untitled ISSN 2 Silver an antibiot Kholoud M Alaa Moha 1 Department of 2 Department of * Corresponding Tel.: +20.64.323 ARTICLE IN DOI: 10.5155/e Received: 09 Ap Received in rev Accepted: 21 M Published onlin Printed: 30 Sep KEYWORDS Antibacterial ef Silver nanopart Precipitation m Zinc oxide nano Antibiotics enh Chemical reduc 1. Introduct Since the choice of p diseases, as the 20th cent the last 60 y cause infecti one of the treatment [2 are resistan treated with The process and expensi million to br problem mor for effective be used as en problem [6]. antimicrobia (NPs) are a f with size ra surface area 2153‐2249 (Prin nd zinc ox tics activit Mohammed ammed Has f Chemistry, Faculty f Botany, Faculty of g author at: Depart 30418. Fax: +20.64. FORMATION eurjchem.7.3.290- pril 2016 vised form: 20 May May 2016 ne: 30 September 2 ptember 2016   S ffect ticles method oparticles ancement ction method tion e discovery, the physicians for they were con tury [1]. They h years. Over tim ions in hospital antibiotic drug 2,3]. The bigges nt to all appro h experimental of producing ve, requiring a ring a new ant re complicated and unconvent nhancers for th . New and prom al effect are n form of nanoma anging from 1n to volume ratio E nt) / ISSN 2153‐ h Euro xide nano ty against Abou El‐No san 2 and Om y of Science, Suez C f Science, Suez Cana tment of Botany, Fa 3230416. E‐mail ad -297.1438 y 2016 2016 e use of antibiot treating mos nsidered the wo have been the me, about 70 % ls have become gs most comm st problem is t oved antibiotic and potentiall a new antibiot approximately tibiotic to mar [5]. So scientis tional antimicro he present antib mising enhance anoparticles [7 aterials that are nm to100 nm o and unique ch uropean Journal Europ 2257 (Online)  http://dx.doi.org/1 pean Jo Journal we oparticles t multi dr our 1, mar Abdulra Canal University, Ism al University, Ismai aculty of Science, Su ddress: prof.abdelw ABSTRACT Some microor outbreak of in antibacterial properties as metal and met chemical redu the precipitati were charac Spectroscopy Microscopy (T different gene Klebsiella pne strong effect a increasing the groups accord recognized via Cite this: Eur. tics is most pro t of the infe onder discover effective weapo of the bacteri e resistant to at monly used for that some orga cs and can on ly toxic drugs tic, however, is ten years and ket which mak ts are now sear obial agents th biotics for solvin rs that have a s 7‐10]. Nanopar e very small par so having a hemical and ph l of Chemistry 7 pean Journal of C 2016 Atlanta Pub 10.5155/eurjchem ournal ebpage: www s as poten rug resista ahman Abdu mailia, 41522, Egyp ilia, 41522, Egypt uez Canal Universit wahid@gmail.com rganisms became nfectious disease properties of s well as the com tal oxide nanop uction method, w ion method foll cterized using (FT‐IR), X‐ray TEM). The antib era: Staphylococ umoniae (Gram against all. Also, e antibacterial a ding to their mec a increasing inhi J. Chem. 2016, 7 obable ctious ries of on for a that t least their anisms nly be [2, 4]. s long $300 ke the rching at can ng the strong rticles rticles larger hysical pro pro anti anti that the to tho pot silv anti for effe zinc stan high inve and 2. E 2.1. (3) (2016) 290‐ Chemistry blishing House LL m.7.3.290-297.14 of Che w.eurjchem.co ntial weap ant micro ulwahid 2,* pt ty, Ismailia, 41522, (O.A. Abdulwahid). e less sensitive t es. Therefore, th silver and zinc mbined effects o articles. Silver n while zinc oxide n owed by calcina UV‐Visible s y diffraction a bacterial effect o ccus aureus (Gra m negative) and the synthesized activity of ten te chanism of actio bition zone diam 7(3), 290‐297 operties [11,1 opensity to stim imicrobial agen ibiotics preven t of nanopartic potential to se antibiotics. C roughly being tential antimicr ver and ZnO n imicrobial field indicating the ect with antibio The objective c oxide nanopa ndard laborato hly expensive estigate the ant d in combination Experimental . Materials ‐297 LC ‐ All rights rese 438 emistry om pons for e oorganism Egypt. to several ordina e present study c oxide nanopa of antibiotics pr nanoparticles (A nanoparticles (Z ation process. T pectroscopy, F analysis (XRD) of AgNPs and Z am positive), Sh Escherichia coli d nanoparticles w ested antibiotic on. Enhancemen meters (mm). 