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Shirley W. I. Siu, Academic Editor, François-Xavier Campbell-Valois, Academic Editor, Watshara Shoombuatong, Academic Editor, and Siti Azma Jusoh, Academic Editor

Abstract

Antibiotics are regarded as a miracle in the medical field as it prevents disease caused by pathogenic bacteria. Since the discovery of penicillin, antibiotics have become the foundation for modern medical discoveries. However, bacteria soon became resistant to antibiotics, which puts a burden on the healthcare system. Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prominent antibiotic-resistant bacteria in the world since 1961. MRSA primarily developed resistance to beta-lactamases antibiotics and can be easily spread in the healthcare system. Thus, alternatives to combat MRSA are urgently required. Antimicrobial peptides (AMPs), an innate host immune agent and silver nanoparticles (AgNPs), are gaining interest as alternative treatments against MRSA. Both agents have broad-spectrum properties which are suitable candidates for controlling MRSA. Although both agents can exhibit antimicrobial effects independently, the combination of both can be synergistic and complementary to each other to exhibit stronger antimicrobial activity. The combination of AMPs and AgNPs also reduces their own weaknesses as their own, which can be developed as a potential agent to combat antibiotic resistance especially towards MRSA. Thus, this review aims to discuss the potential of antimicrobial peptides and silver nanoparticles towards controlling MRSA pathogen growth.

Keywords: antibiotic resistance, antimicrobial peptides, MRSA, silver nanoparticles

1. Introduction

Antibiotics are one of the outstanding discoveries in the medical field in treating infectious diseases caused by pathogenic bacteria. Before the antibiotic discovery era, the lethality and death rate caused by pathogenic microorganisms was high until the accidental rediscovery of penicillin in 1928 by Alexander Fleming [ 1]. This rediscovery grants the exploration of other types of antibiotics such as sulphonamides, lipopeptides, aminoglycosides, fluoroquinolones, and many more [ 1, 2]. Antibiotics also allow modern medical technology to exist as it aids in preventing infection in chemotherapy and various surgical wounds.

Although antibiotics give significant advantages in treating diseases caused by pathogenic bacteria, Alexander Fleming warns of the danger of uncontrolled antibiotic usage where resistance can be developed. The warning appeared to be true as Escherichia coli started to exhibit antibiotic resistance (AR) towards penicillin in 1940 [ 3]. Up until this day, antibiotic resistance has been a significant threat in the healthcare system as more bacteria developed resistance towards various classes of antibiotics. It is predicted that, by 2050, AR related death may reach 10 million per year [ 4, 5].

A recent comprehensive report released in The Lancet [ 6] stated that 4.95 million AR associated death and 1.27 million AR attributed death were estimated from 204 countries in 2019. Highest AR related death can be found in Western Sub-Saharan Africa with estimated 27.3 AR attributed death per 100,000 and 114.8 AR associated death per 100,000. Meanwhile, the lowest death can be found in Australasia where only 6.5 AR attributed deaths per 100,000 and 28 AR associated deaths per 100,000. The same report also lists out six pathogenic bacteria that cause the most death in 2019 [ 6]. In order of the number of deaths, E. coli, S. aureus, K. pneumoniae, A. baumannii, and P. aeruginosa caused 929,000 AR attributed deaths and 3.57 million AR associated deaths.

Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant type of S. aureus that is generally resistant towards beta-lactam antibiotics such as penicillin (methicillin and oxacillin) and cephalosporin [ 7, 8, 9]. Beta-lactam inhibits the bacterial growth by halting the cell wall synthesis process [ 10, 11, 12]. MRSA generally overcomes the beta-lactam effects by producing beta-lactamase and altering the binding site for cell wall synthesis [ 7, 8, 9, 13]. The current clinically approved method to treat MRSA infection involves different antibiotic classes such as vancomycin and teicoplanin [ 14, 15]. These glycopeptide antibiotics act on the bacterial cell wall similar to beta-lactam, but it utilises different target by binding to the peptidoglycan side chain, which prevents peptidoglycan crosslinking [ 13, 14, 15]. However, the newer MRSA strain started to exhibit resistance towards glycopeptide antibiotics, which makes it difficult to treat the infection [ 13, 14]. Other types of antibiotics such as mupirocin, clindamycin, fusidic acid, and co-trimoxazole also used a second line option in treating MRSA [ 16]. However, these antibiotics can only be prescribed when there is no other alternative available due to the risk of resistance [ 16, 17]. Thus, alternatives to treat MRSA without the use of different classes of antibiotics are greatly needed.

Recent scientific development showed some promising potential in inhibiting MRSA through the usage of antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs). These two agents exhibit broad-spectrum antimicrobial properties, which makes them the suitable candidates to combat MRSA threat [ 18, 19, 20, 21]. AMPs are naturally occurring molecules that can be found in all types of life, which are involved in innate immunity defense [ 20, 21]. AMP mainly takes action on the bacterial membrane, and it can be simplified into two mechanisms of action: membranolytic and non-membranolytic action [ 21, 22, 23]. Membranolytic action can be defined as direct AMP action on the bacterial membrane, which greatly alters its structural integrity [ 23, 24, 25]. Meanwhile, non-membranolytic action is when AMPs were internalised into the cells without causing major damage to the membrane, but it targets the vital intracellular components [ 26, 27, 28].

AgNPs are metallic nanoparticles that have unique physicochemical properties including optical, thermal, electrical and high electrical conductivity in comparison to its bulk form due to its nano size [ 29, 30]. Their enhanced antimicrobial properties mainly contributed with their large surface area per volume area, which allows more antibacterial contact with the pathogenic bacteria [ 19, 31, 32]. AgNPs are also steadily gaining interest due to its multiple mechanism of actions. AgNPs generally act on membranes by disrupting it through hole formation, direct adhesion and internalisation into the membrane, excessive ROS generation and alteration in signalling pathways [ 30, 33, 34, 35]. Despite their excellent antimicrobial properties, AMPs and AgNPs have their own limitations, but, through the combination of both agents, a positive synergistic effect can be observed [ 36, 37]. Thus, this review discusses about MRSA, its mechanism of resistance, advantages and limitations of AMPs and AgNPs as its own. This topical review also discusses the combination of AMPs-AgNPs in combating bacteria, particularly MRSA and S. aureus, as no other review has been reported with the combination of the two antimicrobial agents. The review process was done based on a literature search in Google Scholar with the keywords, antimicrobial peptide, antibiotic resistance, MRSA, silver nanoparticles and S. aureus, with most of it published from 2016-2022 and some of the papers published before 2016.

2. Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is Gram-positive bacteria with round shape morphology that commonly can be found in the body as a part of its microbiota. Despite it acting commensally on the human body, it can be opportunistic bacteria since it can cause skin infections and food poisoning. Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant strain of S. aureus that are mainly resistant to beta-lactam antibiotics. MRSA was first identified in 1961 in United Kingdom just a year after methicillin was introduced to treat S. aureus infection [ 38, 39]. Despite methicillin no longer being used clinically, the term methicillin-resistant is still used to reflect S. aureus resistance towards commercial antibiotics such as beta-lactams antibiotics including oxacillin. According to World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), MRSA has been a big and serious threat on the pathogenic bacteria watch list respectively [ 40, 41]. According to recent systematic analysis in the Lancet in 2019, MRSA alone caused more than 100,000 deaths [ 6]. Originally, MRSA are common in the healthcare setting, and this type of MRSA is often dubbed as healthcare-associated or hospital-acquired MRSA (HA-MRSA) [ 42]. The infection can be spread through direct contact with an infected wound or contaminated hands. Untreated infection can cause serious bloodstream infections, surgical site infections, sepsis and pneumonia [ 7, 43]. Other types of MRSA are community-associated (CA-MRSA) and livestock-associated MRSA (LA-MRSA) [ 39, 43].

Beta-lactam antibiotics act on the bacterial cell wall by binding to the penicillin binding protein (PBP), which is responsible for the crosslinking of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) [ 10, 11]. This crosslinking will form a cell wall that protects the bacteria from external threats. MurNAc subunits have pentapeptide chains attached to it, typically with a sequence of l-Ala-γ-d-Glu-l-lysine (or -meso-diaminopimelic acid)-d-Ala-d-Ala [ 11]. Beta-lactam antibiotics such as penicillin, cephalosporin, carbapenem and monobactams have a beta-lactam ring which shared similar structural homology to d-Ala-d-Ala of the pentapeptide chain [ 10, 44]. The d-Ala-d-Ala substrate is responsible for the PBP binding site for crosslinking, and this similarity causes beta-lactam antibiotics bind to PBP, causing the crosslinking between the glycan stands to be halted [ 11]. The binding between beta-lactam and PBP causes the build-up of peptidoglycan precursors which trigger autolytic digestion of old peptidoglycan by hydrolase [ 10]. Without the production of new peptidoglycan, the structural integrity of the cell wall is significantly disrupted and led to cell damage due to high internal osmotic pressure [ 11, 12].

MRSA overcomes this detrimental effect by producing beta-lactamase, an enzyme to break down the antibacterial effect of beta-lactam antibiotics and production of the mecA gene, which changes the penicillin-binding protein (PBP) confirmation. Beta-lactamase is an enzyme produced by bacteria to counteract the effects of beta-lactam antibiotics. This enzyme hydrolyses beta-lactam in the periplasmic space, thus deactivating it before PBP interaction [ 4]. Beta-lactamase production in staphylococci is controlled by the repressor BlaI and the sensor protein BlaR1 ( Figure 1 a) [ 44]. The genes encoding beta-lactamase, the blaZ-blaR1-blaI genes, are repressed by BlaI is from transcribing beta-lactamase when beta-lactam is absent [ 10, 12]. Once beta-lactam is presented, the transmembrane sensor, BlaR1, covalently binds to it and irreversibly acylated at its active site serine. This will activate the intracellular zinc metalloprotease domain of BlaR1 and cause BlaI that are bound to blaI-blaRI operator to proteolytically cleave and dissociate from its binding site [ 12]. The dissociation allows blaZ to be upregulated and transcribed beta-lactamase enzyme. The produced beta-lactamase enzyme later hydrolyses beta-lactam antibiotic by hindering it from binding with PBP [ 10, 12]. Thus, the peptidoglycan synthesis of the bacteria can be initiated as usual.

In MRSA, the PBP responsible for the peptidoglycan cross-linking is altered to novel penicillin-binding protein 2a (PBP2a), which has a lower binding affinity to beta-lactam antibiotics [ 39]. The resistance arose from the mecA gene located in the staphylococcal cassette chromosome mec ( SCCmec), and this resistance gene can be passed to other populations through horizontal gene transfer [ 12]. Upon acquiring the mecA gene, it will be localized in the S. aureus chromosome. The production of PBP2a is controlled by MecI repressor and transmembrane MecR1 sensor protein ( Figure 1 b) [ 10]. In the absence of beta-lactam antibiotics, MecI represses mecA gene expression by binding to the promoter region of mec operon [ 10, 39]. In the presence of beta-lactam antibiotics, the antibiotic binds to the MecR1 sensor protein. It triggers autolytic activation of the metalloproteinase domain in the cytoplasm part of MecR1, causing signal transduction to be activated [ 12]. The latter caused the MecI repressor to be proteolytically cleaved from its binding site, and this allows the expression mecA to produce PBP2a [ 10]. The PBP2a production allows the peptidoglycan wall synthesis to continue without the interaction of beta-lactam antibiotics due to its low binding affinity to the antibiotic [ 7, 39]. Interestingly, the mec operon shared a similar structure and function with the bla operon, which produces beta-lactamase [ 7, 12]. This similarity allows the Blal repressor to bind to the mec operon to repress mecA transcription ( Figure 1 ) [ 10].

