MICROBIOLOGICAL AND ULTRASTRUCTURAL EVALUATION OF BACTERIOPHAGE 191219 AGAINST PLANKTONIC, INTRACELLULAR AND BIOFILM INFECTION WITH STAPHYLOCOCCUS AUREUS

Infections of orthopaedic implants, such as fracture fixation devices and total-joint prostheses, are devastating complications. Staphylococcus aureus ( S. aureus ) is a predominant pathogen causing orthopaedic-implant biofilm infections that can also internalise and persist in osteoblasts, thus resisting antibiotic therapy. Bacteriophages are a promising alternative treatment approach. However, data on the activity of bacteriophages against S. aureus , especially during intracellular growth, and against in vivo biofilm formation on metals are scarce. Therefore, the present study evaluated the in vitro efficacy of S. aureus bacteriophage 191219, alone as well as in combination with gentamicin and rifampicin, to eradicate S. aureus strains in their planktonic stage, during biofilm formation and after internalisation into osteoblasts. Further, the invertebrate model organism Galleria mellonella was used to assess the activity of the bacteriophage against S. aureus biofilm on metal implants with and without antibiotics. Results demonstrated the in vitro efficacy of bacteriophage 191219 against planktonic S. aureus . The phage was also effective against in vitro S. aureus biofilm formation in a dose-dependent manner and against S. aureus internalised in an osteoblastic cell line. Transmission electron microscopy (TEM) analysis showed bacteriophages on S. aureus inside the osteoblasts, with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria mellonella infection model, single administration of phage 191219 failed to show an improvement in survival rate but appeared to show a not statistically significant enhanced effect with gentamicin or rifampicin. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections. are represented as ± deviation from 3 independent


Introduction
Orthopaedic implants, such as fracture fixation devices and total joint prostheses, have proven their positive effect on patients' quality of life. www.ecmjournal.org scarce and further research is required to translate bacteriophage therapy into clinical practice , Ferry et al., 2021Gibb and Hadjiargyrou, 2021). Specifically, evidence on the effectiveness of bacteriophages against intracellular growing S. aureus is missing. Further, infection models are required to evaluate the activity of bacteriophages in vivo. Replacing models from higher mammals with the G. mellonella insect model would seem to be an appropriate alternative for studying implantassociated S. aureus biofilm infections (Mannala et al., 2020a;Tkhilaishvili et al., 2020;Wang et al., 2020). However, the efficiency of bacteriophages on S. aureus biofilm infection in G. mellonella with metallic implants mimicking orthopaedic infections has not yet been investigated. Therefore, the present study aimed to evaluate the in vitro and in vivo effectivity of phage 191219 (D&D Pharma GmbH, Pyrmont, Germany) against S. aureus during its planktonic, biofilm and intracellular growth phases as well as in an implant-associated G. mellonella biofilm infection model.

Materials and Methods
Bacteriophages S. aureus phage 191219 was provided by D&D Pharma GmbH, Pyrmont, Germany and propagated on the S. aureus EDCC 5055 strain. Briefly, overnight grown S. aureus bacteria were sub-cultured into fresh BHI medium and incubated on a shaker at 37 °C until the OD at 600 nm (OD 600 ) reached 1. After that, 5 mL of bacteriophage solution (5 × 10 8 PFU/mL) was added to 25 mL of bacterial solution and further incubated at 37 °C for 4 h. Phages present in bacterial suspension were obtained by centrifugation at 5086 ×g for 10 min, followed by filtering the supernatant through 0.45 µm and 0.2 µm filters. The concentration of phage solution was determined by plaque assay using bacteriophage serial dilutions (Bonilla et al., 2016).

