability for navigating the experimental design space (El-Sayed et al. 2023). Acetone concentration and formic acid percentage emerged as the primary determinants of proanthocyanidin yield, with optimum conditions identified at an acetone: formic acid ratio of 80:0.5, an extraction time of 12 min, and a temperature of 38 °C. These findings are consistent with prior reports demonstrating that acidified aqueous-acetone systems significantly enhance condensed tannin recovery by promoting cell wall disruption, improving solubilization of proanthocyanidin complexes, and stabilizing the extracted compounds against oxidative degradation (Klavins et al. 2022; Urbstaite et al. 2022). The importance of extraction standardization cannot be overstated; inter-study variability in solvent composition, pH, and temperature represents a recognized source of inconsistency in phytochemical research that undermines the comparability and reproducibility of biological activity data (Wang et al. 2022). By applying RSM to define and validate optimal extraction parameters, the present study establishes a reproducible methodological platform for future investigations of cranberry-derived bioactive compounds.

Phytochemical profiling of the optimized extract by UHPLC-PDA revealed a chemically diverse composition dominated by anthocyanins (6274 ± 76 µg/g), triterpenoids (4194 ± 71 µg/g), and proanthocyanidin aglycones (3977 ± 44 µg/g), alongside flavonols, anthocyanidins, chlorogenic acid, and gallic acid. This polyphenolic diversity is of particular mechanistic significance. Cranberry A-type proanthocyanidins are structurally distinctive in bearing doubly-linked interflavanyl bonds that confer conformational rigidity and enhanced cell surface anti-adhesive properties, distinguishing them from the more common B-type proanthocyanidins found in most other botanical sources (Jangid et al. 2025). Beyond anti-adhesion, the co-occurrence of phenolic acids such as chlorogenic and gallic acid, flavonols including quercetin and rutin, and triterpenoid constituents likely underpins the broad-spectrum biological activity observed in this study through complementary and potentially synergistic mechanisms (Tang et al. 2025; Daglia 2012). It should be noted, however, that compound identification in the present study relied on chromatographic retention times and UV spectral characteristics compared with reference standards and published data, and should therefore be regarded as tentative in the absence of mass spectrometric confirmation. Future studies employing UHPLC-MS/MS would substantially strengthen the phytochemical characterization and facilitate structure-activity analysis.

The antimicrobial evaluation was designed to provide a stepwise and methodologically rigorous assessment of the extract’s antibacterial and antifungal potential. Agar well diffusion was employed as a qualitative initial screen to identify susceptible organisms and confirm the absence of inhibitory activity in the solvent control, while broth microdilution, conducted in strict accordance with CLSI guidelines, enabled reproducible, quantitative determination of MIC and MBC values. This tiered approach, in which disc or well diffusion serves as a preliminary filter and microdilution provides definitive endpoint data, is the standard methodological framework recommended for evaluating natural product antimicrobials (Davidova et al. 2024). The inclusion of biofilm formation and inhibition assays, together with scanning electron microscopy, extended the evaluation beyond planktonic susceptibility to address the clinically critical dimension of biofilm-associated resistance. As no single assay fully captures antimicrobial efficacy, the integration of these complementary techniques provides a more complete and translationally relevant characterization of biological activity. It’s noteworthy that direct comparisons between crude plant extracts and purified antimicrobial agents should be interpreted cautiously due to differences in chemical composition, diffusion characteristics, and mechanisms of action.

The antimicrobial results of the present study demonstrate that cranberry extract exerts measurable bactericidal activity (MBC/MIC ratio = 2) against a broad panel of MDR pathogens, with MIC values ranging from 32 µg/mL for susceptible Gram-positive species to 512 µg/mL for the most resistant Gram-negative isolates. The observed differential susceptibility between Gram-positive and Gram-negative organisms is consistent with established differences in bacterial cell envelope architecture. In Gram-negative bacteria, the asymmetric lipopolysaccharide-rich outer membrane presents a substantial permeability barrier that restricts the passive diffusion of hydrophilic polyphenolic compounds, thereby reducing intracellular bioavailability (Sun et al. 2022). Gram-positive organisms lack this outer membrane and are consequently more accessible to membrane-disrupting and enzyme-inhibiting polyphenolic constituents. This differential susceptibility pattern has been widely reported for plant-derived polyphenolic extracts, including those rich in gallic acid, chlorogenic acid, and condensed tannins, and is therefore not unexpected for cranberry-derived preparations (Sharma et al. 2025). Notably, however, the cranberry extract retained measurable inhibitory and bactericidal activity against the intrinsically resistant ESKAPE pathogens Acinetobacter baumannii and Pseudomonas aeruginosa, suggesting that multiple and potentially synergistic mechanisms are involved in the observed antimicrobial activity.

