2. Targeting S. aureus
The S. aureus cell surface components, including extracellular matrix binding proteins, provide targeting sites for the selective and specific delivery of drugs and therapeutic and diagnostic imaging agents. Figure 2 shows a schematic representation of targeting the infection site for drug delivery and imaging. Many compounds that exhibit affinity binding to S. aureus have been identified or developed. When conjugated to therapeutic molecules, these compounds enable targeted delivery to S. aureus infection sites. The primary S. aureus-targeting agents are discussed below.
2.1. Peptides
Peptides have many advantages, including small size, low immunogenicity, better diffusion, and easier target accessibility (Reubi & Maecke, 2008; Staderini et al., 2018). Specific peptides exhibit a strong affinity for binding to the bacterial structure in the advances of targeted therapy and imaging of bacterial infections. Depending on the intended use, these peptides have been conjugated with a therapeutic agent or fluorophore (Aweda et al., 2016). The resulting conjugate offers a precise and effective approach for osteomyelitis management and other S. aureus infections. The potential applications of peptides in bone infections are discussed below.
Ubiquicidin 29-41 (UBI) is a cationic peptide with an affinity for bacterial membrane components (Beiki et al., 2013). UBI is utilized for the targeting of bacterial infections (Ferro-Flores et al., 2016; Welling, De Korne, et al., 2019; Welling, Hensbergen, Bunschoten, Velders, Roestenberg, et al., 2019). UBI has shown high sensitivity, specificity, and accuracy in detecting bacterial infections (Yang et al., 2018). Radiolabeled UBI (Technetium-99m labeled UBI, 99mTc-UBI) scintigraphy was utilized to differentiate between S. aureus-related orthopedic infection and sterile inflammation (Beiki et al., 2013), and with its dynamic imaging format, could accurately differentiate between infectious osteomyelitis and bone tumors (Ayati et al., 2017). Therefore, 99mTc-UBI scintigraphy has the potential to detect diabetic foot osteomyelitis in human subjects accurately (Saeed et al., 2013). In another study, UBI labeled with positron-emitting Gallium-68 (68Ga) radioisotope through a bifunctional chelator showed remarkable uptake of the labeled UBI at the sites of S. aureus infection when compared to sterile inflammation in an in vivo rat model measured through positron emission tomography/computed tomography (Boddeti & Kumar, 2021). Similarly, UBI was conjugated to liposomes (LU) and functionalized with gentamicin-loaded mesoporous silica nanoparticles (Gen@MSN) to yield Gen@MSN-LU. These Gen@MSN-LU nanoparticles inhibited intracellular S. aureus in mouse osteoblasts in vitro due to the cellular uptake of the nanoparticles and the release of gentamicin. Additionally, UBI enabled Gen@MSN-LU's in vivo targeting and delivery of Gen@MSN-LU to S. aureus infection sites in mice (Yang et al., 2018). Similarly, Chloramphenicol conjugate of UBI (CAP-UBI29-41) was effectively utilized for the targeted delivery of chloramphenicol to S. aureus-infected sites in mice (Chen et al., 2015). UBI holds great promise as both a therapeutic delivery and diagnostic agent for S. aureus-associated osteomyelitis. Its ability to penetrate biofilms and potential for rapid and sensitive detection make it a valuable tool in the management of this challenging infection. Continued research efforts are needed to further elucidate the mechanisms of action, optimize therapeutic formulations, and validate diagnostic assays, ultimately translating UBI into clinical practice for the improved management of S. aureus-associated osteomyelitis.
Furthermore, Cationic antimicrobial peptides such as cathelicidin LL-37 also bind to bacterial membranes through electrostatic interactions, and LL-37 is effective intracellularly against S. aureus in osteoblasts (Staderini et al., 2018). The heptapeptide YSPXTNF, an RNAIII-inhibiting peptide (RIP), reduced infection levels in a rabbit osteomyelitis model (Balaban et al., 2000). RIP is a quorum sensor inhibitor that inhibits S. aureus pathogenesis by hindering the synthesis of the accessory gene regulator (agr) transcript RNAIII (Assis et al., 2017; Balaban et al., 2000; Giacometti et al., 2005). The agr quorum-sensing system is associated with S. aureus pathogenesis and biofilm formation (Tan et al., 2018). A chimeric peptide, PpIX-[PEG8-(KLAKLAK)2]2 (PPK), consisting of protoporphyrin IX conjugated to a bacterial binding peptide (KLAKLAK)2, was influential in the photodynamic inactivation of S. aureus in vivo (A. N. Zhang et al., 2019). Besides, a low-cationic heptapeptide called Bacaucin-1a targets and specifically kills S. aureus both in vitro and in vivo (Liu et al., 2019).
