Intrinsic antimicrobial resistance: Molecular biomaterials to combat microbial biofilms and bacterial persisters.

The escalating rise in antimicrobial resistance (AMR) coupled with a declining arsenal of new antibiotics is imposing serious threats to global public health. A pervasive aspect of many acquired AMR infections is that the pathogenic microorganisms exist as biofilms, which are equipped with superior survival strategies. In addition, persistent and recalcitrant infections are seeded with bacterial persister cells at infection sites. Together, conventional antibiotic therapeutics often fail in the complete treatment of infections associated with bacterial persisters and biofilms. Novel therapeutics have been attempted to tackle AMR, biofilms, and persister-associated complex infections. This review focuses on the progress in designing molecular biomaterials and therapeutics to address acquired and intrinsic AMR, and the fundamental microbiology behind biofilms and persisters. Starting with a brief introduction of AMR basics and approaches to tackling acquired AMR, the emphasis is placed on various biomaterial approaches to combating intrinsic AMR, including (1) semi-synthetic antibiotics; (2) macromolecular or polymeric biomaterials mimicking antimicrobial peptides; (3) adjuvant effects in synergy; (4) nano-therapeutics; (5) nitric oxide-releasing antimicrobials; (6) antimicrobial hydrogels; (7) antimicrobial coatings. Particularly, the structure-activity relationship is elucidated in each category of these biomaterials. Finally, illuminating perspectives are provided for the future design of molecular biomaterials to bypass AMR and cure chronic multi-drug resistant (MDR) infections.

Biocide loaded shear-thinning hydrogel with anti-biofilm efficacy cures topical infection.

The continuous intervention of multidrug-resistant (MDR) bacterial infections worsens and slows the dynamicity of natural wound healing processes. Fortunately, antibiotics, metal ions, or metal nanoparticle-loaded antimicrobial hydrogels have been developed to tackle infections at injury sites and speed up the healing process. Despite their success, these marketed released based hydrogels are still limited owing to their lack of broad-spectrum activity, inability to tackle biofilm-associated infections, susceptibility towards resistance development, fast release kinetics, and mild to moderate toxicity. To address these shortcomings, we report the development of a biocompatible, shear-thinning, injectable gellan-gelatin hydrogel loaded with a peptidomimetic potent biocide (ASAM-10). The hydrogel upon sustained biocide release (60% within 72 h), displays a broad-spectrum antibacterial activity with negligible in vitro (hemolysis < 20%) and in vivo toxicity (no adverse effects on dermal layer of mice). Besides tackling bacterial dormant subpopulation (1-6 Log reduction), the optimized hydrogel is able to disrupt the preformed bacterial biofilm and even kill the biofilm-trapped pathogens with enhanced pathogenicity. Above all, the lead hydrogel was proficient in tackling methicillin-resistant Staphylococcus aureus (MRSA) wound infections in a mouse model through its safe topical administration. Overall, the biocide-loaded hydrogel can be considered as a promising candidate to combat MDR chronic infections at the wound site.

Facially Amphiphilic Bile Acid-Functionalized Antimicrobials: Combating Pathogenic Bacteria, Fungi, and Their Biofilms.

We report facially amphiphilic bile acid-based antimicrobials with a broad spectrum of activity against both bacterial and fungal pathogens and negligible detrimental effects on mammalian cells. Two lead compounds eliminated dormant subpopulations of various bacterial species, unlike conventional antibiotics. The lead compounds were also effective in eradicating biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. Additionally, these compounds substantially inhibited the formation of fungal biofilms (C. albicans). Mechanistic investigations revealed the membrane-active nature and endogenous reactive oxygen species (ROS) induction ability of these compounds. Finally, no detectable resistance was developed by the bacterial strains against this class of membrane-targeting antimicrobials.

Facial amphiphilic naphthoic acid-derived antimicrobial polymers against multi-drug resistant gram-negative bacteria and biofilms.

