Microbial strains, chemicals and growth media
Five clinical strains of P. aeruginosa, obtained from Suraksha Diagnostic Centre, Kolkata, were considered as the organism of interest in the current study. The bacteria was cultivated at 37°C for 24 h in sterile Luria Bertani broth (LB; obtained from Sisco Research Laboratories). Cuminaldehyde, a natural compound (Sigma-Aldrich), and ciprofloxacin, an antibiotic (Sisco Research Laboratories), were selected as test compounds. Cuminaldehyde was dissolved in DMSO (Dimethyl Sulfoxide), while ciprofloxacin (10 mg/mL) was dissolved in MilliQ water and further diluted in LB as per the experimental requirement.

Estimating the minimum inhibitory concentration (MIC)
The minimum amount of any anti-microbials that prevents a microorganism to grow visibly is termed as the MIC value of that antimicrobial.¹¹ Here, MIC of ciprofloxacin and cuminaldehyde was estimated against the clinical strains using a 2020 protocol of Chakraborty et al.¹² In this regard, at first, equal numbers (10⁶ CFU/mL) of the test organisms were cultured in autoclaved growth media (5 mL) in which various concentrations of the natural compound, cuminaldehyde (ranging from 0 µg/mL to 600 µg/mL) and ciprofloxacin (ranging from 0 µg/mL to 15 µg/mL) were added individually. After incubation at 37°C for a period of 24 h, the extent of microbial growth was determined at 600 nm using a colorimeter.

Determining Fractional-Inhibitory-Concentration Index (FICI)
The checker board assay was conducted to determine FICI to understand the possible interactions between cuminaldehyde and ciprofloxacin. For this experiment, an equal number (10⁶ CFU/mL) of overnight inoculated cultures of all clinical isolates were further cultured into sterile growth media and subsequently treated with varying concentrations of cuminaldehyde (up to 600 µg/mL) and ciprofloxacin (up to 20 µg/mL), or left unexposed. After incubation at 37°C for a period of 24 h, microbial growth was assessed, and using the following formula the FICI was calculated:

FICI of cuminaldehyde against clinical strains = MIC (cuminaldehyde) while applied in combination / MIC (cuminaldehyde) while applied alone against clinical strains.

FICI of ciprofloxacin against clinical strains = MIC (ciprofloxacin) while applied in combination / MIC (ciprofloxacin) while applied alone against clinical strains

FICI = FIC (cuminaldehyde) + FIC (ciprofloxacin)

The interaction between the two selected compounds was calculated using the FIC index provided in established documents¹³:

When the value of FIC is ≤0.5, it is synergistic;
When the value of FIC is >0.5 and ≤1, it is additive;
When the value of FIC is >1 and ≤4, it is intermediate;
When the value of FIC is >4, it is antagonistic

Crystal-Violet (CV) assay
Assessment of bacterial biofilm using CV assay relies on the ability of the basic dye, CV, to bind to negatively charged cells and surface molecules of the extracellular polymeric substance.¹⁴ Thus, the CV staining assay is widely recognized as a conventional method for quantifying microbial biofilm formation on various surfaces.¹⁵ In this study, the degree of biofilm of both untreated and treated test organisms were analyzed using the CV assay, using the protocol adapted from Das et al.¹⁶ To proceed with the experiment,
10⁶ CFU/mL of the test bacteria were inoculated into autoclaved growth media (5 mL) and subsequently treated with 50 µg/mL of cuminaldehyde (for all five selected isolates). Ciprofloxacin was either added along with cuminaldehyde (0.3 µg/mL for PSA1; 0.1 µg/mL for PSA2; 0.01 µg/mL for PSA3 and 1 µg/mL for PSA4, PSA5) or left unexposed. Following incubation, free-living planktonic cells were removed and the adhered biofilm on the test tubes was CV-stained and kept aside for half an hour. The non-adhered CV solution was discarded post incubation, and the tubes were washed with sterile double-distilled water. Thereafter, 33% acetic acid (glacial) solution (5 mL) was added to the respective tubes to dissolve the stain adhered with the biofilm cells. Finally, OD₆₃₀ was recorded by measuring the intensity of the CV solution using a colorimeter.

