Research ArticleBiosynthesis, Characterization of Some CombinedNanoparticles, and Its Biocide Potency against a BroadSpectrum of Pathogens

Marwa Eltarahony,1 Sahar Zaki ,1 Marwa ElKady ,2,3 and Desouky Abd-El-Haleem 1

1Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Researchand Technological Applications, Borg El Arab, Alexandria, Egypt2Chemical and Petrochemical Engineering Department, Egypt-Japan University for Science and Technology, New Borg El-Arab City,Alexandria, Egypt3Fabrication Technology Researches Department, Advanced Technology and New Materials Research Institute, City of ScientificResearch and Technological Applications, Alexandria, Egypt

Correspondence should be addressed to Sahar Zaki; saharzaki@yahoo.com

Received 4 April 2018; Revised 19 June 2018; Accepted 4 July 2018; Published 28 August 2018

Academic Editor: Ilaria Fratoddi

Copyright © 2018 Marwa Eltarahony et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

The development of environmentally benign procedures for the synthesis of metallic nanoparticles (NPs) is a vital aspect inbionanotechnology applications for health care and the environment. This study describes the biosynthesis of Ag, Co, Ni,and Zn NPs by employing nanobiofactory Proteus mirabilis strain 10B. The physicochemical characterization UV-visiblespectroscopy, scanning electron microscopy-energy-dispersive X-ray microanalysis (EDX), X-ray diffraction analysis (XRD),transmission electron microscopy (TEM), dynamic light scattering (DLS) technique including ζ potential, and polydispersityindex (PDI) confirmed the formation of pure, stable monodisperse quasi-spherical oxide NPs of corresponding metals. Theantimicrobial activity of biofabricated NPs was assessed against Gram-negative and Gram-positive bacteria, biofilm, yeast, mold,and algae via a well diffusion method. The results displayed significant antagonistic activity in comparison to their bulk andcommercial antibiotics. Interestingly, the combined NPs exhibited promising synergistic biocide efficiency against examinedpathogens which encourages their applications in adjuvant therapy and water/wastewater purification for controlling multipledrug-resistant microorganisms. To the best of our knowledge, no previous study reported the synthesis of semiconductor NPsby Proteus mirabilis and the biocide potency of combined NPs against a broad spectrum of pathogens not reported previously.

1. Introduction

Microbial pollutants are the most dreadful cause for a widerange of infectious diseases which lead to an increase inthe rate of hospitalization, morbidity, and mortality. Thedisease-causing agents (bacteria, fungi, viruses, algae, andprotozoa) could be transmitted through water purificationsystems, contaminated medical devices such as cathetersand dental materials, and food manufacturing machines,which create obvious threat for human health and theambient ecosystem [1]. In developing countries, approxi-mately 12 million people die annually due to consumption

of water contaminated with various microbes as pointedout by Oves et al. [2]. Notably, the microbial population isnaturally capable of developing resistance against commer-cial antibiotic drugs, besides their ability to organize biofilmstructures which formed 15% aggregates of microbial cellsembedded in 85% of the extracellular matrix which com-prises glycolipids, polysaccharides, proteins, and DNA whichundoubtedly leads to ineffectiveness of drugs [2, 3].

Currently, nanotechnology which is described as “thesixth revolutionary technology” after the industrial revolu-tion, nuclear energy revolution, green revolution, informa-tion technology revolution, and biotechnology revolution

HindawiJournal of NanomaterialsVolume 2018, Article ID 5263814, 16 pageshttps://doi.org/10.1155/2018/5263814

[4] opens the door for multidrug-resistant microorganism(MDR) defeat by virtue of metal nanomaterials’ leading-edge nature. Recently, membrane and polymers incorporatednanoparticles (NPs) which were developed for the waterpurification system [2], and NP-coated fabrics [5], bandages,walls, bed linen, surfaces, and medical equipment were exam-ined as magic cure against microbial contamination [6, 7].Among others, transition metal oxide NPs were deemedparticularly attractive for the application of a new class ofantimicrobial agents. Interestingly, several studies speculatedthat metals and metal oxide NPs utilize multiple mechanismssimultaneously in the microbial combating battle, placingMDR microorganisms in a critical position to develop resis-tance. However, antibiotics, especially bacterial drugs, inducecell death by cell wall inhibition (β-lactams), RNA synthesis(rifamycins), DNA replication (quinolones), or proteinsynthesis (macrolides) [8]. In this context, The US FDA hasalready approved some metal oxides such as ZnO as safeantimicrobial agents against bacteria, fungi, and virus [9].Additionally, conjugation of antibiotic and metal NPs in“combination therapy” against pathogens exhibited a prom-ising solution to stop MDR crisis [2, 10].