3. Antimicrobial Peptides (AMPs)

Antimicrobial peptides (AMPs) are naturally occurring host defense mechanisms against infections. AMPs can be found in all living organisms such as plants, microorganisms and animals [ 20, 23]. Typical AMPs consist of 5-50 amino acid chains and have amphipathic or cationic structure. Despite AMPs being naturally occurring, synthetic AMPs have been developed by the scientist to overcome the naturally occurring AMP limitations [ 21]. While naturally occurring AMPs are susceptible to proteolytic degradation, synthetic AMPs have a longer half-life, and it is designed to improve their antimicrobial properties. AMPs then can be divided into four main groups based on its secondary structure including amphipathic alpha-helices, beta-sheets, a combination of both alpha and beta structure (mixed) and extended structure (without alpha and beta structure) ( Figure 2 ) [ 21].

Alpha helices' AMPs generally contain two amino acids that are adjacent to each other with the distance of 1.5 Å (0.15 nm) [ 23]. The most studied AMPs in this group is LL-37 (amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), AMPs that can be found in the human body that act as the host defense towards bacterial infections [ 21, 23, 45, 46]. Beta-sheets' AMPs have at least two beta strands with disulfide bonds. Protegrin-1 (amino acid sequence: RGGRLCYCRRRFCVCVGR-NH ₂), which is isolated from pigs, is one of the examples in this group which exhibits antimicrobial activity against fungi, bacteria and some enveloped viruses [ 23, 26]. Mixed structure usually consists of a combination of alpha-helix and beta-sheet that are packed against each other. Human-beta-defensin-2 is one of the well-studied AMPs in this group [ 23, 28]. The extended structure is a unique group of AMPs that consists of two or more tryptophan, arginine, histidine and proline structure in single molecules. Cattle neutrophil isolated AMPs, indolicidin (amino acid sequence: ILPWKWPWWPWRR-NH ₂), are in this group [ 23, 45].

In terms of AMP mechanism of actions, it can be divided into two main categories, membrane disruptive and non-membrane disruptive AMPs [ 22, 26, 28]. Membrane disruptive AMP can be further divided into the toroidal-pore model, barrel-stave model and carpet model ( Figure 3 ) [ 26, 27]. The toroidal-pore model is where AMPs form pores in the membrane (1-2 nm diameter) vertically [ 27]. This will also cause the phospholipid head to bend due to the insertion of AMPs [ 28]. In the barrel-stave model, AMPs bind to the cell membrane and aggregate before penetrating the membrane [ 26]. During this process, hydrophobic regions of AMPs are inserted into the phospholipid membrane, while the hydrophilic regions of AMPs are facing the outer part of the membrane pore [ 27]. This will cause uncontrolled cellular movement for the cell, which will lead to cell death. The carpet-like model destroys the membrane in a detergent-like manner [ 22]. AMPs are first arranged onto the cell membrane by their hydrophobic part facing the phospholipid bilayer, which alters its surface tension. The altered surface tension later causes micelles formation as the results of peptide accumulation and destroys the membrane [ 22, 27].

A non-membrane disruptive mechanism is rarely studied in AMP research, but recent advancement showed that AMPs are internalised into cells and interacts with vital intracellular targets and even inhibits cell wall biosynthesis [ 28]. This includes inhibition of protein and nucleic acid synthesis, cell division and protease activity [ 23, 26, 28]. AMPs inhibit protein synthesis by directly interacting with the transcription and translation process. PR-39 AMPs isolated from a pig's small intestine can inhibit protein synthesis, which causes proteins degradation that are required for DNA synthesis [ 28]. Indolicilin induces degradation of nucleic acid by binding to the double stranded DNA, which causes the DNA synthesis to be halted [ 23, 26]. Teixobactin AMPs bind to lipid II and lipid III (precursors of cell wall), which later inhibits the cell wall synthesis process [ 28]. Based on the promising antimicrobial action of AMPs, Table 1 showed some examples of AMPs that are effective towards MRSA and wild type S. aureus.

Table 1

AMPs Type(s)

AMPs Origin/Type

Amino Acid Sequence

Antibacterial Activity on Staphylococcus aureus

Ref.

Temporin A

Isolated from frog skin secretion, Rana temporaria

FLPLIGRVLSGIL-NH2

Effective toward methicillin-susceptible S. aureus (MSSA) and MRSA. Exhibit MIC value of 4 µg/mL once tested on surgical wound isolated MRSA.

[ 18]

Citropin 1.1

Isolated from frog's dorsal and submental glands Litoria citropa

GLFDVIKKVASVIGGL-NH (2)

Exhibit MIC value of 16-64 mg/mL once tested on 215 isolates of MSSA and MRSA

[ 47]

As the innate immune system, AMPs have broad spectrum antimicrobial properties which are said to be effective towards pathogenic microorganisms [ 21, 23, 45]. These antimicrobial properties are greatly enhanced as AMPs can be found abundantly at the site of the infection, which makes it more time efficient since it can react faster to combat the infection [ 23, 28]. Resistance towards AMPs is also said to be low, which makes it one of the suitable candidates to combat MRSA [ 28]. Besides that, AMPs also have good water solubility and thermal stability [ 23, 45]. However, AMPs do possess weakness as naturally occurring AMPs are susceptible towards proteolytic degradation, which limits their potential [ 23, 28]. In addition, AMP production and purification can be costly sometimes. Despite their broad antimicrobial spectrum, it can be a challenge to be used medically as some AMPs might induce hypersensitivity after application and might cause immunogenicity and toxicity when it is administered in humans [ 21, 23, 45].

4. Silver Nanoparticles (AgNPs)

Silver nanoparticles are the product of nanotechnology which are particles of silver that are ranging in size from 1 to 100 nm [ 38, 39]. Their nanosized greatly enhances its broad-spectrum antibacterial properties as it has larger surface area per volume ratio [ 29, 33, 35, 51]. Due to their unique properties in terms of optical, electrical, magnetic and antibacterial, AgNPs have various applications, which include medical appliances, optical sensors, cosmetics, drug delivery, textiles, keyboards, wound dressings and food packaging [ 35, 51, 52, 53]. AgNPs are gaining popularity due to their multiple mechanisms of action on bacteria ( Figure 4 ), which include direct adhesion of AgNPs on the bacterial surface and altering the membrane structural integrity [ 32, 54]. Next, AgNPs penetrate inside the bacterial cell and interact with its intracellular components, damaging it until it cannot perform vital cellular processes [ 32, 54]. AgNPs are also able to induce reaction oxygen species and free radical generation, thus causing irreversible oxidative damage to the bacteria [ 55, 56]. Alteration of vital signaling transduction, which is necessary for the bacterial life cycle, is also one of the mechanisms exhibited by AgNPs [ 52, 57].

