Nanotargeting of Resistant Infections with a Special Emphasis on the Biofilm Landscape

Abstract

Bacterial resistance to antimicrobial compounds is a growing concern in medical and public health circles. Overcoming the adaptable and duplicative resistance mechanisms of bacteria requires chemistry-based approaches. Engineered nanoparticles (NPs) now offer unique advantages toward this effort. However, most in situ infections (in humans) occur as attached biofilms enveloped in a protective surrounding matrix of extracellular polymers, where survival of microbial cells is enhanced. This presents special considerations in the design and deployment of antimicrobials. Here, we review recent efforts to combat resistant bacterial strains using NPs and, then, explore how NP surfaces may be specifically engineered to enhance the potency and delivery of antimicrobial compounds. Special NP-engineering challenges in the design of NPs must be overcome to penetrate the inherent protective barriers of the biofilm and to successfully deliver antimicrobials to bacterial cells. Future challenges are discussed in the development of new antibiotics and their mechanisms of action and targeted delivery via NPs.

Graphical Abstract

1. EMERGING ISSUE OF ANTIMICROBIAL RESISTANCE

1.1. Development of Antimicrobial Resistance.

Antimicrobials, such as antibiotics, are molecules produced naturally by bacteria for the putative function of inhibiting other bacteria. Antimicrobials also can be synthetically produced in the laboratory. Antimicrobials consume 30% of the total pharmaceutical budget in the US, but surprisingly only 1.6% of drug development allotments by major pharmaceutical companies are spent on antimicrobial development.^1,2 The U.S. Centers for Disease Control (CDC) predicts that by the year 2050, antimicrobial resistant infections (ARIs) will result in over 10 million deaths annually, far exceeding deaths from all cancers and AIDS combined.^3,4 The current economic burden created by ARIs for health-care costs and lost productivity is already estimated at $20 and $35 billion per year, respectively, and is projected to reach $100 trillion by 2050.^5,6

ARIs are emerging as “one of the greatest global threats to human health”. The U.S. CDC reported that over 2 million people in the US are affected annually.⁴ Most recent estimates indicate over 162 000 antimicrobial-resistance deaths per year, with comparable numbers occurring in Europe.⁷ Over 1.3 million deaths occur worldwide due to tuberculosis alone. Infections due to methicillin-resistant Staphylococcus aureus (MRSA) contribute over 19 000 deaths annually and are responsible for 60–90% of hospital-acquired (i.e., nosocomial) infections.⁸ MRSA, as early as 2009, accounted for more deaths than HIV/AIDS and TB combined.⁹ The human and economic tolls of ARIs are already quite substantial and rising.

1.2. Antimicrobial Overuse and Development of Multidrug Resistant Bacterial Strains.

A decades-long reluctance to invest in the discovery and development of novel antibiotics has been coupled with the concurrent overuse of antimicrobials in agriculture and animal farming. For example, tens of millions of pounds per year are given to animals for nontherapeutic purposes, to largely enhance growth. In contrast, only 3 million pounds are used in humans.¹⁰ This has enabled resistance mechanisms to present-day antimicrobials to develop in food-related systems and clinical settings and be rapidly (e.g., days, weeks) transferred among pathogens within both hospital and community settings.¹¹ The escalating crisis of ARIs has recently driven a greater awareness for the exploration and development of innovative antimicrobial therapies.^11,12

The rapid emergence of ARIs and the arms race toward the development of novel bactericidal agents is one of the most urgent public health concerns in the United States. The development, screening, and testing of small-molecule antibiotics is costly and resource-intensive, thus limiting development of alternative treatments against resistant bacteria. As of December 2019, there were approximately 41 novel antimicrobials in clinical trials in the U.S. In general, the success rates of developing any new drug, including antimicrobials, are typically low, in which only approximately 20% of newly developed antimicrobials are approved as treatments. These primary limitations prompt an urgent need for the development of unique antimicrobials with precise targets. Limitations in current ARI treatments motivate a fundamental change in the approach for developing antimicrobials. The major disadvantages of conventional antimicrobial approaches involve the systemic toxicity and the inability of the antimicrobials to reach their desired effective concentration at the localized site of infection. Current drug therapies nonspecifically target the intended pathogen, commensal microbiome flora, and the host’s normal healthy cells.^13,14 Therefore, the development of novel and other antimicrobial agents requires the identification of target pathway’s molecules, the quantity and concentration required, and specificity of the target. The bacteria structural and replication pathways are potential targets for the development of new antimicrobial agents.

Resistance genes for antimicrobials occur in many natural bacteria, as well as in clinical settings. ARI genes are often associated in clusters and therefore can be transferred among bacteria as groups of genes via transposons and other molecular mechanisms.¹⁵ This, coupled with their overuse, has resulted in the emergence of deadly multidrug resistant (MDR) superbugs, which are resistant to most or all available antibiotic therapies. Major MDR superbugs of concern are labeled with the acronym “ESKAPE” pathogens standing for Enterococcus faecium; Staphylococcus aureus; Klebsiella sp. Acginetobacter baumannii; Pseudomonas aeruginosa; and Enterobacter sp. These bacteria cause many US hospital infections and escape antimicrobial therapeutics.⁹ Resistance often originates from natural mechanisms that are already present in certain types of microbes and is spread through horizontal gene transfer (i.e., transformation, conjugation, transduction) to other bacteria when there are increased selective pressures.

The abuse and misuse of antibiotics together have contributed to the emergence of resistant bacteria threatening human health. Antimicrobial resistance has become one of the leading causes of death worldwide in recent years. Currently, strategies such as chemical modification of antibiotics, combinatorial therapy, photothermal agents, antimicrobial peptides, cationic polymers, and nanomaterials have been reported to be useful for conquering antimicrobial resistance.¹⁶

1.3. Nonspecific Effects of Antibiotics on the Beneficial Microbiome.

A relatively recent realization is that humans possess a complex array of over ten thousand different microbial species and strains, collectively called the human microbiome.¹⁷ This microbiome has coevolved with humans and is beneficial and even necessary for human health.^18,19 A challenging issue, one that is most especially pronounced in western societies, is the overuse of antibiotics and their resulting effects of the gut microbiome. Humans are estimated to consist of approximately 37.2 trillion human cells but may possess over one hundred trillion bacterial cells. Given the variabilities in estimates and human size, the ratios of bacterial:human cells are between 3:1 and 1:1.²⁰ This presents a diversity of potentially beneficial bacteria, which can be influenced in an adverse manner by antibiotic exposure and delivery. The nonspecific activities of most antibiotics often impart destructive consequences to the beneficial host flora in humans.

2. NANOPARTICLES FOR DELIVERY OF ANTIMICROBIALS

In the past decade, there have been great advances in nanomedicine that show promise for the treatment of bacterial infections. The inherent plasticity of microbial cells and their capabilities for the rapid evolution of resistance have driven the development of alternative approaches for the delivery of antimicrobials to pathogenic forms. nanoparticles (NPs), owing to their extremely small sizes relative to biological cells and often unique physical/chemical properties, have become efficient vehicles for drug delivery, especially for anticancer drugs, to cells (Figure 1). The continuous emergence of bacterial resistance and MDR strains have generated significant challenges resulting in an urgent need in the development of novel and effective antimicrobial agents. Among the most promising of these novel antimicrobial agents are metal NPs, which have shown strong antibacterial activity in an overwhelming number of studies. It is hypothesized that NPs with antibacterial activities have the potential to reduce or eliminate the evolution of resistant bacteria due to the nature of NPs being designed to target multiple pathways at once avoiding the development of resistant bacterial strains.

Figure 1.

Nanoparticle-based design strategies for intracellular delivery applications [Reprinted with permission from ref 27. Copyright 2011 Royal Society of Chemistry].