Bacteria and G. mellonella
S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains were used. S. aureus EDCC 5055, which was isolated from a human wound infection, is known for its high biofilm formation capacity and its whole genome sequence is available (Mannala et al., 2017;. S. aureus DSM 21979 was isolated from the nasal swab of an infected patient and showed methicillin resistance. BHI broth was used to maintain S. aureus strains aerobically at 37 °C by constant shaking at 180 rpm. G. mellonella larvae were purchased from EVERGREEN GmbH (Augsburg, Germany) and maintained on an artificial diet in an incubator at 30 °C. For each experiment, 10 larvae weighing around 200-250 mg and present in the last instar stage were used. After infection, G. mellonella larvae were maintained at 37 °C.
Metal implants are primarily used based on their biomechanical properties. Despite their known functional benefits, all implants exhibit a certain risk of deep infection (Moriarty et al., 2016;Riebeiro et al., 2012). Earlier studies have identified the so-called "race for the surface" phenomenon between bacteria and host proteins and subsequent biofilm formation by bacteria as a key element for the pathophysiology of implant-associated bone infections (Davidson et al., 2019;Gristina and Costerton, 1985;Romanò et al., 2015).
S. aureus is a clinically significant pathogen that is predominantly involved in implant-associated bone infections, colonising necrotic tissue and abiotic surfaces, thus forming biofilms. S. aureus biofilms are embedded in an extracellular matrix composed of either self-produced extracellular polysaccharides, DNA and proteins or host-derived matrices such as fibrin. Further, S. aureus can invade osteoblasts and enter a "persister" state in which exposure to high levels of antibiotics can be survived due to a lack of metabolic activity (Gimza and Cassat, 2021). Antimicrobial drug diffusion into biofilms is limited by several factors such as the physical barrier of EPS, including polysaccharides, proteins, DNA and RNA. The efficiency of antimicrobial drugs against bacteria embedded in biofilm is reduced with enhanced exchange of antibiotic resistance genes and slower growth rates (Akanda et al., 2018). Hence, biofilm microorganisms are up to 1,000 times more resistant to growth-dependent antimicrobial agents than their planktonic equivalent (Arciola et al., 2015;Gebreyohannes et al., 2019). Therefore, once a biofilm is formed, eradication of bacteria and of the infection is challenging and reinfections can often not be avoided. Thus, treatment concepts for implant-associated bone infections mainly depend on the duration of the infection since maturation of the biofilm is a key factor for the therapeutic opportunity of implant retention (Zimmerli and Sendi, 2017). The remarkable resistance of biofilms to conventional antibiotic therapy has sparked a fair amount of research such as on alternative combination agents, synthetic surfaces and antimicrobial coatings (Alt, 2017;Izakovicova et al., 2019;Ribeiro et al., 2012). One promising approach involves bacteriophages, ubiquitously present in the environment as the most abundant biological agent on Earth (Baltinovic et al., 2019). These viruses infect bacteria, replicate inside them and finally are released through lysis, killing the host. Hence, bacteriophages are potentially potent antimicrobial agents, which were already being used to treat bacterial infections when discovered in 1917 by Felix d´Herelle (d'Hérelle, 1917). Nowadays, especially in light of the increasing multidrugresistant infections, bacteriophages are being reconsidered as a possible therapy option.
Whereas first preclinical studies show promising results, in vitro data on the efficacy of bacteriophages in the treatment of bone and joint infection is GK Mannala et al. Treatment of S. aureus

with bacteriophages
Effect of phage 191219 on planktonic growth of bacterial pathogens The efficiency of the bacteriophages on S. aureus planktonic growth was tested using the double layer agar method. Both S. aureus EDCC 5055 and DSM 21979 strains were used for the planktonic assay. Briefly, logarithmic growth phase S. aureus cultures were diluted to 10 7 CFU/mL. Later, this solution was mixed with bacteriophage dilutions (10 − 1 -10 − 8 ) in 5 mL total volume of semi solid BHI medium (0.7 % agar), poured onto the solid agar medium (BHI with 1.5 % agar) and incubated at 37 °C for plaque formation. The growth inhibition of S. aureus by bacteriophages in liquid medium was assessed by culturing the bacteria for 24 h in a 96-well plate at a concentration of 10 7 CFU/mL with bacteriophages at MOI 10, 1, 0.1 and 0.01. The bacterial growth at 37 °C was monitored after 24 h incubation by measuring the OD 600 . These bacterial cultures were washed 3 times with PBS to remove the bacteriophages in the supernatant, the pellet was suspended in PBS and finally plated onto the BHI agar plates to determine the bacterial concentration. For each group, 3 replicates were performed.