After AgNPs attached on the membrane, its permeability and structural integrity are greatly altered, and this causes some portion of AgNPs to infuse into the cell. This statement is further strengthened by several studies which revealed that AgNPs penetrate the cells through transmission electron microscope analysis on bacteria [ 19, 62, 63]. In addition, in the presence of oxygen and proton, AgNPs dissociate to Ag⁺ ions, which also facilitates the infusion [ 64]. When AgNPs are internalised into the cell, it interacts with cellular molecules and structures such as protein, DNA and lipids. For instance, AgNPs interrupt protein synthesis by interacting with ribosome by denaturing it, which halts the translation process [ 65, 66]. AgNPs also interacts with DNA molecules, which may cause denaturation and shearing of DNA and also cell division interruption [ 32, 67]. The interaction causes the bacteria to lose the ability to undergo division, reproduction and eventually cell death [ 54, 68].

Tyrosine phosphorylation is important in activation of various proteins such as RNA polymerase sigma factor that are essential in the bacterial transcription process [ 34, 57]. Increased dephosphorylation of tyrosine profile might inhibit vital processes in bacteria such as polysaccharide biosynthesis of bacterial capsule in bacteria [ 33, 34]. AgNPs can alter tyrosine phosphorylation, which led to failed regulation of the cellular process and homeostasis, which later destroys the cells [ 52, 57]. Since AgNPs managed to exhibit multiple mechanisms of action on bacteria, it is said to be effective to combat MRSA. Table 2 showed some AgNPs action on inhibiting the growth of MRSA.

Table 2

Silver Nanoparticles' Properties

Antibacterial Action

Ref.

5. AMP and AgNPs Combination on MRSA or MSSA

Despite AMPs and AgNPs having their own weaknesses on their own, the combination of these two, or sometimes with the addition of polymer, enhances its antibacterial properties while greatly reducing their toxicity effects. Synergistic effect in terms of stronger antibacterial activity of these two agents can also be observed once they are administered together.

A study by Jin et al. utlises AMPs, Tet-213 and AgNPs that are loaded onto porous silicon microparticles [ 36]. Tet-213 is a 10 amino acid peptide (sequence: KRWWKWWRRC) that possesses broad spectrum activity due to the presence of thiol group and, with the combination of AgNPs, the antimicrobial effect increases drastically. The presence of porous silicon microparticles (PSiMPs) acts as a carrier for effective delivery of the antimicrobial agent to the infected site [ 36, 81]. PSiMPs was chosen due to its tunable pore size, biocompatibility and decompatibility. However, PSiMPs only dissociate in an alkaline condition as it is normally acidic during the early stage of infection [ 82]. Despite the carrier only being able to dissociate in alkaline conditions, the presence of ROS also allows PSiMPs be to be dissociated easily. When ROS is high during the wound infection, it allows the carrier to be disintegrated and releases silver ions from AgNPs together with Tet-213. The acidic condition also allows a gradual release of AgNPs-AMPs, which allows more effective and stable antimicrobial action. In this study, for the combination of these agents, the MIC value was greatly reduced to 2 mg/mL in comparison to AgNPs-PsiMPs (2.5 mg/mL) and AMPs-PsiMPs (>5 mg/mL) on S. aureus [ 36]. In-vitro testing on mouse fibroblast (NIH3T3) cells and human immortal keratinocyte (HaCaT) showed low toxicity effects as this complex does not affect the cells' proliferation. This AgNPs-AMPs-PSiMPs combination also exhibits low toxicity and faster wound healing on rats infected with S. aureus [ 36]. The faster wound healing contributed with the release of silicon ions in the complex, with the help of AgNPs and AMPs to reduce the bacterial infection in the wound. Note that silicon ions promote wound healing by activating the epidermal growth factor receptor (EGFR), epidermal growth factor (EGF) and extracellular signal-related kinase (ERK) signaling pathway [ 36, 83, 84].

A star conjugated PCL- b-AMPs nanocomposite was also used in stabilising AgNPs and enhancing antimicrobial activity of it with the help of AMPs [ 77]. Star conjugated PCL- b-AMPs consist of polycaprolactone (PCL) and polypeptide (Phe ₈- stat-Lys ₃₂), which are later loaded with AgNPs. This complex is relatively stable at room temperature for three months with any sign of aggregations. In this case, PCL- b-AMPs penetrate the negatively charged membrane since this complex is positively charged. This penetration allows AgNPs to be released in the cytoplasm and the deactivating of vital cellular components. This complex managed to exhibit enhanced inhibition on S. aureus (27.6 mm) when compared to the combination of PCL- b-AMPs (19.1 mm) and AgNPs (12.7 mm) alone. A low MIC value (4 µg/mL) is also observed when PCL- b-AMPs with AgNPs is tested on MRSA [ 77]. This suggests that a synergistic effect of AMPs and AgNPs allows higher inhibition on the bacterial growth. A damaged membrane was also observed on MRSA, which later led to cell death [ 77, 85]. This complex also showed no sign of resistance even after 21 passage exposure with a sub-lethal MIC value of the complex when tested on the wild type S. aureus [ 77]. It also showed low cytotoxicity towards normal mouse fibroblast cells (L929) as it managed to retain up to 80% of cell viability after 48 h. The PCL- b-AMPs managed to reduce AgNPs toxicity by only releasing it to the target site besides from their biocompatibility.

