POTENTIAL APPLICATION OF LYOPHILIZATION IN COMMERCIAL USE OF BACTERIOPHAGE PREPARATIONS IN VETERINARY MEDICINE
DOI:
https://doi.org/10.26873/SVR-396-2017Abstract
Microbial contamination in livestock, resulting in foodborne illnesses, poses a serious problem for veterinary. Rapidly growing number of antibiotic-resistant bacterial strains triggers increased interest in phage therapy, which has already been tested against zoonotic pathogens in animals.
The aim of the study was to investigate the potential application of lyophilization process in commercial processing of phages. We lyophilized three phages active against common animal pathogens E. coli, Salmonella and Enterococcus, in the presence of three different cryoprotectants. The activity of phages was determined after 10 days, 1 month, 3 months, 6 months and 1 year storage at room temperature. All conditions of storage were established to consider practical aspects of phages processing.
Skim milk appeared to be the most effective cryoprotectant, however the obtained results varied for different phages, suggesting that efficiency of this process was phage-dependent. This in turn, may be problematic during optimization of phage lyophilization for commercial processing. Nevertheless, further commercialization of phage preparations seems to be unavoidable and the development of new methods for phage processing and storage is required.
Key words: bacteriophages; lyophilization; freeze-drying; long-term preservation of bacteriophages
MOŽNOSTI UPORABE LIOFILIZACIJE V KOMERCIALNE NAMENE ZA PRIPRAVO BAKTERIOFAGOV V VETERINARSKI MEDICINI
Mikrobiološka kontaminacija hrane v živinoreji, ki povzroča bolezni, predstavlja resen problem v veterinarski medicini. Hitro naraščajoče število bakterijskih sevov, odpornih proti antibiotikom vzbuja vedno večje zanimanje za uporabo t.i. fagoterapije, zdravljenja z bakterijskimi virusi bakteriofagi, ki je bila že preizkušena kot metoda za preprečevanje okužb pri živalih. Cilj raziskave je bil proučiti potencialno uporabo postopka liofilizacije za komercialno obdelavo fagov. Liofilizirali smo tri fage, aktivne proti skupnim živalskim patogenom Escherichia coli, Salmonella sp. in Enterococcus faecalis v prisotnosti treh različnih krioprotektantov. Dejavnost fagov je bila določena po 10 dneh, 1 mesecu, 3 mesecih, 6 mesecih in 1 letu skladiščenja pri sobni temperaturi. Vzpostavljeni so bili vsi pogoji shranjevanja, z namenom upoštevanja praktičnih vidikov obdelave fagov. Posneto mleko je bilo najučinkovitejši krioprotektant, vendar so se dobljeni rezultati razlikovali pri različnih fagih, kar kaže na to, da je učinkovitost postopka odvisna od vrste faga. To bi lahko predstavljalo težavo pri optimizaciji liofilizacije fagov v komercialne namene. Kljub temu je nadaljnja komercialna priprava preparatov s fagi neizogibna in bo potrebno razvijati nove metode za obdelavo in shranjevanje fagov.
Ključne besede: bakteriofagi; liofilizacija; sušenje z zamrzovanjem; dolgoročno ohranjanje bakteriofagov
References
(1) Nobrega FL, Costa AR, Kluskens LD, Azeredo J. Revisiting phage therapy: new applications for old resources. Trends Microbiol 2015; 23: 185–91.
(2) Dini C, Islan GA, de Urraza PJ, Castro GR. Novel biopolymer matrices for microencapsulation of phages: enhanced protection against acidity and protease activity. Macromol Biosci 2015; 12: 1200–8.
(3) Johnson S, Taylor CM. What’s new in haemolytic uraemic syndrome? Eur J Pediatr 2008; 167: 965–71.
(4) Rivero MA, Padola NL, EtcheverrÃa AI, Parma AE. [Enterohemorrhagic Escherichia coli and hemolytic-uremic syndrome in Argentina]. Medicina (B. Aires) 2004; 64: 352–6.
