SURGICAL INFECTIONS

Volume 19, Number 00, 2018 Original Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/sur.2018.135

Evaluation of Bacteriophage Anti-Biofilm Activity

for Potential Control of Orthopedic Implant-Related
Infections Caused by Staphylococcus Aureus

Jodie Morris,1,2 Natasha Kelly,3 Lisa Elliott,4 Andrea Grant,1 Matthew Wilkinson,1
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Kaushik Hazratwala,1 and Peter McEwen1

Abstract

Background: Despite significant advancements in surgical protocols and biomaterials for orthopedics, peri-
prosthetic joint infection (PJI) remains a leading cause of implant failure. Staphylococcus aureus nasal colo-
nization is an established risk factor for PJI, with methicillin-sensitive S. aureus a leading cause of orthopedic
implant-related infections. The purpose of these in vitro studies was to investigate the antibacterial activity of a
tailored bacteriophage cocktail against planktonic and biofilm-associated S. aureus.
Methods: The S. aureus strains (n = 30) were screened for their susceptibility to a library of S. aureus-specific
bacteriophage (n = 31). Five bacteriophage preparations that demonstrated bactericidal activity against >90% of
S. aureus strains tested were combined as a StaPhage cocktail and assessed for their antibacterial activity toward
planktonic and biofilm-associated S. aureus, with biofilms established on three-dimensional-printed porous
titanium scaffolds.
Results: StaPhage treatment immediately after bacterial inoculation inhibited growth of S. aureus by >98% in
eight hour cultures when multiplicity of infection of phages to bacteria was greater than 1:1 (p < 0.01). Viable
bacterial numbers within biofilms on titanium surfaces were significantly reduced (6.8 log10 to 6.2 log10 colony
forming units [CFU]; p < 0.01) after exposure to the StaPhage cocktail, in vitro. No significant reduction was
observed in biofilms exposed to 100 times the minimal inhibitory concentration of cefazolin (log10 6.81 CFU).
Conclusions: Combined, these data demonstrate the in vitro efficacy of S. aureus-specific bacteriophage cock-
tails against S. aureus growing on porous titanium and warrant further in vivo studies in a clinically relevant
animal model to evaluate the potential application of bacteriophage in the management of PJI caused by S. aureus.

Keywords: bacteriophage; biofilm; implant; prosthetic joint infection; Staphylococcus aureus

P rosthetic joint infections (PJI) are a devastating

problem in orthopedic surgical procedures resulting in
significant morbidity and major healthcare costs [1,2]. Al-
most a quarter of revision operations for implant failure are
because of infection [3,4]. Improvements to pre-operative
and surgical protocols have reduced the incidence of PJI to
below 1%; however, the costs for management of this com-
plication can be 10-fold higher than uncomplicated ar-
throplasty [3,4].
The formation of bacterial biofilms is intrinsic to the
pathogenesis of PJI [5–7]. The ability of bacteria such as
S. aureus to form biofilms on orthopedic implants makes
diagnosis and treatment of PJI extremely challenging [3,7].
Biofilms are complex microbial communities encased in a
self-produced polysaccharide matrix that serves to protect
bacteria from the host immune response and antibiotic
agents, thus facilitating persistent infection and increased
likelihood for the emergence of antibiotic-resistant bacterial
strains [5]. The alarming global rise in antibiotic-resistant
bacterial strains is driving an urgent need for the development
of innovative strategies to combat bacterial infections [8].
Bacteriophage therapy offers an alternate strategy for the

1
Orthopaedic Research Institute of Queensland, Townsville, Australia.
2
College of Medicine and 3College of Public Health Medical and Veterinary Sciences, Division of Tropical Health and Medicine, James
Cook University, Townsville, Australia.
4
AusPhage Pty Ltd, Townsville, Australia.

1
 2 MORRIS ET AL.
management of PJI, particularly where there are limited ef-
fective antibiotic agents available.
Bacteriophages, or phages, are naturally occurring viruses
that infect only bacteria without harming eukaryotic cells [9].
Lytic phages infect and replicate within a specific host bac-
terium causing lysis and death of the bacterial cell and release
of large numbers of newly assembled virions that can attack
more target bacteria, perpetuating the cycle for as long as
there are live bacterial target cells present. Therefore, unlike
antibiotic agents that decrease in concentration below the
surface of bacterial biofilms, phages can penetrate into bio-
films making them particularly useful for managing chronic,
persistent bacterial infections [10].
