# Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates

Jean-Baptiste Mascary, Valérie Bordeau, Irène Nicolas, Marie-Clémence Verdier, Pierre Rocheteau, Vincent Cattoir

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Jean-Baptiste Mascary, Valérie Bordeau, Irène Nicolas, Marie-Clémence Verdier, Pierre Rocheteau, et al. Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates. JAC-Antimicrobial Resistance, 2024, Jac-Antimicrobial Resistance, 6 (1), pp.dlae025. 10.1093/jacamr/dlae025. hal-04513158

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# Intracellular activity and *in vivo* efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against *Staphylococcus aureus* clinical isolates

Jean-Baptiste Mascary<fnref n="1" />,², Valérie Bordeau<fnref n="1" />, Irène Nicolas², Marie-Clémence Verdier³, Pierre Rocheteau² and Vincent Cattoir ID ⁴,⁵*

Received 19 January 2024; accepted 2 February 2024

**Objectives:** Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both *in vitro* and *in vivo* for the treatment of septic arthritis caused by *Staphylococcus aureus*.

**Methods:** Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the *in vivo* efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg).

**Results:** MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2×MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (~10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h.

**Conclusions:** Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

## Introduction

*Staphylococcus aureus* remains a problematic pathogen implicated in a spectrum of bone and joint infections (BJIs), which encompass septic arthritis, acute and chronic osteomyelitis and spondylodiscitis, as well as infections related to orthopaedic devices.<fnref n="1" />⁻³ Septic arthritis, in particular, stands out due to its rapid progression and the extensive damage it inflicts on joints and cartilage. Collectively, BJIs can lead to long-lasting disability and place a substantial burden on healthcare systems.⁴

Although traditional antibiotics have been employed in the fight against S. aureus, the emergence of antibiotic resistance, particularly with the widespread prevalence of MRSA isolates, presents a substantial challenge to achieving effective treatment outcomes.⁵ The growing complexity of treating BJIs arises not

only from the ability of S. aureus to invade and persist within professional (e.g. macrophages) and non-professional (e.g. osteoblasts) phagocytic cells⁶ but also from its capacity to form biofilms.⁷ These intricacies significantly compound the challenges of treatment.⁸⁻¹² Consequently, there is an urgent need to explore innovative antibacterial strategies capable of effectively addressing both intracellular and biofilm-associated forms of S. aureus.

Among the emerging alternatives, antimicrobial peptides (AMPs) have become promising therapeutic options due to their established ability to modulate symbiotic bacterial populations.¹³,¹⁴ These small peptides are ubiquitous across all domains of life and play an integral role in an organism immune system.¹⁵ They typically possess broad-spectrum antimicrobial activity against bacteria, viruses and fungi.¹⁶ Recent advancements have led to the engineering of pseudomimetic AMPs, derived

<footnote n="1">¹Inserm U1230 BRM (Bacterial RNAs and Medicine), Université de Rennes, Rennes, France; ²SAS Olgram, Bréhan, France; ³CHU de Rennes, Service de Pharmacologie, Rennes, France; ⁴CHU de Rennes, Service de Bactériologie-Hygiène hospitalière, 2 rue Henri Le Guilloux, 35033 Rennes, France; ⁵CNR de la Résistance aux Antibiotiques (laboratoire associé ‘Entérocoques’), CHU de Rennes, Rennes, France</footnote>

<footnote>*Corresponding author. E-mail: vincent.cattoir@chu-rennes.fr</footnote>
# Intracellular activity and *in vivo* efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against *Staphylococcus aureus* clinical isolates

Jean-Baptiste Mascary<fnref n="1" />,², Valérie Bordeau<fnref n="1" />, Irène Nicolas², Marie-Clémence Verdier³, Pierre Rocheteau² and Vincent Cattoir ID ⁴,⁵*

Received 19 January 2024; accepted 2 February 2024

**Objectives:** Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both *in vitro* and *in vivo* for the treatment of septic arthritis caused by *Staphylococcus aureus*.

**Methods:** Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the *in vivo* efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg).

**Results:** MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2×MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (~10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h.

**Conclusions:** Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

## Introduction

*Staphylococcus aureus* remains a problematic pathogen implicated in a spectrum of bone and joint infections (BJIs), which encompass septic arthritis, acute and chronic osteomyelitis and spondylodiscitis, as well as infections related to orthopaedic devices.<fnref n="1" />⁻³ Septic arthritis, in particular, stands out due to its rapid progression and the extensive damage it inflicts on joints and cartilage. Collectively, BJIs can lead to long-lasting disability and place a substantial burden on healthcare systems.⁴

Although traditional antibiotics have been employed in the fight against S. aureus, the emergence of antibiotic resistance, particularly with the widespread prevalence of MRSA isolates, presents a substantial challenge to achieving effective treatment outcomes.⁵ The growing complexity of treating BJIs arises not

only from the ability of S. aureus to invade and persist within professional (e.g. macrophages) and non-professional (e.g. osteoblasts) phagocytic cells⁶ but also from its capacity to form biofilms.⁷ These intricacies significantly compound the challenges of treatment.⁸⁻¹² Consequently, there is an urgent need to explore innovative antibacterial strategies capable of effectively addressing both intracellular and biofilm-associated forms of S. aureus.

Among the emerging alternatives, antimicrobial peptides (AMPs) have become promising therapeutic options due to their established ability to modulate symbiotic bacterial populations.¹³,¹⁴ These small peptides are ubiquitous across all domains of life and play an integral role in an organism immune system.¹⁵ They typically possess broad-spectrum antimicrobial activity against bacteria, viruses and fungi.¹⁶ Recent advancements have led to the engineering of pseudomimetic AMPs, derived

<footnote n="1">¹Inserm U1230 BRM (Bacterial RNAs and Medicine), Université de Rennes, Rennes, France; ²SAS Olgram, Bréhan, France; ³CHU de Rennes, Service de Pharmacologie, Rennes, France; ⁴CHU de Rennes, Service de Bactériologie-Hygiène hospitalière, 2 rue Henri Le Guilloux, 35033 Rennes, France; ⁵CNR de la Résistance aux Antibiotiques (laboratoire associé ‘Entérocoques’), CHU de Rennes, Rennes, France</footnote>

<footnote>*Corresponding author. E-mail: vincent.cattoir@chu-rennes.fr</footnote>
from a toxin naturally produced by *S. aureus*.¹⁷ These pseudopeptides exhibit bactericidal properties against a range of MDR Gram-negative and Gram-positive pathogens, including MRSA.¹⁸ While their exact mechanism of action remains unclear, they appear to primarily disrupt the integrity of bacterial cell membranes, leading to bacteriolysis.¹⁸ One such peptide of considerable interest is Pep16, which has exhibited promising outcomes in mouse sepsis models caused by MRSA.¹⁸ Importantly, Pep16 administration has shown minimal toxicity and a reduced likelihood of bacterial resistance emergence, positioning it as a favourable candidate for antimicrobial drug development.¹⁸ Given the significant burden posed by BJIs, particularly septic arthritis caused by *S. aureus*, we explored the potential of Pep16 as a viable treatment option for these infections.

In this study, our objective was to assess the efficacy of Pep16, both *in vitro* and *in vivo*, for eradicating *S. aureus* in BJIs. Firstly, we evaluated the *in vitro* activity of Pep16, either alone or in combination with other agents, comparing it with commonly used antibiotics for the BJI treatment. Secondly, we determined the intracellular activity of Pep16 using two relevant cell-based infection models involving osteoblasts and macrophages. Finally, we assessed the *in vivo* efficacy of Pep16 using a mouse model of septic arthritis.

## Materials and methods

### Bacterial strains and antimicrobial susceptibility testing (AST)

A total of seven clinical isolates of *S. aureus* obtained from patients suffering from septic arthritis in 2020–21 were studied, as detailed in Table 1. Additionally, the MSSA reference strain ATCC 29213 was used as control for AST.

AST was conducted through the disc diffusion method, following the guidelines outlined by Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM)/EUCAST (www.sfm-microbiologie.org/boutique/comite-de-lantibiograme-de-la-sfm-casfm/). MIC and MBC values of Pep16 (synthesized by Olgram, France), as well as those of vancomycin, teicoplanin, daptomycin and levofloxacin, were determined using the broth microdilution reference method.

The presence of *in vitro* synergy between Pep16 and clinically relevant antibiotics was assessed through chequerboard synergy assay. Combinations of Pep16 and various antibiotics (vancomycin, teicoplanin, daptomycin, levofloxacin or rifampicin) were tested at concentrations in the ranges 1–64 and 0.03–32 mg/L, respectively. The FIC index was calculated by summing the FICs of Pep16 and the respective antibiotic. Synergy was defined as FIC index < 0.5.

Time-kill curves were performed with Pep16 (80 or 160 mg/L) against four clinical isolates of *S. aureus* from patients suffering from septic arthritis. Bacterial cell suspensions grown overnight in CAMHB (CA-MHB II) and was diluted in pure CAMHB to obtain a suspension calibrated at 0.5 MacFarland, then diluted 1:5 in CAMHB to obtain a suspension of $2 \times 10^7$ cfu/mL. One millilitre of this suspension was inoculated into 20 mL of antibiotic solution (expected final bacterial concentration of $10^6$ cfu/mL). Viable counts were enumerated by plating 100 µL of appropriate culture dilutions onto brain heart infusion (BHI) agar plates after 0, 3, 6, 8, 11 and 24 h of incubation at 37°C and expressed in $log_{10}$ cfu/mL. A bactericidal effect was defined as a $3 \times 10^5$ decrease in cfu counts compared with the initial inoculum.