2]. Also, nan mulate microbia nts [13,14]. The nt microbial gro cles [15‐17]. T erve as an enha urrently; the explored and robials [18‐20 anoparticles ar d. Many investig eir antimicrobia tics has not bee of this study w articles by simp ory equipment and specialize tibacterial effec n with some se erved ‐ Printed in y nhancem ms ary used antibio is carried out to articles. Their p reviously impreg AgNPs) were obt ZnONPs) were sy The formed nano Fourier Transf and Transmi nONPs was test higella boydii (G i (Gram negativ were evaluated s which cover m t in the antibiot noparticles ha al resistance th e mechanisms owth are quite Therefore, nano ncer or even as metallic nan d extensively i 0]. Due to the re being used gations have be al effect, but t en known yet. was to prepare b ple and fast met without the r ed instruments ct of these prep lected antibioti n the USA ment of otics, causing the o investigate the physicochemical gnated with the tained using the ynthesized using oparticles (Nps) forms Infrared ission Electron ted against four Gram negative), ve) and showed for their role in most antibiotics tic activities was ave a lower han many other through which different from oparticles have s an alternative oparticles are nvestigated as ese properties, widely in the een carried out their combined both silver and thods using the requirement of as well as to ared NPs alone ics. e e l e e g ) d n r , d n s s r r h m e e e s , e t d d e f o e El‐Nour et al. / European Journal of Chemistry 7 (3) (2016) 290‐297 291 All chemicals were of the analytical grade and used as received. Silver nitrate (AgNO3), Zn(CH3COO)2 ,Starch and sodium hydroxide NaOH were obtained from Sigma(Sigma, St. Louis, MO, USA). Solutions were prepared in double‐distilled deionized water just before use. All determinations were performed at room temperature and at pH = 7.0. The different antibiotics discs used during this investigation were procured from Oxoid Ltd, Basingstoke, Hampshire, England. 2.2. Preparation of silver nanoparticles (AgNPs) The most common method used for the synthesis of silver nanoparticles is chemical reduction method [21‐24]. In this study we obtained uniform silver nanoparticles by reduction of silver nitrate (5 mM) at 90 °C under atmospheric pressure with 1% starch solution which was used also as a stabilizer, the mixture was stirred at room temperature for 30 min. The transparent colorless solution was converted to pale yellow and then to reddish brown solution after continuous stirring at 90 °C for 60 minutes indicating the formation of silver nano‐ particles [25]. The silver nanoparticles thus prepared in starch were stable for two months without any change in the surface plasmon resonance as indicated from the absorption spectra at room temperatures, showing that the starch was a good reducing and stabilizing agent for the silver nanoparticles. 2.3. Preparation of ZnO nanoparticles (ZnONPs) Synthesis of ZnO nanopowder was achieved by the precipitation method followed by calcination process, that by adding slowly (200 mM) sodium hydroxide (NaOH) solution to (100 mM) zinc acetate Zn(CH3COO)2 solution while stirring. The resulting precipitate of Zn(OH)2 was washed with deioni‐ zed water several times, and then underwent calcination treatment to obtain ZnO nanopowder. The washing process was repeated several times until the pH of the solution was 7 that which was detected by using pH‐ meter. Finally the product was dried in the oven at 150 °C for 8 hours to allow complete dehydration. Zinc Oxide formed by the dehydration was suspended in distilled water to obtain suspension of Zinc oxide nanoparticles. 2.4. Instrumentation The resulting nanoparticles were characterized by Ultraviolet‐visible Spectroscopy (UV‐Vis) which was perfor‐ med at room temperature with samples in a quartz cuvette using a spectrophotometer (SHIMADZU UV‐1650PC, Columbia, MD, USA). The studies of size, morphology and composition of the nanoparticles were performed by means of transmission electron microscopy (TEM; JEOL 1210, JEOL Ltd., Tokyo, Japan). Electron microscope was operated at an accelerating voltage of 90 KV. The samples were prepared by drop‐coating the NPs solutions onto the carbon‐coated copper grid and were loaded onto a specimen holder. X‐ray diffraction was done using equipment Siemens D‐5000 with CuKα radiation operating at 40 kV and 40 mA. Fourier transform Infrared (FT‐ IR) specter were recorded at room temperature on a Bruker Tensor 27 FT‐IR spectrometer. 