Phage cocktails, mixtures of individual phage types, have
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been used to broaden the spectrum of activity against path-
ogenic bacteria such as S. aureus [11]. Phage therapy has
been standard clinical practice for management of bacterial
endocarditis, periodontitis, gastrointestinal, respiratory, bone
and joint, and urinary tract infections in Eastern Europe for
almost a century [12]. Although the clinical application of
phages declined in Western countries with the advent of
antibiotic agents, phage therapy is becoming increasingly
considered globally as an attractive adjunct or alternative
treatment to antibiotic agents in response to the increase in
antimicrobial resistance [9,12].
Staphylococcus aureus nasal colonization is an established
risk factor for PJI after arthroplasty [13]. Molecular studies
have demonstrated the genetic relatedness of S. aureus strains
from surgical infections with those colonizing skin in the pre-
operative period [13,14]. Methicillin-sensitive S. aureus
(MSSA) causes approximately 45% of PJI cases, with MSSA
infections 2.5 times more common than PJI caused by
methicillin-resistant S. aureus (MRSA) [3,15]. Despite an-
tibiotic sensitivity, the ability of MSSA strains to establish
biofilms on the surface of orthopedic materials makes erad-
ication extremely challenging. Therefore, the purpose of the
current study was to investigate the antibacterial activity of
S. aureus-specific phages against planktonic and biofilm-
associated MSSA strains recovered from the skin of patients
undergoing total joint replacement, as proof-of-concept
in vitro studies for future potential therapeutic application of
phage for management of PJI caused by S. aureus.
Methods
Bacteria and bacteriophages
A panel of S. aureus strains (n = 30) recovered from an-
terior nares of patients (n = 94) undergoing total knee or hip
FIG. 1. Transmission electron microscopy (TEM, Tecnai 12, FEI, The Netherlands) image of representative phage
preparations, (a) StaPh_1 and (b) StaPh_4. Anti-Staphylococcus aureus phage preparations were from the Myoviridae
family, with icosahedral heads and contractile tails. Magnification is 100,000. Scale bar is 200 nm. (c) Custom three-
dimensional–printed, porous titanium cylinders were used to assess anti-S. aureus biofilm activity of StaPhage, in vitro.
arthroplasty was used in the current study after approval from
the Institutional Ethics Committee. The S. aureus strains
were isolated from pre-operative nasal swabs and identified
using standard microbiologic and biochemical techniques.
Methicillin resistance was confirmed using a rapid im-
munochromatographic PBP2a culture colony test (Alere,
Australia). Glycerol stocks of S. aureus isolates were stored
at -80C until further analysis.
The S. aureus-specific phages (StaPhage) (n = 31) prepa-
rations used in the current study were a kind gift by Dr. L.
Elliot (AusPhage Pty Ltd). Lytic StaPhage were isolated from
sewage water, triple plaque purified, and characterized using
methods described previously [16]. Host sensitivity range
was determined for each preparation using spot testing of
serially diluted lysates against a panel of veterinary and
clinical isolates of S. aureus as part of the routine isolation
procedure [17]. Phage classification was confirmed by mor-
phology using transmission electron microscopy (TEM,
Tecnai 12, FEI, The Netherlands). The StaPhage preparations
provided were from the family Myoviridae (Fig. 1a, b) and
contained 1 · 107–9 plaque-forming units (PFU) per mL.
S. aureus sensitivity screening
Akin to antimicrobial susceptibility testing of bacterial
isolates, phage sensitivity screening was performed using
standard spot tests [17]. The S. aureus strains (n = 29 MSSA,
n = 1 MRSA) were screened against a panel of individual
StaPhage preparations (n = 31) to identify phage preparations
with a broad spectrum of lytic activity for subsequent in-
clusion in a StaPhage cocktail. Propagating strains of S. au-
reus were used in parallel to test strains to confirm viability,
activity, and concentration of StaPhage preparations. Bac-
terial sensitivity was considered where confluent, semi-
confluent, opaque lysis, or individual plaques were observed
in the bacterial lawn. Data are presented as the percentage of
S. aureus strains tested that demonstrated sensitivity to each
individual StaPhage preparation.
