Two clinical S. epidermidis isolates were used (Department of Microbiology, St. Olavs University Hospital, Trondheim, Norway): one gentamicin susceptible S. epidermidis and one Methicillin-resistant S. epidermidis (MRSE) strain. The MRSE strain is resistant to gentamicin, erythromycin, cloxacillin/dicloxacillin/penicillin. S. epidermidis strain ATCC35984 and ATCC12228 were used as a strong and poor biofilm forming strain, respectively. S. epidermidis strains were cultured in tryptic soy broth (TSB, 1% glucose) or Mueller-Hinton Broth (MHB) for MIC and Time-killing assay. Blood agar plates were utilized for plating and incubated at 37°C with 5% CO₂.

APIM-peptides (Innovagen, Sweden) used here were TFA-salt (>90% pure). The APIM-peptide consists of the APIM-motif (in bold) connected via a linker to an arginine tail (cell penetrating part), containing 10 or 11 arginine; Ac-MD-RWLVK-WKKKRKI-R11/R10 the C-termini on all peptides were amidated (Nedal et al., 2020). Lyophilized peptide from the manufacturer was dissolved in water.

Minimal inhibitory concentration (MIC) was determined following the protocol recommended by National Committee of Laboratory Safety and Standards, NCCLS (NCCLS., 2012) for microtiter broth dilution assay, conducted with modifications suggested by R.E.W. Hancock Lab for cationic peptides¹. Briefly, bacterial suspension in MHB was adjusted to 0.5 McFarland and diluted to 5 × 10⁵ colony forming units (CFU)/mL. 0.1 mL of the suspension (5 × 10⁴ CFU) was seeded in each well in polypropylene microtiter plates (Greiner). APIM-peptide and gentamicin were added as 10% of the total volume, diluted to specific concentrations (not only twofold serial dilution). After 24 h of incubation (37°C), the plates were inspected for visible growth.

Time-kill curves were conducted after NCCLS standards (NCCLS., 1999) by inoculating the MRSE strain in 10 mL MHB, 5 × 10⁵CFU/mL. The cultures were treated at 0 h with 0.5x, 1x, 2x, and 5x MIC concentrations of APIM-peptide (MIC = 5 μM), sub-MIC dose of gentamicin (39 μM, MIC = 309 μM) or 5x MIC vancomycin (10 μM, MIC = 2 μM). 0.5x MIC of APIM-peptide was also combined with gentamicin. The cultures were grown in a shaking incubator (250 rpm, 37°C). Samples were harvested at the time points from 5 min up to 48 h, and plated (duplicates) on blood agar plates in 10-fold dilutions in saline solution (0.9% NaCl). CFUs were counted after 24 h of incubation. The detection limit is calculated to 50 CFU/mL (1.72 log₁₀CFU/mL); however, zero CFU were included to calculate average CFU counts when no colonies was detected in non-diluted culture. The culture density after media exchange was determined for the cultures added x5 MIC with APIM-peptide/vancomycin at the 48 h timepoint to quantify CFU/mL after the removal of the antibacterial agent. In this case, 5 mL of the cultures were centrifuged (2 min, 14,000 rpm) and the pellet re-suspended in fresh MHB (same volume) before plating.

Biofilm was quantified following a modified version of the Christensen’s microtiter plate test (Stepanovic et al., 2000). Briefly, S. epidermidis strain ATCC35984 were seeded in suspension (200 μL/well) adjusted to 0.5 McFarland turbidity standard in TSB in microtiter plates. APIM-peptide was added directly to the culture, 10% of the total volume, to minimum 3 wells per sample. After 24 h incubation 37°C, the wells were washed and vortexed (3 × 1 min, 600 rpm) with saline solution before fixation with 99% methanol for 15 min. The wells were emptied and dried followed by staining (5 min) with crystal violet (0.5–2%). Wells were washed with water and dried before the stain was re-solubilized with 160 μL glacial acetic acid (33%). Finally, absorbance was measured at 570 nm. ATCC12228 and MRSE (clinical isolate) was also tested with this assay to compare biofilm forming ability (Supplementary Figure S2).

