Journal of Microbiology and Biotechnology

  • 제33권7호
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  • Pages.964-972
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  • 2023
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Development of a Magnetic Bead-Based Method for Specific Detection of Enterococcus faecalis Using C-Terminal Domain of ECP3 Phage Endolysin

  • Yoon-Jung Choi (Department of Biomedical Sciences, The Graduate School, Kyungpook National University) ;
  • Shukho Kim (Department of Biomedical Sciences, The Graduate School, Kyungpook National University) ;
  • Jungmin Kim (Department of Biomedical Sciences, The Graduate School, Kyungpook National University)
  • 투고 : 2023.02.27
  • 심사 : 2023.04.10
  • 발행 : 2023.07.28
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초록

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

키워드

과제정보

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education and the Korea government Ministry of Science and ICT (MSIT), grant numbers NRF-2017R1D1A3-B06032486 and NRF-2022R1F1A1073686, respectively.

참고문헌

  1. Fernandez-Guerrero ML, Verdejo C, Azofra J, de Gorgolas, M. 1995. Hospital-acquired infectious endocarditis not associated with cardiac surgery: an emerging problem. Clin. Infect. Dis. 20: 16-23. https://doi.org/10.1093/clinids/20.1.16
  2. Kotsanas D, Tan K, Scott C, Baade B, Cheng MHL, Tan ZV, et al. 2019. A nonclonal outbreak of vancomycin-sensitive Enterococcus faecalis bacteremia in a neonatal intensive care unit. Infect. Control Hosp. Epidemiol. 40: 1116-1122. https://doi.org/10.1017/ice.2019.202
  3. Montalban-Lopez M, Cebrian R, Galera R, Mingorance L, Martin-Platero AM, Valdivia E, et al. 2020. Synergy of the bacteriocin AS-48 and antibiotics against uropathogenic enterococci. Antibiotics 9: 567.
  4. Wang JS, Muzevich K, Edmond MB, Bearman G, Stevens MP. 2014. Central nervous system infections due to vancomycin-resistant enterococci: case series and review of the literature. Int. J. Infect. Dis. 25: 26-31. https://doi.org/10.1016/j.ijid.2014.01.009
  5. Bhardwaj SB, Mehta M, Sood S, Sharma J. 2017. Biofilm formation by drug resistant enterococci isolates obtained from chronic periodontitis patients. J. Clin. Diagn. Res. 11: DC01-DC03. https://doi.org/10.7860/JCDR/2017/24472.9152
  6. Graetz C, Mann L, Krois J, Salzer S, Kahl M, Springer C, et al. 2019. Comparison of periodontitis patients' classification in the 2018 versus 1999 classification. J. Clin. Periodontol. 46: 908-917. https://doi.org/10.1111/jcpe.13157
  7. Ardila CM, Bedoya-Garcia JA. 2020. Antimicrobial resistance of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in periodontitis patients. J. Glob. Antimicrob. Resist. 22: 215-218. https://doi.org/10.1016/j.jgar.2020.02.024
  8. Yoon DL, Kim S, Song H, Kim YG, Lee JM, Kim J. 2015. Detection of bacterial species in chronic periodontitis tissues at different stages of diseases severity. J. Bacteriol. Virol. 45: 364-371. https://doi.org/10.4167/jbv.2015.45.4.364
  9. Santimaleeworagun W, Changpradub D, Thunyaharn S, Hemapanpairoa J. 2019. Optimizing the dosing regimens of daptomycin based on the susceptible dose-dependent breakpoint against vancomycin-resistant enterococci infection. Antibiotics 8: 245.
  10. Roach DR, Donovan DM. 2015. Antimicrobial bacteriophage-derived proteins and therapeutic applications. Bacteriophage 5: e1062590.
  11. Ha E, Son B, Ryu S. 2018. Clostridium perfringens virulent bacteriophage CPS2 and its thermostable endolysin LysCPS2. Viruses 10: 251.
  12. Wang S, Gu J, Lv M, Guo Z, Yan G, Yu L, et al. 2017. The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. J. Microbiol. 55: 403-408. doi:10.1007/s12275-017-6431-6
  13. Love MJ, Coombes D, Ismail S, Billington C, Dobson RCJ. 2022. The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily. Biochem. J. 479: 207-223. https://doi.org/10.1042/BCJ20210701
  14. Park Y, Lim JA, Kong M, Ryu S, Rhee S. 2014. Structure of bacteriophage SPN1S endolysin reveals an unusual two-module fold for the peptidoglycan lytic and binding activity. Mol. Microbiol. 92: 316-325. https://doi.org/10.1111/mmi.12555
  15. Kong M, Na H, Ha NC, Ryu S. 2019. LysPBC2, a novel endolysin harboring a Bacillus cereus spore binding domain. Appl. Environ. Microbiol. 85: e02462-18.
  16. Schmelcher M, Shabarova T, Eugster MR, Eichenseher F, Tchang VS, Banz M, et al. 2010. Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains. Appl. Environ. Microbiol. 76: 5745-5756. https://doi.org/10.1128/AEM.00801-10
  17. Adriaenssens EM, Brister JR. 2017. How to name and classify your phage: an informal guide. Viruses 9: 70.
  18. Kang HY, Kim S, Kim J. 2015. Isolation and characterization of an Enterococcus faecalis bacteriophage. Kor. J. Microbiol. 51: 194-198. https://doi.org/10.7845/kjm.2015.5025
  19. Uchiyama J, Rashel M, Maeda Y, Takemura I, Sugihara S, Akechi K, et al. 2008. Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate. FEMS Microbiol. Lett. 278: 200-2006. https://doi.org/10.1111/j.1574-6968.2007.00996.x
