scholarly journals A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria

Antibiotics ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 92 ◽  
Author(s):  
Gerard Boix-Lemonche ◽  
Maria Lekka ◽  
Barbara Skerlavaj
Keyword(s):  
Antimicrobial Peptides ◽  
Staphylococcus Epidermidis ◽  
Membrane Depolarization ◽  
Membrane Permeabilization ◽  
Microplate Assay ◽  
Membrane Systems ◽  
Gram Positive Bacteria ◽  
Gramicidin D ◽  
Membrane Effects ◽  
Specific Membrane

Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.

scholarly journals Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria

2000 ◽  
Vol 44 (8) ◽  
pp. 2086-2092 ◽  
Author(s):  
Carol L. Friedrich ◽  
Dianne Moyles ◽  
Terry J. Beveridge ◽  
Robert E. W. Hancock
Keyword(s):  
Amino Acid ◽  
Mechanism Of Action ◽  
Cytoplasmic Membrane ◽  
Membrane Depolarization ◽  
Membrane Permeabilization ◽  
Cationic Peptides ◽  
Nuclear Condensation ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Helical Peptide

ABSTRACT Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments withStaphylococcus aureus and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of S. aureus. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid α-helical peptide with a high MIC against S. aureus, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of S. aureus andStaphylococcus epidermidis treated with CP29 (a 26-amino-acid α-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH2 (a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus, whereas cell wall effects and mesosomes were seen with S. epidermidis. Nuclear condensation and abherrent septation were occasionally seen in S. epidermidis. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.

scholarly journals Diversity in Antistaphylococcal Mechanisms among Membrane-Targeting Antimicrobial Peptides

2001 ◽  
Vol 69 (8) ◽  
pp. 4916-4922 ◽  
Author(s):  
Su-Pin Koo ◽  
Arnold S. Bayer ◽  
Michael R. Yeaman
Keyword(s):  
Antimicrobial Peptides ◽  
Cytoplasmic Membrane ◽  
Membrane Permeabilization ◽  
Dependent Manner ◽  
Quantitative Culture ◽  
Cell Permeabilization ◽  
Concentration Dependent Manner ◽  
Gramicidin D ◽  
Platelet Microbicidal Protein ◽  
The Relationship

ABSTRACT Many antimicrobial peptides permeabilize the bacterial cytoplasmic membrane. However, it is unclear how membrane permeabilization and antimicrobial activity are related for distinct peptides. This study investigated the relationship between Staphylococcus aureusmembrane permeabilization and cell death due to the following antistaphylococcal peptides: thrombin-induced platelet microbicidal protein 1 (tPMP-1), gramicidin D, and protamine. Isogenic S. aureus strains ISP479C and ISP479R (tPMP-1 susceptible and resistant, respectively), were loaded with the fluorochrome calcein and exposed to a range of concentrations of each peptide. Flow cytometry was then used to monitor membrane permeabilization by quantifying the release of preloaded calcein. Killing was determined by quantitative culture at time points simultaneous to measurement of membrane permeabilization. Membrane permeabilization and killing caused by tPMP-1 occurred in a time- and concentration-dependent manner, reflecting the intrinsic tPMP-1 susceptibilities of ISP479C and ISP479R. In comparison, gramicidin D killed both S. aureusstrains to equivalent extents in a concentration-dependent manner between 0.5 to 50 μg/ml, but cell permeabilization only occurred at the higher peptide concentrations (25 and 50 μg/ml). Protamine permeabilized, but did not kill, either strain at concentrations up to 10 mg/ml. Regression analyses revealed different relationships between membrane permeabilization and staphylocidal activity for the distinct antimicrobial peptides. Taken together, these findings demonstrate that permeabilization, per se, does not invariably result in staphylococcal death due to distinct antimicrobial peptides. Thus, although each of these peptides interacts with the S. aureus cytoplasmic membrane, diversity exists in their mechanisms of action with respect to the relationship between membrane permeabilization and staphylocidal activity.

scholarly journals Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Marine Drugs ◽  
2021 ◽  
Vol 19 (5) ◽  
pp. 239
Author(s):  
Anguo Li ◽  
Ruihao Huang ◽  
Chaogang Wang ◽  
Qunju Hu ◽  
Hui Li ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Chlamydomonas Reinhardtii ◽  
Penaeus Monodon ◽  
Total Soluble Protein ◽  
Cell Factory ◽  
Gram Positive Bacteria ◽  
Resistance Selection ◽  
Expression Cluster ◽  
Potential Strategy

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.

scholarly journals Plant antimicrobial peptides: structures, functions, and applications