The mechanistic basis of cranberry polyphenol antimicrobial activity is likely multifaceted. Proanthocyanidins and phenolic acids can destabilize bacterial membranes by intercalating into the lipid bilayer, increasing membrane permeability, and causing leakage of intracellular constituents (Ivanov et al. 2022). Additionally, polyphenolic compounds have been reported to inhibit efflux pump activity, an important resistance mechanism in MDR Gram-negative pathogens such as Cranberry polyphenols have also been reported to disrupt quorum-sensing signaling networks that coordinate the transition from planktonic to biofilm growth states, and to attenuate the production of virulence factors including flagella, type IV pili, and exopolysaccharide matrices that stabilize mature biofilms (Santos et al. 2021; Zai et al. 2025). It must be emphasized, however, that the molecular mechanisms underlying the observed antimicrobial and antibiofilm activities were not directly investigated in the present study, and mechanistic inferences remain speculative in the absence of dedicated mechanistic experiments such as membrane integrity assays, efflux pump inhibition studies, or quorum-sensing reporter assays.

The antibiofilm activity of Vaccinium macrocarpon extract observed in the present study represents one of its most clinically significant findings. Biofilm formation is a pivotal virulence strategy in MDR pathogens, conferring tolerance to antimicrobial agents that can be up to 1000-fold higher than the MIC required to inhibit planktonic cells, and creating a persistent reservoir for recurrent and device-associated infections (Sharma et al. 2025). Treatment with cranberry extract at sub-inhibitory concentrations (1/2 MIC and 1/4 MIC) produced concentration-dependent reductions in biofilm biomass ranging from approximately 50% in Candida albicans to approximately 75–80% in A. baumannii, with growth curve analysis confirming that these effects occurred at concentrations that did not appreciably impair bacterial proliferation. The finding that antibiofilm activity was dissociated from direct growth inhibition is of particular significance, as it suggests that the extract may target biofilm-specific regulatory processes rather than exerting a generalized antimicrobial effect. This observation is consistent with previous reports demonstrating that cranberry PACs and phenolic acids can suppress expression of biofilm-associated genes, including bap, ompA, and csu operons in A. baumannii and related pathogens (Sherif et al. 2021). Notably, A. baumannii - which forms biofilms at a rate approaching 90% among clinical isolates - exhibited the highest susceptibility to antibiofilm inhibition in the present study (≈ 75–80%), a finding that carries meaningful clinical relevance given its prominent role in ventilator-associated pneumonia and catheter-related bloodstream infections in intensive care settings (Sherif et al. 2021). The antibiofilm activity of V. macrocarpon extract was assessed through both quantitative (crystal violet microtiter assay) and qualitative (SEM) approaches, each offering distinct and complementary analytical information. The crystal violet assay measures total biofilm biomass, including loosely attached cells and extracellular polymeric substances, providing a global quantitative readout of biofilm inhibition. SEM, by contrast, yields high-resolution visualization of biofilm architecture at the surface level. The SEM observations in the present study - demonstrating reduced bacterial aggregation, disrupted intercellular connectivity, and diminished extracellular matrix deposition in cranberry-treated biofilms relative to the untreated control - qualitatively corroborate the quantitative biofilm inhibition data. While some degree of discrepancy between the two methods is expected and has been well documented in the biofilm literature, the convergent findings across both platforms strengthen confidence in the overall antibiofilm conclusion. It should be noted that SEM does not discriminate between viable and non-viable cells, nor does it permit quantification of biofilm thickness or volumetric biomass; these limitations are best addressed in future studies through confocal laser scanning microscopy (CLSM) combined with LIVE/DEAD fluorescent staining, which provides three-dimensional structural information alongside viability data (Azeredo et al. 2017). Furthermore, SEM analysis in the present study was restricted to A. baumannii as a representative strong biofilm-forming species, which reflects practical and resource constraints inherent to electron microscopy; the antibiofilm effects across the remaining panel of tested pathogens were comprehensively characterized by the quantitative crystal violet assay.