Biofilm killing assay was performed as previously described (Mandell et al., 2017) with some minor modifications. Briefly, stainless steel wire (C-WIRE, 0.89 mm, ConMed) was cut to 6 mm rods and autoclaved. The rods were placed in 200 μL bacterial suspension with S. epidermidis ATCC35984 (10⁶ CFU/mL in MHB) in 96-well polypropylene microtiter plates and grown for 5 days to allow for biofilm formation. Fresh media was exchanged every 24 h. After 5 days the rods were placed into fresh media and treated with APIM-peptides, water (control), or a peptide containing only the cell penetrating arginine tail of the APIM-peptide (R11). The rods were harvested 1, 6, and 24 h after the addition of peptides. Here, the rods were washed once in 0.2 mL saline solution before being transferred to 0.5 mL saline solution in sterile glass tubes. The rods were then vortexed for 20 s (2,500 rpm) followed by sonication for 6 min (40 kHz, 200W, BactoSonic^®). The rods were again vortexed after sonication. Samples plated in duplicates on blood agar plates (10-fold dilutions in saline solution). CFU counts were calculated after 24 h incubation at 37°C. In addition, the rods were put into 3 mL fresh media after sonication and incubated 24 h (37°C, shaking) to check for visible re-growth of bacteria from the rods.

The preparation of the bone grafts and the bone grafts experiments are based on a bone graft model (Witso et al., 2020). Corticospongeous bone grafts were harvested from Norwegian white sheep, delivered from Nortura Malvik, Norway. Rib bones were cut to approximately 1 × 1 cm in size, height ∼0.5 cm, hollowed from end to end with a 3 mm drill through the bone marrow, creating a cylindrical hole through the bone. In addition to this, a smaller hole (2 mm wide) was drilled on the top of the bone grafts, perpendicular to the cylindrical hole in the bone marrow. Periosteum from the bone was removed and sharp edges of the bone grafts were rounded, and the bone grafts were sterilized by autoclaving. The cylindrical hole of the bone grafts was filled with bone cement with a syringe. Bone cement without gentamicin (Biomet Bone^® Cement R) was combined with water (control) or APIM-peptide. Gentamicin-containing bone cement (PALACOSR^® + G) was used as it came from the provider (adjusted with corresponding amount of water) or was combined with APIM-peptide (combination treatment). APIM-peptide was dissolved in water (160 mg/mL) before mixing it with the cement liquid component, enabling a homogenous cement containing 1.6% APIM-peptide (net peptide, 25 mg APIM-peptide/g cement, corresponding to 16 mg net peptide/g cement). The corresponding amount of water was added to the control cement without APIM-peptide. The hole on top of the bone grafts was blocked when added cement, keeping it hollow to allow the bacterial inoculum to reach the cement inside the bone graft.

The bone grafts, containing cement filled with APIM-peptide, gentamicin, or a combination of these, were each added an inoculum of 50 μL TSB containing 1,000 CFU of MRSE in the hole on the top. After 24 h incubation (37°C) the bone grafts were harvested for quantification of bacterial load. The bone grafts were “opened” with a sterile scalpel to separate the cement inside from the bone. The bone and cement pieces were placed in 3 mL saline solution each (0.9% NaCl) in sterile glass tubes. Samples were sonicated for 5 min in a water bath (40 kHz, 200 W, BactoSonic^®, Bandelin GmbH, Berlin) in addition to being vortexed 30 s (2,500 rpm) before and after sonication. Samples were plated in triplicates on blood agar plates diluted 10-fold in saline solution.

The animal study was reviewed and approved by Norwegian Food Safety Authority (Mattilsynet, application ID 11937). The animals were acclimatized for a week after arrival to the Comparative Medicine Core Facility (NTNU, St. Olav’s Hospital). The animals were housed in solitude during the experiment and had access to environmental enrichment and standard food/water ad libitum in a 12 h night-day cycle. The rats were weighed on the first and the last day of the experiment and only minor weight loss was observed.