Inspired by the facial amphiphilic nature and antimicrobial efficacy of many antimicrobial peptides, this work reported facial amphiphilic bicyclic naphthoic acid derivatives with different ratios of charges to rings that were installed onto side chains of poly(glycidyl methacrylate). Six quaternary ammonium-charged (QAC) polymers were prepared to investigate the structure-activity relationship. These QAC polymers displayed potent antibacterial activity against various multi-drug resistant (MDR) gram-negative pathogens such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Polymers demonstrated low hemolysis and high antimicrobial selectivity. Additionally, they were able to eradicate established biofilms and kill metabolically inactive dormant cells. The membrane permeabilization and depolarization results indicated a mechanism of action through membrane disruption. Two lead polymers showed no resistance from MDR-P. aeruginosa and MDR-K. pneumoniae. These facial amphiphiles are potentially a new class of potent antimicrobial agents to tackle the antimicrobial resistance for both planktonic and biofilm-related infections.

Membrane-Active Metallopolymers: Repurposing and Rehabilitating Antibiotics to Gram-Negative Superbugs.

Among multiple approaches to combating antimicrobial resistance, a combination therapy of existing antibiotics with bacterial membrane-perturbing agents is promising. A viable platform of metallopolymers as adjuvants in combination with traditional antibiotics is reported in this work to combat both planktonic and stationary cells of Gram-negative superbugs and their biofilms. Antibacterial efficacy, toxicity, antibiofilm activity, bacterial resistance propensity, and mechanisms of action of metallopolymer-antibiotic combinations are investigated. These metallopolymers exhibit 4-16-fold potentiation of antibiotics against Gram-negative bacteria with negligible toxicity toward mammalian cells. More importantly, the lead combinations (polymer-ceftazidime and polymer-rifampicin) eradicate preformed biofilms of MDR E. coli and P. aeruginosa, respectively. Further, ß-lactamase inhibition, outer membrane permeabilization, and membrane depolarization demonstrate synergy of these adjuvants with different antibiotics. Moreover, the membrane-active metallopolymers enable the antibiotics to circumvent bacterial resistance development. Altogether, the results indicate that such non-antibiotic adjuvants bear the promise to revitalize the efficacy of existing antibiotics to tackle Gram-negative bacterial infections.

Isoamphipathic antibacterial molecules regulating activity and toxicity through positional isomerism.

Peptidomimetic antimicrobials exhibit a selective interaction with bacterial cells over mammalian cells once they have achieved an optimum amphiphilic balance (hydrophobicity/hydrophilicity) in the molecular architecture. To date, hydrophobicity and cationic charge have been considered the crucial parameters to attain such amphiphilic balance. However, optimization of these properties is not enough to circumvent unwanted toxicity towards mammalian cells. Hence, herein, we report new isoamphipathic antibacterial molecules (IAMs: 1-3) where positional isomerism was introduced as one of the guiding factors for molecular design. This class of molecules displayed good (MIC = 1-8 µg mL-1 or µM) to moderate [MIC = 32-64 µg mL-1 (32.2-64.4 µM)] antibacterial activity against multiple Gram-positive and Gram-negative bacteria. Positional isomerism showed a strong influence on regulating antibacterial activity and toxicity for ortho [IAM-1: MIC = 1-32 µg mL-1 (1-32.2 µM), HC50 = 650 µg mL-1 (654.6 µM)], meta [IAM-2: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 98 µg mL-1 (98.7 µM)] and para [IAM-3: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 160 µg mL-1 (161.1 µM)] isomers. Co-culture studies and investigation of membrane dynamics indicated that ortho isomer, IAM-1 exerted more selective activity towards bacterial over mammalian membranes, compared to meta and para isomers. Furthermore, the mechanism of action of the lead molecule (IAM-1) has been characterized through detailed molecular dynamics simulations. In addition, the lead molecule displayed substantial efficacy against dormant bacteria and mature biofilms, unlike conventional antibiotics. Importantly, IAM-1 exhibited moderate in vivo activity against MRSA wound infection in a murine model with no detectable dermal toxicity. Altogether, the report explored the design and development of isoamphipathic antibacterial molecules to establish the role of positional isomerism in achieving selective and potential antibacterial agents.