(5) Boerlin P. Implications of antimicrobial agents as therapeutics and growth promoters in food animal production. In: Sabour PM, Griffiths M, eds. Bacteriophages in the control of food and waterborne pathogens. Washington : ASM Press, 2010: 1–9.
(6) Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage therapy. Antimicrob Agents Chemother 2001; 45: 649–59.
(7) Sillankorva SM, Oliveira H, Azeredo J. Bacteriophages and their role in food safety. Int J Microbiol 2012; 2012: e863945 (13 pp.). https://www.hindawi.com/journals/ijmicro/2012/863945/
(8) Ly-Chatain MH. The factors affecting effectiveness of treatment in phages therapy. Front Microbiol 2014; 5: e51 (7 pp.). https://www.frontiersin.org/articles/10.3389/fmicb.2014.00051/full
(9) Hubálek Z. Protectants used in the cryopreservation of microorganisms. Cryobiology 2003; 46: 205–29.
(10) Merabishvili M, Vervaet C, Pirnay JP, et al. Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization). PLoS One 2013; 8(7): e68797 (7 pp.). http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0068797&type=printable
(11) Carlson K, Miller ES. Working with T4. In: Molecular biology of bacteriophage T4
Washington: American Society for Microbiology Press, 1994: 421–26.
(12) Adams MH. The bacteriophages. New York: Interscience Publishers, 1959: 27–30.
(13) Clark WA. Comparison of several methods for preserving bacteriophages. Appl Microbiol 1962; 10: 466–71.
(14) Dini C, de Urraza PJ. Effect of buffer systems and disaccharides concentration on Podoviridae coliphage stability during freeze drying and storage. Cryobiology 2013; 66: 339–42.
(15) Bedu-addo FK. Understanding lyophilization formulation development. Pharm Pharmacol 2004; 28: 10–8.
(16) Puapermpoonsiri U, Ford SJ, van der Walle CF. Stabilization of bacteriophage during freeze drying. Int J Pharm 2010; 389: 168–75.
(17) Alfadhel M, Puapermpoonsiri U, Ford SJ, McInnes FJ, van der Walle CF. Lyophilized inserts for nasal administration harboring bacteriophage selective for Staphylococcus aureus: in vitro evaluation. Int J Pharm 2011; 416: 280–7. doi:10.1016/j.ijpharm.2011.07.006
(18) Balcão VM, Azevedo AF, Castro LM, et al. Design of a lipid nanovesicle system encapsulating bacteriophages integrated in a multiple emulsion formulation: a proof-of-concept. In: NSTI-Nanotech. Nanotechnology 2010: bio sensors, instruments, medical, environment and energy. Vol. 3. Boca Raton: CRC Press, 2010: Chapter 7, 459–62.
http://www.nsti.org/procs/Nanotech2010v3/7
(19) Puapermpoonsiri U, Spencer J, van der Walle CF. A freeze-dried formulation of bacteriophage encapsulated in biodegradable microspheres. Eur J Pharm Biopharm 2009; 72: 26–33.
(20) Callaway TR, Edrington TS, Brabban A, et al. Evaluation of phage treatment as a strategy to reduce Salmonella populations in growing swine. Foodborne Pathog Dis 2011; 8: 261–6.
(21) Monk AB, Rees CD, Barrow P, Hagens S, Harper DR. Bacteriophage applications: where are we now? Lett Appl Microbiol 2010; 51: 363–9.
(22) Wall SK, Zhang J, Rostagno MH, Ebner PD Phage therapy to reduce preprocessing Salmonella infections in market-weight swine. Appl Environ Microbiol 2010; 76: 48–53.
(23) Heyse S, Hanna LF, Woolston J, Sulakvelidze A, Charbonneau D. Bacteriophage cocktail for biocontrol of Salmonella in dried pet food. J Food Prot 2015; 78: 97–103.