Phage efficacy against planktonic S. aureus
Based on in vitro sensitivity screening assays, five lytic
StaPhage preparations (StaPh_1, StaPh_3, StaPh_4,
StaPh_11, and StaPh_16) were selected for further analysis.
To compare the inhibitory effect of individual StaPhage
preparations with a pooled cocktail of the individual prepa-
rations on bacterial growth, two MSSA strains (OR-
I16_C02N and ORI16_025) were cultured in the presence of
StaPhage at a multiplicity of infection (MOI) of 10 (phage):1
 PHAGE EFFICACY AGAINST S. AUREUS
(bacteria). To compare the effect of MOI on inhibition of
planktonic growth of S. aureus, the StaPhage cocktail was
added to cultures of two additional MSSA strains
(ORI16_002 and ORI16_096) at MOI 0, 0.01:1, 0.1:1, 1:1, or
1:10. Individual StaPhage preparations or a cocktail con-
taining an equal concentration of the five pooled StaPhage
preparations were added to log-phase S. aureus cultures (105
CFU), at the indicated MOI.
Growth kinetics (37C, 50 rpm) were monitored over an
eight hour period with hourly measurement of optical density
(OD, 600 nm). Data are expressed as mean OD or, where
indicated, as percentage reduction in bacterial growth com-
pared with untreated cultures according to the formula:
(ODuntreated - ODtreated)/ODuntreated *100. To enumerate via-
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ble bacteria, parallel culture plates were prepared, and at four
hours, aliquots were removed from untreated and treated
cultures, serially diluted, and plated onto tripticase soy agar
(TSA) with colonies enumerated after overnight incubation.
Biofilm-forming capacity of S. aureus
The S. aureus strains (n = 10) were screened for their
ability to form biofilms, where each isolate was categorized
on strength of biofilm formation in comparison with a ref-
erence S. aureus strain of known biofilm-forming capacity
(ATCC 25923) [18]. Biofilms were established in microtiter
plates using log phase suspensions of S. aureus strains
(5 · 104 CFU/mL, tryptic soy broth (TSB) with 0.5% glu-
cose). After incubation (48 h, 37C), biofilm formation was
assessed using a colorimetric crystal violet technique and
measurement of OD, 590 nm [19]. The cutoff optical density
(ODc) for each plate was defined as three standard deviations
above the mean OD of the blank wells. Thresholds to dis-
tinguish weak, moderate, and strong biofilm formation were
set at 2 · ODc or 4 · ODc, respectively.
Phage efficacy against biofilm-coated titanium
surfaces
Titanium is commonly used in prosthetic joint compo-
nents, with three-dimensional (3D)-printed titanium in-
creasingly being used in orthopedic implants [20]. To assess
the efficacy of phages against biofilms growing on a clini-
cally relevant orthopedic material, two MSSA strains (OR-
I16_C02N and ORI16_C02J) that demonstrated strong
biofilm-forming capacity on polystyrene surfaces were se-
lected for further analysis of their ability to colonize the
surface of customized 3D-printed, porous titanium cylindri-
cal scaffolds (Fig. 1c, 70% porosity, 5 mm · 1.6 mm) de-
signed for use in our rat model of knee implant surgery (a
kind gift from R. Wood, Stryker Orthopaedics).
ORI16_C02N was isolated from the anterior nares of a
patient who had undergone total knee arthroplasty (TKA)
with uncomplicated recovery; however, the patient presented
with acute-onset septic arthritis in the operated knee three
years post-TKA. The ORI16_C02J was recovered from sy-
novial fluid of the implanted knee of this patient before de-
bridement with implant retention and intravenous
flucloxacillin therapy. Staphylococcal single neucleotide
polymorphism based-typing [21] confirmed MSSA status,
with both strains found to be of the same clonal complex,
CC78, with identical binary profiles for pvl, cna, sdrE,
pUB110, and pT181.
3
Biofilms were established by placing sterile implants into
log phase suspensions of S. aureus (104 CFU) for 48 hours,
with a media change performed after four and 24 hours. Ce-
fazolin is frequently used as a first-line antibiotic agent for
management of post-surgical infections caused by MSSA in
orthopedics [3]. To compare in vitro efficacy of cefazolin and
StaPhage cocktail, biofilm-coated implants were left untreated
or treated with cefazolin (50 mg/mL, 100X minimum inhibi-
tory concentration [MIC]) or StaPhage cocktail (2 · 106 PFU)
for 48 hours, with media changed after 24 hours of culture.