Engineering Antimicrobial Polymer Nanocomposites: In Situ Synthesis, Disruption of Polymicrobial Biofilms, and In Vivo Activity.

The increasing incidence of microbial infections and a limited arsenal of effective antibacterial and antifungal agents have entailed the need for new broad-spectrum therapeutics. Polymer-inorganic nanocomposites have emerged as an integral choice of antimicrobials but are limited by complicated synthesis, narrow-spectrum activity, and poor in vivo efficacy. Herein, chloride counterions of a nontoxic, moderately antibacterial polymer have been explored for in situ nanoprecipitation-based synthesis of water-soluble polymer-silver chloride nanocomposites. With the controlled release of silver ions, the nanocomposites were highly active against multidrug-resistant bacteria as well as fluconazole-resistant fungi. Alongside the elimination of metabolically inactive bacterial cells, the nanocomposites disrupted polymicrobial biofilms, unlike antibiotics and only silver-based ointments. This underlined the role of the engineered composite design, where the polymer interacted with the biofilm matrix, facilitating the penetration of nanoparticles to kill microbes. Further, the nanocomposite diminished Pseudomonas aeruginosa burden in mice skin infection (>99.9%) with no dermal toxicity proving its potential for clinical translation.

Macromolecular Nanotherapeutics and Antibiotic Adjuvants to Tackle Bacterial and Fungal Infections.

The escalating rise in the population of multidrug-resistant (MDR) pathogens coupled with their biofilm forming ability has struck the global health as nightmare. Alongwith the threat of aforementioned menace, the sluggish development of new antibiotics and the continuous deterioration of the antibiotic pipeline has stimulated the scientific community toward the search of smart and innovative alternatives. In near future, membrane targeting antimicrobial polymers, inspired from antimicrobial peptides, can stand out significantly to combat against the MDR superbugs. Many of these amphiphilic polymers can form nanoaggregates through self-assembly with superior and selective antimicrobial efficacy. Additionally, these macromolecular nanoaggregrates can be utilized to engineer smart antibiotic-delivery system for on-demand drug-release, exploiting the infection site's micoenvironment. This strategy substantially increases the local concentration of antibiotics and reduces the associated off-target toxicity. Furthermore, amphiphilc macromolecules can be utilized to rejuvinate obsolete antibiotics to tackle the drug-resistant infections. This review article highlights the recent developments in macromolecular architecture to design numerous nanostructures with broad-spectrum antimicrobial activity, their application in fabricating smart drug delivery systems and their efficacy as antibiotic adjuvants to circumvent antimicrobial resistance. Finally, the current challenges and future prospects are briefly discussed for further exploration and their practical application in clinical settings.

Amphiphilic Cationic Macromolecules Highly Effective Against Multi-Drug Resistant Gram-Positive Bacteria and Fungi With No Detectable Resistance.