The cytotoxicity evaluation conducted in the present study, employing the MTT assay on Vero (African green monkey kidney epithelial) cells, demonstrated that cranberry extract exhibits a favorable mammalian cell safety profile, with an IC₅₀ value of approximately 450–500 µg/mL. Critically, effective antimicrobial and antibiofilm concentrations (MIC range: 32–512 µg/mL) were achieved at levels substantially below this cytotoxic threshold, yielding selectivity index (SI) values that range from excellent (SI = 12.5 for Streptococcus viridans and Bacillus spp.) to moderate (SI = 3.1 for MRSA and E. coli) and lower for highly resistant Gram-negative pathogens such as P. aeruginosa (SI = 0.78). The reduced SI values noted for these intrinsically resistant organisms are primarily due to their higher MIC values rather than increased cytotoxic effects of the extract. This likely reflects the barrier function of the Gram-negative outer membrane, which limits the penetration of polyphenolic compounds, rather than indicating a genuinely narrower therapeutic window. This explanation is supported by previous reviews on essential oils and plant extracts against ESKAPE pathogens, where SI values were similarly influenced by inherent Gram-negative resistance rather than by toxicity toward mammalian cells (Belete et al. 2026). Taken together, the cytotoxicity data support the potential therapeutic applicability of cranberry extract, particularly against Gram-positive and fungal pathogens, while recognizing that additional cytotoxicity assessments across a broader range of human cell lines, along with in vivo safety evaluations, are required before considering any potential translational application.

The in vivo component of the present study provided important translational corroboration of the in vitro findings in a rat oral infection model using MDR E. coli. Untreated infected animals (Group II) exhibited a marked reduction in survival to 60%, confirming the virulence of the challenge strain and the biological validity of the infection model. By contrast, cranberry extract-treated animals (Group III) demonstrated survival rates of 100%, numerically surpassing those of the gentamicin-treated positive control group (Group IV; ≈90%), although direct statistical comparison between these two treatment groups should be interpreted cautiously, given the small group size (n = 5) and the short three-day observation window. The selection of E. coli for the in vivo model is justified by its moderate in vitro MIC (128 µg/mL), clinical relevance as a cause of systemic MDR infections, and its established use in oral infection models in the literature (El-Shiekh et al. 2023). Histopathological examination of spleen tissue provided an important surrogate measure of systemic infection severity and host immune activation. The marked splenic architectural disruption, lymphoid depletion, red pulp congestion, and extensive inflammatory cell infiltration observed in the untreated infected group reflect the systemic consequences of MDR E. coli bacteremia, consistent with previously described pathological changes in rodent models of Gram-negative systemic infection. Treatment with cranberry extract was associated with partial restoration of splenic microarchitecture, reduced lymphoid depletion, and attenuated inflammatory infiltration, indicating meaningful mitigation of infection-associated tissue injury. The spleen was prioritized as a target organ for histopathological analysis based on its central role in filtering bacteremic blood and orchestrating systemic adaptive immune responses; however, the absence of parallel histological analysis of intestinal tissues represents a recognized limitation, given that the oral infection route was employed. Future studies employing this model should incorporate gastrointestinal tissue sampling to characterize local infection dynamics alongside systemic responses.

The immunomodulatory data generated in the present study constitute a particularly compelling element of the overall findings. MDR E. coli infection induced a pronounced systemic pro-inflammatory response in untreated animals, characterized by marked elevation of serum TNF-α, IL-6, IL-1β, NF-κB p105, and COX-2 relative to the negative control (p < 0.001–0.0001). Treatment with cranberry extract was associated with significant reductions in all five inflammatory mediators (2.6- to 3.3-fold relative to the untreated infected group; p < 0.05–0.01), with the greatest attenuation observed for IL-6 (3.3-fold reduction). This pattern of broad-spectrum cytokine downregulation is consistent with the capacity of polyphenolic compounds to attenuate NF-κB pathway activation, which serves as the central transcriptional regulator governing expression of TNF-α, IL-6, IL-1β, and COX-2 in response to bacterial pathogen-associated molecular patterns (Liu et al. 2017; Sun et al. 2022). NF-κB-mediated inflammatory signaling is recognized as a key driver of the hyperinflammatory state that characterizes severe MDR bacterial infections, and its pharmacological modulation has been proposed as a complementary therapeutic strategy alongside direct antimicrobial therapy (Yu et al. 2022). Immunohistochemical assessment of IL-6 expression in spleen tissue further corroborated these serum-level findings, with reduced DAB-positive staining in cranberry-treated animals compared with untreated infected controls. It is important to acknowledge, however, that the mechanisms underlying this anti-inflammatory response were not directly investigated in the present study. Whether the observed cytokine reductions reflect direct NF-κB pathway inhibition by polyphenolic compounds, indirect modulation secondary to reduced bacterial burden, or a combination of both remains to be established through dedicated mechanistic experiments including pathway-specific inhibition studies, bacterial load quantification across time points, and transcriptomic analysis of immune cell populations.

