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Plant Science Today (PST)

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Early Access

Investigating the biological activity and biofilm inhibition of purified Pichia kudriavzevii killer toxin (PkKt) against pathogenic bacterial as a promising substance for food preservation

DOI
https://doi.org/10.14719/pst.3970
Submitted
24 May 2024
Published
10-08-2024
Versions

Abstract

Killer toxins (PkKT) produced by yeasts exhibit antimicrobial effects against pathogenic bacteria and fungi. This study aimed to extract, purify, and characterize the killer toxin from Pichia kudriavzevii killer toxin (PkKT) and evaluate its antimicrobial and anti-biofilm potential. PkKT was produced as a killer toxins and was purified through ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The purified PkKT showed a molecular weight of 36 kDa. Antimicrobial testing revealed significant inhibition against Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Acinetobacter baumannii, Escherichia coli, and Candida albicans using well diffusion assays. The minimum inhibitory concentration (MIC) ranged from 16-128 ?g/mL, while the sub-MIC was determined to be 8-64 ?g/mL depending on the pathogen. At sub-MIC levels, PkKT exhibited a 66-89% reduction of biofilm formation in all tested species. S. aureus, S. pyogenes, and A. baumannii showed the highest MIC of 128 and 64 ?g/mL sub-MIC, while E. coli and K. pneumoniae were most susceptible with MIC of 16 ?g/mL and sub-MIC of 8 ?g/mL. In conclusion, the purified PkKT toxin displayed broad-spectrum antimicrobial and anti-biofilm activities against common healthcare-associated pathogens. These findings reveal the potential of PkKT as a therapeutic agent to prevent or treat biofilm-associated infections.

 