Effect of phage 191219 on bacterial biofilms formed in 96-well plate
To check the effect of phage 191219 on biofilms, the biofilm assay for S. aureus EDCC 5055 was performed in a 96-well plate, as described by Mannala et al. (2018). Briefly, bacteria were grown in TSB overnight at 37 °C with constant shaking at 180 rpm. The bacterial cultures were diluted into fresh medium (1:50) and the diluted bacterial cultures were transferred into U-bottom 96-well plate under static conditions and incubated for 1 d at 37 °C. After 1 d, the 96-well plate was washed once with PBS. Bacteriophages (10 6 -10 9 PFU) in TSB medium were added to well-biofilms and incubated for 1 d at 37 °C. Afterwards, the biomass in the wells was washed twice with PBS and stained with 0.01 % crystal violet solution for 10 min. Then, the wells were washed twice with PBS to remove unbound crystal violet. The cell-wall-bound crystal violet was dissolved by using 100 % ethanol and absorbance was measured at 595 nm using a UV spectrophotometer (Bio-Rad GmbH). Further, to count the bacteria before and after treatment with bacteriophage, the wells were washed twice followed by dissolving the biofilm in 200 µL of PBS solution by stirring using a a sterile inoculation loop. The use of the inoculation loop might leave some of the biofilm clumps on the well wall, which is a limitation of the method. This recovered biofilm solution was serially diluted and plated on agar plates. The CFUs were counted and results were analysed between control (t0) and treated groups after 24 h, as previously described (Thieme et al., 2019). 8 replicates were performed for each group.
Phage 191219 effect on intracellular replicating bacteria inside osteoblasts Infection of osteoblasts was performed according to Mohammed et al. (2014). The SAOS-2 osteoblast-like cell line was grown in McCoy's 5A medium. For the infection assay, SAOS-2 cells were cultured to a semi-confluent layer in 24-well plates. S. aureus EDCC 5055 bacteria were incubated overnight. Bacteria were added at a MOI of 10 per well. The number of osteoblasts per well was determined using a Neubauer chamber before starting the infection experiment. After 30 min incubation, McCoy's 5A medium was replaced by medium supplemented with 30 µg/mL gentamicin to kill only the remaining extracellular bacteria without affecting the intracellular bacteria. After gentamicin treatment for 30 min, the medium was removed, the cells were washed with PBS once and McCoy's 5A medium with the bacteriophages (10 9 PFU/well) was added to the wells. Then, cells were incubated at 37 °C for 2, 4 and 24 h, respectively. Next, the supernatants were discarded and the cells were lysed using 0.2 % Triton X-100 in sterile, cold, distilled water for 10 min. The lysates were diluted 10 times in 1× PBS and plated onto BHI agar. After 24 h of incubation at 37 °C, the number of bacterial CFU was determined. These results were compared with the effects of rifampicin (8 µg/ mL) and gentamicin (30 µg/mL) on the intracellular survival of bacteria inside osteoblasts. In addition, synergistic effects of bacteriophages with gentamicin or rifampicin antibiotics to clear intracellular S. aureus were analysed. For each group, 3 replicates were performed. Further, the stability and activity of the bacteriophages were investigated performing a plaque assay on osteoblast lysates that were treated with bacteriophages following the S. aureus infection. For this purpose, the S. aureus-infected osteoblasts were treated with two concentrations of bacteriophages (5 × 10 8 PFU and 1 × 10 9 PFU) and incubated for 24 h. Subsequently, the wells were washed 3 times with PBS and the cells treated with citrate buffer (pH 4) for 5 min to inactivate extracellular phages. Next, the wells were washed twice with PBS and the cells trypsinised, centrifuged and lysed using 0.2 % Triton X-100. This lysate was serially diluted (1:10) in sterile PBS and mixed with bacterial suspension in semisolid-agar medium. Next, the mixed solution was spread onto agar plates. The plates were observed for plaque formation after overnight incubation at 37 °C. A control of osteoblasts treated with only bacteriophages (1 × 10 9 PFU/well) was also included. As a positive control, phage 191219 solution with 4 × 10 9 PFU was used.

