Nanosilver: An Old Antibacterial Agent with Great Promise ...

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Abstract

Antibiotic resistance in bacteria is a major problem worldwide that costs 55 billion USD annually for extended hospitalization, resource utilization, and additional treatment expenditures in the United States. This review examines the roles and forms of silver (e.g., bulk Ag, silver salts (AgNO3), and colloidal Ag) from antiquity to the present, and its eventual incorporation as silver nanoparticles (AgNPs) in numerous antibacterial consumer products and biomedical applications. The AgNP fabrication methods, physicochemical properties, and antibacterial mechanisms in Gram-positive and Gram-negative bacterial models are covered. The emphasis is on the problematic ESKAPE pathogens and the antibiotic-resistant pathogens of the greatest human health concern according to the World Health Organization. This review delineates the differences between each bacterial model, the role of the physicochemical properties of AgNPs in the interaction with pathogens, and the subsequent damage of AgNPs and Ag+ released by AgNPs on structural cellular components. In closing, the processes of antibiotic resistance attainment and how novel AgNP&#;antibiotic conjugates may synergistically reduce the growth of antibiotic-resistant pathogens are presented in light of promising examples, where antibiotic efficacy alone is decreased.

Keywords:

nanosilver, antimicrobial applications, physicochemical properties, antibacterial mechanisms, synergy, antibiotic-resistant bacteria

2. Modern Antimicrobial Applications of Nanosilver

The antimicrobial activity of nanosilver such as colloidal silver nanoparticles (AgNPs) is linked to its unique, size-related physicochemical properties such as the very large surface-to-volume ratios and the potential release of Ag+ ions from the nanosurface under favorable redox conditions. These properties are currently exploited in the manufacturing of everyday consumer products and other antimicrobial applications ( ) [4,5].

Antimicrobial consumer products: In , consumer products have been identified worldwide as containing nanomaterials by the manufacturer, and over of these products exploit the unique properties of nanosilver (e.g., antimicrobial, optical, and catalytical) [45,46]. Antimicrobial consumer products containing silver ( ) can be found in the health (24.08%), textile (17.53%), cosmetic (13.38%), appliance (9.31%), environmental (8.30%), and construction (7.93%) sectors [46]. In the last few decades, the U.S. Food and Drug Administration (FDA) has approved many of these products containing antimicrobial Ag+ and nanosilver such as AgNPs. Examples include wound dressings, facial masks, textile fibers, sanitizers, coatings of surgical tools, dental implants, and urinary catheters ( ) [45,47].

Figure 3

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&#;Silver wound dressings&#; represent the most web-searched (n = &#; ) and one of the most heavily used consumer products containing Ag in the medical sector. A large variety of U.S. FDA-approved (e.g., Silverlon, Aquacel Ag Advantage, and Acticoat) and non-approved wound dressings are offered through prescriptions as well as over the counter [48,49,50]. Silver-based wound dressings are used as both preventative and curative measures against bacterial infection of acute and chronic wounds. Textiles, the second most widespread application of nanosilver, have been used in many types of clothing (e.g., facemasks, socks, shirts, athletic wear, and towels) [45]. An illustrative example associated with nanosilver use is disinfectants in facemasks to prevent the spread of pathogens and the formation of malodor caused by bacterial colonies that inhabit the surface of the skin [51]. Manufacturers of cosmetics, the third largest sector, have employed nanosilver for the same antimicrobial benefits [52]. Nanosilver can be found in lotions, face masks, soaps, sunscreens, etc. [45].

Because the adverse effects of Ag on human health are not yet fully understood, concerns have been raised about the growing exposure to nanosilver during the manufacture or prolonged utilization of nanosilver-based consumer products [53]. Furthermore, the environmental health impacts of nanosilver remain under debate as nanosilver properties can change in the environment, leading to altered toxicity and stability [54,55,56]. The regulation of nanosilver-based consumer products has been compounded by the challenging task of tracking products that do not specify the nanomaterial as an ingredient, especially when in minute quantities, and by the product distribution under different brand names [57]. Nevertheless, the integration of nanosilver into consumer products continues to experience a vertiginous increase. An estimated tons of nanosilver is produced worldwide [58].

Table 2

Product TypeSearch ResultVendor[Ag] and Ag FormPurposeU.S. FDA
ApprovalSilver-based wound dressings&#;silver wound dressing&#;
n =

• Aquacel Ag Advantage&#;Convatec, Berkshire, England

• 1.2% w/w&#;&#;ionic silver&#;

• To prevent and cure infection in acute or hard-to-heal wounds

YESAnkle socks with silver&#;silver textile&#;
n =

• NanoSilver, Denmark, E.U.

• Not Reported&#;&#;Nano-Silver&#;

• To defend from pathogens

NOPlatinum silver nanocolloid cream&#;silver cosmetic&#;
n =

• DHC Skincare, Tokyo, Japan

• Not Reported&#;&#;Nanosilver&#;

• To eliminate bacteria in sweat

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Other antimicrobial applications: Lately, AgNPs and Ag+ have received increased attention due to their potential use in the fight against two major global health threats, namely antibiotic resistance and viral infections, where treatments are either limited or not available [61]. For instance, non-cytotoxic concentrations of AgNPs were reported to act against a broad spectrum of viruses of different families regardless of their tropism, clade, and resistance to antiretrovirals [61,62,63]. Relevant examples include HIV-1, hepatitis B (HBV), Tacaribe virus, herpes simplex virus, mpox, smallpox, H1N1 influenza A, respiratory syncytial viruses, vaccinia virus, and dengue virus (DENV). In these studies, AgNPs were found to bind specifically or nonspecifically to proteins in the envelope of virions and thereby deactivate them (virucidal activity). These target proteins are mainly responsible for the viral interaction with host cells [13,61,62,63]. During the pre-viral entry into host cells, AgNPs competitively attach to the cells and lyse the membrane of the virions (antiviral activity). In the case of the post-viral entry, AgNPs mainly inhibit the viral fusion with the cell membrane, and in several cases interfered with the stages of the viral replication cycle such as the synthesis of viral RNA (antiviral activity). At the molecular level, these mechanisms relied on the chemical interaction of AgNPs or Ag+ ions released by AgNPs with sulfur, nitrogen, or phosphorus-containing biomolecules including proteins and genetic material. Hence, AgNPs have multiple mechanisms of action, which suggests that resistance to AgNPs will be less likely to arise when compared to specific antiviral or antibiotic therapies [64,65,66].

The World Health Organization (WHO) has published a list of high-priority (first tier), antibiotic-resistant pathogens that present the greatest threat to human health. These include strains in the Acinetobacter, Pseudomonas, and various Enterobacteriaceae genera (Klebsiella, Escherichia coli (E. coli), Serratia, and Proteus) [67]. Most of these pathogens are Gram-negative strains that exhibit increased resistance when compared to the Gram-positive strains. Gram-negative bacteria have an outer membrane that contains lipopolysaccharide (LPS), which creates a permeability barrier against external, harmful factors [68]. For example, Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative species that nanosilver-based products are commonly tested against, is listed as Priority 1 because the organism is CRITICAL due to its resistance to carbapenem antibiotics that are used as &#;last line&#; or &#;last resort&#; antibiotics [67,69]. Four of the six multi-drug-resistant (MDR) pathogens that are primarily responsible for infections originating from hospitalization are also Gram-negative bacteria, labeled as ESKAPE pathogens (Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), P. aeruginosa, and Enterobacter species) [70]. Due to the imminent threat posed by Gram-negative and Gram-positive bacterial examples, this review mainly focuses on the potential use of AgNPs in the fight against antibiotic resistance, in these organisms.

5. Antibacterial Mechanisms of AgNPs

Numerous studies report significant cell membrane and DNA damage by nearly all types of AgNPs and Ag+ ions, in both bacterial models [149,150,151]. Examples include but are not limited to GNB such as P. aeruginosa [151], K. pneumoniae [152], V. vulnificus [86], A. baumannii [153], E. coli [151], and Enterobacter species, and GPB such as E. faecium [154], S. aureus [155], S. pneumoniae [156], and B. brevis [157]. As illustrated below, the antibacterial mechanisms of engineered AgNPs are multifaceted and intertwined. This is because they are governed by both the different cellular structures of GNB and GPB, and the PCC properties of AgNPs (e.g., size, aggregation, surface charge, surface area, and surface-to-volume ratio) [155].

5.1. Cell Membrane Damage

The first, and often viewed as the most important, interaction between AgNPs and bacteria involves the plasma membrane and the components outside of this specialized structure ( ). These interactions can cause physical damage through direct membrane contact, depolarization, altered permeability, osmotic collapse, leakage of K+ ions and other intracellular contents, and halted cellular respiration [151,155,158]. In turn, the membrane damage can facilitate additional entry of AgNPs and other cytotoxic, extracellular compounds such as antibiotics that were previously unable to pass through or were ejected by the semipermeable membrane [64].

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GNB-AgNPs: The cell wall of a GNB cell is arranged in four layers: the outer membrane, the thin peptidoglycan layer, the periplasmic space, and the plasma membrane [160].