After 48 hours of treatment, implants were sonicated
(5 · 5 min rounds, 100% power, XUB12 Grant Ultrasonic
bath) in recovery media (phosphate buffered saline [PBS],
pH 7.2 with 0.1% Triton-X-100 and 0.15% ethylenediami-
netetraacetic acid [EDTA]). Recovery media were centri-
fuged (3000g, 10 min, 4C), bacteria re-suspended in sterile
PBS, and serial 10-fold dilutions plated in duplicate onto
TSA to enumerate colonies after 24 hours. Data are expressed
as log10 CFU per implant.
Scanning electron microscopy
StaPhage-treated, cefazolin-treated, and untreated biofilm-
coated titanium implants were fixed in 2.5% glutaraldehyde
in 0.1M sodium cacodylate buffer, sputter-coated with gold,
and visualized by scanning electron microscopy (SEM,
JSM5410LV, Jeol Ltd).
Statistical analysis
Statistical analyses were performed using GraphPad Prism
for Mac software (version 7). All experiments were con-
ducted in triplicate or quadruplicate. Results are expressed as
mean – standard error (SE). Two-way analysis of variance
(ANOVA) with Holm-Sidak multiple comparison test was
used to compare in vitro antibacterial activity of StaPhage
preparations between S. aureus strains. Comparison between
treatment groups was performed using one-way ANOVA
with the Levene test for equality of variances and Tukey post-
hoc analysis for pairwise comparison. Differences corre-
sponding to p < 0.05 were considered significant.
Results
Sensitivity of S. aureus nasal isolates to phage
Phage sensitivity screening was performed to identify
StaPhage preparations with broad activity against S. aureus
strains for use in subsequent in vitro efficacy assays. Ap-
proximately half of the StaPhage preparations tested (17 of
31) demonstrated lytic activity against >90% of S. aureus
strains tested (Fig. 2). Of the 30 S. aureus strains isolated
from the anterior nares of TKA patients, one strain was
identified as MRSA positive (ORI16_041). Twenty-five of
the 31 (80.6%) StaPhage preparations demonstrated lytic
activity against this MRSA strain. Given that MSSA is more
commonly associated with PJI [3,15], however, further in-
vestigations of the effectiveness of StaPhage preparations
were based on the MSSA strains recovered.
Inhibition of S. aureus growth
To compare the inhibitory effect of individual StaPhage
preparations and a pooled cocktail on bacterial growth, two
 4 MORRIS ET AL.
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FIG. 2. Phage sensitivity screening was performed against a panel of Staphylococcus aureus strains (n = 30) isolated from
pre-operative nasal swabs of patients undergoing joint replacement surgical procedures. More than 90% of the S. aureus
strains screened showed sensitivity toward 17 of the 31 phage preparations tested.
MSSA strains (ORI16_C02N and ORI16_25) were cultured
in the presence of StaPhage at an MOI of 10:1. Differences
were observed in the antibacterial activity of individual
StaPhage preparations against the two S. aureus strains
(Fig. 3a). In vitro bacterial growth of ORI16_C02N and
ORI16_025 was inhibited by up to 98.3% and 88.3% in the
presence of at least one StaPhage preparation, respectively.
There was no additive effect observed, however, on the re-
duction in bacterial growth of ORI16_C02N or ORI16_025
under these in vitro conditions when individual StaPhage
preparations were pooled as a cocktail.
A dose-response inhibition effect was observed on in vitro
growth of two additional MSSA skin-associated strains,
ORI16_002 and ORI16_096, after addition of the StaPhage
cocktail at increasing MOI (Fig. 3b,c). Bacterial growth was
reduced by 98.9% and 98.5% for ORI16_002 (p < 0.01) and
ORI16_096 (p < 0.01), respectively, where the StaPhage cock-
tail was added at MOI 1:1. Enumeration of viable bacterial
numbers confirmed a significant decrease in ORI16_002
(p < 0.01) and ORI16_096 (p < 0.01) in the presence of
StaPhage cocktail at an MOI 10:1 (Fig. 3c). Combined, data
demonstrate that StaPhage is effective at inhibiting in vitro
growth of S. aureus when cultured in the presence of MOIs
greater than 1:1.