2.4.1. UV‐Visible spectroscopy The formation of silver and zinc oxide nanoparticles was preliminarily confirmed by visual observation of color change from pale yellow color to deep reddish brown in case of AgNPs and from clear colorless to turbid white in case of ZnONPs. The produced nanoparticles were subjected to characterization by UV‐Vis. spectra at different time intervals. Sharp peak given by UV‐Vis. spectra at the 420 and 370 nm for silver and zinc oxide nanoparticles respectively confirms the nanoparticles forma‐ tion. 2.4.2. Transmission electron microscopy (TEM) Characterization of the synthesized nanoparticles was carried out by TEM to determine the size and shape of nanoparticles. For the TEM measurements; a drop of a solution containing the particles was deposited on a copper grid covered with amorphous carbon. After allowing the film to stand for 2 minutes, the extra solution was removed by means of blotting paper and the grid was allowed to dry before the measurement. From the TEM micrographs sizes and shape of silver and zinc oxide NPs were detected. Histograms of size distribution were calculated from the TEM images by measuring the diameters of at least 50 particles. 2.4.3. X‐ray diffraction analysis (XRD) The synthesized ZnO nanoparticles were characterized by x‐ray diffraction using equipment Siemens D‐5000 with CuKα radiation operating at 40 kV and 40 mA to determine formed phases, lattice parameters, and relative crystallinity. The observed diffraction peaks in all the recorded XRD patterns are in agreement with those of the JCPDS card 89‐7102 for hexagonal ZnO with wurtzite structure. No peaks of any other phase were detected. 2.4.4. Fourier transforms infrared spectroscopy (FT‐IR) For FT‐IR measurements of synthesized nanoparticles, an appropriate amount of the formed nanoparticles were mixed with KBr salt to obtain FT‐IR spectrum. Silver and zinc oxide nanoparticles were subjected to FT‐IR analysis in the range of 4000 to 400 cm−1 2.5. Preparation of inocula Isolates of Staphylococcus aureus (NCMB 6571), Shigilla boydii (ATCC9207), Klebsiella pneumoniae (clinical culture) and Escherichia coli were provided by bacteriology laboratory, faculty of science, Suez Canal University. Firstly, a single colony of the tested organisms were grown overnight in a nutrient broth (NB) medium at 30 °C. The inocula were prepared by diluting the overnight cultures with 0.9% NaCl and detected by UV spectrophotometer at 600 nm to obtain a 0.5 McFarland standard. A 200 μL of these bacterial suspensions was added to the sterile nutrient agar on plates and spread by a glass spreader. 2.5.1. Antibacterial activity of the synthesized AgNPs and ZnONPs The antibacterial activity of synthesized silver and zinc oxide nanoparticles were evaluated by using the disc‐diffusion method. Different volumes (10, 20 and 30 μL) of freshly prepared AgNPs and ZnONPs were injected in sterile filter paper discs using sterile micropipette and placed in the bacterial lawn. The combined effect of AgNPs and ZnO nanoparticles with antibiotics was tested along with 10 various antibiotics covering most of antibiotics groups according to mechanism of action. Each standard antibiotic disc was impregnated with three different volumes (10, 20 and 30 μL) of the two metallic nanoparticles and placed in the bacterial lawn. After incubation at 30 °C for 24 hours, the zone of inhibition was measured, which appear as a clear area around the discs. The assays were performed in triplicate. 3. Results and discussion Silver nanoparticles were synthesized by chemical reduction method with starch at 90 °C as mentioned above. 