In the framework of this research topic, particularly, forbiocompatibility and biosafety in biological systems, the mostimportant criterion is the NP synthesis approach. Classically,metallic NPs have been fabricated by well-establishedphysical, chemical, and hybrid methods. Although NP yieldof these methods was high with controlled size, morphology,and dispersion, the employment of hazard flammable chemi-cals and high temperature limit medical and environmentalapplication due to contamination from precursor residuals.In recent decades, the legislation on waste electrical/electronic equipment (WEEE) and restriction of hazardoussubstances (RoHS) has been issued by the European Union.Thus, the timeline for exploiting the nature’s secret is comingfor the employment of a benign, green, cost-effective, andmedically/environmentally biocompatible approach. Thegreen synthesis of NPs dedicates to the use of biological hostsincluding, but not limited to, bacteria, fungi, actinomycetes,algae, yeast, and plants [11]. The biological method for metalNP fabrication by microorganisms can be either intracellularor extracellular. Generally, in both cases, the proteins that areinvolved in cell metabolism are considered the responsibleinstrument for the reduction and subsequent conversion ofmetal ions into metal NPs [12]. Bacteria attract immenseinterest in NP synthesis by short generation times and easymanipulation [13]. Numerous bacterial genera as Pseudomo-nas sp., Bacillus mojavensis, Achromobacter sp. Rhodobacter,Klebsiella, and Lactobacillus have been used to synthesizecompound NPs [14–17].

In the light of the aforementioned, this study is aimed atthe synthesis of Ag, Co, Ni, and Zn NPs by utilizing Proteusmirabilis strain 10B as a bacterial nanofactory in an eco-friendly approach. The biosynthesized NPs were character-ized using optical observation, UV-Vis spectrophotometry,XRD, EDX, TEM, zeta potential, and PDI. The antagonisticefficiency of the biosynthesized NPs was examined againstpathogenic bacteria (Gram-positive and Gram-negative),biofilms (Gram-positive and Gram-negative), and eukaryotes

(mold, yeast, and algae). No study to the best of the authors’acquaintance has so far been reported regarding the biosyn-thesis of Ag, Co, Ni, and Zn NPs by using Proteus mirabilis.Additionally, no previous report recorded the effect ofcombined as-prepared NPs on broad-spectrum pathogens.

2. Materials and Methods

2.1. Bacterial Strain, Growth Conditions, and Synthesis ofAg/Co/Ni/Zn NPs. The bacterial strain Proteus mirabilis10B was procured from existing indoor strain collectionthat is concerned with denitrification study, which hasbeen submitted to GenBank under the accession numberKY964505 [18].

The bacterium lawn (0.5 McFarland ≈ 108 CFU/ml) wasallowed to grow in nutrient broth (NB) (1.5% peptone,0.3% yeast extract, 0.05% NaCl, and 0.01% glucose, finalpH7.0) followed by addition of equivalent 3mM NP precur-sors Ni(NO3)2·6H2O, Co(NO3)2·6H2O, and Zn(NO3)2·6H2Oand 1.5mM of AgNO3 (Sigma-Aldrich). The cultures wereincubated at 30°C under shaking conditions (Stuart orbitalshaker). The cells at the stationary phase that include NPswere collected by centrifugation at 10,000×g for 20min.The NPs were extracted from cells after disruption by usingTSE buffer [17] through mild osmotic shock in a procedurereported by Samadi et al. [19]. The extracted NPs were driedat 100°C for 2 hr. The dried NPs were purified by washing 3successive times by ethanol 70% and double-distilled wateras described by Metz et al. [20]. The purified NPs weresubjected for subsequent characterization and applications.

2.2. Characterization of Biosynthesized NPs. The biosynthesisof NPs was preliminarily observed optically by the visualcolor change throughout the incubation period. In parallel,control experiments (bacterial growth medium containingmetal precursor and without bacteria) were incubatedtypically as in the test experiments. A UV-visible diffusedreflection spectrum of the bacterially synthesized NPs wasrecorded using a Labomed model UV-Vis double-beamspectrophotometer in a wavelength range of 200–800nm atroom temperature. Scanning electron microscopy-energy-dispersive X-ray microanalysis (EDX) was performed forchemical composition analysis using JEOL JSM-6360LA,from Japan (Faculty of Science, Alexandria University).X-ray diffraction (XRD) analysis of nanoparticles was carriedout on an X-ray diffractometer (Shimadzu 7000, USA) thatoperates with Cu Kα radiation (λ = 0 15406 nm) generatedat 30 kV and 30mA with a scan rate of 2°/min for 2θ valuesover a wide range of Bragg angles 10° ≤ 2θ ≤ 80 for identify-ing and evaluating crystallinity of NPs. Transmissionelectron microscopy (TEM) was employed to determine themorphology and particle size of as-synthesized NPs usingJEOL JEM-1230, from Japan (Faculty of Science, AlexandriaUniversity). Dynamic light scattering (DLS) technique usingZetasizer Nano ZS (Malvern Instruments, Worcestershire,UK; Faculty of Pharmacy, Alexandria University) was usedfor the determination of hydrodynamic diameter and poly-dispersity index (PDI) of diluted samples. The measurementswere performed at 25°C, at a fixed scatter angle of 173°.

2 Journal of Nanomaterials

Additionally, the zeta potential was measured for estimatingcolloidal stability using Zetasizer Nano ZS (Malvern Instru-ments, Worcestershire), and data were analyzed by Zetasizersoftware 6 [21].

