scholarly journals Role of Viscoelasticity in Bacterial Killing by Antimicrobials in Differently GrownPseudomonas aeruginosaBiofilms

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
René T. Rozenbaum ◽  
Henny C. van der Mei ◽  
Willem Woudstra ◽  
Ed D. de Jong ◽  
Henk J. Busscher ◽  
...  
Keyword(s):  
Stress Relaxation ◽  
Environmental Dna ◽  
Luria Bertani ◽  
Matrix Composition ◽  
Growth Media ◽  
Multiple Characteristic ◽  
Bacterial Killing ◽  
Time Constants ◽  
Content Type ◽  
Insoluble Polysaccharides

ABSTRACTPseudomonas aeruginosacolonizes the sputum of most adult cystic fibrosis patients, forming difficult-to-eradicate biofilms in which bacteria are protected in their self-produced extracellular polymeric substance (EPS) matrices. EPS provide biofilms with viscoelastic properties, causing time-dependent relaxation after stress-induced deformation, according to multiple characteristic time constants. These time constants reflect different biofilm (matrix) components. Since the viscoelasticity of biofilms has been related to antimicrobial penetration but not yet bacterial killing, this study aims to relate killing ofP. aeruginosa, in its biofilm mode of growth, by three antimicrobials to biofilm viscoelasticity.P. aeruginosabiofilms were grown for 18 h in a constant-depth film fermenter, with mucin-containing artificial sputum medium (ASM+), artificial sputum medium without mucin (ASM−), or Luria-Bertani (LB) broth; this yielded 100-μm-thick biofilms that differed in their amounts of matrix environmental DNA (eDNA) and polysaccharides. Low-load compression testing, followed by three-element Maxwell analyses, showed that the fastest relaxation component, associated with unbound water, was most important in LB-medium-grown biofilms. Slower components due to water with dissolved polysaccharides, insoluble polysaccharides, and eDNA were most important in the relaxation of ASM+-grown biofilms. ASM−-grown biofilms showed intermediate stress relaxation.P. aeruginosain LB-medium-grown biofilms was killed most by exposure to tobramycin, colistin, or an antimicrobial peptide, while ASM+provided the most protective matrix, with less water and most insoluble polysaccharides and eDNA. In conclusion, stress relaxation ofP. aeruginosabiofilms grown in different media revealed differences in matrix composition that, within the constraints of the antimicrobials and growth media applied, correlated with the matrix protection offered against different antimicrobials.

scholarly journals Ability of Bicarbonate Supplementation To Sensitize Selected Methicillin-Resistant Staphylococcus aureus Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Warren E. Rose ◽  
Ana M. Bienvenida ◽  
Yan Q. Xiong ◽  
Henry F. Chambers ◽  
Arnold S. Bayer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bactericidal Activity ◽  
Ex Vivo ◽  
Rabbit Model ◽  
Luria Bertani ◽  
Growth Media ◽  
Methicillin Resistant ◽  
Content Type ◽  
Mrsa Strains

ABSTRACT Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) increases β-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains (“NaHCO3 responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO3-responsive or NaHCO3-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO3. Intra-SEV MRSA killing was determined over 72-h exposures. In both “responsive” strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO3-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA

2021 ◽  
Vol 70 (4) ◽  
Author(s):  
Sharda Prasad Awasthi ◽  
Nityananda Chowdhury ◽  
Noritoshi Hatanaka ◽  
Atsushi Hinenoya ◽  
Thandavarayan Ramamurthy ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Type Species ◽  
Sandwich Elisa ◽  
Luria Bertani ◽  
Production Level ◽  
Quantitative Assay ◽  
Growth Media ◽  
Mice Model ◽  
Content Type ◽  
Link Type

Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae . However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae . Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis. Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection. Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA. Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml−1 of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml−1 to 1.6 µg ml−1. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20–80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested. Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.

scholarly journals Adaptation and Heterogeneity of Escherichia coli MC1000 Growing in Complex Environments

2012 ◽  
Vol 79 (3) ◽  
pp. 1008-1017 ◽  
Author(s):  
Pilar Eliana Puentes-Téllez ◽  
Martin Asser Hansen ◽  
Søren Johannes Sørensen ◽  
Jan Dirk van Elsas
Keyword(s):  
Escherichia Coli ◽  
Luria Bertani ◽  
Growth Media ◽  
Adaptive Responses ◽  
Complex Environments ◽  
Content Type ◽  
Batch Cultures ◽  
Genomic Analyses ◽  
Oxygen Conditions