The surgery of every rat was performed in a specialized cabinet with laminar airflow and in accordance with standard operating room procedures. 36 male Rattus norvegicus (Albino outbred Wistar) were used, 7–14 weeks old weighing from 300 to 500 g. The rats were shaved on the back before surgery. The rats were anesthetized with inhalation of isofluorane (carrier gas 66% N₂O, 34% O₂) delivered in an induction chamber (3–5% isoflurane) before surgery and by an inhalation mask (1.5–2.5% isoflurane) during surgery. The rats were placed on their stomach on sterile drapes covered with sterile sheets, only exposing the shaved mid part of the back. The exposed area was washed with 70% alcohol and a ∼4 cm long incision was made in the midline of the upper part of the back. One intramuscular cavity was made on each side of the back midline with a scissor. Two bone grafts were each instilled with 5 μL saline, containing 1,000 CFU of S. epidermidis, in the small upper hole of the bone graft, before being placed sub-fascial in the intramuscular pockets, one bone graft in each side. The incision was closed with sutures and the rats were given analgesia (Buprenorphine; Temgesic, Reckitt & Colman), 0.05 mg/kg) at the end of the surgery and postoperatively (8–12 h after the surgery).

The operation wounds of the rats grew without any signs of bacterial infection and the general health of the animals did not seem to be affected by the implanted infected bone grafts. The bone grafts were retrieved after 4 days. The animals were anesthetized, as described above, and the sutures on the back of the rats were removed and the incision made on day 1 was re-opened. The bone grafts were harvested with a sterile tweezer and a muscle biopsy (approximately 5 mm) was taken from the tissue surrounding the bone graft. The bacterial load was quantified as described in the in vitro bone infection model, and random bacterial colonies were analyzed with MALDI-TOF and confirmed to be the same bacterial species as inoculum. The rats were euthanized after the experiment with pentobarbital (Mebumal 10%, 0.1 mL/100 g), by intracardiac injection or intravenously injection (tail). No signs of systemic infection were found, i.e., the spleen biopsies (5–10 mm) of the rats were negative. The tissue around bone grafts was mainly unaffected by the infection and very few CFU were detected in the muscle biopsies. All biopsies were ground in 1 mL saline solution with mortar and pestle and plated on chocolate and blood agar plates.

Gentamicin is a widely used antibiotic against S. epidermidis infections. Here we selected a gentamicin susceptible S. epidermidis strain and a gentamicin resistant MRSE strain for MIC testing of APIM-peptide, gentamicin and the combination of these. In addition, the ATCC 35984 S. epidermidis, which is producing biofilm, was tested for gentamicin and APIM-peptide sensitivity. Table 1 shows that MIC for APIM-peptide was 4 and 5 μM for the gentamicin susceptible S. epidermidis and the MRSE strain, respectively. The MIC for gentamicin was 0.72 μM for the susceptible strain and 309 μM for the MRSE strain. In combination with APIM-peptide; however, the MICs for gentamicin was reduced three and eightfold, respectively. The ATCC 35984 strain also showed reduced MIC for gentamicin when combined with 0.5x MIC of APIM-peptide. This indicates that the combination of APIM-peptide and gentamicin has an additive effect.

To assess the bactericidal activity of APIM-peptide compared to other antibacterial drugs, a time-kill experiment was conducted. CFU counts from the MRSE culture were determined from 5 min and up to 48 h after the addition of APIM-peptides, vancomycin, or gentamicin. The time-kill experiment showed that the addition of all three APIM-peptide doses caused a significant decrease in survival already after 5 min (Figure 1A); 1x MIC (5 μM) of APIM-peptide killed 97% of the bacteria after 5 min and has complete bactericidal effect (defined as ≥ 3log₁₀ decrease of original inoculum) (NCCLS., 1999) after 60 min. APIM-peptides at 2x and 5x MIC have even faster killing efficiencies, i.e., bactericidal effect was seen after 30 min with 5x MIC. In comparison, a bactericidal effect with 5x MIC vancomycin (10 μM) addition was first detected 8 h after addition (Figure 1B). Vancomycin is known to be bacteriostatic at lower doses on staphylococci (Stevens, 2006), and this was also seen in our MIC assay using 1x MIC (Supplementary Figure S1). Both 5x MIC of APIM-peptide and vancomycin inhibit bacterial growth up to 48 h without any re-growth; however, when removing the antibiotics by transferring the bacteria into fresh media after 48 h, CFU determinations revealed bacteria (∼4.5 × 10³ CFU/mL) in the vancomycin culture but not in the APIM-peptide culture (Figure 1C).

