In recent years, there was great interest in nanoparticles with antibacterial and antibiofilm activity as new strategy against multidrug resistant bacteria 11, 29. Silver nanoparticles are known to have antibacterial and antibiofilm activities. Several methods used for synthesis silver nanoparticles has been reported in several previous studies 11. The aim of this study is to synthesize silver nanoparticles using three different chemical procedures and investigating their dual antibacterial and antibiofim activities on both planktonic and sessile bacterial isolates and whether the effect differ according to the type of method or to the bacteria.
Synthesis of silver nanoparticles was made by three ways, ZnO-Ag Xerogel, Ag-NaHB4 / PVP, Ag-Glucose). After synthesis, characterization of nanoparticles was made using UV-visible spectrophotometer, infrared spectra, FTIR (data not shown) and by SEM and AFM. The results revealed that size and shape of nanoparticles were (rod 54 nm, spherical 20 ± 16nm, and spherical 40-80nm, for silver nanoparticles with NaHB4/PVP, Zno-Ag, and Ag with glucose, respectively).
Biofilm formation assay was used to determine the ability of bacteria to form biofilm and the results showed four types of bacteria were able to form biofilm (two of them were gram positive- S. aurues, and S. agaglactaie- and the other two were gram negative- Pseudomonas aeruginosa, and Klebsiella pneumonaie). There were different strategies for defining the bacteria that can produce biofilm, two strategies were applied in the current study (the first is considering 0.1 as a cut-off value and the second is applying the equation: Biofilm formation= the OD at 595 nm of the stain extracted from attached cells – the OD at 595 nm of control wells). The results showed that Pseudomonas produce higher biofilm layer followed by Klebsiella which is indicated by higher optical density value. However, there were no significant difference in biofilm formation among the four bacteria.
The synthesized nanoparticles were tested against biofilm producing bacteria to investigate the effect on planktonic cell and sessile cells. Regarding the antibacterial activity of silver nanoparticles, the results demonstrated that the three forms of silver nanoparticles Significantly reduce the absorbance of the biomass for the tested bacterial isolates as compared with their controls. Additionally, when we compare the antibacterial efficacy of the three forms of silver nanoparticles against bacterial isolates the results showed that silver nanoparticles prepared by glucose had statistically significant lowest effect.
Concerning the antibiofilm activity, the results showed that there was significant inhibition of biofilm formation for all tested bacteria when it compared with control (P value ?0.05). However, there were no significant difference in inhibition among the type of bacteria. This mean that the antibiofilm activity of different silver nanoparticles does not differ according to the type of bacterial isolates but at the same time the results revealed that K. pneumoniae and S. agalactiae biofilm formation is inhibited more likely with ZnO-Ag nanoparticles more than other nanoparticles (99.2%, 94.2%, respectively). Whereas, P. aeruginosa and S. aureus biofilm formation were greatly inhibited by silver nanoparticles prepared by glucose as compared with other types of nanoparticles (98.8%, 97.8%, respectively).
Moreover, the results indicated that 0.0125 µg/ ml concentration for the three types of silver nanoparticles can cause significant inhibition for more than ? 93 % of biofilm and 90% of cell biomass for all types of bacterial isolates. Lower percentage of antibacterial and antibiofilm activity with higher concentration were reported in previous studies. Palanisamy et al., showed that the antibiofilm activity of chemically synthesized silver nanoparticles (at concentration of 20 ?g/ml and size of nanoparticles is about 20-30 nm) was 67% at bacterial concentration of 10-4 for sensitive Pseudomonas and 56% at bacterial concentration 10-5 and 10-6 for multidrug resistant Pseudomonas and there was no inhibition at concentration 10-8 for both strains 10. In other previous study, biologically synthesized silver nanoparticles had antibiofilm activity (more than 90 %) against P. aeruginosa and S. flexneri at the concentration of 0.5 ?g/ml and against S. aureus and S. pneumonaie with the concentration 0.7 ?g/ml 11. In the same study, 0.6 ?g/ml cause 95% reduction in cell viability of P. aeruginosa and S. flexneri and that the concentration of about 0.5 ?g/ml cause 50% inhibition for S. aureus and S. pneumonia (the nanoparticle size was 2-10nm).
Amany et al. showed that MIC of silver nanoparticles prepared by glucose and NaHB4/PVP were 30 µg/mL, 40 µg/mL, respectively against S. aureus ATCC 6538 15.
Its proposed that the antibacterial effect of silver result from the inhibition of enzymatic system of respiratory chain and interference with DNA synthesis30. Another proposed mechanism is by pore formation in cell membrane, result in cell destruction 31.
Yammato et al. and Sawai et al. suggested that the inhibitory effect of ZnO-Ag nanoparticles result from hydrogen peroxide generated from surface of ZnO 32, 33.
Previous study documented that different antibacterial effect of silver nanoparticles against bacteria might possibly leads to various levels of biofilm inhibition10. In the current study, higher antibiofilm activity was observed than antibacterial effect for the tested bacteria. This mean that silver nanoparticles affect sessile cells than planktonic cells when they tested simultaneously.