Cuminaldehyde synergistically enhances the antimicrobial and antibiofilm potential of gentamicin: A direction towards an effective combination for the control of biofilm-linked threats of Staphylococcus aureus

Abstract

Staphylococcus aureus, a Gram-positive, coccus-shaped bacterium often causes several infections on human hosts by exploiting biofilm. This current work investigates a potential strategy to manage the threats of biofilm-linked infections by embracing a combinatorial approach involving cuminaldehyde (phytochemical) and gentamicin (antibiotic). Despite showing antimicrobial properties individually, cuminaldehyde and gentamicin could exhibit enhanced antimicrobial potential when used together against S. aureus. The fractional inhibitory concentration index (FICI = 0.36) suggested that the selected compounds (cuminaldehyde and gentamicin) offered synergistic interaction while showing antimicrobial potential against the same organism. A series of experiments indicated that the selected compounds (cuminaldehyde and gentamicin) showed substantial antibiofilm potential against S. aureus when combined. The increased antibiofilm potential was linked to the accumulation of reactive oxygen species (ROS) and increased cell membrane permeability. Additionally, the combination of the selected compounds (cuminaldehyde and gentamicin) also impeded the cell surface hydrophobicity of S. aureus, aiding in the prevention of biofilm formation. The present study also showed that combining the mentioned compounds (cuminaldehyde and gentamicin) notably reduced the secretion of several virulence factors from S. aureus. Furthermore, the current research showed that these compounds (cuminaldehyde and gentamicin) could also exhibit antibiofilm potential against the clinical strains of Methicillin-Resistant S. aureus (MRSA). Taken together, this innovative approach not only enhances the potential of existing standard antibiotics but also opens up new therapeutic possibilities for combating biofilm-related infections.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-025-01628-7.

Introduction

The rise of antibiotic resistance, particularly among biofilm-forming bacteria poses a critical global health threat [1, 2]. Biofilms are structures comprising bacterial cells embedded in a self-produced slimy matrix known as extracellular polymeric substances (EPS), which aid in the adherence of the biofilm structure to biotic and abiotic surfaces [3]. Staphylococcus aureus, a common opportunistic Gram-positive pathogenic bacterium exhibits resistance to multiple antibiotics. It is commonly found on skin and mucous membranes and linked with chronic infections such as surgical wound infections, blood wound infections, endocarditis, osteomyelitis, meningitis, pneumonia [4]. Staphylococcal infections are rising globally due to the increased ineffectiveness of treatment options [4, 5]. Considering this substantial healthcare burden and mortality rate associated with these persistent infections highlights the urgent need for effective countermeasures. In this scenario, managing the biofilm-linked threats of S. aureus is essential due to their immense impact on public healthcare [5]. The limitation of using mono-antibiotic therapy highlights the need of exploring strategies for the effective management of Staphylococcal biofilm [6]. In this regard, literature reported that combinatorial applications have proven to be a promising approach for better management of microbial biofilm-linked infections [7]. Natural compounds, including bioactive essential oils, have been found to augment the antimicrobial and antibiofilm potential of antibiotics [7]. Hence, in the current study, cuminaldehyde, obtained from cumin seeds (Cuminum cyminum) has been combined with gentamicin (antibiotic) to test the potential of these compounds (either separately or together) in managing the biofilm threats of S. aureus. The results suggested that the effective combinatorial application of cuminaldehyde and the gold standard antibiotic (gentamicin) could provide avenues for the management of Staphylococcal biofilm-associated threats.

Materials and methods

Microbial strain, growth media, and culture conditions

S. aureus (MTCC 96) was used as the test microorganism in this study. The organism was grown in sterile Tryptone soya broth (TSB) (Himedia, India) for 24 h at 37℃. Cuminaldehyde (product number: 135178) and gentamicin (product number: 37636) were purchased from Sigma-Aldrich and Sisco Research Laboratories, respectively. The stock solutions of cuminaldehyde (10 mg/mL) and gentamicin (10 mg/mL) were prepared by using dimethyl sulfoxide (DMSO) and sterile double-distilled water, respectively. In line with the experimental requirements, stock solutions of cuminaldehyde and gentamicin were subsequently diluted in the sterile TSB media.

Determination of minimum inhibitory concentration (MIC)

The MIC of cuminaldehyde and gentamicin was determined against S. aureus by using the broth dilution method [8]. In this regard, a similar number of bacterial cultures (1 × 10⁸ CFU/mL) were separately exposed to cuminaldehyde (30, 50, 100, 200, and 300 µg/mL) and gentamicin (0.5, 1, 2 and 5 µg/mL) in 5 mL of sterile TSB. A control group was also prepared in which cells were not subjected to any of the mentioned compounds. After an incubation of 24 h at 37 °C, the optical density (OD) was estimated at 600 nm using a Systronics µC Colorimeter 115.