A novel composite of AgNPs and designed AMPs P-13 (amino acid sequence: KRWWKWWRRCECG) were tested against S. aureus (ATCC 25923) [ 86]. Based on the MIC values, this composite manages to inhibit bacteria effectively at lower concentration (7.8 ± 0.05 µg/mL) compared to AgNPs and AMPs alone with 7.8 ± 0.05 µg/mL and >500 ± 0.04 µg/mL, respectively. Interestingly, with the addition of P-13 to AgNPs, a drastic toxicity reduction can be observed on mouse fibroblast cells (NIH-3T3) [ 86]. This addition allows AMPs to stabilise AgNPs and reduce its cytotoxicity effect in comparison to AgNPs alone. It is proposed that this complex inhibits bacteria growth by adhesion to the bacteria through electrostatic force and was internalised into the cell reacting with vital cellular components. This causes cellular leakage out of the cell, which led to cell death [ 27, 86].

Another study by Li et al. developed multifunctional peptide (MFP)-coated silver nanoparticles as an alternative to combat antibiotic resistance [ 78]. In this study, AMPs tachyplesin-1 and target peptide N-ac-PGP-PEG were combined to adsorb AgNPs through electrostatic interaction. This complex was proven to be effective at inhibiting S. aureus and MRSA growth with MIC values of 8 µg/mL and 16 µg/mL, respectively [ 78]. Despite the MIC for vancomycin, an antibiotic control in this experiment is much lower than the complex (2 µg/mL); this complex was proven to be a promising agent to inhibit the bacterial growth with future optimisations.

The AMP@PDA@AgNPs nanocomposite was created through polymerisation to inhibit biofilm formation by S. aureus [ 80]. PDA was added as a delivery agent, which allows more effective AMPs and AgNPs delivery to the target site. This allows more effective internalisation into the cell to exhibit its antimicrobial activity. This nanocomposite showed no cytotoxicity effect even at a high concentration (400 µg/mL) when tested on human embryonic kidney (HEK293T) cells. To inhibit S. aureus growth, only a concentration of 25 μg/mL was required, which is much lower than the concentration used in the cytotoxicity assessments. This complex also managed to reduce biofilm formed by the bacteria by reducing the expression of biofilm forming genes (las I and rh II, fim H) [ 80]. Table 3 showed other combinations of AMPs with AgNPs that are able to inhibit S. aureus or MRSA growth effectively.

Table 3

AMP Type

Product Combination

Antibacterial Properties

Ref.

Nisin (antibacterial peptide produced by the Lactococcus lactis, which is commonly used as food preservative)

Silver-nisin nanoparticles (Ag-nisin NP)

Exhibit MIC value of 4 mg/L on MRSA in comparison to silver nitrate (16 mg/L) and nisin (4 mg/L) alone. Inhibit MRSA growth by destroying the biofilm. Ag-nisin NP showed lower cytotoxicity on human skin fibroblasts (Hs 44.Fs, ATCC ® CRL7024™) and human kidney epithelium cell line (HEK) compared to silver nitrate.

[ 87]

Daptomycin (clinically approved AMPs for medical usage)

Daptomycin-silver nanoclusters (D−AgNCs)

Complex exhibits the highest inhibitory effect against S. aureus in comparison to the controls (daptomycin or AgNCs alone). Inhibit growth by inducing DNA damage and ROS generation.

[ 88]

GL13K (amphiphilic AMPs that was developed from BPIFA2 (human salivary protein)

AgNP-dGL13K complexes (AMPs and AgNPs coated with etched Titanium (eTi) for stable nanostructure)

Exhibit excellent antibacterial properties on MRSA through in vitro and in vivo rat models.

[ 89]

G-Bac3.4 (amino acid sequence: CRFRLPFRRPPIRIHPP
PFYPPFRPFL-NH2)

Bioconjugate G-Bac3.4 with silver nanoparticles

These bioconjugate AMPs and AgNPs exhibit antimicrobial action by internalising into MRSA and inhibiting the growth.

[ 90]

MBP-1 (plant antimicrobial peptide)

MBP-1 and silver nanoparticles combination

The MIC of MBP-1 is 0.6 mg/mL while MIC for silver nanoparticles were 6.25 and 12.5 mg/L. MIC of silver nanoparticles and MBP-1 combination was found to be 3.125 mg/mL and 6.25 mg/mL, respectively, on S. aureus. Faster wound healing can be observed on rats infected with S. aureus.

[ 37]

In general, AMPs and AgNPs can be combined together to exhibit synergistic antimicrobial effect or carrier/polymer can be added to the complex to allow more effective AMPs and AgNPs for the target site without exhibiting a high toxicity effect ( Figure 5 ).

6. Conclusions and Future Perspectives

Despite as antibiotic resistance threat that keeps on increasing year by year, scientists are never giving up on finding alternatives to curb the spreading of antibiotic resistance. The emergence of antimicrobial peptides with multiple membranolytic and non-membranolytic mechanism cast light on the antibiotic resistance research. A lower rate of microbial resistance towards AMPs also allows it to be studied intensively in combating MRSA, a worldwide pathogenic threat. The introduction of silver nanoparticles in this modern era also allows its utilisation in combating antibiotic-resistant bacteria including MRSA. Multiple mechanisms, such as direct adherence and internalization of AgNPs, allow it to exhibit an antimicrobial effect effectively by inducing ROS and alteration of signal transduction. Nevertheless, AMPs and AgNPs each possess their own weaknesses, which include toxicity and instability. A combination of these two agents somehow overcomes these weaknesses by stabilising these agents to the target site. A synergistic effect can also be observed once these two agents are combined to inhibit bacterial growth. A lower toxicity effect in vitro and in vivo can also be observed. Although this research is still the beginning, future optimisations can be done, especially in terms of enhanced complex stability, lower dosage required in inhibiting bacteria and lower toxicity, in vitro and in vivo need to be done. Clinical research also needs to be done before the co-application of AMPs and AgNPs can be truly used in combating antibiotic resistance, especially towards MRSA in humans.

Acknowledgments

Special thanks to Research Management Centre and Faculty of Science and Marine Environment, Universiti Malaysia Terengganu for supporting this project.

