scholarly journals Identification and Characterization of the First Cholesterol-Dependent Cytolysins from Gram-Negative Bacteria

2012 ◽  
Vol 81 (1) ◽  
pp. 216-225 ◽  
Author(s):  
Eileen M. Hotze ◽  
Huynh M. Le ◽  
Jessica R. Sieber ◽  
Christina Bruxvoort ◽  
Michael J. McInerney ◽  
...  
Keyword(s):  
Cell Structure ◽  
Bacterial Species ◽  
Cytolytic Activity ◽  
Opportunistic Pathogens ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Positive Type ◽  
Secretion Signal ◽  
Anaerobic Soils

The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from theFirmicutesandActinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which includeDesulfobulbus propionicustype species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) andEnterobacter lignolyticus(formerlyEnterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neitherD. propionicusnorE. lignolyticusis known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.

scholarly journals In VitroActivity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens

2015 ◽  
Vol 59 (10) ◽  
pp. 6053-6063 ◽  
Author(s):  
Douglas J. Biedenbach ◽  
Michael D. Huband ◽  
Meredith Hackel ◽  
Boudewijn L. M. de Jonge ◽  
Daniel F. Sahm ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Dna Gyrase ◽  
Species Group ◽  
Coagulase Negative Staphylococci ◽  
Topoisomerase Iv ◽  
Gram Positive ◽  
Bacterial Dna ◽  
Gram Negative ◽  
Content Type

ABSTRACTAZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed thein vitroactivity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter againstStaphylococcus aureus(n= 11,680), coagulase-negative staphylococci (n= 1,923), streptococci (n= 4,380), andMoraxella catarrhalis(n= 145), 0.5 mg/liter againstStaphylococcus lugdunensis(n= 120) andHaemophilus influenzae(n= 352), 1 mg/liter againstEnterococcus faecalis(n= 1,241), and 2 mg/liter againstHaemophilus parainfluenzae(n= 70). The activity againstEnterococcus faeciumwas more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, β-lactamase-producingHaemophilusspp., andM. catarrhalis. Based on thesein vitrofindings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species.

scholarly journals In VitroAntibacterial Activity of AZD0914, a New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria

2014 ◽  
Vol 59 (1) ◽  
pp. 467-474 ◽  
Author(s):  
Michael D. Huband ◽  
Patricia A. Bradford ◽  
Linda G. Otterson ◽  
Gregory S. Basarab ◽  
Amy C. Kutschke ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Dna Gyrase ◽  
Cross Resistance ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Topoisomerase Inhibitor

ABSTRACTAZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potentin vitroantibacterial activity against key Gram-positive (Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus pneumoniae,Streptococcus pyogenes, andStreptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzaeandNeisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance inS. aureus, and if mutants were obtained, the mutations mapped togyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration andin vitrotime-kill studies. Inin vitrocheckerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potentin vitroantibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.

scholarly journals Nybomycin Inhibits both Fluoroquinolone-Sensitive and Fluoroquinolone-Resistant Escherichia coli DNA Gyrase

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Dmitrii I. Shiriaev ◽  
Alina A. Sofronova ◽  
Ekaterina A. Berdnikovich ◽  
Dmitrii A. Lukianov ◽  
Ekaterina S. Komarova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
Dna Gyrase ◽  
Resistant Bacteria ◽  
Mutant Form ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type

ABSTRACT Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are targets of many antibiotics, including fluoroquinolones (FQs). Unfortunately, a number of bacterial species easily acquire resistance to FQs by mutations in either DNA gyrase or topoisomerase IV genes. The emergence of resistant pathogenic strains is a global problem in health care; therefore, identifying alternative pathways to thwart their persistence is the current frontier in drug discovery. Nybomycins are an attractive class of compounds, reported to be “reverse antibiotics” that selectively inhibit growth of some Gram-positive FQ-resistant bacteria by targeting the mutant form of DNA gyrase while being inactive against wild-type strains with FQ-sensitive gyrases. The strong “reverse” effect was demonstrated only for a few Gram-positive organisms resistant to FQs due to the S83L/I mutation in the GyrA subunit of DNA gyrase. However, the activity of nybomycins has not been extensively explored among Gram-negative species. Here, we observed that in a ΔtolC strain of the Gram-negative Escherichia coli with enhanced permeability, wild-type gyrase and a GyrA S83L mutant, resistant to fluoroquinolones, are similarly sensitive to nybomycin.

scholarly journals Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Omeed Faghih ◽  
Zhongsheng Zhang ◽  
Ranae M. Ranade ◽  
J. Robert Gillespie ◽  
Sharon A. Creason ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Trna Synthetase ◽  
Resistant Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Methionyl Trna Synthetase

ABSTRACT Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

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 Phylogenetic Analysis of Chromosomally Determined Qnr and Related Proteins