NPs can act as antimicrobial agents or the carriers for loading antimicrobial drugs to promote the bioavailability and their effectiveness.²¹ Antibacterial NPs without the need of drugs are developed using diverse materials, including metals, chitosan, and surfactants.²² The net-positive charge of cationic compounds can bind to the negatively charged membrane surface of bacterial cell walls, while the amphiphilic structure of some NPs prompts membrane damage.²³ Another strategy involves the encapsulation of antibiotics into nanocarriers to enhance bacterial eradication and bioavailability. The delivery of drugs from nanosystems improves the efficacy, while reducing toxicity in comparison to conventional therapy. Given the high surface-to-volume ratio, the possibility of surface functionalization, and the capacity to load drug molecules, NPs contribute to the efficient antimicrobial activity with their high affinity to bacteria.¹⁴ The NPs can protect drugs from enzymatic attack and sustain the drug release to increase the half-life and bioavailability.²⁴

Active targeting of NPs against bacteria can provide a suitable strategy to efficiently increase the therapeutic index. The nanocarriers can be functionalized with ligands to the bacterial surface to enhance the targeting to specific pathogens. The design of stimuli-responsive nanosystems is another concept approach for bacterial targeting through recognition of the bacterial microenvironment and the response in a dynamic process.²⁵ NPs can be designed to respond to internal stimuli, including conditions varying in pH, concentrations of specific enzymes, or chemicals, which are associated with pathological conditions of infection and inflammation.²⁶ The antibiotic targeting of bacteria can be also achieved by NP responses to external stimuli, such as magnetic, thermal, light, and ultrasound effects.

2.1. Cell Wall and Membrane Barriers of Bacteria.

Many studies have found that Gram-positive bacteria are more resistant (than Gram-negatives) to NP exposure and their NP-associated mechanisms of action. This has been attributed to the fundamental differences in the thickness and composition of cell walls between Gram-positive and Gram-negative bacteria. The bacterial cell wall is considered a single polymeric molecule that encloses the bacterium and is composed of glycan strands covalently linked to each other with an interconnecting peptide stem attached perpendicularly to alternating saccharides of the glycan strands.²⁸ The alternating saccharides consist of an N-acetylglucosamine (GlcNAc, NAG) and N-acetylmuramic acid (MurNAc, NAM) connected by a β-1,4-glycosidic bond.²⁸

Gram-negative bacteria, such as Escherichia coli, typically have a relatively thin cell wall, with 1–3 layers of peptidoglycan (~3–4 nm thick) and an additional outer membrane consisting of lipopolysaccharide (LPS; 1–3 nm thick).²⁹ This structural arrangement is hypothesized to facilitate the entrance of ions released from NPs into the cell. Alternatively, the cell wall of Gram-positive bacteria, like S. aureus, is relatively thicker and consists of 10–20 layers of peptidoglycan and is 30 nm thick in Bacillus subtills, consisting of covalently attached teichoic and teichuronic acids.^30,31 The proposed mechanism explaining the difference in susceptibility is in the direct interaction between NPs and the cell wall, which may be more detrimental for Gram-negative bacteria since they lack the thick peptidoglycan layer present in Gram-positive bacteria.

The surface charge and electronegativity of the cell wall also are hypothesized to play a significant role in the interactions between the bacterial cell walls and NPs or the ions released from these structures. It is known that both Gram-positive and Gram-negative bacteria have negatively charged cell walls; however, changes in the electronegativity of the cell wall can occur due to growth in different types of culture media.^32,33 Additionally, the Gram-negative cell walls are surrounded by an outer membrane containing negatively charged LPS molecules. This imparts a higher affinity for the positive ions released by most NP constructs and leads to the increased uptake and overall accumulation of ions, which then causes intracellular damage. Several reactive oxygen species (ROS), including hydroxyl radicals, are negatively charged and therefore unable to easily penetrate the negatively charged cell membrane during NP exposure. The electrostatic charge plays an even more important role when charged capping agents are used during NP synthesis, further adding to the overall electrostatic attraction or repulsion.

2.2. Metallic Nanoparticles.

Transition metals are commonly used for antimicrobial NPs and are classified as metals with a density >5 g/cm³ and having valence electrons in two shells, rather than just the outer one. They form coordination bonds and perform a diversity of important biological functions, including hydroxylation, redox reactions, and electron transport.³⁴ While many transition metals are essential to life in very small concentrations, they become toxic at higher concentrations. In metallic NPs, the metal is present in a neutral state and likely incapable of crossing the cell membrane. However, it has been demonstrated that metal NPs slowly release metal ions that are able to penetrate the cell membrane and disrupt the intracellular function and pathways.³⁵ In general, trends in bactericidal activity often exhibit similarities to those observed in eukaryote cytotoxicity. Thus, most potent bactericidal agents are also toxic toward mammalian cells.^36,37

Currently, the most effective antimicrobial metal oxide is ZnO, which exhibits efficacies comparable to Ag.³⁸ Due to photocatalysis, ZnO-NPs are strongly bactericidal upon exposure with UV irradiation. Here, NP shape can influence efficacy. For example, ZnO nanorods were revealed to be better antimicrobials than nanospheres. Although, flower-shaped ZnO-NPs with exposed polar groups had even stronger efficacies.³⁹ TiO₂ NPs possess their antimicrobial activities through the production of OH• radicals. However, these same NPs exhibit minimal antibacterial properties in the absence of UV given their weak interactions with the negatively charged bacterial surface. Recent studies have shown that TiO₂ NPs exhibited a high tendency to bind via van der Waals and receptor–ligand interactions to the OM of E. coli.⁴⁰ A recent strategy involves metal doping of TiO₂ to substantially increase antimicrobial activities and enable visible-light-induced photocatalytic activities.^41,42

Au-NPs, in contrast, are both biocompatible and biologically inert. Internalization by eukaryote cells of Au-NPs is dependent on the size, shape, and charge of the NP. The fastest uptake was observed for 40–50 nm sizes and spherical shapes, when compared to nanorods with positively charged NPs, which penetrated more easily.^43-45 Au-NPs, when functionalized with amino acid residues exhibiting a similar structure to antimicrobial peptides, facilitated strong electrostatic interactions between the cationic functionalization of the Au-NPs and the bacterial membrane. This interfered with membrane compactness and structure leading to its disruption and resulted in antibacterial activities against E. coli and S. aureus.⁴⁶

Metal NPs and their associated antibacterial activities can be attributed to the formation of ROS and their affinity for interacting with thiol groups (R–SH) (Figure 2), especially the amino acid cysteine in proteins. This directly disrupts functioning of specific enzymes and/or breaks disulfide bridges required to maintain the integrity of protein folding and ultimately results in cell death. Metallic NPs have been extensively addressed elsewhere and will not be considered further here.^47-54

Figure 2.

Schematic representation illustrating the antimicrobial mechanisms that have been demonstrated for various metal-based NPs [Reprinted with permission from ref 55. Copyright 2019 Shaikh et al.].

2.3. Noncytotoxic Nanoparticles for Delivery of Antimicrobials.

NPs can be engineered using a variety of materials for different applications. The size, shape, and surface charge of the NPs can be finely tuned by modulation of material types, contents, and preparation methods. Since most NP-based approaches utilize distinct mechanisms, structures, and pathways compared to those administered by conventional antibiotics, combined therapeutic regimens are favorable for tackling the rise of MDR bacteria bypassing their defense mechanisms. The multitarget action of NPs may overcome multidrug resistance by circumventing obstacles encountered by conventional antibiotics.¹⁴

Some commonly used nanosystems for antibiotic drugs include inorganic metal, polymeric, lipid-based, silica, micellar, and cell membrane-coated NPs.⁵⁶ Superparamagnetic iron oxide NPs (SPIONs) have been widely investigated as potent bactericidal agents based on their magnetic hyperthermia properties.⁵⁷ They have also been utilized as bacterial separation agents and bioimaging contrast agents for bacterial diagnoses.⁵⁸ The hyperthermic properties lead to the increased bacterial membrane permeability and result in the killing of targeted bacteria.⁵⁹ In order to potentiate their antibacterial activity, SPIONs can be functionalized with antibodies, antimicrobial peptides, and aptamers for targeting bacteria.⁶⁰

Polymeric NPs can be fabricated with high biocompatibility and biodegradability. Chitosan-based NPs have been broadly used as drug delivery systems and display intrinsic antibacterial and antibiofilm activity due to their polycationic nature capable of disrupting bacterial membranes.⁶¹

The strategy of coating NPs with the lipids of natural cell membranes has gained much attention recently. Lipid-based NPs, including liposomes, nanoemulsions, and solid lipid NPs (SLNs) are widely utilized for the targeted delivery of antibacterial drugs. Lipid NPs easily fuse with the bacterial outer membrane, delivering the antimicrobial agent directly to the bacterial cells.^62,63 As carriers for drug delivery, liposomes can prolong circulation time and accelerate cellular uptake, thus counteracting the selection of resistance. Liposomes can fuse with mammalian cells, tumor cells, and microbes, facilitating the transport of drugs across biological membranes.⁶⁴

Due to their physiochemical stability, uniform porosity, large surface area, and biocompatibility, mesoporous silica NPs (MSNs) are widely used as drug delivery vehicles, biosensors, catalysts, and absorbents.⁶⁵ Their tunable particle pore volume and surface chemistry can be modified so antibacterial agents can be neatly packaged inside the porous matrix and effectively shielded against enzymatic degradation and facilitate their passage through biomembranes.⁶⁶

2.3.1. Surface Chemistry of Nanoparticles.

NPs design can be tailored to target specific cell types or pathways by tuning the nanomaterials parameters based on their size, shape, charge, hydrophobicity, rigidity, roughness, and surface functionalization.^27,67 The physiochemical characteristics of NPs, including size, shape, and surface charge are critical for governing bacterial interactions and antibacterial activities and are summarized in Figure 1.

