Fatty Acid Comprising Lysine Conjugates: Anti-MRSA Agents That Display In Vivo Efficacy by Disrupting Biofilms with No Resistance Development

Methicillin-resistant Staphylococcus aureus (MRSA) has developed resistance to antibiotics of last resort such as vancomycin, linezolid, and daptomycin. Additionally, their biofilm forming capability has set an alarming situation in the treatment of bacterial infections. Herein we report the potency of fatty acid comprising lysine conjugates as novel anti-MRSA agents, which were not only capable of killing growing planktonic MRSA at low concentration (MIC = 3.1–6.3 μg/mL), but also displayed potent activity against nondividing stationary phase cells. Furthermore, the conjugates eradicated established biofilms of MRSA. The bactericidal activity of d-lysine conjugated tetradecanoyl analogue (D-LANA-14) is attributed to its membrane disruption against these metabolically distinct cells. In a mouse model of superficial skin infection, D-LANA-14 displayed potent in vivo anti-MRSA activity (2.7 and 3.9 Log reduction at 20 mg/kg and 40 mg/kg, respectively) without showing any skin toxicity even at 200 mg/kg of the compound exposure. Additionally, MRSA could not develop resistance against D-LANA-14 even after 18 subsequent passages, whereas the topical anti-MRSA antibiotic fusidic acid succumbed to rapid resistance development. Collectively, the results suggested that this new class of membrane targeting conjugates bear immense potential to treat MRSA infections over conventional antibiotic therapy

Lysine-Based Small Molecules That Disrupt Biofilms and Kill both Actively Growing Planktonic and Nondividing Stationary Phase Bacteria

The emergence of bacterial resistance is a major threat to global health. Alongside this issue, formation of bacterial biofilms is another cause of concern because most antibiotics are ineffective against these recalcitrant microbial communities. Ideal future antibacterial therapeutics should possess both antibacterial and anti-biofilm activities. In this study we engineered lysine-based small molecules, which showed not only commendable broad-spectrum antibacterial activity but also potent biofilm-disrupting properties. Synthesis of these lipophilic lysine–norspermidine conjugates was achieved in three simple reaction steps, and the resultant molecules displayed potent antibacterial activity against various Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative bacteria (Escherichia coli) including drug-resistant superbugs MRSA (methicillin-resistant <i>S. aureus</i>), VRE (vancomycin-resistant <i>E. faecium</i>), and β-lactam-resistant Klebsiella pneumoniae. An optimized compound in the series showed activity against planktonic bacteria in the concentration range of 3–10 μg/mL, and bactericidal activity against stationary phase <i>S. aureus</i> was observed within an hour. The compound also displayed about 120-fold selectivity toward both classes of bacteria (<i>S. aureus</i> and <i>E. coli</i>) over human erythrocytes. This rapidly bactericidal compound primarily acts on bacteria by causing significant membrane depolarization and K⁺ leakage. Most importantly, the compound disrupted preformed biofilms of <i>S. aureus</i> and did not trigger bacterial resistance. Therefore, this class of compounds has high potential to be developed as future antibacterial drugs for treating infections caused by planktonic bacteria as well as bacterial biofilms

Versatile and User-Friendly Anti-infective Hydrogel for Effective Wound Healing

Wound dressings play a crucial role in facilitating optimal wound healing and protecting against microbial infections. However, existing commercial options often fall short in addressing chronic infections due to antibiotic resistance and the limited spectrum of activity against both Gram-positive and Gram-negative bacteria frequently encountered at wound sites. Additionally, complex fabrication processes and cumbersome administration strategies pose challenges for cost-effective wound dressing development. Consequently, there is a pressing need to explore easily engineered biocompatible biomaterials as alternative solutions to combat these challenging wound infections. In this study, we present the development of an anti-infective hydrogel, P-BAC (polymeric bactericidal hydrogel), which exhibits simple administration and promotes efficient wound healing. P-BAC is synthesized via a one-step fabrication method that involves the noncovalent cross-linking of poly(vinyl alcohol), N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride-AgCl nanocomposite, and proline. Remarkably, P-BAC demonstrates broad-spectrum antibacterial activity against both planktonic and stationary cells of clinically isolated Gram-positive and Gram-negative bacteria, resulting in a significant reduction of bacterial load (5–7 log reduction). Moreover, P-BAC exhibits excellent efficacy in eradicating bacterial cells within biofilm matrices (>95% reduction). In vivo experiments reveal that P-BAC accelerates wound healing by stimulating rapid collagen deposition at the wound site and effectively inactivates ∼95% of Pseudomonas aeruginosa cells. Importantly, the shear-thinning property of P-BAC simplifies the administration process, enhancing its practicality and usability. Taken together, our findings demonstrate the potential of this easily administrable hydrogel as a versatile solution for effective wound healing with potent anti-infective properties. The developed hydrogel holds promise for applications in diverse healthcare settings, addressing the critical need for improved wound dressing materials

Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation

Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers

Membrane Active Vancomycin Analogues: A Strategy to Combat Bacterial Resistance

The alarming growth of antibiotic resistant superbugs such as vancomycin-resistant Enterococci and Staphylococci has become a major global health hazard. To address this issue, we report the development of lipophilic cationic vancomycin analogues possessing excellent antibacterial activity against several drug-resistant strains. Compared to vancomycin, efficacy greater than 1000-fold was demonstrated against vancomycin-resistant Enterococci (VRE). Significantly, unlike vancomycin, these compounds were shown to be bactericidal at low concentrations and did not induce bacterial resistance. An optimized compound in the series, compared to vancomycin, showed higher activity in methicillin-resistant Staphylococcus aureus (MRSA) infected mouse model and exhibited superior antibacterial activity in whole blood with no observed toxicity. The remarkable activity of these compounds is attributed to the incorporation of a new membrane disruption mechanism into vancomycin and opens up a great opportunity for the development of novel antibiotics

Membrane Active Phenylalanine Conjugated Lipophilic Norspermidine Derivatives with Selective Antibacterial Activity

Natural and synthetic membrane active antibacterial agents offer hope as potential solutions to the problem of bacterial resistance as the membrane-active nature imparts low propensity for the development of resistance. In this report norspermidine based antibacterial molecules were developed that displayed excellent antibacterial activity against various wild-type bacteria (Gram-positive and Gram-negative) and drug-resistant bacteria (methicillin-resistant <i>Staphylococcus aureus</i>, vancomycin-resistant <i>Enterococcus faecium</i>, and β-lactam-resistant <i>Klebsiella pneumoniae</i>). In a novel structure–activity relationship study it has been shown how incorporation of an aromatic amino acid drastically improves selective antibacterial activity. Additionally, the effect of stereochemistry on activity, toxicity, and plasma stability has also been studied. These rapidly bactericidal, membrane active antibacterial compounds do not trigger development of resistance in bacteria and hence bear immense potential as therapeutic agents to tackle multidrug resistant bacterial infections

Cleavable Amphiphilic Biocides with Ester-Bearing Moieties: Aggregation Properties and Antibacterial Activity

The rise of multidrug-resistant bacterial infections and the dwindling supply of newly approved antibiotics have emerged as a grave threat to public health. Toward the ever-growing necessity of the development of novel antimicrobial agents, herein, we synthesized a series of cationic amphiphilic biocides featuring two cationic headgroups separated by different hydrophobic spacers, accompanied by the inclusion of two lipophilic tails through cleavable ester functionality. The detailed aggregation properties offered by these biocides were investigated by small-angle neutron scattering (SANS) and conductivity. The critical micellar concentration of the biocides and the size and shape of the micellar aggregates differed with variation of pendant and spacer hydrophobicity. Furthermore, the aggregation number and size of the micelles were found to vary with changing concentration and temperature. These easily synthesized biocides exhibited potent antibacterial properties against various multidrug-resistant bacteria. The optimized biocides with minimum hematotoxicity and potent antibacterial activity against methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii exhibited rapid killing kinetics against planktonic bacteria. Also, these membrane-active agents were able to eradicate preformed biofilms. The enzymatic and acidic degradation profile further offered proof of gradual degradation. Collectively, these cleavable amphiphilic biocides demonstrated excellent potency for combating the multidrug-resistant bacterial infection