References

  1. Terreni M, Taccani M, Pregnolato M. New antibiotics for multidrug-resistant bacterial strains: latest research developments and future perspectives. Molecules. 2021 May 2;26(9):2671. https://doi.org/10.3390/molecules26092671.
  2. Naimah AK, Al-Manhel AJ, Al-Shawi MJ. Isolation, purification and characterization of antimicrobial peptides produced from Saccharomyces boulardii. International Journal of Peptide Research and Therapeutics. 2018 Sep;24:455-6. https://doi.org/10.1007/s10989-017-9632.
  3. Liu GL, Chi Z, Wang GY, Wang ZP, Li Y, Chi ZM. Yeast killer toxins, molecular mechanisms of their action and their applications. Critical reviews in biotechnology. 2015 Apr 3;35(2):222-34. https://doi.org/10.3109/07388551.2013.833582
  4. Anke T, Schüffler A. Physiology and Genetics, In: R. Schaffrath, F. Meinhardt and R. Klassen (Eds.),Yeast killer toxins: Fundamentals and applications, Springer link, pp. 107-130, 2009. https://doi.org/10.1007/978-3-642-00286-1
  5. Anke T, Schüffler A. Physiology and Genetics, In: R. Schaffrath, F. Meinhardt and R. Klassen (Eds.). Yeast killer toxins: Fundamentals and applications, Springer link, pp. 87-118, 2009. https://doi.org/10.1007/978-3-642-00286-1
  6. Mannazzu I, Domizio P, Carboni G, Zara S, Zara G, Comitini F, Budroni M, Ciani M. Yeast killer toxins: From ecological significance to application. Critical reviews in biotechnology. 2019 Jul 4;39(5):603-17. https://doi.org/10.1080/07388551.2019.1601679
  7. Douglass AP, Offei B, Braun-Galleani S, Coughlan AY, Martos AA, Ortiz-Merino RA, Byrne KP, Wolfe KH. Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: one species, four names. PLoS pathogens. 2018 Jul 19;14(7):e1007138. https://doi.org/10.1371/journal.ppat.1007138
  8. de Ullivarri MF, Mendoza LM, Raya RR. Characterization of the killer toxin KTCf20 from Wickerhamomyces anomalus, a potential biocontrol agent against wine spoilage yeasts. Biological control. 2018 Jun 1;121:223-8. https://doi.org/10.1016/j.biocontrol.2018.03.008
  9. Delali KI, Chen O, Wang W, Yi L, Deng L, Zeng K. Evaluation of yeast isolates from kimchi with antagonistic activity against green mold in citrus and elucidating the action mechanisms of three yeast: P. kudriavzevii, K. marxianus, and Y. lipolytica. Postharvest Biology and Technology. 2021 Jun 1;176:111495. https://doi.org/10.1016/j.postharvbio.2021.111495
  10. Bajaj BK, Raina S, Singh S. Killer toxin from a novel killer yeast Pichia kudriavzevii RY55 with idiosyncratic antibacterial activity. Journal of basic microbiology. 2013 Aug;53(8):645-56. https://doi.org/10.1002/jobm.201200187
  11. Utama GL, Kurniawan MO, Natiqoh N, Balia RL. Species identification of stress resistance yeasts isolated from banana waste for ethanol production. InIOP Conference Series: Earth and Environmental Science 2019 Jul 1 (Vol. 306, No. 1, p. 012021). IOP Publishing. https://doi.org/10.1088/1755-1315/306/1/012021
  12. Utama GL, Rahmah SA, Kayaputri IL, Balia RL. Antibacterial activities of indigenous yeasts isolated from pomegranate peels (Punica granatum L.). Journal of Advanced Pharmaceutical Technology and Research. 2022 Jan 1;13(1):56-60. https://doi.org/10.4103/japtr.japtr_86_21
  13. Lata P, Kumari R, Sharma KB, Rangra S. In vitro evaluation of probiotic potential and enzymatic profiling of Pichia kudriavzevii Y33 isolated from traditional home-made mango pickle. Journal of Genetic Engineering and Biotechnology. 2022 Dec 1;20(1):132. https://doi.org/10.1186/s43141-022-00416-2
  14. Magliani W, Conti S, Salati A, Vaccari S, Ravanetti L, Maffei DL, Polonelli L. Therapeutic potential of yeast killer toxin-like antibodies and mimotopes. FEMS Yeast Research. 2004 Oct 1;5(1):11-8. https://doi.org/10.1016/j.femsyr.2004.06.010
  15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976 May 7;72(1-2):248-54. https://doi.org/10.1006/abio.1976.9999
  16. Bajaj BK, Sharma S. Construction of killer industrial yeast Saccharomyces cerevisiae HAU-1 and its fermentation performance. Brazilian Journal of Microbiology. 2010;41:477-85. https://doi.org/10.1590/S1517-83822010000200030
  17. Ragavan ML, Das N. Production and purification of killer toxin from probiotic yeasts and its effect on foodborne pathogens. Journal of microbiology, biotechnology and food sciences. 2020 Dec 1;10(3):350-3. https://doi.org/10.15414/jmbfs.2020.10.3.350-353
  18. Abdulateef SA, Hussein MH, Al-Saffar AZ. In vitro cytotoxic and genotoxic of lipopolysaccharide isolated from klebsiella pneumoniae as1 on mcf-7 human breast tumor cell line. International Journal of. Drug Delivery Technology. 2021;11(1):184-9.
  19. Lewus CB, Kaiser AL, Montville TJ. Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Applied and Environmental Microbiology. 1991 Jun;57(6):1683-8. https://doi.org/10.1128/aem.57.6.1683-1688.1991
  20. Cazella LN, Glamoclija J, Sokovi? M, Gonçalves JE, Linde GA, Colauto NB, Gazim ZC. Antimicrobial activity of essential oil of Baccharis dracunculifolia DC (Asteraceae) aerial parts at flowering period. Frontiers in plant science. 2019 Jan 29;10:27. https://doi.org/10.3389/fpls.2019.00027
  21. Singh AK, Prakash P, Achra A, Singh GP, Das A, Singh RK. Standardization and classification of in vitro biofilm formation by clinical isolates of Staphylococcus aureus. Journal of global infectious diseases. 2017 Jul 1;9(3):93-101. https://doi.org/10.4103/jgid.jgid_91_16
