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Enhanced eradication of Staphylococcus aureus biofilms by combination disinfectants: comparative analysis of antimicrobial and anti-biofilm efficacy
(2025-10-01)
Dawan, Jirapat
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Pomseethong, Panalee
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Hwang, Jeong Seon
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Kim, Jae Kyu
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Lee, Sang Soo
Biofilms of Staphylococcus aureus pose significant challenges in various environments due to their resilience against antimicrobial agents. This study evaluated the antimicrobial efficacy of single disinfectant including 0.25% calcium hydroxide, 0.25% sodium hypochlorite, 0.25% deoxycholate, 0.25% poly(vinylpyrrolidone)-iodine complex and combination (0.25%) disinfectant suspensions against S. aureus (SAATCC and SACCARM) biofilms on metal disc surfaces. The results revealed that all treatments significantly reduced biofilm formation compared to the untreated control, with the combination treatment exhibiting the most potent and sustained anti-biofilm activity. After 4 h of incubation, biofilm cells were undetectable under the combination treatment (< 20 CFU/mL), indicating complete eradication. Crystal violet staining and biofilm formation index analysis confirmed that the combination treatment resulted in the lowest biofilm biomass. Additionally, extracellular DNA and polysaccharide levels were markedly reduced, further supporting the disruption of biofilm integrity. Gene expression analysis demonstrated a significant downregulation of biofilm-associated genes, including norB, clpP, clpX, and dnaK, under all treatments, with the combination treatment showing the highest inhibitory effect. Transmission electron microscopy analysis revealed severe morphological alterations and biofilm dispersal in the combination-treated samples. Collectively, these findings suggest that the combination disinfectant formulation is the most effective strategy for eradicating S. aureus biofilms, offering a promising approach for biofilm control in medical, industrial, and environmental applications.
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Antibiofilm strategies using mixed disinfectants and cubosome nanocarriers to combat Staphylococcus aureus on sandblasted stainless steel disc surfaces
(2025-09-01)
Dawan, Jirapat
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Guo, Yuyuan
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Lee, Sang Soo
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Kim, Jin Chul
Bacterial biofilms are complex communities in which microorganisms are adhered and encapsulated in a self-produced extracellular polymeric material (EPS) matrix. Biofilm acts as a protective barrier, enhancing the resistance of bacteria to environmental stressors, cleaning agents and antimicrobial treatments. Therefore, this study aimed to evaluated the effectiveness of a mixed disinfectant suspension (MIX) against Staphylococcus aureus biofilms on sandblasted stainless steel disc surfaces. A cubic phase containing MIX was prepared, and its anti-biofilm ability was assessed in comparison to free MIX. The susceptibility of S. aureus ATCC 15,564 (SAATCC) and multidrug-resistant S. aureus CCARM 3080 (SACCARM) biofilms to single and mixed disinfectants (MIX) was evaluated. The incorporation of MIX into cubosomes was performed to evaluate its potential enhancement in antibiofilm activity. 1% MIX demonstrated the most effective anti-biofilm activity. Extracellular DNA (eDNA) and polysaccharides were also significantly reduced in biofilms treated with 1% MIX. TEM analysis revealed that 1% MIX effectively disrupted bacterial cell clusters within the biofilms. The incorporation of 1% MIX into cubosomes enhanced its antibiofilm activity, with cubosome-encapsulated MIX exhibiting superior results compared to free MIX. This study demonstrates the effectiveness of a mixed disinfectant suspension in reducing S. aureus biofilms on sandblasted stainless steel disc surfaces. Furthermore, the incorporation of MIX into lipid-based nanocarriers, such as cubosomes, enhanced its antibiofilm efficacy compared to the free disinfectant.