2.3. Nanoparticles’ Inhibitory Effect against PlanktonicPathogens. The well diffusion assay was applied to assessantibacterial and antifungal activity of as-synthesized NPson bacterial and fungal species. A single colony was grownovernight in nutrient broth for bacterial inoculum prepara-tion, and turbidity was adjusted to 0.5 McFarland standards.The fungal inoculum was cultivated in Sabouraud dextrosebroth for 72 h. Mueller-Hinton agar (MHA) (Sigma-Aldrich)plates were swabbed with 0.1ml of each culture suspension,and bacterially synthesized NPs (100 and 200μg/ml) wereimpregnated to a center well with a diameter of 8mm. Theplates were incubated at 37°C for 24h (bacteria) (Labnet311D incubator) and 25°C for 72h (fungi) (Labnet 311Dincubator). The zone of inhibition (ZOI) was measured bysubtracting the well diameter from the total inhibition zonediameter and expressed in millimeters. The antimicrobialactivity of antibiotics (rifamycin, streptomycin, and tetracy-cline for prokaryotes and nystatin for eukaryotes) in additionto NP precursors (100 and 200μg/ml) was also examinedcomparatively as conventional control for the antimicrobialassay [7, 15, 22].

2.4. Inhibitory Effect against Biofilm Formation. Spectro-photometric tissue culture plate assay was performed toinvestigate the biofilm inhibition of both P. aeruginosaand S. aureus. Sterile 96-well polystyrene microtiter platewells were inoculated with 100μl of bacterial cell suspension.The respective concentrations (150 and 300μg/ml) of NPs,antibiotics, and NP precursors were added into the wells.Two controls were examined in parallel (positive controlwells: medium containing bacterial suspension and negativecontrol wells: sterile medium only). Microtiter plates werecovered and incubated under stationary conditions at 37°Cfor 24 hours. After the incubation time, the well contentwas discarded, washed, processed by crystal violet, andsolubilized with ethanol as in Namasivayam et al. [10]. Theabsorbance of the ethanol-solubilized mixture at 595 nmusing a plate reader (Tecan Infinite M200, Switzerland) was

determined, and biofilm inhibition percentage was calculatedby the following equation:

Biof ilm inhibition% = A − A0A

× 100 , 1

where A represents the absorbance of the positive controlwells and A0 reveals the absorbance of the treated wells withan antimicrobial agent [23].

2.5. Inhibitory Effect against Algae (Chlorella vulgaris). Thealgicidal effect of NPs was studied by adding 150 and300μg/ml compared to exact concentrations of antibioticon C. vulgaris growth. Algae were cultivated and incubatedas presented by Ilavarasi et al. [24]. The cell density of theculture was determined by counting with a hemocytometerunder a light microscope (Olympus BH2, Japan). The inhibi-tion percentage was calculated as in (1).

2.6. Application of Combined NPs in Water and Wastewater.The bacterial suppression potential of combined NPs againstbacterial load present in various samples from water bodiesand wastewater manufactories was studied. The samples(Table 1) were collected in March 2017 and subjected totreatment with combined NPs (150μg/ml) for differentcontact times (30, 60, and 120min) [17]. The bacterial countwas determined using the pour plate method and expressedas CFU/ml. The bacterial load within each sample withoutany treatment was determined as positive control plates.The suppression percentage at each time interval wascalculated according to

Suppression percentage = number of colonies in control− number of colonies in treatment/number of colonies in control ∗ 100

2

3. Results and Discussion

3.1. Biosynthesis and Characterization of NPs. This studyexplores the biogenic synthesis of numerous metallicnanoclusters by P. mirabilis strain 10B. The reaction mixtureof ionic solutions (metal salts) was analyzed primarily alongwith their respective controls by visual observation; the

Table 1: Application of combined NPs in real water and wastewater samples from different sources.

Sample type Sample nameBacterial suppression (%)

30min 60min 120min

Agricultural wastewater Bahig Canal 48.5 54.5 72.1

Industrial wastewater Tolombat, Almax 33.6 49.4 75.0

Municipal wastewaterBorg El Arab sewage plant 73.8 97.1 98.7

Sewage 21 k-region 75.2 83.7 94.7

Freshwater Almahmoudia Canal 67.6 80.7 94.6

Lakewater Mariout Lake 36.7 53.1 74.0

Seawater Al Max Sea 39.3 56.1 74.8

Salt mine water Elmahahat 19.9 32.1 42.4

3Journal of Nanomaterials

solution color changed from pale yellow to black or darkbrown in the cells and surrounding media of Ag, Co, andNi (Figure 1). Alternatively, in the case of Zn diverse starcheslike haziness and white clusters were noticed. In parallelcontrol experiments, no particularly notable changes wereobserved, suggesting that the biotransformation of metal ionsoccurs only in the presence of the reducing agent (bacteria) torelevant NPs. The results obtained are in accordance withThanh et al. [25] and Manokari and Shekhawat [26].

The bacterially synthesized NPs have optical and physicalproperties that are related to shape, size, concentration, andagglomeration state, which were studied by applying valuableanalysis techniques such as UV-Vis spectroscopy, EDX,XRD, TEM, and zeta potential for identifying and charac-terizing as-synthesized NPs.

3.2. UV Spectroscopic Analysis. The signature of colloidalparticles particularly noble metals was monitored throughUV-Vis spectroscopy which deliberates being a preliminarystage for nanocrystal characterization. As illustrated inFigure 2(a), a strong and narrow surface plasmon absorptionpeak (SPR) was observed at wavelengths 400–430nm.According to Mie’s theory, small spherical nanocrystalsshould exhibit a single surface plasmon band; however,anisotropic particles should exhibit two or three bands,depending on their size, morphology, configuration, anddielectric environment of the prepared nanoparticles [16].The SPR phenomenon arises because the metallic NPsphysically absorbed light, and as a result of this absorption,conduction electrons of metal undertake coherent oscillation.