TEM analysis
The effect of bacteriophages on bacteria was visualised using TEM imaging, after normal culture conditions as well as following the osteoblast infection. Under normal culture conditions, S. aureus bacterial suspension (OD 600 : 0.5) was treated with bacteriophages at MOI 50 at 37 °C and samples were collected by centrifugation (457 ×g) for 10 min at the 10 min and 12 h time points. In case of osteoblast infection, SAOS-2 cells were seeded on cell culture Petri dishes (1 × 10 6 cells/dish) and infected with S. aureus EDCC 5055 at MOI 30, as previously described.
After gentamicin treatment, cells were treated with bacteriophages (10 9 PFU/mL), trypsinised and collected by centrifugation (5086 ×g) for 10 min at the 6 h time point for TEM imaging. MOI 30 and 6 h time point were used for the detection of the bacteriophage effect on the bacteria inside the osteoblasts. These pellets were first fixed in Karnovsky fixative (for 15 min at room temperature; Heim et al., 2015) and, after that, they were enclosed with agarose. For the embedding process, cells were fixed again with 1 % osmium tetroxide at pH 7.3 and dehydrated through a graded series of ethanol. Next, the samples were embedded in EMbed-812 epoxy resin (Science Services, Munich, Germany) and polymerised at 60 °C for 48 h. Then, the LYNX microscopy tissue processor (Reichert-Jung, Wetzlar, Germany) was used. Semi-thin-sections (75 µm), for the selection of relevant areas, and ultra-thin sections (80 nm) were cut using the Reichert Ultracut S Microtome (Leica-Reichert, Wetzlar, Germany). Then, the ultra-thinsections were contrasted using aqueous 2 % uranylacetate and 2 % lead-citrate solution for 10 min each.

Testing of phage 191219 effect on implantassociated infections in the G. mellonella model
Stainless steel K-wires with a diameter of 0.8 mm, were purchased from Synthes, Zuchwil, Switzerland. 4-5 mm long pieces were sterilised in 70 % ethanol before performing the experiment. For the infection process, implants were pre-incubated in bacterial growth culture medium with 1 × 10 6 CFU/mL S. aureus EDCC 5055 for 30 min while shaking at 150 rpm. Later, the implants were washed with 10 mL PBS and implanted on the rear side in the abdomen of the larvae by piercing the cuticle of the larvae using the sharp edge of the K-wire (Mannala et al., 2020a). For the control group, the same process was applied but without bacterial contamination of the implants. Before implantation, the number of adhered bacteria was estimated by sonicating the samples and plating them on agar plates. For each testing group, 10 larvae were used.
To determine the effect of bacteriophages on bacterial survival in the early-stage stainless steel implant biofilm infection model, 50 µL of bacteriophages (10 9 PFU/larva) were injected at day 1 into the abdomen of a larva that had already been implanted with an S. aureus-preincubated implant, as previously described (Mannala et al., 2020a). To determine the synergistic effect of antibiotics with bacteriophages against S. aureus biofilm infection, the larvae were injected with 10 µL of gentamicin (120 mg/kg) and rifampicin (70 mg/kg) at their rear part and survival of the larvae was observed for 5 d. All experiments were performed 3 times.

Statistical analysis
Statistical analysis was performed using sigmaP lot 10.0 (SYSSTAT software Inc.,Chicago, IL, USA). For the analysis of bacterial numbers, Student's t-test was applied. For the survival analysis, logrank test was performed. Data were represented as means ± standard deviation from 3 independent experiments, with at least 3 technical replicates in each experiment. Data were considered to be statistically significant for p < 0.05.

Results
Phage 191219 exhibited in vitro activity against planktonic S. aureus with proliferation of bacteriophages within S. aureus First, the activity of phage 191219 was assessed against S. aureus EDCC 5055 and S. aureus DSM 2197 using the double agar overlay method. The results showed clear plaque formation with both strains. Next, the efficiency of phage 191219 against planktonic growth was measured. Phage 191219 and bacteria were cultured at MOI 10, 1, 0.1 and 0.01 and GK Mannala et al. Treatment of S. aureus with bacteriophages bacterial number was measured by CFU analysis. The CFU analysis showed that a significant antimicrobial activity of the bacteriophages was measured at MOI 10 and 1. There was no significant reduction in planktonic growth at MOI 0.1 and 0.01 (Table 1). TEM investigation revealed that numerous phage 191219 particles had attached to the S. aureus after 12 h, with a large number of newly formed bacteriophages within the bacteria (Fig. 1a). Furthermore, the infected bacteria showed disrupted cell walls and even bacterial degradation, with detectable remnants of dead bacteria after 12 h of treatment (Fig. 1b). At the early time point of 10 min after treatment, no significant attachment of phages (191219) to S. aureus bacteria was detected.

