Antibacterial properties ofnanoparticlesMohammad J. Hajipour1, Katharina M. Fromm2, Ali Akbar Ashkarran3,Dorleta Jimenez de Aberasturi4,5, Idoia Ruiz de Larramendi5, Teolo Rojo5,Vahid Serpooshan6, Wolfgang J. Parak4 and Morteza Mahmoudi1,7

1 Laboratory of NanoBio Interactions, Department of Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences,Tehran, Iran2University of Fribourg, Department of Chemistry, Chemin du Muse e 9, CH-1700 Fribourg, Switzerland3Department of Physics, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran4 Fachbereich Physik and WZMW, Philipps Universitat Marburg, Marburg, Germany5Department of Inorganic Chemistry, UPV/EHU, Bilbao, Spain6Division of Pediatric Cardiology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5101, USA7Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Antibacterial agents are very important in the textileindustry, water disinfection, medicine, and food packag-ing. Organic compounds used for disinfection have somedisadvantages, including toxicity to the human body,therefore, the interest in inorganic disinfectants such asmetal oxide nanoparticles (NPs) is increasing. This re-view focuses on the properties and applications of inor-ganic nanostructured materials and their surfacemodications, with good antimicrobial activity. Suchimproved antibacterial agents locally destroy bacteria,without being toxic to the surrounding tissue. We alsoprovide an overview of opportunities and risks of usingNPs as antibacterial agents. In particular, we discuss therole of different NP materials.

Antimicrobial NPsAntibacterial activity is related to compounds that locallykill bacteria or slow down their growth, without being ingeneral toxic to surrounding tissue. Most current antibac-terial agents are chemically modied natural compounds[1], for instance, b-lactams (like penicillins), cephalospor-ins or carbapenems. Also, pure natural products, such asaminoglycosides, as well as purely synthetic antibiotics, forexample, sulfonamides, are often used. In general, theagents can be classied as either bactericidal, which killbacteria, or bacteriostatic, slowing down bacterial growth.Antibacterial agents are paramount to ght infectiousdiseases. However, with their broad use and abuse, theemergence of bacterial resistance to antibacterial drugshas become a common phenomenon, which is a majorproblem. Resistance is most often based on evolutionaryprocesses taking place during, for example, antibiotictherapy, and leads to inheritable resistance. In addition,horizontal gene transfer by conjugation, transduction ortransformation can be a possible way for resistance to buildup [2]. Such antibacterial-resistant strains and species areinformally referred to as superbugs and contribute to the

emergence of diseases that were under good control formany years. One prominent example is bacterial strainscausing tuberculosis (TB) that are resistant to previouslyeffective antibacterial treatment. Indeed, it is estimatedthat nearly half a million new cases of multidrug-resistanttuberculosis (MDR-TB) occur worldwide every year [3];along these lines, the newly identied enzyme, newDelhimetallo-b-lactamase-1 (NDM-1), is responsible forbacterial resistance to a broad range of b-lactam antibac-terials, and it seems that most isolates with NDM-1 en-zyme are resistant to all standard intravenous antibioticsfor treatment of severe infections [4]. Thus, due to the factthat bacteria developed resistance against many commonantibacterial agents, infectious diseases continue to be oneof the greatest health challenges worldwide. In addition,drawbacks for conventional antimicrobial agents are notonly the development of multiple drug resistance, but alsoadverse side effects. Drug resistance enforces high-doseadministration of antibiotics, often generating intolerabletoxicity. This has prompted the development of alterna-tive strategies to treat bacterial diseases [5]. Among them,nanoscale materials have emerged as novel antimicrobialagents. Especially, several classes of antimicrobial NPsand nanosized carriers for antibiotics delivery haveproven their effectiveness for treating infectious diseases,including antibiotic-resistant ones, in vitro as well as inanimal models [6]. Why can NPs offer improved propertiesto classical organic antibacterial agents? One reason liesin their high surface area to volume ratio, resulting inappearance of new mechanical, chemical, electrical,optical, magnetic, electro-optical, and magneto-opticalproperties of the NPs that are different from theirbulk properties [7]. In this case, NPs have been demon-strated to be interesting in the context of combatingbacteria [8]. We rst discuss particular properties of bac-teria and important differences between different strains.The way to destroy bacteria is highly specic to the re-spective bacterial strains. We then describe the toxicitymechanisms of NPs against bacteria, and drug-resistantbacteria and their defense mechanisms. Finally we

Corresponding author: Mahmoudi, M. (Mahmoudi@biospion.com),(Mahmoudi-M@TUMS.ac.ir), (Mahmoudi@Illinois.edu).Keywords: antibacterial agent; nanoparticle; toxicity.