292 The chan brown and indicated th process can b In this r glycose redu the silver m particles, pr larger partic by the preci hydroxide a hydroxide Zn to convert th according to Zn(CH3COO) Zn(OH)2 → Z Figu 3.1. Charact 3.1.1. UV‐Vis Synthesi visible range UV‐Vis spect absorbance p nm for ZnO broad surfac various AgN nm [26‐28]. peak indicati is a relation size and sha particle size, ticles that ar towards long or red shift i related to ob 2AgNO nge of the colo finally to redd e formation of be summarized reaction, starch uces the silver metal forms, s reventing them cles. Synthesis o pitation metho and zinc acetat n(OH)2. Calcina he formed Zn(O the following r 2 + 2NaOH → Zn ZnO + H2O ure 2. ZnO nanopar terization stud sible spectrosc s of AgNPs e e due to the sur tra of the synth peaks at 420 nm NPs (Figure 3B ce Plasmon pe Ps, with sizes On the other h ing the synthes nship between ape of nanopart , the optical abs re dominated b ger wavelength n the waveleng btaining NPs in El‐ Figure 1. 3 (aq) + (C6H10O rless solution t dish‐brown in f AgNPs (Figur d in the redox re h (C6H10O5)n wh cations from t starch coats t m from aggreg of ZnO nanopa od as the reacti te leads to th ation process (1 OH)2 to ZnO nan reaction: n(OH)2 + 2CH3C rticles obtained aft dies of synthesiz copy exhibits strong face Plasmon r esized samples m for AgNPs (F B). Observation eak has been w ranging all the hand ZnONPs sh sis of spherical the UV‐Vis ab ticles [29]. With sorbance spect by surface Plas hs (red shift) [ gth of the absor different shape ‐Nour et al. / Eur Silver nanoparticle O5)n (aq) + H2O to yellow, yello the reaction v re 1). The syn eaction (1). hich is a polym the silver nitra the outsides o gating and fo articles was ach ion between so e formation o 150 °C) was foll noparticles (Figu COONa ter calcination. zed nanopartic g absorption i esonance (SPR) showed a max igure 3A) and a n of this stron well documente e way from 2 t howed single na nanoparticles. bsorbance spec h the increase ra of metal nan mon resonance 29]. Small blue rbance peak cou es and sizes [29 ropean Journal of es obtained (A) aft O (l) → 2Ag (s) + owish‐ vessels nthesis mer of ate. As of the rming hieved odium f zinc lowed ure 2) (2) (3) cles n the ) [26]. ximum at 370 ng but ed for to 100 arrow There ctrum, in the nopar‐ e shift e shift uld be ]. 3.1. mor mis par wel natu (SA nan con 30 pos pro also from syn rev Mos an a of Chemistry 7 (3) ter 30 min and (B) + 2HNO3 (aq) + Figure 3. UV‐Vis s .2. Transmissio The TEM meas rphology and ssion electron rticles (Figure 4 ll dispersed w ure was confirm AED) pattern (F noparticles size ntained 25 part nm. Most of pa ssess an avera ovided evidence o revealed sph m 10 to 40 nm Transmission nthesized zinc o ealed that they st of particles w average size of ) (2016) 290‐297 after 60 min. C6H12O7 (aq) spectra of synthesi on electron mi surements were shape of the f microscope m 4A) revealed t without agglom med by the sele Figure 4B). Fig e histograms o icle unit/mL in articles were be age size of 17 e of synthesis o herical and po [26]. electron m oxide nanopart y were spheric were between 12 nm (Figure 7 (1) ized (A) AgNPs and croscopy (TEM e carried out to formed nanopa micrograph of that they were eration, their ective area elect gure 4C shows of silver nanop n sizes ranging etween 15‐20 n 7 nm. Various of AgNPs by TE olydispersive A microscope m ticles denoted b cal with little 10‐15 nm in si 5B). d (B) ZnONPs. M) o determine the articles. Trans‐ f silver nano‐ e spherical and polycrystalline tron dispersion representative particles which between 10 to nm in size, and reports have EM images that AgNPs ranging micrograph of by (Figure 5A) agglomeration. ze and possess e ‐ ‐ d e n e h o d e t g f ) . s Figure 4. (A) shape of AgNPs polycrystalline synthesized Ag Figure 5. (A) ZnONPs. (B) Hi 3.1.3. X‐Ray XRD pat showed stro 31.72, 34.38 (100), (002) The strong a and good cr reflections ca the pure hex 3.2516 Å, c = standard car Figure Transmission ele s. (B) Selective are nature of AgNPs NPs. Transmission ele stogram shows the diffraction stu ttern of the p ong and narrow 8, 36.26, 47.54 ), (101), (102) and narrow dif rystallinity of t an be assigned xagonal phase = 5.2000 Å. The rd of ZnO powd e 6. X‐Ray diffractio El‐Nour et al ectron microscope ea electron diffract s (C) Histogram ectron microscope e sizes of ZnONPs. udies prepared Zinc w diffraction pe 4 and 56.58 ° and (110) (as ffraction peaks the grown nan to the standard of ZnO with l hkl values are er sample [30]. on patterns of synt l. / European Jou e image shows sp tion (SAED) confir shows the sizes e image shows sh oxide nanopar eaks of ZnO at