The outer membrane possesses proteins, lipids, and LPSs (lipopolysaccharides), where AgNPs and Ag+ ions initially interact with the bacteria [161]. For example, the negative charge of LPS in GNB has a strong attraction to positively charged AgNPs due to the large polysaccharide component [91,150]. Both GNB and GPB have an overall negative outer surface charge; the peptidoglycan components of carboxyl derivatives and phosphate groups in the GPB cell envelope are responsible for this charge [162]. Positively charged AgNPs (e.g., (NH2)-functionalized AgNPs synthesized with ethyleneimine) were found to exhibit a higher attraction to the bacterial cell surfaces than their negatively charged counterparts (e.g., citrate-capped AgNPs) [163]. Generally, neutral and negatively charged AgNPs have showed diminished antibacterial efficacy, with negative AgNPs being the least effective [162,164]. Nevertheless, negatively charged AgNPs can overcome this electrostatic barrier and thereby exhibit antibacterial efficacy. This was related to the formation of a protein corona around AgNPs or the charge reversal of AgNPs caused by the change in surrounding conditions [165,166,167,168]. For example, lowering the pH to acidic changed the charge of AgNPs from negative to positive [165]. This phenomenon potentially allows specific targeting of AgNP therapeutics to wound infection sites that are typically acidic. PCC properties of AgNPs such as size, surface charge, and hydrophobicity were reported to determine the type of protein corona formed around them, within a biological matrix [169]. Once formed, the protein corona improves the stability of AgNPs, promotes their cellular uptake, and generally prevents their aggregation [170,171]. Larger or unstable AgNPs that are prone to aggregation into larger AgNP clusters (&#;100 nm) can exhibit reduced antimicrobial efficacy [64]. This has been demonstrated by contrasting the MIC values of citrate-capped AgNPs of 5 nm and 100 nm in E. coli strains (20and 110 µg mL&#;1, respectively) [64,172]. Like charge, the size of AgNPs is known to greatly influence their antibacterial activity in both GNB and GPB. It is generally accepted that AgNPs of smaller size (&#;10 nm in diameter) have enhanced antibacterial activity when compared to larger AgNPs [61,172]. This was attributed to the larger nanosurface area that is available for direct contact with the bacterial cell, and the increased membrane permeability for smaller AgNPs [61,64]. Overall, the structural damage or alterations of the membrane caused by AgNPs provide a gateway for the other layers to undergo further interactions with Ag. The severity of these interactions depends on the depth and composition of the membrane layer as well as on their PCC properties [159].

The peptidoglycan layer is the second component of the cell envelope but makes up only a small fraction in GNB (i.e., 5&#;10%) [76,173]. In E. coli, the most widely used GNB model, the glycan strands consist of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β-1&#;4 bonds [174]. This structure has many carboxyl groups, giving peptidoglycan a negative charge [159]. This opposition in charges results in a strong attraction to AgNPs+ or Ag+, which then adhere to the cell membrane and disrupt the cellular transport of vital molecules, the membrane potential, and the osmotic equilibrium [159,175]. It is possible that AgNPs may only attach to the outer membrane, but when AgNPs penetrate the membrane, vital intracellular processes are modified (e.g., ATP production, DNA replication, and gene expression) [159,176].

The periplasmic space is what divides the inner membrane from the peptidoglycan layer. Functions of the periplasmic space include cell division regulation, sequestration of enzymes that could be toxic in the cytoplasm, signaling, protein folding, protein oxidation, and protein transport [177]. There are two mechanisms present in the periplasm: catalyzation of thiol oxidation and reduction of disulfides. These pathways dispel electrons after oxidation or translocate the reducing power from the cytoplasm [178]. Thioredoxin and glutaredoxin systems play an essential role in bacteria to upkeep disulfide bonds in their reduced state in cytoplasmic proteins. AgNPs (positively or negatively charged) and Ag+ ions penetrating the periplasm have a very high affinity for these electron-rich groups (especially cis) and therefore, they can interrupt these enzymes and pathways [151,178,179].

The inner membrane is the final separation of the cytoplasm and intracellular parts from the environment in GNB. It is represented by a symmetric bilayer composed of glycerophospholipids [177]. Studies have shown that the inner membrane could be affected without damage to the outer membrane. The inner membrane is rich in ions, so leakage of these materials has been utilized to track membranolytic activity [180]. In E. coli and P. aeruginosa, depolarization of the inner membrane was noticed upon interactions with AgNPs [41]. Furthermore, K+ ions from the Na+ K+ ATPase pump, which helps in maintaining osmotic equilibrium and membrane potential, have been shown to leak from the inner membrane [151,175].

GPB-AgNPs: Like GNB, GPB plasma membranes also exhibit an overall negative charge [162]. In contrast to the GNB, the GPB models have a very thick peptidoglycan layer of 20&#;80 nm, which makes up approximately 90% of the cell wall [76,173]. Thus, most substances including AgNPs and Ag+ ions might pass with more difficulty through the peptidoglycan layers of GPB or become stuck onto the surface of the cell wall [159,181]. However, AgNPs&#; contact with the peptidoglycan layer was associated with reactive oxygen species (ROS) release and the subsequent breakdown of the glycan backbone or other components (e.g., lipoteichoic acid) [182]. Furthermore, the attachment of positively charged AgNPs was found to be enhanced by the larger, negatively charged peptidoglycan [159]. Thus, positively charged AgNPs were reported to be more efficient in killing GPB than negatively charged AgNPs [162]. This is despite the lower susceptibility of GPB when compared to GNB [162].

5.2. Cell DNA Damage

GNB and GPB models: DNA damage is possible through multiple mechanisms that affect the integrity of its structure ( ) [183]. Like the plasma membrane, DNA is negatively charged in both bacterial models. This is mostly due to the sugar-phosphate backbone containing a negative phosphate group (PO43&#;) in each nucleotide [184]. This results in similar electrostatic attractions as also observed in the peptidoglycan layers [159]. These AgNP-DNA interactions ( ) lead to DNA denaturation, DNA breaks, mutations in DNA repair genes (mutY, mutS, mutM, mutT, and nth), and interference with cell division [185]. Ag+ ions disrupt the double-helical structure of DNA by distorting the hydrogen bonds intercalating between base pairs [186]. Another contributor to DNA damage is the presence of ROS. AgNPs and Ag+ ions are foreign bodies in bacterial cells, so host-induced ROS generation will put the cell under oxidative stress and lead to apoptosis [187]. Studies of engineered AgNPs reported that smaller AgNPs have higher antibacterial efficacy and faster ROS production (e.g., 5 min for AgNPs of 1 nm in diameter versus 60 min for AgNPs of 70 nm in diameter) [188]. Under aerobic conditions, smaller AgNPs have been correlated with increased Ag+ ion release when compared to bulk Ag [162]. This is probably due to the larger surface-to-volume ratios characteristic of smaller AgNPs, providing a larger surface footprint for the interaction with bacteria and the subsequent Ag+ release [140]. For example, AgNPs of 30 nm in diameter or larger have 15&#;20% of its atoms on the surface, as opposed to AgNPs of 10 nm in diameter, possessing 35&#;40% of its atoms on the surface [189]. Overall, the oxygen radicals associated with the exposure to AgNPs and Ag+ ions kill pathogens through oxidative damage to amino acids, leading to DNA denaturation [190].

5.3. Collateral Cell Damage

GNB and GPB models: ROS activates other mechanisms ( ) that include autophagy, neutrophil extracellular trap formation, and the triggering of pattern recognition receptors (PRRs) [190]. The oxidation of amino acids from ROS radicals consequentially results in the alteration of protein structure that jeopardizes their function. These changes can alter the protein structures, solubility, conformation, vulnerability to proteolysis, and enzymatic activity [191]. Therefore, bacterial enzymes and ribosomes are susceptible to alteration and/or denaturation as all are composed of various proteins. Bacterial ribosomes (70S) are made of ribosomal RNA and proteins, and the 70S unit is represented by a 50S and a 30S unit bonded together [192]. Ag+ ions are known to bind to the smaller 30S ribosomal unit that ends protein synthesis by shutting down the complex [192]. The resulting immature precursor protein buildup from AgNPs and Ag+ interacting with ribosomes and gene expression can lead to cell death [158].

6. Antibiotics against Bacteria

As is the case with all living organisms, bacteria compete for space, nutrients, and environments that are conducive to their existence and propagation. Antibiotic production is a natural mechanism used by microbes, including bacteria, to inhibit or kill other microbial competitors present in their environment [193]. Antibiotics are generally classified according to how they interfere with bacterial cellular growth and essential molecular processes ( ) [194].