### WGS and bioinformatic analysis

Genomic DNA was isolated using the Quick-DNA fungal/bacterial miniprep kit (Zymo Research, Irvine, CA, USA). DNA libraries were prepared using the NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs,

Ipswich, MA, USA). The libraries were then sequenced as paired-end reads ($2 \times 300$ bp) using an Illumina MiSeq platform and the MiSeq reagent kit version 3. The Illumina reads were assembled using the SPAdes software and the annotation of chromosome and plasmids was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (www.ncbi.nlm.nih.gov/genome/annotation_prok/). MLST and staphylococcal protein A (*spa*) typing were performed using the MLST database version 2.0 (https://cge.food.dtu.dk/services/MLST/) and the spaTyper software version 1.0 (https://cge.food.dtu.dk/services/spaTyper/). The nucleotide sequences were also submitted to ResFinder 4.4.1 (http://genepi.food.dtu.dk/resfinder) and VirulenceFinder 2.0 (https://cge.food.dtu.dk/services/VirulenceFinder/) to identify antimicrobial resistance mechanisms and virulence genes, respectively. Genomic sequences of the seven strains were deposited in GenBank under project PRJNA1040494.

### Cellular-based models of infection

The osteoblast infection model was conducted using MG-63 (CRL-1427) osteoblastic cells, following previously published procedures.¹⁹ Briefly, MG-63 cells were cultured at 37°C in a 5% $CO_2$ environment and passaged weekly for up to 20 weeks. The culture medium used was DMEM supplemented with 10% FBS, 25 mM HEPES, 2 mM $L$-glutamine, with or without antibiotics (100 U/mL penicillin plus 100 mg/L streptomycin). For the assays, MG-63 cells were seeded in 24-well plates until reaching 70%–80% confluence. Bacterial cell suspensions grown overnight in BHI broth were prepared and adjusted to an moi of 50, and then introduced to the osteoblasts. After a 2 h coincubation to facilitate bacterial adhesion, MG-63 cells were rinsed and treated with 100 mg/L gentamicin for 1 h to eliminate extracellular bacterial cells. Infected osteoblasts were subsequently exposed to Pep16 or levofloxacin (at $2 \times MIC$) for 3 h in the absence of FBS. Finally, intracellular bacterial cells were quantified in cellular lysates (following osmotic shock) by plating them onto BHI agar.

The macrophage infection model was performed using the THP-1 human monocytic cell line (ATCC, Rockville, USA). In brief, cells were cultured in the Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS in T75 flasks at 37°C and 5% $CO_2$, and were maintained through weekly transfers within passages 2 to 20. For the assays, THP-1 cells were seeded in 12-well plates and differentiated into macrophages with phorbol myristate acetate (PMA) for 72 h until they reached approximately 80% confluence. Bacterial suspensions, grown overnight in BHI, were prepared and adjusted to an moi of 10, and then introduced to the macrophages. After a 2 h coincubation to facilitate bacterial adherence, the cells were washed and treated with 100 mg/L gentamicin for 1 h. Infected macrophages were subsequently exposed to Pep16 or levofloxacin (at $2 \times MIC$) for 24 h in the absence of FBS. Finally, intracellular bacterial cells were enumerated from cellular lysates (following osmotic shock) by plating them onto BHI agar. Infected but untreated cells were used as a control in all experiments. The number of cfu in treated cells was standardized as a percentage of the controls.

In both models, the viability was assessed using the MTT assay to determine the potential cytotoxicity of antimicrobials on untreated and infected/treated cells. Briefly, an MTT solution (0.5 mg/mL) was added to the cell medium without FBS. After washing with PBS and a 30 min incubation at 37°C, the formazan crystals formed were dissolved in DMSO. Absorbance values at 540 nm were measured using a BioTek Synergy 2 microplate reader.

### Mouse model of septic arthritis

A mouse model of septic arthritis was employed to assess the *in vivo* effectiveness of Pep16 in comparison with vancomycin, following previously described procedures.²⁰ Female Swiss mice, obtained from Janvier Labs and aged between 11 and 13 weeks with an approximate weight of 34 g, were used. The experiments were conducted in the ARCHE animal facility (Biosit) in Rennes. Mice were inoculated IV with 200 µL of a 0.9% NaCl
from a toxin naturally produced by *S. aureus*.¹⁷ These pseudopeptides exhibit bactericidal properties against a range of MDR Gram-negative and Gram-positive pathogens, including MRSA.¹⁸ While their exact mechanism of action remains unclear, they appear to primarily disrupt the integrity of bacterial cell membranes, leading to bacteriolysis.¹⁸ One such peptide of considerable interest is Pep16, which has exhibited promising outcomes in mouse sepsis models caused by MRSA.¹⁸ Importantly, Pep16 administration has shown minimal toxicity and a reduced likelihood of bacterial resistance emergence, positioning it as a favourable candidate for antimicrobial drug development.¹⁸ Given the significant burden posed by BJIs, particularly septic arthritis caused by *S. aureus*, we explored the potential of Pep16 as a viable treatment option for these infections.

In this study, our objective was to assess the efficacy of Pep16, both *in vitro* and *in vivo*, for eradicating *S. aureus* in BJIs. Firstly, we evaluated the *in vitro* activity of Pep16, either alone or in combination with other agents, comparing it with commonly used antibiotics for the BJI treatment. Secondly, we determined the intracellular activity of Pep16 using two relevant cell-based infection models involving osteoblasts and macrophages. Finally, we assessed the *in vivo* efficacy of Pep16 using a mouse model of septic arthritis.

## Materials and methods

### Bacterial strains and antimicrobial susceptibility testing (AST)

A total of seven clinical isolates of *S. aureus* obtained from patients suffering from septic arthritis in 2020–21 were studied, as detailed in Table 1. Additionally, the MSSA reference strain ATCC 29213 was used as control for AST.

AST was conducted through the disc diffusion method, following the guidelines outlined by Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM)/EUCAST (www.sfm-microbiologie.org/boutique/comite-de-lantibiograme-de-la-sfm-casfm/). MIC and MBC values of Pep16 (synthesized by Olgram, France), as well as those of vancomycin, teicoplanin, daptomycin and levofloxacin, were determined using the broth microdilution reference method.

The presence of *in vitro* synergy between Pep16 and clinically relevant antibiotics was assessed through chequerboard synergy assay. Combinations of Pep16 and various antibiotics (vancomycin, teicoplanin, daptomycin, levofloxacin or rifampicin) were tested at concentrations in the ranges 1–64 and 0.03–32 mg/L, respectively. The FIC index was calculated by summing the FICs of Pep16 and the respective antibiotic. Synergy was defined as FIC index < 0.5.

Time-kill curves were performed with Pep16 (80 or 160 mg/L) against four clinical isolates of *S. aureus* from patients suffering from septic arthritis. Bacterial cell suspensions grown overnight in CAMHB (CA-MHB II) and was diluted in pure CAMHB to obtain a suspension calibrated at 0.5 MacFarland, then diluted 1:5 in CAMHB to obtain a suspension of $2 \times 10^7$ cfu/mL. One millilitre of this suspension was inoculated into 20 mL of antibiotic solution (expected final bacterial concentration of $10^6$ cfu/mL). Viable counts were enumerated by plating 100 µL of appropriate culture dilutions onto brain heart infusion (BHI) agar plates after 0, 3, 6, 8, 11 and 24 h of incubation at 37°C and expressed in $log_{10}$ cfu/mL. A bactericidal effect was defined as a $3 \times 10^5$ decrease in cfu counts compared with the initial inoculum.

### WGS and bioinformatic analysis

Genomic DNA was isolated using the Quick-DNA fungal/bacterial miniprep kit (Zymo Research, Irvine, CA, USA). DNA libraries were prepared using the NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs,

Ipswich, MA, USA). The libraries were then sequenced as paired-end reads ($2 \times 300$ bp) using an Illumina MiSeq platform and the MiSeq reagent kit version 3. The Illumina reads were assembled using the SPAdes software and the annotation of chromosome and plasmids was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (www.ncbi.nlm.nih.gov/genome/annotation_prok/). MLST and staphylococcal protein A (*spa*) typing were performed using the MLST database version 2.0 (https://cge.food.dtu.dk/services/MLST/) and the spaTyper software version 1.0 (https://cge.food.dtu.dk/services/spaTyper/). The nucleotide sequences were also submitted to ResFinder 4.4.1 (http://genepi.food.dtu.dk/resfinder) and VirulenceFinder 2.0 (https://cge.food.dtu.dk/services/VirulenceFinder/) to identify antimicrobial resistance mechanisms and virulence genes, respectively. Genomic sequences of the seven strains were deposited in GenBank under project PRJNA1040494.

### Cellular-based models of infection

The osteoblast infection model was conducted using MG-63 (CRL-1427) osteoblastic cells, following previously published procedures.¹⁹ Briefly, MG-63 cells were cultured at 37°C in a 5% $CO_2$ environment and passaged weekly for up to 20 weeks. The culture medium used was DMEM supplemented with 10% FBS, 25 mM HEPES, 2 mM $L$-glutamine, with or without antibiotics (100 U/mL penicillin plus 100 mg/L streptomycin). For the assays, MG-63 cells were seeded in 24-well plates until reaching 70%–80% confluence. Bacterial cell suspensions grown overnight in BHI broth were prepared and adjusted to an moi of 50, and then introduced to the osteoblasts. After a 2 h coincubation to facilitate bacterial adhesion, MG-63 cells were rinsed and treated with 100 mg/L gentamicin for 1 h to eliminate extracellular bacterial cells. Infected osteoblasts were subsequently exposed to Pep16 or levofloxacin (at $2 \times MIC$) for 3 h in the absence of FBS. Finally, intracellular bacterial cells were quantified in cellular lysates (following osmotic shock) by plating them onto BHI agar.