2021 ◽  
Vol 62 (1) ◽  
Author(s):  
Junpeng Li ◽  
Shuping Hu ◽  
Wei Jian ◽  
Chengjian Xie ◽  
Xingyong Yang
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Agricultural Production ◽  
Food Additives ◽  
Antimicrobial Activities ◽  
Gram Positive Bacteria ◽  
Potential Applications ◽  
Potential Applicability ◽  
Bacteria And Fungi ◽  
Positively Charged

AbstractAntimicrobial peptides (AMPs) are a class of short, usually positively charged polypeptides that exist in humans, animals, and plants. Considering the increasing number of drug-resistant pathogens, the antimicrobial activity of AMPs has attracted much attention. AMPs with broad-spectrum antimicrobial activity against many gram-positive bacteria, gram-negative bacteria, and fungi are an important defensive barrier against pathogens for many organisms. With continuing research, many other physiological functions of plant AMPs have been found in addition to their antimicrobial roles, such as regulating plant growth and development and treating many diseases with high efficacy. The potential applicability of plant AMPs in agricultural production, as food additives and disease treatments, has garnered much interest. This review focuses on the types of plant AMPs, their mechanisms of action, the parameters affecting the antimicrobial activities of AMPs, and their potential applications in agricultural production, the food industry, breeding industry, and medical field.

scholarly journals Antimicrobial Activities of Mefloquine and a Series of Related Compounds

2000 ◽  
Vol 44 (4) ◽  
pp. 848-852 ◽  
Author(s):  
C. M. Kunin ◽  
W. Y. Ellis
Keyword(s):  
Staphylococcus Epidermidis ◽  
Antimicrobial Activities ◽  
Walter Reed Army Institute ◽  
Gram Negative Bacteria ◽  
Antimicrobial Drugs ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Resistant Strains ◽  
Vancomycin Resistant ◽  
Related Compounds

ABSTRACT Mefloquine was found to have bactericidal activity against methicillin- and fluoroquinolone-susceptible and -resistant strains ofStaphylococcus aureus and Staphylococcus epidermidis and gentamicin- and vancomycin-resistant strains ofEnterococcus faecalis and Enterococcus faecium. The MICs were 16 μg/ml, and the minimal bactericidal concentrations (MBCs) were 16 to 32 μg/ml. These concentrations cannot be achieved in serum. Mefloquine was active at a more achievable concentration against penicillin-susceptible and -resistant Streptococcus pneumoniae, with MICs of 0.2 to 1.5 μg/ml. Mefloquine was not active against gram-negative bacteria and yeasts. In an attempt to find more active derivatives, 400 mefloquine-related compounds were selected from the chemical inventory of The Walter Reed Army Institute of Research. We identified a series of compounds containing a piperidine methanol group attached to pyridine, quinoline, and benzylquinoline ring systems. These had activities similar to that of mefloquine against S. pneumoniae but were far more active against other gram-positive bacteria (MICs for staphylococci, 0.8 to 6.3 μg/ml). They had activities similar to that of amphotericin B againstCandida spp. and Cryptococcus neoformans. Combinations of the compounds with gentamicin and vancomycin were additive against staphylococci and pneumococci. The MIC and MBC of gentamicin were decreased by four- to eightfold when this drug was combined with limiting dilutions of the compounds. There was no antagonism with other antimicrobial drugs. The compounds were rapidly bactericidal. They appear to act by disrupting cell membranes. Combinations of the compounds with aminoglycoside antibiotics may have potential for therapeutic use.

scholarly journals In Vitro Evaluation of Five Antimicrobial Peptides against the Plant Pathogen Erwinia amylovora

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 554
Author(s):  
Rafael J. Mendes ◽  
Laura Regalado ◽  
João P. Luz ◽  
Natália Tassi ◽  
Cátia Teixeira ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Peptides ◽  
Erwinia Amylovora ◽  
Growth Curves ◽  
Fruit Trees ◽  
Membrane Permeabilization ◽  
Control Measures ◽  
Minimal Bactericidal Concentration ◽  
Pome Fruit

Fire blight is a major pome fruit trees disease that is caused by the quarantine phytopathogenic Erwinia amylovora, leading to major losses, namely, in pear and apple productions. Nevertheless, no effective sustainable control treatments and measures have yet been disclosed. In that regard, antimicrobial peptides (AMPs) have been proposed as an alternative biomolecule against pathogens but some of those AMPs have yet to be tested against E. amylovora. In this study, the potential of five AMPs (RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) together with BP100, were assessed to control E. amylovora. Antibiograms, minimal inhibitory, and bactericidal concentrations (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), growth and IC50 were determined and membrane permeabilization capacity was evaluated by flow cytometry analysis and colony-forming units (CFUs) plate counting. For the tested AMPs, the higher inhibitory and bactericidal capacity was observed for RW-BP100 and CA-M (5 and 5–8 µM, respectively for both MIC and MBC), whilst for IC50 RW-BP100 presented higher efficiency (2.8 to 3.5 µM). Growth curves for the first concentrations bellow MIC showed that these AMPs delayed E. amylovora growth. Flow cytometry disclosed faster membrane permeabilization for CA-M. These results highlight the potential of RW-BP100 and CA-M AMPs as sustainable control measures against E. amylovora.