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Antibacterial and antibiofilm activities of kaffir lime essential oils and their active constituents against Staphylococcus aureus focusing on sortase A
(2025-01-30)
Pontanayodsakorn, Chayapol
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Eurtivong, Chatchakorn
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Jiamboonsri, Pimsumon
Staphylococcus aureus biofilm has become a global medical concern due to rising antibiotic resistance. This study aimed to evaluate the potential activities of kaffir lime essential oils and their active compounds as alternative anti-S. aureus biofilm agents. The compositions of the essential oils were identified by gas chromatography/mass spectrometry (GC/MS), and their antibacterial activity was determined through broth-microdilution and time-kill assays. Antibiofilm activities were assessed using Congo red agar (CRA) well diffusion method, pre-biofilm inhibition resazurin assay, and post-biofilm inhibition assay. Sortase A (SrtA) inhibition was also investigated using in silico and in vitro approaches. This is followed by morphological studies to observe change in biofilm formations using light and scanning electron microscopy (SEM). Phytochemical analysis revealed that the kaffir lime essential oils from leaves (KLL) and peels (KLP) were primarily composed of the monoterpene aldehyde citronellal (59.13 %) and the monoterpene hydrocarbon (−)-limonene (25.69 %). However, the monoterpenoid alcohols, β-citronellol and terpinen-4-ol, which were the third most abundant compounds in KLL (5.35 %) and KLP (10.87 %), respectively, were selected for further study. All test compounds exhibited anti-Staphylococcal activity with a minimum inhibitory concentration (MIC) range of 0.1–0.3 % v/v. Their inhibition above the MIC levels showed time- and concentration-independence. Among the test compounds, terpinen-4-ol revealed good antibiofilm activity by inhibiting biofilm formation rather than eradicating the established biofilm. However, terpinen-4-ol exhibited weak SrtA inhibition with docking score of 32.58 and in vitro SrtA inhibition of 46.14 ± 3.58 % at 1 % v/v. Interestingly, terpinen-4-ol caused visible damage to the bacterial cell barrier, as revealed by SEM micrographs. These findings suggest the potential use of kaffir lime oils and their active compounds to combat biofilm-forming S. aureus infection.
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Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis
(2024-12-01)
Wongchai, Mathira
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Wongkaewkhiaw, Saharut
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Kanthawong, Sakawrat
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Roytrakul, Sittiruk
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Aunpad, Ratchaneewan
Background: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. Methods: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. Results: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. Conclusions: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.
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Anti-oral streptococci and anti-biofilm properties of Etlingera pavieana essential oil and its bioactive compounds proposed for an alternative herbal mouthwash
(2024-05-30)
Wongsariya, Karn
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Lapirattanakul, Jinthana
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Chewchinda, Savita
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Kanchanadumkerng, Pimpikar
Oral streptococci are the major group of bacteria in the oral cavity. Some of their species cause oral diseases that may lead to tooth loss and quality-of-life reduction, such as dental caries. One of prevention techniques to promote oral health is rinsing mouthwash after toothbrushing. This study aimed to determine the potential uses of local food, also remedy, plant in Thailand called Reaw-Horm or Etlingera pavieana for alternative herbal mouthwash. The essential oil from E. pavieana rhizome (Eo) is used for anti-streptococci including Streptococcus mutans and Streptococcus sobrinus and anti-biofilm activities. The main components of Eo are methyl chavicol (MC) and trans-anethole (TA). The disk diffusion method showed the inhibition zone of Eo in a dose-dependent manner. The minimum inhibitory concentration (MIC) of Eo and TA was >1.6 % v/v, and 0.4 % v/v of MC. Regarding anti-biofilm activities, MC showed nearly equal anti-biofilm formation of S. mutans and S. sobrinus, whereas Eo and TA acted toward S. sobrinus more than S. mutans biofilm. Sub-MIC killing effects on cells under biofilm were observed in Eo and MC. Therefore, MC was recommended as an active compound for anti-streptococci activities. Biocompatibility of Eo and MC were shown to be safe for epidermal cell lines. Herbal mouthwashes containing Eo were developed and had antioxidant and antimicrobial actions with established for 3 months. This study provides in vitro support on the use of herbal mouthwash with antioxidant and antimicrobial activities for dental caries prevention and well-being of individuals.
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Effects of natural sugar on acidogenic potential, biofilm biomass, and antiseptic resistance of oral streptococci
(2021-01-01)
Kanchanadumkerng, Pimpikar
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Wongsariya, Karn
Natural sugar is deliberated as ordinary, non-chemical, and healthy alternatives. However, a diet rich in sugar is well documented as a causative agent for dental caries. The purpose of this study was to investigate the effects of natural sugars, including raw cane, palmyra palm and coconut sugar on the acidogenic profiles, biofilm formation and antiseptic treatment efficacy compared with refined sugar. The study was based on single-species and dual-species of Streptococcus mutans ATCC 25175 and Streptococcus sobrinus ATCC 33402. Our results showed that sucrose was a major component of all samples, with percentages of relative content higher than 89.0. Palmyra sugar gave the least pH change at 180 min of 4.84-4.93, which indicated it was the least acidogenic. Coconut sugar formed the lowest level of biofilm biomass compared to refined sugar (p < 0.05) and other samples. Antiseptic treatment was performed to study the level of percent eradication of bacterial plaque using MTT assay to determine cell viability under biofilm in each sugar medium. Biofilm derived from coconut sugar had a susceptibility to antiseptic treatment with 56.2-61.99% eradication which was higher than the biofilm from palmyra and raw cane sugar. As a result, this study points out the effects of various natural sugars (especially different sources of plant material) on cariogenic potential. However, further experiment should be done to confirm the results in vivo and further study the cariogenic effects of diet supplementation of these fermentable sugars.
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