scholarly journals Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Ryan D. Heselpoth ◽  
Chad W. Euler ◽  
Raymond Schuch ◽  
Vincent A. Fischetti
Keyword(s):  
Outer Membrane ◽  
Receptor Binding ◽  
Standard Of Care ◽  
Multidrug Resistant ◽  
Gram Negative ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Surface Receptor ◽  
Inability To Work ◽  
Protein Channels

ABSTRACTThe prevalence of multidrug-resistantPseudomonas aeruginosahas stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins ofP. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol- or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineeredlysin-bacteriocinfusion molecules capable of periplasmic import. In our proof-of-concept studies, components from theP. aeruginosabacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing ofP. aeruginosawith minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice fromP. aeruginosachallenge in a bacteremia model. In addition to targetingP. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.

scholarly journals Outer Membrane Interaction Kinetics of New Polymyxin B Analogs in Gram-Negative Bacilli

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Noushin Akhoundsadegh ◽  
Corrie R. Belanger ◽  
Robert E. W. Hancock
Keyword(s):  
Outer Membrane ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Intact Cells ◽  
Membrane Interaction ◽  
Gram Negative ◽  
Content Type ◽  
Interaction Kinetics ◽  
Hill Numbers ◽  
Multiple Strains

ABSTRACT Infections caused by drug-resistant Gram-negative bacilli are a severe global health threat, limiting effective drug choices for treatment. In this study, polymyxin analogs designed to have reduced nephrotoxicity, direct activity, and potentiating activity were assessed for inhibition and outer membrane interaction kinetics against wild-type (WT) and polymyxin or multidrug-resistant (MDR) Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In MIC assays, two polymyxin B (PMB) analogs (SPR1205 and SPR206) and a polymyxin E analog (SPR946), with shortened peptide side chains and branched aminobutyryl N termini, exhibited promising activity compared with PMB and previously tested control polymyxin analogs SPR741 and polymyxin B nonapeptide (PMBN). Using dansyl-polymyxin (DPX) binding to assess the affinity of interaction with lipopolysaccharide (LPS), purified or in the context of intact cells, SPR206 exhibited similar affinities to PMB but higher affinities than the other SPR analogs. Outer membrane permeabilization measured by the 1-N-phenyl-napthylamine (NPN) assay did not differ significantly between the polymyxin analogs. Moreover, Hill numbers were greater than 1 for most of the compounds tested on E. coli and P. aeruginosa strains which indicates that the disruption of the outer membrane by one molecule of compound cooperatively enhances the subsequent interactions of other molecules against WT and MDR strains. The high activity demonstrated by SPR206 as well as its ability to displace LPS and permeabilize the outer membrane of multiple strains of Gram-negative bacilli while showing cooperative potential with other membrane disrupting compounds supports further research with this polymyxin analog.

scholarly journals Inactivation of Polymyxin by Hydrolytic Mechanism

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Jianhua Yin ◽  
Gang Wang ◽  
Dan Cheng ◽  
Jianv Fu ◽  
Juanping Qiu ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Peptide Antibiotics ◽  
Peptide Bonds ◽  
Gram Negative ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Polymyxin E ◽  
Cyclic Heptapeptide ◽  
Hydrolytic Mechanism

ABSTRACTPolymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxin-inactivating enzyme fromBacillus licheniformisstrain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other betweenl-Thr andl-α-γ-diaminobutyric acid (l-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, includingBacillusandPaenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis.

scholarly journals Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Mary Kate Alexander ◽  
Anh Miu ◽  
Angela Oh ◽  
Mike Reichelt ◽  
Hoangdung Ho ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Drug Targets ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Transport Pathway ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
New Antibiotics ◽  
Outer Membrane Biogenesis

ABSTRACTThere is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that killEscherichia colithrough inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

scholarly journals Potentiation of Antibiotic Activity by a Novel Cationic Peptide: Potency and Spectrum of Activity of SPR741

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Corbett ◽  
Andrew Wise ◽  
Tara Langley ◽  
Kirsty Skinner ◽  
Emily Trimby ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Fusidic Acid ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Conventional Antibiotics