Inhibition of cell wall synthesis: The majority of globally produced antibiotics in use today are those that target and disrupt the bacterial cell wall. Both GPB and GNB contain layers of peptidoglycan as a constituent of the cell wall structure [195]. Each peptidoglycan layer is cross-linked to the next enveloping layer by a process called transpeptidation [195]. During bacterial growth, transpeptidases catalyze this cross-linking, resulting in a relatively strong and stable wall structure [196,197]. The β-lactam class of antibiotics (e.g., penicillin, cephalosporin, carbapenem, monobactam, and their derivatives) are so named because they all share a characteristic as part of their molecular structure&#;a β-lactam ring [196]. The β-lactam antibiotics bind to and inactivate the bacterial transpeptidases during new cell wall synthesis, causing loss of the cell wall entirety [198,199]. Vancomycin, a non β-lactam antibiotic, also disrupts the cell wall structure. Vancomycin belongs to a group of glycopeptide antibiotics that target the bacterial cell wall by inhibiting the synthesis of penta-peptidoglycan precursor molecules [200]. Bacitracin, a broad-spectrum cyclopeptide antibiotic, interferes with the translocation of peptidoglycan precursors across the cell membrane, so they are unable to reach, or add to, the structure of the growing cell wall [201,202,203]. In all these cases, a weaker wall results in cell death [196].

Figure 10

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Cell membrane disruption: Two main groups of antibiotics can disrupt bacterial cell membranes. Polymyxins (polymyxin B and E) bind to the outer LPS membrane surface of GNB [205]. The result is an ionic imbalance across the membrane, which then becomes porous and eventually collapses [205]. This then facilitates further antibiotic entry and similar damage to the inner cytoplasmic membrane [205]. Cyclic lipopeptide antibiotics, such as daptomycin, disrupt the cytoplasmic membranes of GPB. As daptomycin binds, depolarization of the membrane occurs, resulting in a porous &#;leaky&#; barrier [206]. Membrane damage in either of these scenarios is irreparable, and so the bacterial cell dies.

Inhibition of protein synthesis: The aminoglycosides (streptomycin, gentamycin, kanamycin, and their derivatives) inhibit protein synthesis by interfering with bacterial ribosome function [207]. Specifically, aminoglycosides bind to the bacterial 30S (small) ribosomal subunit and cause blocking and/or misreads during translation [207]. The tetracycline group of antibiotics, such as tetracycline and doxycycline, also act via the 30S subunit, inhibiting tRNA translocation activity, and/or polypeptide chain elongation processes [204,208]. The macrolides (erythromycin, clarithromycin, and azithromycin), lincosamides (lincomycin), and streptogammin B are collectively termed the MLSB group of antibiotics [209]. Despite different molecular structures and origins, all MLSBs interact with the bacterial 50S (large) ribosomal subunit to specifically cause tRNAs, that are in the process of translocation, to prematurely drop-off the ribosome [209]. Ultimately, a drop in protein synthesis occurs that can be lethal for a growing bacterial cell [204].

Disruption of nucleic acid synthesis and function: Bacteria use a class of enzymes, known collectively as type II topoisomerases, to supercoil and effectively compact their DNA within the cellular space [210]. These enzymes also play an important role during DNA replication by removing supercoils upstream of the replication machinery. Once DNA synthesis is complete, the new bacterial chromosome is separated from the original and directed toward the new daughter cell. The topoisomerase that is vital for regulating the supercoiling process is DNA gyrase (topoisomerase II), while topoisomerase IV is the enzyme necessary at the end of DNA replication, since its role is to unlink newly synthesized DNA from the original [210]. The quinolone antibiotics (such as ciprofloxacin) disrupt these processes by inhibiting the DNA gyrase function in GNB and by targeting topoisomerase IV in GPB [210]. Attenuation of DNA unwinding, supercoiling, and processes imperative to its replication will ultimately lead to the cell&#;s demise [210].

RNA synthesis can also be interrupted by the action of antibiotics. Rifamycins (rifamycin B, SV, and derivatives) all have a characteristic macrocyclic ring structure that targets the DNA-dependent RNA polymerase enzyme (RNAP) [211]. Binding of rifamycin to the β-subunit of the RNAP stalls the transcription of DNA to RNA, which results in a significant decrease in protein production and leads to cell death [211].

Disruption of metabolic pathways: In addition to naturally derived antibiotics, a number of synthetic antibiotics have been developed to limit growth (bacteriostatic) or destroy (bactericidal) bacterial cells. Some, such as the sulfonamides and trimethoprim, are growth factor analogs that interrupt pathways involved in bacterial metabolism [212,213]. The sulfonamides are structural analogs of para-amino benzoic acid (PABA)&#;a vital substrate required by bacterial cells to synthesize folic acid [214]. Folic acid itself is an important vitamin used by cells to create nucleic acids. Typically, PABA forms a complex with an enzyme (dihyropteroate synthase), which then converts PABA to the folic acid precursor dihydopteric acid [214,215]. Because sulfonamide is an analog of PABA, it will also bind to this crucial enzyme, effectively outcompeting PABA and inhibiting the enzymatic production of dihydopteric acid [214,215]. Trimethoprim is a structural analog of a subsequent enzyme (dyhydrofolate reductase) in this pathway. Trimethoprim outcompetes the binding of dihydopteric acid to this secondary enzyme, thus inhibiting its activity and limiting the production of additional metabolites required for folic acid synthesis [214,215]. The synergistic effects of sulfonamides and trimethoprim cause folic acid levels to drop, which in turn inhibits nucleic acid synthesis and bacterial cell growth [214,215].

8. Synergistic Effects of AgNP-Antibiotic Conjugates on Antibiotic-Resistant Bacteria

Synergy is defined as the phenomenon that combines two or more compounds, leading to a response with more potency than an individual compound can exert alone [242]. Currently, extensive efforts have been dedicated to exploiting the synergistic effects of core AgNPs functionalized with antibiotics (denoted AgNP&#;antibiotic conjugates) in preventing antibiotic-resistant and non-resistant pathogens from spreading or multiplying [243,244,245]. This section will focus on summarizing the advances and challenges encountered in the fabrication, characterization, and evaluation of the synergistic effects of AgNP&#;antibiotic conjugates on bacterial growth. Within this context, illustrative examples are presented for both antibiotic-resistant and non-resistant bacterial strains with respect to the type of AgNP&#;antibiotic conjugates used, and their PCC properties.

Strategies to conjugate antibiotics to AgNPs: Core AgNPs are fabricated chemically through the reduction of Ag+ from a silver salt with chemical reagents (e.g., citrate or sodium borohydride) or biogenic reagents (e.g., bacterial, fungal, or plant extracts). Antibiotics are then conjugated to AgNPs via one of the following methods: (i) conjugation of antibiotics after the AgNP synthesis or (ii) conjugation of antibiotics during the AgNP synthesis. Both methods can use the antibiotic as either a reducing agent, a functionalization agent, or both [244]. Each of these strategies have been reported to produce a wealth of AgNP&#;antibiotic conjugates with synergistic effects against both non-resistant and antibiotic-resistant pathogens [244]. For example, AgNP&#;gentamycin conjugates capped with PVP were shown to be potent antibacterial agents against S. aureus, E. coli, and gentamycin-resistant E. coli [246]. The mechanism of synergy was attributed to a multistep process: gentamycin, a neutral aminoglycoside, lowers the negative charge of AgNPs, and thereby promotes the membrane&#;AgNP interaction and release of Ag+ ions at the site of membrane attachment. Additionally, complex nanostructures such as cyanographene Ag nanohybrids (GCN/AG) conjugated with gentamicin reduced the original MIC of gentamicin by 32-fold and had an average fractional inhibitory concentration (FIC) of 0.39 [247]. Partial synergy was also found for GCN/Ag conjugated with ceftazidime against E. coli. Several studies address the significance of AgNP size, shape, and surface charge on synergy. These include demonstrations that positively charged AgNPs (e.g., amine-capped AgNPs) have a stronger inhibitory effect against bacteria than negatively charged AgNPs (e.g., citrate-capped AgNPs), while both AgNPs have the same nanocore (sodium borohydride reducing agent) and are conjugated with the same antibiotic (e.g., vancomycin; Van-AgNPs) [248,249]. The reported MIC values against S aureus were 5.7 fmol mL&#;1 for positively charged AgNPs (+50 mV), 4 nmol mL&#;1 for neutral AgNPs (0 mV), and 97 nmol mL&#;1 for negatively charged AgNPs (&#;38 mV). AgNP&#;ampicillin conjugates that were synthesized using ampicillin as a reducing agent had significantly reduced MIC values (3&#;28 µg mL&#;1) when compared to ampicillin (12&#;720 µg mL&#;1) or AgNPs alone (280&#;640 µg mL&#;1). Antibiotic-resistant (E. coli and S. aureus) and MDR (P. aeruginosa and K. pneumonia) bacterial strains were susceptible to ampicillin but did not develop resistance to the AgNP&#;ampicillin conjugates even after 15 growth cycles [250].