Fractional inhibitory concentration index (FICI) determination

The interaction between cuminaldehyde and gentamicin was determined by calculating the FICI score via checkerboard analysis [9]. S. aureus cells (1 × 10⁸ CFU/mL) were allowed to grow in autoclaved TSB media along with various combinations of cuminaldehyde (0, 30, 50, 100, 150, 200, and 300 µg/mL) and/or gentamicin (0, 0.5, 1, 2 and 5 µg/mL) at 37 °C for 24 h. Thereafter, the following formula was used to calculate the FICI.

$$\begin{array}{c}\mathrm{FICI}={\mathrm{FICI}}_{\mathrm{of}\mathrm{cuminaldehyde}}+{\mathrm{FICI}}_{\mathrm{of}\mathrm{gentamicin}}\hfill \\ {\mathrm{FICI}}_{\mathrm{of}\mathrm{cuminaldehyde}}={\mathrm{MIC}\mathrm{of}\mathrm{cuminaldehyde}}_{\mathrm{in}\mathrm{combination}}/{\mathrm{MIC}\mathrm{of}\mathrm{cuminaldehyde}}_{\mathrm{alone}}\hfill \\ {\mathrm{FICI}}_{\mathrm{of}\mathrm{gentamicin}}={\mathrm{MIC}\mathrm{of}\mathrm{gentamicin}}_{\mathrm{in}\mathrm{combination}}/{\mathrm{MIC}\mathrm{of}\mathrm{gentamicin}}_{\mathrm{alone}}\hfill \end{array}$$ 1

FICI ≤ 0.5 indicates synergistic impact (the combination of compounds is more effective than the individual compounds alone). FICI > 0.5 and ≤ 1 indicate additive impact (the combination is as effective as the individual compounds alone). FICI > 1 and ≤ 4 indicate indifferent interaction between two compounds (there are no such combinatorial interactions between those two compounds). FICI > 4 indicates an antagonistic impact (the combination is less effective than the individual compounds alone).

Analysis of microbial biofilm inhibition

This study sought to estimate the microbial biofilm inhibition of S. aureus under the influence of selected sub-MIC dosages of cuminaldehyde (30 µg/mL) and/or gentamicin (0.3 µg/mL) by following a series of well-established biofilm estimation assays. These included the crystal violet (CV) assay, time-lapse biofilm formation, analysis of the microbial adherence through light microscopy, estimation of total biofilm protein, measurement of extracellular polysaccharides, and the analysis of extracellular DNA (eDNA) profile. To perform these experiments, S. aureus cells (10⁸ CFU/mL) were inoculated into different test tubes containing sterile TSB media {either challenged with the test compounds (individually as well as in combination) or left untreated}. Post incubation (24 h at 37 °C), the following experiments were carried out to analyze the extent of biofilm formation under different conditions.

CV assay

The CV assay was performed to estimate the amount of biofilm formation by S. aureus under both treated and untreated growth conditions [9]. Specifically, planktonic cells were discarded, and tubes were washed with sterile double-distilled water, air-dried, and incubated with 0.4% (v/v) CV solution for 30 min. Furthermore, after washing out the unbound CV solution, the CV-bound biofilm was dissolved in 33% glacial acetic acid, vortexed, and the OD was measured at 630 nm.

Analysis of time-lapse biofilm formation

A time-lapse biofilm formation assay was carried out to analyze the Staphylococcal biofilm development over time under both treated and untreated culture conditions. To address the same, the CV assay was performed at 4 h time intervals to measure the extent of Staphylococcal biofilm formation over a 24 h time period [10, 11].

Analysis of microbial adherence through light microscope

To analyze the extent of microbial colonization under different treatment conditions, the coverslips were retrieved from both compound-exposed and unexposed culture conditions. After that, the collected coverslips were washed with sterile double-distilled water, stained with 0.4% (v/v) CV solution, and air-dried for 30 min. Furthermore, these coverslips were visualized under a light microscope.

Estimation of total biofilm protein

To estimate the total biofilm protein, adhered biofilm cells under both treated and untreated culture conditions were aseptically scrapped and treated with 5 mL of 0.3 M NaOH, followed by boiling the test tubes at 100 °C for 30 min. After centrifugation (8000 rpm for 10 min), the supernatant containing the total biofilm protein was collected. Finally, the supernatant was subsequently combined with the Bradford reagent in a 4:1 ratio. Following incubation (room temperature for 30 min), the OD was measured at 595 nm to estimate the total biofilm protein.

Measurement of extracellular polysaccharides

The phenol–sulphuric acid method was employed to measure the amount of extracellular polysaccharides of the biofilm matrix under different culture conditions [12]. The extracellular polysaccharides of the biofilm were extracted from both compounds treated and untreated sets by following the protocol of Chakraborty et al. 2020 [12]. Furthermore, the collected extracellular polysaccharides underwent hydrolysis and condensation by treating them with phenol and concentrated sulphuric acid in a 2:1:5 ratios. Then, the suspension was subsequently boiled at 100 °C for 10 min to facilitate color development. Finally, the OD was measured at 490 nm to measure the extracellular polysaccharide content.

Analysis of extracellular DNA (eDNA) of S.aureus

The protocol described by Das et al. (2024) was followed to analyze the effect of the sub-MIC dosages of the selected compounds on the eDNA profile of S. aureus [13]. The biofilm cells were scrapped from both treated and untreated culture conditions and centrifuged at 8000 rpm for 10 min. After that, supernatant was collected, and the eDNA present in supernatant was analysed through an agarose gel electrophoresis [13].