The macrophage infection model was performed using the THP-1 human monocytic cell line (ATCC, Rockville, USA). In brief, cells were cultured in the Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS in T75 flasks at 37°C and 5% $CO_2$, and were maintained through weekly transfers within passages 2 to 20. For the assays, THP-1 cells were seeded in 12-well plates and differentiated into macrophages with phorbol myristate acetate (PMA) for 72 h until they reached approximately 80% confluence. Bacterial suspensions, grown overnight in BHI, were prepared and adjusted to an moi of 10, and then introduced to the macrophages. After a 2 h coincubation to facilitate bacterial adherence, the cells were washed and treated with 100 mg/L gentamicin for 1 h. Infected macrophages were subsequently exposed to Pep16 or levofloxacin (at $2 \times MIC$) for 24 h in the absence of FBS. Finally, intracellular bacterial cells were enumerated from cellular lysates (following osmotic shock) by plating them onto BHI agar. Infected but untreated cells were used as a control in all experiments. The number of cfu in treated cells was standardized as a percentage of the controls.

In both models, the viability was assessed using the MTT assay to determine the potential cytotoxicity of antimicrobials on untreated and infected/treated cells. Briefly, an MTT solution (0.5 mg/mL) was added to the cell medium without FBS. After washing with PBS and a 30 min incubation at 37°C, the formazan crystals formed were dissolved in DMSO. Absorbance values at 540 nm were measured using a BioTek Synergy 2 microplate reader.

### Mouse model of septic arthritis

A mouse model of septic arthritis was employed to assess the *in vivo* effectiveness of Pep16 in comparison with vancomycin, following previously described procedures.²⁰ Female Swiss mice, obtained from Janvier Labs and aged between 11 and 13 weeks with an approximate weight of 34 g, were used. The experiments were conducted in the ARCHE animal facility (Biosit) in Rennes. Mice were inoculated IV with 200 µL of a 0.9% NaCl
<table><caption>**Table 1.** *In vitro* activity of Pep16 and comparators against the seven clinical isolates of S. aureus responsible for septic arthritis</caption><thead><tr><th rowspan="2">S. aureus<br>strain</th><th rowspan="2">Origin (location, date)</th><th rowspan="2">Resistance<br>phenotype</th><th rowspan="2">ST</th><th rowspan="2">spa type</th><th rowspan="2">Resistance<br>mechanisms</th><th rowspan="2">Virulence genes</th><th colspan="5">MIC/MBC (mg/L)</th></tr><tr><th>Pep16</th><th>VAN</th><th>TEC</th><th>DAP</th><th>LVX</th></tr></thead><tbody><tr><td>1333</td><td>Clinical, joint fluid<br>(Rennes, September 2020)</td><td>MSSA, multi<sup>5</sup></td><td>398</td><td>t34</td><td>blaZ</td><td>aur, hlgA, hlgB, hlgC</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1334</td><td>Clinical, joint fluid<br>(Rennes, November 2020)</td><td>MSSA, multi<sup>5</sup></td><td>New<sup>6</sup></td><td>t548</td><td>—</td><td>aur, splA, spIB, hlgA,<br>hlgB, hlgC, lukD, lukE,<br>seg, sei, sem, sen,<br>seo, seu, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.25/0.5</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1814</td><td>Clinical, joint fluid<br>(Rennes, September 2020)</td><td>MRSA, cip<sup>R</sup></td><td>30</td><td>t363</td><td>blaZ, mecA, dfrG<br>Mutations in gyrA<br>(S84L) and parC<br>(S80Y)</td><td>aur, splE, hlgA, hlgB,<br>hlgC, lukF-PV, lukS-PV,<br>seg, sei, sem, sen,<br>seo, seu, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>4/4</td></tr><tr><td>1815</td><td>Clinical, joint fluid<br>(Rennes, March 2021)</td><td>MRSA, gen<sup>R</sup>, ery<sup>R</sup></td><td>8</td><td>t1476</td><td>blaZ, mecA,<br>aac(6')-aph(2'),<br>erm(C), dfrG<br>Mutation in parC<br>(S80Y)</td><td>aur, splA, spIB, spIE,<br>hlgA, hlgB, hlgC, lukD,<br>lukE, tst, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1816</td><td>Clinical, joint fluid<br>(Rennes, January 2021)</td><td>MSSA, multi<sup>5</sup></td><td>398</td><td>t6608</td><td>—</td><td>aur, hlgA, hlgB, hlgC, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.25/0.5</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>1817</td><td>Clinical, joint fluid<br>(Rennes, February 2021)</td><td>MSSA, ery<sup>R</sup></td><td>398</td><td>t571</td><td>blaZ, erm(T)</td><td>aur, hlgA, hlgB, hlgC, SCN</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>1818</td><td>Clinical, joint fluid<br>(Rennes, February 2021)</td><td>MSSA, multi<sup>5</sup></td><td>12</td><td>t156</td><td>—</td><td>aur, splA, spIB, spIE,<br>hlgA, hlgB, hlgC, lukD,<br>lukE, sec, sel, sep, sak,<br>scn</td><td>8/16</td><td>0.5/0.5</td><td>1/2</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>ATCC 29213</td><td>Reference</td><td>MSSA, multi<sup>5</sup></td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr></tbody></table>

VAN, vancomycin; TEC, teicoplanin; DAP, daptomycin; LVX, levofloxacin; CIP, ciprofloxacin resistant; ERY, erythromycin resistant (inducible MLS<sub>B</sub> phenotype); GEN<sup>R</sup>, gentamicin resistant (KTG phenotype); MULTI<sup>5</sup>, multi-susceptible; nd, not determined.

<footnote n="6"><sup>6</sup>New ST close to ST2975.</footnote>
<table><caption>**Table 1.** *In vitro* activity of Pep16 and comparators against the seven clinical isolates of S. aureus responsible for septic arthritis</caption><thead><tr><th rowspan="2">S. aureus strain</th><th rowspan="2">Origin (location, date)</th><th rowspan="2">Resistance phenotype</th><th rowspan="2">ST</th><th rowspan="2">spa type</th><th rowspan="2">Resistance mechanisms</th><th rowspan="2">Virulence genes</th><th colspan="5">MIC/MBC (mg/L)</th></tr><tr><th>Pep16</th><th>VAN</th><th>TEC</th><th>DAP</th><th>LVX</th></tr></thead><tbody><tr><td>1333</td><td>Clinical, joint fluid (Rennes, September 2020)</td><td>MSSA, multi<sup>5</sup></td><td>398</td><td>t34</td><td>blaZ</td><td>aur, hlgA, hlgB, hlgC</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1334</td><td>Clinical, joint fluid (Rennes, November 2020)</td><td>MSSA, multi<sup>5</sup></td><td>New<sup>6</sup></td><td>t548</td><td>—</td><td>aur, splA, spIB, hlgA, hlgB, hlgC, lukD, lukE, seg, sei, sem, sen, seo, seu, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.25/0.5</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1814</td><td>Clinical, joint fluid (Rennes, September 2020)</td><td>MRSA, cip<sup>R</sup></td><td>30</td><td>t363</td><td>blaZ, mecA, dfrG</td><td>aur, splE, hlgA, hlgB, hlgC, lukF-PV, lukS-PV, seg, sei, sem, sen, seo, seu, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>4/4</td></tr><tr><td>1815</td><td>Clinical, joint fluid (Rennes, March 2021)</td><td>MRSA, gen<sup>R</sup>, ery<sup>R</sup></td><td>8</td><td>t1476</td><td>blaZ, mecA, aac(6')-aph(2'), erm(C), dfrG</td><td>aur, splA, spIB, spIE, hlgA, hlgB, hlgC, lukD, lukE, tst, sak, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr><tr><td>1816</td><td>Clinical, joint fluid (Rennes, January 2021)</td><td>MSSA, multi<sup>5</sup></td><td>398</td><td>t6608</td><td>—</td><td>aur, hlgA, hlgB, hlgC, scn</td><td>8/16</td><td>0.5/0.5</td><td>0.25/0.5</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>1817</td><td>Clinical, joint fluid (Rennes, February 2021)</td><td>MSSA, ery<sup>R</sup></td><td>398</td><td>t571</td><td>blaZ, erm(T)</td><td>aur, hlgA, hlgB, hlgC, SCN</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>1818</td><td>Clinical, joint fluid (Rennes, February 2021)</td><td>MSSA, multi<sup>5</sup></td><td>12</td><td>t156</td><td>—</td><td>aur, splA, spIB, spIE, hlgA, hlgB, hlgC, lukD, lukE, sec, sel, sep, sak, SCN</td><td>8/16</td><td>0.5/0.5</td><td>1/2</td><td>0.25/0.5</td><td>0.25/0.25</td></tr><tr><td>ATCC 29213</td><td>Reference</td><td>MSSA, multi<sup>5</sup></td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>8/16</td><td>0.5/0.5</td><td>0.5/1</td><td>0.25/0.5</td><td>0.5/0.5</td></tr></tbody></table>

VAN, vancomycin; TEC, teicoplanin; DAP, daptomycin; LVX, levofloxacin; CIP, ciprofloxacin resistant; ERY, erythromycin resistant (inducible MLS<sub>B</sub> phenotype); GEN<sup>R</sup>, gentamicin resistant (KTG phenotype); MULTI<sup>5</sup>, multi-susceptible; nd, not determined.