are associated shown in Figu indicate high p nostructures. A d powder patte lattice constant agreed well wi . thesized ZnONps. urnal of Chemistr pherical rms the of the hape of rticles t 2θ = d with ure 6). purity All the ern for ts a = ith the 3.1. cm‐ FT‐ AgN ZnO 103 cou asy stre at 2 the ban gro sho corr met and H‐O at 1 ban and ban nan 3.2. to h anti diff pos Kleb Esch aga pne ry 7 (3) (2016) 2 .4. Fourier tran Various vibrat ‐1 of AgNPs and IR spectrum r NPs at 3455, 2 ONPs spectrum 35 and 650 cm− The well‐defin uld be due to mmetric) of a etching vibratio 2256 cm−1 is as peak observed nd of H2O and up (C=O) result FT‐IR spectru owed in Figure responds to st tric) of aliphatic d symmetric str O‐H vibration ba 1632 cm‐1 may a nd of carbonyl g d 1380 cm‐1 ar nd at 650 ind noparticle. Figure 7. F . Antibiotic sus Both silver m have antimicrob ibacterial effec ferent genera S sitive), Shigell bsiella pneumo herichia coli (G ainst Staphyloco eumonia (clinica 90‐297 nsforms infrar tional frequenc d ZnONPs were revealed the p 256, 1638 and m showed band 1 (Figure 7B). ned peak, in Figu (i) stretching liphatic hydrox onal band of H ssigned for alip d at 1638 cm−1 c a stretching v ting from the re um of the syn e 7B exhibited tretching vibra c hydroxyl (OH retching H‐O‐H and is observed also be corresp group (C=O). B re due to the C dicates the st FT‐IR spectra of (A sceptibility pat etal and zinc ox bial role since ct of AgNPs w Staphylococcus a boydii (AT oniae (clinical c ram negative) w occus aureus ( al culture). red spectroscop cies, in the rang e shown in (Fig resence of ban d 542 cm‐1 (Fig s at 3420, 163 ure 7A, observe g vibration (s xyl (OH) grou 2O molecules. B hatic (C–H) str could be assigne vibrational ban esidue of the sta nthesized ZnO d bands at 342 ation (symmet ) group as well H vibration whi d at 1632 cm‐1. ponds to stretch Bands observed C‐O stretching tretching vibra A) AgNPs and (B) Zn ttern xide nanopartic many years. In was investigate aureus (NCMB TCC9207) (Gra culture) (Gram while ZnONPs w (NCMB 6571) 293 py (FT‐IR) ge of 4000‐400 gure 7A and B). nds in case of gure 7A), while 32, 1380, 1116, ed at 3455 cm‐1 symmetric and up and (ii) the Band observed retching , while ed as a bending nd of carbonyl arch molecule. nanoparticles 20 cm‐1 which tric and asym‐ l as asymmetric ile the bending Peak observed hing vibrational d at 1035, 1116 vibration. The ations of ZnO nONPs. cles are known n this study the d against four B 6571) (Gram am negative), negative) and was tested only and Klebsiella 0 . f e , 1 d e d e g l s h ‐ c g d l 6 e O n e r m , d y a 294 El‐Nour et al. / European Journal of Chemistry 7 (3) (2016) 290‐297 Table 1. The antibacterial effect of AgNPs against S. aureus and their combined affect with ten various antibiotics. Antibiotic Inhibition zone diameter (mm) Different concentrations of AgNps 0 μL +10 μL +20 μL +30 μL No antibiotic ‐ 11 14 16 Norfloxacin 10 34 38 40 42 Ciprofloxacin 5 35 37 38 39 PenicillinG 10 27 31 34 37 Cefotoxime 30 30 32 34 35 Erythromycin 15 20 22 23 24 Tetracyclin 30 11 14 16 21 Gentamycin 10 20 23 25 25 Nalidixic acid 30 24 26 27 30 Ampicillin 10 20 22 23 25 Amoxycillin 10 9 10 12 14 Table 2. The antibacterial effect of AgNPs against Shigella boydii and their combined affect with ten various antibiotics. Antibiotic Inhibition zone diameter (mm) Different concentrations of AgNps 0 μL +10 μL +20 μL +30 μL No antibiotic ‐ 14 16 17 Norfloxacin 10 23 25 27 31 Ciprofloxacin 5 24 26 28 30 PenicillinG 10 16 17 18 22 Cefotoxime 30 12 13 14 15 Erythromycin 15 15 17 19 20 Tetracyclin 30 26 30 33 36 Gentamycin 10 14 14 15 16 Nalidixic acid 30 23 25 26 27 Ampicillin 10 10 12 13 14 Amoxycillin 10 10 11 12 14 Table 3. The antibacterial effect of AgNPs against K. pneumoniae and their combined affect with ten various antibiotics. Antibiotic Inhibition zone diameter (mm) Different concentrations of AgNps 0 μL +10 μL +20 μL +30 μL No antibiotic ‐ 9 11 12 Norfloxacin 10 00 12 14 15 Ciprofloxacin 5 14 16 18 20 PenicillinG 10 00 11 13 14 Cefotoxime 30 26 28 30 31 Erythromycin 15 00 13 14 16 Tetracyclin 30 15 18 20 22 Gentamycin 10 15 17 17 19 Nalidixic acid 30 21 23 25 26 Ampicillin 10 00 9 11 12 Amoxycillin 10 00 9 11 12 Table 4. The antibacterial effect of AgNPs against E. coli and