Other cationic antimicrobial peptides, hLF1-11 (Human lactoferricin), loaded into calcium phosphate orthopedic cement, effectively reduced osteomyelitis development in rabbit models (Stallmann et al., 2004). A 9 amino acid cyclic peptide is another cationic form called the CARG peptide conjugated to vancomycin-loaded silicon nanoparticles to target S. aureus infections. CARG-conjugated nanoparticles homed into and accumulated in infected mouse lung tissue and skin, thereby releasing therapeutics at the site of infection (Hussain et al., 2018). In another study, the peptide sequence SVPLNSWSIFPR displayed on a bacteriophage showed high specificity for S. aureus. The peptide SVPLNSWSIFPR was expressed on vacuoles isolated from yeasts and loaded with the antibiotic daptomycin. The peptide vacuoles encapsulating daptomycin formed an efficient system that targeted and killed S. aureus (J. Lee et al., 2023). Thus, cationic antimicrobials could be another potential route in osteomyelitis management for the site-specific delivery of therapeutics, thereby reducing systemic toxicity and dosage, and it may have potential benefits in treating localized bone infections.
2.2. Antibodies
Antibodies may constitute an alternative means of antibiotic therapy due to increased antibiotic resistance. Additionally, vaccination can protect individuals from future bacterial infections. Monoclonal antibodies target specific bacterial epitopes and have little cross-reactivity. Antibodies help in the host defense mechanism by (i) neutralizing bacterial toxins and limiting their binding to host cells, (ii) binding to bacterial surfaces and rendering them recognizable by macrophages and neutrophils that ingest bacteria, and (iii) triggering the classical pathway due to the formation of an antibody‒pathogen complex leading to binding of complement C3b on the bacterial surface, leading to opsonization and phagocytosis (Speziale et al., 2018).
According to a study by Wang et al., neutralizing monoclonal antibodies against S. aureus α-toxin and clumping factor A inhibited biofilm formation in vitro as well as in a hematogenous orthopedic implant infection mouse model (Wang et al., 2017). The effects of monotherapies using anti-glucosaminidase (anti-gmd) monoclonal antibodies (against the glucosaminidase subunit of S. aureus autolysin) or vancomycin were compared to those of a combination therapy consisting of anti-gmd and vancomycin in a murine 1-stage exchange model of methicillin-resistant S. aureus (MRSA) contaminated femoral implants. Anti-gmd therapy inhibited MRSA abscess communities, whereas vancomycin reduced the CFUs on the femoral implants. However, combination therapy with anti-gmd and vancomycin was effective and achieved sterile implant levels by day 12 of treatment (Yokogawa et al., 2018). Anti-IsaA antibodies targeting IsaA (Immunodominant S. aureus antigen A), an S. aureus cell wall-bound lytic transglycosylase, were found to be effective at reducing the S. aureus load in mouse models due to greater opsonophagocytosis and effective intracellular killing (Lorenz et al., 2011). Monoclonal antibodies targeting the multiple peptide resistance factor (MprF) of the S. aureus cell membrane have been developed. MprF is responsible for the virulence and increased resistance of MRSA and other pathogens to host defense systems and antibiotics. The MC7.1 antibody targeted the flippase domain of MprF and thereby blocked the function of the flippase; this rendered S. aureus susceptible to antimicrobial peptides and antibiotics, according to in vitro studies. Therefore, utilizing antibodies to inhibit Mprf could be an efficient antivirulence strategy against antibiotic-resistant pathogens such as S. aureus (Slavetinsky et al., 2022). An engineered human-derived anti-S. aureus monoclonal antibody-centyrin fusion protein called mAbtyrin (SM1B74) was developed that exhibited superior properties, including targeting bacterial adhesins, counteracting pore-forming leukocidins, and resisting proteolysis by bacterial enzymes. Compared with parental monoclonal antibodies, SM1B74 enhanced phagocyte-mediated killing and protected phagocytes. In animal models, SM1B74 synergized with the antibiotic vancomycin, resulting in enhanced clearance of S. aureus (Buckley et al., 2023).