Funding Statement

This research received no external funding.

Author Contributions

Conceptualization, M.A.A.M. and W.I.W.I.; writing-original draft preparation, M.A.A.M.; writing-review and editing, M.A.A.M., N.A.H., M.M. and W.I.W.I. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data sharing not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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# data[].attributes.attachment_link.description
Antibiotics are regarded as a miracle in the medical field as it prevents disease caused by pathogenic bacteria. Since the discovery of penicillin, antibiotics have become the foundation for modern medical discoveries. However, bacteria soon became resistant ...

# data[].attributes.attachment_link.readable_text
Abstract

Introduction

Invasive Staphylococcus aureus infections are common and cause considerable mortality, particularly at the margins of the age continuum. They are the most common infective reason for admission to an ICU in both adults and children, with high mortality identified amongst these cohorts: 23%-33% in adults and 9% in children. Contributing to this severe phenotype is the ability of S. aureus to express multiple extracellular toxins that cause additional tissue damage.

Despite this disease burden, fewer than 3000 adults and 300 children have been randomly assigned into clinical trials to assess the efficacy of treatments for S. aureus bacteraemia. Hence, opinions on best management vary widely, with an adjunctive protein synthesis inhibitor antibiotic suggested by some experts in severe, toxin-mediated S. aureus infections. The most frequently used protein synthesis inhibitor in this setting, clindamycin, decreases exotoxin production by binding to the 50S bacterial ribosomal subunit. The evidence to support adjunctive protein synthesis inhibitor use is currently limited to in vitro, animal and observational human case-series data, with no previous randomized controlled trials (RCTs).

In this multicentre pilot RCT, we aimed to determine feasibility and to inform the design of a definitive RCT, the S. aureus network adaptive platform (SNAP) trial ( https://www.snaptrial.com.au/). One of the aims of the SNAP trial will be to examine if the addition of clindamycin to standard antibiotic therapy (β-lactam, vancomycin or daptomycin) has greater clinical efficacy than standard therapy alone in adults and children with S. aureus infections. In addition, we aimed to provide proof of concept of including adults and children in the same RCT.

Methods

Study design and setting

We performed an investigator-initiated, open-label, multicentre, parallel-group, pilot RCT across four states in Australia, at six adult and three paediatric tertiary referral hospitals in adults and children with severe S. aureus infections from July 2018 to October 2020. This trial was endorsed by the Australasian Society of Infectious Diseases Clinical Research Network (ASID-CRN) and registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12617001416381p). The published protocol was prospectively approved at each participating site with ethical approval (HREC/17/HNE/411). Written legal guardian (<18 years) or participant/surrogate decision-maker (≥18 years) informed consent was required for all participants before enrolment by the site principal investigator (PI).

Participants

Eligibility criteria were: hospital inpatients ≥28 days old expected to remain an inpatient at the study site for ≥7 days post-randomization; S. aureus cultured from ≥1 clinically relevant site (defined as any specimen where the site investigator determines the organism to be contributing to the patient's clinical syndrome); and index tissue (including blood) culture drawn within ≤48 h of hospital admission (to exclude nosocomial infections) and ≤72 h pre-randomization. Evidence of severe S. aureus disease was defined as at least one of the following: (i) septic shock ( Appendix S1, available as Supplementary data at JAC-AMR Online); (ii) severe lung (necrotizing or multifocal pneumonia) or pleural space infection ( Appendix S2); or (iii) multifocal disease (>1 non-contiguous site or >1 contiguous anatomical site involved).

To facilitate recruitment, after 12 months, the eligibility criteria for multifocal infection was broadened (initially it only included >1 non-contiguous site) to also include >1 contiguous anatomical site. The rationale for this change was supported in the literature, with an association of Panton-Valentine leucocidin (PVL)-positive S. aureus disease and complicated osteomyelitis (thrombosis and pyomyositis).

Exclusion criteria related to patients who: had significant immunosuppression (defined as prednisolone >0.5 mg/kg/day for ≥14 days in the last 30 days, other immunosuppressive medication, known HIV with CD4 cell count <200 cells/mm ³ or congenital immunodeficiency); were expected to die within 24 h with or without treatment; had current severe diarrhoea (defined as >6 stools per day or clinician-determined severe diarrhoea in children) or onset of Clostridioides difficile-associated diarrhoea within 48 h prior to enrolment; had necrotizing fasciitis; were known to be pregnant; had a history of severe allergy to β-lactams, glycopeptides, lincosamides or daptomycin; had previously participated in the trial or were currently receiving a protein synthesis inhibitor antibiotic that could not be ceased or substituted. Other exclusions were the presence of polymicrobial culture containing other clinically significant isolates and where the primary clinician was unwilling to enrol the patient.

Randomization

Participants were randomized by a computerized central randomization schedule generated by a statistician in a 1:1 ratio to standard therapy (control group) or standard therapy plus clindamycin (adjunctive clindamycin group), stratified by age (<18 years versus ≥18 years), in permuted blocks of variable size. We pre-specified inclusion of one-third of children in our target sample.

Procedures

Standard therapy comprised IV flucloxacillin, 4-6 hourly or cefazolin, 6-8 hourly (both 50 mg/kg/dose for MSSA) or IV vancomycin [with dosing adjustments to maintain trough levels at 15-20 mg/L], daptomycin or ceftaroline ( Appendix S3) for MRSA. Clindamycin was given IV 10 mg/kg/dose up to 600 mg four times daily or oral 450 mg three times daily in adults (10 mg/kg/dose in children) for 7 days. The choice and duration of standard antibiotic therapy and the decision to switch to oral clindamycin were clinician determined.