To further investigate the additive effect of the combination, APIM-peptide (2.5 μM, 0.5x MIC) was combined with gentamicin (sub-MIC, 39 μM) in a time-kill experiment with the MRSE strain. The combination gave a 1.5-log₁₀ decrease in bacterial growth compared to single-agent addition with APIM-peptide at 24 h (Figure 1D).

Because biofilm formation is an important parameter in chronic S. epidermidis infections, the effects of APIM-peptide on the formation and eradication of biofilm were tested. The MRSE strain used in the previous in vitro studies described above is a relatively poor biofilm former (Supplementary Figure S2); thus, for these experiments the biofilm-forming reference strain ATCC 35984 was applied. Christensen’s microtiter plate biofilm assay showed that APIM-peptide reduces the formation of biofilm up to ∼80% at 2 μM, which equals ∼0.5x MIC (Figure 2A). To test if APIM-peptide has an effect on already existing biofilm, we grew ATCC 35984 biofilm on C-wire steel rods, added APIM-peptide and quantified CFU after sonication of the rods. Here, 25 μM (∼8x MIC) of APIM-peptide completely eradicated the biofilm (Figure 2B) and no re-growth after transferring the rods to fresh media was detected (data not shown). Lower doses of the peptide also significantly reduced the biofilm, i.e., the doses of 5 and 10 μM APIM-peptide resulted in ∼0.5 and ∼3 log decrease in CFU/mL, respectively (Figure 2B). To exclude that this anti-biofilm effect was due to the cell penetrating part of the APIM-peptide, we included the 11-arginine cell penetrating peptide, R11, as a control. However, 60 μM of the R11 peptide gave only ∼1 log decrease in CFU/mL, showing that, similarly to the antibacterial and anti-mutagenic effects (Nedal et al., 2020), the full-length peptide containing APIM is needed for maximal anti-biofilm effect. Preliminary data also shows that coating steel rods with APIM-peptide dissolved in polyethylene glycol (PEG) inhibited biofilm formation (data not shown); thus, APIM-peptides could potentially be exploited for further use in coating of surgical implants.

To test APIM-peptide in a clinically relevant in vivo model related to PJI, i.e., an infected prosthesis, a variant of a bone graft model in rats was used (Material and Methods; Witso et al., 2020). Hollow bone grafts were filled with cement containing APIM-peptide, gentamicin, or a combination of these, thereby functioning as a local vehicle for delivery of the antibacterial agents. The bone grafts were infected with MRSE to mimic the event of bacterial infection colonizing the grafts at the time of surgery.

In initial in vitro experiments, APIM-peptide was shown to be rapidly released/eluted from the cement (see Supplementary Figure S3A). Further, in vitro studies using bone grafts infected with MRSE also showed that the APIM-peptide alone and in combination with gentamicin abolished bacterial growth. The cement was separated from the bone to quantify the bacterial load on each part of the bone grafts. All the treatments reduced bacterial growth in the bone compared to the control in vitro; however, the cement containing only gentamicin had some bacterial growth (Supplementary Figures S3B,C).

Next, infected bone grafts were next implanted into the back of rats and harvested after 4 days. The quantification showed that the bacterial load was 96% reduced in bone grafts containing cement with the APIM-peptide-gentamicin combination, compared to control (Figure 3A). In bone grafts treated with APIM-peptide, the bacterial load was reduced by 68% compared to control, whereas gentamicin alone reduced the bacterial load by 66%. Calculating the fraction of animals with detectable bacterial growth vs. no growth in the bone grafts, showed that 77% of the bone grafts containing cement with the combination had no detectable growth, vs. 38% in the groups containing either APIM-peptide or gentamicin.

The bacterial load on the bone cement (Figure 3B) correlates with the bacterial load found in the bone (Figure 3A), except for the detection of an even lower bacterial load on the cement containing only APIM-peptide. All the additions to the cement significantly reduced bacterial load compared to control (Figure 3B). There is a tendency that the cement containing APIM-peptide (either alone or in combination) have lower bacterial load than cement with only gentamicin; however, this is significant (p < 0.05) only if the outlier in the gentamicin group is removed. Removing the similar outlier in the gentamicin group in Figure 3A, also yields a significant difference between gentamicin and the combination.