ABSTRACT
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.
ABSTRACT
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.