Outcomes and measurements

The primary outcome was number of days alive and free of systemic inflammatory response syndrome (SIRS) (defined as meeting <2 simultaneous SIRS criteria on a calendar day) ≤14 days post-randomization ( Appendix S2). The secondary outcomes were: (i) all-cause mortality at 14, 42 and 90 days; (ii) time to first resolution of SIRS (number of days until the patient meets <2 simultaneous SIRS criteria); (iii) proportion with microbiological relapse by Day 90 (positive blood culture for S. aureus ≥72 h after a preceding negative culture); (iv) proportion with microbiological treatment failure by Day 90 (positive sterile site culture for S. aureus ≥14 days after randomization); (v) number of surgical procedures needed to achieve source control; (vi) duration of IV antibiotic treatment; (vii) C. difficile-associated diarrhoea (three or more loose stools per day with a positive laboratory test for C. difficile toxin); (viii) all cause diarrhoea (three or more loose stools per day); and (ix) time to a ≥50% decrease in C-reactive protein (CRP) in the first 14 days post-randomization (excluding participants missing a baseline CRP level).

Routine haematological parameters, repeat cultures, surgery for source control for S. aureus, patient progress and treatment were gathered from medical records, pathology results and treating teams onto an electronic case report form on Days 1, 3, 7, 10 and 14 and then weekly thereafter if an inpatient, and out to 90 days post-randomization (REDCap database). All-cause mortality data were collected through participant contact at 90 days or retrieved from medical records. Adverse events (AEs) were captured daily whilst an inpatient and up until 90 days post-randomization. All serious adverse events (SAEs) as defined in the protocol to any of the study drugs reported.

Laboratory methods

Identification of S. aureus was based on routine phenotypic and/or genotypic methods used in clinical laboratories of participating hospitals.

Statistical methods

The principal analysis of both primary and secondary endpoints was according to intention-to-treat principles. All participants with data available for the endpoint were analysed according to the treatment allocation, regardless of what treatment they received. Analyses of aggregated outcomes was initially performed by a statistician blinded to treatment groups, who was subsequently unblinded. Summary statistics were calculated for each outcome by trial arm and differences and 95% CIs were calculated using appropriate regression models. Given that this is a pilot study which is not powered for formal hypothesis testing, P values were not reported. All regression analyses were adjusted by the randomization covariate of age group (<18 years versus ≥18 years), and both unadjusted and adjusted estimates are reported.

For the primary outcome, days alive and free of SIRS, the difference between treatment arms was estimated using a zero-inflated negative binomial regression model, adjusted by days observed to account for participants with missing data. Binary secondary outcomes were assessed using logistic regression to estimate differences in proportions. All-cause mortality in both treatment arms was illustrated in a Kaplan-Meier survival curve. Competing risks regression was used to estimate HR for time-to-event outcomes. Difference in number of surgical procedures was estimated with negative binomial regression adjusted for days observed. All analyses were performed using Stata v16.0 (StataCorp LLC, College Station, TX, USA).

We planned to enrol 60 patients, at least 20 of whom were aged <18 years. As a pilot study, the sample size was based on the number achievable and the number needed to determine feasibility and to refine assumptions and study design. No assumptions were made about the rate of occurrence of the primary outcome in the control group nor for anticipated treatment effects of the intervention.

Results

Over 28 months (July 2018-October 2020), across nine sites, 127 individuals were screened and 34 randomized from 40 eligible participants (85% consent rate). The recruitment rate was 1.2 participants/per month. Key reasons for exclusion included: inability to enrol participants within 72 h of the index culture (38/127, 30%) and not meeting the severe S. aureus disease definition (27/127, 21%). Consented participants were randomized to receive either standard therapy ( n = 17) or standard therapy plus adjunctive clindamycin ( n = 17) (Figure 1).

The trial was stopped prior to the anticipated sample size of 60 patients due to the slow recruitment rate and because the SNAP trial was planned to commence in 2021 and includes a domain for adjunctive treatment with clindamycin.

Baseline characteristics were similar between groups (Table 1). One third of the cohort were children <18 years (11/34, 32%) and female (11/34, 32%). All S. aureus infections (34/34, 100%) were community acquired and 24% (8/34) were caused by MRSA.

The majority of participants (32/34, 94%) met SIRS criteria on Day 1 of randomization (Table 1). The median SOFA score in adults was 7 (IQR 5-11) and underlying comorbidities were present in 35% (8/23) of adults and 9% (1/11) of children. Half (18/34, 53%) developed septic shock and 65% (22/34) required admission to the ICU, which was less frequent in children (4/11, 36%) compared with adults (18/23, 78%). Receipt of antibiotics prior to randomization was frequent overall (32/34, 94%), including with a β-lactam (32/34, 94%), vancomycin (28/34, 82%) or protein synthesis inhibitor antibiotic (16/34, 47%).

The primary endpoint, number of days alive and free of SIRS, was similar between the clindamycin group (median 3 days [IQR 1-6]) and standard therapy group (4 days [IQR 0-8]; adjusted difference [aD] −0.31 days [CI −3.20, 2.57]) (Table 2). For the secondary outcome of all-cause mortality, there were no deaths (0/17, 0%) up to 90 days in the clindamycin group. In the standard therapy group, 14 day mortality was 18% (3/17) and 42 and 90 day mortality (all in adults) were 24% (4/17) (Figure 2). There were no differences in microbiological relapse (3/17, 18% versus 1/17, 6%; aD 12% [CI −10%, 33%]) or microbiological treatment failure (1/17, 6% in both; aD 0% [CI −16%, 16%]) in the clindamycin compared with standard therapy group.

All-cause diarrhoea occurred in 47% (8/17) of the clindamycin group compared with 35% (6/17) of the standard therapy group (aD 12% [CI −21%, 45%]), with one case (1/17, 6%) of C. difficile-associated diarrhoea occurring in the standard therapy group. The median days to first resolution of SIRS was equivalent between groups; 9 days (CI 2-10) in the clindamycin and 9 days (CI 5-12) in the standard therapy group; (adjusted hazard ratio [aHR] 1.57 [CI 0.75, 3.31]). The median days until CRP decreased by over 50% were similar between groups: 7 days (CI 3-14) in the clindamycin group and 5 days (CI 3-7) in the standard therapy group (aHR 0.59 [CI 0.30, 1.13]) (Table 2).