Characterization of AgNP&#;antibiotic conjugates: The two components (AgNPs and antibiotics) must be chemically conjugated to exhibit synergistic effects. The AgNP&#;antibiotic interactions (e.g., the surface functionalization) and other PCC properties are typically characterized following the U.S. EPA standards in conjunction with the techniques described in . For example, the UV-Vis absorption spectrophotometry analysis of AgNP&#;vancomycin conjugates exhibited a consistent red shift in the characteristic localized surface plasmon resonance (SPR) peak of the citrate-capped AgNPs (392 nm) upon binding to antibiotics [251]. This AgNP&#;antibiotic conjugate demonstrated synergistic antibacterial potential, rather than additive effects, against GPB (S. aureus) and GNB (E. coli). FT-IR measurements of citrate-capped AgNPs synthesized using Bacillus sp. SJ14 showed peaks characteristic to the bending and stretching motions of primary amines at cm&#;1 and cm&#;1, respectively [252]. The spectroscopic measurements helped characterize the surface chemistry and stability of AgNPs when attached to microbial sourced proteins [252]. The subsequent conjugation of these AgNPs to antibiotics (i.e., ciprofloxacin, methicillin, and gentamicin) was confirmed through the broadening of the localized SPR absorption peaks at 420 nm, and the significant Raman shifts in the marker amine vibrational peaks (e.g., from cm&#;1 to cm&#;1) [252]. All AgNP&#;antibiotic conjugates showed synergy; most notably, the MIC of methicillin was reduced from 250 μg mL&#;1 to 7.8 μg mL&#;1 against an MDR-biofilm-forming coagulase-negative S. epidermidis. Raman spectroscopy can be utilized as a complementary tool to IR spectroscopy to characterize the change in the nanosurface chemistry upon the AgNP-antibiotic chelation. For example, the UV-Vis absorption and Raman spectra of four classes of antibiotics, β-lactam (ampicillin and penicillin), quinolone (enoxacin), aminoglycoside (kanamycin and neomycin), and polypeptide (tetracycline), could be collected with minimum to no sample preparation, before and after complexation with citrate-capped AgNPs [253]. Both analytical techniques confirmed the interaction between AgNPs and antibiotics, after the replacement of the citrate coating with antibiotics. All AgNP&#;antibiotic conjugates showed synergistic growth inhibition against MDR Salmonella Typhimurium DT 104, except for ampicillin and penicillin [253]. Specifically, no SERS enhancements were observed when AgNPs were combined with ampicillin and penicillin at any test concentrations (i.e., minimal to no AgNP&#;antibiotic interaction for these antibiotics) [253]. In contrast, distinct Raman marker bands were observed for all other antibiotics complexed to the nanosurface. For example, kanamycin was identified through vibrational modes at 270 cm&#;1 (Ag-O stretching), 620 cm&#;1, and 890 cm&#;1 (skeletal deformation and stretching of the tetrahydropyran rings) [253]. AgNPs alone reduced the bacterial growth of MDR Salmonella by 10%. Furthermore, tetracycline enhanced the binding of AgNPs to Salmonella by 21% and the Ag+ release by 26%, when compared to AgNPs alone. This Raman study further confirmed the relationship between the synergistic, antibacterial effects and the necessity of prior AgNP-antibiotic binding.

Quantifying growth inhibition and synergy of AgNP&#;antibiotic conjugates: Three basic procedures are typically utilized to study growth inhibition. One of the oldest methods to determine growth inhibition is the Kirby&#;Bauer (disk diffusion) test that maintains its popularity because it requires small volumes of sample (10&#;20 μL), no specialized equipment, and gives a quick turnaround [245,254]. In these assays, 6 mm filter disks are soaked with antimicrobials and placed on an agar plate coated with bacteria at 108 CFU mL&#;1. Following overnight growth, the antibiotic concentration that produces a distinctive halo (mm diameter) around the disc is considered the zone of inhibition (CLSI protocol). This varies according to the antibiotic used and bacteria being tested. In the solution-based growth inhibition assay, bacterial cultures at 105 CFU mL&#;1 are mixed with antimicrobials and are grown for 20&#;24 h [245]. The cell growth is then assessed by monitoring the optical density (OD) at 600 nm. The antibiotic concentration corresponding to OD values of ~50% below that of the untreated cells is considered the MIC. This type of assay also has a relatively fast turnaround, but it must be supplemented with colony counting to establish that the OD 600 values correspond to viable cell counts. The growth inhibition assay based on colony counting is perhaps the most labor-intense of the three methods. It is a solution-based growth inhibition, in which cells at 105 CFU mL&#;1 are grown for 2 h with antimicrobials, then plated [245]. Viable colonies are then counted after 24 h of growth.

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To establish a common standard for synergy, a FIC can be calculated by dividing the MIC of the AgNP&#;antibiotic conjugate with the MIC of antibiotic alone (Equation (1)). An FIC value of 0.5 is considered synergistic [243].

FIC=MIC of AgNP&#;antibiotic conjugateMIC of antibiotic alone

(1)

Selective examples of synergy against resistant and non-resistant bacterial strains: There is an extensive collection of evidence highlighting the potency of AgNP&#;antibiotic conjugates against both GPB and GNB [243,244,255,256]. collates available data listing the method used for AgNP synthesis (biological versus chemical), the properties of AgNPs (i.e., size, charge, and shape, if available), and the antibiotic&#;bacteria pairs with demonstrated synergy.

AgNP&#;antibiotic synergy with potential for clinical applications on MDR pathogens: A recently published seminal study used A. baumannii to produce biogenic AgNPs and test their antibacterial efficacy on carbapenemase-producing Gram-negative bacteria (CPGB) alone and conjugated to various antibiotics [257]. The study found potent antimicrobial activity against CPGB, with MICs ranging from 64 to 8 μg mL&#;1. Among the conjugates, AgNP-ceftriaxone showed the highest synergistic effect with the MIC lowered by 250-fold (from μg mL&#;1 to 4 μg mL&#;1) against A. baumannii. This result is significant because carbapenems are considered the &#;last resort&#; option, while β-lactams are often the go-to first line of treatment against severe bacterial infections. Hence, developing new treatment options against carbapenem-resistant bacteria is imperative. Post-surgical ointments: Biogenic AgNPs were produced by reduction of Ag+ with extracts of the fungus Fusarium oxysporum. The potency of these AgNPs was enhanced with waxes and natural oils and used as a post-surgical ointment on goats that were infected with Caseous lymphadenitis. The goats receiving treatment healed faster and had fewer wound infections compared to the control group [258]. AgNP coating prevents biofilm formation: Biofilm formation on surgical implants is a major cause of post-surgical infections. AgNPs that were coated with polydopamine, chitosan, and hydroxyapatite on titanium implants resulted in 90&#;92% of antibiofilm efficiency against S. aureus, S. epidermidis, and E coli [259].

