Long term behaviour of lyophilized phages in relation to its morphological stability and titre

Long term behaviour of lyophilized phages in relation to its morphological stability and titre

Authors: VinodKumar C.S [1], Suneeta Kalasuramath [1,2], Srinivasa H [3]

Article Received: 14/07/2021
Received: 16/08/2021
Accepted: 24/08/2021


Bacteriophage therapy is one of the hopeful alternatives to combat the snowballing problem of antibiotic resistance. Lyophilization of bacteriophage could improve their stability in lLONG-term storage. The aim of this study was to assess the stability of lyophilized bacteriophages over the period of 20 years. 6-7 log decrease in the phage titre and morphological changes were observed when lyophilized phages were cultured after 20 years.

Keywords: St abilit y, Phage t it r e, L yophilizat ion


The emergence and spread of drug-resistant patho-gens that have acquired new resistance mechanisms, leading to antimicrobial resistance, continues to threaten our ability to treat common infections. Especially alarming is the rapid global spread of multi- and pan-resistant bacteria that cause infections that are not treatable with existing antimicrobial medicines such as antibiotics. Anti-biotics are becoming increasingly ineffective as drug-resistance spreads globally leading to more difficult to treat infections and death. New antibacterials are urgently needed 2 – for example, to treat carbapenem-resistant gram-negative bacterial infections as identified in the WHO priority pathogen list. The clinical pipeline of new antimicrobials is dry. In 2019 WHO identified 32 antibiotics in clinical development that address the WHO list of priority pathogens, of which only six were classified as innovative. The one of the alternative to treat drug resistant pathogen is bacteriophage. Bacteriophages are a sustainable alternative to state in order to maintain structural integrity and extend the lLONGevity of the microorganisms stored at either room or refrigeration temperatures. There is a paucity of information on the lLONG-term viability of preserved bacteriophages. Since 2 decades, various phages were isolated against MDR bacteria. The stability and the phage titre was estimated yearly and the results are presented in this article.


The phages were isolated from different sources against MDR bacteria isolated from clinical specimens by the method of Smith and Huggins [3].

In-vitro confirmation of bacteriophage activity of all the phages isolated were done by the standard technique [3].The bacteriophage isolated were lyophilized and stored at -20°C [4,5]. The morphology of the phages were studied before and after lyophilisation using transmission electron microscopy. The Recovery of lyophilized phages were done by reconstituting phage with 1 mL of buffer and quantifying using double-layer plaque assay. The phage stability and titre was estimated once in a year.


tudy period included from October 2001 to September 2021. Over 20 years 124 bacteriophages were isolated against various MDR bacteria.bacteriophages were lyophilized (Approximate concentration PFU/ml) and maintained in the stock (Table-1). All the phages isolated had an icosahedral head, measuring about 70-100 nm in diameter, and a 100-120nm lLONG tail. Based on the morphology and the guidelines provided by International Committee on Taxonomy of Viruses (ICTV, Bethesda MD, USA) all the phages are tentatively placed in the Siphov iridae, Myoviridae and Podoviridae family [5].In-vitro confirmation study revealed all the phages isolated were lytic phages.

An annual titre determination revealed most of lyophilized phages retained original post lyophilisation titres. After five years’ retention [34], phages showed 1log reduction and 5 phages showed approximately the initial concentration. At the end of the 10 years, 32 phages showed 3 log reduction, 7 phages showed 2 log reduction in the phage titre. At the end of 15 years, 27 phages showed 4 log reduction and 10 phages showed 5 log reduction and 2 phages showed 6 log reduction. At the end of 20 years, 19 phages showed 5 log reduction, 16 phages showed 6 log reduction and 4 phages showed 7 log reduction in the phage titre (Table-1). Transmission electron microscopy imaging of the selected phages revealed that at the end of the 1st year, most of the phages retained their morphologies. The minimum damage to morphology was observed at the end of the 5 year and maximum damage to morphology was observed at the end of the 20th year.


1. de Lencastre H, Oliveira D, Tomasz A. Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive power. Curr. Opin. Microbiol.2007;10:428–35

2. Livermore DM. The need for new antibiotics. Clin. Microbiol. Infect. 2004;10(Suppl 4):1–9.

3. Smith HW, Huggins MB. Successful treatment of experimental E. coli infections in mice using phage; its superiority over antibiotics. Journal of General Microbiology 1982; 128:307-318.

4. Zierdt, C. H. Preservation of staphylococcal bacteriophage by means of lyophilization. Am. J. Clin. Pathol. 1959;31:326-331.

5. Ackermann, H. W., Tremblay, D. & Moineau, S. LLONG-term bacteriophage preservation. World. Fed. Cult. Collect. Newslett. 2004;38, 35–40.

Mirzaei MK, Eriksson H, Kasuga K, HaggårdLjungquist E, Nilsson AS. Genomic, proteomic, morphological, and phylogenetic analyses of vB_EcoP_SU10, a podoviridae phage with C3 morphology. PLoS One. 2014; 9(12):e116294

Nelson D. Phage taxonomy: We agree to disagree. J Bacteriol 2004;186:7029– 7031.

Phages Lyophilized concentration 1 Year 5 Year 10 Year 15 Year 20 Year
Staphylococcus aureus Ø1 9.544 9.041 8.944 6.964 3.914 2.813
Staphylococcus aureus Ø2 9.431 9.342 8.813 6.653 5.886 3.886
MRSA Ø1 9.602 9.505 8.716 6.505 4.748 3.765
MRSA Ø2 9.491 9.462 8.681 6.763 4.643 3.643
MRSA Ø3 9.255 9.204 8.46 6.342 5.505 3.944
MRSA Ø4 9.322 9.255 8.740 6.556 5.672 3.602
Staphylococcus epidermidis Ø1 9.580 9.556 8.845 6.778 5.653 2.279
Staphylococcus epidermidis Ø2 9.613 9.623 8.869 6.924 4.892 2.881
Enterococci faecalis Ø1 9.591 9.531 8.833 6.991 4.681 3.792
Enterococci faecalis Ø2 9.204 9.146 8.505 6.079 4.255 3.643
Enterococci faecalis Ø3 9.431 9.380 8.763 6.886 4.580 3.771
Enterococci faecium Ø1 9.519 9.491 8.771 6.462 4.833 3.792
Enterococci faecium Ø2 9.613 9.519 8.708 6.531 4.806 3.672
Enterococci faecium Ø3 9.322 9.279 8.623 6.568 4.591 3.886
Enterococci faecium Ø4 9.857 9.732 8.672 6.833 4.602 3.716
Streptococcus pneumococci Ø 9.820 9.653 8.964 6.892 3.991 2.653
E.coli Ø1 9.380 9.342 8.556 6.568 5.663 3.643
E.coli Ø2 9.146 9.041 8.462 7.041 5.255 3.892
E.coli Ø3 9.230 9.079 8.544 7.672 5.505 3.813
How to cite this article?

VinodKumar C.S, Suneeta K, Srinivasa H. Titre LLONG term behaviour of lyophilized phages in relation to its morphological stability and titre. Int J Bacteriophage Res 2021:1:37-9