Phage 191219 was active against in vitro S. aureus biofilm
To evaluate the role of bacteriophages against in vitro S. aureus biofilm, bacteriophages at various concentrations were added to the biofilm formed on the plastic surface of the 96-well plate. After 24 h of bacteriophage treatment, the biofilm was analysed using both crystal violet and CFU analysis methods.  Fig 2. Antimicrobial activity of phage 191219 on S. aureus biofilms measured using the crystal violet method and CFU analysis. The antimicrobial activity of the bacteriophages on biofilms in vitro was measured by incubating for 24 h the several fold dilutions of bacteriophages in TSB medium with biofilms formed on 96-well U-bottom plate. After 24 h, the wells were washed twice with PBS, stained with 0.1 % crystal violet solution and incubated at room temperature for 10 min. Later, unbound colour was removed using PBS washes, dissolved in ethanol and absorbance was measured at 595 nm. A concentration of 10 6 PFU/well already resulted in a significant reduction in the biofilm biomass on the 96-well plate and the reduction in biomass was profound at increasing phage concentrations. (a,b) Visualisation of the outcome of the crystal violet assay. For the bacterial count determination, after 24 h treatment, wells were washed twice with PBS and bacterial cell number was determined by CFU analysis. (c) Representation of the obtained CFU/well for each tested condition. Bacteriophage concentrations of 10 7 ,10 8 and 10 9 PFU showed 2.6, 2.8 and 3-log reduction in biofilm-embedded bacteria compared to control (t0), respectively (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; t-test). Data are represented as mean ± standard deviation from 3 independent experiments. www.ecmjournal.org Results showed that the bacteriophage dose of 10 9 PFU was effective in eradicating the biofilm as evidenced by a decrease in crystal violet absorbance as well as a significant reduction in CFUs for the treated group (9.59 × 10 2 CFUs/well) when compared to control at time point t0 (1.29 × 10 6 CFUs/well) (*** p ≤ 0.001) (Fig. 2). Treatment of biofilm with bacteriophages at a concentration of 10 8 and 10 7 PFU/ well showed 2.8 and 2.6 log reduction in S. aureus in biofilms, respectively.
Phage 191219 improved the low effect of gentamicin on intracellular growing S. aureus inside osteoblasts but not the high effect of rifampicin SAOS-2 osteoblasts were infected with S. aureus and treated with bacteriophages at 10 9 PFU/well. The bacterial number was determined at 2, 4 and 24 h using cell lysis and CFU analysis (Fig. 3). Results showed that bacteriophages significantly affected the intracellular growing bacteria compared to control after 4 and 24 h (*** p ≤ 0.001). The additional use of bacteriophages with gentamicin showed further inhibition of S. aureus intracellular growth at 4 and 24 h compared to solely gentamicin treatment (*** p ≤ 0.001). The combination of bacteriophages with rifampicin could not further improve the good intracellular effect of rifampicin alone.
The plaque assay with osteoblast lysates showed the presence of bacteriophages inside the osteoblasts after 24 h as well as phage propagation inside the S. aureus-infected osteoblasts. The plaque assay with serial dilution of osteoblast lysates showed that there was no difference in the number of bacteriophages between the 1 × 10 9 PFU/well-and 5 × 10 8 PFU/well-treated groups (data not shown). The number of bacteriophages inside S. aureus-infected osteoblasts was many folds increased compared to the control non-infected osteoblasts treated with bacteriophages (1 × 10 9 PFU/well). The cell lysate of the control group non-infected osteoblasts treated with bacteriophages revealed the internalisation of phages (5 × 10 2 PFU/ well) into osteoblasts (data not shown).
Analysis of TEM images showed the effect of the bacteriophages in S. aureus inside the osteoblasts (Fig. 4). Phages were detected in the osteoblasts in all stages of the lytic cycle, such as binding to the bacterial cell wall, proliferation inside the bacteria and lysis of the bacteria (Fig. 5). Remnants of lysed bacteria with deteriorated cell walls were also found with newly formed bacteriophages in close vicinity (Fig. 4,5).