Anti-biofilm activity of StaPhage cocktail
Having demonstrated the efficacy of StaPhage in inhibiting
planktonic growth of S. aureus in vitro, we subsequently
sought to investigate the antibacterial activity of StaPhage
toward S. aureus biofilms. Five of 10 (50%) of the S. aureus
strains screened demonstrated strong in vitro biofilm-forming
capacity on polystyrene surfaces (Fig. 4). Two MSSA strains
derived from a TKA patient in whom PJI had developed
(ORI16_C02N, ORI16_C02J) were selected for further ana-
lyses based on previous demonstration of their in vitro sus-
ceptibility to the StaPhage cocktail in planktonic form, the
strength of in vitro biofilm formation, and in vitro suscepti-
bility to cefazolin in planktonic form (Fig. 5a).
After 48 hours in culture with titanium implants, levels of
ORI16_C02N and ORI16_C02J within biofilms reached 6.3
and 6.2 log10 CFU, respectively. Although both S. aureus
strains were shown to be sensitive to cefazolin, viable bac-
terial numbers recovered from untreated and cefazolin-
treated (50 mg/mL, 100X MIC) biofilm-coated titanium
implants were comparable for ORI16_C02N (6.81 log10 CFU,
p = 0.52) and ORI16_C02J (6.44 log10 CFU, p = 0.49)
(Fig. 5b). Despite demonstrating effectiveness of the StaPh-
age cocktail in inhibition of planktonic growth of OR-
I16_C02J and ORI16_C02N, differences were observed in
the anti-biofilm activity of StaPhage toward the two S. aureus
strains. Bacterial numbers in ORI16_C02J biofilms exposed
to the StaPhage cocktail in vitro or untreated were similar
(p = 0.86). In contrast, a significant decrease (3.3-fold,
p = 0.004) was observed in bacterial numbers remaining on
ORI16_C02N-coated implants after exposure to the StaPh-
age cocktail compared with untreated implants. Micro-
biological data were confirmed with SEM images of
untreated, cefazolin-treated, and StaPhage treated biofilms of
ORI16_C02N on titanium implants (Fig. 5c–e), demonstrat-
ing reduction in the thickness and area covered by S. aureus
biofilm after 48 hours for implants exposed to the StaPhage
cocktail.
Discussion
Despite significant improvements in surgical techniques
and advancements in biomaterials for orthopedics, infection
remains a leading cause of revision of total knee and hip
arthroplasty [4]. Methicillin-sensitive S. aureus is the or-
ganism most commonly associated with PJI, with skin col-
onization an established risk factor for infection after
arthroplasty [3,13,15]. The global threat of antibiotic resis-
tance has ignited interest in the use of bacteriophages as an
alternate management option for biofilm-related infections,
including PJI. We have conducted proof-of-principle in vitro
studies demonstrating potential application of lytic bacte-
riophages in the management of S. aureus biofilms growing
on porous titanium, a material commonly used in the man-
ufacture of orthopedic implants.
Phages are highly abundant in the environment and are
relatively inexpensive to isolate and purify [9]. Despite the
 PHAGE EFFICACY AGAINST S. AUREUS
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FIG. 3. (a) Percentage reduction in the eight hour growth
of Staphylococcus aureus strains, ORI16_C02N and
ORI16_025 when exposed to StaPh_1, StaPh_3, StaPh_4,
StaPh_11 and StaPh_16 alone or combined as a StaPhage
cocktail compared with untreated bacterial cultures.
(b) Percentage reduction in the eight hour growth of S.
aureus strains, ORI16_002 and ORI16_096 when exposed
to StaPhage cocktail at increasing multiplicity of infection
(MOI), compared with untreated bacterial cultures. (c)
Bacterial numbers expressed as log10 colony forming units
(CFU) in four hour cultures of ORI16_002 and ORI16_096
exposed to StaPhage at an MOI of 0.1:1 or 10:1. Data
shown are representative of three or more independent
experiments and displayed as mean – standard error.
* p < 0.05, ** p < 0.01. A and v indicate p < 0.05, for
within-group comparison with MOIs 0.01:1 and 0.1:1,
respectively.