ABSTRACTIn a study aiming to assess bacterial evolution in complex growth media, we evaluated the long-term adaptive response ofEscherichia coliMC1000 in Luria-Bertani (LB) medium. Seven parallel populations were founded and followed over 150 days in sequential batch cultures under three different oxygen conditions (defined environments), and 19 evolved forms were isolated. The emergence of forms with enhanced fitness was evident in competition experiments of all evolved forms versus the ancestral strain. The evolved forms were then subjected to phenotypic and genomic analyses relative to the ancestor. Profound changes were found in their phenotypes as well as whole-genome sequences. Interestingly, considerable heterogeneity was found at the intrapopulational level. However, consistently occurring parallel adaptive responses were found across all populations. The evolved forms all contained a mutation ingalR, a repressor of the galactose operon. Concomitantly, the new forms revealed enhanced growth on galactose as well as galactose-containing disaccharides. This response was likely driven by the LB medium.

scholarly journals Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

2012 ◽  
Vol 80 (11) ◽  
pp. 3921-3929 ◽  
Author(s):  
Donporn Riyapa ◽  
Surachat Buddhisa ◽  
Sunee Korbsrisate ◽  
Jon Cuccui ◽  
Brendan W. Wren ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Neutrophil Extracellular Traps ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Predisposing Factor ◽  
Bacterial Killing ◽  
Content Type ◽  
Extracellular Traps ◽  
Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

scholarly journals Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl

2013 ◽  
Vol 80 (1) ◽  
pp. 247-256 ◽  
Author(s):  
Janja Zajc ◽  
Tina Kogej ◽  
Erwin A. Galinski ◽  
José Ramos ◽  
Nina Gunde-Cimerman
Keyword(s):  
Biomass Production ◽  
Osmotic Shock ◽  
Model Organism ◽  
Salinity Range ◽  
Growth Media ◽  
Optimal Salinity ◽  
Content Type ◽  
Batch Cultures ◽  
Specific Growth Rates ◽  
Optimum Salinity

ABSTRACTWallemia ichthyophagais a fungus from the ancient basidiomycetous genusWallemia(Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilicW. ichthyophagaand to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates ofW. ichthyophagawere higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth ofW. ichthyophagawas between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition,W. ichthyophagamaintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence,W. ichthyophagashould become well established as a novel model organism for studies of halophily in eukaryotes.

scholarly journals Activity of Meropenem-Vaborbactam againstPseudomonas aeruginosaandAcinetobacter baumanniiin a Neutropenic Mouse Thigh Infection Model

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Mojgan Sabet ◽  
Ziad Tarazi ◽  
David C. Griffith
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Clinical Isolates ◽  
Infection Model ◽  
Model Data ◽  
Bacterial Killing ◽  
Content Type

ABSTRACTWe have evaluated the activity of meropenem-vaborbactam against clinical isolates ofPseudomonas aeruginosaandAcinetobacter baumanniiin a neutropenic mouse thigh infection model. Data show that meropenem-vaborbactam regimens equivalent to 3-h infusions every 8 h with 2 g meropenem and 2 g vaborbactam produced bacterial killing against strains with MICs of 2 to 16 mg/liter and suggests that this combination may have utility in the treatment of infections caused byP. aeruginosaandA. baumannii.

scholarly journals Meropenem Combined with Ciprofloxacin Combats Hypermutable Pseudomonas aeruginosa from Respiratory Infections of Cystic Fibrosis Patients

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Vanessa E. Rees ◽  
Rajbharan Yadav ◽  
Kate E. Rogers ◽  
Jürgen B. Bulitta ◽  
Veronika Wirth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Time Course ◽  
Respiratory Infections ◽  
Resistant Bacteria ◽  
Infection Model ◽  
Time Curve ◽  
Bacterial Killing ◽  
Content Type ◽  
Concentration Time

