Effects of Phenyl Isothiocyanate on Biofilm Formation and Violacin Production in Chromobacterium violaceum

Öz

In this study, the anti-quorum sensing (anti-QS) effects of phenyl isothiocyanate (PITC) on the virulence factors of Chromobacterium violaceum ATCC 12472 were examined. The antibacterial activity of PITC was evaluated, revealing inhibition zones within the range of 8-1 mM. The minimum inhibitory concentration (MIC) was determined to be 1 mM, while the minimum bactericidal concentration (MBC) was found to be greater than 2 mM. At sub-MIC concentrations (0.5-0.0625 mM), it was identified through the crystal violet assay that PITC inhibited biofilm formation by 41% at 0.5 mM and by 21% at 0.25 mM. Additionally, spectrophotometric analysis indicated a reduction in violacein production of 55% in the presence of 0.5 mM PITC and 31% at 0.25 mM. These findings suggest that PITC is capable of suppressing biofilm formation and violacein production in C. violaceum without inducing cell death, thereby positioning it as a potential anti-virulence agent. However, comprehensive studies on toxicity and the mechanism of action are necessary prior to clinical applications. This research indicates that PITC could represent an alternative approach to the antibiotic resistance crisis and may illuminate new strategies for the control of biofilm-related infections.

Anahtar Kelimeler

• Phenyl isothiocyanate
• Quorum sensing
• Chromobacterium violaceum
• Biofilm
• Violacein

Proje Numarası

8139

Chromobacterium violaceum'da Biyofilm Oluşumu ve Viyolasin Üretimi Üzerine Fenil İzotiyosiyanatın Etkileri

Öz

Bu çalışmada, fenil izotiyosiyanatın (PITC) Chromobacterium violaceum ATCC 12472'nin virülans faktörleri üzerindeki anti-quorum sensing (anti-QS) etkileri incelenmiştir. PITC'nin antibakteriyel aktivitesi değerlendirildiğinde ve 8-1 mM aralığında inhibisyon zonları gözlemlenmiştir. Minimum inhibitör konsantrasyon (MİK) 1 mM, minimum bakterisidal konsantrasyon (MBK) ise >2 mM olarak belirlenmiştir. Sub-MİK konsantrasyonlarda (0.5-0.0625 mM), PITC'nin biyofilm oluşumunu 0.5 mM için %41, 0.25 mM için %21 oranında inhibe ettiği kristal viyole yöntemiyle tespit edilmiştir. Viyolasin üretiminin 0.5 mM PITC varlığında %55, 0.25 mM için %31 azaldığı spektrofotometrik olarak belirlenmiştir. Elde edilen bulgular, PITC'nin C. violaceum için hücre ölümüne neden olmadan biyofilm oluşumunu ve viyolasin üretimini baskılayabildiğini ve anti-virülans ajan olarak potansiyel taşıdığını ortaya koymuştur. Ancak, klinik uygulamalar öncesinde toksisite ve etki mekanizmasına yönelik detaylı çalışmalar gereklidir. Bu çalışma, PITC'nin antibiyotik direnci krizine alternatif bir yaklaşım olarak değerlendirilebileceğini ve biyofilm kaynaklı enfeksiyonların kontrolünde yeni stratejilere ışık tutabileceğini göstermektedir.

Anahtar Kelimeler

• Fenil izotiyosiyanat
• Quorum sensing
• Chromobacterium violaceum
• Biyofilm
• Viyolasin

Destekleyen Kurum

Süleyman Demirel Üniversitesi Bilimsel Araştırma Projeleri (BAP)

Proje Numarası

8139

Etik Beyan

Bu çalışmada, Yükseköğretim Kurumları Bilimsel Araştırma ve Yayın Etiği Yönergesi'nde belirtilen tüm kurallara tam olarak uyulduğunu ve aynı yönergenin 'Bilimsel Araştırma ve Yayın Etiğine Aykırı Eylemler' başlığı altında tanımlanan herhangi bir eylemin gerçekleştirilmediğini beyan ederiz.

Teşekkür

Bu çalışma, Süleyman Demirel Üniversitesi Bilimsel Araştırma Projeleri (BAP) tarafından desteklenmiştir (Proje ID: 8139). Destekleri için SDÜ BAP'a teşekkür ederiz.