In vivo testing of phage 191219 effect on S. aureus biofilm in the G. mellonella infection model
The effect of phage 191219 was assessed against earlystage S. aureus biofilm formation on the implanted K-wire inside the larvae. The adhered bacteria on pre-incubated K-wires were 5,300 ± 480 CFUs per K-wire, as determined by sonication. In the G. mellonella early-stage biofilm infection model, the singular bacteriophage application was not effective against S. aureus infection, as survival rates were comparable to those of the untreated control group (Fig. 6). Further, the treatment with rifampicin against S. aureus biofilm infection significantly improved the larval survival (** p ≤ 0.01), whereas gentamicin did not. Simultaneous treatment with bacteriophages and either gentamicin or rifampicin appeared to slightly aureus-infected osteoblasts with 10 9 PFU/well, followed by CFU analysis at 2, 4 and 24 h time points. In addition, the synergistic effect of gentamicin and rifampicin to clear intracellular S. aureus was analysed. The results showed a significant antimicrobial effect on the intracellular survival of S. aureus compared to control after 4 h. Similarly, bacteriophages showed an improved effect in combination with gentamicin at 4 and 24 h time points, with significant inhibition of intracellular bacterial growth, but not in combination with rifampicin (*** p ≤ 0.001; t-test). Data are represented as mean ± standard deviation from 3 independent experiments. GK Mannala et al. Treatment of S. aureus with bacteriophages enhance the effect of the respective antibiotic, despite the results not being statistically significant.

