Chapter 20

Bacteriophage Attack as an Anti-biofilm Strategy

Sanna Sillankorva and Joana Azeredo

Abstract
Bacteriophages are bacterial viruses. Bacteriophages replicate inside their target host whether this is in
planktonic or biofilm forms. Here, we describe the methods used to control readily formed biofilms using
bacteriophages.

Key words Bacteriophage, Biofilm, Control

1 Introduction

Bacteriophages are bacterial viruses that infect bacteria and can be

used as biocontrol agents targeting bacterial cells either in suspen-
sion or in biofilm. The vast majority of known bacteriophages
(96 %) belong to the Caudovirales order which encompasses
dsDNA-tailed bacteriophages [1]. These phages start their infec-
tion cycles upon specifically binding to bacterial receptors displayed
at the cell surface and inject their nucleic acid into the cell
cytoplasm. There are two main bacteriophage life cycles—lytic and
lysogenic. Lytic or virulent bacteriophages reproduce within the
host and induce lysis, resulting in the release of progeny bacterio-
phages that start another round of infection. Temperate bacterio-
phages integrate their prophage into the chromosome or other
replicon of the host bacteria which, through cell division, pass the
bacteriophage genome (prophage) to daughter cells [2]. Only lytic
bacteriophages are of interest for bacterial biocontrol [3]. The bac-
teriophage plaques can vary in turbidity and size depending on the
bacteriophage characteristics, the host physiological state, and the
growth conditions. Furthermore, plaques can sometimes be sur-
rounded by a halo, an indicative of genes encoding for extracellular
polymeric substances (EPS) depolymerase within the bacterio-
phage genome [4]. Bacteriophages that exhibit this type of charac-
teristic are particularly useful for biofilm control because the

Gianfranco Donelli (ed.), Microbial Biofilms: Methods and Protocols, Methods in Molecular Biology, vol. 1147,
DOI 10.1007/978-1-4939-0467-9_20, © Springer Science+Business Media New York 2014

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278 Sanna Sillankorva and Joana Azeredo

depolymerases released after cell lysis, by disrupting the biofilm

matrix, help bacteriophage to gain access to the inner layers of the
biofilm [5]. Besides the diffusional barrier to bacteriophage pene-
tration imposed by the biofilm matrix, the presence of slow-grow-
ing cells constitutes an extra challenge to bacteriophage efficacy in
biofilms since bacteriophages need the enzymatic machinery of
actively growing cells, replicating faster in exponentially growing
cells [6]. Nevertheless, there are bacteriophages that display an
active life cycle in stationary growing cells, such as T7-like phages,
which is undoubtedly a useful characteristic for biofilm control [7].
Biofilm control can be done by adding a bacteriophage sus-
pension to already formed biofilm. Bacteriophages will diffuse
through the biofilm and cause cell lysis. Along with cell lysis, the
number of bacteriophages increases, and frequently, small portions
of biofilms detach to the planktonic phase. The infection pro-
gresses both in the remaining biofilm and detached cells [8].
Biofilm control can also be made prior to biofilm formation, and in
this case, the substratum surface needs to be coated with bacterio-
phages. The virus particles on the surfaces prevent the colonization
of bacterial cells and thus prevent biofilm formation [9].
In this chapter, we describe all necessary steps for biofilm con-
trol and prevention assays using bacteriophages.

2 Materials

In order to use bacteriophages for biofilm control, one must pro-

duce them to have high-titer bacteriophage stocks. Therefore, the
procedure to obtain these stocks has been included in this chapter.

2.1 Bacteriophage 1. Bacteriophage stock.

Propagation 2. Tissue culture flask(s) (100 cm2) containing a thin agar layer
of LB: Weigh 8.0 g of LB and 4.8 g of agar and pour in a
500 mL bottle. Adjust to 400 mL with distilled water and
autoclave at 121 °C for 15 min. Once the LB agar has cooled
to 42 °C, pour on the tissue culture flasks (see Note 1).
3. Overnight-grown bacteria: Transfer a loopful of the host bac-
terium to 100 mL Erlenmeyers containing 25 mL of sterile LB
and incubate 16 h at the proper host growth temperature.
4. Sterile molten top agar (MTA): Weigh 8.0 g of LB and 2.4 g
of agar and pour in a 500 mL bottle. Adjust to 400 mL with
distilled water and autoclave at 121 °C for 15 min and store
accordingly (see Note 2).
5. Sterile saline magnesium buffer (SM buffer): Prepare 1 M
Tris–HCl buffer (pH 7.5) in a 100 mL bottle. Weigh 6.06 g
of Tris base, add 50 mL of water, and adjust the pH with HCl
 Bacteriophage as Anti-biofilm Strategy 279

to 7.5. To a 1 L bottle, add 5.8 g of NaCl, 2.0 g of

MgSO4·7H2O, and 50 mL of the prepared 1 M Tris–HCl
(pH 7.5) and make up to 1 L with water (see Note 3).
Autoclave at 121 °C for 15 min.
6. Sterile 50 mL Falcon tubes.
7. Sterile 200 mL bottle.
8. Filters 0.2 μm.