On the other hand, two absorption bands were observedin wavelength ranges of 250–350 and 400–580nm of acobalt-containing sample (Figure 2(b)). As in Farhadi et al.[27], the first band can be assigned to the O2−→Co2+ chargetransfer process and the second one to the O2−→Co3+

charge transfer, which suggested formation of cobalt oxide.However, the UV-Vis optical absorption spectra of Ni andZn reaction mixture appeared at 370 and 380nm, respec-tively, indicating an almost uniform size of the NPs ashighlighted by Sathyavathi et al. [21] and Selvarajan andMohanasrinivasan [28].

3.3. Energy-Dispersive X-Ray Analysis (EDX). EDX is acompositional analysis approach which gives a qualitativeas well as quantitative status of elements that may be involvedin formation of NPs [29]. As can be seen in Figure 3, theelemental profile of as-fabricated NPs exhibited typicalcharacteristic elemental peaks at approximately 3 keV, 7 keV,6–7 keV, and 8–10 keV which was attributed to Ag, Co,Ni, and Zn, with atomic percentages 50, 21, 9, and 39%,respectively, and confirms the formation of their correspond-ing nanomaterial. The existence of other elements accompa-nied with biosynthesized NPs could also be noticed in highpercentage specially sulfur and phosphorus, suggesting con-jugation of bacterial biomolecules that contain polar phos-phorus backbones as DNA, RNA, ATP, and phospholipids.However, sulfur is an important structural and functionalcomponent of amino acids as methionine and cysteine [17].Furthermore, some signals of Na, K, Ca, and Cu weredetected, proposing that they are constituents of an aminoacid functional group that still adhered to the nanoparticles.Additionally, a peak for Al was also revealed due to the Alstub used to place the sample in the instrument [30].

3.4. X-Ray Diffraction (XRD). The crystallographic identityand crystalline nature quality in addition to the phase purityof the examined material were determined by X-ray diffrac-tion (XRD). A comparison of the XRD pattern of surveyedsamples with the standard Joint Committee of Powder Dif-fraction Standards (JCPDS) file confirmed that the particlesformed in our experiments were Ag2O, Co3O4, NiO, andZnO nanocrystals as elucidated in Table 2. The X-ray difrac-tograms of biosynthesized NPs are illustrated in Figure 4.Generally, the diffraction peaks of all as-synthesized NPsappeared sharp, clearly distinguishable, and broad, whichindicates the ultrafine nature and small crystallite size. TheXRD spectrum containing no other phase indicates thepurity of the sample [31].

3.5. Dynamic Light Scattering (DLS) and ξ Potential. DLStechnique was employed to evaluate the time-dependentoscillation of scattered light in dispersed nanoparticlesowing to Brownian motion, which is mainly based on theirhydrodynamic diameters (size distribution). Moreover, it

Ctrl A B C D

Figure 1: Visual inspection of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs. Ctrl: control (mediacontaining metal ions without bacteria).

4 Journal of Nanomaterials

also describes the degree of uniformity, either homogeneityor heterogeneity of particle size distribution through thepolydispersity index (PDI). PDI is a dimensionless parame-ter which can also estimate NP aggregation. Its values rangefrom 0 (highly uniform, monodisperse, and finer particlesize distribution) to 1 (highly polydisperse with a very broadparticle size distribution). In general, values of 0.3 andbelow are considered to be acceptable and reveal a homog-enous distribution and values higher than 0.7 refer toheterogeneous dispersion of the samples and are usuallynot suitable to be measured by DLS [36]. In this study,the particle size distribution curves of biosynthesizedAgNPs, CoNPs, NiNPs, and ZnNPs are illustrated inFigure 5. It shows the Z-average of 5, 57, 93, and 48nmfor AgNPs, CoNPs, NiNPs, and ZnNPs, respectively. It isnotable that Z-average is the mean hydrodynamic size ofthe collection of particles measured by DLS [37].

Notably, the size measurement by DLS seems to be largerthan TEM measurement (as seen later), which could beattributed to that DLS assesses the size of overall aqueous

medium accompanying with NPs as referred by Vendittiet al. [38]. On the other hand, PDI values recorded 0.329,0.381, 411, and 0.337 for AgNPs, CoNPs, NiNPs, and ZnNPs,respectively, which implies homogenous dispersity of NPsregarding to the small size of monodisperse NPs.

The zeta potential considers being a pivotal criterion forthe determination of colloidal stability and gives an insinua-tion about the degree of repulsion between adjacent similarlycharged particles in dispersion. The ξ potential magnitude isindicative of long-term stability, since the stability range liesbetween +30 and −30mV which means that particles with ξpotentials more positive than +30mV or more negative than−30mV are considered stable [39]. In this study, the ξpotential value of dispersed biosynthesized NPs appearedclearly to be advantageous by recording −54, −52.5, −43.1,and −53.4mV for AgNPs, CoNPs, NiNPs, and ZnNPs,respectively (Figure 6). Based on the ranking table of colloidstability behavior in relation to the zeta potential referred byVishwakarma [40], our as-prepared NPs exhibited goodstability by the considerable repulsive force that is present

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Figure 2: UV-Vis absorption spectra of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

5Journal of Nanomaterials

between ultrafine particles which results in Brownian motionthat retains particles away from disposition to come intoaggregates or flocculates.