2.3.1.1. Size and Surface Charge.

NP size influences its interactions with bacterial cells. Typically, smaller-sized NPs exhibit a higher probability of interacting with a bacterial cell. The higher surface-to-mass ratio of smaller nanocomposites demonstrates increasing adaption and binding to bacterial surfaces.⁶⁸ Additionally, smaller size NPs can more easily permeate into the bacterial membrane and enhance the overall antibacterial activity.⁶⁹ In metallic NPs, smaller sizes increase particle dissolution rates into ions.⁷⁰ Increased dissolution rates raise the potency of bacterial killing. NPs may aggregate into larger complexes before and after attachment on bacterial surfaces.⁷¹ NP aggregates of larger sizes interact with bacterial surfaces but in different manners when compared with dispersed NPs.⁷² However, aggregation decreased the overall reactivity with bacterial cells and resulted in lower antibacterial efficacies.⁷³ It is generally recognized that smaller-sized NPs exhibit increased interactions and activities.

2.3.2. Increased Efficacies of Antimicrobials Delivered by NPs and Possible Mechanisms of Action.

A major goal of nanoengineering is to produce and utilize nontoxic NPs that avoid cross-reactivity with mammalian cells. The use of green chemistry to synthesize NPs may be advantageous over more common physical/chemical methods, owing to their ecofriendly, less toxic, and more economical syntheses.^74,75 Applications of nontoxic NPs to act as carriers for antibiotics is especially relevant to the emerging global scale of antibiotic resistance. Nanocarriers can be designed for drug delivery to a specific site, organ, or cell type. Targeting capability can be achieved by coupling of antibodies, ligands, or other targeting moieties, such as sugars to the surface of the nanocarrier.⁶⁷

An important aspect to consider when studying the interactions of nanomaterials in vivo and with cells is the dispersion of NPs in biological media and the potential agglomeration and stability over time. Agglomeration can impact and affect the corona composition and interactions with cells. NPs incubated with cells in the absence of serum or other biological media may lead to conclusions that are entirely irrelevant to their biological applications and physiochemical properties in vivo.⁷⁶

2.4. Antimicrobial Peptides and Antibiotics Using Nanobased Strategies.

Approximately 60–70% of current antibiotics when delivered by conventional means are not effective against infections due to their inability to cross the bacterial cell surface and/or low retention times once in the cell. Many common antibiotics, such as the β-lactams and aminoglycosides, are hydrophilic in nature and unable to easily penetrate the bacterial cell wall.

Antimicrobial peptides (AMPs) have garnered interest as potential attractive alternatives to conventional antibiotics based on their unique physiochemical properties and broad or narrow spectrum activities against bacteria. Typically, AMPs are cationic amphipathic molecules that target and kill bacteria using two major mechanisms of actions. In the first, AMPs induce membrane disruption, which leads to lysis and cell death. In the second, AMPs are internalized into bacterial cells without membrane disruption and target key intracellular functions.⁷⁷ AMPs including their mechanisms of action are further elaborated in a recent review.⁷⁸

In a recent study, a class of antimicrobial polymers derived from bile acids were developed possessing facial amphiphilicity for enhanced interactions with the bacterial cell membrane.⁷⁹ It was observed that the cholic acid–based cationic polymers exhibited higher effectivities against Gram-negative bacteria, particularly P. aeruginosa, compared to Gram-positive bacteria (e.g., S. aureus). The three cationic charges present on each cholic acid unit enable localization in the outer membrane via electrostatic interactions while the hydrophobic face of cholic acid inserts into the membrane.⁷⁹

Some infections occur when bacteria reside within human cells, called intracellular pathogens. Most current antibacterial agents exhibit limited activity against intracellular bacteria. This is because antibiotic concentrations within the intracellular compartments of host cells, where pathogens are located, are typically less than the minimum inhibitory concentration (MIC) and contribute to the development of drug resistance of intracellular pathogens.⁸⁰

Several types of NPs, including polymeric NPs, lipid-based NPs, and silica NPs, have been used for facile internalization into eukaryote host cells to eradicate intracellular micro-organisms.⁸¹ Such NPs can be designed having an increased affinity for eukaryote host cells (containing the intracellular pathogens) or having ligand conjugation on the NP surface for active targeting of cells. The absorption of drug molecules onto NP surfaces enables the delivery of relatively high concentrations of antimicrobials to a targeted site.⁸² Antimicrobials can either be passively sorbed, covalently bound to, or incorporated into a polymer matrix generating polymeric NPs with high biocompatibility and biodegradability.

3. ENTRY OF NANOPARTICLES INTO BACTERIA AND POTENTIAL FOR THE INTRACELLULAR DELIVERY OF DRUGS AND THERAPEUTICS

3.1. Cellular Barriers to Nanoparticles in Bacteria.

Entering NPs directly into a bacterium to control or kill the cell represents a new approach for antimicrobial drug delivery. The bacterial cell envelope consists of the cell membrane(s) and cell wall (CW) and represents a sophisticated and complex permeability barrier that can hinder the internalization/accumulation of antibiotics in bacterial cells. This results in selection toward acquiring bacterial resistance.⁸³ Poor internalization could be potentially overcome if antibiotics were “carried” into bacterial cells.¹³

To reach a cytoplasmic target (e.g., such as proteins, nucleic acids) inside a Gram-negative bacterium, soluble antibiotics must cross three barriers: the outer membrane (OM), the cell wall (CW), and inner plasma membrane (PM).⁸⁴ This tripartite barrier functions to protect the cell against exogeneous toxic compounds. The CW provides protection against osmotic pressures and mechanical damage, while maintaining the permeability of key molecules for bacterial metabolism and communication with the environment.¹³ Membrane transporters, e.g., porins or efflux pumps, function as primary penetration strategies regulating the internal accumulation of various hydrophilic molecules. Regulation of membrane permeability, involving the processes of influx and efflux, is modulated by the bacterial cell to regulate the intracellular concentration of various molecules and maintenance of a positive turgor pressure.⁸⁵

3.2. Uptake by Cells and Synergistic Interactions of Small Molecules.

To be active against a bacterial pathogen, an antibiotic is required to reach a critical concentration threshold to inhibit the corresponding target. Diffusion of antibiotics through bacterial membranes must be facilitated and must overcome efflux pumps that remove antibiotics once in the periplasm, to attain a sufficient cytoplasmic concentration^86,87 (Figure 3). Membranotropic compounds, such as polymyxin B and polymyxin E (colistin), perturb the integrity of bacterial membranes and enhance the bacterial susceptibility to various compounds. However, they can also act as opsonins for targeted killing by phagocytic cells. Polymyxin B nonapeptide (PMBN) lacks a fatty acid tail and plays a critical role by increasing the permeability of the bacterial plasma membrane (PM). This sensitizes bacterial cells to hydrophobic antibiotics while exhibiting minimal toxicity to eukaryotic cells.⁸⁴

Figure 3.

Antimicrobial drug resistance mechanisms in bacteria. (1) Prevention of antimicrobial drug penetration into the bacterial cell by bacterial cell envelope. (2) Removal of antimicrobial drug from bacterial cell by nonspecific efflux pumps. (3) Acquisition of new genetic material from drug resistant bacterial strain. (4) Inactivation of antimicrobial drug by intracellular modification [Reprinted with permission from ref 70. Copyright 2018 Elsevier].