Analysis of bacterial biofilm under light microscope
Microscopic images offer detailed visual presentation of biofilm formation of a microorganism under a given condition.¹⁷ Therefore, to analyze the biofilm profiles of test organisms in the absence or presence of selected doses (as mentioned previously) of test compounds, light microscopic image analysis was considered. To perform this analysis, clinical strains of P. aeruginosa (10⁶ CFU/mL) were inoculated in LB media containing coverslips that supported the formation of biofilm clusters either in the absence or presence of the chosen concentrations of test compounds (ciprofloxacin and/or cuminaldehyde) under same experimental conditions. Following overnight incubation, coverslips from each experimental set were aseptically collected from each experimental set, gently washed with MilliQ water, subsequently stained CV (0.4%) in solution form for half an hour and observed under a light microscope (Olympus CX21i).

Assessment of total biofilm protein
As an indirect measure, quantifying total biofilm protein content offers a reliable method for the relative assessment of biofilm profile.¹⁸ To this end, the Bradford assay was conducted to analyze the total biofilm protein content of all clinically isolated P. aeruginosa strains, both in the absence and presence of test compounds, using the protocol outlined by Malik et al.⁷ For this assay, cells were either challenged with selected doses of cuminaldehyde and/or ciprofloxacin (as mentioned previously), or left untreated as a control set. After incubation at 37°C for a period of 24 h, biofilm cells adhered to the tube were considered. NaOH solution (5 mL, 0.3 M) was added in each tube and boiled for half an hour to extract the total protein of the biofilm present on the glass tube surface. Then the supernatant was collected by centrifuging the solution for 10 min at 8000 rpm. Bradford reagent was mixed with the supernatant in a 1:1 ratio and kept aside for 30 min at room temperature (RT). Finally, the OD at 595 nm was recorded to measure the intensity of the color developed by the biofilm protein.

Measurement of extracellular polysaccharides (EPS)
To measure the EPS of all clinical isolates, the phenol-sulfuric acid method, as outlined by Dubois et al.¹⁹ was followed. The EPS profile, consisting of primary and major constituents, reflects the degree of biofilm formation on the given surface. For this assay, 10⁶ CFU/mL of test organism were cultivated to form biofilm on the glass surface in the presence or absence of the chosen concentrations of cuminaldehyde and/or ciprofloxacin (as mentioned previously). A control set was also prepared where 10⁶ CFU/mL of test organism was left untreated. Post incubation, free-living planktonic cells were removed, and the attached biofilm cells on the glass tube were suspended in phosphate-buffered saline (PBS; pH 7.4). This prepared suspension was then subjected to centrifugation at a rate of 6000 rpm for around 10 min. The supernatant obtained from this step was the cell-free EPS and the pellet (re-suspended in 10 mM EDTA) obtained was re-centrifuged (10 min, 6000 rpm) to extract the cell-bound EPS matrix. Then, the supernatant obtained from each of the sets was dissolved in chilled ethanol (1:2.2 ratios) for an hour at 4°C. Then, this suspension was subjected to 6000 rpm centrifugation for 20 min. The pellet obtained from this step was termed as the total EPS content. To measure the degree of EPS extracted in both treated and untreated samples, the extracted pellets were re-suspended in 1 mL of PBS and mixed with phenol and concentrated sulfuric acid in a ratio of 2:1:5 (sample: phenol: sulfuric acid), respectively. The prepared mix solution was then boiled at 100°C for 10 min and cooled for 10 min at RT. Finally, the OD at 490 nm was recorded to measure the amount of EPS in each set.