<footnote n="9">⁹New ST close to ST2975.</footnote>
solution containing approximately 10⁸ cfu of the 1334 strain (MSSA). Three hours after inoculation, infected mice received a subcutaneous dose of either a saline solution (0.9% NaCl), Pep16 (10 mg/kg) or vancomycin (100 mg/kg). These treatments were administered daily for a duration of 7 days. Each experimental group included a maximum of five mice and the experiments were repeated twice for the vancomycin group and three times for the 0.9% NaCl and Pep16 groups. The mice were monitored on a daily basis, and on the seventh day, they were humanely euthanized using CO₂. Subsequently, knee joints, spleen and kidneys were excised, weighed and homogenized in 0.9% NaCl using a Polytron PT 3100D homogenizer (Kinematica, Switzerland). Bacterial loads were determined by plating serial dilutions onto BHI agar and expressed as log₁₀ cfu/g.

## Pep16 pharmacokinetic analysis

Following subcutaneous injection of Pep16 (10 mg/kg), plasma concentrations were assessed at various timepoints including 0, 0.5, 1, 3, 6, 12, 15 and 24 h in non-infected mice. Blood samples were collected, subjected to centrifugation and the resulting plasma was stored at -80°C. Quantification was carried out utilizing a previously described LC-MS/MS method.²¹

## Statistical methods

For continuous variables, means and their standard deviations (SDs) were presented and compared using the Mann-Whitney U-test. Proportions were compared using a Fisher's exact or chi-squared test, when appropriate. Statistical analyses were conducted using Prism software v9.0 (GraphPad, San Diego, CA, USA). A P value of <0.05 was considered statistically significant.

## Ethics

All experimental protocols were approved by the Adaptive Therapeutics Animal Care and Use Committee (APAFIS #4508-201603111554125_v5). The animals were housed in compliant cages and provided with free access to food and water.

## Results

### Characterization of clinical isolates

Out of the seven clinical isolates of S. aureus, two were MRSA (1814 and 1815) while the remaining five were MSSA (1333, 1334, 1816, 1817 and 1818) (Table 1). One MRSA isolate displayed ciprofloxacin resistance, while the other MRSA isolate exhibited resistance to gentamicin (KTG phenotype) and erythromycin (inducible MLS<sub>B</sub> phenotype) but remained susceptible to fluoroquinolones. Among the five MSSA isolates, four displayed susceptibility to multiple antibiotics, with only one (1817) exhibiting a resistance trait (inducible MLS<sub>B</sub> phenotype).

Thanks to WGS analysis, we determined that the seven isolates belonged to five STs (including three strains being ST398) and seven different spa types. As expected, both MRSA isolates harboured a mecA gene and the presence or absence of other resistance genes correlated with resistance phenotypes in all cases. Interestingly, several virulence genes were detected for all strains including exoenzymes, toxins and immunomodulatory proteins. Notably, the MRSA strain 1814 possessed *lukF-PV* and *lukS-PV* genes coding for Panton-Valentine leucocidin (PVL) while the MSSA strain was positive for the *tst* gene coding for toxic shock syndrome toxin-1 (TSST-1).

## In vitro activity of Pep16

Subsequently, we assessed the *in vitro* activity of Pep16 and compared it with commonly used antibiotics for treating BJIs caused by S. aureus. This assessment involved determining both MIC and MBC values. Notably, the MICs of Pep16 for all S. aureus isolates were consistently 8 mg/L. In contrast, the MICs for vancomycin, teicoplanin, daptomycin and levofloxacin were found to be 0.5, 0.25-1, 0.25 and 0.25-4 mg/L, respectively (Table 1). Although Pep16 exhibited MICs 2-to-32-fold higher than those of the comparator antibiotics, the MBC/MIC ratios indicated bactericidal activity, with ratios consistently at or below 4 for all strains (Table 1). This bactericidal activity was confirmed by establishing time-kill curves with concentrations of 10×MIC and 20×MIC (Figure S1, available as Supplementary data at JAC-AMR Online). Furthermore, we employed a cheuerboard synergy assay to explore combinations of Pep16 with various clinically relevant antibiotics (vancomycin, teicoplanin, daptomycin, levofloxacin or rifampicin). Unfortunately, this analysis did not reveal any synergy (Figure S2).

## Intracellular activity of Pep16

We assessed the potential intracellular activity of Pep16 using two cellular infection models, one involving non-professional phagocytic cells (osteoblasts) and the other using professional phagocytic cells (macrophages), by comparison with levofloxacin.

In the acute model of osteoblast infection, we observed that both Pep16 and levofloxacin (administered at 2×MIC) significantly reduced the bacterial load of all four tested S. aureus isolates (2 MSSA and 2 MRSA) 3 h after treatment, in comparison with untreated cells (Figure 1). Pep16 notably decreased the intracellular inoculum of both MRSA and MSSA, with reductions ranging from 46.5% to 86.6% for MRSA isolates and approximately 63% for MSSA isolates (Figure 1). Although levofloxacin appeared to be significantly more effective than Pep16 against MSSA isolates, no significant differences were observed between these compounds against intracellular MRSA (Figure 1). Importantly, it is worth noting that we did not observe any significant direct cytotoxicity for either Pep16 or levofloxacin (administered at 2×MIC) on uninfected osteoblasts (MG-63) after 3 h of incubation (Figure S3).

In the acute model of macrophage infection, both Pep16 and levofloxacin (administered at 2×MIC) were effective in significantly reducing the intracellular inoculum of the same four S. aureus clinical isolates (MRSA 1814, MRSA 1815, MSSA 1333 and MSSA 1334) 24 h after treatment, compared with untreated cells (Figure 2). Pep16 produced substantial reductions in the intracellular inoculum, with decreases of 83% (±1.8%), 91.9% (±2.2%), 90.1% (±2.6%) and 90.1% (±8.0%) for MRSA 1814, MRSA 1815, MSSA 1333 and MSSA 1334, respectively (Figure 2). No significant differences were found between levofloxacin and Pep16 in reducing intracellular MRSA and MSSA isolates (Figure 2). Similar to osteoblasts, no significant direct cytotoxicity was observed for either Pep16 or levofloxacin (at 2×MIC) on uninfected macrophages (THP-1) after 24 h of incubation (Figure S3).

Altogether, we demonstrated that Pep16 exhibits substantial intracellular activity against both MRSA and MSSA strains in osteoblasts and macrophages, with comparable efficacy to
solution containing approximately 10⁸ cfu of the 1334 strain (MSSA). Three hours after inoculation, infected mice received a subcutaneous dose of either a saline solution (0.9% NaCl), Pep16 (10 mg/kg) or vancomycin (100 mg/kg). These treatments were administered daily for a duration of 7 days. Each experimental group included a maximum of five mice and the experiments were repeated twice for the vancomycin group and three times for the 0.9% NaCl and Pep16 groups. The mice were monitored on a daily basis, and on the seventh day, they were humanely euthanized using CO₂. Subsequently, knee joints, spleen and kidneys were excised, weighed and homogenized in 0.9% NaCl using a Polytron PT 3100D homogenizer (Kinematica, Switzerland). Bacterial loads were determined by plating serial dilutions onto BHI agar and expressed as log₁₀ cfu/g.

## Pep16 pharmacokinetic analysis

Following subcutaneous injection of Pep16 (10 mg/kg), plasma concentrations were assessed at various timepoints including 0, 0.5, 1, 3, 6, 12, 15 and 24 h in non-infected mice. Blood samples were collected, subjected to centrifugation and the resulting plasma was stored at -80°C. Quantification was carried out utilizing a previously described LC-MS/MS method.²¹

## Statistical methods

For continuous variables, means and their standard deviations (SDs) were presented and compared using the Mann-Whitney U-test. Proportions were compared using a Fisher's exact or chi-squared test, when appropriate. Statistical analyses were conducted using Prism software v9.0 (GraphPad, San Diego, CA, USA). A P value of <0.05 was considered statistically significant.

## Ethics

All experimental protocols were approved by the Adaptive Therapeutics Animal Care and Use Committee (APAFIS #4508-201603111554125_v5). The animals were housed in compliant cages and provided with free access to food and water.

## Results

### Characterization of clinical isolates

Out of the seven clinical isolates of S. aureus, two were MRSA (1814 and 1815) while the remaining five were MSSA (1333, 1334, 1816, 1817 and 1818) (Table 1). One MRSA isolate displayed ciprofloxacin resistance, while the other MRSA isolate exhibited resistance to gentamicin (KTG phenotype) and erythromycin (inducible MLS<sub>B</sub> phenotype) but remained susceptible to fluoroquinolones. Among the five MSSA isolates, four displayed susceptibility to multiple antibiotics, with only one (1817) exhibiting a resistance trait (inducible MLS<sub>B</sub> phenotype).

Thanks to WGS analysis, we determined that the seven isolates belonged to five STs (including three strains being ST398) and seven different spa types. As expected, both MRSA isolates harboured a mecA gene and the presence or absence of other resistance genes correlated with resistance phenotypes in all cases. Interestingly, several virulence genes were detected for all strains including exoenzymes, toxins and immunomodulatory proteins. Notably, the MRSA strain 1814 possessed *lukF-PV* and *lukS-PV* genes coding for Panton-Valentine leucocidin (PVL) while the MSSA strain was positive for the *tst* gene coding for toxic shock syndrome toxin-1 (TSST-1).