A proposed strategy now offered is to combine low (subinhibitory) concentrations of polymyxin with other clinical antibiotics to synergize their activities.⁸⁴ As an example, the combination of meropenem and colistin demonstrated synergistic killing of Acinetobacter baumannii and P. aeruginosa.⁸⁸ Similar results have been reported using a combination of colistin with a high dose of tigecycline, rifampin, and vancomycin.^89-91 Interestingly, it has been demonstrated that PMBN (polymyxin B nonapeptide) and polymyxin B can act synergistically as a new family of antibacterial agents, peptide deformylase inhibitor against Gram-negative bacteria, such as E. coli, Enterobacter aerogenes, K., and P. aeruginosa.⁹² The increase in bacterial susceptibility was associated with an increase in entry of the new antibacterial agents using subinhibitory concentrations of polymyxin.⁹² Importantly, the use of polymyxin compounds in combination with another antibiotic may reduce the risk of resistant emergence.⁸⁴

Interestingly, certain natural compounds can act as membrane permeabilizers. Eugenol, a terpenic compound and a major component of clove oil from Eugenia aromatica was reported to disrupt cytoplasmic membranes.⁹³ When applied in combination with eugenol, ten antibiotics were shown to have minimum inhibitory concentrations (MICs) that were 5 to 1000-fold lower than when used alone, indicating a synergistic effect.⁹⁴ The effect on bacterial membranes was also demonstrated through the observed enhancement of nitrocefin uptake associated with a sensitization of bacterial cells to lysis by lysozyme or detergents in the presence of eugenol.

An additional strategy to circumvent the membrane barrier is through destabilization of the LPS layer using chaotropic agents or detergents that consequently aid in the diffusion of hydrophilic compounds through the membrane lipid bilayer.⁹⁵ EDTA has been shown to increase the susceptibility of bacteria producing metallo-β-lactamases toward β-lactams.⁹⁶ Recently, squalamine, a natural aminosterol, has been characterized based on its antimicrobial activity and its ability to destabilize the OM of Gram-negative.^97,98 More interestingly, squalamine in combination with other conventional antibiotics was demonstrated to be capable of restoring bacterial susceptibility to various antibiotic classes including β-lactams, fluoroquinolones, macrolides, phenicols, and cyclines in resistant isolates that lack the expression of porins.⁹⁹ This class of amphiphilic derivatives has been suggested for direct use in the development of novel antibacterial agents, or alternatively to produce original adjuvants for rejuvenating activities of old conventional antibiotics on MDR bacteria.^100,101 Finally, Tang and colleagues have developed amphiphilic antimicrobials that effectively slice open the outer membranes of Gram-negative bacterial to demonstrate their efficacy.⁷⁹

3.3. Can Nanoparticle Design Facilitate Antimicrobial and Cellular Uptake into Bacterial Cells?

Different strategies have been proposed to overcome the bacterial envelope barrier (i.e., the OM, CW, and PM) and most involve altering the influx/efflux mechanisms of the cell, in order to restore the activity of antibiotics against resistant strains.⁸⁵ NP and molecular transporters able to successfully interact with bacterial envelopes are unique approaches to carry oligonucleotides and poorly internalized antibiotics across the envelope.¹³ Also, a key advantage of NP carriers is potentially provided by a reduction of drug efflux from bacterial cells. This is due to the intracellular delivery of a high concentration of drug into cytoplasm that may overwhelm the efflux pumps.^41,81 Consequently, the overall potential of NPs and molecular transporters to circumvent the bacterial envelop barrier depends upon the ability of the NP/transporters to efficiently interact with the different bacterial envelope structures.¹³

Key ideal features of drug delivery systems are biodegradability, biocompatibility, controlled drug transport, and targeted delivery to a tissue/organ.¹⁰² Drug absorption efficiency is directly proportional to the surface area of the absorbent and inversely proportional to NP particle size. Given their large surface-to-volume ratio and the diverse functionalization, NPs can be utilized as transporters for targeted drug delivery. Their use provides distinct advantages over alternative drug carrier systems due to a reduction in side effects observed for antibiotics and other antimicrobial agents. A study showed that superparamagnetic FeOH-NPs interact with microbial cells by directly penetrating the cell membrane and interfering with the transfer of transmembrane electrons.¹⁰³

NPs that are surface loaded with antibiotics often exhibit enhanced killing efficiencies of bacteria, even those strains exhibiting resistance to the antibiotic.¹⁰⁴ Recently, Decho and colleagues observed that when the beta lactam antibiotic penicillin G (PenG) was passively sorbed to NPs, their killing efficiencies dramatically increased against several types of penG-resistant bacteria, including MRSA strains.¹⁰⁵ They postulated the “grenade hypothesis” suggesting that each NP contained a sufficiently large number of PenG molecules, and constituted a powerful package (i.e., a high concentration) delivered to a single bacterial cell, which overwhelmed the natural beta lactamase-based resistance mechanism.¹⁰⁵

NPs themselves can lead to cell membrane damage caused by NP absorption, which sometimes is followed by penetration into the cell and cell death. Several studies have suggested that absorption on the CW followed by its disintegration was the primary mechanism of toxicity.^49,106 Absorption of NPs results in CW depolarization, which alters the negatively charged CW and becomes more permeable. Using laser scanning confocal microscopy, the microbial cell membrane became visibly “blurred” upon exposure to AgNPs, indicative of CW degradation.¹⁰⁷ The authors proposed that AgNPs followed a two-step mechanism of action. The first step involved disruption of the cell membranes and penetration of the AgNPs. In the second step, ROS are subsequently formed that inhibit the production of ATP and DNA replication. It was also noted that the production of ROS may be a primary step as well since its production counteracts the innate antioxidant defense mechanism and results in disruption of the cell wall.¹⁰⁸

Developing novel antibacterial agents to combat the membrane barrier, antibiotic-efflux pump inhibitors (EPIs) and affect the multidrug efflux pumps of bacteria that have been a recent focus of investigation, with the goal of developing new agents having the capability to rejuvenate activities of traditional antibiotics against MDR bacteria.

4. PENETRATION OF BIOFILMS: THE MAJOR BARRIER OF IN SITU INFECTIONS

4.1. Why do Infections Preferentially Occur As Biofilms?

It has been realized for several decades that bacteria occur in two major forms or states.¹⁰⁹ They occur as free-living suspended (i.e., planktonic) cells and as attached groups of cells called “biofilms”.¹¹⁰ Biofilms are multicellular, densely packed communities of bacteria that are surrounded by a self-secreted matrix of extracellular polymeric substances (EPSs).¹¹¹ EPS is an operational term, which refers to a wide range of secreted polymeric molecules such as polysaccharides, proteins, DNA, and lipids.^112-115

