Protected by the host cells, the hidden intracellular bacteria are typically difficult to kill by common antibiotics and cannot be visualized without complex cellular pretreatments. Herein, we ...successfully developed a bacteria‐metabolizable dual‐functional probe TPEPy‐d‐Ala, which is based on d‐alanine and a photosensitizer with aggregation‐induced emission for fluorescence turn‐on imaging of intracellular bacteria in living host cells and photodynamic ablation in situ. Once metabolically incorporated into bacterial peptidoglycan, the intramolecular motions of TPEPy‐d‐Ala are inhibited, leading to an enhanced fluorescent signal, which allows the clear visualization of the intracellular bacteria. Moreover, TPEPy‐d‐Ala can effectively ablate the labeled intracellular bacteria in situ owing to covalent ligation to peptidoglycan, yielding a low intracellular minimum inhibitory concentration (MIC) of 20±0.5 μg mL−1, much more efficient than that of a commonly used antibiotic, vancomycin.
Search and destroy: A bacteria‐metabolizable probe, TPEPy‐d‐Ala, was designed to covalently label intracellular bacteria in living host cells. Additionally, the probe can effectively ablate the labeled bacteria in situ owing to covalent ligation to peptidoglycan, yielding a minimum inhibitory concentration of 20±0.5 μg mL−1, which is lower than that of vancomycin.
High concentrations of non-essential heavy metals/metalloids (arsenic, cadmium, and lead) in soils and irrigation water represent a threat to the environment, food safety, and human and animal ...health. Microbial bioremediation has emerged as a promising strategy to reduce the concentration of heavy metals in the environment due to the demonstrated ability of microorganisms, especially bacteria, to sequester and transform these compounds. Although several bacterial strains have been reported to be capable of remediation of soils affected by heavy metals, published information has not been comprehensively analyzed to date to recommend the most efficient microbial resources for application in bioremediation or bacterial-assisted phytoremediation strategies that may help improve plant growth and yield in contaminated soils. In this study, we critically analyzed eighty-five research articles published over the past 15 years, focusing on bacteria-assisted remediation strategies for the non-essential heavy metals, arsenic, cadmium, and lead, and selected based on four criteria: i) The bacterial species studied are part of a plant microbiome, i.e., they interact closely with a plant species ii) these same bacterial species exhibit plant growth-promoting characteristics, iii) bacterial resistance to the metal(s) is expressed in terms of the Minimum Inhibitory Concentration (MIC), and iv) metal resistance is related to biochemical or molecular mechanisms. A total of sixty-two bacterial genera, comprising 424 bacterial species/strains associated with fifty plant species were included in our analysis. Our results showed a close relationship between the tolerance level exhibited by the bacteria and metal identity, with lower MIC values found for cadmium and lead, while resistance to arsenic was widespread and significantly higher. In-depth analysis of the most commonly evaluated genera,
Agrobacterium, Bacillus, Klebsiella, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus,
and
Mesorhizobium
showed significantly different tolerance levels among them and highlighted the deployment of different biochemical and molecular mechanisms associated with plant growth promotion or with the presence of resistance genes located in the
cad
and
ars
operons. In particular, the genera
Klebsiella
and
Enterobacter
exhibited the highest levels of cadmium and lead tolerance, clearly supported by molecular and biochemical mechanisms; they were also able to mitigate plant growth inhibition under phytotoxic metal concentrations. These results position
Klebsiella
and
Enterobacter
as the best potential candidates for bioremediation and bacteria-assisted phytoremediation strategies in soils contaminated with arsenic, cadmium, and lead.
Inoculum effect of antimicrobial peptides Loffredo, Maria Rosa; Savini, Filippo; Bobone, Sara ...