### In vitro activity of Pep16

Subsequently, we assessed the *in vitro* activity of Pep16 and compared it with commonly used antibiotics for treating BJIs caused by S. aureus. This assessment involved determining both MIC and MBC values. Notably, the MICs of Pep16 for all S. aureus isolates were consistently 8 mg/L. In contrast, the MICs for vancomycin, teicoplanin, daptomycin and levofloxacin were found to be 0.5, 0.25-1, 0.25 and 0.25-4 mg/L, respectively (Table 1). Although Pep16 exhibited MICs 2-to-32-fold higher than those of the comparator antibiotics, the MBC/MIC ratios indicated bactericidal activity, with ratios consistently at or below 4 for all strains (Table 1). This bactericidal activity was confirmed by establishing time-kill curves with concentrations of 10×MIC and 20×MIC (Figure S1, available as Supplementary data at JAC-AMR Online). Furthermore, we employed a cheuerboard synergy assay to explore combinations of Pep16 with various clinically relevant antibiotics (vancomycin, teicoplanin, daptomycin, levofloxacin or rifampicin). Unfortunately, this analysis did not reveal any synergy (Figure S2).

### Intracellular activity of Pep16

We assessed the potential intracellular activity of Pep16 using two cellular infection models, one involving non-professional phagocytic cells (osteoblasts) and the other using professional phagocytic cells (macrophages), by comparison with levofloxacin.

In the acute model of osteoblast infection, we observed that both Pep16 and levofloxacin (administered at 2×MIC) significantly reduced the bacterial load of all four tested S. aureus isolates (2 MSSA and 2 MRSA) 3 h after treatment, in comparison with untreated cells (Figure 1). Pep16 notably decreased the intracellular inoculum of both MRSA and MSSA, with reductions ranging from 46.5% to 86.6% for MRSA isolates and approximately 63% for MSSA isolates (Figure 1). Although levofloxacin appeared to be significantly more effective than Pep16 against MSSA isolates, no significant differences were observed between these compounds against intracellular MRSA (Figure 1). Importantly, it is worth noting that we did not observe any significant direct cytotoxicity for either Pep16 or levofloxacin (administered at 2×MIC) on uninfected osteoblasts (MG-63) after 3 h of incubation (Figure S3).

In the acute model of macrophage infection, both Pep16 and levofloxacin (administered at 2×MIC) were effective in significantly reducing the intracellular inoculum of the same four S. aureus clinical isolates (MRSA 1814, MRSA 1815, MSSA 1333 and MSSA 1334) 24 h after treatment, compared with untreated cells (Figure 2). Pep16 produced substantial reductions in the intracellular inoculum, with decreases of 83% (±1.8%), 91.9% (±2.2%), 90.1% (±2.6%) and 90.1% (±8.0%) for MRSA 1814, MRSA 1815, MSSA 1333 and MSSA 1334, respectively (Figure 2). No significant differences were found between levofloxacin and Pep16 in reducing intracellular MRSA and MSSA isolates (Figure 2). Similar to osteoblasts, no significant direct cytotoxicity was observed for either Pep16 or levofloxacin (at 2×MIC) on uninfected macrophages (THP-1) after 24 h of incubation (Figure S3).

Altogether, we demonstrated that Pep16 exhibits substantial intracellular activity against both MRSA and MSSA strains in osteoblasts and macrophages, with comparable efficacy to
<figure><img src="image_1.png" /><figcaption>**Figure 1.** Intracellular activity of Pep16 and levofloxacin against intra-osteoblastic S. aureus MRSA 1814 (a), MRSA 1815 (b), MSSA 1333 (c) and MSSA 1334 (d). At 3 h post-infection, MG-63 cells were treated with Pep16 or levofloxacin at 2×MIC for 3 h. Data represent the mean±SD from three independent experiments. Intracellular cfu counts were normalized relative to untreated cells (depicted by black histograms), and differences between treated and untreated cells, as well as between the two compounds, were assessed using the Mann-Whitney U-test. \*P<0.05; \*\*P<0.01; \*\*\*P<0.001; \*\*\*\*P<0.0001.</figcaption></figure>

levofloxacin, making it a promising candidate for the treatment of S. aureus septic arthritis.

**In vivo efficacy of Pep16 in a mouse model of septic arthritis**

Based on these promising results, we proceeded to assess the efficacy of Pep16 in a mouse model using only one isolate (MSSA 1334) due to ethical reasons and since all four isolates had similar *in vitro* and intracellular activities. Note that all mice, untreated or treated by Pep16 or vancomycin, had survived at Day 7 (Figure S4). After subcutaneous treatment with Pep16 at a concentration of 10 mg/kg, a significant (~10-fold) reduction in cfu counts was observed within the knee joints of infected mice 7 days after infection, in comparison with untreated mice. However, this reduction was not observed in kidneys or spleen (Figure 3). A similar reduction in cfu counts was noted in knee joints, kidneys and spleen of mice treated with vancomycin (at 100 mg/kg), compared with control mice (Figure 3).

Additionally, a pharmacokinetic analysis of Pep16 following a single subcutaneous administration of 10 mg/kg revealed a gradual increase in plasma concentrations over the course of 12 h, with a $C_{max}$ of 5.6 mg/L (Figure 4).

Here, we demonstrate that Pep16, administered subcutaneously, significantly reduces load in the knee joints of infected mice, underscoring its potential as an effective treatment for S. aureus septic arthritis, while its peculiar pharmacokinetic profile needs further investigation.

**Discussion**

The aim of this research was to explore Pep16's antimicrobial potential against S. aureus, a key pathogen implicated in BJIs, by using a comprehensive investigation, designed to reflect clinical scenarios.

Notably, Pep16 exhibited a substantial reduction in the intracellular burden of S. aureus within both osteoblasts and macrophages, demonstrating comparable efficacy to the reference antibiotic, levofloxacin—a fluoroquinolone antibiotic known for its *in vitro*
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Intracellular activity of Pep16 and levofloxacin against intra-osteoblastic S. aureus MRSA 1814 (a), MRSA 1815 (b), MSSA 1333 (c) and MSSA 1334 (d). At 3 h post-infection, MG-63 cells were treated with Pep16 or levofloxacin at 2×MIC for 3 h. Data represent the mean±SD from three independent experiments. Intracellular cfu counts were normalized relative to untreated cells (depicted by black histograms), and differences between treated and untreated cells, as well as between the two compounds, were assessed using the Mann-Whitney *U*-test. \*P<0.05; \*\*P<0.01; \*\*\*P<0.001; \*\*\*\*P<0.0001.</figcaption></figure>

levofloxacin, making it a promising candidate for the treatment of S. aureus septic arthritis.

**In vivo efficacy of Pep16 in a mouse model of septic arthritis**

Based on these promising results, we proceeded to assess the efficacy of Pep16 in a mouse model using only one isolate (MSSA 1334) due to ethical reasons and since all four isolates had similar *in vitro* and intracellular activities. Note that all mice, untreated or treated by Pep16 or vancomycin, had survived at Day 7 (Figure S4). After subcutaneous treatment with Pep16 at a concentration of 10 mg/kg, a significant (~10-fold) reduction in cfu counts was observed within the knee joints of infected mice 7 days after infection, in comparison with untreated mice. However, this reduction was not observed in kidneys or spleen (Figure 3). A similar reduction in cfu counts was noted in knee joints, kidneys and spleen of mice treated with vancomycin (at 100 mg/kg), compared with control mice (Figure 3).

Additionally, a pharmacokinetic analysis of Pep16 following a single subcutaneous administration of 10 mg/kg revealed a gradual increase in plasma concentrations over the course of 12 h, with a $C_{max}$ of 5.6 mg/L (Figure 4).

Here, we demonstrate that Pep16, administered subcutaneously, significantly reduces load in the knee joints of infected mice, underscoring its potential as an effective treatment for S. aureus septic arthritis, while its peculiar pharmacokinetic profile needs further investigation.

**Discussion**

The aim of this research was to explore Pep16's antimicrobial potential against S. aureus, a key pathogen implicated in BJIs, by using a comprehensive investigation, designed to reflect clinical scenarios.

Notably, Pep16 exhibited a substantial reduction in the intracellular burden of S. aureus within both osteoblasts and macrophages, demonstrating comparable efficacy to the reference antibiotic, levofloxacin—a fluoroquinolone antibiotic known for its *in vitro*
<figure><img src="image_2.png" /><figcaption>**Figure 2.** Intracellular activity of Pep16 and levofloxacin against intra-macrophagic *S. aureus* MRSA 1814 (a), MRSA 1815 (b), MSSA 1333 (c) and MSSA 1334 (d). At 3 h post-infection, TH-P1 cells were treated with Pep16 or levofloxacin at 2×MIC for 24 h. Data represent the mean ± SD of three independent experiments. Intracellular cfu counts were normalized relative to untreated cells (depicted by black histograms), and differences between treated and untreated cells, as well as between the two compounds, were assessed using the Mann-Whitney *U*-test. ***P*<0.001; ****P*<0.0001.</figcaption></figure>

effectiveness against intracellular Gram-positive bacteria, including *S. aureus*.²² Importantly, no drug-induced cytotoxicity was observed at the tested concentrations. In comparison with newer tenders like dalbavancin and rifamycins, Pep16 showcased remarkable intracellular activity. Chauvelot *et al*.¹¹ showed that dalbavancin, a recently introduced extended-duration lipoglycopeptide, significantly reduced the presence of *S. aureus* in osteoblasts starting at its MIC (0.125 mg/L), demonstrating a dose-dependent effectiveness. However, Pep16 outperformed dalbavancin, achieving reductions of 86.6% for MRSA isolates and approximately 63% for MSSA isolates. In contrast, the peak of reduction by dalbavancin at 100× its MIC was only 51.6%.¹¹ In another study, conducted by Abad *et al*.¹⁹ on rifamycins, a group of antibiotics that inhibit bacterial RNA polymerase, the potential intracellular activity of these drugs against *S. aureus* was highlighted. When examining concentrations that were 10× or 100× their respective MICs, all rifamycins exhibited significant intracellular activity. However, the extent of reduction in intracellular bacteria varied: rifampicin reduced it by approximately 54.0%, rifapentine by 45.3%, and rifabutin stood out with a reduction of around 62.1%. The intracellular activity of Pep16 against *S. aureus*, compared with dalbavancin and rifamycins, further

underscores its potential as an effective agent. Pep16's unique structural composition, which includes non-natural amino acids,¹⁸ suggests enhanced cellular penetration, potentially supported by its amphiphilic properties. However, it is crucial to recognize that intracellular antimicrobial activity relies on factors beyond cellular concentration, including distribution within the intracellular compartment and the phagolysosomal acidic environment.²³

