Antibacterial activity means either killing bacteria or slowing down their growth which are named as bactericidal or bacteriostatic respectively. It should be non-toxic to surrounding tissue . Antibacterial agents are very useful in water disinfection, textile industry, medicine, and food packaging. Antibacterial compounds are divided into natural and chemically modified natural compounds. However, the development of bacterial resistance to antibacterial compounds is increasing and becoming a major problem . Therefore, development of alternative compounds to treat bacterial diseases is necessary .
Nanoparticles (NPs) as a new alternative for antimicrobial compounds have developed. The main characteristic of NPs is high surface area to volume ratio which causes new properties such as chemical, mechanical, electrical, optical, and magnetic as different from their bulk properties . In this case, NPs attracted the interest of Scientifics to apply them for combating bacteria . The precise mechanisms of NP toxicity on the various bacteria have not been understood entirely. As it obvious, NPs would attach to the bacteria membrane by electrostatic interaction and disrupt the integrity of the bacterial membrane . NPs with antibacterial activity included metallic and metal oxide NPs, semiconductors, polymers, and carbon-based materials against gram-positive and gram-negative bacteria . Some NPs such as NiO nanoplates show good zone of inhibition against B. subtilis, S. aureus, E. coli and P. vulgaris . And also NiNPs was reported to show potential antimicrobial activity against E. coli, K. pneumoniae, (Gram negative) B. cereus and S. aureus (Gram positive) . Therefore, NiNPs and Ni(OH)2-NPs have been revealed antibacterial activity. Recent data from the U.S. National Healthcare Safety Network indicate that gram-negative bacteria are responsible for more than 30% of hospital-acquired infections. Also, U. S. data indicate that E. coli is the most common etiologic gram-negative organism, followed in descending order of frequency by P. aeruginosa, klebsiella species, enterobacter species, and A. baumannii . There is no report in calculating two significant parameters as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Finally, it is important to note that until now, the antimicrobial properties NiNPs and Ni(OH)2-NPs have not been studied on multidrug resistance K. pneumonia and E. coli. Therefore, the purposes of this study were the synthesis and characterization of NiNPs and Ni(OH)2-NPs as well as investigate their antimicrobial activity [a1] against two selected standard pathogenic bacteria and real clinical multidrug resistance K. pneumonia and E. coli.
Materials and Methods
Materials and apparatus
The materials were purchased and used as received without any further treatment. All materials including Nickel chloride (NiCl2⋅6H2O), sodium hydroxide (NaOH) ,polyvinilprolydon (PVP), ethanol (99%), H2O2 (30%), phenol, 4-Aminoantipyrine (4-AAP), trypic soy agar (TSA), trypic soy broth (TSB) were obtained from Merck (Germany). Hydrazine hydrate (N2H4⋅H2O), 98%, was purchased from Roialex (India). Tetracycline (TE), ampicillin (AM) and medium were obtained from Sigma. The pH measurements were carried out with a pH meter Elmeiron model CP-501. Dynamic light scattering (DLS) (Brookhaven, USA) were used to measure size the nanoparticles. The Surface properties of the NiNPs were recorded using atomic force microscopy (AFM, Nanosurf easyScan, Swiss) in semi contact mode through silicon tip. SEM image of Ni(OH)2-NPs were obtained by SEM (Phenom ProX, Netherlands). Attenuated total reflection (ATR) of nanoparticles was carried out using a Perkin-Elmer UATR Two (USA). All solutions were prepared in DI water [11, 12].