An innovative novel approach for the treatment of bacterial infections has emerged that combines the specificity of an antibody and the antibacterial effects of an antibiotic in a single entity called an antibody‒antibiotic conjugate (AAC), which presents superior absorption, distribution, metabolism, and elimination (ADME) properties attributed to the antibody part of the conjugate, along with slow clearance as well as a long half-life (Mariathasan & Tan, 2017). It was demonstrated that intracellular S. aureus is protected from antibiotics and host immune mechanisms, which results in the spread of infection. An AAC was created by conjugating an anti-S. aureus antibody to a cleavable antibiotic rifalogue, which is similar to rifampicin. AAC opsonizes S. aureus, which, when taken up by the host cell, causes the cleavage and release of the antibiotic by intracellular proteases, thereby resulting in the intracellular killing of S. aureus (Lehar et al., 2015).
THIOMAB™ is a term for antibodies that consist of cysteine residues, which enable the site-selective conjugation of a drug, label, or any other payload directed to specific tissues for therapeutic purposes (Adhikari et al., 2020). DSTA4637S (lyophilized form) is a THIOMAB™ antibody comprising a monoclonal human immunoglobulin against S. aureus conjugated to a rifamycin-class antibiotic (4-dimethylaminopiperidino-hydroxybenzoxazino rifamycin [dmDNA31]) via a protease-cleavable linker (Deng et al., 2019; Peck et al., 2019). DSTA4637S is an innovative antibody-antibiotic conjugate that targets intracellular S. aureus (Peck et al., 2019). DSTA4637A, the liquid form of DSTA4637S, showed prolonged antibacterial activity and substantial S. aureus load reduction in the kidney, heart, and bones of mice infected with S. aureus (Zhou et al., 2016).
S. aureus-specific antibodies linked to near-infrared (NIR) fluorophores or PET tracers were used to examine S. aureus infections. A human monoclonal antibody against S. aureus IsaA (Immunodominant S. aureus antigen A), called 1D9, was conjugated to the NIR dye IRDye 800CW for imaging of S. aureus infection in a human postmortem model or in vivo mouse models. S. aureus was implanted subdermally on the tibia of a human cadaver, and in the mouse study, a skin infection model was used. Moreover, the utilization of 89Zr-labeled 1D9 facilitated imaging of the mouse thigh infection model via positron emission tomography (PET), providing a valuable tool for non-invasive monitoring of infections. A 1D9 antibody was found to bind and accumulate at the site of S. aureus infection, enabling imaging of infected sites. A dual-labeled 1D9 probe labeled with 89Zr and NIR680 (89Zr-1D9-NIR680) facilitated multimodal imaging involving both positron emission tomography-computed tomography (PET-CT) and fluorescence imaging (FLI), aiding in the accurate diagnosis of infection and fluorescence image-guided surgery for infected tissue debridement in a mouse model (Zoller et al., 2019).
The utilization of antibodies presents a promising alternative to traditional antibiotic therapy, particularly in the face of increasing antibiotic resistance. Through their specificity and multifaceted mechanisms, monoclonal antibodies offer targeted approaches to combatting bacterial infections, as demonstrated by their efficacy in inhibiting biofilm formation, enhancing phagocytosis, and synergizing with antibiotics. Additionally, the innovative strategies of antibody‒antibiotic conjugates showcase the potential for combining the antibacterial effects of antibodies with the therapeutic properties of antibiotics, paving the way for advanced treatments with improved efficacy and targeting capabilities in the fight against bacterial pathogens like S. aureus.