Analysis of the underlying mechanisms of biofilm inhibition of S.aureus under the influence of cuminaldehyde and gentamicin

A series of biochemical assays were conducted to elucidate the mechanisms behind the microbial biofilm inhibition of S. aureus under different treatment condition. To elucidate the same, similar number of cells (1 × 10⁸ CFU/mL) was inoculated into TSB media challenged with cuminaldehyde (30 µg/mL) and gentamicin (0.3 µg/mL) (both individually as well as in combination). After the required incubation, several experiments like the estimation of intracellular reactive oxygen species (ROS) generation, assessment of cell membrane permeability, determination of cell surface hydrophobicity, analysis of cell auto-aggregation, and analysis of sliding motility were carried out by following the protocols outlined below.

Measurement of intracellular reactive oxygen species (ROS) generation

The intracellular ROS accumulation was estimated by using the DCFDA (2’, 7’-dichlorodihydrofluorescein diacetate) assay [14]. Post incubation (24 h at 37ºC), cells from both treated and untreated growth conditions were collected, supplemented with the DCFDA solution (1:2000 dilution) and incubated for 30 min at 37ºC. In a separate set, ascorbic acid (50 µg/mL) (a well-established ROS quencher) was added along with the selected combinatorial dosages of the test compounds. Finally, the amount of intracellular ROS accumulation was quantified using a fluorescence spectrophotometer (excitation 488 nm and emission 535 nm) [14].

Estimation of cell membrane permeability

The ethidium bromide (EtBr) influx method was employed to determine the membrane permeability of the test organism under different treatment conditions [10]. In this regard, similar number of cells were collected from both treated and untreated growth conditions, treated with EtBr (0.5 µg/mL) solution and incubated at 37 °C for 30 min. After that, the fluorescence intensity (if any) was measured by using a fluorescence spectrophotometer (excitation 520 nm and emission 590 nm) [10].

Determination of cell surface hydrophobicity

The cell surface hydrophobicity of S. aureus was estimated by measuring the OD of cell suspensions before and after chloroform treatment [15]. In this regard, an equivalent number of cells were collected from different treatment conditions followed by centrifugation at 8000 rpm for 10 min. The collected cell pellets were resuspended in sterile double-distilled water (3.4 mL), and the OD was recorded at 420 nm (initial OD). Then, chloroform (0.6 mL) was added to the cell suspension and incubated at room temperature for 30 min. After phase separation, the OD of the aqueous phase was measured at 420 nm (final OD). Finally, the cell surface hydrophobicity was estimated by adhering to the formula:

$$\mathrm{Cell}\mathrm{surface}\mathrm{hydrophobicity}=\{(\mathrm{initial}\mathrm{OD}-\mathrm{final}\mathrm{OD})/\mathrm{initial}\mathrm{OD}\}\times 100$$

Analysis of auto-aggregation profile of S.aureus

The auto-aggregation profile of S. aureus under different treatment conditions was analyzed by following the protocol stated by Askoura et al. (2022) [16]. In this regard, similar number of cells were collected from different experimental sets and centrifuged at 8000 rpm for 10 min. The cell pellets were re-suspended in sterile PBS (20 mM) and incubated for 20 h at 37 °C. Following incubation, the OD of the upper phase was recorded at 600 nm using a UV–Vis spectrophotometer [16].

Analysis of sliding motility

Equivalent number of cells collected from both treated and untreated conditions was spot inoculated onto the middle of the semi-solid agar plates (30 g/L tryptone soya broth, 5 g/L glucose, and 8 g/L agar). Following incubation (24 h at 37ºC), motility was assessed by measuring the diameter (mm) of the growth zone of the bacteria from the inoculation point [13, 17].

Analysis of virulence factors

Virulence factors (measurement of metabolic activity, proteolytic activity, Staphyloxanthin production, hemolytic activity, and the fibrinogen clumping ability) of S. aureus under the influence of cuminaldehyde (30 µg/mL) and gentamicin (0.3 µg/mL) (individually as well as in combination) was analyzed by inoculating similar number of S. aureus cells (10⁸ CFU/mL) into TSB media challenged with different concentrations of cuminaldehyde and gentamicin. After that, all the sets were incubated for 24 h at 37ºC.

Estimation of metabolic activity

Microbial metabolic activity of the test organism in treated (both combination as well as individually) and untreated conditions was conducted by following the fluorescein diacetate (FDA) hydrolysis test [10]. After the incubation (24 h at 37ºC), a similar number of biofilm cells from each condition were collected by scrapping with sterile PBS (pH 7.4), and supplemented with FDA (5 mg/mL) solution. Post incubation (4 h at room temperature), each set was centrifuged (8000 rpm for 12 min), and the amount of fluorescein generated in each condition was estimated by measuring the OD of supernatant at 490 nm.

Analysis of proteolytic activity

Azo-casein degradation assay was carried out to analyze the proteolytic activity of the cells [18]. In this regard, cells were collected from both treated and untreated growth conditions, and centrifuged for 10 min at 8000 rpm. The obtained supernatant was further mixed with 500 µL of a 0.3% (w/v) azo-casein solution. After 1 h of incubation at 37 °C, trichloroacetic acid (10%) was added to stop the reaction. The reaction mixture was further centrifuged at 10,000 rpm for 5 min, and the OD of the supernatant was measured at 440 nm.

Estimation of Staphyloxanthin production

Staphyloxanthin, a virulent pigment produced by S. aureus was estimated under different treatment conditions. In brief, cells were collected from each treatment conditions and subjected to centrifugation twice at 5000 rpm for 5 min. Afterwards, the resulting cell pellets were dissolved in 420 µL of methanol. Following incubation (55 °C for 5 min), the cell suspensions were centrifuged at 12,000 rpm for 5 min. Then, methanol was added to the supernatant to achieve a final ratio of 7:13 followed by the measurement of the OD at 465 nm [19].