5
use of phages for medical, veterinary, agricultural, and
aquaculture application for almost a century, there have been
no reported adverse safety concerns for mammalian cells
[9,12,22]. Their specificity, safety for mammalian cells, and
self-propagating characteristics are key characteristics for
which phages are being increasingly being considered for the
management of biofilm-related infections outside of Eastern
Europe, where phage therapy has been used since 1921 [9].
The first report of clinical application of staphylococcal
phages was for the successful management of skin infections
[9]. Since then, staphylococcal phages have also been in-
vestigated for the management of bacteremia, eye, respira-
tory, gastrointestinal, urinary tract, and bone infections [23].
More than half of the individual phage preparations screened
in the current study possessed lytic activity against the S.
aureus strains recovered from arthroplasty patients. Five
representative phages preparations were shown to prevent the
planktonic, in vitro growth of MSSA and an MRSA strain.
The wide host spectrum of many of the individual phage
preparations screened suggests their polyvalent nature and
potential use in anti-staphylococcal cocktails. Notably, dif-
ferences were observed in the spot test screening and the lytic
activity of the phage preparations in broth cultures, high-
lighting the importance of the screening technique selected
for determining the phage host range. These differences may
reflect distinct mechanisms of action contributing to bacterial
cell lysis. Plasticity in phage host range has also been dem-
onstrated and may evolve over time or under different se-
lective pressures, rather than being a fixed characteristic [24].
Often phage cocktails containing three or more individual
phage preparations are used to widen the spectrum of anti-
bacterial activity and the success rate of phage therapy for
chronic bacterial infections [9,10,12]. In the current in vitro
studies, we did not observe an additive effect on inhibition of
planktonic S. aureus growth when individual phage prepa-
rations were combined as a cocktail. Although the most ef-
fective phage preparation differed for each S. aureus strain
investigated, typically one of the five individual preparations
tested exerted >90% reduction in bacterial growth when ad-
ded alone at the time of bacterial seeding, suggesting an in-
creased adsorption efficiency for the given strain of S. aureus.
Inclusion of a ‘‘high-performing’’ preparation within a
cocktail format would eliminate target bacterial cells rapidly,
thus masking less obvious effects of additional phages on
inhibition of planktonic S. aureus growth, in vitro. Never-
theless, the highly lytic impact of phages against planktonic
growth of S. aureus strains was demonstrated with a 6 log
decline of CFU within 4 hours of phage delivered at an MOI
of 10:1. The reported optimal MOI for in vivo effectiveness
of phages is between 1 and 10 [22], which is in accordance
with our observations in the current study.
While the pathogenesis of PJI remains poorly understood,
biofilm formation is considered pivotal [6,7]. Biofilm-
forming capacity of S. aureus is thought to be influenced by
clonal variation, rather than gene regulation, and is not
influenced by antibiotic resistance [25,26]. Consistent with
the literature [25], we observed differences in the capacity of
S. aureus associated with skin carriage to form biofilms
in vitro using a polystyrene microtiter assay. Porous titanium
is used widely in orthopedic implants, and 3D-printing is
being increasingly used for manufacture of patient-specific
orthopedic implants [20]. To investigate the anti-biofilm
 6 MORRIS ET AL.
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FIG. 4. Biofilm formation of S.taphylococcus aureus strains on polystyrene surfaces after 48 hour growth at 37C,
in vitro. Biofilm formation was quantified by measuring the optical density at 590 nm (OD590) of dissolved crystal violet.
Means and standard error of the mean values of OD590 values of five replicate wells for each bacterial strain are shown. A S.
aureus reference strain (ATCC25923) with known biofilm-forming ability was included as a positive control. The cutoff OD
(ODc) was defined as three standard deviations above the mean OD of the negative control. Biofilm production was
classified as follows: ODc < OD <2 · ODc = weak biofilm production; (2 · ODc) < OD <4 · ODc = moderate biofilm pro-
duction; and 4 · ODc < OD = strong biofilm production.

activity of StaPhage against biofilms growing on clinically
relevant orthopedic materials, we utilized 3D-printed porous
titanium implants designed for a rat model of knee implant
surgery.