2.3. Bacteriophages
Bacteriophages are viruses that kill bacteria and are considered as therapeutics due to their high specificity, nontoxicity, and natural abundance. (Abatángelo et al., 2017; Cobb et al., 2019; Z. Tang et al., 2015). The successful clinical use of bacteriophages in the treatment of antibiotic-resistant infections has been carried out for many years (Cobb et al., 2020; Furfaro et al., 2018; Kutateladze & Adamia, 2008), and the outcome of these clinical studies have pointed out the effectiveness of phage therapy in humans worldwide from a historical perspective (Abedon et al., 2011; Chan et al., 2013; Kutter et al., 2010). The utilization of bacteriophages in orthopedic infections can be attributed to three key factors. Firstly, phage therapy has been shown to be safe with no reported adverse effects or tissue toxicity, while having no impact on eukaryotic cells. Secondly, phages have the unique ability to self-reproduce and self-dose, with in vivo high titers established in animal tissue as long as the host bacterium is present. Lastly, the straightforward and cost-effective methods of phage production make it a practical option (Kaur et al., 2014).
Compared with sterile inflammation, 99mTc-labeled M13 phage enabled the imaging of S. aureus and E. coli in an in vivo mouse model with infected thighs. It produced stronger signals in bacteria-infected mice (Rusckowski et al., 2004). Phage-displayed linear dodecapeptide A9 conjugated with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) via a lysine linker and labeled with 68Ga to form 68Ga-A9-K-DOTA was found to have the desirable features of a novel S. aureus-specific positron emission tomography (PET) imaging agent for S. aureus-related infections (Nielsen et al., 2016).
Several clinical trials have emphasized the significance of phage therapy in the management of osteomyelitis. This therapeutic approach has shown promising results and is being considered as a potential alternative to traditional antibiotics. It is reported, that topical application of phage Sb-1 was successful in treating a group of six patients with poorly vascularized diabetic toe ulcers that involved exposed bone, after antibiotic therapy had failed (Fish et al., 2016). Also, Staphylococcal phage Sb-1 was effectively used for the long-term treatment of a 63-year-old female patient who had distal phalangeal osteomyelitis (Fish et al., 2018).
Besides osteomyelitis due to orthopedic implants, the lytic bacteriophage MR-5 and linezolid were effective against S. aureus on orthopedic K-wires in an in vitro study (Kaur et al., 2014). S. aureus-specific bacteriophage cocktails comprising seven strains (SA-BHU1, SA-BHU2, SA-BHU8, SA-BHU15, and SA-BHU21, SA-BHU37, SA-BHU47) were successfully used for the eradication of MRSA in a rabbit osteomyelitis model (Kishor et al., 2016). In an implant-related infection model induced in the tibia of rats, treatment with a Sb-1 bacteriophage combined with the antibiotic teicoplanin resulted in the eradication of MRSA biofilms (Yilmaz et al., 2013). A CRISPR-Cas9-modified bacteriophage (with or without fosfomycin) utilized for treating S. aureus-induced osteomyelitis and soft tissue infection in an in vivo rat model was able to reduce soft tissue infection but not bone infection (Cobb et al., 2019). JD419, a Staphylococcus phage, was shown to lyse 61 out of 138 clinical S. aureus strains in studies conducted in vitro. JD419 can be utilized as a therapeutic phage for S. aureus infections if genetic engineering can modify the phage to remove prophage-associated genes to prevent lysogeny (Feng et al., 2021). The S. aureus bacteriophage phage VW reduced S. aureus biofilms to a greater extent than the antibiotic streptomycin. The combination of phage VW and streptomycin had a more significant antibiofilm effect than either phage VW or streptomycin alone in the in vitro studies. These phages could be potential candidates for targeted therapy for S. aureus infections (Y. Jiang et al., 2021). Bacteriophages represent a promising avenue in the therapeutic landscape of infectious diseases, particularly in the management of S. aureus-associated osteomyelitis. Their safety profile, ability to target specific bacterial strains, and cost-effective production methods underscore their potential as alternatives to traditional antibiotics. Clinical trials have shown encouraging results, suggesting that bacteriophage therapy may offer effective treatment options for challenging infections such as osteomyelitis, paving the way for further exploration and development in this field.
2.4. Peptide Nucleic Acids
Peptide nucleic acids (PNAs) are synthetic oligonucleotides in which a pseudopeptide substitutes for the sugar-phosphodiester backbone. PNAs hybridize with complementary RNA or DNA and form complexes with high affinity and specificity (Menchise et al., 2003; Montazersaheb et al., 2018; Pellestor & Paulasova, 2004; Swenson & Heemstra, 2020; Wojciechowska et al., 2020).