Discussion
In the present study, the in vitro efficacy of phage 191219 to eradicate planktonic stage S. aureus strains -following biofilm formation as well as after internalisation into osteoblasts -alone as well as in combination with gentamicin and rifampicin was tested. Furthermore, to the best of the authors' knowledge, the present was the first work to study the effects of bacteriophages in a G. mellonella biofilm model with an already incorporated metal implant mimicking orthopaedic infections, as previous studies with phages in Galleria models have only looked at planktonic bacterial infections. Two S. aureus strains (MSSA and MRSA) were treated during their planktonic stage with distinct  Results showed that bacteriophages administered at a MOI 1 were sufficient to kill both MSSA and MRSA strains. Morris et al. (2019) tested a cocktail of distinct bacteriophage types against S. aureus strains in their planktonic stage, reporting a reduction in bacterial growth of 90-100 %. In another study, bacteriophages were modified using CRISPR-Cas9 to remove staphylococcal cytotoxin and enterotoxin genes. The bacteriophage killed 1 × 10 5 CFU S. aureus culture within 6 h of treatment, being more effective than the unmodified type . TEM investigations confirmed the attachment of numerous bacteriophages to the bacterial cell after 12 h and attacked bacteria showed disrupted cell walls and even bacterial degradation, confirming the effect of the phages on S. aureus.
From the biofilm assay, it became evident that increasing doses of phage 191219 led to decreased bacterial biofilm burden on plastic surfaces. Further, the treatment with 1 × 10 9 PFU bacteriophages resulted in a 3 log 10 reduction in bacterial biofilm inoculum after 24 h. Morris et al. (2019) showed that treatment of established biofilm on titanium scaffolds with the bacteriophage cocktail could reduce 6.8 log 10 to 6.2 log 10 CFU compared to a control, however, not after exposure to cefazolin (50 mg/mL). Further research has shown the effectiveness of a temperate bacteriophage genetically modified with CRISPR/Cas9, removing major virulence genes and expanding the host specificity through modification of the tail fibre protein. The modified bacteriophage was effective at a dose of 1 × 10 8 and 2.5 × 10 8 PFU, with a total clearance of bacterial biofilm, whereas vancomycin (1,024 µg/mL) did not affect the biofilm (Cobb et al., 2019). Similarly, several other in vitro studies (Alves et al., 2014;Tkhilaishvili et al., 2020) have confirmed the efficacy of bacteriophages against S. aureus biofilm, which could be corroborated by the results of the present study. Alves et al. (2014) revealed a significant biofilm reduction after 48 h treatment using a novel phage DRA88. Tkhilaishvili et al. (2020) showed complete eradication of biofilmembedded bacteria on glass beads after treatment with Sb-1 and PYO phages for 5-7 d. The work conducted by Kumaran et al. (2018) targeted the question whether treatment order matters, showing significantly more S. aureus reduction when treatment with the bacteriophage SATA-8505 precedes the antibiotic (cefazolin, vancomycin, dicloxacillin, tetracycline and linezolid). The apparent improved effect in the present study with simultaneously administered antibiotics enhancing bacteriophage activity in the G. mellonella assay endorsed previously published data (Chaudry et al., 2017;Kolenda et al., 2020;Tkhilaishvili et al., 2018). Kolenda et al. (2020) showed a synergistic effect with simultaneous treatment of S. aureus biofilms with a cocktail of 3 phages along with either vancomycin or rifampicin antibiotics.
Analyses of treated S. aureus-infected osteoblasts revealed that bacteriophages were more effective in comparison to gentamicin alone, whereas a combination of both agents further but slightly reduced bacterial CFU counts. Rifampicin alone seemed to be the most efficient in eradicating internalised S. aureus; a combination of bacteriophages with rifampicin cleared most of the bacteria after 24 h. The combination of gentamicin or rifampicin and phage 191219 failed to show significant effects on the intracellular growth of bacteria compared to the antibiotic alone. This might be due to the different time durations required for the antimicrobial activity of phage/antibiotic combinations and the intracellular For the biofilm infection model, the larvae were implanted with pre-incubated K-wires, followed by treatment with bacteriophages alone and in combination with rifampicin and gentamicin 24 h post-implantation. The synergy between the phages and the antibiotics was compared with their respective controls. Results showed a significant improvement in survival rate for the larvae treated with rifampicin compared to the untreated control group. The combination of rifampicin and phages appeared to have slightly improved the larval survival, although not significantly. Neither phages, gentamicin nor the combination of both treatments significantly improved the larval survival compared to the untreated control group (ns, nonsignificant, *** p ≤ 0.001; ** p ≤ 0.01; log-rank test). In each experiment, 10 larvae were used for each group. Data are represented as mean ± standard deviation from 3 independent experiments. GK Mannala et al. Treatment of S. aureus with bacteriophages conditions. In the present study, ultrastructural analysis of the phages in S. aureus-infected osteoblasts showed the activity of phages, with binding of the phages to the bacteria, proliferation of new phage particles and subsequent lysis of the bacterial cell and release of the phages into the cell cytoplasm. Phages are found ubiquitously in nature and the human body harbours a large number of bacteriophages together with their bacterial host. Moreover, they constitute the key component of the gut microbiome (Bichet et al., 2021). Phages are detected in the circulatory systems, organs and tissue indicating that they are capable of translocating from the gut and penetrating throughout the body (Ghose