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Extracellular Synthesis and Characterization of Silver Nanoparticles-Antibacterial Activity against Multidrug-Resistant Bacterial Strains.

ABSTRACT: Herein, we report the use of a cell-free extract for the extracellular synthesis of silver nanoparticles (AgNPs) and their potential to address the growing threat of multidrug-resistant (MDR) pathogenic bacteria. The reproducibility of AgNP synthesis was good and AgNP formation kinetics were monitored as a function of various reaction factors via ultraviolet-visible absorption spectroscopy. This green method was dependent on the alkaline pH of the reaction mixture. With the addition of dilute sodium hydroxide, well-dispersed AgNPs could be produced in large quantities via the classical nucleation and growth route. The new biosynthetic route enabled the production of AgNPs within a narrow size range of 4 to 17 nm. The AgNPs were characterized using various techniques and their antibacterial activity against MDR pathogenic bacteria was evaluated. Field-emission scanning electron microscopic imaging revealed prominent morphological changes in Staphylococcus aureus cells due to mechanical damage, which led to cell death. Escherichia coli cells showed signs of contraction and intracellular fluid discharge as a consequence of disrupted cell membrane function. This new biologically-assisted extracellular strategy is potentially useful for the decontamination of surfaces and is expected to contribute to the development of new products containing AgNPs.

SUBMITTER: Ghodake G

PROVIDER: S-EPMC7075330 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Extracellular Synthesis and Characterization of Silver Nanoparticles-Antibacterial Activity against Multidrug-Resistant Bacterial Strains.

Ghodake Gajanan G Kim Min M Sung Jung-Suk JS Shinde Surendra S Yang Jiwook J Hwang Kyojung K Kim Dae-Young DY

Nanomaterials (Basel, Switzerland) 20200219 2

Herein, we report the use of a cell-free extract for the extracellular synthesis of silver nanoparticles (AgNPs) and their potential to address the growing threat of multidrug-resistant (MDR) pathogenic bacteria. The reproducibility of AgNP synthesis was good and AgNP formation kinetics were monitored as a function of various reaction factors via ultraviolet-visible absorption spectroscopy. This green method was dependent on the alkaline pH of the reaction mixture. With the addition of dilute so ...[more]

PMID: 32092941

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Project description:Bacterial resistance to conventional antibiotics has become a clinical and public health problem, making therapeutic decisions more challenging. Plant compounds and nanodrugs have been proposed as potential antimicrobial alternatives. Studies have shown that oregano (Origanum vulgare) essential oil (OEO) and silver nanoparticles have potent antibacterial activity, also against multidrug-resistant strains; however, the strong organoleptic characteristics of OEO and the development of resistance to these metal nanoparticles can limit their use. This study evaluated the antibacterial effect of a two-drug combination of biologically synthesized silver nanoparticles (bio-AgNP), produced by Fusarium oxysporum, and OEO against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. OEO and bio-AgNP showed bactericidal effects against all 17 strains tested, with minimal inhibitory concentrations (MIC) ranging from 0.298 to 1.193 mg/mL and 62.5 to 250 μM, respectively. Time-kill curves indicated that OEO acted rapidly (within 10 min), while the metallic nanoparticles took 4 h to kill Gram-negative bacteria and 24 h to kill Gram-positive bacteria. The combination of the two compounds resulted in a synergistic or additive effect, reducing their MIC values and reducing the time of action compared to bio-AgNP used alone, i.e., 20 min for Gram-negative bacteria and 7 h for Gram-positive bacteria. Scanning electron microscopy (SEM) revealed similar morphological alterations in Staphylococcus aureus (non-methicillin-resistant S. aureus, non-MRSA) cells exposed to three different treatments (OEO, bio-AgNP and combination of the two), which appeared cell surface blebbing. Individual and combined treatments showed reduction in cell density and decrease in exopolysaccharide matrix compared to untreated bacterial cells. It indicated that this composition have an antimicrobial activity against S. aureus by disrupting cells. Both compounds showed very low hemolytic activity, especially at MIC levels. This study describes for the first time the synergistic and additive interaction between OEO and bio-AgNP produced by F. oxysporum against multidrug-resistant bacteria, such as MRSA, and β-lactamase- and carbapenemase-producing Escherichia coli and Acinetobacter baumannii strains. These results indicated that this combination can be an alternative in the control of infections with few or no treatment options.