Accordingly, the negative singe of the zeta potential indi-cates that the particles are warped with anionic biomoleculesas nucleic acid residues (DNA-RNA) and also negativelycharged amino acids as glutamate and aspartate [39], whichprovide constancy by acting as capping, stabilizing, andfunctionalizing agents.

3.6. Transmission Electron Microscopy (TEM). For decades,TEM has been a powerful tool in microbiological researchesfor high-resolution ultrastructural studies of microorganismsand their components [41]. The size, shape, and morphol-ogies of the as-prepared NPs and its producing biofactorywere characterized by TEM. Particles with higher electrondensity will appear darker in the TEM-negative film. Ashighlighted in Figure 7, strain 10B behaved differentlywith metal precursors. Remarkably, the AgNP micrograph

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Figure 3: EDX profile of biosynthesized NPs by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

Table 2: XRD diffraction peaks and corresponding crystallographic planes matched with JCPDS.

NP type Bragg reflections (2θ degree) and corresponding Miller indices Crystalline phaseStandard JCPDScard number

Reference

Ag2O27.94° (100), 32.27° (110), 46.34° (200), 54.9° (211), 57.6° (211),

and 67.48° (220)Face-centered cubic (FCC) 76–1393 [30]

Co3O4 19.2° (111), 31.1° (220), 44.9° (400), and 55.9° (422) Face-centered cubic (FCC) 073–1701 [32, 33]

NiO 37.1° (111), 43.5° (200), and 62.4° (220) Face-centered cubic (FCC) 47–1049 [34, 35]

ZnO31.6° (100), 34.35° (002), 36.2° (101), 47.5° (102), 56.4° (110),

62.8° (103), 67.8° (200), 68.9° (112), and 76.9° (004)Hexagonal (wurtzite structure) 89–1397 [15, 28]

6 Journal of Nanomaterials

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Figure 4: XRD crystallographic pattern of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

7Journal of Nanomaterials

(Figure 7(a)) exhibited numerous, tiny, roughly globular,uniform NPs with particle size 8.86 nm scattered as seeds likein a monodisperse pattern at the periplasmic space of bacte-rial cells. Nonetheless, large aggregates of quasi-sphericalCoNPs, NiNPs, and ZnNPs appeared to be engulfed in thecytoplasmic compartment and held by cytoplasmic proteinsin a spider-net thread-like shape recording 22.1, 35.9, and19.1 nm, respectively, in diameter.

Interestingly, strain 10B was easily handled and manipu-lated with different heavy metals disparately and withoutmuch difficulty according to their toxicity or benefiting.This selective interaction with metals resulted in produc-tion of NPs in different cellular localizations [42]. Generally,the acquisition of heavy metals takes place in bacteriathrough harnessing of uptake systems (unspecific or specific)which proceeded by the chemiosmosis gradient across the

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Figure 5: Particle size distribution curve of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

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Figure 6: Zeta potential analysis of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

8 Journal of Nanomaterials

100 nm

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Figure 7: TEM micrograph of strain 10B-synthesized NPs: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.

9Journal of Nanomaterials

cytoplasmic membrane of bacteria. The expression of theseuptake systems is either constitutive or inducible. ATP-binding cassette transporters (ABC system) coordinate ametal traffic process of nearly every biologically requiredtransition metal ion from the extracellular milieu to thecell cytosol interiorly [43]. Consequently, the accumulationof heavy metal ions within the microbial cell occurs bymetabolic-dependent biosorption processes, which includesintracellular compartmentalization [44].

Once metal ions enter the cell, it begins transformationreactions to encounter their toxicity. Oxidation reductionreaction is the most pivotal enzymatic detoxification mecha-nism for bacterial transformation of metals to their nanoscale[42]. The respiratory enzymes (oxidoreductase) which weremembrane-bounded proteins such as NADH-dependentnitrate reductase or NAD-linked dehydrogenases wereproposed to initiate the reduction process.

In both enzymes with different scenarios, the finalresult was analogous, where NADH and NADPH arecoenzymes which act as electron carriers mediating severaland reversible electron transfers (accepting or losing) inthe electron transfer chain throughout the metabolic pro-cess. During the biological oxidation/reduction reaction,the enzymes may shuttle electrons to the metal ions thatare capable of undergoing redox reaction via multiplechanges in their oxidation state, which finally leads tometal NP formation [45]. This mechanism seems to besimilar to the formation of magnetic Fe3O4 particles bymagnotatic bacteria [46]. In our study, the oxide form ofNPs was produced due to the oxygen present in themedium and incubation condition or other oxidizingagents produced by bacterial cells as reported by Dhoondiaand Chakraborty [47].

