INTRODUCTION:

Biofilms are microbial communities irreversibly attached to a surface and protected by a matrix of Extracellular Polymeric Substances (EPS) that provide protection for bacteria from the body's immune system and antibiotic treatment. Biofilms are also a contributing factor to healthcare-associated infections with a percentage of about 70-80%^1–3.

Catheter-associated urinary tract infection (UTI) is one of the healthcare-associated infections that has increased significantly. Catheterization-associated UTI is the most common type of nasocomial infection with 1 million cases annually, which is equivalent to 40% of all nasocomial infections⁴.

According to Chandra et al. (2014), urine culture results from patients using urethral catheters revealed that S. aureus was the most frequently detected bacterium, with an incidence rate of 45%. This bacterium can adhere to medical device surfaces and form biofilms, positioning S. aureus as a major contributor to biofilm development on catheter surfaces^5,6. Biofilm formation on these catheters is also linked to the death of approximately 7,500 people annually⁷. Therefore, controlling biofilm is a significant challenge in clinical practice.

Turmeric (Curcuma longa Linn.) has long been used in traditional medicine for its content of curcuminoids, including demethoxycurcumin. Demethoxycurcumin is reported to have strong antimicrobial activity. Based on research conducted by Hamzah et al (2020) showed that demethoxycurcumin has the potential to inhibit microbial growth and biofilm formation, making it an interesting candidate for further study in a medical context⁸. Although many studies have examined the activity of curcuminoids against various microbes, especially curcumin, there are limited studies that specifically examine the effect of demethoxycurcumin on S.aureus biofilms. This gap offers a research opportunity to explore the potential of demethoxycurcumin in biofilm control on medical devices such as catheters. Through this research, it is hoped that a new strategy that is effective in controlling biofilm formation on catheters can be found.

MATERIALS AND METHODS:

Materials:

Materials used were Demethoxycurcumin compound from isolation of (Curcuma longa L.), Standart biofilm-forming S.aureus ATCC 25923 from the Microbiology Laboratory, Faculty of Pharmacy, UGM, Chloramphenicol, DMSO 1%, NaCL, McFarland 0.5 standard, sterile aquadest, Tryptic soy north (TSB) (Merck, Germany), PBS (Phosphate Buffer Saline) solution, catheter, and crystal violet 1% (Merck, Germany). Some equipment’s used in this research were incubator (IF-2B) (Sakura, Japan), analytical scales (AB204-5, Switzerland), autoclave (Sakura, Japan), Laminar air flow, micropipette pipetman (Gilson, France), spectrophotometry (Genesys 10UV Scanning, 335903) (Thermo Sci-entific Spectronic, USA), incubator with orbital shaker S1500 (Stuart, UK), multichannel micropipette (Socorex, Swiss), microplate flat-bottom polystyrene 24 well (Iwaki, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain).

Bacterial Strains:

The standard strain of Staphylococcus aureus (ATCC 25923) was cultured in tryptic soy broth (TSB) and incubated at 37°C for 72hours. The optical density (OD600) of the microbial culture was adjusted to 0.1, corresponding to a McFarland standard of 0.5 (~1.5 x 10^8 CFU/ml)⁹.

Catheter biofilm inhibition activity:

The impact of demethoxycurcumin on S. aureus biofilms (ATCC 25923) was evaluated through a series of steps. Initially, the catheter was cut into one-centimeter segments, sterilized with 70% ethanol, and dried. Each well of a microplate was filled with 200μL of media and incubated at approximately 37°C for 24 and 48hours. Following incubation, the plates were washed with PBS. Different concentrations of media containing pure isolates (ranging from 1% b/v to 0.125% b/v) were added to the washed wells. Controls included media with 1% ethanol as the solvent control, a microbial suspension as the negative control, and media with 1% chloramphenicol as the positive control. Media without microbial growth served as the media control. The plates were then incubated at 37°C for 24hours for intermediate phase biofilms and 48hours for mature phase biofilms, followed by another PBS wash. Subsequently, 125μL of a 1% crystal violet solution was added to each well and incubated at room temperature for 15minutes. The microplate was washed with PBS, and 200μL of 96% ethanol was used to dissolve the biofilm. Optical density (OD) measurements were taken with a microplate reader at 595nm⁹.