Synthesis of NiNPs
The NiNPs were synthesized by reduction of NiCl2⋅6H2O in an aqueous solution with hydrazine hydrate acting as the reductant . Briefly, 0.5 g NiCl2⋅6H2O was dissolved in 60mL distilled water. In a separate beaker, 0.2 g of PVP dissolved in 3mL of absolute ethanol and then added to 1.0 g of NaOH in 20mL DI water and then 20mL of hydrazine hydrate. This hydrazine hydrate solution then added to nickel solution. Subsequently, the combined solution stirred for 2 minutes until royal blue color appears, in a pH∼12.5. According to Chen and Zhou`s method, the reaction temperature needs to stay below 70∘C in order to prepare ultrafine NiNPs. The solution was sonicated for 15 minutes to ∼55∘C, allowed to sit particles for 15 minutes in order to control the temperature, and then sonicated again for 15 minutes to ∼65∘C, to activate the precipitation of the NiNPs. During the activation period, the solution initially became grey and coated along the side and bottom of the beaker and then precipitated out to the bottom as a black powder. The nickel powder solution was centrifuged and washed with DI water and was allowed to dry at room temperature.
Synthesis of Ni(OH)2-NPs
The synthesis of Ni(OH)2-NPs was done base on previously reported method . The Ni(OH)2-NPs was prepared by two steps: firstly, solution A was prepared by dissolving 1.19 g NiCl2⋅6H2O in 50mL DI water; then 200mL of absolute ethanol was added to it, forming a green solution. The solution was transferred into a 500mL conical flask. Secondly, solution B was prepared in a 200mL volumetric flask by dissolving 0.4 g of sodium hydroxide in 100mL of DI water; 1.5mL of hydrazine hydrate was added and then diluted up to 200 ml with DI water. Subsequently, solution B was slowly dropped into solution A at a rate of ∼0.2mL per minute and mixed with a magnetic stirrer. The resulting solution became cloudy green with precipitate forming as soon as the pH reached 6.8 and then turned to a cloudy blue green. The mixed solution finally became a grey blue with the addition of 80–90mL of solution B at a final pH of ∼10. The solution was sonicated for 15 minutes to ∼55∘C, allowed to sit for 15 minutes in order to control the temperature, and then sonicated again for 15 minutes to ∼65∘C. The precipitated solution was centrifuged. The Nickel Oxide powder solution was centrifuged and washed with DI water and was allowed to dry at room.
Preparation of nanoparticle suspensions
The synthesized Ni and Ni(OH)2 NPs were added to 1mL of double distilled water to obtain a final concentration of 20 mg/ml and shaken vigorously and dispersed by ultrasonication ( BANDELIN, Germany)
Spot on thelawn test
The Antimicrobial propertices of NiNPs and Ni(OH)2-NPs were investigated using E. coli (PTCC1394as gram negative) and S. aureus (PTCC1431 as gram positive) and 8 clinical multidrug resistance K. pneumonia and E. coli ( Isolated urinary tract, Milad Laboratory, Yazd, Iran, Table 4) by the spot on the lawn method . The bacterial suspension was rubbed uniformly on the surface of a Tryptic Soy Agar (TSA, Merck, Germany) plate at a concentration of 107 CFU/ml. For antibacterial investigation, Antibacterial activity of NiNPs and Ni(OH)2-NPs were tested by spotting from 2.5 to 100 µl (presented in Table 1 and 2) of nanoparticle suspensions (20 mg/ml) on TAS plate seeded with 107 cell/mL E. coli , S. aureus and 8 clinical pathogenic samples respectively. Antibiotic disks including Tetracycline (TE) and Ampicillin (AM) were used as positive control for to comparing the inhibition of growth of bacteria with NiNPs and Ni(OH)2-NPs respectively. For negative control water was used. The control and sample plates were incubated at 37°c for 24h, after which the average diameter of the inhibition zone surrounding the spot was measured with a ruler.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination
The MIC is defined as the minimum inhibitory concentration of antibacterial material sufficient to prevent bacterial growth in vitro. The MICs were determined using the broth dilution technique as recommended by CLSI. Briefly, serial dilutions of Ni and Ni (OH)2-NPs (13- 0.5 of mg/ml) in TSB (Trypic Soy Broth, Merck, Germany) were prepared. Bacterial suspensions were then added to each tube to achieve final inoculums of 8 logs CFU/mL. Tubes containing growth media alone or bacterial culture without Ni and Ni(OH)2-NPs suspension were included as negative and positive controls, respectively. The cultures were inspected for bacterial growth after incubation at 37°C for 24 h and the MICs of Ni and Ni(OH)2-NPs suspension were recorded by measuring optical density at 620 nm in a spectrophotometer. The amount of NPs that lead to growth inhibition of bacteria was regarded as MIC. The MBC (minimum bactericidal concentration) was defined as the lowest concentration of Ni and Ni (OH)2-NPs suspension that resulted in more than 99.9% reduction of the initial inoculums. MBC was assessed by sub-culturing 50 µl on to TSA, from each tube in which no growth was viewable. After incubation for 24 h at 37°C, the number of grown colonies will be enumerated. The growth of one colony showed a 99.9% fall for in viable numeration .