Table 5

Fabrication Method and
Reducing AgentAgNP Size and
CoatingSynergy, Bacterial Model, and Method of EvaluationBiological
Bacillus sp. [260]14&#;42 nm
primary and aromatic aminesZoI; all combinations of fusidic acid, gentamycin, ciprofloxacin, erythromycin, penicillin, chloramphenicol, levofloxacin, nalidixic acid, and ampicillin against S. epidermidis, S. aureus, V. cholerae, S. aureus, Salmonella Typhi, and Salmonella ParatyphiChemical [261]10&#;30 nm
nanosilver colloidMIC; allicin against MRSABiological
Trichoderma viride
Aspergillus flavus [262,263]5&#;40 nmZoI; ampicillin, kanamycin, erythromycin, and chloramphenicol against E. coli, S. Typhi, S. aureus, Micrococcus luteus, P. aeruginosa, E. faecalis, A. baumanii, K. pneumoniae, and Bacillus spp.Biological
Phytophthora
Infestans [264]5&#;30 nmZoI, MIC, mupirocin, neomycin, vancomycin against S aureus; cefazolin, mupirocin, gentamycin, vancomycin against P. aeruginosa; and cefazolin, mupirocin, gentamycin, neomycin, tetracycline against E. coli.Chemical
Maltose [265]28 nm and 8 nmMIC; amoxycillin, colistin, and gentamycin against A. pleuropneumoniae, and P. multocida. Penicillin G against A. pleuropneumoniaeBiological
Mukia
Maderaspatana [266]N. A.ZoI; biofilm microplate; cefriaxone with B. subtilis, K. pneumoniae, S. aureus, S. Typhi, and Pseudomonas fluorescensChemical
ascorbic acid [267]20 nmMIC; amoxicillin against E. coliBiological
E. hermannii, C. sedlakii, and
P. putida [268]4&#;12 nmZoI; gentamicin against P. aeruginosa and vancomycin against S. aureus and MRSAChemical
solid silver [269]N. A.MIC, FIC, biofilm, and hydroxyl radical assay; E. faecium, S. mutans, and E. Coli with ampicillin; E. faecium and P. aeruginosa with chloramphenicol; S. aureus, S. mutans, E. coli, and P. aeruginosa with kanamycinBiological
K. pneumoniae [270]50 nmZoI; penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin against S. aureus and E. coliBiological
Dioscorea bulbifera [271]8&#;20 nmZoI; chloramphenicol and vancomycin against P. aeruginosa, streptomycin with E. coliChemical
sodium citrate and garlic [272]-
citrate-coatedZoI; S. Typhi, E. coli, P. aeruginosa, M. luteus, S. aureus with amoxclav and S. Typhi with ampicillinChemical [273]3.0 nmFI; E. faecium, ampicillin and chloramphenicol; S. mutans, ampicillin and kanamycin; E. coli, ampicillin and kanamycin; P. aeruginosa, chloramphenicol and kanamycinChemical
NaBH4/citrate [274]5.0&#;12.0 nm
citrate-coatedA. baumannii with polymyxin B and rifampicinChemical
NaBH4/maltose [265]8.0 nm
gelatin-coatedFIC; A. pleuropneumoniae, penicillin G; E. coli, colistin; S. aureus, gentamicinChemical
Gallic acid [275]8.6 nm
Gallic-acid-coatedFIC; E. faecium, A. baumannii, K. pneumoniae, Morganella morganii, and P. aeruginosa, ampicillin and amikacin; S. aureus, E. coli, and Enterobacter cloacae, amikacin (FIC)Chemical
NaBH4/citrate/hydrazine [276]10 nm
PVP- and
citrate-coatedZoI; S. aureus, cephalexinChemical
NaBH4/citrate [277]16 nm
PVP-coatedZoI; E. coli, streptomycin, ampicillin, and tetracycline; S. aureus, streptomycin, ampicillin, and tetracyclineChemical
NaBH4/citrate [278]19.3 nm
SDS-coatedZoI; E. coli, streptomycin, ampicillin, and tetracycline; S. aureus, streptomycin, ampicillin, and tetracyclineChemical
ascorbic acid [279]20.0 nmMIC; E. coli, amoxicillinChemical
NaBH4 [280]20.0 nm
PVP-coatedZoI; all combinations of vancomycin and amikacin and S. aureus and E. coliChemical
Tween 80 [281]20.0&#;40.0 nm
Tween 80-coatedFIC; S. epidermidis and gentamicinChemical
citrate [246]23.0 nm
citrate-coatedMIC and inhibition (plate counting); S. Typhimurium, tetracycline, neomycin, and penicillin GChemical
ethylene glycol [251]25.0 nm
PVP-coatedFIC; E. coli and S. aureus, gentamicinChemical
Maltose [282]26.0 nm
gelatinMIC; E. coli, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefoxitin, cefuroxime, cotrimoxazole, colistin, gentamicin, ofloxacin, oxolinic acid, and tetracycline; P. aeruginosa, amikacin, aztreonam, cefepime, cefoperazone, ceftazidime, ciprofloxacin, colistin, gentamicin, meropenem, ofloxacin, piperacillin, and piperacillin/tazobactam; S. aureus, ampicillin/sulbactam, chloramphenicol, ciprofloxacin, clindamycin, cotrimoxazole, erythromycin, gentamicin, oxacillin, penicillin, teicoplanin, tetracycline, and vancomycinChemical
NaBH4/maltose [265]28.0 nm
gelatinAmoxycillin, penicillin G, gentamicin, and colistinChemical
Maltose [283]28.0 nm
MaltoseFIC; E. coli and K. pneumoniae with cefotaxime, ceftazidime, meropenem, ciprofloxacin, and gentamicin; synergism in all resistant strains except to K. pneumonia carbapenemaseChemical
Citrate [253]29.8 nm
citrate-coatedAmpicillin, penicillin, enoxacin, kanamycin, neomycin, and tetracycline, and S. TyphimuriumChemical
Citrate [284]29.8 nm
citrate-coatedInhibition (plate counting); S. Typhimurium, enoxacin, kanamycin, neomycin, and tetracyclineChemical
NaBH4/citrate [271]38.3 nm
citrate-coatedZoI; E. coli, streptomycin, ampicillin, and tetracycline; S. aureus, streptomycin, ampicillin, and tetracyclineChemical
Citrate [251]70.0 nm
Citrate-coatedZoI; vancomycin and S. aureus and E. coliBiological
Streptomyces cali- diresistants IF17 strain [284]5.0&#;20.0 nm
biomolecules from actinobacterial strainsFIC: E. coli, tetracycline; S. aureus, ampicillin, kanamycin, and tetracycline; B. subtilis, ampicillin, kanamycin, and tetracyclineBiological
Klebsiella pneumoniae extract
[271]5.0&#;32.0 nm
proteins from biomassZoI; E. coli, amoxicillin, erythromycin, penicillin, and vancomycin; S. aureus, amoxicillin, erythromycin, penicillin, and vancomycinBiological
Streptomyces calidiresistants IF11
[284]5.0&#;50.0 nm
biomolecules from reducing strainsFIC; B. subtilis, kanamycinBiological
Actinomycetes strains [277]17.0 nm
proteins from biomassMIC and ZoI; E. coli, K. pneumoniae, and P. aeruginosa, ampicillinBiological
Klebsiella pneumoniae [285]20.0 nm
-ZoI; E. faecalis, chloramphenicol and gentamicinBiological
silver-resistant estuarine
P. aeruginosa strain
[286]35.0&#;60.0 nm
biomolecules from reducing strainsZoI; all combinations of ampicillin and ciprofloxacin with resistant S. aureus strain VN3 and ciprofloxacin-resistant V. cholera strain VN1Biological
Trichoderma viride
[287]5.0&#;40.0 nm
proteins from biomassZoI; E. coli, ampicillin, chloramphenicol, erythromycin, and kanamycin; M. luteus, ampicillin, chloramphenicol, and kanamycin; S. Typhi, ampicillin, chloramphenicol, erythromycin, and kanamycin; S. aureus, ampicillin, chloramphenicol, erythromycin, and kanamycinBiological
Acinetobacter calcoaceticus [288]8.0&#;12.0 nm
biomolecules from reducing strainsZoI or MIC; A. baumannii, amikacin, amoxicillin, ampicillin, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, tetracycline, trimethoprim, and vancomycin; Klebsiella (previously known as Enterobacter) aerogenes, amikacin, amoxicillin, ampicillin, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycin; E. coli, amikacin, amoxicillin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycin; P. aeruginosa, amikacin, amoxicillin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycin; S. Typhimurium, amikacin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycin; Shigella sonnei, amikacin, amoxicillin, ampicillin, ceftazidime, ceftriaxone, ciprofloxacin, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, tetracycline, trimethoprim, and vancomycin; S. aureus, amikacin, amoxicillin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, gentamicin, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycin; S. mutans, amikacin, amoxicillin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, kanamycin, penicillin, tetracycline, trimethoprim, and vancomycinBiological
Cryphonectria sp.
[289]30&#;70 nmZoI; S. aureus, S. Typhi, and E. coli, streptomycinBiological
Emericella nidulans
[290]66.7 nm
biomolecules from biomassFIC; E. coli, amikacin and streptomycinBiological
Aspergillus flavus
[290]81.1 nm
biomolecules from biomassFIC; E. coli, amikacin and streptomycin; S. aureus, kanamycin, oxytetracycline, and streptomycinBiological
Dioscorea bulbfera
[272]2.0 nm
biomolecules from biomassZoI; all combinations of treptomycin, rifampicin, chloramphenicol, novobiocin, and ampicillin in E. coli, P. aeruginosa, and S. aureusBiological
Dioscorea bulbifera
[291]5.0&#;30.0 nm
proteins from biomassZoI; A. baumannii, amoxicillin, ampicillin, cefotaxime, erythromycin, gentamycin, kanamycin, nalidixic acid, nitrofurantoin, penicillin, piperacillin, rifampicin, and rimethoprim; B. subtilis, ampicillin, cefotaxime, chloramphenicol, nalidixic acid, nitrofurantoin, penicillin, piperacillin, streptomycin, trimethoprim, and vancomycin; E. cloacae, amikacin, amoxicillin, erythromycin, nalidixic acid, and penicillin; E. coli, amikacin, erythromycin, kanamycin, nalidixic acid, polymyxin, streptomycin, and trimethoprim; Haemophilus influenzae, cefotaxime, ceftriaxone, nitrofurantoin, and trimethoprim; K. pneumoniae, amoxicillin, ampicillin, chloramphenicol, erythromycin, feropenem, nitrofurantoin, penicillin, rifampicin, trimethoprim, and vancomycin; Neisseria mucosa, amikacin, ampicillin, erythromycin, feropenem, gentamycin, nitrofurantoin, penicillin, polymyxin, tetracycline, trimethoprim, and vancomycin; Proteus mirabilis, erythromycin, nalidixic acid, and vancomycin; P. aeruginosa, amikacin, amoxicillin, ampicillin, chloramphenicol, doxycycline, erythromycin, feropenem, gentamycin, kanamycin, nalidixic acid, nitrofurantoin, penicillin, streptomycin, trimethoprim, and vancomycin; S. Typhi, amikacin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, chloramphenicol, erythromycin, gentamycin, kanamycin, nalidixic acid, nitrofurantoin, penicillin, piperacillin, polymyxin, streptomycin, trimethoprim, and vancomycin; Serratia odorifera, ceftazidme, erythromycin, nalidixic acid, nitrofurantoin, trimethoprim, and vancomycin; S. aureus, amikacin, amoxicillin, ampicillin, ceftazidme, erythromycin, kanamycin, nalidixic acid, polymyxin, streptomycin, and trimethoprim; Vibrio parahemolyticus, ampicillin, cefotaxime, ceftriaxone, kanamycin, nalidixic acid, nitrofurantoin, polymyxin, and trimethoprimBiological
Argyreia nervosa [292]5.0&#;40.0 nm
biomolecules from biomassZoI; S. aureus, amoxicillin/clavulamic acid, ciprofloxacin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin; E. coli, amoxicillin/clavulamic acid, erythromycin, streptomycin, tetracycline, and vancomycinBiological
Gum kondagogu [293]5.8 nm
biomolecules from biomassFIC; S. aureus, gentamicin and streptomicin; S. aureus, streptomicin; E. coli, streptomicin; P. aeruginosa, streptomicinBiological
Rosa damascenes [294]7.4&#;18.3 nmZoI; cefotaxime with E. coli and MRSABiological
Ulva fasciata [295]15.0 nmZoI; E. coli, cefotaxime, cefuroxime, fosfomycin, chloramphenicol, azithromycin, and gentamicin; Salmonella enterica, azithromycin, gentamicin, oxacillin, cefotaxime, neomycin, ampicillin/sulbactam, cefuroxime, fosfomycin, chloramphenicol, and oxytetracycline; S. aureus, azithromycin, oxacillin, cefotaxime, neomycin, ampicillin/sulbactam, cefuroxime, fosfomycin, chloramphenicol, and oxytetracyclineBiological
Eurotium cristatum
[296]15.0&#;20.0 nm
biomolecules from biomassZoI; all combinations of vancomycin, oleandomycin, ceftazidime, rifampicin, penicillin G, neomycin, cephazolin, novobiocin, carbenicillin, lincomycin, tetracycline, and erythromycin, and Candida albicans, P. aeruginosa, and E. coliBiological
Urtica dioica Linn.
[297]20.0&#;30.0 nm
biomolecules from biomassZoI; B. cereus, streptomycin, amikacin, kanamycin, vancomycin, tetracycline, ampicillin, cefepime, amoxicillin, and cefotaxime; S. epidermidis, streptomycin, amikacin, kanamycin, tetracycline, ampicillin, cefepime, and amoxicillin; S. aureus, streptomycin, amikacin, kanamycin, vancomycin, tetracycline, cefepime, amoxicillin, and cefotaxime; B. subtilis, streptomycin, amikacin, kanamycin, vancomycin, tetracycline, ampicillin, cefepime, amoxicillin, and cefotaxime; E. coli, streptomycin, amikacin, vancomycin, tetracycline, ampicillin, cefepime, amoxicillin, and cefotaxime; S. Typhimurium, streptomycin, amikacin, kanamycin, vancomycin, tetracycline, ampicillin, cefepime, amoxicillin, and cefotaxime; K. pneumoniae, streptomycin, amikacin, kanamycin, vancomycin, tetracycline, ampicillin, cefepime, amoxicillin, and cefotaxime; Serratia marcescens, streptomycin, kanamycin, tetracycline, ampicillin, amoxicillin, and cefotaximeBiological
Zea may [298]45.3 nm
biomolecules from biomassZoI; B. cereus, E. coli, Listeria. monocytogenes, Salmonella Typhimurium, and S. aureus, kanamycin and rifampicinCommercial
N. A. [299]10.0&#;15.0 nm
N. A.Optical density; ampicillin, kanamycin, gentamycin, and clindamycin with A. baumanniiCommercial
N. A.
[300]15.2 nm
starchFIC; Burkholderia pseudomallei with meropenem and gentamicin sulfateCommercial
N. A. [301]35.0 nm
PVPFIC; E. coli, Salmonella Typhimurium, and S. aureus, kanamycinOpen in a separate window