AEs were more common in the clindamycin group (10/17, 59%) compared with standard therapy (5/17, 29%) ( Appendix S4). Study investigators determined most events to be unrelated to the study treatment and to be mild in severity.

Discussion

This is the first clinical trial to assess adjunctive clindamycin for severe S. aureus infections. Key findings from the CASSETTE trial include that it is feasible to conduct an RCT examining adjunctive clindamycin therapy, adults and children can be incorporated into one trial using harmonized endpoints, and there were no serious safety concerns evident from this preliminary cohort.

This study took almost 2.5 years to recruit a relatively small number of participants despite a large cohort being screened (34/127, 27%). This was due, in part, to difficulty identifying patients that met the severe S. aureus infection definition within 72 h of the index culture (Figure 1), with trial recruitment performed during the working week by clinician study investigators. This information has provided future direction for the planned SNAP trial, which will expand inclusion criteria to all individuals with S. aureus bacteraemia, regardless of illness severity and contain an adjunctive clindamycin domain. Importantly, clinician equipoise and patient acceptability were high, with 85% of eligible participants providing consent and no participant withdrawals or discontinued interventions noted (Figure 1). This trial experience confirms feasibility and has progressed the partnerships needed for a robust adult and paediatric infectious diseases trial network.

This pilot RCT has also provided insights into the utility of trial endpoints. The primary endpoint (number of days alive and free of SIRS), was pragmatically selected, given the availability of a validated scoring system that aligns in both adults and children. It was reasoned it would reflect decreased systemic inflammation due to decreased exotoxin production. Despite the rationale, the primary outcome, days alive and free of SIRS, were comparable, yet a difference in mortality was demonstrated (0% [0/17] 90 day mortality: clindamycin group versus 24% [4/17]: standard therapy group), although small numbers limit any definitive conclusions. Given these hypothesis-generating findings, and the much larger planned sample size for the SNAP trial, the more clinically relevant endpoint of all-cause mortality has been selected as the primary outcome for this planned RCT.

In the CASSETTE trial, mortality in the standard treatment arm occurred only in adults and was consistent with mortality in other adult S. aureus bacteraemia trials. To have no deaths in the clindamycin arm despite balanced groups with severe disease suggests a possible benefit of clindamycin, but this could have occurred by chance given the small numbers. In the literature the human clinical studies previously examining adjunctive clindamycin include only four case series in the setting of necrotizing pneumonia ( n = 92 adults/adolescents), skin and soft tissue infections (SSTIs) ( n = 269 adults), severe influenza-MRSA pneumonia ( n = 29 children) and PVL-positive invasive S. aureus infections ( n = 141 adults). Two case series in the severe pneumonia setting demonstrated lower mortality with adjunctive protein synthesis inhibitor antibiotics compared with standard therapy alone, but all have major study design limitations. A definitive trial is needed to objectively determine if this observed mortality difference reflects a true benefit or a chance finding.

The burden of daily data collection for the primary endpoint was evident. This has since been refined to a single study timepoint (Day 5) for the equivalent secondary outcome in SNAP. In addition, the statistical model for SNAP acknowledges the infrequent occurrence of trial endpoints in children (particularly death, 0/11, 0%) and will incorporate Bayesian hierarchical models that specify borrowing of data from adult patients to children. This pilot RCT has importantly enabled significant refinement of trial design in preparation for the definitive SNAP trial.

Limitations of this study include a small sample size, limiting conclusions regarding efficacy and mortality between treatment groups. However, this was a pilot RCT designed primarily to assess feasibility. There were some missing data points, however primary and key secondary outcomes including death and relapse were complete. We were unable to assess the total duration of IV antibiotic therapy or timing of IV to oral switch due to incomplete data. Some of the standard therapy group received adjunctive clindamycin prior to enrolment, which may have impacted results. There was no placebo available in the standard therapy group.

Lessons from the CASSETTE trial will inform the SNAP trial, which includes an adjunctive clindamycin domain and will be powered to determine whether clindamycin reduces the likelihood of death. Incorporating both adults and children with S. aureus infections in an RCT is feasible, despite challenges in analysis and data interpretation.

Acknowledgements

Preliminary results of this study were presented virtually as an oral presentation (number 131) at the Australian Society of Infectious Diseases Annual Scientific Virtual Meeting on 25 March 2021.

We would like to acknowledge the participants and their families involved in this study, as well as the study-site clinical and microbiology laboratory staff.

Funding

This work was supported by a National Health and Medical Research Council HOT NORTH grant (grant number 1131932), a Telethon Kids Institute, Wesfarmers Centre for Vaccines and Infectious Diseases Seed Fund grant, a National Health and Medical Research Council PhD scholarship to A.J.C (grant number APP1133670), National Health and Medical Research Council fellowship to A.C.B (grant number APP1175509) and a National Health and Medical Research Council fellowship to S.Y.C.T. (grant number 1145033).

Transparency declarations

S.Y.C.T has received consultancy fees from Roivant Sciences for advising on a clinical trial design. All other authors: none to declare.

Author contributions

J. S. Davis, S. Y. C. Tong and A. C. Bowen were involved in the conceptualization, methodology and supervision of this clinical trial. J. S. Davis, S. Y. C. Tong, A. C. Bowen, R. Dotel and A. J. Campbell were involved in the trial methodology. Statisticians N. Meagher and D. J. Price were involved in trial methodology, formal analysis and data visualization. Study coordinators J. Nelson and A. Whelan contributed to trial resources, data curation and project administration. A. J. Campbell, J. S. Davis, S. Y. C. Tong and A. C. Bowen wrote the original draft manuscript. All authors contributed to methodology, trial investigation and reviewing and editing of the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. A. C. Bowen was involved in funding acquisition for the project.

Supplementary data

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

References

Author notes

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Clindamycin adjunctive therapy for severe Staphylococcus aureus treatment evaluation (CASSETTE)-an open-labelled pilot randomized controlled trial

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AbstractBackground. Combination antibiotic therapy with an antitoxin agent, such as clindamycin, is included in some guidelines for severe, toxin-mediated