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

Authors

DOI:

https://doi.org/10.14719/pst.3970

Keywords:

Antibiofilm, antimicrobial activity, gel filtration, ion exchange chromatography, Pichia kudriavzevii, protein precipitation, toxin

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.

 

Downloads

Download data is not yet available.

References

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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.

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

Published

10-08-2024 — Updated on 11-08-2024

Versions

How to Cite

1.
Noor NH, Shatha AS, Zaid A. Investigating the biological activity and biofilm inhibition of purified Pichia kudriavzevii killer toxin (PkKt) against pathogenic bacterial as a promising substance for food preservation . Plant Sci. Today [Internet]. 2024 Aug. 11 [cited 2024 Nov. 23];11(3). Available from: https://horizonepublishing.com/journals/index.php/PST/article/view/3970

Issue

Section

Research Articles