SYNERGISTIC EFFECT OF COPPER OXIDE NANOPARTICLES FOR ENHANCING ANTIMICROBIAL ACTIVITY AGAINST K. PNEUMONIAE AND S. AUREUS

This study was aimed to assess the antimicrobial activity of copper oxide nanoparticles (CuO NPs) created by method of thermal green way using basically a maize starch. Mucoid were appeared of Klebsiella pneumoniae bacterial colonies and the positive results with some biochemical tests . On the other hand, Staphylococcus aureus appeared pigmented colonies surrounded by a yellow halo because of mannitol fermentation. According to the 24 time incubation period, the CuO NPs antimicrobial activity showed of bacterial growth pathogenic K. pneumonia was 0.52 ± 0.04 cell/ml than control 1.60 ± 0.01 cell/ml .Aven as S. aureus appeared the number of bacterial growth as follow 0.79 ± 0.07 cell/ml compared with control 1.90 ± 0.01 cell/ml. The biologically effect for enhancing antimicrobial activity the percentage of resistant was decreasing from 66.6% to 22.2% when used copper oxide nanoparticles. Also, S. aureus sensitivity test showed resistant percentage was decreased from 55.5% to 33.3% at 24 hours.

) with moved at 10 min toward arrive at white color starch suspension.Then the suspension color was altered to a bold blue when added starch solution to copper sulphate solution with continues stirring.Later added 0.5M (15 drop wise) an abundance amount of ammonia solution until pH solution reached 10, later than 20 min.Then for 2min put the mixture in microwave (LG/Korea), the mixture set was formatted is CuO nanoparticles that changed to a suspension black color.Then centrifugation at ten min (8000 rpm) was used.Finally, wash by DDW and ethanol many times the precipitant to make it free impurities organic and from sulphate, ammonia.By oven for 2 hours precipitant was dried at 150ºC.Finally, obtained black expected CuO NPs powder (18).In previous study, the properties nanoparticles showed were very pure, globular shape, the ranging diameter size of particles was from (47.41 to 109.49)nm and constant, in addition average crystallite measure is 9.8nm.However, the average distribution (d50) is71.17nm(1).To prepare a stock CuO suspension using0.001gprepared black powdered dissolving with 5ml DDW (stock solution).Than was used Sonicatar instrument (Heraeus/Germany) to homogenizing and fine dissolving for more analysis.

Bacterial isolation K. pneumoniae
isolation was done by streaking loopful from brain heart infusion broth culture previously isolated from pus specimen on MacConky agar and on EMB agar for primary selection of pathogenic K. pneumoniae (3,4,5,17).According to MacFaddin (21), the biochemical tests were employed Catalase, Oxidase, Motility, Urease, Indol, Methyl Red(MR), Voges-Proskauer and Citrate utilization.Beside these tests, Api 20E system kit (Bio-Meriaux, France) and VITEK 2 system kit (Bio-Meriaux, France) were also checked for identification of K. pneumoniae.On the other hand, S. aureus also previously identified from purulent wound and on manitol salt agar.The bacterial isolated were activated on brain heart infusion broth(himedia) and incubated over night at 37 ᵒC.

Antibacterial activity tests
The activity antimicrobial of 40μg/ml concentration CuONPs was investigated against two strains; K. pneumonia is represented gram-negative organisms and S. aureus is represented gram positive bacteria.Activated bacterial strains on nutrient broth sterile (Himedia/India).Prepared bacteria suspension by alone colony was inoculated for 24h in nutrient broth with turbidity adjust using 0.5 McFarland standards.in brief, (100 µl /40 μg/ml) of CuO NPs were made was added to NB media sterile (5ml).Then inoculated with activated bacterial strains(0.1 ml).Later at37°C incubated these tubes for 24 hours.Control was involved; 0.1 ml of nanoparticles inoculated with nutrient broth media only at 37ºC.Finally, measured the bacterial growth after incubation using uv-vis spectrophotometer (DRG/ USA) at 625nm (18).The triplicate reading values of mean for each bacterial strain was recorded.CuO NPs synergistic effect with diverse antibacterial agents K. pneumonia and S. aureus incubated with the CuO NPs (40μg/ml) was subjected to evaluate the synergistic effect with diverse antimicrobials agents usually was used in present test.The Kirby-Bauer disk diffusion method was used on Mueller-Hinton agar(MHA) plates for determined synergistic effect (7).Antimicrobial disks (Bioanalyse/Turkey) involve; Methicillin (ME) (10μg), Trimethoprim/ sulphamethoxazole (SXT) (25μg), Amoxillin/ clavulanic acid (AMC) (30μg), Gentamicin (CN) (10μg), Levofloxacin (LEV) (5μg), Ciprofloxacin (CIP) ( 10μg), Cefixime (CFM) (5μg), Cefotaxime (CTX) (5μg) and Amikacin (AK) (30 μg) were used (Table 1).CuO NPs (40 μg/ml) with activated bacterial strain suspension (1.5 X 10 8 ) CFU/ml at McFarland 0.5 were mixed.0.1 ml of a mixture were inoculated by dispersed regularly in Mueller Hinton agar (Himedia /India) plates via swab, then antimicrobial disks were dispensed.Afterward incubated these plates for 24 h.at 37°C.After that calculated the zone of inhibition(ZOI) in millimeters (mm) around each antimicrobial disk, and compared to the activated bacterial (0.1 ml) inoculated directly on plate of Mueller-Hinton agar (Control).In addition, the degree sensitivity was determined relation to rules of National Committee for Clinical and Laboratory Standards Institute (NCCLs) (37)