The ever increasing threats of Gram-positive superbugs such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococccus faecium (VRE) are serious matter of concern worldwide toward public health. Such pathogens cause repeated recurrence of infections through the formation of biofilms which consist of metabolically inactive or slow growing dormant bacterial population in vast majority. Concurrently, dispersal of biofilms originates even more virulent dispersed cells responsible for pathogenesis. Along with this, fungal infections most commonly associated with Candida albicans also created a major complicacy in human healthcare. Moreover, concomitant survival of C. albicans and MRSA in a multispecies biofilms created extremely complicated polymicrobial infections. Surprisingly, infections associated with single species biofilm as well as multiple species biofilm (co-existence of MRSA and C. albicans) are almost untreatable with conventional antibiotics. Therefore, the situation demands an urgent development of antimicrobial agent which would tackle persistent infections associated with bacteria, fungi and their biofilms. Toward this goal, herein we developed a new class of branched polyethyleneimine based amphiphilic cationic macromolecules (ACMs) bearing normal alkyl, alkyl ester and alkyl amide moieties. An optimized compound with dual activity against drug-resistant bacteria (MIC = 2-4 µg/mL) and fungi (MIC = 4-8 µg/mL) was identified with minimal toxicity toward human erythrocytes (HC50 = 270 µg/mL). The lead compound, ACM-AHex (12) displayed rapid bactericidal and fungicidal kinetics (>5 log CFU/mL reduction within 1-4 h). It also killed metabolically dormant stationary (MRSA and VRE) and persister (S. aureus) cells. Moreover, this compound was able to disrupt the preformed biofilm of MRSA and reduced the bacterial burden related to the dispersed cells. It showed significant proficiencies to eliminate polymicrobial biofilms of MRSA and C. albicans. Bacteria also could not develop any resistant against this class of membrane active molecules even after 15 days of successive passages. Taken together this class of macromolecule can be developed further as a dual therapeutic agent to combat infections associated with bacterial and fungal co-existence.

Hydrophobicity-Modulated Small Antibacterial Molecule Eradicates Biofilm with Potent Efficacy against Skin Infections.

The role of molecular arrangement of hydrophobic and hydrophilic groups for designing membrane-active molecules remains largely ambiguous. To explore this aspect, herein we report a series of membrane-active small molecules by varying the spatial distribution of hydrophobic groups. The two terminal amino groups of linear triamines such as diethylene triamine, bis(trimethylene)triamine, and bis(hexamethylene)triamine were conjugated with cationic amino acids bearing variable side chain hydrophobicity (such as diaminobutyric acid, ornithine, and lysine). The hydrophobicity was also modulated through conjugation of different long chain fatty acids with the central secondary amino group of the triamine. Molecules with constant backbone hydrophobicity displayed an enhanced antibacterial activity and decreased hemolytic activity upon increasing the side chain hydrophobicity of amino acids. On the other hand, increased hydrophobicity in the backbone introduced a slight hemolytic activity but a higher increment in antibacterial activity, resulting in better selective antibacterial compounds. The optimized lead compound derived from structure-activity-relationship (SAR) studies was the dodecanoyl analogue of a lysine series of compounds consisting of bis(hexamethylene)triamine as the backbone. This compound was active against various Gram-positive and Gram-negative bacteria at a low concentration (MIC ranged between 3.1 and 6.3 µg/mL) and displayed low toxicity toward mammalian cells (HC50 = 890 µg/mL and EC50 against HEK = 85 µg/mL). Additionally, it was able to kill metabolically inactive bacterial cells and eradicate preformed biofilms of MRSA. This compound showed excellent activity in a mouse model of skin infection with reduction of ∼4 log MRSA burden at 40 mg/kg dose without any sign of skin toxicity even at 200 mg/kg. More importantly, it revealed potent efficacy in an ex vivo model of human skin infection (with reduction of 85% MRSA burden at 50 µg/mL), which indicates great potential of the compound as an antibacterial agent to treat skin infections.

Cyclam-based antibacterial molecules eradicate Gram-negative superbugs with potent efficacy against human corneal infection.

Cyclam-based antibacterial molecules (CAMs) that display potent activity against both the planktonic and stationary phase of multidrug-resistant Gram-negative bacteria were rationally designed. The optimized compound retained its activity in human plasma and eradicated preformed biofilms. It also revealed excellent potency in an ex vivo model of human corneal infections with negligible propensity of resistance development. This indicated the potential of this class of compound as a future antibacterial agent to tackle human corneal infections.

Amino-Acid-Conjugated Polymer-Rifampicin Combination: Effective at Tackling Drug-Resistant Gram-Negative Clinical Isolates.