ABSTRACTNovel approaches for the treatment of multidrug-resistant Gram-negative bacterial infections are urgently required. One approach is to potentiate the efficacy of existing antibiotics whose spectrum of activity is limited by the permeability barrier presented by the Gram-negative outer membrane. Cationic peptides derived from polymyxin B have been used to permeabilize the outer membrane, granting antibiotics that would otherwise be excluded access to their targets. We assessed thein vitroefficacies of combinations of SPR741 with conventional antibiotics againstEscherichia coli,Klebsiella pneumoniae, andAcinetobacter baumannii. Of 35 antibiotics tested, the MICs of 8 of them were reduced 32- to 8,000-fold againstE. coliandK. pneumoniaein the presence of SPR741. The eight antibiotics, azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, retapamulin, rifampin, and telithromycin, had diverse targets and mechanisms of action. AgainstA. baumannii, similar potentiation was achieved with clarithromycin, erythromycin, fusidic acid, retapamulin, and rifampin. Susceptibility testing of the most effective antibiotic-SPR741 combinations was extended to 25 additional multidrug-resistant or clinical isolates ofE. coliandK. pneumoniaeand 17 additionalA. baumanniiisolates in order to rank the potentiated antibiotics. SPR741 was also able to potentiate antibiotics that are substrates of the AcrAB-TolC efflux pump inE. coli, effectively circumventing the contribution of this pump to intrinsic antibiotic resistance. These studies support the further development of SPR741 in combination with conventional antibiotics for the treatment of Gram-negative bacterial infections.

scholarly journals Increasing the Antimicrobial Activity of Nisin-Based Lantibiotics against Gram-Negative Pathogens

2018 ◽  
Vol 84 (12) ◽  
Author(s):  
Qian Li ◽  
Manuel Montalban-Lopez ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Rational Design ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Lipid Ii

ABSTRACTLantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e.,Escherichia coli,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,Enterobacter aerogenes) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens.IMPORTANCELantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

scholarly journals Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Yves Briers ◽  
Maarten Walmagh ◽  
Victor Van Puyenbroeck ◽  
Anneleen Cornelissen ◽  
William Cenens ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Viruses ◽  
Cell Wall Hydrolysis

ABSTRACTThe global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, includingPseudomonas aeruginosaandAcinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strainsin vitrowith a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effectivein vitro(human keratinocytes) andin vivo(Caenorhabditis elegans).IMPORTANCEBacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active againstPseudomonas aeruginosaandAcinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

scholarly journals Clinically Relevant Gram-Negative Resistance Mechanisms Have No Effect on the Efficacy of MC-1, a Novel Siderophore-Conjugated Monocarbam

2012 ◽  
Vol 56 (12) ◽  
pp. 6334-6342 ◽  
Author(s):  
Craig J. McPherson ◽  
Lisa M. Aschenbrenner ◽  
Brian M. Lacey ◽  
Kelly C. Fahnoe ◽  
Margaret M. Lemmon ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Negative Resistance ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Pharmacologically Active

ABSTRACTThe incidence of hospital-acquired infections with multidrug-resistant (MDR) Gram-negative pathogens is increasing at an alarming rate. Equally alarming is the overall lack of efficacious therapeutic options for clinicians, which is due primarily to the acquisition and development of various antibiotic resistance mechanisms that render these drugs ineffective. Among these mechanisms is the reduced permeability of the outer membrane, which prevents many marketed antibiotics from traversing this barrier. To circumvent this, recent drug discovery efforts have focused on conjugating a siderophore moiety to a pharmacologically active compound that has been designed to hijack the bacterial siderophore transport system and trick cells into importing the active drug by recognizing it as a nutritionally beneficial compound. MC-1, a novel siderophore-conjugated β-lactam that promotes its own uptake into bacteria, has exquisite activity against many Gram-negative pathogens. While the inclusion of the siderophore was originally designed to facilitate outer membrane penetration into Gram-negative cells, here we show that this structural moiety also renders other clinically relevant antibiotic resistance mechanisms unable to affect MC-1 efficacy. Resistance frequency determinations and subsequent characterization of first-step resistant mutants identified PiuA, a TonB-dependent outer membrane siderophore receptor, as the primary means of MC-1 entry intoPseudomonas aeruginosa. While the MICs of these mutants were increased 32-fold relative to the parental strainin vitro, we show that this resistance phenotype is not relevantin vivo, as alternative siderophore-mediated uptake mechanisms compensated for the loss of PiuA under iron-limiting conditions.

scholarly journals Prevalence of SUL(1,2), GYR(A, B) and OXA genes among multidrug resistance Klebsiella pneumoniae isolates recovered from women suffering urinary tract infection

2020 ◽  
Vol 11 (2) ◽  
pp. 2424-2432
Author(s):  
Nabil Salim Saaid Tuwaij ◽  
Huda Jameel Baker Al-khilkhali ◽  
Haneen Mohamed Mohsen
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Vitek 2 ◽  
Resistant Microorganism ◽  
Urine Specimens

