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 N-(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.

Biocompatible Injectable Hydrogel with Potent Wound Healing and Antibacterial Properties.

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 ß-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.

Chitosan Derivatives Active against Multidrug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials.

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 (HC50 = >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.

Membrane Disruption and Enhanced Inhibition of Cell-Wall Biosynthesis: A Synergistic Approach to Tackle Vancomycin-Resistant Bacteria.

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.

An extensive analysis of Codon usage pattern, Evolutionary rate, and Phylogeographic reconstruction in Foot and mouth disease (FMD) serotypes (A, Asia 1, and O) of six major climatic zones of India: A comparative study.

Foot and mouth disease (FMD) is a major economically important viral disease of cloven-hoofed livestock globally. The FMD virus (FMDV) spreads widely in confined, cool, and humid climatic conditions. Being an RNA virus, FMDV is genetically unstable, and its genome evolution is highly influenced by mutational pressure. The climatic and environmental conditions have a significant impact on mutational pressure. The present study is a primary effort to establish a comprehensive relationship between climatic factors and the molecular evolutionary pattern of serotypes FMDV circulating in India. In this study, isolates of three serotypes (A, Asia 1, and O) were selected from six major climatic zones of India (Montane, Humid subtropical, Tropical wet and dry, Tropical wet, Semi-arid and Arid). Based on the full genome nucleotide sequence data, the codon usage bias, evolutionary and phylogeographic analysis was carried out. The study revealed that the codon use bias indicators in the FMDV serotypes differed significantly depending on the climatic zones. It implies that the selection and mutational pressure influence the codon usage pattern indices, with mutational pressure taking precedence in determining the codon usage bias of the FMDV genome. The tMRCA was estimated to be 1977, 1956, and 1953 for Indian FMD virus serotype-A, Asia 1, and O respectively which is around 32, 60, and 61 years before its actual identification in the field. Based on the evolutionary rates the serotype O is evolving rapidly compare to other serotypes in India. Virus transmission across the region was evident from the phylogeographic analysis. The integrated analysis of codon usage bias, evolutionary rate, and phylogeography analysis signifies the major role of mutational and selection pressure, implying that the FMD virus co-evolution and adaptations are highly influenced by climatic/environmental factors.

Bovine babesiosis: An insight into the global perspective on the disease distribution by systematic review and meta-analysis.

Bovine babesiosis is continuing as a great threat to the livestock sector causing havoc production losses with significant morbidity and mortality. Being a tick-borne disease, the great complexity in the agent-host- vector relationship has severely hampered the sincere efforts towards the development of an effective vaccine against bovine babesiosis. In these circumstances, assessing the global scenario of disease prevalence is a prerequisite to strategize the available control measures. Keeping this in view, the objective of this study was to estimate the pooled prevalence of bovine babesiosis globally. The literature search was conducted to identify all relevant published articles reporting the prevalence of bovine babesiosis and a total of 163 studies were found eligible for final systematic review and meta-analysis. Meta-analysis was conducted using meta package of R software and summary estimates of the prevalence were calculated. Meta analysis of 81099 samples from 62 countires representing six continents revealed pooled global prevalence of bovine babesiosis as 29% (95% CI = 24%-34%) with estimated prevalence of active infection as 16% (95% CI = 13%-20%) and seroprevalence as 50% (95% CI = 45%-56%) using random effects model. Continent wise highest prevalence of bovine babesiosis in South America 64% (95% CI = 49%-77%) and lowest in Asia 19% (95% CI = 14%-25%). Highest prevalence was estimated with B. bigemina 22% (95% CI = 18%-27%) and least prevalence was recorded with B. divergens 12% (95% CI = 2%-46%). The pooled prevalence estimates generated in the study is revealing an increase in disease trend and the need for immediate planning of mitigation strategies paralleled with the development of early diagnostic methods to reduce the impact of disease throughout the world.

Charge-Switchable Polymeric Coating Kills Bacteria and Prevents Biofilm Formation in Vivo.

Preventing bacterial biofilm formation on medical devices and implants in vivo still remains a daunting task. Current antibacterial coatings to combat implant-associated infections are generally composed of toxic metals or nondegradable polymers and involve multistep surface modifications. Here, we present a charge-switchable antibacterial and antibiofilm coating based on water-insoluble cationic hydrophobic polymers that are soluble in organic solvents and can be noncovalently coated onto different surfaces. Toward this, a library of quaternary polyethylenimine (QPEI) polymers with an amide or ester group in their pendant alkyl chain was developed. These QPEIs are shown to hydrolyze from active cationic to nontoxic zwitterionic polymers under acidic or enzymatic conditions. Notably, polymers with both zwitterionic and cationic groups, obtained upon partial hydrolysis of QPEIs, are shown to retain their antibacterial activity with much lower toxicity toward mammalian cells. Furthermore, the zwitterionic polymer, a fully hydrolyzed product of the QPEIs, is shown to be nontoxic to mammalian cells in vitro as well as in vivo. The QPEIs, when coated onto surfaces, kill bacteria and prevent formation of biofilms. In an in vivo mice model, the QPEI-coated medical grade catheter is shown to reduce methicillin-resistant Staphylococcus aureus contamination both on the catheter surface and in the adjacent tissues (99.99% reduction compared to a noncoated catheter). Additionally, biofilm formation is inhibited on the catheter surface with negligible inflammation in the adjacent tissue. The above results thus highlight the importance of these polymers to be used as effective antibacterial coatings in biomedical applications.

