Biogenic silver nanoparticles reduce adherence, infection, and proliferation of toxoplasma gondii RH strain in HeLa cells without inflammatory mediators induction.

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity - depending on size and concentration - and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 µM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

Evaluation of the Antibiotic Resistance and Virulence of Escherichia coli Strains Isolated from Chicken Carcasses in 2007 and 2013 from Paraná, Brazil.

The frequent use of antimicrobials in commercial poultry production has raised concerns regarding the potential impact of antimicrobials on human health due to selection for resistant bacteria. Several studies have reported similarities between extraintestinal pathogenic Escherichia coli (ExPEC) strains isolated from birds and humans, indicating that these contaminant bacteria in poultry may be linked to human disease. The aim of our study was to analyze the frequency of antimicrobial resistance and virulence factors among E. coli strains isolated from commercial chicken carcasses in Paraná, Brazil, in 2007 and 2013. A total of 84 E. coli strains were isolated from chicken carcasses in 2007, and 121 E. coli strains were isolated in 2013. Polymerase chain reaction was used to detect virulence genes (hlyF, iss, ompT, iron, and iutA) and to determine phylogenetic classification. Antimicrobial susceptibility testing was performed using 15 antimicrobials. The strains were also confirmed as extended-spectrum ß-lactamase (ESBL)-producing E. coli with phenotypic and genotypic tests. The results indicated that our strains harbored virulence genes characteristic of ExPEC, with the iutA gene being the most prevalent. The phylogenetic groups D and B1 were the most prevalent among the strains isolated in 2007 and 2013, respectively. There was an increase in the frequency of resistance to a majority of antimicrobials tested. An important finding in this study was the large number of ESBL-producing E. coli strains isolated from chicken carcasses in 2013, primarily for the group 2 cefotaximase (CTX-M) enzyme. ESBL production confers broad-spectrum resistance and is a health risk because ESBL genes are transferable from food-producing animals to humans via poultry meat. These findings suggest that our strains harbored virulence and resistance genes, which are often associated with plasmids that can facilitate their transmission between bacteria derived from different hosts, suggesting zoonotic risks.

Antibacterial Activity of Biodegradable Films Incorporated with Biologically-Synthesized Silver Nanoparticles and the Evaluation of Their Migration to Chicken Meat.

The food industry has been exploring the association of polymers with nanoparticles in packaging production, and active products are essential to increase the shelf life of food and avoid contamination. Our study developed starch-poly (adipate co-terephthalate butyl) films with silver nanoparticles produced with Fusarium oxysporum components (bio-AgNPs), intending to control foodborne pathogens. The bio-AgNPs showed activity against different Salmonella serotypes, including multidrug-resistant Salmonella Saint Paul and Salmonella Enteritidis, with minimum bactericidal concentrations ranging from 4.24 to 16.98 µg/mL. Biodegradable films with bio-AgNPs inhibited the growth of up to 106Salmonella isolates. Silver migration from the films to chicken was analyzed using electrothermal atomic absorption spectrophotometry, and the results showed migration values (12.94 mg/kg and 3.79 mg/kg) above the limits allowed by the European Food Safety Authority (EFSA) (0.05 mg/kg). Thus, it is necessary to improve the technique to avoid the migration of silver to chicken meat, since these concentrations can be harmful.

Cellulose Hydrogel with Hyaluronic Acid and Silver Nanoparticles: Sustained-Release Formulation with Antibacterial Properties against Pseudomonas aeruginosa.

Pathogenic bacteria resistant to conventional antibiotics represent a global challenge and justify the need for new antimicrobials capable of combating bacterial multidrug resistance. This study describes the development of a topical hydrogel in a formulation composed of cellulose, hyaluronic acid (HA), and silver nanoparticles (AgNPs) against strains of Pseudomonas aeruginosa. AgNPs as an antimicrobial agent were synthesized by a new method based on green chemistry, using arginine as a reducing agent and potassium hydroxide as a carrier. Scanning electron microscopy showed the formation of a composite between cellulose and HA in a three-dimensional network of cellulose fibrils, with thickening of the fibrils and filling of spaces by HA with the presence of pores. Ultraviolet-visible spectroscopy (UV-vis) and particle size distribution for dynamic light scattering (DLS) confirmed the formation of AgNPs with peak absorption at ~430 nm and 57.88 nm. AgNPs dispersion showed a minimum inhibitory concentration (MIC) of 1.5 µg/mL. The time-kill assay showed that after 3 h of exposure to the hydrogel containing AgNPs, there were no viable cells, corresponding to a bactericidal efficacy of 99.999% in the 95% confidence level. We obtained a hydrogel that is easy to apply, with sustained release and bactericidal properties against strains of Pseudomonas aeruginosa at low concentrations of the agent.