Klebsiella pneumoniae is a significant concern multidrug-resistant microorganism and a one common gram negative bacteria associated with infections of women urinary tract. Therefore, this work aimed to the molecular screening of Sul(1and 2), Gyr(A and B) and OXA genes among K. pneumoniae isolates in Najaf City, Iraq. Out of 250 urine specimens were collected from women showing symptoms of urinary tract infection during five months January to of May 2019, bacterial growth was157 isolates, included 133 gram negative compared with  24 gram positive bacteria while 98 specimens were no growth. According to the Vitek-2 system, 30 K. pneumoniae isolates were obtained.Data on current work revealed that the 26-35 age group was the highest 14 K. pneumoniae isolates. Results of antimicrobial susceptible recorded all isolates were multi-drug resistant (MDR) and they have a different range of resistance. However, all 30 isolates(100%) resistant to ampicillin drugs, while the lowest rate was 1(3.33%) forImipenemdrug. PCR assay revealed exist of oxa, sul-1, sul-2, gyr-A and gyr-B genes among K. pneumoniae isolates with rates 20(66.66%), 11(36.66%), 22(73.33%), 3(10%) and 17(56.66%) respectively.

scholarly journals In VitroActivity of RX-P873 against Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii

2015 ◽  
Vol 59 (4) ◽  
pp. 2280-2285 ◽  
Author(s):  
Robert K. Flamm ◽  
Paul R. Rhomberg ◽  
Ronald N. Jones ◽  
David J. Farrell
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Active Agent ◽  
Multidrug Resistant ◽  
Surveillance Program ◽  
Gram Negative ◽  
Content Type ◽  
Highly Active ◽  
Bacterial Ribosome

ABSTRACTRX-P873 is a novel antibiotic from the pyrrolocytosine series which exhibits high binding affinity for the bacterial ribosome and broad-spectrum antibiotic properties. The pyrrolocytosines have shownin vitroactivity against multidrug-resistant Gram-negative and Gram-positive strains of bacteria known to cause complicated urinary tract, skin, and lung infections, as well as sepsis.Enterobacteriaceae(657),Pseudomonas aeruginosa(200), andAcinetobacter baumannii(202) isolates from North America and Europe collected in 2012 as part of a worldwide surveillance program were testedin vitroby broth microdilution using Clinical and Laboratory Standards Institute (CLSI) methodology. RX-P873 (MIC90, 0.5 μg/ml) was >32-fold more active than ceftazidime and inhibited 97.1% and 99.5% ofEnterobacteriaceaeisolates at MIC values of ≤1 and ≤4 μg/ml, respectively. There were only three isolates with an MIC value of >4 μg/ml (all were indole-positiveProtea). RX-P873 (MIC50/90, 2/4 μg/ml) was highly active againstPseudomonas aeruginosaisolates, including isolates which were nonsusceptible to ceftazidime or meropenem. RX-P873 was 2-fold less active againstP. aeruginosathan tobramycin (MIC90, 2 μg/ml; 91.0% susceptible) and colistin (MIC90, 2 μg/ml; 99.5% susceptible) and 2-fold more potent than amikacin (MIC90, 8 μg/ml; 93.5% susceptible) and meropenem (MIC90, 8 μg/ml; 76.0% susceptible). RX-P873, the most active agent againstAcinetobacter baumannii(MIC90, 1 μg/ml), was 2-fold more active than colistin (MIC90, 2 μg/ml; 97.0% susceptible) and 4-fold more active than tigecycline (MIC90, 4 μg/ml). This novel agent merits further exploration of its potential against multidrug-resistant Gram-negative bacteria.

scholarly journals In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Susanne Paukner ◽  
Astrid Gruss ◽  
Jørgen Skov Jensen
Keyword(s):  
Chlamydia Trachomatis ◽  
Bacterial Pathogens ◽  
Mycoplasma Genitalium ◽  
Standard Of Care ◽  
Multidrug Resistant ◽  
In Vitro Activity ◽  
First Line ◽  
Content Type ◽  
Sexually Transmitted

ABSTRACT The pleuromutilin antibiotic lefamulin demonstrated in vitro activity against the most relevant bacterial pathogens causing sexually transmitted infections (STI), including Chlamydia trachomatis (MIC 50/90 , 0.02/0.04 mg/liter; n = 15), susceptible and multidrug-resistant Mycoplasma genitalium (MIC range, 0.002 to 0.063 mg/liter; n = 6), and susceptible and resistant Neisseria gonorrhoeae (MIC 50/90 , 0.12/0.5 mg/liter; n = 25). The results suggest that lefamulin could be a promising first-line antibiotic for the treatment of STI, particularly in populations with high rates of resistance to standard-of-care antibiotics.

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