Project description:Objectives:Due to the expanded bacterial genetic tolerance to antibiotics through different mechanisms, infectious diseases of MDR bacteria are difficult for treatment. Consequently, we synthesized drug conjugated nanoparticles to dissolve this problem. Moreover, the present study aims to display the cell death status treated with cefotaxime-CS-AgNPs and also, apoptosis pathways of human RPE-1 normal cells and human MCF-7 breast cancer cells. Methods:Here, we demonstrate the possibility to synthesize AgNPs and conjugate them with cefotaxime to survey the probability of cefotaxime-CS-AgNPs as an antimicrobial agent against cefotaxime-resistant strains E. coli and MRSA. Results:TEM showed the size of AgNPs, CS-AgNPs and cefotaxime-CS-AgNPs ranged from 7.42 to 18.3 nm, 8.05-23.89 nm and 8.48-25.3 nm, respectively, with a spherical shape. The cefotaxime-CS-AgNPs enhanced the high antimicrobial properties compared to AgNPs or pure antibiotic. The MIC of Cefotaxime-CS-AgNPs ranged from 3 µg/mL to 8 µg/mL against tested E. coli and MRSA bacteria. Consequently, the highest reduction in the MIC of cefotaxime-CS-AgNPs was noted against tested strains ranging from 22% to 96%. Comparing cefotaime-CS-AgNPs to AgNPs we showed that cefotaime-CS-AgNPs have no cytotoxic effect on normal cells at even 12 µg/mL for 24 hrs. The IC50 for the AgNPs and cefotaxime-CS-AgNPs was 12 µg/mL for human RPE-1 normal cells and human MCF-7 breast cancer cell lines. The pro-apoptotic genes p53, p21, and Bax of cancer cell lines significantly upregulated followed by downregulated by anti-apoptotic gene Bcl-2 after 48 hrs at 24 µg/mL, and this concentration represents the most effective dose. Conclusion:Results enhanced the conjugating utility in old unresponsive cefotaxime to AgNPs to restore its efficiency against previous strains and demonstrated potential therapeutic applications of cefotaxime-CS-AgNPs. Moreover, this research gives remarkable insights for designing nanoscale delivery and curative systems that have a pronounced cytotoxic activity on cancer cells and are safe to normal cells.

Project description:Multidrug-resistant bacteria have become a public health problem worldwide, reducing treatment options against several pathogens. If we do not act against this problem, it is estimated that by 2050 superbugs will kill more people than the current COVID-19 pandemic. Among solutions to combat antibacterial resistance, there is increasing demand for new antimicrobials. The antibacterial activity of binary combinations containing bioAgNP (biogenically synthesized silver nanoparticles using Fusarium oxysporum), oregano essential oil (OEO), carvacrol (Car), and thymol (Thy) was evaluated: OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy. This study shows that the mechanism of action of Thy, bioAgNP, and Thy plus bioAgNP involves damaging the membrane and cell wall (surface blebbing and disruption seen with an electron microscope), causing cytoplasmic molecule leakage (ATP, DNA, RNA, and total proteins) and oxidative stress by enhancing intracellular reactive oxygen species and lipid peroxidation; a similar mechanism happens for OEO and Car, except for oxidative stress. The combination containing bioAgNP and oregano derivatives, especially thymol, shows strategic antibacterial mechanism; thymol disturbs the selective permeability of the cell membrane and consequently facilitates access of the nanoparticles to bacterial cytoplasm. BioAgNP-treated Escherichia coli developed resistance to nanosilver after 12 days of daily exposition. The combination of Thy and bioAgNP prevented the emergence of resistance to both antimicrobials; therefore, mixture of antimicrobials is a strategy to extend their life. For antimicrobials alone, minimal bactericidal concentration ranges were 0.3-2.38 mg/ml (OEO), 0.31-1.22 mg/ml (Car), 0.25-1 mg/ml (Thy), and 15.75-31.5 μg/ml (bioAgNP). The time-kill assays showed that the oregano derivatives acted very fast (at least 10 s), while the bioAgNP took at least 30 min to kill Gram-negative bacteria and 7 h to kill methicillin-resistant Staphylococcus aureus (MRSA). All the combinations resulted in additive antibacterial effect, reducing significantly minimal inhibitory concentration and acting faster than the bioAgNP alone; they also showed no cytotoxicity. This study describes for the first time the effect of Car and Thy combined with bioAgNP (produced with F. oxysporum components) against bacteria for which efficient antimicrobials are urgently needed, such as carbapenem-resistant strains (E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) and MRSA.