Characteristics of bacterial isolates
The colonies of K. pneumoniae were appeared mucoid on MacConky agar (Figure 1) and the positive results with sugar catalase, Simmons' citrate, urease and Voges-Proskauer except negative results in MR, Indol, oxidase and motility tests were identified in biochemical testing (Table 2).Result of Api-20E system was agreed with previous biochemical tests (Figure 2).The consequences similar to Patel, et al. (25).On the other hand, the results mannitol fermentation of S. aureus on mannitol salt agar appearance pigmented colonies surrounded by a yellow halo and lush (Figure 3).

Figure 1. A result of K. pneumoniae on MacConky agar Table 2. The biochemical tests of K. pneumoniae bacterial isolates Figure 2. A positive result of Api20E for K. pneumoniae Figure 3. A result of S. aureus on mannitol salt agar Antibactericidal tests
The results bactericidal impact of biosynthesis 40μg/ml (200μl from stock solution added to 5ml DW)concentrations of copper oxide nanoparticles against pathogenic K. pneumonia and S.aureus strains.According to the 24 time incubation period, showed of pathogenic K. pneumonia was recorded 0.52 ± 0.04 cell/ml compared with control 1.60 ± 0.01 cell/ml (Table3) (Figure4) .While S.aureus showed the number of bacterial growth absorption as 0.79 ± 0.07 compared with control 1.90 ± 0.01 cell/ml (Table3) (Figure5).From consequences exposed previous, the 40mg/ml concentration of copper oxide nanoparticles has impact antimicrobial against G-ve and G+ve bacteria, and all were recorded significant alteration than control.Structure of cell wall bacteria, particle size, in addition to the degree of bacterial cell suitability test was achieved that impact these consequences represent and to settle on the impact of bactericidal assorted attachment the organisms with NPs (20).The great quantity of amines and carboxyl groups on cell surface of bacteria, that raise the Cu ions attraction towards both bacteria and ascribed to the template CuO NPs that lead to the greater sensitivity of these groups to the CuO NPs(19).Mechanisms diverse have been planned to translate the antibacterial conduct of metal oxides.(6) agreement with our current study, all have behavior antimicrobial higher, which is represented that nanoparticle smaller sizes that helped the nanoparticles access during membrane of bacteria and react with component of its.

S.aureus investigation CuO NPs synergistically impact with diverse antibacterial agents
The 40μg/ml of CuONPs incubation with bacterial strains (K.pneumonia, and S. aureus) during period 24h.Than the bacterial strains sensitivity test against diverse antimicrobial was done suggested by (13), using method of disc diffusion.Depending on guideline of the (NCCLs).The consequences sensitivity of K. pneumoniae to copper oxide nanoparticles and biologically effect for enhancing antimicrobial activity were converted from resistance to sensitive for Methicillin, Ciprofloxacin, Cefataxime and Cefixime at 24hours compared with the resistance control.Furthermore, K. Pneumoniae isolates showed a high sensitive rate to Gentamicin, Levofloxacin and Amikacin (100%), and the percentage of resistant was decreasing from 66.6% to 22.2% when used Copper oxide nanoparticles at 24 hours(Table4) (Figure6).Also, sensitivity test of S. aureus appeared change against antimicrobial agents in the level of resistant involve: Trimethoprim sulphamethox and Cefataxime which was converted from resistant to sensitive at24 hours that copper oxide nanoparticles enhancing antimicrobial activity compared with the resistance control.Thus the resistant percentage was decreased from 55.5% to 33.3%.Whereas S. aureus (MRSA) results showed high sensitivity rate to Gentamicin, Levofloxacin, Ciprofloxacin and Amikacin at a percentage (100%).As well as, S. aureus was showed high degree of resistant to Methicillin, Amoxillin/ clavulanic acid and Cefixime at a percentage (100%) (Table5)(Figure 6).In addition to, sensitivity of K. pneumoniae to copper oxide nanoparticles and biologically effect for enhancing antimicrobial activity higher than S. aureus.The structural of the cell membrane in addition to the compositional contrasts could be ascribed to variation in the antimicrobial agents impact as well as CuO nanoparticles against K. pneumonia and S. aureus (15).Thicker peptidoglycan cell membranes for Gram-positive bacteria contrasted with thin peptidoglycan cell membranes for Gramve bacteria and a low antibacterial effect of CuO of Gram-positive bacteria for the reason that CuO NPs are difficult to enter thicker peptidoglycan cell (32).Multidrug resistance between bacteria or/and some genetic mutations were occurred and randomly used of antibacterial agents may be that due to rise of resistance to most recently antibacterial agents as results in the present study elicited that, and mentioned by Stock and Wiedemann (30).In addition to mutation, The highly resistant organisms was producing from that present genes of resistance in plasmid , and creation

CONCLUSON
According to the findings of the experiments, copper nanoparticles is beneficial as antibacterial agent and resistance rates to antibiotics were decreased after 24

Fig. 4 .Fig. 5 .
Fig. 4. Antibacterial impact of green produced CuO NPs 40μg/ml concentrations against K. pneumonia the similar letters stated to non significant differences