The mouse model of septic arthritis further confirmed Pep16's therapeutic potential, resulting in a 10-fold reduction in cfu counts within knee joints. These results build upon previous findings, indicating the promise of Pep16 as a therapeutic option.¹⁸,²¹ Indeed, the study by Nicolas *et al*. illustrated Pep16 significant efficacy in animal models of MRSA infections (sepsis and skin abscesses) and its ability to limit resistance emergence in MDR pathogens. This robust defence against MRSA infections corroborates our results and extends Pep16 applicability beyond septic arthritis to include potential treatments for severe sepsis.¹⁷,¹⁸ The findings of Chosidow *et al*. present another dimension of Pep16's therapeutic promise. In their peritonitis mouse model, Pep16 alone was most in its impact. However, in combination with colistin it showcased a dramatic reduction in bacterial counts, also staving off
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Intracellular activity of Pep16 and levofloxacin against intra-macrophagic *S. aureus* MRSA 1814 (a), MRSA 1815 (b), MSSA 1333 (c) and MSSA 1334 (d). At 3 h post-infection, TH-P1 cells were treated with Pep16 or levofloxacin at 2×MIC for 24 h. Data represent the mean ± SD of three independent experiments. Intracellular cfu counts were normalized relative to untreated cells (depicted by black histograms), and differences between treated and untreated cells, as well as between the two compounds, were assessed using the Mann-Whitney *U*-test. ***P*<0.001; ****P*<0.0001.</figcaption></figure>

effectiveness against intracellular Gram-positive bacteria, including *S. aureus*.²² Importantly, no drug-induced cytotoxicity was observed at the tested concentrations. In comparison with newer tenders like dalbavancin and rifamycins, Pep16 showcased remarkable intracellular activity. Chauvelot *et al*.¹¹ showed that dalbavancin, a recently introduced extended-duration lipoglycopeptide, significantly reduced the presence of *S. aureus* in osteoblasts starting at its MIC (0.125 mg/L), demonstrating a dose-dependent effectiveness. However, Pep16 outperformed dalbavancin, achieving reductions of 86.6% for MRSA isolates and approximately 63% for MSSA isolates. In contrast, the peak of reduction by dalbavancin at 100× its MIC was only 51.6%.¹¹ In another study, conducted by Abad *et al*.¹⁹ on rifamycins, a group of antibiotics that inhibit bacterial RNA polymerase, the potential intracellular activity of these drugs against *S. aureus* was highlighted. When examining concentrations that were 10× or 100× their respective MICs, all rifamycins exhibited significant intracellular activity. However, the extent of reduction in intracellular bacteria varied: rifampicin reduced it by approximately 54.0%, rifapentine by 45.3%, and rifabutin stood out with a reduction of around 62.1%. The intracellular activity of Pep16 against *S. aureus*, compared with dalbavancin and rifamycins, further

underscores its potential as an effective agent. Pep16's unique structural composition, which includes non-natural amino acids,¹⁸ suggests enhanced cellular penetration, potentially supported by its amphiphilic properties. However, it is crucial to recognize that intracellular antimicrobial activity relies on factors beyond cellular concentration, including distribution within the intracellular compartment and the phagolysosomal acidic environment.²³

The mouse model of septic arthritis further confirmed Pep16's therapeutic potential, resulting in a 10-fold reduction in cfu counts within knee joints. These results build upon previous findings, indicating the promise of Pep16 as a therapeutic option.¹⁸,²¹ Indeed, the study by Nicolas *et al*. illustrated Pep16 significant efficacy in animal models of MRSA infections (sepsis and skin abscesses) and its ability to limit resistance emergence in MDR pathogens. This robust defence against MRSA infections corroborates our results and extends Pep16 applicability beyond septic arthritis to include potential treatments for severe sepsis.¹⁷,¹⁸ The findings of Chosidow *et al*. present another dimension of Pep16's therapeutic promise. In their peritonitis mouse model, Pep16 alone was most in its impact. However, in combination with colistin it showcased a dramatic reduction in bacterial counts, also staving off
<figure><img src="image_3.png" /><figcaption>**Figure 3.** Bacterial loads (in log cfu/g) at Day 7 in knee joints, kidneys and spleen from mice infected by MSSA 1334. Mice were initially inoculated with approximately $1 \times 10^8$ cfu of MSSA 1334. At 3 h post-infection, mice were treated with either with 10 mg/kg of Pep16 or 100 mg/kg of vancomycin, while untreated control mice received a saline solution without antibiotics. Treatments were administered via subcutaneous injection and continued daily until the conclusion of the experiment. Individual dots on the graph represent cfu counts for single animals (enumerated at Day 7), with black horizontal lines indicating median values. The experiments were performed with five mice per condition, in triplicate for Pep16 and saline solution, and in duplicate for vancomycin. Statistical analysis was conducted using the Mann-Whitney *U*-test. ***P*<0.001; ****P*<0.0001.</figcaption></figure>

colistin-resistant mutants.²¹ This underscores the potential of leveraging combination therapies with Pep16 to achieve better clinical outcomes and manage drug resistance.

Nonetheless, our study has certain limitations, with one being the absence of standardized methodologies for assessing intracellular activity and *in vivo* efficacy. This poses a challenge when comparing results across different studies. Methodological discrepancies, such as treatment timing, can significantly impact findings. In particular, the majority of studies opt for immediate post-infection treatment while only a few consider delayed treatments, such as 12 h or 7 days post-infection. This methodological choice can affect the relevance of the findings, especially in chronic osteomyelitis contexts, where patients are often treated long after the infection onset.²⁴ Moreover, a crucial distinction observed among existing studies is the cell model selected. Numerous studies utilize macrophages and cell lines, which exhibit contrasting behaviours to bone cells, particularly primary cells, in the context of *S. aureus* infection. Macrophages, being professional phagocytes, harbour substantially higher numbers of *S. aureus* compared with osteoblasts. Notably, primary osteoblasts, in comparison with other non-professional phagocytic cells, internalize fewer bacteria but present a higher rate of cell survival post-infection. To address this disparity, we employed both macrophages and osteoblasts to provide a comprehensive understanding of the infection dynamics of *S. aureus*. However, there are significant behavioural differences between osteoblastic cell lines, like MG-63 used in this study, and primary osteoblasts, with the latter showing reduced cell death upon infection and greater bacterial persistence in

<figure><img src="image_4.png" /><figcaption>**Figure 4.** Plasma pharmacokinetics of Pep16 in mice after a single subcutaneous administration of 10 mg/kg. Dots from represent the mean ± SEM of three replicates.</figcaption></figure>

phagosomes.²⁵ While primary cells often present more clinically pertinent results, cell lines offer advantages in terms of consistency and availability. This emphasizes the importance of selecting a suitable bone cell model in evaluating the efficacy of BJI treatments. Also, variability in treatment durations and timings, especially in *in vitro* models involving osteoblasts and macrophages, could influence outcomes.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Bacterial loads (in log cfu/g) at Day 7 in knee joints, kidneys and spleen from mice infected by MSSA 1334. Mice were initially inoculated with approximately $1 \times 10^8$ cfu of MSSA 1334. At 3 h post-infection, mice were treated with either with 10 mg/kg of Pep16 or 100 mg/kg of vancomycin, while untreated control mice received a saline solution without antibiotics. Treatments were administered via subcutaneous injection and continued daily until the conclusion of the experiment. Individual dots on the graph represent cfu counts for single animals (enumerated at Day 7), with black horizontal lines indicating median values. The experiments were performed with five mice per condition, in triplicate for Pep16 and saline solution, and in duplicate for vancomycin. Statistical analysis was conducted using the Mann-Whitney *U*-test. ***P*<0.001; ****P*<0.0001.</figcaption></figure>

colistin-resistant mutants.²¹ This underscores the potential of leveraging combination therapies with Pep16 to achieve better clinical outcomes and manage drug resistance.

Nonetheless, our study has certain limitations, with one being the absence of standardized methodologies for assessing intracellular activity and in vivo efficacy. This poses a challenge when comparing results across different studies. Methodological discrepancies, such as treatment timing, can significantly impact findings. In particular, the majority of studies opt for immediate post-infection treatment while only a few consider delayed treatments, such as 12 h or 7 days post-infection. This methodological choice can affect the relevance of the findings, especially in chronic osteomyelitis contexts, where patients are often treated long after the infection onset.²⁴ Moreover, a crucial distinction observed among existing studies is the cell model selected. Numerous studies utilize macrophages and cell lines, which exhibit contrasting behaviours to bone cells, particularly primary cells, in the context of *S. aureus* infection. Macrophages, being professional phagocytes, harbour substantially higher numbers of *S. aureus* compared with osteoblasts. Notably, primary osteoblasts, in comparison with other non-professional phagocytic cells, internalize fewer bacteria but present a higher rate of cell survival post-infection. To address this disparity, we employed both macrophages and osteoblasts to provide a comprehensive understanding of the infection dynamics of *S. aureus*. However, there are significant behavioural differences between osteoblastic cell lines, like MG-63 used in this study, and primary osteoblasts, with the latter showing reduced cell death upon infection and greater bacterial persistence in

phagosomes.²⁵ While primary cells often present more clinically pertinent results, cell lines offer advantages in terms of consistency and availability. This emphasizes the importance of selecting a suitable bone cell model in evaluating the efficacy of BJI treatments. Also, variability in treatment durations and timings, especially in *in vitro* models involving osteoblasts and macrophages, could influence outcomes.