  20. Xu X, Zhang D, Zhou B, Zhen X, Ouyang S. 2021. Structural and biochemical analyses of the tetrameric cell binding domain of Lys170 from enterococcal phage F170/08. Eur. Biophys J. 50: 721-729. https://doi.org/10.1007/s00249-021-01511-x
  21. Proenca D, Velours C, Leandro C, Garcia M, Pimentel M, Sao-Jose CA. 2015. A two-component, multimeric endolysin encoded by a single gene. Mol. Microbiol. 95: 739-753. https://doi.org/10.1111/mmi.12857
  22. Kim S, Lee DW, Jin JS, Kim J. 2020. Antimicrobial activity of LysSS, a novel phage endolysin, against Acinetobacter baumannii and Pseudomonas aeruginosa. J. Glob. Antimicrob. Res. 22: 32-39. https://doi.org/10.1016/j.jgar.2020.01.005
  23. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10: 845-858. https://doi.org/10.1038/nprot.2015.053
  24. Bae M, Park J, Seong H, Lee H, Choi W, Noh J, et al. 2022. Rapid extraction of viral nucleic acids using rotating blade lysis and magnetic beads. Diagnostics 12: 1995.
  25. Kretzer JW, Lehmann R, Schmelcher M, Banz M, Kim KP, Korn C, et al. 2007. Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells. Appl. Environ. Microbiol. 73: 1992-2000. https://doi.org/10.1128/AEM.02402-06
  26. Rahman MU, Wang W, Sun Q, Shah JA, Li C, Sun Y, et al. 2021. Endolysin, a promising solution against antimicrobial resistance. Antibiotics 10: 1277.
  27. Kunstmann S, Scheidt T, Buchwald S, Helm A, Mulard LA, Fruth A, et al. 2018. Bacteriophage Sf6 tailspike protein for detection of Shigella flexneri pathogens. Viruses 10: 431.
  28. Eugster MR, Haug MC, Huwiler SG, Loessner MJ. 2011. The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid. Mol. Microbiol. 81: 1419-1432. https://doi.org/10.1111/j.1365-2958.2011.07774.x
  29. Kong M, Shin JH, Heu S, Park JK, Ryu S. 2017. Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin. Biosens. Bioelectron. 96: 173-177. https://doi.org/10.1016/j.bios.2017.05.010
  30. Garde S, Calzada J, Sanchez C, Gaya P, Narbad A, Meijers R, et al. 2020. Effect of Lactococcus lactis expressing phage endolysin on the late blowing defect of cheese caused by Clostridium tyrobutyricum. Int. J. Food Microbiol. 329: 108686.
  31. Ma H, O'Fagain C, O'Kennedy R. 2020. Antibody stability: a key to performance - analysis, influences and improvement. Biochimie 177: 213-225. https://doi.org/10.1016/j.biochi.2020.08.019
  32. Langley G, Besser J, Iwamoto M, Lessa FC, Cronquist A, Skoff TH, et al. 2015. Effect of culture-independent diagnostic tests on future emerging infections program surveillance. Emerg. Infect. Dis. 21: 1582-1588. https://doi.org/10.3201/eid2109.150570
  33. Ha SM, Kim CK, Roh J, Byun JH, Yang SJ, Choi SB, et al. 2019. Application of the whole genome-based bacterial identification system, TrueBac ID, using clinical isolates that were not identified with three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Ann. Lab. Med. 39: 530-536. https://doi.org/10.3343/alm.2019.39.6.530
  34. da Silva DAV, Brendebach H, Grutzke J, Dieckmann R, Soares RM, de Lima JTR, et al. 2020. MALDI-TOF MS and genomic analysis can make the difference in the clarification of canine brucellosis outbreaks. Sci. Rep. 10: 19246.
  35. Law JW, Ab Mutalib NS, Chan KG, Lee LH. 2014. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front. Microbiol. 5: 770.
  36. Giuliano C, Patel CR, Kale-Pradhan PB. 2019. A guide to bacterial culture identification and results interpretation. P T 44: 192-200.
  37. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M. 2010. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin. Microbiol. Rev. 23: 235-251. https://doi.org/10.1128/CMR.00043-09
  38. Bhattacharya S. 2013. Early diagnosis of resistant pathogens: how can it improve antimicrobial treatment? Virulence 4: 172-184. https://doi.org/10.4161/viru.23326
  39. Batra A, Cottam D, Lepesteur M, Dexter C, Zuccala K, Martino C, et al. 2023. Development of a rapid, low-cost portable detection assay for enterococci in wastewater and environmental waters. Microorganisms 11: 381.
  40. Zhu B, Hu J, Li X, Li X, Wang L, Fan S, et al. 2022. Rapid and specific detection of Enterococcus faecalis with a visualized isothermal amplification method. Front. Cell. Infect. Microbiol. 12: 991849.
  41. Dreier M, Berthoud H, Shani N, Wechsler D, Junier P. 2020. SpeciesPrimer: a bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species. Peer J. 8: e8544.
  42. Gigante AM, Olivenca F, Catalao MJ, Leandro P, Moniz-Pereira J, Filipe SR, et al. 2021. The mycobacteriophage Ms6 LysB N-terminus displays peptidoglycan binding affinity. Viruses 13: 1377.
  43. Muhammad Jai HS, Dam LC, Tay LS, Koh JJW, Loo HL, Kline KA. 2020. Engineered lysins with customized lytic activities against enterococci and staphylococci. Front. Microbiol. 11: 574739.
  44. Regulski K, Courtin P, Kulakauskas S, Chapot-Chartier MP. 2013. A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins. J. Biol. Chem. 288: 20416-20426. https://doi.org/10.1074/jbc.M112.446344