Hemolysis assay

Hemolysis assay was conducted to evaluate the hemolytic activity of S. aureus biofilm cells under the treated (cuminaldehyde and/or gentamicin) and untreated conditions [20]. Biofilm cells from each condition were harvested, centrifuged at 8000 rpm for 10 min at 4 °C, and cells were resuspended in cold PBS to achieve a final ratio of 1:12. Then, the cell suspension was mixed with goat blood (collected from a slaughterhouse) to prepare a suspension of 1:1 (cell suspension: goat blood). The cell suspension was maintained at 37 °C for 1 h, after which the OD of each sample was measured at 650 nm to determine the erythrocyte density in all groups. After completing the experiment, any leftover blood was disposed off at the slaughterhouse.

Analysis of fibrinogen clumping ability

Biofilm cells were collected from the treated and untreated groups by scrapping with sterile double-distilled water. The cells were subsequently suspended in 3 mL of sterile cold PBS, together with 1.25% (v/v) plasma derived from the goat blood. During the 2 h incubation period, the OD of the upper portion of the cell suspensions was measured at 600 nm every 30 min. Subsequently, the fibrinogen clumping ability of the Staphylococcal biofilm cells was determined using the formula given below:

$$\%\mathrm{of}\mathrm{clumping}=100\times \{({\mathrm{OD}600}_{\mathrm{t}=0}-{\mathrm{OD}600}_{\mathrm{t}})/{\mathrm{OD}600}_{\mathrm{t}=0})\}$$

Analysis of the effect of cuminaldehyde and gentamicin against clinical isolates of MRSA

The antimicrobial potential of either cuminaldehyde or gentamicin or their combination against the clinical isolates of MRSA (MRSA 1–7) was analyzed through a checkerboard assay by following the protocol described above. The FICI score was calculated to study the interaction between the said compounds. Furthermore, efforts were given to understand the biofilm-inhibiting and disintegrating potential of the compounds (either individual or in combination) against the clinical isolates of MRSA by performing CV assay, total biofilm protein estimation assay and FDA assay (protocol explained above). The non-biofilm cells (planktonic and disintegrated cells) under both treated (cuminaldehyde and/or gentamicin) and untreated conditions were estimated by measuring the viable cell counts (CFU/mL) [10].

Analysis of the potential of the test compounds in controlling the biofilm development on catheter tubes

Cells were grown in sterile TSB media along with different sub-MIC dosages of the compounds (cuminaldehyde or gentamicin) either alone or in combination. To it, equal number of sterile catheter tubes (each measuring 0.3 cm in width and 1 cm in length) were added. All the tubes were then incubated at 37 °C for 24 h. After the incubation, catheter tubes were collected from the experimental sets and the extent of biofilm developed on the catheter tubes were analyzed through several experiments such as CV assay, estimation of extracellular polysaccharides, and total protein estimation by following the protocol mentioned above. Besides, the disintegration of pre-existing biofilm was also carried out on the catheter tubes. In this regard, microbial biofilm of clinical isolates of MRSA was developed on catheter tubes by growing cells for 24 h at 37ºC. Post incubation, catheter tubes were collected from each set and subsequently treated with different sub-MIC dosages of cuminaldehyde and/or gentamicin for 6 h at 37ºC. After that, the previously mentioned experiments were conducted to assess the residual biofilm on catheter surface.

Statistical analysis

To perform all the statistical analyses for the current research, a one-way analysis of variance (ANOVA) was utilized to determine if there were any significant differences between the groups. Following the ANOVA, Tukey’s honestly significant difference (HSD) test (post-hoc analysis) was performed to compare the mean value of each experimental group with the mean value of the control group across three repetitions of the test. The error bars indicated the standard deviation of each experiment. Statistical significance was indicated with p-values: p-values < 0.05 were noted with (*), < 0.01 with (**), and < 0.001 with (***) when compared to the control group. The p-values which were greater than 0.05 denoted as N.S. (no statistical difference).

Result and discussion

Combination of cuminaldehyde and gentamicin showed promising antimicrobial activity against S.aureus

The opportunistic pathogen S. aureus can cause a range of infections and has acquired resistance to antibiotics due to its biofilm-forming ability [10]. These healthcare-associated challenges have inspired researchers to investigate effective combinatorial applications involving antibiotics with natural phytochemicals. As the resistance to well-established antibiotics continues to rise, leveraging the properties of cuminaldehyde alongside antibiotics may herald a pivotal advancement in managing bacterial infections. Cuminaldehyde has been reported to augment the potential of ciprofloxacin in addressing infections caused by S. aureus and E. coli [21]. Cuminaldehyde has demonstrated substantial effectiveness against Pseudomonas aeruginosa when used in combination with aminoglycoside antibiotics [7, 9]. In this regard, gentamicin emerges as a promising option for use in combination with cuminaldehyde against the test organism. Therefore, the objective of this present study is to evaluate the antimicrobial potential of cuminaldehyde and gentamicin (separately as well as in combination) against S. aureus. The MIC of cuminaldehyde and gentamicin was found to be 300 µg/mL and 5 µg/mL, respectively (Fig. 1A and B). The experimental findings revealed that the combination of 50 µg/mL of cuminaldehyde and 1 µg/mL of gentamicin was found to show significant antimicrobial potential against S. aureus (Fig. 1C). Notably, a sixfold decrease in the required concentration of cuminaldehyde and a fivefold decrease in the concentration of gentamicin were noted when the compounds were used together. To elucidate the interaction between cuminaldehyde and gentamicin, a checkerboard assay was conducted following standard procedures. Consequently, the FICI score between the selected compounds was calculated to be 0.36 (< 0.5), signifying a prominent synergistic effect. This implies that the combined use of these compounds (cuminaldehyde and gentamicin) could serve as an effective strategy to address the microbial infections caused by S. aureus.

