Skip to main content

Lytic Bacteriophage as a Biomaterial to Prevent Biofilm Formation and Promote Neural Growth

Abstract

Background

Although non-lytic filamentous bacteriophages have been made into biomaterial to guide tissue growth, they had limited ability to prevent bacterial infection. In this work a lytic bacteriophage was used to make an antibacterial biomaterial for neural tissue repair.

Methods

Lytic phages were chemically bound to the surface of a chitosan film through glutaraldehyde crosslinking. After the chemical reaction, the contact angle of the sample surface and the remaining lytic potential of the phages were measured. The numbers of bacteria on the samples were measured and examined under scanning electron microscopy. Transmission electron microscopy (TEM) was used to observe the phages and phage-infected bacteria. A neuroblast cell line was cultured on the samples to evaluate the sample’s biocompatibility.

Results

The phages conjugated to the chitosan film preserved their lytic potential and reduced 68% of bacterial growth on the sample surface at 120 min (p < 0.001). The phage-linked surface had a significantly higher contact angle than that of the control chitosan (p < 0.05). After 120 min a bacterial biofilm appeared on the control chitosan, while the phage-linked sample effectively prevented biofilm formation. The TEM images demonstrated that the phage attached and lysed the bacteria on the phage-linked sample at 120 min. The phage-linked sample significantly promoted the neuroblast cell attachment (p < 0.05) and proliferation (p < 0.01). The neuroblast on the phage-linked sample demonstrated more cell extensions after day 1.

Conclusion

The purified lytic phages were proven to be a highly bioactive nanomaterial. The phage-chitosan composite material not only promoted neural cell proliferation but also effectively prevent bacterial growth, a major cause of implant failure and removal.

Graphical abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol. 2005;175:7512–8.

    CAS  Article  Google Scholar 

  2. Williams DL, Epperson RT, Ashton NN, Taylor NB, Kawaguchi B, Olsen RE, et al. In vivo analysis of a first-in-class tri-alkyl norspermidine-biaryl antibiotic in an active release coating to reduce the risk of implant-related infection. Acta Biomater. 2019;93:36–49.

    CAS  Article  Google Scholar 

  3. Shiels SM, Bouchard M, Wang H, Wenke JC. Chlorhexidine-releasing implant coating on intramedullary nail reduces infection in a rat model. Eur Cell Mater. 2018;35:178–94.

    CAS  Article  Google Scholar 

  4. Hegde V, Park HY, Dworsky E, Zoller SD, Xi W, Johansen DO, et al. The use of a novel antimicrobial implant coating in vivo to prevent spinal implant infection. Spine (Phila Pa 1976). 2020;45:E305–11.

  5. Ghosh C, Sarkar P, Issa R, Haldar J. Alternatives to conventional antibiotics in the era of antimicrobial resistance. Trends Microbiol. 2019;27:323–38.

    CAS  Article  Google Scholar 

  6. Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. 2019;25:730–3.

    CAS  Article  Google Scholar 

  7. Romero-Calle D, Guimarães Benevides R, Góes-Neto A, Billington C. Bacteriophages as alternatives to antibiotics in clinical care. Antibiotics (Basel). 2019;8:138.

  8. Markoishvili K, Tsitlanadze G, Katsarava R, Morris JG Jr, Sulakvelidze A. A novel sustained-release matrix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing wounds. Int J Dermatol. 2002;41:453–8.

  9. Cheng W, Zhang Z, Xu R, Cai P, Kristensen P, Chen M, et al. Incorporation of bacteriophages in polycaprolactone/collagen fibers for antibacterial hemostatic dual-function. J Biomed Mater Res B Appl Biomater. 2018;106:2588–95.

    CAS  Article  Google Scholar 

  10. Hay ID, Lithgow T. Filamentous phages: masters of a microbial sharing economy. EMBO Rep. 2019;20:e47427.

  11. Zhou N, Li Y, Loveland CH, Wilson MJ, Cao B, Qiu P, et al. Hierarchical ordered assembly of genetically modifiable viruses into nanoridge-in-microridge structures. Adv Mater. 2019;31:e1905577.