Proceedings of the National Academy of Sciences - PNAS,
05/2021, Volume:
118, Issue:
21
Journal Article
Peer reviewed
Open access
Significance
Bacterial drug resistance is a crucial threat to global health, and antimicrobials with novel mechanisms of action are urgently needed. Antimicrobial peptides are natural molecules that ...kill bacteria mostly by perturbing their membranes, and they constitute promising compounds for fighting resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here, we show that the minimum peptide concentration needed for bacterial growth inhibition can vary by more than 100-fold with an increase in the density of cells in the initial inoculum of the assay (a phenomenon termed the “inoculum effect”). These findings question the utility of the currently used activity screening assays.
The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 10
5
cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide–cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.
A novel series of substituted 4,6-dimethyl-2-oxo-1-(thiazol-2-ylamino)-1,2-dihydropyridine-3-carbonitrile derivatives 6, 9, 13, 15, and 17 was synthesized in a good to excellent yield from the ...reaction of 1-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl)thiourea with 2-oxo-N'-arylpropanehydrazonoyl chloride, chloroacetone, α-bromoketones, ethyl chloroacetate, and 2,3-dichloroquinoxaline, respectively. The potential DNA gyrase inhibitory activity was examined using in silico molecular docking simulation. The novel thiazoles exhibit dock score values between - 6.4 and - 9.2 kcal/mol and they were screened for their antimicrobial activities. Compound 13a shown good antibacterial activities with MIC ranged from 93.7-46.9 μg/mL, in addition, it shown good antifungal activities with MIC ranged from 7.8 and 5.8 μg/mL.
In this study, we describe the synthesis and characterization of silver nanoparticles (Ag-NPs) of different sizes and evaluated their antibacterial activity. Particles size and morphology were ...characterized by transmission electron microscopy. Evaluation of the bacteriostatic effects was performed by ultraviolet-visible spectrophotometry and comet assays. The smaller the particle size of Ag-NPs, the smaller the value of the minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC), indicating the greater the antibacterial activity. The antibacterial activity was determined by the generation of reactive oxygen species (ROS) by bacteria and by bacterial membrane damage. In this study, we determined ROS-induced damage of bacteria caused by Ag-NPs. In conclusion, our findings indicated that Ag-NPs were effective at different particle sizes and concentrations and that the smaller the particle size of Ag-NPs, the greater the antibacterial activity.
Introduction: The adequate protocol for treatment of an infection is often determined on the basis of the minimum inhibitory concentration (MIC) of the causative organism. Traditional methods (agar ...dilution, microbroth dilution, and gradient diffusion) are labour intensive and time consuming (they are usually take over 48 hours to report the results). On the other hand, automated systems (VITEK™, Phoenix™, MicroScan WalkAway™) and rapid methods of MIC detection (using dielectrophoresis (DEP), magnetic bead rotation sensors and microfluidic incubation) require expensive instruments. This study is aimed to develop a rapid MIC detection method with the ability to applied to a resource limited setting. Methods: Agar dilution method and a novel broth dilution method (containing indicator solution) were simultaneously performed using amikacin, ceftriaxone, piperacillin-tazobactam, imipenem, cefoxitin and azithromycin. Results: Isolates of Escherichia coli, Enterobacter spp, Klebsiella spp, Staphylococcus aureus, Proteus mirabilis, Acinetobacter spp and Pseudomonas spp were used. The MIC values for Enterobacteriaceae and S. aureus isolates for each antibiotic were obtained within 4 to 5 hours by a novel broth dilution method. The obtained MIC values were corresponded with the MIC shown on the following day by agar dilution method. Conclusion: Broth dilution method with indicator solution is effective in rapid determination of the MIC for cephalosporins, penicillin, carbapenems, cephamycin, aminoglycosides and macrolides for most isolates of Enterobacteriaceae and S. aureus. Unfortunately this method did not work for the non-fermenter group of organisms like Pseudomonas spp and Acinetobacter spp, as their results could not be obtained before 24 hours. The method is time saving, relatively inexpensive and is applicable to resource limited settings.