Both cranberry extract and gentamicin treatment were associated with significant improvements in survival, inflammatory biomarker profiles, and splenic histopathology relative to the untreated infected group, indicating that the protective effects of the cranberry extract are broadly comparable to those of a standard aminoglycoside antibiotic under the conditions of this model. This parallel is noteworthy, particularly given that the cranberry extract is a chemically complex, multi-constituent preparation without the nephrotoxic or ototoxic liabilities associated with aminoglycoside use. Nonetheless, these observations must be framed within the inherent limitations of the experimental design: the study employed a small sample size (n = 5 per group), a single bacterial species, a single route of infection, and a short three-day observation period. These constraints limit the statistical power and generalizability of the in vivo results, and independent replication in larger, more rigorously powered experiments - ideally using multiple MDR species, extended observation periods, and serial bacterial load quantification - will be essential before substantive claims of therapeutic equivalence can be made.

From a broader antimicrobial resistance perspective, the multitarget and pleiotropic nature of plant-derived polyphenolic preparations may confer a strategic advantage over single-target antibiotics in the development of resistance. Unlike conventional antibiotics, which exert selective pressure through inhibition of a defined molecular target and thereby create conditions highly favorable for resistance mutation or gene acquisition, polyphenolic compounds are theorized to act simultaneously on multiple cellular processes - including membrane integrity, enzymatic activity, metal ion chelation, and cell-to-cell signaling - making it intrinsically more difficult for bacteria to acquire simultaneous resistance mutations across all relevant targets (Tyers and Wright 2019). Furthermore, the demonstrated antibiofilm activity of cranberry extract at sub-inhibitory concentrations is particularly relevant in this context, as biofilm formation represents one of the principal mechanisms by which MDR pathogens persist under antibiotic pressure and serve as reservoirs for resistance gene dissemination (Sharma et al. 2025). Targeted disruption of biofilm formation without exerting bactericidal pressure could therefore represent a resistance-sparing antimicrobial strategy. Nevertheless, these potential advantages in terms of resistance dynamics remain largely theoretical in the context of the present study, and dedicated experimental validation - including serial passaging of bacteria under sub-inhibitory extract exposure and comparative assessment of spontaneous resistance frequencies -will be required to substantiate this hypothesis (Tyers and Wright 2019).

The integration of RSM-guided extraction optimization with phytochemical characterization and biological evaluation addresses a recognized methodological gap in natural product research, where inconsistent extraction procedures often undermine the reproducibility and cross-study comparability of reported biological activities (Wang et al. 2022). By establishing and reporting the precise extraction conditions that maximize proanthocyanidin yield, the present study provides a well-characterized and reproducible biological material as the basis for all subsequent assays, strengthening the internal validity of the findings. Despite these strengths, several important limitations must be acknowledged. The study employed UHPLC-PDA for phytochemical characterization; while this permitted reliable quantification of major compound classes, mass spectrometric identification was not performed, and compound assignments based solely on retention time and UV spectra should be regarded as tentative. The mechanistic basis of the observed antimicrobial, antibiofilm, and immunomodulatory activities was not directly investigated, and inferences regarding membrane disruption, efflux pump inhibition, quorum-sensing interference, and NF-κB pathway modulation remain speculative. The in vivo study was limited in sample size, duration, and scope, and the absence of gastrointestinal tissue histology and serial bacterial load quantification constrains the mechanistic interpretation of the survival and biomarker data. Future research should prioritize: (i) UHPLC-MS/MS-based structural confirmation of bioactive constituents; (ii) mechanistic in vitro studies targeting membrane integrity, efflux pump activity, quorum-sensing inhibition, and NF-κB signaling; (iii) larger, longer-duration in vivo studies with serial bacterial enumeration in target organs; (iv) pharmacokinetic and bioavailability profiling of cranberry polyphenols relevant to systemic infection; (v) evaluation of synergistic interactions between cranberry extract and clinically used antibiotics; and (vi) assessment of resistance development potential under sub-inhibitory extract concentrations. The evidence generated by such investigations will be essential for establishing whether cranberry-derived proanthocyanidins can be translated into viable therapeutic or adjunct candidates for the management of MDR infections.

Conclusion

In conclusion, the present study indicates that optimally extracted Vaccinium macrocarpon proanthocyanidins exhibit measurable antibacterial, antibiofilm, and immunomodulatory activities against clinically relevant MDR pathogens under the experimental conditions. The observed effects may be associated with multiple biological processes, including modulation of microbial growth, biofilm formation, and inflammatory responses; however, these mechanisms were not directly investigated and should be considered speculative. The integration of RSM-guided extraction with in vitro and in vivo evaluation provides a reproducible framework for assessing the biological activity of plant-derived compounds. While the findings suggest potential applicability of cranberry-derived constituents as adjunct candidates in the context of MDR infections, they should be interpreted within the limitations of the experimental design. Therefore, further mechanistic, pharmacokinetic, and clinical studies are required to validate these observations and clarify their translational relevance.