Catheter biofilm eradication activity:

This method is similar to the biofilm inhibition process on the catheter, with the key difference being that the testing duration was extended to 6 days for catheter biofilm degradation, following the approach adapted from Hamzah et al. (2020) and Hola and Ruzick (2011)^8,10. Biofilms were inoculated into microplates following the described procedure. After incubation at 37°C for 48 hours, the culture in each well was discarded, and planktonic cells were removed by washing with PBS. The biofilm cells were then treated with demethoxycurcumin at various concentrations, ranging from 1% b/v to 0.125% b/v, and incubated again at 37°C for 48hours. Chloramphenicol at 1% b/v was used as a positive control. Following incubation, the plates were washed three times with 200mL of sterile PBS to remove any adherent cells. Biofilm degradation was assessed by adding 125μL of 1% crystal violet solution to each well and incubating at room temperature for 15 minutes. The microplate was then washed with PBS, and 96% ethanol was added to each well to dissolve the biofilm. Optical density (OD) measurements were conducted using a microplate reader at a wavelength of 595nm.

Scanning electron microscopy (SEM) study:

The catheter was inserted into a 24-well polystyrene microtiter plate with a round bottom, containing the test suspension that was prepared similarly to the biofilm inhibition assay. Following this, the catheter was incubated at 37°C for 24 to 48hours. The catheter was then thoroughly rinsed three times with sterile distilled water and fixed with a 2.5% (v/v) glutaraldehyde solution in kakodilate buffer for about 24hours to preserve cell structure while killing the cells. Subsequently, the catheter underwent a dehydration process with methanol for 30 minutes to minimize water content, ensuring no disruption to the observation. Finally, the samples were examined using scanning electron microscopy (SEM) at 10 kV⁹.

Statistical Analysis:

Statistical analysis starts with testing for normality and data homogeneity. If the normality test yields a p-value <0.05, indicating that the data is not normally distributed, the data is then analyzed using One Way Anova. This is followed by the Bonferroni post-hoc test to compare the control group with the treatment group, with a significance threshold set at p<0.005.

RESULT:

Antibacterial Effect of Demethoxycurcumin Against S.aureus:

The antibacterial activity of demethoxycurcumin against S.aureus showed an inhibitory activity of (88.17%± 0.01). In comparison, chloramphenicol 1% had an inhibitory activity of (86.23%±0.01) (Figure 1).

Figure 1. Antibacterial Effect of Demethoxycurcumin Against S.aureus

This finding indicates that demethoxycurcumin is slightly more effective against S.aureus compared to the positive control chloramphenicol. This indicates that demethoxycurcumin has strong antibacterial activity and potential to be developed into a new antibiofilm candidate.

Inhibitory Activity of Demethoxycurcumin on Mid Phase (24hours) S.aureus Biofilm on Catheter:

In this study, we assessed the potential of demethoxycurcumin as an antibiofilm in inhibiting S.aureus biofilm on catheters. The results showed that demethoxycurcumin with a concentration of 1% had an activity of (81.72%±0.01) while the positive control had an inhibitory activity of (81.63%±0.01) (Figure 2).

Figure 2. Inhibitory Activity of Demethoxycurcumin on Mid Phase (24hours) S.aureus Biofilm on Catheter

The results of this study showed that demethoxycurcumin was able to inhibit up to 50% of S.aureus biofilm growth on catheters with demethoxycurcumin being slightly more effective than the positive control chloramphenicol. This finding provides evidence that demethoxycurcumin is more effective in inhibiting S.aureus biofilm growth and has the potential to be used as an antibiofilm candidate in catheters.

Inhibitory Activity of Demethoxycurcumin on Maturation Phase (48 hours) S.aureus Biofilm on Catheter:

In the Maturation phase (48hours), the compound demethoxycurcumin 1% showed inhibition of (61.48%± 0.01), while the positive control chloramphenicol 1% gave inhibition of (79.49%±0.01) (Figure 3).

Figure 3. Inhibitory Activity of Demethoxycurcumin on Maturation Phase (48 hours) S.aureus Biofilm on Catheter

This data shows that the inhibitory activity of demethoxycurcumin decreased in the 48hour phase. Nevertheless, demethoxycurcumin was still able to inhibit biofilm growth well, reaching more than 50%. These results suggest that despite the decrease in inhibitory activity, demethoxycurcumin is still effective in controlling the growth of S.aureus biofilm on catheters.

Inhibitory Activity of Demethoxycurcumin on Eradication Phase S.aureus Biofilm on Catheter:

The eradication phase is the longest phase compared to the mid-phase (24hours) and maturation (48hours), even so the demethoxycurcumin compound is still able to provide inhibitory activity of more than 50%, namely (60.16% ±0.01), while chloramphenicol shows higher inhibitory activity with a value of (71.10% ±0.01) (Figure 4).

Figure 4. Inhibitory Activity of Demethoxycurcumin on Maturation Phase (48 hours) S.aureus Biofilm on Catheter

In this phase, the 1% demethoxycurcumin compound experienced a decrease in inhibitory activity. This is because the matrix structure formed on the catheter is more numerous, complex and has a strong defense, making it difficult for demethoxycurcumin compounds to penetrate this structure. Not only that, in this phase, biofilm structures form communities and communicate between cells known as quorum sensing⁹.