Introduction: The adequate protocol for treatment of an infection is often determined on the basis of the minimum inhibitory concentration (MIC) of the causative organism. Traditional methods (agar ...dilution, microbroth dilution, and gradient diffusion) are labour intensive and time consuming (they are usually take over 48 hours to report the results). On the other hand, automated systems (VITEK™, Phoenix™, MicroScan WalkAway™) and rapid methods of MIC detection (using dielectrophoresis (DEP), magnetic bead rotation sensors and microfluidic incubation) require expensive instruments. This study is aimed to develop a rapid MIC detection method with the ability to applied to a resource limited setting. Methods: Agar dilution method and a novel broth dilution method (containing indicator solution) were simultaneously performed using amikacin, ceftriaxone, piperacillin-tazobactam, imipenem, cefoxitin and azithromycin. Results: Isolates of Escherichia coli, Enterobacter spp, Klebsiella spp, Staphylococcus aureus, Proteus mirabilis, Acinetobacter spp and Pseudomonas spp were used. The MIC values for Enterobacteriaceae and S. aureus isolates for each antibiotic were obtained within 4 to 5 hours by a novel broth dilution method. The obtained MIC values were corresponded with the MIC shown on the following day by agar dilution method. Conclusion: Broth dilution method with indicator solution is effective in rapid determination of the MIC for cephalosporins, penicillin, carbapenems, cephamycin, aminoglycosides and macrolides for most isolates of Enterobacteriaceae and S. aureus. Unfortunately this method did not work for the non-fermenter group of organisms like Pseudomonas spp and Acinetobacter spp, as their results could not be obtained before 24 hours. The method is time saving, relatively inexpensive and is applicable to resource limited settings.
Display omitted
•For the first time biosynthesis of silver nanoparticles (AgNPs) obtained using of Gelidium corneum.•Determination of physochemical properties of AgNPs by UV–vis, TEM, TEM-EDS, XRD, ...FTIR and ICP-MS.•Comparison of ultrastructurel effects of biosynthesized AgNPs on E.coli and C. albicans.•Potential Antimicrobial and Antibiofilm Activity of AgNPs.
In our study, green synthesis of silver nanoparticles was carried out using a red algae Gelidium corneum extract as reducing agent. The obtained silver nanoparticles were characterized by UV–vis, TEM, XRD, FTIR and ICP-MS measurements. FTIR measurements indicated the possible functional groups responsible for the stabilization and reduction of nanoparticles, while XRD analysis results explained the crystalline structure of the particles with centric cubic geometry. TEM micrographs showed that the size of the nanoparticles was between 20–50 nm. According to the broth microdilution test results, AgNPs showed a high antimicrobial activity with very low MIC values (0.51 μg/ml for Candida albicans yeast and 0.26 μg/ml for Escherichia coli bacteria). The different ultrastructural effects of silver nanoparticles on yeast and bacterial cells were observed by TEM. Antibiofilm efficacy studies were also examined in two stages as prebiofilm and postbiofilm effect. In prebiofilm effect studies, AgNPs (0.51 μg/ ml) exhibited 81% reducing effect on biofilm formation. The highest reduction rate in postbiofilm studies was 73.5% and this was achieved with 2.04 μg/ml AgNPs. Our data support that the silver nanoparticles obtained by this environmentally friendly process have potential to be used for industrial and therapeutic purposes.
Abstract
We aimed to isolate
Acinetobacter baumannii
(
A. baumannii
) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on ...the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy.
A. baumannii
(n = 200) were isolated and identified. Resistance pattern was determined and virulence genes (
afa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE
and
traT)
were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes (
bap, OmpA, abaI, csuA/B, A1S_2091, A1S_1510, A1S_0690, A1S_0114
) were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of
A. baumannii
with human fibroblast skin cell line HFF-1 or Vero cell lines.
A. baumannii
had high level of resistance to antibiotics. Most of the isolates harbored the
fimH
,
afa/draBC
,
cnf1
,
csgA
and
cnf2,
and the majority of
A. baumannii
produced strong biofilms. AgNPs inhibited the growth of
A. baumannii
efficiently with MIC ranging from 4 to 25 µg/ml.
A. baumannii
showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of
kpsMII
,
afa/draBC,bap, OmpA,
and
csuA/B
genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular
A. baumannii
. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.
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