2.2 Bacteriophage 1. Sterile 15 and 50 mL Falcon tubes.

Concentration and 2. Sterile 500 mL Erlenmeyers.
Purification
3. Sterile 100 and 200 mL bottles.
4. Filters 0.2 μm.
5. DNase I.
6. RNase A.
7. Solid NaCl.
8. Solid polyethylene glycol 8000 (PEG 8000).
9. Chloroform (≥99.8 %).
10. Sterile saline magnesium buffer (SM buffer).

2.3 Biofilm 1. 24-well microplates (see Note 4).

Formation 2. LB medium: Weigh 8.0 g of LB and adjust to 400 mL with
distilled water. Autoclave at 121 °C for 15 min.
3. Overnight-grown bacteria: Transfer a loopful of the host bac-
terium to 100 mL Erlenmeyers containing 25 mL of sterile LB
and incubate 16 h at the proper host growth temperature.
4. Sterile cell scrapers.
5. Sonication bath.

2.4 Prevention of 1. Bacteriophage-conditioned 24-well microplates: Add a high

Biofilm Formation concentration of bacteriophage (>1 × 109 PFU per mL) to
Using Bacteriophages 24-well microplates and leave at least 1 h at room tempera-
ture. Remove all bacteriophage prior to use and wash the
24-well microplate twice with SM buffer.
2. LB media.
3. Overnight-grown bacteria.
4. Sterile cell scrapers.
5. Sonication bath.

2.5 Biofilm Control 1. 24-well microplates containing biofilms.

with Bacteriophages 2. Bacteriophage.
3. LB medium.
280 Sanna Sillankorva and Joana Azeredo

4. Sterile cell scrapers.

5. Sonication bath.

2.6 Bacteriophage 1. Sterile SM buffer.

Titration 2. 96-well microplates.
3. LB agar plates.
4. Overnight-grown bacteria.

2.7 Biofilm Cell 1. LB agar plates (20 plates).

Enumeration 2. 96-well microplates.
3. Sterile saline solution: To a 1 L bottle, add 9.0 g of NaCl and
make up to 1 L with water. Autoclave at 121 °C for 15 min.

3 Methods

Carry out all procedures at room temperature unless otherwise

specified.

3.1 Bacteriophage 1. Add 1 mL of an overnight-grown culture and 1 mL of diluted

Propagation bacteriophage (approximate titer 1 × 105 PFU per mL) in 5 tissue
culture flasks (100 cm2) containing an agar layer of LB and mix
gently.
2. Incubate for 15 min at the proper temperature to allow bacte-
riophages to adsorb to the host bacterium.
3. Add 30 mL of MTA (47 °C) and let it harden.
4. Incubate overnight, without inverting, at the proper tempera-
ture (see Note 5).
5. Add 30 mL of SM buffer to the tissue culture flask(s) (100 cm2)
and incubate a minimum of 5 h at 4 °C under slow agitation
(see Note 6).
6. Remove the SM buffer with the eluted bacteriophages to
50 mL Falcon tubes.
7. Centrifuge (9,000 × g, 4 °C, and 10 min).
8. Carefully collect the supernatant, and filter (0.2 µm) to sterile
or 200 mL bottles.
9. Store at 4 °C until further use (see Note 7).