Structure–Activity Relationship of Amino Acid Tunable Lipidated Norspermidine Conjugates: Disrupting Biofilms with Potent Activity against Bacterial Persisters

The emergence of bacterial resistance and biofilm associated infections has created a challenging situation in global health. In this present state of affairs where conventional antibiotics are falling short of being able to provide a solution to these problems, development of novel antibacterial compounds possessing the twin prowess of antibacterial and antibiofilm efficacy is imperative. Herein, we report a library of amino acid tunable lipidated norspermidine conjugates that were prepared by conjugating both amino acids and fatty acids with the amine functionalities of norspermidine through amide bond formation. These lipidated conjugates displayed potent antibacterial activity against various planktonic Gram-positive and Gram-negative bacteria including drug-resistant superbugs such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae. This class of nontoxic and fast-acting antibacterial molecules (capable of killing bacteria within 15 min) did not allow bacteria to develop resistance against them after several passages. Most importantly, an optimized compound in the series was also capable of killing metabolically inactive persisters and stationary phase bacteria. Additionally, this compound was capable of disrupting the preformed biofilms of S. aureus and E. coli. Therefore, this class of antibacterial conjugates have potential in tackling the challenging situation posed by both bacterial resistance as well as drug tolerance due to biofilm formation

Dual Function Injectable Hydrogel for Controlled Release of Antibiotic and Local Antibacterial Therapy

We present vancomycin-loaded dual-function injectable hydrogel that delivers antibiotic locally suitable for treatment of infections in avascular or necrotic tissues. The syringe-deliverable gels were developed using polydextran aldehyde and an inherently antibacterial polymer <i>N</i>-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride along with vancomycin. The antibiotic was primarily encapsulated via reversible imine bonds formed between vancomycin and polydextran aldehyde in the hydrogel which allowed sustained release of vancomycin over an extended period of time in a pH-dependent manner. Being inherently antibacterial, the gels displayed excellent efficacy against bacteria due to dual mode of action (killing bacteria upon contact as well as by releasing antibiotics into surroundings). Upon subcutaneous implantation, the gel was shown to kill methicillin-resistant Staphylococcus aureus (>99.999%) when bacteria were introduced directly into the gel as well as at distal site from the gel in a mice model. These materials thus represent as novel noninvasive drug-delivery device suitable for local antibiotic therapy

Dual-Function Polymer–Silver Nanocomposites for Rapid Killing of Microbes and Inhibiting Biofilms

Polymer–silver nanocomposites have emerged as an integral weapon to combat device-related infections. However, synthesis of the nanocomposites still remains a major challenge that often involves two-step process in which silver nanoparticles are synthesized ex situ. Additionally, polymers used in the nanocomposites are commonly not antimicrobial and biodegradable thus often lack bioactivity and biocompatibility. Herein we report highly active dual-function polymer-silver nanocomposites consisting of an inherently antimicrobial and biodegradable polymer in one-pot. A simple method of in situ reduction of a silver salt was employed to synthesize the silver nanoparticles (5–15 nm) from silver <i>para</i>-toluenesulfonate in which the intrinsically biodegradable and antimicrobial polymer <i>N</i>,<i>N</i>-dimethyl-<i>N</i>-hexadecyl ammonium chitin tosylate acted as reducing as well as stabilizing agent. The nanocomposite with the water-insoluble and organo-soluble polymer was simply painted onto surfaces via facile noncovalent immobilization. Notably, composite-coated surfaces inactivated both drug-sensitive and drug-resistant bacteria including pathogenic fungi at a much faster rate than polymer alone. The composites released active silver ions over an extended period of time and displayed remarkably long-lasting activity. In addition, surfaces coated with composites effectively inhibited both bacterial and fungal biofilm formation. Further, upon coating on catheter, the nanocomposites reduced methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) burden both on catheter (>99.99% reduction) and in tissues surrounding the catheter (>99.999% reduction) in a mice model. These novel nanomaterials that showed negligible hemolysis toward human erythrocytes might be used as safe and effective antimicrobial coatings in biomedical device applications