Characterizations of NiNPs and Ni (OH)2-NPs
The formation of the NiNPs was confirmed by studying two dimensional AFM image. The AFM image for NiNPs at glass plate, is shown in Fig 1. SEM image of Ni (OH)2-NPs is presented in Fig 2. ATR would be useful for investigation of the synthesized NiNPs and Ni(OH)2-NPs. ATR spectra for NiNPs in KBr disc and that gotten for free PVP were compared and shown in Fig. 3. The ATR spectrum of and Ni (OH)2-NPs was illustrated in Fig. 5. The ATR results showed a strong peak at 1654 cm−1 which is related for the carbonyl stretch of pure PVP. The C=O stretch was made weaker and also red shifts from pure PVP to 1581 cm−1 as nickel is protected with PVP. Additionally the peaks around 1280 to 1180 cm−1 indicated the C–N stretch in PVP. As it could be seen, the peaks are strong with three different peaks for each of the C–N bonds in pure PVP. But as NiNPs was synthesized, this peak became weaker in the region around 1214 cm−1. Hence, these changes in ATR spectra revealed that NiNPs synthesized by PVP (16).
The ATR spectrum of Ni (OH)2-NPs in Fig 4 was demonstrated a hydroxyl stretching band related to Ni(OH)2 lattice at 3300 cm−1. Also, an H–O–H bend is observed at 1635 cm−1 from the vibration of free water molecules. The spectrum also shows a sharp O–H stretch at 608 cm−1 from the hydroxyl lattice vibration and a weak peak around 480 cm−1 indicating a Ni–O lattice vibration . As a whole, based on data obtained from ATR spectra for NiNPs and Ni (OH)2-NPs and compared in literature, it would be resulted that NiNPs and Ni(OH)2-NPs were synthesized completely.
DLS technique was applied for measuring the average sizes of NiNPs and Ni (OH) 2-NPs. The results showed in Fig 5. It can be seen that the average diameter for in deionized water were 5 and 75 nm respectively.
Antibacterial assay based on spot on thelawn method
The antibacterial activity of nanoparticles was taken on the basis of diameter of zone of inhibition of bacterial growth which was measured at cross-angles after 24hr of incubation. The results are shown in Tables 2 and 3 illustrated the inhibition of the E. coli, S. aureus and 8 clinical multidrug resistance K. pneumonia and E. coli on the agar plates by using different concentrations of NiNPs and Ni(OH)2-NPs respectively. As it shown, in the presence of NPs the zone of inhibition of bacterial growth was increased which is related to increasing antibacterial activity. Based on decreasing the bacterial growth in present of nanoparticle, it would be concluded that these nanoparticles show antibacterial activity on E. coli, S. aureus and 8 clinical multidrug resistances K.
pneumonia and E. coli
MBC and MIC assay
MBC and MIC concentration which is related to power of nanoparticles to settle down the E. coli , S. aureus and 8 clinical multidrug resistance K. pneumonia and E. coli growth determined and demonstrated in Table 4. It could be seen that the lower concentration of NiNPs is enough for prevention S. aureus bacterial growth than E. coli. But for Ni(OH)2-NPs almost the same concentration was needed for inhibiting the E. coli and S. aureus bacteria growth. These data confirm that NiNPs and Ni(OH)2-NPs presence to have an antimicrobial activity on E. coli and S. aureus. Among the NiNPs and Ni(OH)2-NPs , the treatment of NiNPs was the most effective in inhibition of the E. coli and S. aureus bacteria growth. Additionally, MBC and MIC concentration for 8 clinical multidrug resistance K. pneumonia and E. coli showed that NiNPs and Ni(OH)2-NPs have good antibacterial activity against four groups as categorized: sensitive (NDR), sensitive (MDR), resistant (XDR) and completely resistant (PDR).