The silver ions were described as “oligodynamic” owingto their higher bactericidal competency at minute concentra-tions [48]. Accordingly, strain 10B during the stationarygrowth phase accumulated the biosynthesized AgNPs at theperiplasm for external extrusion by the efflux system tomaintain homeostasis [46]. As recorded by Ma et al. [43],the periplasm of Gram-negative bacteria has dual functionaccording to bacterial sensitivity and ambient condition;under favorable circumstances, the periplasm behaves asstorage compartment for biologically essential metal ionsdetached from the cytosol. However, under acute toxicity, iteliminates metals away from the cell through the Czc-ABCefflux pumping process.

Remarkably, formation of metal-protein complexessequestered in the cytoplasm considers being another basicmechanism of heavy metal resistance according to Cerasiet al. [49] which proposed to be exploited by strain 10Bin cytoplasmic synthesis of CoNPs, NiNPs, and ZnNPs.Metallothioneins (MTs) and glutathione (GSH) are impor-tant metal-binding proteins that conduct efficiently in metalscavenging (detoxification), storage, and metal homeostasismaintenance [50, 51]. The nanofactory 10B seems to storeCoNPs, NiNPs, and ZnNPs as essential micronutrientswhich play vital catalytic, regulatory, and structural roles inproteins [52]. It is noteworthy to mention the significanceof nickel ion (Ni2+) in nickel-metallo enzyme synthesis such

as urease, Ni superoxide dismutase, hydrogenase, methylcoenzyme reductase, and carbon monoxide dehydrogenasewhich resides in the cytoplasm [53].

Besides, more than 300 known bacterial enzymesrequire zinc for their catalytic functions as protein struc-ture stabilization/folding, management of gene expression,DNA replication/repair, response to oxidative stress, biosyn-thesis of amino acids, extracellular peptidoglycan synthesis,cofactor of virulence-related proteins, and maintenance ofthe intracellular redox buffering of the cell [49, 54]. On theother hand, cobalt is known as a constituent of cobalamincofactor (B12) that is crucial for fatty acid catabolism andmethyl transfer reactions [43].

The conclusive outcome is ascertained that the bacterialmechanism for metal resistance/detoxification is somehowinvolved in the NP biosynthesis process. The silver detoxifi-cation machinery could typically bind and reduce otherdivalent ion systems and thus resulted in NP formation[55]. Also, such metal-resistant nanobiofactory participatesmainly in biogeochemical cycling of those metal ions [52]and could be utilized in bioremediation of metal pollutedareas as well [56].

3.7. Inhibitory Effect against Planktonic Pathogens. Theantimicrobial activities of examined NPs in differentconcentrations (100–200μg/ml) which were assessed on thebasis of clearance zone in comparison with standardantibiotics and metal precursors are presented in Table 3.The diameter of inhibition zone ranged from 4.0 to 9.5mm,1.8 to 10.3mm, and 3.2 to 13.4mm for fungi, Gram-negative bacteria, and Gram-positive bacteria, respectively.Notably, the antimicrobial action of all examined biogenicNPs was rated “good” since the zone of inhibition was>1mm as reported by Prasad et al. [11]. It is evidentfrom Table 3 that Gram-positive bacteria intrinsically weremore susceptible to all examined antimicrobial agents thanGram-negative ones were. This could be attributed to thestructural and compositional differences of the outer bacte-rial wall; Gram-negative bacteria have an additional lipopoly-saccharide layer comparable to Gram-positive ones. Theuniqueness of the penetration mechanism of this extra layercould dramatically alter the suppression caused by antimi-crobial agents. It is plausible to speculate that the presenceof powerful resistance mechanisms like multiple efflux pumpin Gram-negative bacteria such as the CzcCBA system [11]contributes in cobalt/zinc/cadmium resistance as mentionedby Lee et al. [57]. Also, plenty of negative charges in thelipopolysaccharide layer repel the negatively charged NPsand hence block the availability of cell wall-binding sites forNPs. Moreover, the outer thick peptidoglycan layer of theGram-positive bacteria cell wall has better permeability thanthe Gram-negative one which makes the bacteria moresusceptible to harmful substances such as toxins [58]. Conse-quently, the bacterial resistance/susceptibility rate is gov-erned by both cell wall structure and resistance of bacteriato the reactive oxygen species produced by the action of anantimicrobial agent [59]. Therefore, Gram-negative bacteriarequire a significantly higher concentration of antimicrobialagent to be eradicated.

10 Journal of Nanomaterials

Table3:Antim

icrobialactivity

throughthemaxim

uminhibition

zone

ofdifferentcon

centration

sof

biogenicNPs,metalprecursors,and

antibioticsin

paralleltoacollectiveactivity

effect

againstplankton

icpathogens.

Microorganism

Con

centration

(μg/ml)

Zon

eof

inhibition

(ZOI,mm)

Class

Strain

NPtype

Metalprecursor

Collectiveactivityeffect

(120

μg/ml)

Antibiotics

Ag

Co

Ni

Zn

Ag

CO

Ni

Zn

Rif.

Tet.

Strp.

Nyst.