A peptide nucleic acid conjugated to HIV-TAT cell-penetrating peptide (PNA-TAT) targeted against the bacterial RNA polymerase α subunit gene rpoA, which is involved in bacterial transcription, inhibited the growth of MRSA in vitro as well as in infected cells and in vivo in a C. elegans model (Abushahba et al., 2016). A tetrahedral DNA nanostructure (TDN) was developed to deliver antisense PNA targeted against the specific ftsZ gene required for MRSA cell division. The TDN antisense PNA delivery system effectively inhibited MRSA cell growth and has the potential for targeted drug delivery in antibacterial therapy (Y. Zhang et al., 2018). A peptide conjugate-peptide nucleic acid (PPNA) in which the PNA was conjugated to the cell-penetrating peptide KFFKFFKFFK [(KFF)3K, K = lysine and F = phenylalanine] targeted against the essential S. aureus gene gyrA (encoding subunit A of the DNA topoisomerase gyrase). Anti-gyrA-PPNA was effective at inhibiting S. aureus growth in Mueller Hinton broth as well as in infected mammalian cell lines, suggesting that anti-gyrA-PPNA could be a potential candidate for antisense therapeutics for the treatment of S. aureus infections (Tavakoli et al., 2019). Additionally, antisense PNA conjugated to the peptide (KFF)3K, which is targeted against the S. aureus cmk gene responsible for the cytidine monophosphate kinase, effectively inhibited S. aureus in mouse models (H. T. Lee et al., 2019). In a porcine implant-associated osteomyelitis model, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) was employed to detect S. aureus on the implant surface from a porcine model of implant-associated osteomyelitis employing S. aureus-specific probes (Jensen et al., 2017). PNA-FISH detected S. aureus in the bone samples of diabetic foot osteomyelitis patients utilizing confocal laser scanning microscopy and S. aureus-specific PNA-FISH probes (Johani et al., 2019).
2.5. Aptamers
Single-stranded DNA or RNA binding to cognate molecular targets with high specificity and affinity are commonly known as aptamers (Chandola et al., 2016; Li et al., 2020; Zhu & Chen, 2018). Nucleic acid aptamers are identified and selected from a pool of oligonucleotide libraries by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are regarded as artificial antibodies and have gained considerable attention due to their specificity, high affinity, stability, easy chemical modification, low production cost, repeatability, and usability (Li et al., 2020). The conjugation of aptamers to small interfering RNA (siRNA) therapeutics contributes to the accumulation of siRNAs in certain cell types (Catuogno et al., 2018; Chernikov et al., 2019). Hence, aptamers are potential targeting agents for the delivery of therapeutic molecules.
Aptamers are effective tools for targeted drug delivery due to their low toxicity and immunogenicity (Rabiee et al., 2020). Targeting aptamers have been employed to detect S. aureus (Baumstummler et al., 2014; Stoltenburg et al., 2016). An S. aureus-targeted drug delivery system was designed in which silica nanoparticles were loaded with vancomycin, and the pores of the nanoparticles were capped with S. aureus-targeting SA20hp aptamers. The binding of the aptamer to the target resulted in the release of the drug and the killing of S. aureus (Kavruk et al., 2015). Four aptamers, AT-27, AT-33, AT-36, and AT-49, neutralized S. aureus α-toxin and inhibited α-toxin-mediated cell death. These aptamers are potential therapeutics for the treatment of S. aureus infections (Vivekananda et al., 2014). The aptamers SA20, SA23, and SA34 specific to S. aureus were radiolabeled with 99mTc and utilized to detect S. aureus-induced infection in the tibia of Wistar rats. The infected site showed increased uptake of the radiolabeled aptamers and enabled the identification of the infection foci (Santos et al., 2015). Additionally, 99mTc-labeled aptamers SA20, SA23, and SA34 resulted in a high uptake of the radiolabeled aptamer in S. aureus-induced localized infection sites in mouse models (dos Santos et al., 2015). Thus, aptamers represent a highly versatile and promising tool for targeted drug delivery, offering advantages such as low toxicity and immunogenicity. With their specific binding properties, aptamers have shown great potential in detecting and treating S. aureus-associated infections, paving the way for innovative therapeutic approaches in combating bacterial diseases.