Rapid emergence of multidrug-resistant Gram-negative pathogens coupled with their biofilm-forming capability have set a clinical ultimatum to global public health with an increasing rate of mortality. Recently, the World Health Organization (WHO) identified Acinetobacter baumannii, Pseudomonas aeruginosa, and enterobacteriaceae (Klebsiella pneumoniae, E. coli, etc.) as the pathogens of top priority because of their ability to cause difficult-to-treat life-threatening infections insusceptible to conventional antibiotic therapy. Hence, the severity of the current scenario necessitates the development of a potent therapeutic agent with a smart strategy. Toward this goal herein, we have explored the potency of membrane-active, amino-acid-conjugated polymers (ACPs) to combat notorious Gram-negative pathogens in combination with intrinsically resistant antibiotic rifampicin. The polymers were able to enhance the antibacterial potency of rifampicin against different drug-resistant Gram-negative bacteria by 4-66 fold. The combination, which consisted of glycine-conjugated polymer, ACP-1 (Gly), and rifampicin was rapidly bactericidal in nature. This combination also exhibited significant potency to disrupt the preformed biofilms of drug-resistant strains of P. aeruginosa and E. coli. More importantly, a negligible propensity of resistance development was observed against this combination, whereas a high level of resistance development was observed against the last-resort antibiotic, colistin. Furthermore, ACP-1 (Gly) displayed noticeably good 50% lethal dosage in different administration routes (LD50 (subcutaneous) > 179 mg/kg and LD50 (intraperitoneal) = 100 mg/kg) in a mouse model. Additionally, ACP-1 (Gly) did not show any adverse effect on mouse skin even at 200 mg/kg. Therefore, the results ensured that the ACP-1 (Gly) is suitable for both topical as well as systemic application. Altogether, the results indicated significant promises of the combination for further development as a therapeutic regimen to tackle the outbreak of critical Gram-negative bacteria.

Amino Acid Conjugated Polymers: Antibacterial Agents Effective against Drug-Resistant Acinetobacter baumannii with No Detectable Resistance.

An optimum hydrophilic/hydrophobic balance has been recognized as a crucial parameter in designing cationic polymers that mimic antimicrobial peptides (AMPs). To date, this balance was achieved either by hydrophilicity variation through altering the nature and the number of cationic charges or by hydrophobicity modulation through incorporation of alkyl groups of different chain lengths. However, how the hydrophobicity variation through AMPs' building blocks-amino acids-influences the antibacterial efficacy of AMP-mimicking cationic polymers has rarely been explored. Toward this goal, herein we report a class of amino acid conjugated polymers (ACPs) with tunable antibacterial activity through a simple post-polymer-functionalization strategy. Our polymeric design comprised a permanent cationic charge in every repeating unit, whereby the hydrophobicity was tuned through incorporation of different amino acids. Our results revealed that the amino acid alteration has a strong influence on antibacterial efficacy. Upon increasing the amino acid side-chain hydrophobicity, both the antibacterial activity (against broad spectrum of bacteria) and toxicity increased. However, the distinct feature of this class of polymers was their good activity against Acinetobacter baumannii-the top most critical pathogen according to WHO, which has created an alarming situation worldwide, causing the majority of infections in humans. A nontoxic (no hemolysis even at 1000 µg/mL) ACP including a glycine residue (ACP-1 (Gly)) showed very good activity (MIC = 8-16 µg/mL) against both drug-sensitive and drug-resistant strains of A. baumannii, including clinical isolates. This polymer not only was rapidly bactericidal against growing planktonic A. baumannii but also killed nondividing stationary-phase cells instantaneously (<2 min). Moreover, it eradicated the established biofilm formed by drug-resistant A. baumannii clinical isolates. No propensity for bacterial resistance development against this polymer was seen even after 14 continuous passages. Taken together, the results highlight that hydrophobicity modulation through incorporation of amino acids in cationic polymers will provide a significant opportunity in designing new ACPs with potent antibacterial activity and minimum toxicity toward mammalian cells. More importantly, the excellent anti-A. baumannii efficacy of the optimized lead polymer indicates its immense potential for being developed as therapeutic agent.