Overcoming bacterial antibiotic resistance using AgNP&#;antibiotic conjugates: AgNP-antibiotic conjugates are less likely to promote the emergence of bacterial antibiotic resistance because they use a multifaceted approach to attack bacteria. In contrast, chemical antibiotics target a specific process in the bacterial cell such as protein synthesis or DNA replication, making it easier for the bacteria to counter their action. summarizes how AgNP&#;antibiotic conjugates evade bacterial defenses.

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The inherent antibacterial properties of AgNPs promote antibiotic function by overcoming various drug resistance mechanisms.

AgNPs trigger the production of ROS that destabilize the bacterial membrane, allowing entry of antibiotics: AgNPs alone are potent weapons against MDR-resistant bacteria. AgNPs synthesized with extracts of Areca catechu exhibited potency against vancomycin-resistant E. faecalis (MIC of 11.25 µg mL&#;1) and MDR A. baumannii (MIC of 5.6 µg mL&#;1) [301]. AgNPs fabricated with Convolvulus fruticosus extracts were effective against MDR E. coli (17.1 µg mL&#;1), K. pneumoniae (4 µg mL&#;1), and P. aeruginosa (2 µg mL&#;1). Studies indicated that biogenic AgNPs accumulate at the surface of bacteria, leading to the concomitant release of Ag+ ions and production of ROS. AgNPs facilitate ROS release by competing with amino acids (especially cis) that coordinate Fe-S clusters in complexes of the electron transport chain [302]. While some ROS may be eliminated by bacteria, large amounts of ROS destabilize the bacterial cell membrane due to lipid peroxidation [303]. As a result, antibiotics that would otherwise be prevented from entering the cell are allowed to seep through the damaged membrane. Likewise, antibiotics that interfere with cell wall synthesis can provide an entryway for AgNPs. Ampicillin&#;AgNP synergy was found to be based on this mechanism, where ampicillin interfered with cell wall synthesis, facilitating a porous entryway for AgNPs [264].

AgNPs interfere with the action of efflux pumps: The efflux pump mechanism is one of the most common defenses against tetracycline antibiotics. Gold NPs were observed to downregulate efflux pump production using EtBr (a common efflux pump substrate) as well as the production of other membrane proteins that play a crucial role in membrane stability [304].

Evading enzymes that destroy antibiotics: One of the best-known resistance mechanisms is the enzymatic destruction of antibiotics through the action of β-lactamase (against ampicillin) and chloramphenicol acyltransferase. In this scenario, AgNPs carrying antibiotics act as a Trojan-horse delivery vehicle camouflaging their cargo [303]. Treatment of intracellular bacteria by antibiotic conjugates: Intracellular bacteria are species that produce infection by residing in mammalian cells, as is seen with the bacterial pathogen M. tuberculosis. Treatment of these conditions is challenging because most antimicrobials cannot easily enter mammalian cells [302]. In contrast, the mechanism used by mammalian cells to internalize NPs via receptor-mediated endocytosis is well established [302]. AgNPs chelated by antibiotics may provide an avenue for treating ailments caused by intracellular bacteria. One example is Ag/ZnNPs encapsulated in poly(lactic-co-glycolic acid) that delivered rifamycin to mammalian cells infected with M. tuberculosis [305]. These AgNP&#;antibiotic conjugates can employ multiple mechanisms to combat bacterial infections, making it difficult for bacteria to develop effective resistance.

Challenges and future directions: Even though AgNP&#;antibiotic conjugates have demonstrated potential in treating infections caused by MDR bacteria, there are few U.S. FDA-approved treatments that use AgNP&#;antibiotic conjugates [303]. To address this, improvements must be made in batch-to-batch reproducibility of AgNP during synthesis (especially biological), developing dose&#;response relationships, and advancing the use of organism models (in vitro and in vivo) to bridge the gap between promising potential and the establishment of AgNP&#;antibiotic conjugates as effective treatments. Specific targeting is expected to reduce dosage and limit toxicity: AgNP&#;drug complexes that do not specifically target their cargo can interact with non-target cells, while targeting strategies can lead to enhanced therapeutic effects. Two common strategies for targeting AgNP-antibiotic cargo are antibody and aptamer targeting. In the antibody method, AgNPs are covalently labeled with antibodies (e.g., AuNPs labeled with S. aureus antibodies) to build a construct that can selectively kill bacteria [305]. In the aptamer strategy, antibodies made of 20&#;80 nucleotide ssDNA or RNA (i.e., aptamers) are first developed in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) to target specific ligands such as small molecules, membrane proteins, peptidoglycans, and whole cells. The aptamers are then chemically attached at one end to the AgNPs through covalent bonds. The advantages of aptamers over protein antibodies are numerous and include the ease of chemical attachment to AgNPs, potential labeling with fluorophores for target detection, higher thermal stability, and bypassing of the immune system. For example, conjugated gold nanorods have been successfully targeted against MRSA&#;s surface [306]. Furthermore, silver nanoclusters (AgNC) bridged by DNA aptamers specific to S. aureus targeted its cargo six-times more effectively compared to AgNC containing scramble (non-specific) DNA sequences [306]. Overall, specific targeting of pathogens can reduce the therapeutic dosage of antibiotics and AgNPs.