Biofilm formation plays a key role in the development of bacterial antibiotic resistance and in chronic and persistent infections.^116-119 This is due to the many protective roles they afford to cells.¹²⁰ While free-living bacterial cells are exposed to their immediate surrounding environment, biofilm cells embedded within an EPS matrix are provided many protective advantages. (1) The EPS matrix, secreted by microbial cells, is the primary emergent property of a biofilm; one that facilitates many subsequent advantages.¹¹⁰ The majority of biomass in a biofilm is contributed by EPSs, rather than cells.^111,121 (2) Diffusion-slowing properties of EPSs. When EPSs form dense gels, there is a measurable reduction in the diffusivities of molecules and ions within a biofilm.^122-124 This can slow (or prevent) antibiotics from reaching cells enclosed in the biofilm.¹²⁵ (3) Localization of extracellular enzymes (e-enzymes). e-Enzymes are secreted by cells and localized within EPS. This affords cells the benefits of the enzymatic hydrolysis products. Certain e-enzymes, such as β-lactamases, hydrolyze lactam antibiotics (e.g., penicillin, ampicillin). Their concentrated extracellular presence can create a “minefield” for inbound diffusing antibiotics.^126,127 Studies have also shown that when e-enzymes are localized within EPS, their denaturation over time is reduced.¹¹¹ (4) Enhanced gene exchange. Plasmid DNA (i.e., containing various genetic capabilities) is released by cells into the EPS matrix. Localization in EPSs allows for more efficient (subsequent) uptake by cells (i.e., via transformation). Gene exchange also occurs more efficiently via conjugation since cells can be held in place by EPSs to allow transfer through pili. Both transformation and conjugation within biofilms are used to transfer important AR genes among cells.^128,129 Additionally, the overall dynamic of biofilms represents a “breeding ground” for frequent resistant mutants.^130,131 (5) Quorum sensing. Cell to cell chemical communication, called quorum sensing (QS), occurs most efficiently within biofilms. The diffusion-slowing properties of EPSs result in the sustained buildup of signals above threshold concentrations where gene expression changes are triggered and occur within groups of cells.¹³² QS allows the cells to act in a coordinated manner in their activities, such as production/secretion of e-enzymes and the upregulation of virulence genes in bacteria. (6) Persister cells. A relatively small portion of cells within a biofilm (i.e., and even within actively growing cultures) are physiologically inactive and called persister cells.^133,134 Since most antibiotics target actively metabolizing cells, persisters are largely immune to antibiotic effects. If a biofilm community is effectively removed by antibiotics, persister cells remain viable (but inactive) and will become active again to repopulate once the antibiotic is no longer present. Many persistent and chronic infections are thought to occur via this mechanism. Persister cells are thought to be an inherent strategy for survival employed by bacteria against sudden stressors. Recently, a new class of antibiotics was discovered that are effective against MRSA and persister cells encompassing the dormant bacteria associated with troublesome chronic, recurring infections.¹³⁵ In this study, two synthetic retinoids, CD437 and CD1530, were demonstrated to be able to kill both growing and persister cells of MRSA individually and in combination with the antibiotic gentamicin. The retinoids exhibited the capability to disrupt the bacterial membrane lipid bilayers, which was suggested to enhance the diffusion of gentamicin into the cells when coadministrated. Further investigation led to the development of an analog of CD437 that maintained the ability to efficiently kill persisters while reducing the toxicity observed to mammalian cells.¹³⁵ (7) Amyloid proteins and structural stability. Amyloids are fibril-shaped proteins having a cross-β-sheet structure, where the β-sheets are stacked on top of each other.¹³⁶They are well-known for their involvement in human pathological conditions such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. For over a decade, however, it has been realized that bacteria also produce amyloids, which are multifunctional components of the EPS and therefore referred to as “functional amyloids (FA)”.^137,138 Major FAs include CsgA (i.e. curli protein) found in a range of bacteria¹³⁹ and FapC produced by members of the genus Pseudomonas. FA biogenesis is a tightly regulated process, and its release from the cell involves a series of related proteins, which transport the FA to the cell surface.¹⁴⁰ Once outside the cell, the individual proteins undergo fibrillation, where imperfect repeat portions of the protein aggregate to form a cross-β-sheet structure.^141,142 The interaction of FapC with membrane components such as the outer membrane lipopolysaccharide and a rhamnolipid (surfactant) enhance fibrillation.¹⁴³ Bacterial amyloids often extend from the cell surface into the EPS and survival properties,¹⁴⁴ acting as a form of structural rebar enhancing the physical architecture of biofilms. The genes for amyloids production and secreted formed appear to be present in a wide range of bacteria. Additionally, amyloids enhance the localization of quorum sensing signals and other small molecules within the biofilm matrix.^145,146 Given the refractory nature of amyloids, they have likely evolved as a collective adaptation, which contributes to the overall resiliency of biofilms¹⁴⁷ and likely serve other important roles as well. This presents amyloids as a target for potential antimicrobial drugs¹⁴⁸ and for the development of antibiofilm technologies, as discussed below.

Finally, biofilm bacteria often produce superantigens to evade host immune systems. They are less restrained by the selective pressures associated with antibacterial agents then their respective planktonic bacterial cells.¹⁴⁹ The combination of the protective synergistic effects of a biofilm constitute a formidable barrier to antibiotics. Their unique composition and structure shelter and protect the embedded microorganisms and facilitate their overall survival, whether in natural environments or in the human body. Biofilm bacteria display a 10- to 1000-fold higher resistance to antibiotic treatment than the planktonic form.¹⁶

4.2. Extracellular Bastion against Nanoparticle Delivery of Antibiotics?

While antibiotic delivery by NPs is a promising approach to overcome the biofilm barriers the ability of antimicrobials to penetrate and reach cells within a protective biofilm remains a prime limiting factor in controlling infections.^63,129,150 Numerous studies have demonstrated that NPs can disrupt the membranes of individual bacterial cells.^151-153 Also, NPs can be designed to contain a concentrated antibiotic package that can be released upon a single bacterial cell under the proper conditions. Therefore, the potential exists for NPs as an alternative approach to target bacterial biofilms depends upon them reaching cells.

Applications of metallic NPs in preventing and/or overcoming biofilm formation have included the use of AuNPs, AgNPs, Mg-NPs, ZnO-NPs, CuO-NPs, Fe₃O₄-NPs, and YF-NPs.^151-158 In order to obtain greater prevention of biofilms, smaller size and a higher surface-to-mass ratio has been recommended. However, while size limitation effects on NP penetration of biofilms appears reasonable, they have been generalized based on few detailed data and insufficient characterization of the actual biofilms.¹⁵⁹ In addition, NP shape also is considered to influence biofilm destruction. For example, NPs with rodlike shapes have appeared more effective than NPs with a spherical shape.¹⁵²

Fusogenic NPs, NP targeting, and triggered drug release from nanocarriers are ideal strategies to maximize the exposure of the biofilm to drugs. Enzymes, such as deoxyribonuclease (DNase) and protease, have been loaded into NPs to hydrolyze the biofilm structure for enhanced penetration of antibacterial drugs or NPs.⁶³ Magnetic NPs composed of iron oxide are also effective in the deep penetration into biofilms.¹⁶⁰

4.3. Penetrating the Extracellular Matrix to Reach Pathogen Cells.

The EPS surrounding cells consists of a meshwork of (i.e., mostly charged) polymer molecules linked together in varying densities.¹⁶¹ However, they also contain much water, the majority of which (i.e., approximately 99%) is not bound to EPSs (Figure 4).¹⁶² For this reason, the water within the EPS is considered as “immobilized” water.¹⁶² The polymers may be densely packed to form gels, or more-loosely spread to form pliant moveable slime matrices.¹⁶³ Lectin studies of EPSs in natural aggregates, coupled with confocal microscopy have shown the matrix to vary in composition over small (i.e., μm) spatial scales. Such small-scale variations in EPSs have been termed EPS microdomains.^164-166

Figure 4.

Protective attributes of biofilms. Bacteria within biofilms secrete a wide range of extracellular polymeric substances, collectively called EPS, which secure attachment, slow the diffusion of antimicrobials such as antibiotics and NPs, and protect bacterial cells from external stressors. Within a biofilm, there is enhanced gene exchange (e.g., AR genes) and secretion of extracellular enzymes such as β-lactamases, that can degrade antibiotics. Together, these attributes enhance resistance of biofilm infections to antibiotics and other antimicrobial approaches.

4.3.1. Density.

Molecules, such as antibiotics, diffuse through water-filled pore spaces between adjacent polymers. Therefore, the relative sizes of the pore spaces become a limiting constraint in the diffusion-slowing properties of a biofilm.¹⁶¹ When considering the movement of (larger) nanosized particles, this relative spacing becomes especially important. Estimates of NP diffusion in biofilms have been made.^167,168 Looser and less dense EPSs offer larger spacing between adjacent polymers and perhaps weaker polymer–polymer linkages, while more dense gels possess relatively smaller pore-spaces.¹⁶⁹ One of the first insightful laboratory studies to address this directly manipulated the densities and relative pore spacing of artificial hydrogels of EPSs. This was found to directly influence the penetration and diffusive movement of NPs as determined using super-resolution fluorescence microscopy.¹⁷⁰ More dense EPS gels reduce the movements of NPs compared with less dense EPSs, all else being constant. However, in situ biofilms exhibit much small-scale variability in the composition and density of EPSs making such direct measurements difficult. When linkages are abundant, the matrix is condensed to understand the porosity and exposed functional groups within the pores.

The effects of size, shape, and surface coatings of NPs on their penetration into biofilm matrices have not yet been thoroughly tested. To understand these effects more completely, it will require the characterization of different types of EPS matrices. EPS polymers are linked to each other through ion bridges and H-bonding.