3.2 Concentration This protocol was adapted from Sambrook and Russell [10]; how-
and Purification of ever, it includes some minor modifications and does not include an
Bacteriophage Lysates ultracentrifugation step.
1. To a 200 mL bottle, add 100 mL of bacteriophage lysate
(see Subheading 3.1 step 9) and DNase I and RNase A (1 μg/mL
each).
 Bacteriophage as Anti-biofilm Strategy 281

2. Incubate for 30 min at room temperature.

3. Add 5.84 g of NaCl and mix gently until the NaCl has
dissolved.
4. Incubate at 4 °C or on ice under agitation (50–90 rpm) for 1 h.
5. Pour onto 50 mL Falcon tubes and centrifuge (9,000 × g,
4 °C, 10 min).
6. Collect the supernatant to a 500 mL Erlenmeyer.
7. Add PEG to the supernatant (10.0 g PEG per 100 mL
solution).
8. Incubate 5 h to overnight at 4 °C under gentle agitation
(50–90 rpm).
9. Pour onto 50 mL Falcon tubes and centrifuge (9,000 × g,
4 °C, 10 min).
10. Discard the supernatant and resuspend the pellet, containing
the precipitated bacteriophage particles, in SM buffer contain-
ing 2 % gelatin. Use 6 mL of SM buffer for each 50 mL of
centrifuged sample.
11. Transfer the bacteriophage solution to 15 mL Falcon tubes
and add chloroform (see Note 8) in a proportion of 1:4 (v/v).
Vortex for 30 s.
12. Centrifuge (3,500 × g, 4 °C, 5 min).
13. Recover and filter (0.2 μm) the aqueous phase (upper phase)
which contains the bacteriophage to a 100 mL Falcon tube
and store at 4 °C.
14. Determine the bacteriophage titer as described in
Subheading 3.6.

3.3 Biofilm 1. Add to a 24-well microplate 1 mL of sterile LB media (see Note 4).
Formation on 2. Add 10 μL of a bacterial culture adjusted to an OD600 of 1.0.
Microplates
3. Incubate at appropriate temperature conditions and under
agitation (120 rpm or at static conditions (0 rpm)) during the
desired period of time (e.g., 24 h—7 days), replacing the
media every 12 h to remove planktonic bacteria and enhance
biofilm formation.
4. At the end of the desired biofilm formation period, remove all
media.
5. Wash twice the wells with 1 mL of sterile LB media.
6. Add 1 mL of LB media.
7. Use a cell scraper to scrape the biofilm from the well surface.
8. Put the 24-well microplate on a sonication bath for 30 min.
9. Quantify the viable cells present in the biofilms as described
below (see Subheading 3.7) (see Note 9).
282 Sanna Sillankorva and Joana Azeredo

3.4 Prevention of 1. Add 1 mL of LB to a 24-well microplate conditioned with

Biofilm Formation with bacteriophages.
Bacteriophages 2. Follow all steps described above for biofilm formation (see
Subheading 3.3).
3. Quantify the number of bacteriophages and viable cells as
described in Subheadings 3.6 and 3.7.

3.5 Biofilm Control After quantifying the numbers of viable cells in the biofilms, in at
with Bacteriophages least three independent assays performed in triplicate, the efficacy
of bacteriophages for biofilm control can be evaluated. In order to
maintain the infection parameters identical along the experiments,
a constant initial multiplicity of infection (MOI) must be used.
MOI is calculated according to the number of bacteriophages per
number of viable host cells, and, for instance, an MOI of 1 repre-
sents that there is 1 bacteriophage to each host cell (see Note 10).
1. After biofilm formation and washing (Subheading 3.3 step 4),
add 950 µL of LB media and 50 µL of bacteriophage at a
proper concentration in order to have the desired constant
multiplicity of infection (MOI).
2. Incubate at the proper temperature during at least 4 h (see
Note 11).
3. Take samples (Fig. 1a) to quantify the numbers of bacterio-
phages and viable cells (see Subheadings 3.6 and 3.7), respec-
tively, present in the planktonic stage (see Note 12).
4. Remove the spent media and wash twice with sterile LB media.
5. Add 1 mL of sterile LB media.
6. Use a cell scraper to scrape the biofilm from the surface
(Fig. 1b).
7. Put the 24-well microplate on a sonication bath for 30 min.
8. Take samples to quantify the numbers of bacteriophages and
viable cells (see Subheadings 3.6 and 3.7), respectively, present
in the biofilm stage (Fig. 1c).

3.6 Bacteriophage After infection, bacteriophages can be found in both planktonic

Titration and biofilms phases (Fig. 1) and should be quantified.
For bacteriophages producing small plaques (<1–2 mm in
diameter), use the double agar overlay method (Subheading 3.6.1)
[11], and for bacteriophages producing large plaques (>2 mm in
diameter), use the small drop plaque assay (Subheading 3.6.2) [12].