Fungi

Aspergillu

sbracelleuse(ATCC16404)

100

4.8

3.9

4.0

4.5

2.2

0.9

0.8

0.8

9.3

ND

ND

ND

5.3

200

9.0

7.3

6.9

8.5

5.6

1.5

1.3

1.5

ND

ND

ND

9.5

Can

dida

albicans

(ATCC10231)

100

5.3

4.3

4.1

4.9

4.5

1.0

0.9

1.2

9.8

ND

ND

ND

6.1

200

9.5

7.5

7.6

9.1

6.5

1.8

1.8

2.1

ND

ND

ND

10.2

Gram-negativebacteria

Pseudomonas

aeruginosa

(ATCC27853)

100

4.8

2.4

2.5

2.1

3.2

0.9

1.0

0.9

7.8

5.3

6.6

5.8

ND

200

10.3

5.1

4.9

4.8

5.5

1.4

1.4

1.7

9.1

10.7

9.3

ND

Salm

onellatyphi(ATTC700931)

100

4.2

2.5

2.4

2.2

3.5

1.0

0.8

1.0

7.9

5.2

6.9

5.9

ND

200

8.7

4.1

4.5

4.2

5.8

1.8

1.8

1.4

8.8

10.5

9.2

ND

E.coli(ATCC25922)

100

4.6

2.4

2.3

1.8

3.0

1.2

0.8

0.8

7.4

5.3

6.8

5.7

ND

200

9.7

4.8

4.7

4.3

6.2

2.1

1.8

1.7

8.4

9.8

9.2

ND

Gram-positivebacteria

Clostridium

perfringens(ATCC13124)

100

5.5

3.8

4.1

3.9

4.7

1.5

1.8

1.7

10.3

7.0

8.5

7.5

ND

200

12.0

7.2

6.9

7.0

7.6

3.4

3.2

3.2

13.3

14.5

12.4

ND

Enterococcus

faecalis(ATCC29212)

100

4.5

2.4

2.3

2.5

3.7

0.8

0.8

0.8

6.3

7.4

6.1

ND

200

9.3

5.3

5.0

5.1

7.4

2.1

2.3

2.3

8.6

9.8

12.3

10.3

ND

Bacillus

cereus

(ATCC7464)

100

6.3

4.0

4.3

4.2

4.7

1.8

1.4

1.5

12.8

7.5

8.7

8.1

ND

200

13.4

8.8

8.1

8.5

8.0

4.1

4.0

4.2

14.8

15.8

13.7

ND

Staphylococcus

aureus

(ATCC25923)

100

5.8

3.2

3.5

3.3

4.8

1.8

1.3

1.5

11.5

6.7

8.0

7.3

ND

200

11.7

7.0

7.1

6.9

8.0

3.9

4.0

4.3

12.6

14.7

13.6

ND

11Journal of Nanomaterials

It is noteworthy that an efficient antimicrobial activitywas more pronounced by all types of as-synthesized NPs ascompared to their precursors. Besides, a potent antimicrobialpattern of AgNPs was observed against all examined patho-genic prokaryotes and eukaryotes comparable to the others.Absolutely, the larger surface area (surface/volume ratio)associated with ultrafine AgNPs permits more closely inter-actions with microbial cells; hence, it enhances the cytotoxic-ity to the microorganisms than the large-sized nanoclustersdo [1, 59]. Undoubtedly, the extent of NPs’ lethal effectrelies on the concentration of applied NPs and the initialmicrobial concentration. In contrast to our results, a neg-ligible inhibitory effect of 100μg/ml AgNPs with a particlesize of 12–40 nm was recorded on E. coli batch cultures(105–108CFU/ml) [60]. However, AgNPs (120μg/ml) witha particle size range of 2.26–10.34 nm were sufficient enoughto inhibit the growth of E. coliMTCC-1302 at initial concen-tration (103–104CFU/ml) [61].

Further, CoNPs, NiNPs, and ZnNPs showed moderate,reproducible, and almost equal biocide activity suggestingthat they have dominant inhibitory targets focused on diversemicrobial metabolic pathways. Briefly, the NPs’ inhibitorymechanisms include disorganization of the building compo-sition of the cell wall/cell membrane, increasing themembrane permeability, causing dysfunction of essentialproteins by reacting with thiol groups, impairing DNA repli-cation, and producing elevated levels of reactive oxygenspecies (ROS) such as hydrogen peroxide which in turninduce oxidative stress [2, 7, 22]. Interestingly, our resultssuggested that ZnNPs preferentially exhibited antifungalrather than antibacterial properties which come into agree-ment with previous studies by Manokari et al. [26] andRoberson et al. [62].

Moreover, the biocide activity of NPs increases linearlywith increase in NP concentration. Thus, it is obviousfrom the data that the antimicrobial activities are dose-dependent. Our result is coincident with Pandian et al. [22]who reported the dose-dependent manner of Ni-NPs thatexerted antibacterial and antifungal activities against a widerange of pathogens. Lastly, the collective activity of four bio-synthesized NP types with equivalent participation (30μg/mlfor each) resulted in enhancement of antagonistic activityagainst all studied pathogens. From our perspective, thesynergetic effect of combined NPs imitates and approximatesthe antibiotic influence which boosts its application in adju-vant therapy to defeat several multiple antibiotic-resistantmicroorganisms. As indicated by Ashajyothi et al. [63], theability of metals to target multiple sites in an organismmakesthem superior to conventional antibiotics.