Fig. 1.

Gentamicin and cuminaldehyde showed significant antimicrobial activity against S. aureus. A MIC determination of cuminaldehyde. The microbial growth of S. aureus in the presence and absence of cuminaldehyde was estimated by measuring the optical density (OD) at 600 nm. B MIC determination of gentamicin. The microbial growth of S. aureus under the influence of gentamicin was estimated by recording OD at 600 nm. Each experiment was repeated three times, using error bars to indicate the standard deviation of the mean. The statistical analysis was performed by using one-way ANOVA and the Tukey’s HSD post-hoc test to compare the control groups to the antimicrobial treatment groups. Significant differences were indicated by asterisks on p-values, whereas p-values > 0.05 were labelled as N.S. (no significant difference), and p-values < 0.01 and < 0.001 were denoted as ** and ***, respectively. C Checkerboard assay. A checkerboard was constructed to analyse the interaction between gentamicin and cuminaldehyde. A clear box denoted combinations of the two compounds exhibited visible microbial growth, while the grey boxes indicated no visible growth

Sub-MIC dosages of gentamicin and cuminaldehyde showed biofilm inhibition against S.aureus

Bacterial biofilm constitutes an enormous challenge in public healthcare management due to its complex structure. Furthermore, biofilm structure confers the staggering antimicrobial resistance to these microbial communities [5, 22, 23]. This intrinsic resistance complicates the treatment of numerous infections such as cystic fibrosis and chronic wounds, among others [23]. To address this pressing issue, several combinatorial approaches are being explored that strategically influence the synergistic effects of antibiotics in combination with natural compounds. Such combinatorial strategies oftentimes yield promising outcomes in mitigating biofilm-associated infections [24]. Cuminaldehyde, a plant-derived compound, was selected for this research due to its considerable ability to inhibit biofilm formation when used alongside traditional antibiotics [25]. Conversely, gentamicin has also demonstrated notable antibiofilm effects, specifically in hindering the initial adhesion of bacteria to surfaces, a crucial step in biofilm formation [26]. Prior research indicated that a combinatorial approach employing gentamicin alongside temporin A and short lipopeptides has successfully targeted S. aureus biofilms, demonstrating their potential to disrupt these complex architectures in an effective manner [27]. In light of the above discussion, our present research aims to investigate the combined efficacy of cuminaldehyde and gentamicin in combating Staphylococcal biofilms.