Scanning Electron Microscopy (SEM) Results of S.aureus Biofilm on Catheter Without Treatment and With Treatment:

In the SEM test results below, the S. aureus biofilm on the untreated catheter showed very high cell density and significant buildup, and produced a very thick EPS matrix (Figure 5 A).


(A)	(B)

Figure 5. SEM image A) Without Treatment B) With Treatment

In the SEM image (figure 5 A), S.aureus is seen forming a highly structured and complex biofilm on the catheter surface. These bacteria adhere to and alter the catheter surface by blocking receptor areas for uropathogens^9-11. As a result, it is difficult for a compound to provide maximum inhibition because the thickness of the EPS matrix produced is able to protect the biofilm on the catheter well. This complex biofilm structure, starting with S.aureus coordinates biofilm formation and virulence factor expression through Quorum Sensing to enhance their ability to survive on specific surfaces^10–15.

In the SEM image (Figure 5 B), it shows that demethoxycurcumin is proven to be able to disrupt S.aureus biofilms by causing a decrease in cell attachment and density, and it can also be observed in the image that there is an inhibition of biofilm formation indicated by lysis, this is due to the active compound attacking and damaging the EPS matrix that protects S.aureus biofilms. Demetoxycurcumin penetrates through the polysaccharide matrix of the biofilm, and dissolves the lipids within the biofilm matrix¹⁶.

DISCUSSION:

Biofilms are complex assemblages of microbial cells, consisting of multicellular cell aggregates permanently attached to a substrate and encased in a matrix of self-produced Extracellular Polymeric Substances (EPS) consisting of polymeric mucus with polysaccharides, proteins, and nucleic acids, and have a phenotype distinct from planktonic bacteria such as changes in growth stages and gene transcription^17–20. Biofilm formation on catheters stems from urea-producing bacteria that cause an increase in urine pH and when interacting with biofilm, cause precipitation and struvite crust formation of magnesium and calcium along the catheter surface⁸.

Biofilm formation on the catheter creates a strong defense for the bacteria, making it difficult for inhibitory compounds such as demethoxycurcumin to penetrate. In this process, S.aureus bacteria form a complex synergistic community and increase the biofilm's resistance to intervention. The biofilm structure formed allows the bacteria within it to communicate and coordinate in the process of cell detachment and attachment. This is the answer to why compounds such as demethoxycurcumin have difficulty in penetrating the S.aureus biofilm and inhibiting its growth.

Demethoxycurcumin showed decreased activity in the maturation phase compared to the mid-phase. This decrease was due to the growth of mature S.aureus biofilm with better protection. Biofilms in the maturation phase produce a more abundant mucosal layer and EPS matrix compared to the middle phase²¹. This result is in line with the presentation of Rather et al. (2021), which indicated that in the biofilm maturation phase, antimicrobial agents have greater difficulty penetrating the biofilm defense layer²².

The inhibitory activity of S.aureus biofilm also showed a decrease in activity from the maturation phase to the eradication phase on the catheter. This is due to the longer duration of the biofilm growth phase compared to the mid- and maturation phases, so that the biofilm on the catheter becomes thicker and more complex^23–26. In this phase, the EPS matrix acts as a shield, preventing the penetration of candidate antibiofilm compounds into biofilm cells and facilitating the development of antibiotic resistance^26–28. This decrease in inhibitory activity reflects the growing challenge of overcoming mature, well-protected biofilms.

The inhibitory activity values in the mid-, maturation, and eradication phases reached >50% ±0.01, indicating that Demethoxycurcumin has potential as an antibiofilm drug candidate. These results indicated that although its effectiveness decreased in the maturation and degradation phases, this compound still showed significant ability in inhibiting biofilm formation. With this ability, Demethoxycurcumin is worth considering for further development as an antibiofilm therapy, especially to address infections caused by complex S. aureus biofilms on catheters.

CONCLUSION:

Demethoxycurcumin compound showed superior ability in inhibiting and destroying S.aureus biofilm, with inhibitory activity that was not significantly different from the positive control chloramphenicol. These findings suggest that Demethoxycurcumin has great potential as a new candidate antibiofilm agent and could be an effective alternative in the treatment of S.aureus biofilms on catheters of urinary tract infection patients.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We would like to thank Gadjah Mada University for funding and support for this research.This research was funded by Post-Doctoral Gadjah Mada University, grant no. 1688/UN1/DITLIT/PT.01.00/2024

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Received on 19.08.2024 Revised on 21.12.2024

Accepted on 18.03.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):283-288.

DOI: 10.52711/0974-360X.2026.00040

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