2013 ◽  
Vol 57 (4) ◽  
pp. 1930-1934 ◽  
Author(s):  
George A. Jacoby ◽  
David C. Hooper
Keyword(s):  
Phylogenetic Analysis ◽  
Bacterial Species ◽  
Strictly Anaerobic ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Repeat Protein ◽  
Protein Encoding ◽  
Related Proteins

ABSTRACTqnrgenes were discovered on plasmids by their ability to reduce quinolone susceptibility, but homologs can be found in the genomes of at least 92 Gram-negative, Gram-positive, and strictly anaerobic bacterial species. The related pentapeptide repeat protein-encodingmfpAgene is present in the genome of at least 19 species ofMycobacteriumand 10 otherActinobacteriaspecies. The native function of these genes is not yet known.

scholarly journals Antibacterial Activity of Sphingoid Bases and Fatty Acids against Gram-Positive and Gram-Negative Bacteria

2011 ◽  
Vol 56 (3) ◽  
pp. 1157-1161 ◽  
Author(s):  
Carol L. Fischer ◽  
David R. Drake ◽  
Deborah V. Dawson ◽  
Derek R. Blanchette ◽  
Kim A. Brogden ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Antibacterial Activity ◽  
Lauric Acid ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Sphingoid Bases ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Corynebacterium Striatum

ABSTRACTThere is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity—the sphingoid basesd-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid—against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P< 0.0001) for each bacterial species exceptSerratia marcescensandPseudomonas aeruginosa.d-Sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria exceptS. marcescensandP. aeruginosa(MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active againstStreptococcus sanguinis,Streptococcus mitis, andFusobacterium nucleatumbut not active againstEscherichia coli,Staphylococcus aureus,S. marcescens,P. aeruginosa,Corynebacterium bovis,Corynebacterium striatum, andCorynebacterium jeikeium(MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria exceptE. coli,S. marcescens, andP. aeruginosa(MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.

scholarly journals Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Joel D. Ernst ◽  
Amber Cornelius ◽  
Miriam Bolz
Keyword(s):  
Protein Secretion ◽  
Immunosorbent Assay ◽  
Bacterial Population ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Protein ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

scholarly journals Evaluation of MicroScan Bacterial Identification Panels for Low-Resource Settings

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 349
Author(s):  
Sien Ombelet ◽  
Alessandra Natale ◽  
Jean-Baptiste Ronat ◽  
Olivier Vandenberg ◽  
Liselotte Hardy ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Ease Of Use ◽  
Bacterial Identification ◽  
Gram Positive ◽  
Gram Negative ◽  
Low Resource Settings ◽  
Low Resource ◽  
Bacillus Spp ◽  
Gram Negative Organisms ◽  
Instructions For Use

Bacterial identification is challenging in low-resource settings (LRS). We evaluated the MicroScan identification panels (Beckman Coulter, Brea, CA, USA) as part of Médecins Sans Frontières’ Mini-lab Project. The MicroScan Dried Overnight Positive ID Type 3 (PID3) panels for Gram-positive organisms and Dried Overnight Negative ID Type 2 (NID2) panels for Gram-negative organisms were assessed with 367 clinical isolates from LRS. Robustness was studied by inoculating Gram-negative species on the Gram-positive panel and vice versa. The ease of use of the panels and readability of the instructions for use (IFU) were evaluated. Of species represented in the MicroScan database, 94.6% (185/195) of Gram-negative and 85.9% (110/128) of Gram-positive isolates were correctly identified up to species level. Of species not represented in the database (e.g., Streptococcus suis and Bacillus spp.), 53.1% out of 49 isolates were incorrectly identified as non-related bacterial species. Testing of Gram-positive isolates on Gram-negative panels and vice versa (n = 144) resulted in incorrect identifications for 38.2% of tested isolates. The readability level of the IFU was considered too high for LRS. Inoculation of the panels was favorably evaluated, whereas the visual reading of the panels was considered error-prone. In conclusion, the accuracy of the MicroScan identification panels was excellent for Gram-negative species and good for Gram-positive species. Improvements in stability, robustness, and ease of use have been identified to assure adaptation to LRS constraints.

scholarly journals Factors Influencing the Microbial Composition of Metalworking Fluids and Potential Implications for Machine Operator's Lung

2011 ◽  
Vol 78 (1) ◽  
pp. 34-41 ◽  
Author(s):  
Jean-Benjamin Murat ◽  
Frédéric Grenouillet ◽  
Gabriel Reboux ◽  
Emmanuelle Penven ◽  
Adam Batchili ◽  
...  
Keyword(s):  
Metal Cutting ◽  
Hypersensitivity Pneumonitis ◽  
Environmental Parameters ◽  
Metalworking Fluids ◽  
Microbial Composition ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Kinetics Of

ABSTRACTHypersensitivity pneumonitis, also known as “machine operator's lung” (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especiallyMycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces ofM. immunogenumwere sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by furtherin vitrosimulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.

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