Eradication of biofilms with conventional antibiotic ther-
apy is extremely difficult, with MICs of 100 to 1000 times
higher required for effectiveness against bacteria growing in
biofilms than against bacteria in planktonic form [27], in-
creasing the risk for collateral tissue damage. Data demon-
strated that StaPhage more efficiently killed bacteria on the
surface of implants than cefazolin, despite use of 100X MIC
and despite demonstrating cefazolin sensitivity of these

FIG. 5. Anti-biofilm activity of StaPhage cocktail. (a) Percentage reduction in the eight hour growth of Staphylococcus
aureus strains, ORI16_C02J and ORI16_C02N, when exposed to increasing concentrations of cefazolin (0.02 to 2 mcg/mL)
compared with untreated bacterial cultures. (b) 48 hour biofilms of ORI16_C02J and ORI16_C02N on titanium implants
were exposed to cefazolin (50 mcg/mL) or StaPhage cocktail (106 plaque-forming units, PFU) for 48 hours and bacterial
numbers enumerated. Data show mean log10 colony forming units (CFU) – standard error of four independent samples
conducted for each treatment condition. * p < 0.01. Representative scanning electron microscopy images of (c) untreated,
(d) cefazolin-treated and (e) StaPhage-treated ORI16_C02N biofilms on titanium implants. Disruption to the S. aureus
biofilm structure was evident in StaPhage-treated implants. The magnification level is X1000. The scale bar is 10 mm.
 PHAGE EFFICACY AGAINST S. AUREUS
strains in planktonic form. The tolerance of MSSA biofilms
to cefazolin is consistent with findings of Urish et al. [28],
who demonstrated persister bacterial cells at supra-
therapeutic levels of cefazolin, irrespective of biofilm depth.
Cefazolin is used widely for pre- and peri-operative pro-
phylaxis in orthopedic surgical procedures and is the antibi-
otic agent of choice for management of post-operative
infections caused by MSSA. Clinical failure of cefazolin has
been described for high inoculum (>107 CFU) endocarditis
and osteomyelitis infections, is associated with type-A and C
beta-lactamase-producing MSSA strains, and is independent
of clonal type [29,30]. A significant proportion of MSSA
strains recovered from patients with osteomyelitis possesses
the type A beta-lactamase variant, although cefazolin pro-
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phylaxis itself does not appear to exert a selective pressure for
persistence of these MSSA strains in vivo [31,32].
While beta-lactamase typing of these strains was not per-
formed, it is possible that the dramatic contrast in cefazolin
sensitivity of planktonic and biofilm-associated OR-
I16_C02N and ORI16_C02J observed in the current study is
a reflection of this high inoculum effect. Nevertheless, these
data highlight the challenges of eradicating MSSA biofilms
on orthopedic materials with cefazolin and warrant explora-
tion of novel strategies to manage PJI.
Importantly, the StaPhage cocktail significantly lowered the
bacterial biomass on titanium implants and the viability of
biofilm-embedded bacteria, suggesting it also exerts lytic ac-
tivity against slow-growing, persister cells. Reduced activity
of the StaPhage cocktail was observed against ORI16_C02J
compared with ORI16_C02N biofilms, highlighting S. aureus
strain differences in susceptibility to StaPhage. This may re-
flect differences in phage infectivity rates, dependent on the
mode of bacterial growth and presence and accessibility of
phage adsorption receptors. The complexity of these interac-
tions and the implications they have on therapeutic dosing of
phages in terms of the tailoring of MSSA strain-specific phage
cocktails, concentration administered, frequency of dosing,
and route of administration highlight the importance of clini-
cally relevant in vivo studies to more accurately evaluate
efficacy of this approach [33].
Emergence of bacterial resistance and host immunoge-
nicity has been suggested as potential limitations for phage
therapy. Importantly, phage efficacy is independent of the
antibiotic resistance phenotype of the target bacterial strain
[12]. While there is potential for bacteria to develop resis-
tance to phage through modification or shielding of cell
surface receptors, these responses are often detrimental to
bacterial virulence and survival in vivo [12].
Administration of phages has been shown to stimulate host
immune responses, with the strength and type of response
influenced by the route of administration and the dosing
protocol used [9]. Oral and topical administration may induce
the production of anti-phage neutralizing antibodies, while
intravenous administration of phages has potential to stimu-
late innate and adaptive immune responses [9]. Nevertheless,
safety has been demonstrated in patients with immunosup-
pression and autoimmune disorders [22] and indeed, indirect
stimulation of cell-mediated immune responses after ad-
ministration of phage may further facilitate bacterial clear-
ance in vivo [9].