Proteins and/or DNA are sometimes actively secreted by bacteria and comprise part of the EPS matrix. These components have been shown to provide structural stability and even rigidity to the biofilm.¹¹¹ DNA often forms complexes with specific proteins and polysaccharides to act as a structural rebar to reinforce the EPS against expansion and contraction.¹⁷¹ Amyloid proteins may also be used in this manner since their β-sheet configurations make them less easily degraded.^172,173

4.3.2. Size.

The size of NPs most certainly will limit their penetration into the EPS matrix (Figure 5). However, the surface properties or coatings of NPs have been shown to influence their migration into cells, biofilm matrices, and even human colonic mucus.^{76,135,174,175} Hydrophilic coatings on NPs such as polyethylene glycol (PEG) are being explored to penetrate different types of polymeric mucins. Functionalized gold NPs were found to be efficiently bound to natural estuarine biofilms.¹⁷⁶ The EPSs act as an efficient sponge to concentrate NPs.¹⁷⁷ This implies that certain NPs partially penetrate and are retained by the matrix. It additionally offers the potential for photodynamic activation. This uses infrared (IR) irradiation coupled with Au-NPs to treat and destroy superficial wound infections or other infections provided NPs can be specifically localized to the biofilm; a process that can also be potentially useful in removing biofilms from medical devices and implants.¹⁷⁸ Designing NPs with relatively small sizes and coronas (i.e., coatings) that can facilitate penetration of EPSs represents the major hurdle in the successful treatment of infections.^179,180

Figure 5.

Control of NP entry into biofilms. Small-scale changes in the relative density of EPS influences the penetration of NPs (red) into a biofilm. (A) Water-filled pore spaces (i.e., white spaces) in EPS (gray) are interconnected through which NPs diffuse into the matrix. It is hypothesized that (A) when EPS pore spacing is large NPs more easily penetrate the matrix. (B) However, when EPS are densely packed, pore spaces between adjacent polymers on average are small and limit their diffusivity. Therefore, only small diameter NPs (e.g., 10–30 nm dia), may penetrate to reach bacterial cells. The design of NPs with specific hydrated coatings (i.e., coronas) or surface properties may enhance their diffusion through EPS to reach infectious bacterial cells in a biofilm. NPs indicated “A” and “B” represent NPs with small diameters with different functional groups attached.

4.3.3. Surface Chemistry.

A large diversity of functionality has been imparted to the surfaces of NPs. Typically, the strategy of converting an inorganic surface to an organic surface with desired functionality relies on using a difunctional additive which contains a group which can bind or react with the inorganic surfaces and also has the desired organic functionality. For inorganic particles such as silica and related oxides, there are many commercially available silane coupling agents that have been developed over many years to perform this task and include large numbers of organic ligand chemistries.¹⁸¹ When the inorganic surface chemistry is less reactive with the silane coupling agents, commercially available titanate, zirconate, and aluminate coupling agents are available. There is extensive literature concerning the specific binding groups that are used for bioconjugation to many common NP surfaces including those for gold, silver, silica, carbons, quantum dots, magnetic NPs, and others.^182-185

In addition to the attachment of small organic functionalities, the attachment of proteins, polymers, and other macromolecular species is very important and extends the range of surface properties that can be added to NPs. The grafting of polymers to NPs relies on four general strategies, physisorption, grafting-to, grafting-from, and grafting through, each having its own benefits and drawbacks. Physisorption is achieved through an attractive (noncovalent) physical association between a portion of a polymer chain and a surface. Grafting-to is similar in that the polymer chain must diffuse to a surface, but it is then attached through a covalent or stronger chemical linkage. These grafting strategies both have the advantage of attaching polymer chains directly to the surface, but physisorbed chains easily desorb from the surface. Only low graft densities can be achieved with these methods because surface-associated chains sterically limit additional polymer chains from diffusing to the surface. Higher graft densities can be achieved using a grafting-from approach where polymer chains are grown from a tethered initiating group. In the grafting-through approach, the polymerization is not initiated at the surface, but polymerizable functional groups on the surface are quickly incorporated into the growing polymer, resulting in a tethered chain (though not always at the chain end). For many preformed proteins and biological macromolecules, grafting-to is the dominant approach while grafting-from is often used for synthetic polymers due to the degree of control and broader range of graft densities that can be obtained.^186,187

5. ONGOING APPROACHES USING NANOPARTICLE-BASED DELIVERY TO OVERCOME INFECTIONS

5.1. Can Old Antibiotics Be Reused with New Nanobased Delivery Approaches?

Most present-day antibiotics that have been introduced during the past 30 years are synthetic modifications of previously isolated natural forms.² The use of antibiotic-loaded NPs for bacterial targeting and delivery is an ideal strategy given its numerous advantages over the conventional formulations, including improved stability, controlled antibiotic release, targeted capability, and increased bioavailability.¹⁸⁸

Antibiotic-conjugated NPs exhibit higher antibacterial activities compared to free antibiotic or NPs alone. This suggests a synergistic effect between the NPs and antibiotics revealing different antibacterial mechanisms that are at play for these compounds.^49,189,190 In a study using AgNPs in combination with several different antibiotics, penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin demonstrated enhanced efficacies against E. coli and S. aureus, respectively.¹⁸⁹ This was performed in the absence of any direct intended associations of NPs with antibiotics. However, unintentional binding of the NP and antibiotic may have still occurred. Targeted delivery of cationic antibiotics, tetracycline and lincomycin, has been accomplished using bPEI-coated polyacrylic copolymer nanogels.¹⁹¹

5.2. Repurposing Bacterial Defenses into Offensive Attacks Using Nanobased Delivery of CRISPR-Cas Systems to Control Bacterial Metabolism.

Discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) and their functional role in the prokaryotic adaptive immune system (CRISPR-associated system, Cas) has led to significant advances in genetic engineering. CRISPR-Cas is a site-specific gene editing tool, utilizing a naturally occurring RNA-guided endonuclease that functions to protect against viral infection in bacteria and archaea.¹⁹² More than 6000 research publications have appeared since applications began just a few years ago.

CRISPR-Cas systems have been extensively studied and modified for use in genetic modification by selective insertion, deletion, and/or mutation of targeted genes in most cellular organisms. A key difference in this system is the use of a short single-guided RNA sequences (sgRNA) as the specificity-determining factor in the formation of a dsDNA break at a targeted site in the genome compared to the other protein-based DNA-binding gene-editing techniques. The use of CRISPR/Cas9 avoids the requirement of protein engineering methods to develop site-specific nucleases against a specific DNA target sequence. While not discussed here, there are numerous published reviews providing an in-depth summary of the other biotechnological advances and uses of CRISPR-Cas genome engineering.¹⁹²

Currently, applications of CRISPR-based antibacterials will provide several advances or modifications to the present methods available including the capability to target specific bacterial species within complex bacterial populations, delivering antibacterial agents to pathogenic bacteria, and delivery of therapeutic agents to infected host cells. Delivery of CRISPR-Cas9 antibacterial agents pose a unique challenge given in that the active protein–RNA complex (approximately 160-kDa) must be able to be transported across the bacterial membrane to be effective.¹⁹³

To direct the CRISPR-Cas9 machinery to attack rather than provide a defense for bacteria, CRISPR guided RNAs must be designed to target either virulence factors and/or essential chromosomal genes specific to a particular bacterial species. These CRISPR guided RNAs will subsequently induce dsDNA breaks and typically result in lethality, especially if they are chromosomally targeted.