their combined affect with ten various antibiotics. Antibiotic Inhibition zone diameter(mm) Different concentrations of AgNps 0 μL +10 μL +20 μL +30 μL No antibiotic ‐ 9 11 14 Norfloxacin 10 21 24 26 29 Ciprofloxacin 5 29 30 31 32 PenicillinG 10 00 9 11 14 Cefotoxime 30 19 20 21 21 Erythromycin 15 00 11 14 16 Tetracyclin 30 11 12 12 13 Gentamycin 10 12 14 15 16 Nalidixic acid 30 15 19 21 23 Ampicillin 10 00 9 11 14 Amoxycillin 10 10 11 12 14 The antibacterial effect was observed by measuring the inhibition zone diameters around the discs impregnated with different volumes. The combined effect of AgNPs and ZnONPs with 10 various antibiotics using disc‐diffusion method was also examined. The enhanced effect was also observed with the increase in the inhibition zone diameters around the antibiotics discs that previously impregnated with nanopar‐ ticles. The antibiotic susceptibility test showed that the appli‐ cation of silver and ZnO nanoparticles increase the antibac‐ terial effect of all antibiotics used against both Gram (+ve) and Gram (‐ve) bacteria up to varying extents. This showed that the method is applicable for both kinds of bacteria. Their enhancement in the combined effect was preferably due to the difference in the mechanism of inhibition followed by nano‐ particles and antibiotics. 3.2.1. Silver nanoparticles antibacterial and combined effect results Tables 1‐4 and Figures 8 and 9 represent the antibacterial effect of AgNPs which was observed by using disc‐diffusion method and measuring the inhibition zone diameters around the discs impregnated with different volumes and also their combined affect with ten various antibiotics. Figure 8. Effec Figure 9. (I) A acid, (B) Nalidi (D) Nalidixic ac S. boydii (A) C loxacin + 20 μL robial activity o 10 μL AgNPs, ( μL AgNPs. 3.2.2. Zinc ox effect result Tables 5 antibacterial disc‐diffusion diameters ar and also thei Accordin of silver nan and the teste and 30 μL) i highest antim boydii follow and E. coli is is harmless t [31]. The exa to cause ant under discus action agains to act on th where silve proteins in c affinity to su According to silver nanop [35,36]. Thes negatively ch and subsequ respiration, t many enzym death [37]. antibiotics h ticles agains which was n against S. boy ct of different AgNP Antimicrobial activi xic acid +10 μL Ag cid + 30 μL AgNPs. Ciprofloxacin, (B) L AgNPs and (D) C of AgNPs against E (C) Erythromycin + xide nanoparti ts 5 and 6, and l effect of ZnON n method and round the disc i ir combined aff ng to the results noparticles diff ed isolates. Inc increases the a microbial activi wed by S. aureu s nearly equal. to human cells act mechanism timicrobial effe ssion, however st microbes [32 he surface are er nanoparticl cell membrane ulfur compound o electron spin particles releas se ions have ap harged microbi uently disrupt it then react with mes inactivating Also this stud has increased i st the tested o not enhanced w ydii but the larg El‐Nour et al Ps concentrations ity of AgNPs again gNPs, (C) Nalidixic (II) Antimicrobial Ciprofloxacin + 1 Ciprofloxacin + 30 E.coli (A) Erythrom + 20 μL AgNPs an icles antibacte Figures 10 an NPs which was d measuring t impregnated wi fect with ten var s of this study a fers according t reasing the AgN ntimicrobial ac ty of AgNPs wa us and its effec In very small c but it is biocid which silver n ect is not clea r there are man 2,33]. Silver nan ea of the micr les react wit e, as they are k ds, affecting cell n resonance sp se free radicals ppositive charge ial cell membra ts main functio h DNA moieties g them, all lead dy shows that in the presence organisms exc when combined gest volumes di l. / European Jou against the four is nst S. aureus (A) N c acid + 20 μL AgN activity of AgNPs 0 μL AgNPs, (C) μL AgNPs. (III) A mycin; (B) Erythrom d (D) Erythromyc erial and comb nd 11 represen s observed by the inhibition ith different vo rious antibiotic antimicrobial ac to the volumes NPs volumes (1 ctivity gradually as observed aga ct on K. pneum concentrations, dal to microbia nanoparticles em arly known and ny theories for noparticles is k roorganisms [1 th sulfur‐conta known to have l membrane via pectroscopy st s specially Ag+ e so interact wi ane, making it p ns; permeabilit s and thiol grou ding to bacteria