  12. Chung WJ, Merzlyak A, Yoo SY, Lee SW. Genetically engineered liquid-crystalline viral films for directing neural cell growth. Langmuir. 2010;26:9885–90.

    CAS  Article  Google Scholar 

  13. Sultankulov B, Berillo D, Sultankulova K, Tokay T, Saparov A. Progress in the development of chitosan-based biomaterials for tissue engineering and regenerative medicine. Biomolecules. 2019;9:470.

    CAS  Article  Google Scholar 

  14. Pallavali RR, Degati VL, Lomada D, Reddy MC, Durbaka VRP. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections. Plos One. 2017;12:e0179245.

    Article  Google Scholar 

  15. Bryan D, El-Shibiny A, Hobbs Z, Porter J, Kutter EM. Bacteriophage T4 infection of stationary phase E. coli: life after log from a phage perspective. Front Microbiol. 2016;7:1391.

  16. Koskella B. Bacteria-phage interactions across time and space: Merging local adaptation and time-shift experiments to understand phage evolution. Am Nat. 2014;184:S9–21.

    Article  Google Scholar 

  17. Shen HY, Liu ZH, Hong JS, Wu MS, Shiue SJ, Lin HY. Controlled-release of free bacteriophage nanoparticles from 3D-plotted hydrogel fibrous structure as potential antibacterial wound dressing. J Control Release. 2021;331:154–63.

  18. Jamal M, Hussain T, Rajanna Das C, Andleeb S. Isolation and characterization of a myoviridae MJ1 bacteriophage against multi-drug resistant Escherichia coli 3. Jundishapur J Microbiol. 2015;8:e25917.

    Article  Google Scholar 

  19. Cheung DT, Nimni ME. Mechanism of crosslinking of proteins by glutaraldehyde I: reaction with model compounds. Connect Tissue Res. 1982;10:187–99.

    CAS  Article  Google Scholar 

  20. Ertürk G, Lood R. Bacteriophages as biorecognition elements in capacitive biosensors: Phage and host bacteria detection. Sensor Actuators B Chem. 2018;258:535–43.

    Article  Google Scholar 

  21. Janczuk-Richter M, Marinović I, Niedziółka-Jönsson J, Szot-Karpińska K. Recent applications of bacteriophage-based electrodes: A mini-review. Electrochem Commun. 2019;99:11–5.

  22. Lin HY, Chen SH, Chang SH, Huang ST. Tri-layered chitosan scaffold as a potential skin substitute. J Biomater Sci Polym Ed. 2015;26:855–67.

  23. Subramanian A, Krishnan UM, Sethuraman S. Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration. J Biomed Sci. 2009;16:108.

    Article  Google Scholar 

  24. Menzies KL, Jones L. The impact of contact angle on the biocompatibility of biomaterials. Optom Vision Sci. 2010;87:387–99.

    Article  Google Scholar 

  25. Wiegand C, Zieger M, Rode C, Schroter K, Krahmer A, Wyrwa R, et al. JIS L 1902 and ISO 22196 for determination of antifungal properties of textiles and ceramic surfaces. Mycoses. 2011;54:416–7.

    Google Scholar 

  26. Mottaghitalab F, Farokhi M, Mottaghitalab V, Ziabari M, Divsalar A, Shokrgozar MA. Enhancement of neural cell lines proliferation using nano-structured chitosan/poly(vinyl alcohol) scaffolds conjugated with nerve growth factor. Carbohydr Polym. 2011;86:526–35.

  27. Alhosseini SN, Moztarzadeh F, Mozafari M, Asgari S, Dodel M, Samadikuchaksaraei A, et al. Synthesis and characterization of electrospun polyvinyl alcohol nanofibrous scaffolds modified by blending with chitosan for neural tissue engineering. Int J Nanomedicine. 2012;7:25–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. King AM, Lefkowitz E, Adams MJ, Carstens EB. Virus taxonomy: ninth report of the international committee on taxonomy of viruses. Amsterdam: Elsvier; 2011.

