Application Note 34

Bacteriophage storage, transportationand delivery in nanofibrillar cellulose
Sheetal Patpatia 1, Saija Kiljunen 1, Lauri Paasonen 2, Mikael Skurnik 1
1
Department of Bacteriology and Immunology, Faculty of Medicine, University of Helsinki, Finland
2
UPM Kymmene Corporation, Finland

INTRODUCTION

The emergence of antibiotic resistant bacteria due to the misuse of antibiotics presents
a global challenge in terms of increased morbidity, mortality, and healthcare costs [1].
According to the World Health Organization (WHO), antibiotic resistance is one of thebiggest threats to global health, food security, and development of our time [2].
Bacteriophages (phages) are viruses that infect only bacteria. They take over the hostbacterium metabolism, use it to reproduce themselves, and kill the host bacterium. Phages
have high host specificity; a given phage can only infect a certain bacterial species. One
promising alternative to treat infections caused by antibiotic resistant bacteria is phagetherapy (PT), where phages are used to kill the pathogens [3]. The positive outcome fromthe specificity of phages is that the adverse effects of PT are minimal, whereas the broad
spectrum antibiotics cause adverse effects related to their effect also on the normal humanmicrobiome.
The strict host specificity requires the therapy to be targeted individually for each pathogenic
strain. In order to find an effective phage, fast and efficient screening of large bacteriophagelibraries with the infecting strain is needed. The ability to store phages reliably for
prolonged times is a prerequisite for their effective diagnostic and therapeutic use (Fig. 1).
Bacteriophages need to remain viable during storage and shipping between laboratories.
In this study, we demonstrated the use of nanofibrillar cellulose hydrogel, GrowDex®, as astorage, transportation and delivery matrix for the phages.

Figure 1. Schematic illustration of
personalised treatment of bacterial
infection with phage therapy

MATERIALS• GrowDex® (Cat No. 100103002, UPM, Finland)• LB agar plates for bacterial culture (1.5% agar)• SM buffer (100 mM NaCl, 10mM MgSO4, 50 mM Tris-HCl, pH 7.5, 0.01% gelatin)• Bacterial strains• Staphylococcus aureus Storage no. #5676 (HUSlab, Helsinki, Finland)		
Storage no. #6433 (unpublished)• Escheria coli
Storage no. #5521 (HUSlab)		
Storage no. #5507 (HUSlab)• Bacteriophages infecting• Staphylococcus aureus fRuSau02 [4] and DSMZ phage (DSMZ, Germany)• Escherichia coli 		fHoEco02 [5] and fTuEco01 (unpublished)

METHODS

1. Effect of GrowDex on bacteria viability
1. 10 µl drops of 1.5% GrowDex were aliquoted into 96-well microplate.
2. 200 µl of overnight culture of S. aureus #5676 diluted in LB medium was added tothe wells and the plate incubated at 37°C.
3. Bacterial growth was monitored by measuring the absorbance at 600 nm for 6 h.
2. Stability and release of phages diluted in GrowDex andapplied to a bacteria culture plate
1. GrowDex 1.5% was mixed with phage lysate to give a final GrowDex concentrationof 1% or 0.5%, respectively.
2. Different dilutions of phage lysate were tested from 104 to 107.
3. 10 µl drops of GrowDex with phages were pipetted onto the bacteria culture plateor alternatively the phages were sprayed on culture plate and then incubated at37°C.
4. Release of phages from GrowDex to infect and kill the bacteria on the plates wasanalyzed visually.
3. Viability and stability of phages in GrowDex tested in liquid cultures
1. GrowDex 1.5% was mixed with phage lysate to give a final GrowDex concentrationof 1% or 0.5%, respectively.
2. The mixture was stored in tubes or in wells of a 96-well microplate in 10 µl drops.
3. 200 µl of overnight bacterial culture diluted in LB medium was added to each of thewells and the plate incubated at 37°C.
4. Absorbance was measured at 600 nm for 5 h at 1 h intervals.

RESULTS

1. Effect of GrowDex on bacteria viability
If GrowDex is to be used as a storage matrix for phages it is important to show that it does
not have an independent impact on bacteria viability. When S. aureus bacteria was cultured
in wells containing GrowDex, the rate of bacteria proliferation was seen to be similar to thecontrol condition without GrowDex (Fig. 2).

Figure 2. Viability ofStaphylococcus aureus
bacteria (#5676) cultured+/- GrowDex (1.5%).