To minimize the potential for phage resistance and host
immunogenicity, phage therapy centers in Eastern Europe
7
deliver patient-tailored therapy within a controlled environ-
ment, use customized phage cocktails containing three or
more individual preparations to broaden target specificity,
and adopt short-term dosing regimes to avoid extended phage
exposure and thus selective pressure for phage-tolerant bac-
terial strains [11]. Regulatory framework for the therapeutic
application of phages in countries outside of Eastern Europe
is yet to be established [11].
Discordance in the classification of phages underpins con-
tinued debate as to whether their use in medicine should adhere
to current stringent legislative frameworks in place for phar-
maceuticals or whether frameworks should be adapted to en-
able inclusion of customized, patient-specific phage
preparations that are not marketed, but are distributed in a
controlled way through approved national treatment centers
[11]. Currently, application for administration of phages can
be sought for compassionate use for patients with life-
threatening bacterial infections. There is growing impetus,
however, for the approval of phages for wider medical appli-
cation with clinical trials currently under way to evaluate S.
aureus and Pseudomonas aeruginosa phages to manage burn,
rhinosinusitis, and cystic fibrosis-associated infections [12].
Given the well-documented disparity between antimicro-
bial efficacy using in vitro and in vivo systems [34], an ex-
tensive comparison of StaPhage with additional antibiotic
agents and antibiotic combinations was not conducted in the
current study. Substantive in vivo work using a clinically
relevant animal model of PJI will be essential to characterize
efficacy, the potential for development of bacterial and host
resistance, and safety before consideration of clinical appli-
cation. These complexities cannot be understood using
in vitro studies.
Each phage preparation used in the current study was
distinct as determined by a unique host range susceptibility
pattern using spot testing of serially diluted lysates. While
sequencing of each lysate has not been performed, formal
genetic characterization of the phage preparations would
form an essential component of the safety evaluation before
progression to clinical application. An additional limitation
of the current in vitro study was the small number of S. aureus
strains screened for sensitivity to the StaPhage cocktail. The
purpose of the current study, however, was to demonstrate
feasibility of customizing phage cocktails to a given strain,
rather than create a one-size-fits-all therapy.
Conclusion
The current proof-of-principle studies demonstrated anti-
bacterial activity of phages against planktonic and biofilm-
associated S. aureus strains growing on porous titanium
scaffolds. Compared with cefazolin treatment, bacterial
numbers were significantly reduced within S. aureus biofilms
growing on titanium surfaces after exposure to a phage
cocktail, in vitro. These preliminary data provide support for
in vivo studies to further investigate the potential use of
custom phage cocktails for management of orthopedic
implant-related infections caused by S. aureus.
Acknowledgment
This research was supported by internal funds of the Or-
thopaedic Research Institute of Queensland and James Cook
University.
 8 MORRIS ET AL.
Author Disclosure Statement
Titanium implants used in this study were provided in-kind
by Stryker Orthopaedics. Bacteriophage preparations used in
this study were provided in-kind by AusPhage Pty Ltd. LE is
the Director of AusPhage Pty Ltd. LE was not directly in-
volved in the conduct of experiments, data acquisition, or
data analysis. The Orthopaedic Research Institute of
Queensland ( JM, AG, KH, MW, PM) receives annual
funding donations from ARGO to support the offer of a
surgical Research Fellowship, and from Stryker and Arthrex
to support the salary of a Research Coordinator and Ortho-
paedic PHO position.
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Address correspondence to:
pedic infection are not selected by the use of cefazolin in
Dr. Jodie Morris
prophylaxis. Diagn Microbiol Infect Dis 2016;84:266–
267. Orthopaedic Research Institute of Queensland
33. Ryan EM, Gorman SP, Donnelly RF, Gilmore BF. Recent 7 Turner Street, Pimlico
advances in bacteriophage therapy: How delivery routes, Queensland 4812
formulation, concentration and timing influence the suc- Australia
cess of phage therapy. J Pharm Pharmacol 2011;63:1253–
1264. E-mail: jodie.morris@oriql.com.au
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