2.6. Affimers
Affimers are a class of small proteins that can bind or be engineered to target proteins with high affinity and selectivity (Klont et al., 2018; A. A. S. Tang et al., 2017). Affimers have a size of ~ 12 kDa and are heat-stable. Affimers are being developed as functional replacements for antibodies (size ~ 150 kDa) for many applications (Caudwell et al., 2022). Affimers consist of two variable regions or loops and a conserved Cystatin scaffold region. A diagrammatic representation of an affimer is shown in Fig. 3. The amino acid sequence of the two variable regions defines the affinity of the affimer for its target (Caudwell et al., 2022; Klont et al., 2018; A. A. S. Tang et al., 2017). Affimers were developed for different S. aureus strains that can be used for targeting. These affimers strongly bind to biofilms formed by different S. aureus strains (Alsulaimani, 2021). In one study, a novel affimer, AClfA1, with an affinity for the virulence factor Clumping Factor A (ClfA) on the cell surface of S. aureus was utilized for targeting. In another study, lipid-coated microbubbles were surface functionalized with AClfA1 and showed enhanced binding to S. aureus biofilms (Caudwell et al., 2022). Such affimer-functionalized treatment strategies could pave the way for the development of targeted delivery of therapeutics for S. aureus-associated osteomyelitis treatment.
2.7. Other Compounds
Bithionol, an organic compound, was effective in killing MRSA and was more selective in disrupting bacterial cell membranes than in disrupting mammalian cell membranes. Additionally, bithionol in combination with gentamicin significantly reduced the MRSA burden in a mouse model of deep-seated MRSA infection (Kim et al., 2019). Bithionol, 2,2’-thiobis (4,6-dichlorophenol) is an anthelmintic utilized for the treatment of diseases such as metagonimiasis and paragonimiasis in humans (Keiser & Utzinger, 2007). Bithionol could be a potential candidate for developing delivery vehicles for targeting S. aureus infections.
The coumarin derivative 3,3′-(3,4-dichlorobenzylidene)-bis-(4-hydroxycoumarin), termed DCH, showed potent activity against MRSA and biofilm formation. The inhibitory activity of DCH occurs through its ability to target and bind to the bacterial arginine repressor (ArgR). ArgR is absent in eukaryotes, and thus, bacterial ArgR is an essential target for the development of therapeutics against MRSA infections (Qu et al., 2020).
Daptomycin, a lipopeptide antibiotic, was utilized for the targeting of daptomycin-loaded liposomes in a mouse model. Daptomycin can bind to the S. aureus cell wall via its hydrophobic tail. Daptomycin conjugated to the surface of daptomycin-loaded liposomes facilitated MRSA targeting and the delivery of the encapsulated daptomycin to the infection site (H. Jiang et al., 2016). Vancomycin, a glycopeptide with potent antibacterial activity against gram-positive bacteria, is ineffective in treating deep infections due to low tissue penetration (Staderini et al., 2018). At high concentrations, vancomycin alone was ineffective against intracellular S. aureus infection in SAOS-2 osteoblast-like cell lines. However, vancomycin, in combination with the bacterial efflux pump inhibitors piperine or carbonyl cyanide m-chlorophenyl hydrazine, improved the killing of intracellular S. aureus by increasing the permeability of SAOS-2 cells (Dusane et al., 2018). Vancomycin was conjugated to the near-infrared fluorophore IRDye 800CW for the preferential detection of gram-positive bacterial infections. The IRDye 800CW-labeled vancomycin (Vanco-800CW) enabled real-time in vivo imaging of S. aureus-induced myositis in a mouse model and facilitated the discrimination of S. aureus infection from E. coli infection or sterile inflammation. Additionally, Vanco-800CW enabled the imaging of a biomaterial-associated infection in the lower leg of a human cadaver (Van Oosten et al., 2013). In another study, fenoprofen, a nonsteroid anti-inflammatory drug (NSAID) was able to inhibit the regulator protein SaeR protein involved in the virulence of S. aureus. Fenoprofen attenuated S. aureus virulence, alleviated osteolysis and improved walking ability in an implant-associated osteomyelitis mouse model (Jiang et al., 2023). Thus, compounds, including certain antibiotics, facilitate the targeting of S. aureus infections for therapeutics as well as targeted infection imaging.