2. Release of phages from GrowDex
In order to infect and kill target bacteria the phages need to remain viable in the storage
matrix. However, equally important is the need for easy release of phages from the matrix
so they are free to infect the host. Here it was shown that phage release from GrowDex not
only resulted in infection of the bacteria (Fig. 3A,) but also gave comparable results to thoseobtained with release of phages directly from SM buffer or NaCl (Fig. 3B). The diluent,
SM buffer (upper row) and NaCl (lower row), did not have an effect on phage infection
capability.

Figure 3. Titration of DSMZ phagelysate stored 2 h in 1% GrowDex(A). Phage storage in NaCl or SM
buffer were used as controls (B).
Bacteria strain used was S. aureus(#6433), and numbers -4, -5, etc.
indicate the 104 and 105 etc. dilutionsof the phage lysate respectively.

3. Viability of phages stored in GrowDex
Storage tests performed at +4°C showed that phages can remain stable and viable in
GrowDex for extended periods of time. When stored in microcentrifuge tubes, all four ofthe phages studied were seen to retain their infectivity even after 6 months. The results withfRuSau02 phage are shown in Fig. 4.
When phages were stored in 10 µl drops of GrowDex at +4°C in 96-well microplates,
some differences in the stability were observed. The small podoviruses, fTuEco01 and DSMZ
phage, retained their activity throughout the 6 month study, fTu-Eco01 results at the 6 monthtime point is shown in Fig. 5A. However, large myoviruses such as fRuSau02 were shown tobe infective at 8 weeks (Fig. 5C) but were inactivate at the 6 month time point (Fig. 5D). It isthought that storage in small volumes for greater than eight weeks may result in drying of thesample resulting in inactivation of large phages.
Figure 4. Six month storage of
fRuSau02 phage in 0.5% and 1%
GrowDex and in SM buffer. Timepoints
were 24 h (A), 8 weeks (B), and 6months (C). Panel D indicates the growthof the host S. aureus strain #5676
without the phage.

Figure 5. Phage activity after storage
in 10 µl drops in a 96-well microplatefor 6 months. fTu-Eco01 retained
its infectivity (A), host E. coli strain#5507 culture without phage wasused as a control (B). Large myovirus
fRuSau02 (C) was active at 8 weeks
but inactive at 6 months (D).

4. Transportation of a ready to use phage testing plate
In the search for phages active against bacterial strains isolated from patients, phagelibraries often need to be transported between different laboratories. Here a 96-well
microplate containing phages dispensed in 10 µl drops of 1% GrowDex was shippedusing the normal postal service between two sites. On delivery viability of the phages wasmeasured using the protocols described previously. Results showed that all four phages
remained viable after postal transportation indicating that a ready-to-screen plate may
be likely possibility and hold great potential for the diagnosis and subsequent treatment ofpatients.
5. Treatment of topical infections
With topical infections delivery of phages to the infected area could be done by spraying
phages directly onto the skin. Using GrowDex as a carrier for the phages could increase
contact time with the skin, resulting in better efficacy and less pain for the patient. The shearthinning property of GrowDex means it becomes temporarily more fluid when a force isapplied, thus it can be sprayed easily with e.g. finger-operated pump bottles (Fig 6A). Using
this approach we are able to demonstrate that the phages remained active after spraying
and were able to effectively kill the target bacteria (Fig 6B).
Figure 6. Delivery of phagesin GrowDex hydrogel onto solid
surfaces by spraying. Due to its
shear-thinning property GrowDex canbe sprayed using a finger-operated
pump bottle (A). fRuSau02 phages in
0.5% GrowDex were sprayed onto
S. aureus #5676 plates (B), 0.5%
GrowDex without phages was used asa control (C).

CONCLUSIONSREFERENCES

Treatment of antibiotic resistant bacterial infections is very challenging and new innovative
approaches are needed to treat infected patients quickly and easily. Phages offer great
potential for treatment of these infections however as mentioned, would benefit froma robust way to store, distribute, diagnose and deliver phages to the patient. Here wehave demonstrated the versatility of GrowDex as a matrix for phage storage, host range
screening, and preparation of ready-to-screen phage plates. GrowDex can also be used
as carrier for topical application of phages directly to the infected site. The robust nature,temperature stability and shear-thinning properties of GrowDex make it an ideal matrix foruse in multiple bacteriophage applications.
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UPM Biomedicals
Alvar Aallon katu 1
P.O. Box 38000101 Helsinki, Finland
biomedicals@upm.com
www.upmbiomedicals.com
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