NiNPs and Ni(OH)2-NPs were prepared and characterized by DLS and spectroscopic technique. Experimental investigations have shown encouraging results about antibacterial activity of NiNPs and Ni(OH)2-NPs on E. coli and S. aureus bacteria. The inhibition zone diameters of bacterial growth and MBCs and MIC parameters determined. Furthermore, 8 clinical bacteria from two different bacteria strain as E. coli and K. pneumoniae in 4 groups such as sensitive (NDR), semi-sensitive (MDR), resistant (XDR) and completely resistant (PDR) was investigated for confirming antibacterial activity of NiNPs and Ni(OH)2-NPs. As reported in literature, NiNPs (20-25nm diameter) showed antibacterial activity against gram positive and negative bacteria. And also the MBC factor was 100 mg.mL-1 but the MCI factor has not been determined yet . In another study the effect of NiNPs (25-100 nm) on different bacteria and fungi was investigated. These studies showed the inhibition zone diameters of bacterial growth by NiNPs against E. coli and K. pneumoniae was 15.3-25.1 and 9.5-23.3 mm respectively. And also, the antibacterial effect of NiNPs was approved based on MBC and MIC parameters. The MBC parameter was determined for NiNPs against E. coli and K. pneumoniae were 25±0.3 and 25±2.8 mg.mL-1. And the MIC parameters were 25±2.4 and 25±0.7 mg.mL-1 for E. coli and K. pneumoniae respectively . Santhoshkumar et al. reported the antibacterial activity of Ni(OH)2-NPs (96 nm in diameter) against K. pneumoniae. The results showed that inhibition zone diameters of bacterial growth was 23 mm . Up to now there is no report for calculating two significant parameters as MIC and MBC for NiNPs and Ni(OH)2-NPs. In this study we reported inhibition zone diameters of bacteria growth, MIC and MBC parameters for two standard gram negative and positive bacteria as E. coli and S. aureus bacteria respectively. The MBC parameter for NiNPs and Ni(OH)2-NPs against E. coli were 3.25 mg.mL-1 and 6.5 mg.mL-1 respectively. These results demonstrated that the MBC parameter was so lower compare to literature. Additionally the MIC parameter for E. coli was lower compare to literature too. It would be concluded that the antibacterial effect of nanoparticles depends on their size and morphology. Since, in this study the size of NiNPs and was 5 nm which was lower than reported in literature.
And for confirming the antibacterial activity of NiNPs and Ni(OH)2-NPs, 8 pathogenic clinical were tested. For these clinical pathogens three parameters as inhibition zone diameters of bacterial growth, MBCs and MIC showed that NiNPs and Ni(OH)2-NPs possess good antibacterial activity.
NiNPs and Ni(OH)2-NPs were synthesized and characterized by AFM, SEM, ATR, DLS. NiNPs and Ni(OH)2-NPs show an antibacterial activity against S. aureus, E. coli and 8 clinical bacteria from two different bacteria strain as E. coli and K. pneumoniae in 4 groups such as NDR, MDR, XDR and PDR. The recommended daily amounts of nickel are not fixed, but it is suggested that it is enough to intake about 100 micrograms per day. However, some studies show that it can be consumed daily about 200-750 micrograms of nickel (21). So, this study reveals the potential of NiNPs and Ni(OH)2-NPs as an antimicrobial agent at as antimicrobial coatings on surface of materials for various environmental and as the antiseptic agent for environment.
The financial support of Payam e Noor university of Taft branch is greatly acknowledged. Also authors gratefully acknowledge the generous cooperation of the Nano Structured Coatings Institute, Yazd Payame Noor University, Yazd, Iran.