Preliminary investigations of light microscopy revealed that the application of cuminaldehyde and gentamicin led to a noteworthy reduction in microbial colonization compared to untreated control sets (Supplementary Fig. 1). The result suggested that the reduction in biofilm formation occurred without negatively impacting the growth profiles of S. aureus cells (Supplementary Fig. 2A and 2B). To further corroborate these observations, a series of experiments were conducted in connection to the biofilm inhibition studies. The findings indicated that cuminaldehyde at a concentration of 30 µg/mL inhibited biofilm formation by approximately 13% (Fig. 2A). Gentamicin at a concentration of 0.3 µg/mL also demonstrated notable impact, achieving a reduction of ~ 19% (Fig. 2A). When these two compounds were combined, however, the resulting reduction in Staphylococcal biofilm formation was markedly enhanced, reaching approximately 55% (Fig. 2A). The time-lapse analysis of biofilm formation by S. aureus indicated that the combinatorial treatment considerably inhibited biofilm formation of S. aureus (Fig. 2B). To support these results, a total biofilm protein estimation assay was conducted, which acted as an indirect indication of the microbial biomass within the biofilm, as the amount of total protein recovered increases along with the growth of microbial biofilm. The results demonstrated that gentamicin and cuminaldehyde could separately show a notable decrease in the total protein of S. aureus biofilm (Fig. 2C). However, when these compounds were used in combination, they led to a greater decrease in total protein content, approximately 57% relative to the control (Fig. 2C). Extracellular polysaccharides, a vital structural component of biofilm matrix, were also assessed to analyze the biofilm inhibition under the influence of cuminaldehyde and gentamicin. It was observed that the extracellular polysaccharide content of the Staphylococcal biofilm was diminished by ~ 57% upon treatment with the combined application of cuminaldehyde and gentamicin, compared to the untreated control group (Fig. 2D). The reduction in the amount of extracellular polysaccharide was anticipated to correlate with a reduction in the total amount of extracellular DNA (eDNA), which functions as another structural element of the EPS matrix and is crucial for maintaining the integrity of the biofilm [28, 29]. It was predicted that a decrease in the total amount of eDNA, another structural component of the EPS matrix would coincide with a decrease in the amount of extracellular polysaccharide. To test the same, the eDNA was isolated from the treated and untreated microbial cultures and analyzed through an agarose gel electrophoresis. The results showed a significant reduction in band intensity linked to eDNA in the group that received the combined treatment, signifying a notable decrease in eDNA content (Fig. 2E). This reduction endorses the efficacy of combining cuminaldehyde and gentamicin in inhibiting the development of S. aureus biofilm formation. Taken together, our findings emphasize the synergistic effects of cuminaldehyde and gentamicin as innovative therapeutic strategies that considerably control the biofilm-associated infections. These results necessitate further investigation into the potential applications of this combinatorial approach in clinical settings. Furthermore, the efforts were targeted to investigate the underlying cause of biofilm inhibition of S. aureus under the influence of the combination of the selected compounds. Previous studies indicated that the accumulation of ROS in bacteria could show the biofilm inhibition by affecting the cellular biomolecules like proteins, lipids and nucleic acids [30]. The results indicated that intracellular ROS accumulation was increased when treated with either gentamicin or cuminaldehyde (Fig. 3A). However, compared to the control, the amount of ROS accumulation increased by ∼100% when gentamicin (0.3 µg/mL) and cuminaldehyde (30 µg/mL) were applied together (Fig. 3A). Ascorbic acid (50 µg/mL), recognized for its ability to scavenge ROS, was capable of decreasing the level of ROS that accumulated as a result of exposure to cuminaldehyde and gentamicin (Fig. 3A). ROS buildup can affect the bacterial cell membrane, possibly impacting elements like proteins and lipids [31]. In line with this, an EtBr influx assay was carried out to analyze the cell membrane permeability of both treated and untreated sets of S. aureus. The experimental observations revealed that the combination of cuminaldehyde and gentamicin effectively increased the cell membrane permeability of S. aureus by ∼65% compared to untreated cells (Fig. 3B). This enhanced permeability of cell membrane and the buildup of intracellular ROS could aid in the prevention of microbial biofilm formation (Supplementary Fig. 3). The effect of the chosen compounds on cell surface hydrophobicity, auto-aggregation of cells, and microbial motility was also evaluated, as these factors can greatly influence the formation of microbial biofilms [10]. Cell surface hydrophobicity and cellular auto-aggregation were reported to play a critical role in the early stages of biofilm formation [10]. The result revealed that the combination of cuminaldehyde and gentamicin led to a significant reduction of both cell surface hydrophobicity and auto-aggregation by ∼50% and ∼60%, respectively (Fig. 3C and 3D). To test the effect of the combination on microbial motility, the sliding motility of S. aureus was analyzed under different conditions [32]. The results showed that the microbial motility was reduced by ∼55% when the cells were treated with cuminaldehyde and gentamicin together (Fig. 3E). Taken together, these findings collectively demonstrated that cuminaldehyde in combination with gentamicin could effectively target multiple cellular biochemical mechanisms, including the buildup of intracellular ROS, alterations in cell membrane permeability, changes in cell surface hydrophobicity, cell auto-aggregation, and the motility of microorganisms during biofilm inhibition. This multifaceted approach holds potential for developing novel therapeutic strategies to combat biofilm-associated infections by targeting key cellular processes using the combinatorial application of cuminaldehyde and gentamicin.

Fig. 2.

Cuminaldehyde and gentamicin could synergistically forestall biofilm formation of S. aureus. A similar number of S. aureus cells were allowed to grow under the influence of selected sub-MIC dosages of cuminaldehyde and gentamicin. After incubation, a series of experiments were performed to estimate the number of cells adhered to the surfaces. A CV assay. The bacterium, challenged with the test compounds in combination, was subjected to the CV assay after 24 h incubation. B Analysis of the time-lapse biofilm formation. The CV assay was used to quantify the amount of biofilm developed by S. aureus at regular time intervals up to 24 h. C Estimation of total biofilm protein. The amount of bacterial biofilm protein under different treatment conditions was estimated using the Bradford method. D Estimation of the extracellular polysaccharides. The amounts of extracellular polysaccharides in S. aureus biofilm under the influence of the selected compounds were measured by the phenol–sulphuric acid method. All experiments were conducted three times, and the error bars displayed the standard deviation of the mean. To determine significant differences between control and treated groups, a one-way ANOVA was conducted followed by Tukey HSD post-hoc test. Asterisks against the p-values indicated significant differences, while *, **, and *** denoted p-values less than 0.05, less than 0.01, and less than 0.001, respectively. E Agarose gel electrophoresis. An agarose gel electrophoresis was performed to analyze the band intensity of the eDNA under treated and untreated conditions

Fig. 3.

Gentamicin in combination with cuminaldehyde could affect multiple targets to inhibit biofilm formation of S. aureus. A Intracellular ROS accumulation measurement. A S. aureus cells were recovered from both treated and untreated conditions to quantify ROS accumulation in each case by DCFDA method. B Cell membrane permeability estimation. EtBr influx method was followed to determine the permeability of the cell membrane in both the treated and untreated growth conditions. C Cell surface hydrophobicity analysis. The BATH assay was used to determine the cell surface hydrophobicity profile of S. aureus cell under both treated and untreated condition. D Microbial auto-aggregation. The aggregation profile of S. aureus cells was estimated in presence and absence of the test compounds. E Microbial motility. The zone-diameter of the bacterial motility was measured in each case, starting from the point of inoculation. Each experiment was repeated thrice. Error bars signified the standard deviation of the mean. To determine significant differences between control and treated groups, a one-way ANOVA was conducted followed by Tukey HSD post-hoc test. Significant differences were denoted by asterisks with * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively. No significant difference was observed compared to the control when the p-value was less than 0.05