9. Toxicity of AgNPs

While all bacteria are classified as prokaryotes, eukaryotes such as animals, plants, fungi, and humans are made up of cells that possess a membrane-bound nucleus containing genetic material and membrane-bound organelles [307]. Although AgNPs are recognized as efficient antibacterial agents, they can also be toxic to eukaryotic cells and larger organism models. The toxicity of AgNPs in eukaryotes remains a topic of debate due to the high complexity of multicellular organisms and the large variations among the PCC properties of AgNPs. Although a significant amount of toxicity work has been completed for both in vitro and in vivo eukaryotic models, no U.S. EPA National Primary Drinking Water Guidelines have been established yet for AgNPs [308]. National Primary Drinking Water Regulations are primary standards and treatment techniques for public water systems that are enforceable by law for the protection of public health. National Secondary Drinking Water Guidelines were determined for potential contaminants such as Ag+ ions, which are not considered a human health risk and are only tested for aesthetic considerations (e.g., taste, odor, and color), on a voluntary basis [309]. Human consumption of water containing Ag+ ions in amounts higher than the secondary maximum contaminant level (SMCL) of 0.1 mg L&#;1 was found to cause skin discoloration and graying of the white part of the eye [309]. According to the WHO, a dose of 10 g of silver nitrate (AgNO3) containing Ag+ ions can be lethal to humans, but 0.6&#;0.9 g of Ag+ may only cause argyria ( ) [310]. With the low degree of regulation regarding AgNPs, further research is necessary to determine the safe levels of AgNP exposure through consumer products, medical treatments, involuntary ingestion (water and food), or work-related inhalation. To address this, numerous in vitro, in vivo, and human-related studies are being conducted to define the toxicity of AgNPs.

9.1. In Vitro Studies

The in vitro toxicity of AgNPs is influenced by the cell type, the PCC properties of AgNPs, and the exposure conditions (e.g., pH, concentration, and duration) [53]. The difficulty in addressing the cytotoxicity of AgNPs lies in the large variety of eukaryotic cells and the unique way each cell type absorbs, distributes, metabolizes, and excretes AgNPs. Pathways of AgNP-mediated damage found in bacteria (e.g., ROS formation and DNA damage&#; ) are shared with eukaryotic cells, but the additional organelles and differing structural organization require further understanding. Overall, these key damage mechanisms ( ) have resulted in cell cycle arrest and ceased proliferation in eukaryotes [311]. Additional damage mechanisms exclusive to eukaryotes originate from the organelle uptake of intracellular AgNPs. The mitochondrion is one of the most researched organelles with respect to AgNP&#;organelle interactions because of its key role in cellular energy production and various cellular activities. AgNP&#;mitochondrial contact induces dysregulation of ATP production, ROS formation, and mitochondrial-mediated apoptosis [312,313]. For example, exposure of PC-12 cells, a variety of rodent neuronal cell, to 10 µg mL&#;1 of spherical AgNPs (57.2 ± 21.6 nm), for 6 h, caused mitochondrial structural changes and subsequent disruption of mitochondrial function [314]. Intracellular ROS then interfered with the function of the endoplasmic reticulum (ER), which has been correlated with the accumulation of AgNPs. This has been utilized as a potential biomarker for in vitro and in vivo nanotoxicity studies [315]. For instance, multiple forms of ER stress were observed after 18 h of exposure of human retinal pigment epithelium cells to 5 µg mL&#;1 of spherical AgNPs (6.3 ± 0.62 nm) [316]. In another study, the secretory pathway of Wistar rat neuronal cells was found to be overloaded with additional protein secretion from the ER after 21 days of exposure to spherical AgNPs (10 ± 4 nm) [317]. This cellular attempt of excreting AgNPs led to the enlargement of the Golgi apparatus in response to the ER stress [317]. Lysosomes serve as another potential destination for AgNPs upon cellular entry. The AgNP&#;lysosome interactions modified the constitution of the lysosome including the intra-lysosomal pH and the structural integrity of the lysosomal membrane [318,319]. The biochemical degradation of AgNPs within the acidic environment of lysosomes can induce significant releases of Ag+ in the eukaryotic cell and cause high oxidative stress [53,319]. Such changes were reported for the endolysosomal environment of neural PC-12 cells after 1 h of contact with 10 µg mL&#;1 of spherical AgNPs (57 ± 21 nm) [314]. In another study, the highest accumulation of spherical AgNP (50 ± 20 nm, PVP-coated) was observed within lysosomal structures rather than the nucleus, Golgi complex, or ER of human mesenchymal cells. However, AgNPs were agglomerated around the eukaryotic nucleus at concentrations of 20 µg mL&#;1 or higher [320]. Altogether, these in vitro studies allow for a better understanding of the toxicity of AgNPs in specific cell lines and within controlled physical and chemical environments. The in vitro results can then be extrapolated to in vivo models of higher relevance and reliability, dealing with an internal, more complex environment of a living organism.

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9.2. In Vivo Studies

Most in vivo toxicity studies of AgNPs addressed their interactions with different structures of a living organism under physiological conditions. AgNPs were found to be toxic to the skin, liver, lung, brain, vascular system, and reproductive structures of animal test subjects [321,322]. The breaching of other important biological barriers has also been a long-time concern and a subject of animal testing with AgNPs. For example, AgNPs were observed to cross the blood&#;testis barrier (10 nm, citrate-coated) [323], the placental barrier (18&#;20 nm, spherical) [324], and the blood&#;brain barrier (49.7 ± 10.5 nm, spherical, citrate-coated) in mice [325]. Significant efforts continue to be dedicated to identifying nontoxic AgNP concentrations and the organs with the most AgNP accumulation after exposure. In these studies, the most common techniques of AgNP administration to animal subjects (i.e., inhalation, oral dosages, and dermal absorption) resemble those found in humans.

Injection and inhalation: One such study using male and female Wistar rats established that spherical AgNPs (13&#;35 nm, dispersed in ethylene glycol), which were injected intravenously, induced cellular stress responses with later recovery at doses &#;10 µg mL&#;1, and exhibited accumulation in organ tissues at doses &#;20 µg mL&#;1 of AgNPs [326]. Data from another study of 90-day inhalation (low dose: 49 µg m&#;3, medium dose: 133 µg m&#;3, and high dose: 515 µg m&#;3) in male and female Sprague Dawley rats indicated that the lungs and liver accumulated the largest amounts of spherical AgNPs (18&#;19 nm). In the same study, oral ingestion of AgNPs had a low impact on the respiratory tract. AgNPs were also detected in the brain, yet the area with the most build-up was recorded in the olfactory bulb [327]. Another report on 10 days of inhalation (4 h daily) of 3.3 mg m&#;3 of AgNPs (5 ± 2 nm, PVP-coated) revealed low pulmonary inflammation in male C57BL/6 mice and a threefold reduction in the amount of lung accumulation over time. This decrease was from 31 µg g&#;1 of AgNPs per dry weight immediately after the final exposure down to 10 µg g&#;1 of AgNPs at three weeks after the final exposure [328].

Oral ingestion: Oral intake of AgNPs is another widely used technique for AgNP exposure testing on animals [329,330,331]. For example, 28 days of ingestion through oral gavage of 12.6 mg of AgNPs kg&#;1 of body weight (14 ± 4 nm, spherical, PVP-coated) led to the following accumulation levels in female Wistar Hannover Galas rats: 27 µg g&#;1 in the small intestine, 5 µg g&#;1 in the stomach, 2.5 µg g&#;1 in the kidneys, and 1 µg g&#;1 in the liver [329]. In contrast, male CD-1(ICR) mice (10 nm, spherical, citrate-coated) exhibited the highest AgNP accumulation in the brain, followed by the testis, liver, and spleen after 4 weeks of oral gavage [331].

Dermal absorption: Dermal penetration has been observed in both animal and human subjects with varying results of both considerable and negligible effects [332]. For example, the pig&#;s skin was reported to have higher permeability to spherical AgNPs (20 nm, PEG, citrate, and branched polyethyleneimine (bPEI) coatings) than human cadaver skin [333]. For the citrate-coated AgNPs, these levels were 14.02 ± 8.01 µg of AgNPs g&#;1 of skin in pigs versus 3.14 ± 1.97 µg of AgNPs g&#;1 of skin in humans [333]. However, the majority of AgNPs were left unabsorbed by both the human and pig skin and the surface charge of AgNPs (positive versus negative) did not appear to enhance penetration in human skin. In contrast, negatively charged, citrate- or PEG-capped AgNPs had a slightly higher permeability than positively charged, bPEI-capped AgNPs in pig skin [333]. In another dermal study on male and female adult zebrafish (Danio rerio), 24 h of exposure to 30 and 120 mg L&#;1 of spherical AgNPs (5&#;20 nm) contributed to ROS formation and DNA damage to hepatocytes, and enhanced expression of p53 as a response to DNA damage leading to apoptosis and subsequent necrotic sites [334]. The liver has been another principal organ of testing because of its key detoxification role. In the same zebra fish study, the metallothioneins&#;heavy-metal-complexing proteins within the liver&#;also experienced an enhanced dose-dependent expression after exposure to 120 µg mL&#;1 of AgNPs, i.e., up to 7.1-fold higher levels than the basal level for excretion [334]. The 24 h median lethal concentration (LC50) was established at 250 mg Ag L&#;1 for the zebra fish, and the silver buildup within the harvested liver tissues was 0.29 ng mg&#;1 for 30 mg L&#;1 of AgNP exposure and 2.4 nm mg&#;1 for 120 mg L&#;1 of AgNP exposure [334]. Overall, these in vivo studies on animal models can serve as a reference point to human studies.