New type non-lantibiotic bacteriocins: circular and leaderless bacteriocins

2012 ◽  
Vol 3 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Y. Masuda ◽  
T. Zendo ◽  
K. Sonomoto
Keyword(s):  
Antimicrobial Peptides ◽  
Production Systems ◽  
Antimicrobial Compounds ◽  
Gram Positive Bacteria ◽  
Precursor Peptide ◽  
Circular Structure ◽  
Tail Structure ◽  
Circular Bacteriocin ◽  
New Type ◽  
Therapeutic Alternatives

Bacteriocins are antimicrobial peptides that are ribosomally synthesised by bacteria. Bacteriocins produced by Gram-positive bacteria, including lactic acid bacteria, are under focus as the next generation of safe natural biopreservatives and as therapeutic alternatives to antibiotics. Recently, two novel types of non-lantibiotic class II bacteriocins have been reported with unique characteristics in their structure and biosynthesis mechanism. One is a circular bacteriocin that contains a head-to-tail structure in the mature form, and the other is a leaderless bacteriocin without an N-terminal extension in the precursor peptide. A circular structure can provide the peptide with remarkable stability against various stresses; indeed, circular bacteriocins are known to possess higher stability than general linear bacteriocins. Leaderless bacteriocins are distinct from general bacteriocins, because they do not contain N-terminal leader sequences, which are responsible for the recognition process during secretion and for inactivation of bacteriocins inside producer cells. Leaderless bacteriocins do not require any post-translational processing for activity. These two novel types of bacteriocins are promising antimicrobial compounds, and their biosynthetic mechanisms are expected to be applied in synthetic biology to design new peptides and for new mass production systems. However, many questions remain about their biosynthesis. In this review, we introduce recent studies on these types of bacteriocins and their potential to open a new world of antimicrobial peptides.

scholarly journals Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Paul A. Granato ◽  
Melissa M. Unz ◽  
Raymond H. Widen ◽  
Suzane Silbert ◽  
Stephen Young ◽  
...  
Keyword(s):  
Blood Culture ◽  
Staphylococcus Epidermidis ◽  
Bloodstream Infections ◽  
Blood Cultures ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Sequencing Method ◽  
Resistance Markers ◽  
Gram Positive Cocci

ABSTRACT The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

scholarly journals Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of External Ventricular and Lumbar Drainage-Associated Ventriculitis and Meningitis

2020 ◽  
Vol 11 ◽  
Author(s):  
Lingye Qian ◽  
Yijun Shi ◽  
Fangqiang Li ◽  
Yufei Wang ◽  
Miao Ma ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Staphylococcus Epidermidis ◽  
Coincidence Rate ◽  
Next Generation ◽  
Lumbar Drainage ◽  
Gram Positive Bacteria ◽  
Neurosurgical Patients ◽  
Generation Sequencing ◽  
Guided Therapy

Metagenomic next-generation sequencing (mNGS) has become a widely used technology that can accurately detect individual pathogens. This prospective study was performed between February 2019 and September 2019 in one of the largest clinical neurosurgery centers in China. The study aimed to evaluate the performance of mNGS on cerebrospinal fluid (CSF) from neurosurgical patients for the diagnosis of external ventricular and lumbar drainage (EVD/LD)-associated ventriculitis and meningitis (VM). We collected CSF specimens from neurosurgical patients with EVD/LD for more than 24 h to perform conventional microbiological studies and mNGS analyses in a pairwise manner. We also investigated the usefulness of mNGS of CSF for the diagnosis of EVD/LD-associated VM. In total, 102 patients were enrolled in this study and divided into three groups, including confirmed VM (cVM) (39), suspected VM (sVM) (49), and non-VM (nVM) (14) groups. Of all the patients, mNGS detected 21 Gram-positive bacteria, 20 Gram-negative bacteria, and five fungi. The three primary bacteria detected were Staphylococcus epidermidis (9), Acinetobacter baumannii (5), and Staphylococcus aureus (3). The mNGS-positive coincidence rate of confirmed EVD/LD-associated VM was 61.54% (24/39), and the negative coincidence rate of the nVM group was 100% (14/14). Of 15 VM pathogens not identified by mNGS in the cVM group, eight were negative with mNGS and seven were inconsistent with the conventional microbiological identification results. In addition, mNGS identified pathogens in 22 cases that were negative using conventional methods; of them, 10 patients received a favorable clinical treatment; thus, showing the benefit of mNGS-guided therapy.

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