Cuminaldehyde and gentamicin reduced the secretion of virulence factors

Besides leading to various chronic infections, Staphylococcal biofilm could also promote the secretion of several virulence factors [10]. In this context, FDA assay was taken into consideration as it is based on the transformation of non-fluorogenic fluorescein diacetate into fluorogenic fluorescein, making it possible to quantify the number of metabolically active cells [10]. In comparison to the untreated control group, the experimental results indicated that the selected sub-MIC dosages of cuminaldehyde and gentamicin led to a reduction of nearly 45% in the availability of metabolically active cells of biofilm (Fig. 4A). Proteases can break down the peptide bonds in proteins, which may result in tissue damage and facilitate the proliferation of microbial infections [10, 18]. Protease secretion by Staphylococcal cells embedded in the biofilm decreased by ~ 10% and ~ 18% when treated with cuminaldehyde and gentamicin, respectively (Fig. 4B). However, protease secretion was decreased by ~ 55% when both the compounds (cuminaldehyde and gentamicin) were applied together (Fig. 4B). Staphyloxanthin (a secondary metabolite that plays a key role in the pathogenicity of S. aureus) synthesis was also assessed under the presence and absence of the compounds. The results revealed that Staphyloxanthin synthesis was reduced by ~ 55% under the combined use of cuminaldehyde and gentamicin (Fig. 4C). Another virulence factor that S. aureus produces is hemolysin, which targets the host's erythrocytes to increase pathogenicity [10]. The results showed that the combined application of the test compounds could reduce hemolysin production by ~ 50% compared to untreated controls, reflecting a substantial reduction in hemolysin secretion (Fig. 4D). Furthermore, when cuminaldehyde and gentamicin were applied together, cell’s ability to form macroscopic clumps with fibrinogen was reduced by nearly 26% (Fig. 4E). In summary, these results underscore the potential of the combined treatment of cuminaldehyde and gentamicin to diminish Staphylococcal virulence factors, paving the way for new therapeutic strategies in treating infections caused by Staphylococcal biofilm.

Fig. 4.

Cuminaldehyde in combination with gentamicin reduced the secretion of the virulence factors of S. aureus. An array of experiments was performed to estimate the secretion of the virulence factors from S. aureus. A Estimation of the metabolic profile. The FDA assay was performed to determine the metabolic profile under treated and untreated growth conditions. B Protease profile analysis. The amount of protease secretion from treated and untreated growth conditions was estimated using the azo-casein degrading assay. C Quantification of staphyloxanthin. The amount of staphyloxanthin produced under various treatment conditions was estimated by measuring the extracted staphyloxanthin at 495 nm. D Erythrocyte density measurement. The amount of hemolysin secreted from treated and untreated conditions, was measured by following the methodology mentioned in materials and methods section. E Analysis the fibrinogen clumping factor. The clumping property under a range of treated and untreated conditions was estimated by following the methodology mentioned in materials and methods section. Each experiment was conducted three times. Error bars signified the standard deviation of the mean. To determine significant differences between control and treated groups, a one-way ANOVA was conducted followed by Tukey HSD post-hoc test. Significant differences were denoted by asterisks with * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively. No significant difference was observed when the p-value was less than 0.05 compare to the control set

The combination of cuminaldehyde and gentamicin showed antibiofilm potential against the clinical isolates of MRSA

MRSA is more difficult to treat since it has developed resistance to some widely used drugs. To analyze the effectiveness of the combined application of cuminaldehyde and gentamicin against the clinical isolates, seven MRSA (1–7) strains were tested. The checkerboard assay indicated that the combination of cuminaldehyde and gentamicin displayed strong synergy (with a FICI index of ≤ 0.5) in antimicrobial potential against the clinical isolates of MRSA (MRSA 1–7) (Table 1; Fig. 5A, B, C, D, E, F and G). Furthermore, the sub-MIC doses of the compounds (cuminaldehyde and gentamicin) were evaluated individually or in combination for their effectiveness against the biofilm profiles of MRSA, as biofilms can increase the resistance of MRSA-related infections to various antibiotics. Existing literature documented that biofilm-related infections could be mitigated by either inhibiting biofilm formation or disrupting the matured biofilm structure [10, 28]. Towards this direction, the results of the CV assay, total biofilm protein measurement, and extracellular polysaccharides estimation suggested that the sub-MIC dosages of cuminaldehyde and gentamicin could significantly inhibit biofilm development of all the MRSA strains (Fig. 6A). Besides, the same combination of the compounds was tested against the developed biofilm of MRSA. The results of the CV assay, together with the measurements of residual protein and extracellular polysaccharides, showed significant biofilm disintegration for all the MRSA clinical strains when exposed to the selected compounds together (Fig. 6B). Previous studies suggested that the increased biofilm disintegration could lead to the augmentation of the numbers of non-biofilm cells [10, 28]. In line with this, treatment using the combination of cuminaldehyde and gentamicin led to a notable increase (about 15% to 30%) of non-biofilm cells across all selected MRSA clinical strains compared to the control group (Fig. 6C). This further reinforced that the synergistic impact of cuminaldehyde and gentamicin was effective in disintegrating the pre-existing biofilm. This research revealed that the combined use of sub-MIC dosages of cuminaldehyde and gentamicin could manage biofilm-related challenges posed by clinical MRSA strains, by inhibiting the formation of new biofilm and disintegrating existing ones.