ABSTRACT Hypermutable Pseudomonas aeruginosa organisms are prevalent in chronic respiratory infections and have been associated with reduced lung function in cystic fibrosis (CF); these isolates can become resistant to all antibiotics in monotherapy. This study aimed to evaluate the time course of bacterial killing and resistance of meropenem and ciprofloxacin in combination against hypermutable and nonhypermutable P. aeruginosa. Static concentration time-kill experiments over 72 h assessed meropenem and ciprofloxacin in mono- and combination therapies against PAO1 (nonhypermutable), PAOΔmutS (hypermutable), and hypermutable isolates CW8, CW35, and CW44 obtained from CF patients with chronic respiratory infections. Meropenem (1 or 2 g every 8 h [q8h] as 3-h infusions and 3 g/day as a continuous infusion) and ciprofloxacin (400 mg q8h as 1-h infusions) in monotherapies and combinations were further evaluated in an 8-day hollow-fiber infection model study (HFIM) against CW44. Concentration-time profiles in lung epithelial lining fluid reflecting the pharmacokinetics in CF patients were simulated and counts of total and resistant bacteria determined. All data were analyzed by mechanism-based modeling (MBM). In the HFIM, all monotherapies resulted in rapid regrowth with resistance at 48 h. The maximum daily doses of 6 g meropenem (T>MIC of 80% to 88%) and 1.2 g ciprofloxacin (area under the concentration-time curve over 24 h in the steady state divided by the MIC [AUC/MIC], 176), both given intermittently, in monotherapy failed to suppress regrowth and resulted in substantial emergence of resistance (≥7.6 log10 CFU/ml resistant populations). The combination of these regimens achieved synergistic killing and suppressed resistance. MBM with subpopulation and mechanistic synergy yielded unbiased and precise curve fits. Thus, the combination of 6 g/day meropenem plus ciprofloxacin holds promise for future clinical evaluation against infections by susceptible hypermutable P. aeruginosa.

scholarly journals A Distinguishable Role of eDNA in the Viscoelastic Relaxation of Biofilms

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Brandon W. Peterson ◽  
Henny C. van der Mei ◽  
Jelmer Sjollema ◽  
Henk J. Busscher ◽  
Prashant K. Sharma
Keyword(s):  
Relaxation Time ◽  
Stress Relaxation ◽  
Time Constant ◽  
Extracellular Polymeric Substances ◽  
Principal Component ◽  
Mechanical Stresses ◽  
Extracellular Dna ◽  
Viscoelastic Relaxation ◽  
Content Type ◽  
Relaxation Time Constants

ABSTRACTBacteria in the biofilm mode of growth are protected against chemical and mechanical stresses. Biofilms are composed, for the most part, of extracellular polymeric substances (EPSs). The extracellular matrix is composed of different chemical constituents, such as proteins, polysaccharides, and extracellular DNA (eDNA). Here we aimed to identify the roles of different matrix constituents in the viscoelastic response of biofilms.Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus mutans, andPseudomonas aeruginosabiofilms were grown under different conditions yielding distinct matrix chemistries. Next, biofilms were subjected to mechanical deformation and stress relaxation was monitored over time. A Maxwell model possessing an average of four elements for an individual biofilm was used to fit the data. Maxwell elements were defined by a relaxation time constant and their relative importance. Relaxation time constants varied widely over the 104 biofilms included and were divided into seven ranges (<1, 1 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, and >500 s). Principal-component analysis was carried out to eliminate related time constant ranges, yielding three principal components that could be related to the known matrix chemistries. The fastest relaxation component (<3 s) was due to the presence of water and soluble polysaccharides, combined with the absence of bacteria, i.e., the heaviest masses in a biofilm. An intermediate component (3 to 70 s) was related to other EPSs, while a distinguishable role was assigned to intact eDNA, which possesses a unique principal component with a time constant range (10 to 25 s) between those of EPS constituents. This implies that eDNA modulates its interaction with other matrix constituents to control its contribution to viscoelastic relaxation under mechanical stress.IMPORTANCEThe protection offered by biofilms to organisms that inhabit it against chemical and mechanical stresses is due in part to its matrix of extracellular polymeric substances (EPSs) in which biofilm organisms embed themselves. Mechanical stresses lead to deformation and possible detachment of biofilm organisms, and hence, rearrangement processes occur in a biofilm to relieve it from these stresses. Maxwell analysis of stress relaxation allows the determination of characteristic relaxation time constants, but the biofilm components and matrix constituents associated with different stress relaxation processes have never been identified. Here we grew biofilms with different matrix constituents and used principal-component analysis to reveal that the presence of water and soluble polysaccharides, together with the absence of bacteria, is associated with the fastest relaxation, while other EPSs control a second, slower relaxation. Extracellular DNA, as a matrix constituent, had a distinguishable role with its own unique principal component in stress relaxation with a time constant range between those of other EPSs.

scholarly journals In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens

2021 ◽  
Author(s):  
Catrina Olivera ◽  
Vuong Van Hung Le ◽  
Catherine Davenport ◽  
Jasna Rakonjac
Keyword(s):  
Growth Inhibition ◽  
Type Species ◽  
Sodium Deoxycholate ◽  
Gram Positive ◽  
Bacterial Killing ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Time Kill

Introduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens. Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy. Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria. Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively. Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli , Klebsiella pneumoniae and Acinetobacter baumannii , and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner. Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.

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