Analysis of metabolic activity
The degree of metabolically active clinical isolates of the test bacteria was measured by employing FDA hydrolysis assay.²⁰ In this regard, equal numbers of biofilm cells of the clinical strains of P. aeruginosa were either untreated or treated with the compounds and following incubation, the cells were collected by scrapping. The cells collected from the previous step were suspended in PBS (5 mL, pH 7.4). Subsequently, 500 µL (5 mg/mL) FDA in solution form was dispensed in each tube for an hour in darkness. All the prepared experimental sets were then subjected to 10,000 rpm centrifugation for a time span of 10 min. Then, fluorescein, collected from the obtained supernatant was recorded at 490 nm.

ROS accumulation estimation
Intracellular ROS accumulation was assessed for all the selected clinical isolates of P. aeruginosa. DCFDA method was employed for all the various sample conditions (treated and untreated) by following Das et al.²¹ To conduct this experiment, cells were either treated with 50 µg/mL of the natural test compound, cuminaldehyde and/or various concentrations of the antibiotic, ciprofloxacin (0.3 µg/mL for PSA1; 0.1 µg/mL for PSA2; 0.01 µg/mL for PSA3 and 1 µg/mL for PSA4 and PSA5). A separate untreated control set was prepared where only the clinical strains were grown over a similar incubation temperature for 6 h. Then, each set was supplemented with 0.5 µL of DCFDA to achieve a final ratio of 1:2000 (v/v), and incubation at 37°C for half an hour. Finally, the quantity of accumulated ROS produced under each condition was estimated at 488 nm (excitation), and at 535 nm (emission).

Estimation of cell membrane permeability by Ethidium Bromide (EtBr) influx assay
The cell membrane permeability of the clinical strains of the test bacteria was analyzed in response to the test compounds by following the EtBr influx assay.²² For this experiment, overnight-grown cells were centrifuged and the collected cell pellet (1 mL) was suspended in PBS (pH 7.4). The cell pellet was further treated with 50 µg/mL of cuminaldehyde and/or various concentrations of ciprofloxacin (0.3 µg/mL for PSA1; 0.1 µg/mL for PSA2; 0.01 µg/mL for PSA3 and 1 µg/mL for PSA4 and PSA5) at 37°C for 6 h. Following treatment, both the untreated and treated cell suspensions were supplemented with EtBr (0.5 µg/mL) and kept aside without exposure to light for 5 min. Finally, the fluorescence intensity was measured in a fluorometer (excitation at 520 nm, emission at 590 nm).

Analysis of biofilm disintegration
The disintegrating property of the clinically isolated strains of the test bacteria was assessed under the presence of the test compounds. Similar cells were permitted to develop biofilm for a period of 24 h, 37°C. Following incubation, overnight-grown biofilm was either treated with cuminaldehyde or ciprofloxacin or both at similar concentrations stated previously for a span of another 6 h. The degree of residual biofilm denoted the biofilm disintegration percent compared to the control set. Thus, this residual biofilm was subjected to various experiments (CV assay, total biofilm-protein estimation, EPS measurement and FDA assay) already discussed in previous sections.

Analysis of biofilm management on catheter tubes
To analyze the test compounds in managing microbial biofilm formation on catheter surfaces, two sets of experiments were conducted. In the first set, sterile catheter tubes measuring 1 cm in length and 0.3 cm in width were exposed to the clinical strains of the test bacteria under the presence of the selected concentrations of the natural compound, cuminaldehyde and/or the antibiotic, ciprofloxacin. In the second set, a control set was considered without any compound exposure. After similar conditions of incubation time and temperature, catheter tubes from the second set were treated with the chosen concentrations of ciprofloxacin and/or cuminaldehyde for another 6 h. Following treatment, catheter tubes from both sets were aseptically collected, and CV assay, protein estimation, and EPS measurement assay were conducted by following previously established protocol was carried out.

Statistical analysis
To statistically analyze the key observations, ANOVA, One-way analysis of variance was explored (ANOVA). The average recorded value of all the experiment(s) was estimated post a three times repetition. An error bar of each data set represents the standard deviation of each experiment in comparison to the control set. The p values less than 0.05 represented with (*), less than 0.01 represented with (**), and less than 0.001 represented with (***) in comparison to the control. Whereas, p values beyond 0.005 were denoted as N.S. (statistically no difference).