  29. Bibi Z, Abbas Z, Rehman SU. The phage P.E1 isolated from hospital sewage reduces the growth of Escherichia coli. Biocontrol Sci Technol. 2016;26:181–8.

    Article  Google Scholar 

  30. Abedon ST. Lysis of lysis-inhibited bacteriophage T4-infected cells. J Bacteriol. 1992;174:8073–80.

    CAS  Article  Google Scholar 

  31. Pallavali RR, Degati VL, Lomada D, Reddy MC, Durbaka VRP. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections. PLoS One. 2017;12:e0179245.

    Article  Google Scholar 

  32. Kim SG, Jun JW, Giri SS, Yun S, Kim HJ, Kim SW, et al. Isolation and characterisation of pVa-21, a giant bacteriophage with anti-biofilm potential against Vibrio alginolyticus. Sci Rep. 2019;9:6284.

  33. Melo LDR, Ferreira R, Costa AR, Oliveira H, Azeredo J. Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model. Sci Rep. 2019;9:6643.

    Article  Google Scholar 

  34. Roberts GAF, Taylor KE, Gels C. The formation of gels by reaction of chitosan with glutaraldehyde. Makromol Chem. 1989;190:951–60.

    CAS  Article  Google Scholar 

  35. Budianto E, Muthoharoh SP, Nizardo NM. Effect of crosslinking agents, pH and temperature on swelling behavior of cross-linked chitosan hydrogel. Asian J Appl Sci. 2015;3:581–8.

  36. Tolba M, Minikh O, Brovko LY, Evoy S, Griffiths MW. Oriented immobilization of bacteriophages for biosensor applications. Appl Environ Microbiol. 2010;76:528–35.

    CAS  Article  Google Scholar 

  37. Bennett AR, Davids FG, Vlahodimou S, Banks JG, Betts RP. The use of bacteriophage-based systems for the separation and concentration of Salmonella. J Appl Microbiol. 1997;83:259–65.

    CAS  Article  Google Scholar 

  38. Bumgardner JD, Wiser R, Elder SH, Jouett R, Yang Y, Ong JL. Contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium. J Biomater Sci Polym Ed. 2003;14:1401–9.

  39. Wang X, Zhang Q. Role of surface roughness in the wettability, surface energy and flotation kinetics of calcite. Powder Technol. 2020;371:55–63.

    CAS  Article  Google Scholar 

  40. Khan S, Newaz G. A comprehensive review of surface modification for neural cell adhesion and patterning. J Biomed Mater Res A. 2010;93:1209–24.

    Article  Google Scholar 

  41. Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials. 2005;26:2603–10.

    CAS  Article  Google Scholar 

  42. Khan SP, Auner GG, Newaz GM. Influence of nanoscale surface roughness on neural cell attachment on silicon. Nanomedicine. 2005;1:125–9.

    CAS  Article  Google Scholar 

Download references

Acknowledgement

This work is supported by a grant from the Ministry of Science and Technology, Taiwan. Grant number: MOST 110-2637-E-027-006.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hsin-Yi Lin.

Ethics declarations

Conflict of interest

The authors have no financial conflicts of interest to declare.

Informed consent

No informed consents are obtained.

Ethical statement

This study did not involve any human or animal subjects and have no ethical issues for human and animal right.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, ZH., Chiang, MT. & Lin, HY. Lytic Bacteriophage as a Biomaterial to Prevent Biofilm Formation and Promote Neural Growth. Tissue Eng Regen Med (2022). https://doi.org/10.1007/s13770-022-00462-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13770-022-00462-4

Keywords

  • Lytic bacteriophage
  • Antibacterial biomaterial
  • Chitosan biopolymer
  • Neural cell growth