One approach that has been utilized by several groups already employs the species-level specificity of bacteriophages for CRISPR-Cas delivery. Using inert phages specific to the bacterial species of interest, the CRISPR-Cas9-targeting specific bacterial genes can be encoded in a phagemid enabling for packaging of CRISPR-Cas machinery into phage capsid.¹⁹⁴ In 2014, a research study using phage-encoded CRISPR-Cas9 targeting antibiotic resistance in virulent strains of S. aureus concluded that the CRISPR-Cas9 antibacterials demonstrated species-specific pathogenic efficacy while sparing the non-targeted bacterial species This is an essential feature that is ideal for the development of novel, effective antibacterial agents, especially in the presence of the helpful human microbiome^.195

By tuning the chemical properties of drug delivery of the CRISPR-Cas9, the targeting of healthy, uninfected cells can be prevented, allowing for the usage of lower dosage levels and thereby, reducing the overall cost of the treatment. Due to the structural diversity and wide range of sizes of phages, the traditional NP delivery strategies used are not currently reasonable nor practical. The use of different porous NPs to absorb phage-based treatments has been previously demonstrated; however, these methods are ineffective for large, nonsymmetrical phages, as the pore sizes are too small to effectively sorb the phages.¹⁹⁶

One of the primary applications of the CRISPR-Cas9 system that has immense potential for use in metabolic engineering is by controlling the regulatory signaling pathways involved in the complex chemical communications for most cellular functions and synthesis pathways. The CRISPR-Cas9 gene-editing method has been used to facilitate the synthesis of valuable metabolic compounds, while concurrently reducing production of unwanted compounds and byproducts. This gene-editing system utilizes dCas9 to generate multiple tools that can block activities at a single site along complex multistep signaling pathways with no crosstalk. Since the “instructions” for the multiple steps in each signaling pathway are encoded in the genomic DNA, these processes are able to be controlled at each step—by either blocking or enabling transcription of the DNA at the promoter.¹⁹⁷

5.3. Strategies in Disrupting Bacterial Cell–Cell Communication.

5.3.1. Nanoparticles as Signal Sponges.

To combat the increasing development of antibiotic resistance, one of the noncytotoxic approaches that has been proposed to control bacterial infections involves the use of NPs targeting the disruption of bacterial cell–cell chemical communication mechanisms and prevent the acquisition of resistance mechanisms toward antimicrobial therapies. The primary strategy that has been proposed for inhibiting biofilm formation involves targeting and interfering with quorum sensing (QS) molecules (Figure 6).¹⁹⁸

Figure 6.

Targeting quorum sensing. Schematic of QS in bacteria as well as methods to block this signaling mechanism. AHL dependent QS within biofilms (left) can be blocked using competitive QS inhibition that outcompetes AHL for AHL receptors (middle) or quorum quenching enzymes that inactivate AHL signals (right) [Reprinted from ref 210. Copyright 2018 Beitelshees et al.].

QS communication involves the production, detection, and response to chemical environmental signaling molecules called autoinducers (AIs).¹⁹⁹ AIs are secreted chemical signaling factors that are intracellularly synthesized and released into the extracellular environment, disperse through diffusion, and are recognized by adjacent cells and result in the alteration of gene expression accordingly dependent on population density. QS signals accumulate in the local environment as bacterial population density increases requiring threshold levels to be reached to produce a transcriptional response. The minimum behavioral unit, described as a quorum, corresponds to a particular bacterial cell density required to produce signals above threshold levels of target the signal and elicit a transcriptional response.

QS mechanisms are known to modulate a diversity of biological processes in bacteria involved in bioluminescence, sporulation, competence, antibiotic production, biofilm formation, and virulence factor secretion.^200-202 In P. aeruginosa, the QS system is well-characterized and known to regulate the production of several compounds identified to play key roles in biofilm formation, including rhamnolipids, lectins (LecA/LecB), pyochelin, and pyoverdine siderophores.^203-206 Different QS systems have been identified and are classified into several groups according to the type of autoinducer used, such as acyl-homoserine lactones (AHLs or AI-1), furanosyl borate diesters (AI-2s) and autoinducing peptides (AIPs), diffusible signal factors (DSFs), and diketopiperazines (DKPs).²⁰⁰

Based on the key roles that QS plays in bacterial virulence and pathogenesis, the disruption of bacterial communication systems is an emerging approach/strategy that has been described for the development of novel antimicrobial agents. This approach is referred to as “quorum quenching” (QQ) and includes any strategy targeted to interfere and/or disrupt microbial QS signaling (Figure 6). Inhibition can be done at several different steps in the signaling process: inhibition of autoinducer synthesis, degradation of autoinducer, and interception of its interaction(s) with the cognate receptor(s).²⁰⁷ Based upon the type of regulation elicited by QS signal(s), the agents used for targeting QS signaling should either inhibit or stimulate the QS-regulated gene expression, accordingly. As observed in the human pathogen Vibrio cholerae, QS signaling was demonstrated to repress biofilm formation and virulence factor production.^208,209

NPs and related nanomaterials have been designed and utilized as QS inhibitors and NP-based carriers of compounds and/or molecules that possess QS quenching activity. In Vibrio fischeri, initial efforts using SiO₂-NPs functionalized with β-cyclodextrin (β-CD) demonstrated an ability to bind AHLs and quench QS signaling, determined at the transcriptional level by the down-regulation of bacterial QS genes.²¹¹

Surface functionalized NPs with β-CD or N-acylated homoserine lactonase proteins (AiiA) can interfere with QS signaling molecules preventing them from reaching their designated target cognate receptor and thereby inhibiting the signal/receptor interaction.¹⁴ However, the hydrophobic cores of a β-CD are nonspecific in their binding of AHLs and/or other organic molecules. Future work should target incorporations of the binding domains for LuxR receptor proteins onto NPs to increase the specificity of AHL chelation by NPs. Bacterial QS systems function as major regulatory mechanisms of pathogenesis and are involved in the formation of biofilm structures. Using QS, bacterial populations are able to coordinate their gene expression, acquiring a competitive advantage to respond to environmental changes.²⁰⁰ Consequently, QS systems have promoted the formation of antibiotic tolerant communities. Therefore, bacterial biofilms pose a significant challenge to the efficacy of conventional antibiotics given that they serve as a recalcitrant form of bacterial growth that increases bacterial resistance to conventional antibiotics.¹⁴

Several studies demonstrated that bacteria are able to sense and store information regarding surface chemical exposure through the molecule cyclic adenosine monophosphate (cAMP). More recently, it was shown that the coupled cycling of cAMP levels and type IV pili (TFP) activities are coordinated to allow bacteria to adaptively adhere to surfaces. Interestingly, the same cAMP pattern was displayed by descendent cells across multiple generations allowing encompassed cells to persist in a coordinated, nonmotile state and remain as a biofilm.²¹²

5.3.2. Signaling Analogs.

The identification and development of natural or synthetic chemical compounds and/or enzymes that facilitate quorum-sensing inhibition (QSI) has been an emerging approach to combat bacterial pathogenesis. Disruption of QS signaling mechanisms has been demonstrated by targeting the signaling molecules, signal biogenesis, or signal detection (Figure 6).²¹³

Interfering with QS may provide a powerful, nonlethal strategy that does not select for resistance, and ultimately a tool for controlling virulence and antibiotic tolerance in QS pathogens. Such a tool can also be applied within antimicrobial chemotherapy to overcome bacterial infections. Currently, the methods that have been used to disrupt QS include: (1) antagonizing signal binding to LuxR-family receptor, (2) inhibition of signal production, (3) degrading signals, (4) trapping signals, and (5) suppression of synthase and receptor activities, stabilities, or productions (Figure 7).²¹⁴

Figure 7.

Quorum quenching potential of nanomaterials. The agents interrupt QS signaling in different ways: (a) suppressing the production of autoinducers via reduction the activity of LuxI-type cognate receptor synthase and ATP binding cassettes, which produces AHLs and oligopeptides respectively; (b) degrading the autoinducers; (c) inhibiting the binding of autoinducers to the LuxR-type receptor protein; and (d) inhibiting biofilm formation [Reprinted with permission from ref 220. Copyright 2017 Taylor & Francis].

A diversity of compounds for QSI “signal analogs” have been identified and reported to inhibit bacterial communication processes.^215-218 For example, analogs containing alterations in the acyl side chains of 3-oxo-C6-AHL for V. fischeri, 3-oxo-C12-AHL for P. aeruginosa, and 3-oxo-C8-AHL for Agrobacterium tumefaciens inhibited the binding of native AHLs.^215,219 The cognate receptors were able to bind analogs at higher affinities compared to native AHL ligands, which resulted in the analogs inactivating AHL-mediated gene expression. The most extensively utilized QSI strategy that has been currently investigated involves the degradation and modification of QS signals in addition to the use of autoinducer antagonists. Smith and colleagues constructed a library of synthetic analogs to the P. aeruginosa las QS molecule, 3-oxo-C12-AHL, by substituting the homoserine lactone moiety with different alcohols, amines, and/or a 5- or 6-membered ring.^216,217 Using a lasI promoter-fused gfp reporter strain for high-throughput screening of the compounds, three compounds were identified as antagonists. 3-Oxo-C12-(2-aminophenol) and 3-oxo-C12-(aminocyclopentanol) were demonstrated to inhibit the LasR activation attributed to the 3-oxo-C12-AHL–LasR interaction. Alternatively, 3-oxo-C12-(aminocyclohexanone) was observed to target not only LasR but also RhlR, which is an additional QS receptor in P. aeruginosa.

