Two component injectable hydrogels that cross-link in situ have been used as noninvasive wound-filling devices, i.e., sealants. These materials carry a variety of functions at the wound sites, such as sealing leaks, ceasing unwanted bleeding, binding tissues together, and assisting in wound healing processes. However, commonly used sealants typically lack antibacterial properties. Since bacterial infection at the wound site is very common, bioadhesive materials with intrinsic antibacterial properties are urgently required. Herein, we report a biocompatible injectable hydrogel with inherent bioadhesive, antibacterial, and hemostatic capabilities suitable for wound sealing applications. The hydrogels were developed in situ from an antibacterial polymer, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and a bioadhesive polymer, polydextran aldehyde. The gels were shown to be active against both Gram-positive and Gram-negative bacteria, including drug-resistant ones such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiela pneumoniae. Mechanistic studies revealed that the gels killed bacteria upon contact by disrupting the membrane integrity of the pathogen. Importantly, the gels were shown to be efficacious in preventing sepsis in a cecum ligation and puncture (CLP) model in mice. While only 12.5% of animals survived in the case of mice with punctured cecam but with no gel on the punctured area (control), 62.5% mice survived when the adhesive gel was applied to the punctured area. Furthermore, the gels were also shown to be effective in facilitating wound healing in rats and ceasing bleeding from a damaged liver in mice. Notably, the gel showed negligible toxicity toward human red blood cells (only 2-3% hemolysis) and no inflammation to the surrounding tissue upon subcutaneous implantation in mice, thus proving it as a safe and effective antibacterial sealant.

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Aim:  To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results:  A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <103 CFU ml−1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion:  The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study:  The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.

Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

The emergence of drug resistance along with a declining pipeline of clinically useful antibiotics has made it vital to develop more effective antimicrobial therapeutics, particularly against difficult-to-treat Gram-negative pathogens (GNPs). Many antibacterial agents, including glycopeptide antibiotics such as vancomycin, are inherently inactive toward GNPs because of their inability to cross the outer membrane of these pathogens. Here, we demonstrate, for the first time, lipophilic cationic (permanent positive charge) vancomycin analogues were able to permeabilize the outer membrane of GNPs and overcome the inherent resistance of GNPs toward glycopeptides. Unlike vancomycin, these analogues were shown to have a high activity against a variety of multidrug-resistant clinical isolates such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the murine model of carbapenem-resistant A. baumannii infection, the optimized compound showed potent activity with no observed toxicity. The notable activity of these compounds is attributed to the incorporation of new membrane disruption mechanisms (cytoplasmic membrane depolarization along with outer and inner (cytoplasmic) membrane permeabilization) into vancomycin. Therefore, our results indicate the potential of the present vancomycin analogues to be used against drug-resistant GNPs, thus strengthening the antibiotic arsenal for combating Gram-negative bacterial infections.

Resistance to glycopeptide antibiotics, the drugs of choice for life-threatening bacterial infections, is on the rise. In order to counter the threat of glycopeptide-resistant bacteria, we report development of a new class of semi-synthetic glycopeptide antibiotics, which not only target the bacterial membrane but also display enhanced inhibition of cell-wall biosynthesis through increased binding affinity to their target peptides. The combined effect of these two mechanisms resulted in improved in vitro activity of two to three orders of magnitude over vancomycin and no propensity to trigger drug resistance in bacteria. In murine model of kidney infection, the optimized compound was able to bring bacterial burden down by about 6 logs at 12 mg kg(-1) with no observed toxicity. The results furnished in this report emphasize the potential of this class of compounds as future antibiotics for drug-resistant Gram-positive infections.

The continuous rise of antimicrobial resistance and the dearth of new antibiotics in the clinical pipeline raise an urgent call for the development of potent antimicrobial agents. Cationic chitosan derivatives, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides (HTCC), have been widely studied as potent antibacterial agents. However, their systemic structure-activity relationship, activity toward drug-resistant bacteria and fungi, and mode of action are very rare. Moreover, toxicity and efficacy of these polymers under in vivo conditions are yet to be established. Herein, we investigated antibacterial and antifungal efficacies of the HTCC polymers against multidrug resistant bacteria including clinical isolates and pathogenic fungi, studied their mechanism of action, and evaluated cytotoxic and antimicrobial activities in vitro and in vivo. The polymers were found to be active against both bacteria and fungi (MIC = 125-250 μg/mL) and displayed rapid microbicidal kinetics, killing pathogens within 60-120 min. Moreover, the polymers were shown to target both bacterial and fungal cell membrane leading to membrane disruption and found to be effective in hindering bacterial resistance development. Importantly, very low toxicity toward human erythrocytes (HC = >10000 μg/mL) and embryo kidney cells were observed for the cationic polymers in vitro. Further, no inflammation toward skin tissue was observed in vivo for the most active polymer even at 200 mg/kg when applied on the mice skin. In a murine model of superficial skin infection, the polymer showed significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) burden (3.2 log MRSA reduction at 100 mg/kg) with no to minimal inflammation. Taken together, these selectively active polymers show promise to be used as potent antimicrobial agents in topical and other infections.

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla NDM-1 Klebsiella pneumonia and bla NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.