Published in "7UHQGVLQ%LRWHFKQRORJ\GRLMWLEWHFK"which should be cited to refer to this work.

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provide an outlook on NPs in the environment andecosystems.

Properties of bacteria, and thus the way to destroythem, are highly specic to the respective bacterialstrainsRole of the cell wallThe bacterial cell wall is designed to provide strength,rigidity, and shape, and to protect the cell from osmoticrupture and mechanical damage [9]. According to theirstructure, components, and functions, the bacteria cell wallcan be divided into the two main categories: Gram positive(+) and Gram negative (). The wall of Gram-positive cellscontains a thick layer (i.e., 2050 nm) of peptidoglycan(PG), which is attached to teichoic acids that are uniqueto the Gram-positive cell wall (Figure 1a) [10]. By contrast,Gram-negative cell walls are more complex, both structur-ally and chemically. More specically, in Gram-negativebacteria, the cell wall comprises a thin PG layer andcontains an outer membrane, which covers the surfacemembrane. The outer membrane of Gram-negative bacte-ria often confers resistance to hydrophobic compoundsincluding detergents and contains as a unique component,lipopolysaccharides, which increase the negative charge of

cell membranes and are essential for structural integrityand viability of the bacteria (Figure 1b) [11].

The structure of the cell wall plays an important role intolerance or susceptibility of bacteria in the presence of NPs.For instance, vancomycin (van)-functionalized Ag@TiO2NPs have the capacity to target van-sensitive bacteria[12]. In the van-sensitive bacterium, Desulfotomaculum,the D-Ala-D-Ala structure on the surface of the cell wallcan be recognized by vancomycin. By contrast, it is impossi-ble for vancomycin to penetrate into van-resistant bacteriaand access the D-Ala-D-Ala structure moiety. This is due tothe fact that van-resistant bacteria have an additional outermembrane, which covers the cell surface. Bacterial cell wallproperties can play a crucial role in diffusion of NPs insidebiolm matrixes [13]. The expression of the major cell-wall-anchored proteinase PrtP is responsible for altering thesurface of Lactococcus lactis from a hydrophilic to an ex-tremely hydrophobic one. In fact, the expression of PrtP inL. lactis 2 changes the physicochemical properties withoutarchitectural modications during biolm formation.

Role of the NP type and surfaceSpecies sensitivity is not only related to the structure of thecell wall in Gram-positive and Gram-negative bacteria

Pepdoglycan

Cytoplasmic membrane

Lipopolysaccharide

Porin

Outer membrane

Pepdoglycan

Cytoplasmic membrane

Periplasm

Lipoprotein

Teichoicacid

Lipoteichoicacid

(a)

(b)

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Figure 1. Bacterial cell structure. (a) A Gram-positive bacterial cell wall is composed of a thick and multilayered peptidoglycan (PG) sheath outside of the cytoplasmic

membrane. The teichoic acids, as seen, are connected to and embedded in the PG, and lipoteichoic acids extend into the cytoplasmic membrane. (b) A Gram-negative

bacterial cell wall is composed of an outer membrane linked by lipoproteins to thin and single-layered PG. The PG is placed within the periplasmic space that is formed

between the outer and inner membranes. The outer membrane includes porins and lipopolysaccharide molecules [64].

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[12]. Several additional factors can inuence the suscepti-bility or tolerance of bacteria to NPs. For example, Escher-ichia coli () is highly susceptible, whereas Staphylococcusaureus (+) and Bacillus subtilis (+) are less susceptible toCuO NPs [13]. The antibacterial effect of Ag NPs is higherthan Cu NPs against E. coli () and S. aureus (+) bacteria[14]. S. aureus (+) and B. subtilis (+) are more susceptiblethan E. coli () to NiO and ZnO NPs [13].

Role of growth rateAnother factor that can inuence the tolerance of bacteriaagainst NPs is the rate of bacterial growth. Fast-growingbacteria are more susceptible than slow-growing bacteria toantibiotics and NPs [15,16]. It is possible that the toleranceproperty of slow-growing bacteria is related to the expres-sion of stress-response genes [14,17]. Consequently, anti-bacterial effects highly depend on the particular strain.