Similarly, a synthetic analog of C4-AHL, N-decanoyl cyclopentyl-amide (C10-CPA) was also reported to target both LasR and RhlR.²²¹ C10-CPA inhibits lasB and rhlA gene activation by 3-oxo-C12-AHL and C4-AHL resulting in reductions in the elastase, pyocyanin, and rhamnolipid levels and biofilm formation. It was shown that changes in amide function coupled between the lactone ring and the fatty acid influence the AHL binding activity to receptor proteins, due to the amide forming hydrogen bonds with conserved tyrosine and aspartic acid in the AHL binding pocket.²²² Compounds containing this modification have shown antagonistic behaviors with the V. fischeri LuxR receptor.

Selective and broad-spectrum antagonists effective across multiple species have been developed. C8-AHL, C10-AHL, 4-bromophenylpropionyl-AHL, and 4-iodophenylacetyl-AHL have been demonstrated to antagonize the AHL-binding to the TraR, LuxR, and LasR receptor proteins of A. tumefaciens, V. fischeri, and P. aeruginosa, respectively.²²³ It is still not well understood how the conformational change in the receptor occurs to distinguish between a “true ligand” (agonist) and “fake ligand” (antagonist). The crystal structures of the receptor–AHL binding form are available for a few species; however, it is imperative that we obtain both structures: an antagonist-binding form and a ligand-free form. Targeting the production or synthesis of AHL signals is an additional strategy for quorum sensing inhibition. Consequently, if no AHL signals are produced, no QS occurs, and therefore, bacterial communication is prevented from taking place. In an earlier study, several analogs of S-adenosyl-methionine (SAM), which is the second substrate for LuxI synthases, was demonstrated to inhibit the LuxI reaction.²²⁴ Recently a C8-AHL analog was identified to bind to AHL synthase resulting in the inhibition of its enzymatic activity.²²⁵ AHL synthesis inhibitors that are transition state analogs of MTAN (5′-methylthioadenosine/S-adenosylhomocysteine hydrolase) provide a strategy of inhibition that blocks not only AHL production but also the generation of AI-2 and pathogenicity.²²⁶ Coupling the recent results in QS disruptors with nanotargeted designs could provide fruitful approaches for future improvements in disrupting chemical communication in biofilms and ultimately in controlling infection-based pathogenicity itself.

5.3.3. Degradation of AHLs by Nanoparticle-Based Enzymes or Oxidants.

An alternative approach to the use of signal analogs involves the enzymatic breakdown of AHL signals (Figure 6). In natural bacteria, this occurs by two major classes of enzymes, called lactonases and acylases, both of which are produced by Gram-positive strains. The majority of work has focused on the lactonases, which hydrolyzes the lactone of an AHL. A lactonase enzyme from a Gram-positive Bacillus strain and is designated “AiiA”.²²⁷ Some lactonases can be specific in the AHLs that they target for hydrolysis. In Rhodococcus erythropolis, QsdA is a phophotriesterase (PTE) like protein capable of degrading AHLs with acyl chains of C6–C14 in length.²²⁸ AiiM protein in Microbacterium testaceum, an isolate from a potato leaf, was determined by an α/β hydrolase exhibiting a preference of C6 to C12-AHLs with 3-oxo substitutions compared to those without the substitution.²²⁹ A series of BpiB proteins (i.e., designated BpiB01, BpiB04, and BpiB07) isolated from soil-derived metagenomic libraries with traI-lacZ A. tumefaciens reporter strain, hydrolyzed the 3-oxo-C₈-AHL. This also inhibited P. aeruginosa swarming motility and biofilm formation, which is regulated by C₄-AHL and 3-oxo-C₁₂-AHL.²³⁰ More recently, a new type of AHL lactonase isolated from the marine bacterium Pseudoalteromonas byunsanensis was described to encode a hybrid membrane protein containing GDSL hydrolase domain at the N-terminal of an RND (resistance-nodulation-cell-division)-type multidrug efflux transporter.²³¹ The truncated form containing the GDSL hydrolase function was designated as QsdH and catalyzed hydrolyses of C₄ to C₁₂-AHLs (with or without 3-oxo substitution). The use of hydrolytic enzymes presents a signal specific tool to inhibit specific AHL signals, which can be potentially linked to NPs.

5.4. Degradation of Biofilms by Near-Infrared (NIR) Induced Oxidant Photoinactivation and Amyloid Inhibition.

As a promising strategy for antibacterial therapy, photodynamic therapy (PDT) has gained considerable attention recently. Photosensitizers (PSs) can be used to generate ROS upon visible light or infrared illumination in the presence of oxygen.^178,232 More recently, considerable progress has been made on lanthanide-doped upconversion NPs (UCNPs) due to their unique properties of converting NIR light to ultraviolet or visible emission and activating PSs nearby for antibacterial applications. Sun and colleagues²¹¹ designed and synthesized a series of antimicrobial nanocomposite membranes containing novel UCNPs, which demonstrated potent antimicrobial activity upon activation by NIR against both Gram-positive S. aureus and Gram-negative E. coli. These nanocomposite membranes were revealed to provide potential practical applications in antibacterial wound dressing materials that are capable of sterilizing wound areas multiple times via external NIR irradiation.

As alluded to previously (section 4.1), amyloid proteins provide structural resiliency for the biofilm matrix. Therefore, the ability to decrease amyloid components or reduce fibrillation are being explored to reduce the structural efficacy of the biofilm itself. Recently, two well studied examples of plant-derived polyphenols are epigallocatechin gallate (EGCG) and penta-O-galloyl-β-d-glucose (PGG). EGCG was first shown to reduce amyloidogenic polypeptides.²³³ Subsequently, both EGCG and PGG have been shown to redirect the aggregation of FapC monomers oligomeric species, which ultimately results in a reduction in biofilm formation and susceptibility to antibiotic treatments.^143,234 This demonstrates the biotechnologic potential for development of NP surfaces to act as carriers for delivery of such small molecules to bacterial cells, especially during the early stages of biofilm formation to reduce/prevent their resiliency to removal.

6. CONCLUSION

The applications of engineered NPs directly as antimicrobial agents or as carriers for the targeted delivery of antimicrobials offers much potential for removal of biofilm-related infections. However, fundamental issues must be understood and addressed before the resilient EPS matrix of biofilms can be successfully navigated. Significant hurdles remain in designing NPs to penetrate dense gel EPS matrices that often protect biofilms and in the controlled release of the antimicrobial agents. While smaller diameter (<20 nm) NPs with hydrophilic coatings may, at present, appear to have greater facility in penetrating EPS, the adaptable nature of bacteria and the biofilm itself will likely limit this as the only way to penetrate the biofilm. Optimizing delivery platforms and intelligent design of NPs will be essential for long-term efficacy. Finally, the precision, targeted delivery of antibiotics into specific biofilm infection sites is of paramount importance in the control of human infections but presents substantial challenge in NP development. At present, targeted activation may offer the most efficient solution. Further studies using controlled forms of hydrogels are needed to understand the natural variability present in biofilm matrices, and how these can be penetrated using NPs.

ABBREVIATIONS

AgNPs

silver nanoparticles

AHL

acyl-homoserine lactone

AiiA

N-acylated homoserine lactonase proteins

AIPs

autoinducing peptides

AIs

autoinducers

AMPs

antimicrobial peptides

AMR

antimicrobial resistance

ARIs

antibiotic resistant infections

β-CD

β-cyclodextrin

CPP

cell penetrating peptide

CRISPR

clustered regularly interspaced short palindromic repeats

CW

cell wall

DKPs

diketopeptides

DSFs

diffusible soluble factors

EPIs

efflux pump inhibitors

EPSs

extracellular polymers

LPS

lipopolysaccharide

MIC

minimum inhibitory concentration

MRSA

methicillin-resistant Staphylococcus aureus

NPs

nanoparticles

OM

outer membrane

PDT

photodynamic therapy

PM

plasma membrane

PMBN

polymyxin B nonapeptide

PSs

photosensitizers

QQ

quorum quenching

QS

quorum sensing

QSI

quorum sensing inhibition

ROS

reactive oxygen species

sgRNA

single-guided RNA sequences

SLNs

solid lipid nanoparticles

UCNPs

upconversion nanoparticles