  22. Shrivastava A, Pal M, Sharma RK. Pichia as yeast cell factory for production of industrially important bio-products: Current trends, challenges, and future prospects. Journal of Bioresources and Bioproducts. 2023 May 1;8(2):108-24. https://doi.org/10.1016/j.jobab.2023.01.007
  23. Ciani M, Comitini F. Yeast interactions in multi-starter wine fermentation. Current Opinion in Food Science. 2015 Feb 1;1:1-6. https://doi.org/10.1016/j.cofs.2014.07.001
  24. Moura VS, Pollettini FL, Ferraz LP, Mazzi MV, Kupper KC. Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens. Journal of Basic Microbiology. 2021 Feb;61(2):77-87. https://doi.org/10.1002/jobm.202000164
  25. Kulakovskaya E, Zvonarev A, Farofonova V. Characteristics of killer toxin of the yeast Cryptococcus pinus. Journal of Biosciences and Medicines. 2019 Mar 6;7(4):73-82. https://doi.org/10.4236/jbm.2019.74008
  26. Jandera P. Comparison of various modes and phase systems for analytical HPLC: in Valkó, (Ed.), Handbook of Analytical Separations, Elsevier Science, pp. 1-91, 2020. https://doi.org/10.1016/B978-0-444-64070-3.00001-1
  27. Alsoufi MA, Aziz RA. Use killer toxin extracted from bakery yeast for extending shelf life of fruits. Pakistan Journal of Biotechnology. 2017 Jan 25;14(1):23-7.
  28. Bajaj BK, Singh S. Biology of killer yeast and technological implications. Yeast Diversity in Human Welfare. 2017:163-90. https://doi.org/10.1007/978-981-10-2621-8_7
  29. Labbani FZ, Turchetti B, Bennamoun L, Dakhmouche S, Roberti R, Corazzi L, Meraihi Z, Buzzini P. A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts. Antonie Van Leeuwenhoek. 2015 Apr;107:961-70. https://doi.org/10.1007/s10482-015-0388-4
  30. Younis G, Awad A, Dawod RE, Yousef NE. Antimicrobial activity of yeasts against some pathogenic bacteria. Veterinary world. 2017 Aug;10(8):979. https://doi.org/10.14202/vetworld.2017.979-983
  31. Belda I, Ruiz J, Alonso A, Marquina D, Santos A. The biology of Pichia membranifaciens killer toxins. Toxins. 2017 Mar 23;9(4):112. https://doi.org/10.3390/toxins9040112
  32. Giovati L, Ciociola T, De Simone T, Conti S, Magliani W. Wickerhamomyces yeast killer toxins’ medical applications. Toxins. 2021 Sep 15;13(9):655. https://doi.org/10.3390/toxins13090655
  33. Gier S, Schmitt MJ, Breinig F. Analysis of yeast killer toxin K1 precursor processing via site-directed mutagenesis: implications for toxicity and immunity. Msphere. 2020 Feb 26;5(1):10-128. https://doi.org/10.1128/mSphere.00979-19
  34. Abu-Mejdad NM, Al-Badran AI, Al-Saadoon AH, Minati MH. A new report on gene expression of three killer toxin genes with antimicrobial activity of two killer toxins in Iraq. Bulletin of the National Research Centre. 2020 Dec;44:1-9. https://doi.org/10.1186/s42269-020-00418-5
  35. Carboni G, Fancello F, Zara G, Zara S, Ruiu L, Marova I, Pinna G, Budroni M, Mannazzu I. Production of a lyophilized ready-to-use yeast killer toxin with possible applications in the wine and food industries. International journal of food microbiology. 2020 Dec 16;335:108883. 0. https://doi.org/10.1016/j.ijfoodmicro.2020.108883
  36. Robledo-Leal E, Rivera-Morales LG, Sangorrin MP, González GM, Ramos-Alfano G, Adame-Rodriguez JM, Alcocer-Gonzalez JM, Arechiga-Carvajal ET, Rodriguez-Padilla C. Identification and susceptibility of clinical isolates of Candida spp. to killer toxins. Brazilian journal of biology. 2018 Feb 1;78(04):742-9.
  37. Giovati L. Candidacidal Activity of a Novel Killer Toxin from Wickerhamomyces anomalus against Fluconazole-Susceptible and -Resistant Strains, Toxins. 2018;10:492. https://doi.org/10.3390/toxins10020068
  38. Ahmadi F, Najafpour GD, Mohammadi M. Production of bio-fungicide from sugarcane bagasse using Pichia membranifaciens yeast and its activity against post-harvest pathogenic fungi. Biointerface Res Appl Chem. 2021;11:10435-45. https://doi.org/10.33263/BRIAC113.1043510445
  39. Agarbati A, Ciani M, Esin S, Agnolucci M, Marcheggiani F, Tiano L, Comitini F. Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts. International Journal of Molecular Sciences. 2023 Jan 9;24(2):1309. https://doi.org/10.3390/ijms24021309
  40. Kowalska-Krochmal B, Dudek-Wicher R. The minimum inhibitory concentration of antibiotics: Methods, interpretation, clinical relevance. Pathogens. 2021 Feb 4;10(2):165. https://doi.org/10.3390/pathogens10020165
  41. Abebe GM. The role of bacterial biofilm in antibiotic resistance and food contamination. International journal of microbiology. 2020;2020(1):1705814. https://doi.org/10.1155/2020/1705814
  42. Gouka L, Raaijmakers JM, Cordovez V. Ecology and functional potential of phyllosphere yeasts. Trends in Plant Science. 2022;27(11):1109-23. https://doi.org/10.1016/j.tplants.2022.06.007
  43. Calazans GF, SILVA JC, Delabeneta MF, Paris AP, Yassuda Filho P, Auler ME, Menolli RA, Paula CR, Simão RD, Gandra RF. Antimicrobial activity of Wickerhamomyces anomalus mycocins against strains of Staphylococcus aureus isolated from meats. Food Science and Technology. 2020 Oct 9;41:388-94. https://doi.org/10.1590/fst.39319
  44. Tan HW, Tay ST. Anti-Candida activity and biofilm inhibitory effects of secreted products of tropical environmental yeasts. Tropical Biomedicine 2011; 28(1):175–180.
  45. Bumunang EW, Ateba CN, Stanford K, Niu YD, Wang Y, McAllister TA. Activity of bacteriophage and complex tannins against biofilm-forming shiga toxin-producing Escherichia coli from Canada and South Africa. Antibiotics. 2020 May 15;9(5):257. https://doi.org/10.3390/antibiotics9050257

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