Prevalence of Anaplasma species in India and the World in dairy animals: A systematic review and meta-analysis.

In the present study, the prevalence of Anaplasma species in diary animals from India and World was estimated using meta-analysis. Based on systematic review of studies on Anaplasma species from India [35] and World [66] from 1988 to 2017 and 1978-2017, respectively, using online databases and offline literatures, meta-analysis using meta package in R-Software was done. Prevalence of Anaplasma species in India and World were 11% [95% level, Confidence Interval[CI] 7-16%, Prediction Interval[PI] 1-69%] and 39% [95% level, CI 30-49%, PI 2-95%], and these were obtained using 31,117 and 46,365 samples, respectively. Period-wise analysis revealed high Anaplasma species prevalence before 2011 for India and the World than from 2011 through 2017. Zone-wise prevalence indicated high prevalence in Central zone [61%] and low in West and South zones [6%] in India, and continent-wise, it was high in South America [82%]. The studies used methods including blood smear examination, serology and nucleic acid-based techniques and revealed high prevalence in serology for India [34%] and World [46%], low prevalence by blood smear for India [7%] and World [21%], but higher sensitivity using nucleic acid-based techniques. Species-wise indicated higher prevalence in cattle [12%] than buffaloes [2%] in India. Prevalence was lower in India compared to the World and higher in South America. Overall, anaplasmosis causes low productivity in dairy animals and economic loss to dairy farmers. Hence, there is a need to control Anaplasma infections in high risk areas by adopting effective therapeutic and preventive measures so as to improve the economic benefits in dairy farming.

Vancomycin Analogue Restores Meropenem Activity against NDM-1 Gram-Negative Pathogens.

New Delhi metallo-ß-lactamase-1 (NDM-1) is the major contributor to the emergence of carbapenem resistance in Gram-negative pathogens (GNPs) and has caused many clinically available ß-lactam antibiotics to become obsolete. A clinically approved inhibitor of metallo-ß-lactamase (MBL) that could restore the activity of carbapenems against resistant GNPs has not yet been found, making NDM-1 a serious threat to human health. Here, we have rationally developed an inhibitor for the NDM-1 enzyme, which has the ability to penetrate the outer membrane of GNPs and inactivate the enzyme by depleting the metal ion (Zn2+) from the active site. The inhibitor reinstated the activity of meropenem against NDM-1 producing clinical isolates of GNPs like Klebsiella pneumoniae and Escherichia coli. Further, the inhibitor efficiently restored meropenem activity against NDM-1 producing K. pneumoniae in a murine sepsis infection model. These findings demonstrate that a combination of the present inhibitor and meropenem has high potential to be translated clinically to combat carbapenem-resistant GNPs.

Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.

Bacterial biofilms represent the root-cause of chronic or persistent infections in humans. Gram-negative bacterial infections due to nosocomial and opportunistic pathogens such as Acinetobacter baumannii are more difficult to treat because of their inherent and rapidly acquiring resistance to antibiotics. Due to biofilm formation, A. baumannii has been noted for its apparent ability to survive on artificial surfaces for an extended period of time, therefore allowing it to persist in the hospital environment. Here we report, maleic anhydride based novel cationic polymers appended with amide side chains that disrupt surface established multi-drug resistant A. baumannii biofilms. More importantly, these polymers significantly (p < 0.0001) decrease the bacterial burden in mice with chronic A. baumannii burn wound infection. The polymers also show potent antibacterial efficacy against methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VRE) and multi-drug resistant clinical isolates of A. baumannii with minimal toxicity to mammalian cells. We observe that optimal hydrophobicity dependent on the side chain chemical structure of these polymers dictate the selective toxicity to bacteria. Polymers interact with the bacterial cell membranes by causing membrane depolarization, permeabilization and energy depletion. Bacteria develop rapid resistance to erythromycin and colistin whereas no detectable development of resistance occurs against these polymers even after several passages. These results suggest the potential use of these polymeric biomaterials in disinfecting biomedical device surfaces after the infection has become established and also for the topical treatment of chronic bacterial infections.

Aryl-alkyl-lysines: Membrane-Active Small Molecules Active against Murine Model of Burn Infection.

Infections caused by drug-resistant Gram-negative pathogens continue to be significant contributors to human morbidity. The recent advent of New Delhi metallo-ß-lactamase-1 (blaNDM-1) producing pathogens, against which few drugs remain active, has aggravated the problem even further. This paper shows that aryl-alkyl-lysines, membrane-active small molecules, are effective in treating infections caused by Gram-negative pathogens. One of the compounds of the study was effective in killing planktonic cells as well as dispersing biofilms of Gram-negative pathogens. The compound was extremely effective in disrupting preformed biofilms and did not select resistant bacteria in multiple passages. The compound retained activity in different physiological conditions and did not induce any toxic effect in female Balb/c mice until concentrations of 17.5 mg/kg. In a murine model of Acinetobacter baumannii burn infection, the compound was able to bring the bacterial burden down significantly upon topical application for 7 days.