9.3. Human Studies

There are very few reports on the short- and long-term effects of AgNP exposure in human subjects. These studies are mostly related to antibacterial applications of AgNPs [335].

Excretion of AgNPs: Ag present within a human body can be expelled in-part over time due to the multiple excretion pathways (e.g., biliary fecal, urinary, hair, and nail growth) [336]. AgNPs can still experience chemical interactions and transformations with cells within the human body before excretion. Ag metabolism is controlled by the induction and binding to metallothionein proteins, which protect cells and tissues against toxicity from heavy metals [336]. If Ag is ingested, it can enter the blood circulatory system as a protein complex and can be later excreted by the liver and kidneys [337]. AgNP excretion was demonstrated by a clinical study, where nanosilver-based dressings were applied to n = 40 patients (3 withdrew) with chronic inflammatory wounds. Half of the subjects displayed elevated Ag levels in the blood serum after 1 month of treatment and no toxicity was detected along with the slow removal of silver from the body [338].

AgNPs in human blood: Numerous studies have confirmed the interactions between AgNPs and human blood components such as red blood cells (RBCs), lymphocytes, and leukocytes [339,340,341,342,343,344]. Yet, little is known about the AgNP&#;RBC interaction mechanisms (e.g., hemolysis, coagulation, and platelet activity) [339]. The inflammatory response within the bloodstream, that is mediated by many cellular components, also contributes to the complexity of these interactions [340]. The PCC properties of AgNPs, such as size, also affect the toxicity to human RBCs. For instance, exposure to 20 µg mL&#;1 of spherical AgNPs of 15 nm in diameter (citrate-coated) induced higher levels of hemolysis (60% hemolysis) and membrane damage when compared to larger AgNPs of 50 nm and 100 nm in diameter (&#;12%) [341]. For a specific size of spherical AgNPs (43.9 nm, PVP-coated), structural damage of RBCs was observed to increase with the increase in concentration from 100 µg mL&#;1 to 500 µg mL&#;1 (major membrane damage) [342]. Other cells within the bloodstream can also be damaged by AgNPs. For example, the proliferation and viability of lymphocytes (white blood cells), which help fight disease and infection, were negatively impacted by the exposure to spherical AgNPs (20 nm, PVP- and citrate-coated), and this toxicity was concentration-dependent (10, 20, 30, and 40 µg mL&#;1) [339]. Neutrophils, the most populous cell in the bloodstream, are one of the first lines against pathogens that migrate to sites of inflammation [343]. Exposure to 2, 5, and 20 µg mL&#;1 of spherical AgNPs (20 nm) for 4&#;20 h activated and increased the population of immunosuppressive neutrophils in circulation [344]. Thus, it was proposed that AgNPs stimulate an anti-inflammatory response by increasing the apoptotic deaths of neutrophils [344].

Dermal exposure to AgNPs: The human skin has recently become the subject of numerous toxicity studies due to the widespread use of AgNP-based dermal products. Dermal applications of AgNPs (e.g., wound dressings, wound gels, textiles, and cosmetics&#; ) are widely used by the public and healthcare providers. Many wound dressings (e.g., Acticoat) contain nanosilver at concentrations of ~50&#;100 mg mL&#;1 that are above the toxic threshold levels for both fibroblasts and keratinocytes [345]. Skin or immune cells present in topical wounds experience different interactions with and have different tolerance thresholds to AgNPs [346]. For example, human keratinocytes displayed a notably lower viability after 24 and 48 h of exposure to 25 and 50 µg mL&#;1 of AgNPs (30 nm, citrate-coated) [347]. Under identical experimental conditions with citrate-coated AgNPs (30 nm), the viability was significantly lower (&#;20%) at concentrations higher than 50 µg mL&#;1, after 24 h [347]. Different responses are also observed in between healthy and damaged tissue samples. For instance, damaged skin exhibited a fivefold higher penetration (2.32 ng cm&#;2) to AgNPs (25 ± 7.1 nm, PVP-coated) than healthy, intact skin (~0.46 ng cm&#;2) [348].

With the pressing dilemma of antibiotic resistance, AgNPs have been explored as a supplement or alternative to antibiotics [150]. Even though there are silver-based products approved by the U.S. FDA (e.g., Silverlon, Aquacel, and Acticoat wound dressings&#; ), motions have been previously put forward to address the lack of scientific data on the long-term safety of these products [349]. One such proposition, which originated in and was updated in , recognizes the lack of scientific data on the effectiveness and safety of over-the-counter products containing colloidal silver or silver salts that claim to cure disease. Thus, new products might be mislabeled and require further toxicity research before marketing approval [349]. Potential solutions to the observed toxicity of AgNPs incorporated in consumer products include green synthesis reagents and more biocompatible capping agents of high antibacterial activity.

10. Conclusions and Perspectives

The use of silver in its elemental form has been omnipresent in society since historical records began. Before the commercial production of antibiotics, silver compounds were routinely used as treatment for infections and during surgery to prevent infections. Since the 20th century, chemically and biogenically produced nanosilver has been employed not only in medical remedies, but also in commercially available, U.S.-FDA-approved or non-approved products such as clothing, facemasks, and cosmetics. However, the antibacterial mechanistic details of nanosilver and its dependence on the physicochemical properties of nanosilver need further characterization. This review aimed to bridge this gap by focusing on the antibacterial benefits of silver nanoparticles without (AgNPs) and with antibiotic functionalization (AgNP&#;antibiotic conjugates) against relevant Gram-positive and Gram-negative bacteria. This included ESKAPE pathogens and high-priority antibiotic-resistant bacteria as listed by the World Health Organization.

With over 100 years of nanosilver production, strategies to fabricate AgNPs using chemical reducing agents and bacterial, fungal, or plant extracts are well understood. The antibacterial effects of AgNPs and the subsequent synergy with antibiotics are similarly well established. It is apparent that synergy allows the use of lower antibiotic concentrations, which in turn may mitigate the increase in emergence of antibiotic-resistant microorganisms. In addition, older drugs might be repurposed by conjugating them to AgNPs, as the AgNP&#;drug constructs can more easily evade microbial defenses.

Before AgNP-conjugates can become a more common antibacterial treatment, the environmental and health effects of prolonged exposure to Ag and its possible chemical transformations (e.g., from AgNPs to Ag+ ionic forms) must also be investigated. Monitoring Ag accumulation in organs such as the skin, liver, kidneys, cornea, and spleen necessitate the development of animal models to establish guidelines for appropriate silver use in order to avoid the negative consequences of overexposure (e.g., Argyria). Certainly, limiting the amounts of antibiotics and silver used to treat infections would be beneficial to the patient and the environment alike. To further lower the dosage and limit the environmental harm of AgNP&#;antibiotic conjugates, targeted delivery of AgNPs is the next logical step. Targeted delivery is well established in cancer therapies: an antibody, aptamer, peptide, or polysaccharide that is specific to a cell surface receptor is attached to the NP and employed to deliver the NP cargo to only the target cells. Thus, harm to the host and beneficial bacteria can be limited.

AgNPs and AgNP&#;antibiotic conjugates have great potential as the next generation of antibacterial agents in the post-antibiotic area. Despite solid research on the antibacterial efficacy of AgNPs and their conjugates, there are relatively few U.S.-FDA-approved therapeutics that use this strategy. To realize their true potential, more research is needed. Improving the batch-to-batch reproducibility of AgNP synthesis and the functionalization process, characterizing the PCC properties of AgNPs and AgNP&#;antibiotic conjugates, and increasing our understanding of how Ag and what forms of Ag affect vital organs are important. Knowledge-based regulatory guidelines are necessary prerequisites before mass production and deployment of AgNP and AgNP&#;antibiotic conjugates become the go-to antimicrobial therapeutics of the future.

Funding Statement

Texas A&M University Corpus Christi, Texas, and Winthrop University, South Carolina are highly acknowledged for their support. The SC INBRE #5P20GM, SC EPSCoR, and NSF # awards are highly acknowledged.

Author Contributions

Conceptualization, K.G.K., V.D., V.J.F., T.G.F. and I.E.P.; methodology: K.G.K., V.D., V.J.F., G.W.B., T.G.F. and I.E.P.; validation: K.G.K., V.D., V.J.F., G.W.B., J.V.P., T.G.F. and I.E.P.; formal analysis, K.G.K., V.D., V.J.F., G.W.B., J.V.P., T.G.F. and I.E.P.; investigation, K.G.K., V.D., V.J.F., G.W.B., J.V.P., T.G.F. and I.E.P.; data curation: K.G.K., V.D., V.J.F., G.W.B., J.V.P., T.G.F. and I.E.P.; writing&#;original draft preparation, K.G.K., V.D., V.J.F., G.W.B., T.G.F. and I.E.P.; writing&#;review and editing, K.G.K., V.D., V.J.F., G.W.B., T.G.F. and I.E.P.; supervision, V.J.F., T.G.F. and I.E.P.; project administration, T.G.F. and I.E.P.; funding acquisition, T.G.F. and I.E.P. 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

All manuscript figures were created with BioRender.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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• All Antimicrobial agents must be environmentally-friendly. 

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