Kaynakça

1. [1] Durán, N., & Menck, C. F. M. (2001). Chromobacterium violaceum: A review of pharmacological and industiral perspectives. In Critical Reviews in Microbiology ,27(3), 201-222.
2. [2] Alves De Brito, C. F., Carvalho, C. M. B., Santos, F. R., Gazzinelli, R. T., Oliveira, S. C., Azevedo, V., & Teixeira, S. M. R. (2004). Chromobacterium violaceum genome: Molecular mechanisms associated with pathogenicity. Genetics and Molecular Research, 3(1), 148-61.
3. [3] Chattopadhyay, A., Kumar, V., Bhat, N., & Rao, P. L. N. G. (2002). Chromobacterium violaceum infection: A rare but frequently fatal disease. Journal of Pediatric Surgery, 37(1), 108-110.
4. [4] Lichstein, H. C., & Van de Sand, V. F. (1946). The antibiotic activity of violacein, prodigiosin, and phth iocol. Journal of Bacteriology, 52(1), 145-146.
5. [5] McClean, K. H., Winson, M. K., Fish, L., Taylor, A., Chhabra, S. R., Camara, M., Daykin, M., Lamb, J. H., Swift, S., Bycroft, B. W., Stewart, G. S. A. B., & Williams, P. (1997). Quorum sensing and Chromobacterium violaceum: Exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology, 143(12), 3703-3711.
6. [6] Anahas, A. M. P., Kumaran, S., Kandeel, M., Muralitharan, G., Silviya, J., Adhimoolam, G. L., ... & Prasannabalaji, N. (2022). Applications of natural violet pigments from halophilic Chromobacterium violaceum PDF23 for textile dyeing with antimicrobial and antioxidant potentials. Journal of Nanomaterials, 2022(1), 3885396.
7. [7] Anahas, A. M. P., Kumaran, S., Kandeel, M., Muralitharan, G., Silviya, J., Adhimoolam, G. L., Panagal, M., Pugazhvendan, S. R., Suresh, G., Wilson Aruni, A., Rethinam, S., & Prasannabalaji, N. (2022). Applications of Natural Violet Pigments from Halophilic Chromobacterium violaceum PDF23 for Textile Dyeing with Antimicrobial and Antioxidant Potentials. Journal of Nanomaterials, 7(13).
8. [8] Konzen, M., De Marco, D., Cordova, C. A., Vieira, T. O., Antonio, R. V., & Creczynski-Pasa, T. B. (2006). Antioxidant properties of violacein: possible relation on its biological function. Bioorganic & medicinal chemistry, 14(24), 8307-8313.