t the activity e of silver nan cept for Gentam d with 10 μL A id. urnal of Chemistr olates. alidixic NPs and against Ciprof‐ Antimic‐ mycin + in + 30 ined nt the using zone lumes cs. ctivity s used 10, 20 y. The ainst S. moniae silver al cells mploy d still r their known 19,34], aining e high ability. tudies, + ions ith the porous ty and ups of al cell of all nopar‐ mycin AgNPs Figu isola Figu Eryt ZnO ZnO ZnO ZnO (GR 899 hav or acti use (10 gra aur pne ZnO mec anti affe con adh cha cell dea rele ions cell pho cau follo ZnO O2‐ of Pen aga sam did ry 7 (3) (2016) 2 ure 10. Effect of di ates. ure 11. (I) Antim thromycin, (B) Ery NPs and (D) Eryth NPs against K. pn NPs (C) Gentamy NPs. Zinc oxide (Zn RAS) by the U.S 91) as many st ve selective tox even no effect ivity of ZnO nan ed and the teste , 20 and 30 dually. The an reus was highe eumoniae. The O nanoparticles chanisms whic imicrobial acti ect bacterial ce ntact of ZnONP here to the mic anges, resulting l membrane tra ath [39,40]. Th easing of hydro s and release o l membrane and osphorus and using the micr owing equation O + O2 → O2‐ + 2H+ → H2O2 The activity of ZnONPs again nicillin G and Am ainst S. aureus me volume agai . 90‐297 ifferent ZnONPs co microbial activity ythromycin + 10 μL hromycin + 30 μL Z neumonia (A) Gen ycin + 20 μL ZnO nO) is listed as “ . Food and Dru tudies have sho xicity to bacteri ts on human c noparticles diff ed isolates. Inc μL) increase ntimicrobial ac er than its anti mechanism of s is not well und ch have been ivity. Studies ell in two leve Ps with the mi crobial cell wa in increasing p ansport system he other level ogen peroxide of superoxide d interact with sulfur contai robial cell de ns: f all antibiotics nst the two t mpicillin when and Ciprofloxa inst K. pneumo oncentrations agai of ZnONPs again L ZnONPs, (C) Eryt ZnONPs. (II) Antim ntamycin (B) Gen ONPs and (D) Gen “generally reco ug Administrati own that these ia but exhibit m cells [38]. The fers according t creasing the Zn s the antimic ctivity of ZnON imicrobial acti the antimicro derstood but th proposed to suggested that ls, one of them crobial cell wa all so membran permeability an and finally lea l is the oxida (H2O2) from it ions (O2‐) whic intracellular co ining compoun ath as repres has increased i ested organism combined with acin was not en oniae but the la 295 inst the two tested nst S. aureus (A) thromycin + 20 μL microbial activity of ntamycin + 10 μL ntamycin + 30 μL ognized as safe” on (21CFR182. e nanoparticles minimal effects e antimicrobial to the volumes nONPs volumes crobial activity NPs against S. vity against K. bial activity of here are several explain their t ZnONPs can m is the direct all. As ZnONPs ne morphology nd disruption in ding to the cell ative stress by ts surface, Zn2+ ch can damage ontents such as nds like DNA sented by the (4) (5) in the presence ms except for h 10 μL ZnONPs nhanced at the argest volumes d ) L f L L ” . s s l s s y . K. f l r n t s y n l y + e s A e e r s e s 296 Table 5. The an Antibiotic No antibiotic Norfloxacin 10 Ciprofloxacin 5 PenicillinG 10 Cefotoxime 30 Erythromycin 1 Tetracyclin 30 Gentamycin 10 Nalidixic acid 3 Ampicillin 10 Amoxycillin 10 Table 6. The an Antibiotic No antibiotics Norfloxacin 10 Ciprofloxacin 5 PenicillinG 10 Cefotoxime 30 Erythromycin 1 Tetracyclin 30 Gentamycin 10 Nalidixic acid 3 Ampicillin 10 Amoxycillin 10 The res antibiotics P were all effe enhanced by pneumoniae to the other occurred as volumes onl Penicillin G a volumes, but enhancemen were sensit represented and enhance sensitive to N The thr Erythromyci against S. au Tetracycline pneumoniae Synergism o organisms as Figure 12. Syn K. pneum produce exte ntibacterial effect o 15 0 ntibacterial effect o 15 0 sults show th Penicillin G, Cef ective against S y adding AgNP was sensitive o r three antibio the inhibition z ly. On the othe and Ampicillin t was sensitive nt occurred. Al tive to all DN by Norfloxacin ement occurre Norfloxacin and ree antibiotics in, Tetracycline ureus, S. boydi and Gentam and E.coli but occurred with s shown in Figu nergism between a moniae and E. c ended‐spectrum El‐ of ZnONPs against In D 0 ‐ 34 3 2 3 2 1 2 24 2 9 of ZnONPs against In D 0 ‐ 0 14 0 2 0 1 1 2 0 0 at cell wall fotoxime, Ampi S. aureus and S Ps and