3.8. Inhibitory Effect against Biofilm Formation and C.vulgaris. Currently, the biofilm is considered to be amongthe most serious issues which medical devices and waterdisinfection applications are facing in particular, since itexerts several mechanisms simultaneously to resist differentstress factors up to 1000 times in comparison to planktoniccells [64]. Thus, NP strategies were developed to replace orenhance antibiotic treatment in solving this medical andenvironmental problem. In this study, nearly all features that

were observed on planktonic pathogen upon exposure tovarious treatments (individual NPs, combined NPs, metalprecursors, and antibiotics) are considered to be predomi-nant with biofilm pathogens and even algae as evidentfrom Table 4. Generally, sufficient antibiofilm and algicidalactivities were exhibited by all types of as-prepared NPsespecially AgNPs. The inhibition percentage ranged frommoderate to potent and increased with gradual increasein NP concentration.

Notwithstanding that the sessile microbial cells of biofilmprotect themselves by embedding in a self-produced extracel-lular polymeric matrix (DNA, proteins, and polysaccha-rides), NPs inhibit biofilm efficiently through the damage ofa planktonic phase, a chemical communication mechanismthat held sessile state in aggregated form (quorum sensing),and increase in hydrophobicity which blocks the initial adhe-sion of free living cells in the biofilm as referred by Lee et al.[57] and Franci et al. [65]. In consistency with our results,Vincent et al. [66] and Sangani et al. [67] reported the potentantibiofilm effects of Ni-NPs and ZnO-NPs. Otherwise,the enhancement of V. cholerae biofilm by addition ofZnO-NPs was mentioned by Salem et al. [68].

What is more, in contrast to our study, the stimulatoryinfluence of ZnO-NPs (200mg/l) was noticed with 35% cellviability of C. vulgaris growth after 72 h incubation asreferred by Miazek et al. [69]. The drastic alteration in thecellular division processes of C. vulgaris accompanied withapplication of NPs was recorded by Gong et al. [70]. Thismoves in accordance to our result, where 75% reduction inalgal growth was observed by employing combined NPs.Consequently, the exploitation of NPs in controlling algalblooms will restrict their consecutive adverse environmentalproblems such as odoriferous, unsightly scums, toxicity ofwater bodies, and eutrophication [71].

On the other hand, considerable stimulation in algalgrowth was observed with some metal precursors (Ni andZn) and weak inhibition caused by cobalt salt even at highconcentrations. These micronutrients are indispensable ele-ments for algal metabolism and physiological function suchas photosynthesis and respiration [72].

In consideration of the foregoing, the robust synergeticeffect of combined NPs against a wide range of pathogenicmicrobial forms opens promising avenues for their exploi-tation as antimicrobial agents in unlimited biomedicine,pharmaceutical product, and environmental applications.

3.9. Application of Combined NPs in Water and Wastewater.As observed in Table 1, a noticeable bacterial suppressionwas exhibited by combined NPs predominantly with increas-ing contact time. Virtually, the biocide activity of combinedNPs in municipal wastewater, freshwater, and agriculturalwastewater was higher than in salt water, particularly Elmala-hat. This could be attributed to the presence of suspendedsolids which adversely influence on binding strength betweenNPs and bacterial surface. Obviously, the existence of Cl− inthe samples may interact with different metal ions (Ni2+,Co2+, Ag+, and Zn2+) that are released from combined NPswhich resulted in production of NiCL2, CoCL2, AgCl, andZnCL2. In addition, the presence of cations such as K+,

12 Journal of Nanomaterials

Na+, Ca2+, and Mg2+ could be adsorbed on negativelycharged NPs which neutralize their surface forming largeflocculates and thus losing monodispersity. Consequently,the effective dose that was designed for bacterial eradicationrequired to be adjusted according to the type of the samplesand their content [73].

4. Conclusions

To summarize, this study for the first time demonstrates theemployment of Proteus mirabilis strain 10B as a biotemplatein fabrication of multiple compound NPs (Ag, Co, Ni, andZn). The biosynthesized NPs were physicochemically charac-terized using UV-Vis spectroscopy, XRD, EDX, TEM, ζpotential, and PDI. The biosynthesized NPs displayed anantagonistic activity against a wide range of microbial patho-gens (Gram-positive, Gram-negative, and anaerobic bacteria,mold, yeast, biofilm, and algae). The present study evaluatesthe promising potency of combined NPs against variouspathogens which sparks an immense interest in multipleapplications. Also, up to our knowledge, the synergisticbiocidal activity of combined NPs on various microbialpopulations has not been studied before.

Data Availability

The data used to support the findings of this study areincluded within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the City of Scientific Researchand Technological Applications. Also, the authors gratefullythank Engineer Ayman Kamal for his efforts in imaging thesamples by electron microscopy.

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Table 4: Biocide activity of NPs, metal precursors, and antibiotics in addition to collective activity influence on biofilm and algal growth.

Biofilm type P. aeruginosa S. aureus C. vulgarisTreatment (μg/ml) Inhibition (%)

NP type

Ag150 60.25 70.62 56.37

300 89.16 94.57 94.56

Co150 44.77 48.09 24.40

300 76.78 80.65 49.28

Ni150 47.82 49.98 39.71

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Ni150 27.69 38.54 −16.75300 51.98 65.14 4.31

Zn150 21.46 30.11 −46.41300 50.76 59.04 −11.00

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Streptomycin150 50.18 57.69

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ND53.46

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