Glycopeptide Antibiotic To Overcome the Intrinsic Resistance of Gram-Negative Bacteria.

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.

In vivo efficacy and pharmacological properties of a novel glycopeptide (YV4465) against vancomycin-intermediate Staphylococcus aureus.

Infections caused by vancomycin-intermediate Staphylococcus aureus (VISA) are associated with high rates of vancomycin treatment failure. The lipophilic vancomycin-carbohydrate conjugate YV4465 is a new glycopeptide antibiotic that is active against a variety of clinically relevant multidrug-resistant Gram-positive pathogens in vitro. YV4465 was 50- and 1000-fold more effective than vancomycin against VISA and vancomycin-resistant enterococci, respectively. This study evaluated the in vivo efficacy against VISA as well as the pharmacokinetics and toxicology of YV4465. A neutropenic mouse thigh infection model was used for the determination of efficacy and pharmacodynamic properties against VISA. YV4465 produced a dose-dependent reduction in VISA titres in thigh muscle; bacterial titres were reduced by up to ca. 2log(10)CFU/g from the pre-treatment titre at a dosage of 8 mg/kg. Single-dose pharmacokinetic studies demonstrated an increase in drug exposure to the animal following linear kinetics with a prolonged half-life (t(1/2)) compared with vancomycin. The peak plasma concentration (C(max)) following an intravenous dose of 12 mg/kg was 703 µg/mL. Acute toxicology studies revealed that YV4465 did not cause any significant alterations in biochemical parameters related to major organs such as the liver and kidneys at its pharmacodynamic endpoint (>ED(2-log kill)). These studies demonstrate that YV4465 has the potential to be developed as a next-generation glycopeptide antibiotic for the treatment of infections caused by VISA.

Aryl-Alkyl-Lysines: Agents That Kill Planktonic Cells, Persister Cells, Biofilms of MRSA and Protect Mice from Skin-Infection.

Development of synthetic strategies to combat Staphylococcal infections, especially those caused by methicillin resistant Staphyloccus aureus (MRSA), needs immediate attention. In this manuscript we report the ability of aryl-alkyl-lysines, simple membrane active small molecules, to treat infections caused by planktonic cells, persister cells and biofilms of MRSA. A representative compound, NCK-10, did not induce development of resistance in planktonic cells in multiple passages and retained activity in varying environments of pH and salinity. At low concentrations the compound was able to depolarize and permeabilize the membranes of S. aureus persister cells rapidly. Treatment with the compound not only eradicated pre-formed MRSA biofilms, but also brought down viable counts in bacterial biofilms. In a murine model of MRSA skin infection, the compound was more effective than fusidic acid in bringing down the bacterial burden. Overall, this class of molecules bears potential as antibacterial agents against skin-infections.

Correction: Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics.

Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-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 blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates.Organismic studies showed that bacteria had an increased and faster uptake of tetracyclinein 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 blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics.

Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-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 blaNDM-1 Klebsiella pneumonia and blaNDM-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 blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

In vivo antibacterial activity and pharmacological properties of the membrane-active glycopeptide antibiotic YV11455.

The membrane-active glycopeptide antibiotic YV11455 is a lipophilic cationic vancomycin analogue that demonstrates rapid and concentration-dependent killing of clinically relevant multidrug-resistant (MDR) Gram-positive bacteria in vitro. YV11455 was 2-fold and 54-270-fold more effective than vancomycin against clinical isolates of vancomycin-sensitive and vancomycin-resistant bacteria, respectively. In this study, the in vivo efficacy, pharmacodynamics, pharmacokinetics and acute toxicology of YV11455 were investigated. In vivo activity and pharmacodynamics were determined in the neutropenic mouse thigh infection model against meticillin-resistant Staphylococcus aureus (MRSA). YV11455 produced dose-dependent reductions in MRSA titres in thigh muscle. When administered intravenously, the 50% effective dose (ED(50)) for YV11455 against MRSA was found to be 3.3 mg/kg body weight, and titres were reduced by up to ca. 3log(10)CFU/g from pre-treatment values at a dosage of 12 mg/kg with single treatment. Single-dose pharmacokinetic studies demonstrated linear kinetics and a prolonged half-life, with an increase in drug exposure (area under the concentration-time curve) compared with vancomycin. The peak plasma concentration following an intravenous dose of 12 mg/kg was 543.5 µg/mL. Acute toxicology studies revealed that YV11455 did not cause any significant alterations in biochemical parameters or histological pictures related to major organs such as the liver and kidney at its pharmacodynamic endpoint (ED(3-log kill)). These findings collectively suggest that YV11455 could be used clinically for the treatment of infections caused by MDR Gram-positive bacteria.