Table 1.

Types of interaction between cuminaldehyde and gentamicin against the clinical isolates of MRSA

Isolate No	MIC of cuminaldehyde (µg/mL)	MIC of gentamicin (µg/mL)	Type of interaction (between cuminaldehyde and gentamicin)
MRSA1	400	12	Synergistic
MRSA2	300	12	Synergistic
MRSA3	400	2	Synergistic
MRSA4	300	12	Synergistic
MRSA5	400	12	Synergistic
MRSA6	400	12	Synergistic
MRSA7	400	3	Synergistic

Fig. 5.

Checkerboard assay to determine the combinatorial potential of cuminaldehyde and gentamicin against the clinical isolates of MRSA. A Checkerboard assay of MRSA isolate 1. B Checkerboard assay of MRSA isolate 2. C Checkerboard assay of MRSA isolate 3. D Checkerboard assay of MRSA isolate 4. E Checkerboard assay of MRSA isolate 5. F Checkerboard assay of MRSA isolate 6. G Checkerboard assay of MRSA isolate 7

Fig. 6.

Combinatorial application of cuminaldehyde and gentamicin showed promising antibiofilm effects against clinical isolates of MRSA. A Biofilm inhibition profile. To determine the biofilm inhibition profile, similar numbers of the cells of the clinical strains of MRSA were subjected to grow in different treatment conditions for 24 h at 37 °C. To analyze the extent of biofilm inhibition under different conditions, a series of experiments were performed, including the CV assay, estimation of the total biofilm protein, and measurement of the extracellular polysaccharide of the biofilm matrix. B Biofilm disintegration profile. MRSA cells were cultivated for 24 h at 37 °C to establish a matured biofilm. Next, the selected dosages of cuminaldehyde and gentamicin were added to each experimental set, followed by 6 h incubation period. After that, the amount of residual biofilm in each experimental set was measured by using the CV assay, biofilm protein estimation, and measurement of extracellular polysaccharides. C Measurement of non-biofilm cells. The number of non-biofilm cells was determined under the treated and untreated conditions. Each experiment was conducted three times. Error bars signified the standard deviation of the mean. To find out significant differences between control and treated groups, a one-way ANOVA was conducted followed by Tukey HSD post-hoc test. Significant differences were denoted by asterisks with * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively. No significant difference was observed compared to the control when the p-value was less than 0.05

The combination of cuminaldehyde and gentamicin could effectively prevent microbial biofilm on catheter tubes

The enhanced antimicrobial and antibiofilm properties of the combination of cuminaldehyde and gentamicin could mark a notable step forward in controlling the unrelenting threats posed by MRSA. Catheter tubes are common causes of infections linked to healthcare settings. Hence, it highlights the critical demand of new strategies to combat MRSA biofilm threats on catheter tubes. Since the combination of cuminaldehyde and gentamicin could effectively control the formation of microbial biofilm and disintegrate the existing one, the potential of said combination (cuminaldehyde and gentamicin) were tested against the biofilm threats of MRSA on catheter tubes. In this regard, specifically the CV assay, estimation of total protein and measurement of extracellular polysaccharides were undertaken. All experimental results indicated that the combination of cuminaldehyde and gentamicin could demonstrate a promising inhibition in biofilm formation (ranging from approximately 30% to 58%) by the clinical strains of MRSA on the catheter surfaces (Fig. 7A). Besides, the experimental outcome also showed that the combination of cuminaldehyde and gentamicin could effectively disintegrate the pre-existing MRSA biofilm on catheter tubes, with efficiency ranging from ~ 30% to ~ 71% (Fig. 7B). Hence, this study indicated that the combined application of cuminaldehyde and gentamicin showed a promising synergistic effect in controlling microbial biofilm challenges on catheter tubes.

Fig. 7.

Cuminaldehyde and gentamicin effectively reduced the biofilm formation of MRSA on the catheter surface. A Analysis of biofilm inhibition. The extent of biofilm inhibition of MRSA on the catheter tubes was analyzed by following the CV assay, estimation of the amount of total biofilm protein and extracellular polysaccharides in the biofilm matrix. B Analysis of biofilm disintegration. The matured biofilm cells of MRSA were exposed to both treated and untreated conditions, and subsequently assessed them using the CV assay, protein quantification, and extracellular polysaccharide estimation. Each experiment was conducted three times. Error bars signified the standard deviation of the mean. To determine significant differences between control and treated groups, a one-way ANOVA was conducted followed by Tukey HSD post-hoc test. Significant differences were denoted by asterisks with * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively. No significant difference (N.S.) was observed when the p-value was less than 0.05 compared to the control

Conclusion

The present study explores a promising direction to mitigate the threats of Staphylococcal biofilm by utilizing cuminaldehyde in combination with gentamicin. The combination of the compounds was also found to attenuate the secretion of several virulence factors. Therefore, the combined effects of cuminaldehyde and gentamicin may suggest novel and effective therapeutic options for Staphylococcal biofilm infections, leading to enhanced clinical strategies.

Supplementary Information

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Data availability

The data associated with the manuscript shall be made available upon a reasonable request.

Declarations

Complete of interest

The authors’ declare that they do not have any conflict of interest for this work.