et al., 2019;Huh et al., 2019). Recently, Nguyen et al. (2017) have shown that epithelial cells ingest phages through non-specific mechanisms, such as macropinocytosis, and transcytose them from the apical surface toward the basolateral side of the cell (Nguyen et al., 2017). Thus, it is possible that the osteoblasts can internalise bacteriophages into macropinocytotic vesicles. Those vesicles might then fuse with phagolysosomes containing the bacteria and make it possible for the phages to attach to those bacteria. However, data on bacteriophage-mediated killing of S. aureus internalised in osteoblast are scarce. So far, only Kolenda et al. (2020) investigated three different bacteriophages (PP1493, PP1815 and PP1957), alone and in combination with vancomycin and rifampicin, against S. aureus biofilm as well as in an osteoblast infection model. The authors showed that the bacteriophages were able to internalise; however, the data suggest a lack of activity against intracellular bacteria in MG63 osteoblasts. The authors hypothesise that bacteriophages might be shuttled inside the osteoblasts by the bacteria, as it was previously indicated that bacteriophage MR-5 does not affect the natural intracellular killing of S. aureus by macrophages (Kaur et al., 2014). This contrasts with the present study results, which showed a significant inhibition of S. aureus growth in osteoblasts already 4 h following bacteriophage treatment. The present study showed bacteriophages inside S. aureus within osteoblasts with destruction of bacterial walls and newly formed bacteriophages within the osteoblasts. The discrepancy compared to the results of Kaur et al. (2014) might be explained by i) the use of distinct S. aureus strains, which might differ in intracellular metabolic activity; ii) the use of distinct osteoblastic cells possibly influencing bacterial dormancy; iii) different activity of the phages used. G. mellonella was recently shown to be a suitable non-vertebrate animal model to study biofilm infections, with the insertion of contaminated metal implants (stainless steel, titanium) mimicking orthopaedic biofilm infections (Mannala et al., 2020a). To the best of the authors' knowledge, G. mellonella was previously only used for planktonic bacterial infection experiments with the addition of bacteriophages and multiple studies showed enhanced larval survivability due to bacteriophage treatment (Jeon and Yong, 2019;Manohar et al., 2018;Thiry et al., 2019;Tkhilaishvili et al., 2020;Wang et al., 2020). For instance, Tkhilaishvili et al. (2020) showed that the administration of the bacteriophages Sb and PYO results in reduced mortality, whereby survival rates are higher when the bacteriophages are applied before infection with S. aureus and further increases with a higher bacteriophage dose. Analogous effects are observed following treatment with vancomycin. Wang et al. (2020) characterised the activity of the new phage ɸWL-3, which was isolated from hospital sewage, against E. coli biofilms in vitro and in a planktonic infection in vivo model using G. mellonella. ɸWL-3 was effective against a planktonic E. coli infection in G. mellonella and the effectiveness was significantly enhanced by simultaneous treatment with phosphomycin . The present study results of the G. mellonella biofilm model suggested that a single bacteriophage treatment alone did not result in a significantly increased survival rate of the larvae compared to untreated controls, whereas rifampicin significantly increased the survival rates. Phage treatment appeared to exhibit enhanced effects in combination with gentamicin or rifampicin, despite this result not being statistically significant. The bacteriophage treatment might have failed because only one treatment was applied. The coexistence of bacteriophages and bacteria could result in the rise of phage-resistant bacterial strains through various mechanisms, such as point mutations in receptor genes, CRISPR/Cas9 system and restriction-modification systems (Onsea et al., 2020). However, phage resistance might not be the reason for the failure of bacteriophage therapy in G. mellonella, as the phage resistant bacterial strains face reduced fitness and loss of virulence with mutations in receptor genes. Capparelli et al. (2010) showed that the virulence of a phage-resistant S. aureus strain was decreased in a mouse infection model and used an attenuated strain to induce immunity against S.

aureus.
There are several limitations to the present study. First, only one phage (191219) was used and results for this phage cannot be generalised for other phages. Further, only one single dose of the bacteriophages was used and multiple and/or repetitive treatment might enhance their activity, which was not investigated. Finally, the G. mellonella biofilm infection model also has its limitations due to the absence of a humoral immune system. Therefore, the results have to be interpreted with caution for any potential clinical translation.

Conclusions
While bacteriophages exhibited strong activity against planktonic and intracellular S. aureus in an osteoblastic cell line, only high concentrations of bacteriophages were effective against S. aureus www.ecmjournal.org biofilms in vitro. Ultrastructural analysis highlighted the activity of phages on intracellular growing S. aureus, which is responsible for relapse and chronic implant-associated bone infections. For implant-associated biofilm infections tested in the G. mellonella infection model, single administration of bacteriophages failed to show improvement of larval survival rates. However, an improvement of the bacteriophage effect with the addition of gentamicin and rifampicin was suggested, albeit the result not being statistically significant. From the clinical perspective, the data suggested that the administration of bacteriophages might be a potential adjuvant treatment strategy for implant-associated biofilm infections, which has to be proven in further preclinical and clinical trials.