9. [9] Nakamura, Y., Sawada, T., Morita, Y., & Tamiya, E. (2002). Isolation of a psychrotrophic bacterium from the organic residue of a water tank keeping rainbow trout and antibacterial effect of violet pigment produced from the strain. Biochemical Engineering Journal, 12(1), 79-86.
10. [10] Abinaya, K., Gopinath, S. C., Kumarevel, T., & Raman, P. (2023). Anti-quorum sensing activity of selected cationic amino acids against Chromobacterium violaceum and Pseudomonas aeruginosa. Process Biochemistry, 133, (75-84).
11. [11] Durán, N., Justo, G. Z., Durán, M., Brocchi, M., Cordi, L., Tasic, L., ... & Nakazato, G. (2016). Advances in Chromobacterium violaceum and properties of violacein-Its main secondary metabolite: A review. Biotechnology advances, 34(5), 1030-1045.
12. [12] Fitriani, A., Ayuningtyas, D. P., & Kusnadi, K. (2016). Inhibition of quorum sensing in Chromobacterium violaceum cv026 by violacein produced by Pseudomonas aeruginosa. (2016): 103-108.
13. [13] Mah, T. F. C., & O'Toole, G. A. (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends in microbiology, 9(1), 34-39.
14. [14] Pena, R. T., Blasco, L., Ambroa, A., González-Pedrajo, B., Fernández-García, L., López, M., ... & Tomás, M. (2019). Relationship between quorum sensing and secretion systems. Frontiers in microbiology, 10, 1100.
15. [15] Saeki, E. K., Kobayashi, R. K. T., & Nakazato, G. (2020). Quorum sensing system: Target to control the spread of bacterial infections. Microbial pathogenesis, 142, 104068.
16. [16] Smith, A. W. (2005). Biofilms and antibiotic therapy: is there a role for combating bacterial resistance by the use of novel drug delivery systems. Advanced drug delivery reviews, 57(10), 1539-1550.
17. [17] Smith, A. W. (2005). Biofilms and antibiotic therapy: Is there a role for combating bacterial resistance by the use of novel drug delivery systems? In Advanced Drug Delivery Reviews 2022(1), 3885396.
18. [18] De la Fuente-Núñez, C., Reffuveille, F., Fernández, L., & Hancock, R. E. (2013). Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies. Current opinion in microbiology, 16(5), 580-589.
19. [19] Sharma, D., Misba, L., & Khan, A. U. (2019). Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrobial Resistance & Infection Control, 8(1), 76.
20. [20] Subhadra, B., Kim, D. H., Woo, K., Surendran, S., & Choi, C. H. (2018). Control of biofilm formation in healthcare: recent advances exploiting quorum-sensing interference strategies and multidrug efflux pump inhibitors. Materials, 11(9), 1676.
21. [21] Choo, J. H., Rukayadi, Y., & Hwang, J. K. (2006). Inhibition of bacterial quorum sensing by vanilla extract. Letters in applied microbiology, 42(6), 637-641.
22. [22] Fleitas Martínez, O., Rigueiras, P. O., Pires, Á. D. S., Porto, W. F., Silva, O. N., De la Fuente-Nunez, C., & Franco, O. L. (2019). Interference with quorum-sensing signal biosynthesis as a promising therapeutic strategy against multidrug-resistant pathogens. Frontiers in cellular and infection microbiology, 8, 444.
23. [23] Fleitas Martínez, O., Rigueiras, P. O., Pires, Á. da S., Porto, W. F., Silva, O. N., de la Fuente-Nunez, C., & Franco, O. L. (2019). Interference With Quorum-Sensing Signal Biosynthesis as a Promising Therapeutic Strategy Against Multidrug-Resistant Pathogens. In Frontiers in Cellular and Infection Microbiology, 8 (2009), 444.
24. [24] Rasmussen, T. B., & Givskov, M. (2006). Quorum sensing inhibitors: a bargain of effects. Microbiology, 152(4), 895-904.
25. [25] Dangl, J. L., & Jones, J. D. (2001). Plant pathogens and integrated defence responses to infection. nature, 411(6839), 826-833.
26. [26] Dixon, R. A. (2001). Natural products and plant disease resistance. Nature, 411(6839), 843-847.
27. [27] Fahey, J. W., Stephenson, K. K., Wade, K. L., & Talalay, P. (2013). Urease from Helicobacter pylori is inactivated by sulforaphane and other isothiocyanates. Biochemical and Biophysical Research Communications, 435(1). 1-7.
28. [28] de Saravia, S. G. G., & Gaylarde, C. C. (1998). The antimicrobial activity of an aqueous extract of Brassica negra. International biodeterioration & biodegradation, 41(2), 145-148.
29. [29] Saavedra, M. J., Borges, A., Dias, C., Aires, A., Bennett, R. N., Rosa, E. S., & Simões, M. (2010). Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria. Medicinal Chemistry, 6(3), 174-183.
30. [30] Borges, A., Simões, L. C., Saavedra, M. J., & Simões, M. (2014). The action of selected isothiocyanates on bacterial biofilm prevention and control. International Biodeterioration & Biodegradation, 86, 25-33.
31. [31] Ganin, H., Rayo, J., Amara, N., Levy, N., Krief, P., & Meijler, M. M. (2013). Sulforaphane and erucin, natural isothiocyanates from broccoli, inhibit bacterial quorum sensing. MedChemComm, 4(1), 175-179.
32. [32] Abreu, A. C., Borges, A., Simões, L. C., Saavedra, M. J., & Simões, M. (2013). Antibacterial activity of phenyl isothiocyanate on Escherichia coli and Staphylococcus aureus. Medicinal Chemistry, 9(5), 756-761.
33. [33] Venkatramanan, M., Sankar Ganesh, P., Senthil, R., Akshay, J., Veera Ravi, A., Langeswaran, K., ... & Shankar, E. M. (2020). Inhibition of quorum sensing and biofilm formation in Chromobacterium violaceum by fruit extracts of Passiflora edulis. ACS omega, 5(40), 25605-25616.
34. [34] Bakht, J., Islam, A., & Shafi, M. (2011). Antimicrobial potential of Eclipta alba by well diffusion method. Pak. J. Bot, 43, 161-166.
35. [35] Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of applied microbiology, 86(6), 985-990.
36. [36] Im, H., Choi, S. Y., Son, S., & Mitchell, R. J. (2017). Combined application of bacterial predation and violacein to kill polymicrobial pathogenic communities. Scientific reports, 7(1), 14415.
37. [37] Girennavar, B., Cepeda, M. L., Soni, K. A., Vikram, A., Jesudhasan, P., Jayaprakasha, G. K., ... & Patil, B. S. (2008). Grapefruit juice and its furocoumarins inhibits autoinducer signaling and biofilm formation in bacteria. International journal of food microbiology, 125(2), 204-208.
38. [38] Scarafile, G. (2016). Antibiotic resistance: current issues and future strategies. Reviews in Health Care, 7(1), 3-16.
39. [39] Christaki, E., Marcou, M., & Tofarides, A. (2020). Antimicrobial Resistance in Bacteria: Mechanisms, Evolution, and Persistence. In Journal of Molecular Evolution 88(1), 26-40.
40. [40] Dickschat, J. S. (2010). Quorum sensing and bacterial biofilms. In Natural Product Reports 27( 3), 343-369.
41. [41] White, C. E., & Winans, S. C. (2007). The quorum-sensing transcription factor TraR decodes its DNA binding site by direct contacts with DNA bases and by detection of DNA flexibility. Molecular Microbiology, 64(1), 245-256.
42. [42] Zhu, H., He, C. C., & Chu, Q. H. (2011). Inhibition of quorum sensing in Chromobacterium violaceum by pigments extracted from Auricularia auricular. Letters in Applied Microbiology, 52(3), 269-274.
43. [43] Çevikbaş, H., Ulusoy, S., & Kaya Kinaytürk, N. (2024). Exploring rose absolute and phenylethyl alcohol as novel quorum sensing inhibitors in Pseudomonas aeruginosa and Chromobacterium violaceum. Scientific Reports, 14(1), 15666.
44. [44] Ahmad, A., Viljoen, A. M., & Chenia, H. Y. (2015). The impact of plant violatiles on bacterial quorum sensing. Letters in Applied Microbiology, 60(1), 8-19.