Antibiofilm Effect of Biogenic Silver Nanoparticles Combined with Oregano Derivatives against Carbapenem-Resistant Klebsiella pneumoniae.

Resistant bacteria may kill more people than COVID-19, so the development of new antibacterials is essential, especially against microbial biofilms that are reservoirs of resistant cells. Silver nanoparticles (bioAgNP), biogenically synthesized using Fusarium oxysporum, combined with oregano derivatives, present a strategic antibacterial mechanism and prevent the emergence of resistance against planktonic microorganisms. Antibiofilm activity of four binary combinations was tested against enteroaggregative Escherichia coli (EAEC) and Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC): oregano essential oil (OEO) plus bioAgNP, carvacrol (Car) plus bioAgNP, thymol (Thy) plus bioAgNP, and Car plus Thy. The antibiofilm effect was accessed using crystal violet, MTT, scanning electron microscopy, and Chromobacterium violaceum anti-quorum-sensing assays. All binary combinations acted against preformed biofilm and prevented its formation; they showed improved antibiofilm activity compared to antimicrobials individually by reducing sessile minimal inhibitory concentration up to 87.5% or further decreasing biofilm metabolic activity and total biomass. Thy plus bioAgNP extensively inhibited the growth of biofilm in polystyrene and glass surfaces, disrupted three-dimensional biofilm structure, and quorum-sensing inhibition may be involved in its antibiofilm activity. For the first time, it is shown that bioAgNP combined with oregano has antibiofilm effect against bacteria for which antimicrobials are urgently needed, such as KPC.

Hydrogel-Containing Biogenic Silver Nanoparticles: Antibacterial Action, Evaluation of Wound Healing, and Bioaccumulation in Wistar Rats.

Wound infections are feared complications due to their potential to increase healthcare costs and cause mortality since multidrug-resistant bacteria reduce treatment options. This study reports the development of a carbomer hydrogel containing biogenic silver nanoparticles (bioAgNPs) and its effectiveness in wound treatment. This hydrogel showed in vitro bactericidal activity after 2 h, according to the time-kill assay. It also reduced bacterial contamination in rat wounds without impairing their healing since the hydrogel hydrophilic groups provided hydration for the injured skin. The high number of inflammatory cells in the first days of the skin lesion and the greater degree of neovascularization one week after wound onset showed that the healing process occurred normally. Furthermore, the hydrogel-containing bioAgNPs did not cause toxic silver accumulation in the organs and blood of the rats. This study developed a bioAgNP hydrogel for the treatment of wounds; it has a potent antimicrobial action without interfering with cicatrization or causing silver bioaccumulation. This formulation is effective against bacteria that commonly cause wound infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, and for which new antimicrobials are urgently needed, according to the World Health Organization's warning.

Biogenic Silver Nanoparticles Strategically Combined With Origanum vulgare Derivatives: Antibacterial Mechanism of Action and Effect on Multidrug-Resistant Strains.

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.

Biogenic silver nanoparticles reduce Toxoplasma gondii infection and proliferation in RAW 264.7 macrophages by inducing tumor necrosis factor-alpha and reactive oxygen species production in the cells.

Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 µM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis.

Synergistic and Additive Effect of Oregano Essential Oil and Biological Silver Nanoparticles against Multidrug-Resistant Bacterial Strains.

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 ß-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.

Comparison of Antibiotic Resistance and Virulence Factors among Escherichia coli Isolated from Conventional and Free-Range Poultry.

Microbiological contamination in commercial poultry production has caused concerns for human health because of both the presence of pathogenic microorganisms and the increase in antimicrobial resistance in bacterial strains that can cause treatment failure of human infections. The aim of our study was to analyze the profile of antimicrobial resistance and virulence factors of E. coli isolates from chicken carcasses obtained from different farming systems (conventional and free-range poultry). A total of 156 E. coli strains were isolated and characterized for genes encoding virulence factors described in extraintestinal pathogenic E. coli (ExPEC). Antimicrobial susceptibility testing was performed for 15 antimicrobials, and strains were confirmed as extended spectrum of ß-lactamases- (ESBLs-) producing E. coli by phenotypic and genotypic tests. The results indicated that strains from free-range poultry have fewer virulence factors than strains from conventional poultry. Strains from conventionally raised chickens had a higher frequency of antimicrobial resistance for all antibiotics tested and also exhibited genes encoding ESBL and AmpC, unlike free-range poultry isolates, which did not. Group 2 CTX-M and CIT were the most prevalent ESBL and AmpC genes, respectively. The farming systems of poultries can be related with the frequency of virulence factors and resistance to antimicrobials in bacteria.