Project description:BackgroundMultidrug-resistant (MDR) bacteria are acknowledged as one of the main factors contributing to chronic illnesses and fatalities globally. Numerous diseases, including bloodstream infections, pneumonia, urinary tract infections, and surgical site infections, can be brought on by MDR bacteria. Therefore, a crucial topic of continuing research is the development of a novel and different treatment for MDR microbial pathogens. This work is introduce an alternative method for elimination of MDR bacterial isolates which are causative agents of urinary tract infection among people in Egypt. In our study, we need a novel strategy to combat MDR bacteria by green-synthesized metal nanoparticles (MNPs). That is due to the ability of MNPs to penetrate the cell wall and the cell membrane of gram-positive and gram-negative bacteria.MethodsClinical isolates of MDR bacteria had their antibiotic susceptibility assessed before being molecularly identified using 16 s rRNA, sequencing, and phylogenetic analysis. Also, genetic profiles of isolated strains were performed using ISSR and SDS-PAGE. Finally, characterized plant-mediated silver nanoparticles derived from lemon and pomegranate peel extracts were evaluated against isolated multidrug-resistant bacterial stains.ResultsIn our present trial, one-hundred urine samples were collected from 71 females and 29 males complaining of UTI (urinary tract infection) symptoms. One-hundred microbial isolates were isolated, including 88-g negative and only 8-g positive bacteria in addition to four yeast isolates (Candida species). A total of 72% of the isolated bacteria showed MDR activity. The most prevalent MDR bacterial isolates (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, and Klebsiella pneumoniae) were identified through 16S rDNA PCR sequencing as with accession numbers OP741103, OP741104, OP741105, OP741106, and OP741107, respectively. Lemon and pomegranate-mediated silver nanoparticles [Ag-NPs] were characterized by UV spectroscopy, FTIR, XRD, and TEM with average size 32 and 28 nm, respectively. Lemon and pomegranate-mediated silver nanoparticles [Ag-NPs] showed an inhibitory effect on the selected five MDR isolates at MIC 50 and 30 µg/mL, respectively. These common bacterial isolates were also genetically examined using ISSR PCR, and their total protein level was evaluated using SDS-PAGE, showing the presence of distinct genetic and protein bands for each bacterial species and emphasizing their general and protein composition as a crucial and essential tool in understanding and overcoming MDR behavior in UTI patients.ConclusionsLemon and pomegranate-mediated silver nanoparticles [Ag-NPs] were found to have an inhibitory effect on MDR isolates. Therefore, the study suggests that [Ag-NPs] could be a potential treatment for MDR UTI infections caused by the identified bacterial species.

Project description: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.

Dataset Information

Extracellular Synthesis and Characterization of Silver Nanoparticles-Antibacterial Activity against Multidrug-Resistant Bacterial Strains.

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Extracellular Synthesis and Characterization of Silver Nanoparticles-Antibacterial Activity against Multidrug-Resistant Bacterial Strains.

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