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Plasma pharmacokinetics of Pep16 in mice after a single subcutaneous administration of 10 mg/kg. Dots from represent the mean ± SEM of three replicates.</figcaption></figure>
Additionally, our *in vivo* model involved a single clinical isolate, highlighting the need for broader validation across diverse clinical isolates. Strain-dependent responses may introduce variability, emphasizing the importance of determining the MIC of Pep16 across a wider spectrum of *S. aureus* isolates. Regarding the pharmacokinetic analysis on mice, after administering Pep16 subcutaneously at a dose of 10 mg/kg in mice, there was a steady increase in Pep16 plasma concentration, with a peak at 12 h. Still, the pharmacokinetic profile did not attain concentrations surpassing the MICs required to inhibit the studied strains. This discrepancy might be attributed to factors like metabolism, distribution or the potential binding of the compound to other plasma proteins, which warrants further investigation to elucidate the exact mechanism. Given the inherent variability in *in vivo* responses, it is imperative to evaluate Pep16 against a broader collection of clinical isolates in mice. Even though MICs of Pep16 are relatively higher than those of established antibiotics like vancomycin or levofloxacin, it is interesting to note that intracellular activity and *in vivo* efficacy are similar.

In summary, our study demonstrates that Pep16 could be a promising therapeutic option for treating septic arthritis caused by *S. aureus*. Furthermore, its substantial intracellular activity in both non-professional and professional phagocytic cells could also be relevant for treating chronic infections caused by *S. aureus*.

## Acknowledgements

We thank Professor Pascal Guggenbuhl for providing the MG-63 osteoblastic cell line and Dr Claire Lallement for her help with the chequerboard synergy assay. We warmly thank Dr Alexander Kettner and Dr Charlotte Michaux for their critical reading of the manuscript.

## Funding

This work was supported by internal funding from 'BRM' and by external funding from Olgram SA. Notably, J.B.M. was the recipient of a CIFRE grant from Olgram SA, supporting his 3 year PhD fellowship. This work was also supported by a grant from the French Agence Nationale pour la Recherche (NAILR project, ANR-20-PAMR-0006) to V.C.

## Transparency declarations

J.B.M. and I.N. are employed by Olgram SA, which synthesized and developed Pep16. P.R. serves as the CEO and co-founder of Olgram. Pep16 is protected under European Patent Application #EP14191238.6, titled 'Cyclic antimicrobial pseudopeptides and uses thereof'. All remaining authors declare that they have no conflict of interest to disclose.

## Author contributions

All authors made substantial contributions to the study's conception and design. Material preparation, data collection and analysis were carried out collectively by all authors. The initial draft of the manuscript was written by J.B.M. and V.C., with subsequent input and revisions provided by all authors on versions of the manuscript. Finally, all authors reviewed and gave their approval to the final manuscript.

## Supplementary data

Figures S1-S4 are available as Supplementary data at JAC-AMR Online.

## References

1. Sendi P, Banderet F, Graber P et al. Periprosthetic joint infection following *Staphylococcus aureus* bacteremia. *J Infect* 2011; **63**: 17-22. https://doi.org/10.1016/j.jinf.2011.05.005

2. Kapadia BH, Berg RA, Daley JA et al. Periprosthetic joint infection. *Lancet* 2016; **387**: 386-94. https://doi.org/10.1016/S0140-6736(14)61798-0

3. He M, Arthur Vithran DT, Pan L et al. An update on recent progress of the epidemiology, etiology, diagnosis, and treatment of acute septic arthritis: a review. *Front Cell Infect Microbiol* 2023; **13**: 1193645. https://doi.org/10.3389/fcimb.2023.1193645

4. Masters EA, Ricciardi BF, Bentley KLM et al. Skeletal infections: microbial pathogenesis, immunity and clinical management. *Nat Rev Microbiol* 2022; **20**: 385-400. https://doi.org/10.1038/s41579-022-00686-0

5. Darby EM, Trampari E, Siasat P et al. Molecular mechanisms of antibiotic resistance revisited. *Nat Rev Microbiol* 2023; **21**: 280-95. https://doi.org/10.1038/s41579-022-00820-y

6. Josse J, Velard F, Gangloff SC. *Staphylococcus aureus* vs. osteoblast: relationship and consequences in osteomyelitis. *Front Cell Infect Microbiol* 2015; **5**: 85. https://doi.org/10.3389/fcimb.2015.00085

7. Valour F, Rasigade J-P, Trouillet-Assant S et al. Delta-toxin production deficiency in *Staphylococcus aureus*: a diagnostic marker of bone and joint infection chronicity linked with osteoblast invasion and biofilm formation. *Clin Microbiol Infect* 2015; **21**: 568.e1-e11. https://doi.org/10.1016/j.cmi.2015.01.026

8. Couderc M, Bart G, Coiffier G et al. Recommandations françaises 2020 sur la prise en charge des arthrites septiques sur articulation native de l'adulte. *Rev Rhum* 2020; **87**: 428-38. https://doi.org/10.1016/j.rhum.2020.05.004

9. Barberán J. Management of infections of osteoarticular prosthesis. *Clin Microbiol Infect* 2006; **12** Suppl 3: 93-101. https://doi.org/10.1111/j.1469-0691.2006.01400.x

10. Wang J, Wang L. Novel therapeutic interventions towards improved management of septic arthritis. *BMC Musculoskelet Disord* 2021; **22**: 530. https://doi.org/10.1186/s12891-021-04383-6

11. Chauvelot P, Dupieux-Chabert C, Abad L et al. Evaluation of intraosteoblastic activity of dalbavancin against *Staphylococcus aureus* in an *ex vivo* model of bone cell infection. *J Antimicrob Chemother* 2021; **76**: 2863-6. https://doi.org/10.1093/jac/dkab299

12. Abad L, Tafani V, Tasse J et al. Evaluation of the ability of linezolid and tedizolid to eradicate intraosteoblastic and biofilm-embedded *Staphylococcus aureus* in the bone and joint infection setting. *J Antimicrob Chemother* 2019; **74**: 625-32. https://doi.org/10.1093/jac/dky473

13. Mergaert P. Role of antimicrobial peptides in controlling symbiotic bacterial populations. *Nat Prod Rep* 2018; **35**: 336-56. https://doi.org/10.1039/C7NP00056A

14. Erdem Büyükkiraz M, Kesmen Z. Antimicrobial peptides (AMPs): a promising class of antimicrobial compounds. *J Appl Microbiol* 2022; **132**: 1573-96. https://doi.org/10.1111/jam.15314

15. Koehbach J, Craik DJ. The vast structural diversity of antimicrobial peptides. *Trends Pharmacol Sci* 2019; **40**: 517-28. https://doi.org/10.1016/j.tips.2019.04.012

16. Buda De Cesare G, Cristy SA, Garsin DA et al. Antimicrobial peptides: a new frontier in antifungal therapy. *mBio* 2020; **11**: e02123-20. https://doi.org/10.1128/mBio.02123-20

17. Solecki O, Mosbah A, Baudy Floc'h M et al. Converting a *Staphylococcus aureus* toxin into effective cyclic pseudopeptide antibiotics. *Chem Biol* 2015; **22**: 329-35. https://doi.org/10.1016/j.chembiol.2014.12.016

18. Nicolas I, Bordeau V, Bondon A et al. Novel antibiotics effective against Gram-positive and -negative multi-resistant bacteria with limited resistance. *PLoS Biol* 2019; **17**: e3000337. https://doi.org/10.1371/journal.pbio.3000337
Additionally, our *in vivo* model involved a single clinical isolate, highlighting the need for broader validation across diverse clinical isolates. Strain-dependent responses may introduce variability, emphasizing the importance of determining the MIC of Pep16 across a wider spectrum of S. aureus isolates. Regarding the pharmacokinetic analysis on mice, after administering Pep16 subcutaneously at a dose of 10 mg/kg in mice, there was a steady increase in Pep16 plasma concentration, with a peak at 12 h. Still, the pharmacokinetic profile did not attain concentrations surpassing the MICs required to inhibit the studied strains. This discrepancy might be attributed to factors like metabolism, distribution or the potential binding of the compound to other plasma proteins, which warrants further investigation to elucidate the exact mechanism. Given the inherent variability in *in vivo* responses, it is imperative to evaluate Pep16 against a broader collection of clinical isolates in mice. Even though MICs of Pep16 are relatively higher than those of established antibiotics like vancomycin or levofloxacin, it is interesting to note that intracellular activity and *in vivo* efficacy are similar.

In summary, our study demonstrates that Pep16 could be a promising therapeutic option for treating septic arthritis caused by S. aureus. Furthermore, its substantial intracellular activity in both non-professional and professional phagocytic cells could also be relevant for treating chronic infections caused by S. aureus.

## Acknowledgements

We thank Professor Pascal Guggenbuhl for providing the MG-63 osteoblastic cell line and Dr Claire Lallement for her help with the chequerboard synergy assay. We warmly thank Dr Alexander Kettner and Dr Charlotte Michaux for their critical reading of the manuscript.

## Funding

This work was supported by internal funding from 'BRM' and by external funding from Olgram SA. Notably, J.B.M. was the recipient of a CIFRE grant from Olgram SA, supporting his 3 year PhD fellowship. This work was also supported by a grant from the French Agence Nationale pour la Recherche (NAILR project, ANR-20-PAMR-0006) to V.C.