ZnONPs only to Cefotoxi otics. With pen zone diameter er hand E.coli w and the effect e to Cefotoxime so, S. aureus, S NA synthesis‐i n, Ciprofloxacin ed for all. K. p d synergism occ s that inhibit e and Gentam i and enhance mycin were e t Erythromycin Erythromycin ures 12‐14. ntibiotics and AgN coli are the two m beta‐lactama ‐Nour et al. / Eur S. aureus and their nhibition zone dia Different concentr μL 4 5 7 0 0 1 0 4 0 K. pneumoniae and nhibition zone dia Different concentr μL 4 6 5 5 1 synthesis‐inhib cillin and Amoy S. boydii. They s volumes. Kleb ime but not sen nicillin G syne was wider tha was not sensit was only for A e and Amoycilli S. boydii and E inhibitors‐antib n and Nalidixic pneumoniae wa curred. t Protein syn mycin were eff ment occurs fo effective again n was not effe n for both the NPs against K. pneu o main bacteri se enzymes ena ropean Journal of r combined affect w ameter (mm) rations of ZnONps +10 μL 10 36 36 27 32 23 14 21 25 20 11 d their combined a ameter (mm) rations of ZnONps +10 μL 8 12 14 14 27 10 16 17 24 8 8 bitors‐ ycillin were bsiella nsitive ergism n NPs tive to AgNPs in and E. coli biotics c acid as not nthesis fective or all. nst K. ective. e two umonia. a that abling them oth was syn diff syn Am Figu pneu of Chemistry 7 (3) with ten various an s affect with ten vari s m to be resista er types of ant s resistant to pe nergism occurre ferent volumes nergism occurr picillin when tr Figure 13. Synerg ure 14. Synergis umoniae. ) (2016) 290‐297 ntibiotics. +20 μL 12 38 37 28 34 25 15 22 26 21 13 ious antibiotics. +20 μL 9 13 15 16 29 11 17 19 25 9 9 ant to pencillins tibiotics. Result encillin G, Eryth ed with these a of AgNPs and Z red with peni reated with diff gism between antib sm between ant 7 +30 μL 13 40 38 29 36 27 17 23 27 22 15 +30 μL 11 14 16 20 31 12 21 21 27 11 11 s, cephalosporin ts showed that hromycin and N antibiotics whe ZnONPs. While icillin G, Eryt ferent volumes biotics and AgNPs tibiotics and Zn ns and often to K. pneumoniae Norfloxacin and en treated with in case of E.coli thromycin and of AgNPs. against E. coli. ONPs against K. o e d h i d K. El‐Nour et al. / European Journal of Chemistry 7 (3) (2016) 290‐297 297 From results data AgNPs and ZnONPs may inhibit the beta‐ lactamase enzymes causing disruption of the resistance mechanism of K. pneumoniae and E. coli and enables antibio‐ tics to be functional against this two types of bacteria. 4. Conclusion In this study silver and zinc oxide nanoparticles were synthesized by chemical methods. The formation of these nanoparticles was confirmed by UV‐vis spectroscopy, Trans‐ mission electron microscope (TEM), X‐Ray diffraction analysis and Fourier Transform Infrared Spectroscopy (FT‐IR). Also the antimicrobial activity of the synthesized nanoparticles against four different genera Staphylococcus aureus (NCMB 6571), Shigella boydii (ATCC9207), Klebsiella pneumoniae (clinical culture) and Escherichia coli alone and in conjugation with 10 various antibiotics representing most of antibiotics groups according to function were studied. The antibacterial activity of different volumes (10, 20 and 30 μL) of synthesized silver and zinc oxide nanoparticles were evaluated by using the disc‐ diffusion method. Also the combined effect of different volumes (10, 20 and 30 μL) of Ag and ZnO nanoparticles with antibiotics were tested along with ten various antibiotics covering most of antibiotics groups according to mechanism of action. Antimicrobial activity of AgNPs and ZnONPs differ according to the volumes used and the tested isolates. The highest antimicrobial activity of AgNPs was observed against S. boydii followed by S. aureus and its effect on K. pneumoniae and E.coli was nearly equal. In case of ZnONPs the antimicro‐ bial activity against S. aureus was higher than that against K. pneumonia. Acknowledgements The authors are thankful to Faculty of Science, Suze Canal University, for providing us all the laboratory facilities and chemicals. Also we would like to deeply thank Dr. Hesham Abdullah, bacteriology laboratory, Faculty of Science, Suze Canal University for providing us the needed bacteria for antimicrobial tests. References [1]. Davies, J.; Davies, D. Microbiol. Mol. Biol. 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