3.6.1 Bacteriophage 1. Prepare successive serial dilutions (1:10) in SM buffer of the

Enumeration by Double bacteriophage solutions (add 20 μL of bacteriophage solution
Agar Overlay and 180 μL of SM buffer to a 96-well microplate).
2. Add to a test tube 100 µL of bacteriophage solution, 100 µL
of overnight-grown bacteria, and 3–5 mL of MTA (47 °C)
and tap gently.
 Bacteriophage as Anti-biofilm Strategy 283

Fig. 1 Final stage of a biofilm control with bacteriophages. (a) Removal of samples
to quantify the bacteriophages and bacteria in the planktonic phase; (b) using a
cell scraper to scrap the biofilm; (c) removal of samples to quantify the bacterio-
phages and bacteria that were present only in the biofilm phase

3. Pour on an agar plate with LB and swirl carefully.

4. Let the plates dry for 1–2 min.
5. Incubate inverted overnight under optimal temperature
conditions.
6. Count the bacteriophage plaques in the dilution which resulted
in 20–200 plaques.
7. Determine the titer of triplicate preparations according to Eq. 1:
No. of plaques ´ Dilution factor
Bacteriophage titre ( PFU per mL ) = (1)
Volume of phage sample ( mL )
284 Sanna Sillankorva and Joana Azeredo

3.6.2 Bacteriophage 1. In 96-well microplates, serially dilute the bacteriophage sam-

Enumeration Using the ples in sterile SM buffer (20 µL of sample in 180 µL of SM
Small Drop Plaque Assay buffer).
2. To a 96-well microplate, add 20 µL of diluted bacteriophage
solution and 20 µL of an overnight-grown bacterial culture
and pipet up and down a couple of times.
3. Incubate during 5–15 min at the proper growth temperature
and under agitation (120 rpm) to allow bacteriophages to
adsorb to the bacteria.
4. Pipet up and down a few times and add a drop of 20 µL of the
dilution mixture onto an agar plate with LB.
5. Allow the drops to dry completely.
6. Incubate overnight at the proper growth temperature.
7. Count the plaques formed in the drop of the dilution with
3–30 bacteriophage plaques.
8. Calculate the titer of the bacteriophage of triplicate prepara-
tions using Eq. 1.

3.7 Biofilm Cell 1. In 96-well plates, serially dilute the bacterial samples in sterile
Enumeration saline solution (20 µL of sample in 180 µL of SM buffer).
2. Add a drop of 20 µL of sample on an agar plates containing LB.
3. Allow the drop to dry completely.
4. Incubate overnight at the proper growth temperature.
5. Count the colonies formed in the drop of the dilution with
3–30 colonies.
6. Calculate the number of viable cells using Eq. 2:
No. of colonies ´ Dilution factor
Nr. of viable cells (CFU per mL ) = (2)
Volume of sample ( mL )

4 Notes

1. Bacteriophage propagation can alternatively be done using

Petri dishes. Additionally, the propagation can also be
done by infecting mid-exponential bacterial cells with bac-
teriophages. LB is the suggested medium, however alter-
native media can be used.
2. Leave the bottle with MTA at 65 °C if the media is to be used
within 24 h. If planned to be used after 24 h, it is preferable to
let the MTA solidify. Use a water bath (100 °C) or microwave
oven to melt the MTA.
3. Optional: 2 % of gelatin (w/v) can be added to SM buffer.
Gelatin is known to preserve bacteriophages and thus can be
used in the later steps of bacteriophage purification.
 Bacteriophage as Anti-biofilm Strategy 285

4. Biofilms can also be formed on a variety of different substrata.

In this case, coupons of the different materials (stainless steel,
rubber, silicone, acrylic, etc.) can be cut and placed on the
wells. Alternatively, other microplates can be used (e.g., 6-well,
48-well, and even 96-well microplates).
5. In some circumstances, for example, with T7-like phages, 7 h
is enough, and after this, SM buffer can be added.
6. The bottle can alternatively be left overnight at 4 °C.
7. Frequently lysates, such as those obtained after this step, are
used without any further purification.
8. Chloroform is used to extract PEG; however, it should be used
with caution as some bacteriophages may be sensitive to it.
9. It is necessary to quantify the number of viable cells in order
to allow the researcher to use a constant MOI throughout all
the infection assays.
10. The MOIs commonly used vary between 0.1 and 1,000, but
other bacteriophage–host ratios can be tested.
11. According to the results from different authors, the maximum
cell lysis is obtained after 4–5 h, and after this point there can
be observed an increase of cell growth due to the present of
phage-resistant phenotypes.
12. The infection of biofilms with bacteriophages results often in
a release of cell clusters to the planktonic phase, and therefore,
this should be assessed.

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