## Transparency declarations

J.B.M. and I.N. are employed by Olgram SA, which synthesized and developed Pep16. P.R. serves as the CEO and co-founder of Olgram. Pep16 is protected under European Patent Application #EP14191238.6, titled 'Cyclic antimicrobial pseudopeptides and uses thereof'. All remaining authors declare that they have no conflict of interest to disclose.

## Author contributions

All authors made substantial contributions to the study's conception and design. Material preparation, data collection and analysis were carried out collectively by all authors. The initial draft of the manuscript was written by J.B.M. and V.C., with subsequent input and revisions provided by all authors on versions of the manuscript. Finally, all authors reviewed and gave their approval to the final manuscript.

## Supplementary data

Figures S1-S4 are available as Supplementary data at JAC-AMR Online.

## References

1. Sendi P, Banderet F, Graber P et al. Periprosthetic joint infection following *Staphylococcus aureus* bacteremia. *J Infect* 2011; **63**: 17-22. https://doi.org/10.1016/j.jinf.2011.05.005

2. Kapadia BH, Berg RA, Daley JA et al. Periprosthetic joint infection. *Lancet* 2016; **387**: 386-94. https://doi.org/10.1016/S0140-6736(14)61798-0

3. He M, Arthur Vithran DT, Pan L et al. An update on recent progress of the epidemiology, etiology, diagnosis, and treatment of acute septic arthritis: a review. *Front Cell Infect Microbiol* 2023; **13**: 1193645. https://doi.org/10.3389/fcimb.2023.1193645

4. Masters EA, Ricciardi BF, Bentley KLM et al. Skeletal infections: microbial pathogenesis, immunity and clinical management. *Nat Rev Microbiol* 2022; **20**: 385-400. https://doi.org/10.1038/s41579-022-00686-0

5. Darby EM, Trampari E, Siasat P et al. Molecular mechanisms of antibiotic resistance revisited. *Nat Rev Microbiol* 2023; **21**: 280-95. https://doi.org/10.1038/s41579-022-00820-y

6. Josse J, Velard F, Gangloff SC. *Staphylococcus aureus* vs. osteoblast: relationship and consequences in osteomyelitis. *Front Cell Infect Microbiol* 2015; **5**: 85. https://doi.org/10.3389/fcimb.2015.00085

7. Valour F, Rasigade J-P, Trouillet-Assant S et al. Delta-toxin production deficiency in *Staphylococcus aureus*: a diagnostic marker of bone and joint infection chronicity linked with osteoblast invasion and biofilm formation. *Clin Microbiol Infect* 2015; **21**: 568.e1-e11. https://doi.org/10.1016/j.cmi.2015.01.026

8. Couderc M, Bart G, Coiffier G et al. Recommandations françaises 2020 sur la prise en charge des arthrites septiques sur articulation native de l'adulte. *Rev Rhum* 2020; **87**: 428-38. https://doi.org/10.1016/j.rhum.2020.05.004

9. Barberán J. Management of infections of osteoarticular prosthesis. *Clin Microbiol Infect* 2006; **12** Suppl 3: 93-101. https://doi.org/10.1111/j.1469-0691.2006.01400.x

10. Wang J, Wang L. Novel therapeutic interventions towards improved management of septic arthritis. *BMC Musculoskelet Disord* 2021; **22**: 530. https://doi.org/10.1186/s12891-021-04383-6

11. Chauvelot P, Dupieux-Chabert C, Abad L et al. Evaluation of intraosteoblastic activity of dalbavancin against *Staphylococcus aureus* in an *ex vivo* model of bone cell infection. *J Antimicrob Chemother* 2021; **76**: 2863-6. https://doi.org/10.1093/jac/dkab299

12. Abad L, Tafani V, Tasse J et al. Evaluation of the ability of linezolid and tedizolid to eradicate intraosteoblastic and biofilm-embedded *Staphylococcus aureus* in the bone and joint infection setting. *J Antimicrob Chemother* 2019; **74**: 625-32. https://doi.org/10.1093/jac/dky473

13. Mergaert P. Role of antimicrobial peptides in controlling symbiotic bacterial populations. *Nat Prod Rep* 2018; **35**: 336-56. https://doi.org/10.1039/C7NP00056A

14. Erdem Büyükkiraz M, Kesmen Z. Antimicrobial peptides (AMPs): a promising class of antimicrobial compounds. *J Appl Microbiol* 2022; **132**: 1573-96. https://doi.org/10.1111/jam.15314

15. Koehbach J, Craik DJ. The vast structural diversity of antimicrobial peptides. *Trends Pharmacol Sci* 2019; **40**: 517-28. https://doi.org/10.1016/j.tips.2019.04.012

16. Buda De Cesare G, Cristy SA, Garsin DA et al. Antimicrobial peptides: a new frontier in antifungal therapy. *mBio* 2020; **11**: e02123-20. https://doi.org/10.1128/mBio.02123-20

17. Solecki O, Mosbah A, Baudy Floc'h M et al. Converting a *Staphylococcus aureus* toxin into effective cyclic pseudopeptide antibiotics. *Chem Biol* 2015; **22**: 329-35. https://doi.org/10.1016/j.chembiol.2014.12.016

18. Nicolas I, Bordeau V, Bondon A et al. Novel antibiotics effective against Gram-positive and -negative multi-resistant bacteria with limited resistance. *PLoS Biol* 2019; **17**: e3000337. https://doi.org/10.1371/journal.pbio.3000337
19 Abad L, Josse J, Tasse J et al. Antibiofilm and intraosteoblastic activities of rifamycins against *Staphylococcus aureus*: promising *in vitro* profile of rifabutin. *J Antimicrob Chemother* 2020; **75**: 1466-73. https://doi.org/10.1093/jac/dkaa061

20 Bremell T, Lange S, Yacoub A et al. Experimental *Staphylococcus aureus* arthritis in mice. *Infect Immun* 1991; **59**: 2615-23. https://doi.org/10.1128/iai.59.8.2615-2623.1991

21 Chosidow S, Fantin B, Nicolas I et al. Synergistic activity of Pep16, a promising new antibacterial pseudopeptide against multidrug-resistant organisms, in combination with colistin against multidrug-resistant *Escherichia coli*, *in vitro* and in a murine peritonitis model. *Antibiotics* 2023; **12**: 81. https://doi.org/10.3390/antibiotics12010081

22 Smith RP, Baltch AL, Franke MA et al. Levofloxacin penetrates human monocytes and enhances intracellular killing of *Staphylococcus aureus*

and *Pseudomonas aeruginosa*. *J Antimicrob Chemother* 2000; **45**: 483-8. https://doi.org/10.1093/jac/45.4.483

23 Abad L, Chauvelot P, Audoux E et al. Lysosomal alkalization to potentiate eradication of intra-osteoblastic *Staphylococcus aureus* in the bone and joint infection setting. *Clin Microbiol Infect* 2022; **28**: 135.e1-e7. https://doi.org/10.1016/j.cmi.2021.04.030

24 Zelmer AR, Nelson R, Richter K et al. Can intracellular *Staphylococcus aureus* in osteomyelitis be treated using current antibiotics? A systematic review and narrative synthesis. *Bone Res* 2022; **10**: 53. https://doi.org/10.1038/s41413-022-00227-8

25 Strobel M, Pförtner H, Tuchscherr L et al. Post-invasion events after infection with *Staphylococcus aureus* are strongly dependent on both the host cell type and the infecting *S. aureus* strain. *Clin Microbiol Infect* 2016; **22**: 799-809. https://doi.org/10.1016/j.cmi.2016.06.020
19 Abad L, Josse J, Tasse J et al. Antibiofilm and intraosteoblastic activities of rifamycins against *Staphylococcus aureus*: promising *in vitro* profile of rifabutin. *J Antimicrob Chemother* 2020; **75**: 1466-73. https://doi.org/10.1093/jac/dkaa061

20 Bremell T, Lange S, Yacoub A et al. Experimental *Staphylococcus aureus* arthritis in mice. *Infect Immun* 1991; **59**: 2615-23. https://doi.org/10.1128/iai.59.8.2615-2623.1991

21 Chosidow S, Fantin B, Nicolas I et al. Synergistic activity of Pep16, a promising new antibacterial pseudopeptide against multidrug-resistant organisms, in combination with colistin against multidrug-resistant *Escherichia coli*, *in vitro* and in a murine peritonitis model. *Antibiotics* 2023; **12**: 81. https://doi.org/10.3390/antibiotics12010081

22 Smith RP, Baltch AL, Franke MA et al. Levofloxacin penetrates human monocytes and enhances intracellular killing of *Staphylococcus aureus*

and *Pseudomonas aeruginosa*. *J Antimicrob Chemother* 2000; **45**: 483-8. https://doi.org/10.1093/jac/45.4.483

23 Abad L, Chauvelot P, Audoux E et al. Lysosomal alkalization to potentiate eradication of intra-osteoblastic *Staphylococcus aureus* in the bone and joint infection setting. *Clin Microbiol Infect* 2022; **28**: 135.e1-e7. https://doi.org/10.1016/j.cmi.2021.04.030

24 Zelmer AR, Nelson R, Richter K et al. Can intracellular *Staphylococcus aureus* in osteomyelitis be treated using current antibiotics? A systematic review and narrative synthesis. *Bone Res* 2022; **10**: 53. https://doi.org/10.1038/s41413-022-00227-8

25 Strobel M, Pförtner H, Tuchscherr L et al. Post-invasion events after infection with *Staphylococcus aureus* are strongly dependent on both the host cell type and the infecting *S. aureus* strain. *Clin Microbiol Infect* 2016; **22**: 799-809. https://doi.org/10.1016/j.cmi.2016.06.020