Role of biolm formationOne of the major shortcomings of antibacterial drugs andNPs, is their failure to ght with bacteria [e.g., S. aureus(+)] that have the capability to produce biolms [18,19].Biolms are a complex microbial community that form byadhesion to a solid surface and by secretion of a matrix(proteins, DNA, and extra-polysaccharide), which cover thebacterial cell community. Biolms are known as a signi-cant problem because biolm formation protects pathogen-ic bacteria against antibiotics and is one of the main causesof development of chronic infections (Figure 2) [20]. Theelectrostatic properties of both NPs and biolms inuencehow they interact. The majority of bacteria have negativelycharged biolm matrixes but Staphylococcus epidermidis

(+) has a polycationic biolm [21]. The uptake and bioac-cumulation of Ag NPs to biolms is increased in thepresence of Suwannee River fulvic acid (SRFA) [22]. How-ever, surprisingly, Ag NPs are able to impact biolms onlyin the absence of SRFA. In all cases, the viability ofbacteria is unchanged. SRFA may protect bacteria againstNPs by covering the NPs and/or by intrinsic antioxidantactivity, which protects the bacterial membrane from sig-nicant damage [23]. The Ag NP uptake by marine biolmsand reduction of marine biolms are dependent on theconcentration of Ag NPs [24]. Exposure to Ag NPs mayprevent colonization of new bacteria onto the biolm anddecrease the development and succession of the biolm.MgF2 NPs have antimicrobial activity and are able toprevent the biolm formation of common pathogens suchas E. coli and S. aureus [25]. Furthermore, MgF2 NP-modied catheters are able to restrict the biolm formationof these bacteria signicantly [26]. Moreover, they havedemonstrated that glass surfaces coated with ZnO NPs areable to produce reactive oxygen species (ROS) that inter-fere with E. coli and S. aureus biolm formation [27].Among various types of NPs, superparamagnetic ironoxide NPs (SPIONs) with different surface coatings (e.g.,gold and silver) show highest antibacterial activity againstbiolms [18,19] (Figure 3). It is notable that magnetic NPshave considerable capability to penetrate into biolms,using external magnetic elds [18,19].

The toxicity mechanisms of NPs against bacteriaThe exact mechanisms of NP toxicity against various bacte-ria are not understood completely. NPs are able to attach tothe membrane of bacteria by electrostatic interaction and

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Figure 2. The stages of biofilm development [65]; (for additional information on dynamic processes of biofilm formation, see the following link: https://

www.biofilm.montana.edu/biofilm-basics-section-1.html.)

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disrupt the integrity of the bacterial membrane [28]. Nano-toxicity is generally triggered by the induction of oxidativestress by free radical formation, that is, the ROS, followingthe administration of NPs (Figure 4) [29,30]. Tables 1 and 2summarize recently published work on antibacterial prop-erties of nanostructured materials ranging from metallicand metal oxide NPs to semiconductors, polymers, andcarbon-based materials against Gram-positive and Gram-negative bacteria.

The mechanisms of NP toxicity depend on composition,surface modication, intrinsic properties, and the bacterialspecies. There are many reports about the antibacterialeffects of various NPs, but some reports contradict eachother (for more information compare the summaries ofprevious reports in recent reviews [22,24,25,31,32]). Thesereports indicate that the mechanisms of NP toxicity arevery complicated and depend on several factors (e.g., phys-icochemical properties of NPs). Therefore, we are not able

to classify the NPs as benecial NPs and/or adverse NPsfor killing bacteria.

In the following, we describe some mechanisms of tox-icity effects of NPs against bacteria. TiO2 and ZnO NPshave weak mutagenic potential that induces frameshiftmutation in Salmonella typhimurium () (TA98 andTA1537) [33]. The ability of ZnO NPs to induce frameshiftmutation is dependent on the presence of S9 fraction. It ispossible that the S9 fraction increases the internalizationof NPs and then increases the generation of ROS thatinduce frameshift mutation in the bacteria. However,TiO2 NPs induce frameshift mutation in Sal. typhimurium(TA98 and TA1537) independent of S9 fraction. TiO2 NPsare toxic to Pseudomonas aeruginosa (), Enterococcus hire(+), E. coli (), S. aureus (+), and Bacteroides fragilis (),only under UV illumination and killed approximately allbacteria in 60 min. These NPs have no toxicity in the dark[34]. TiO2 NPs photocatalysis can increase peroxidation of

SPIONs

ROS

Ag+

Ag+

e

e

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Figure 3. Schematic representation of toxicology effect of multifunctional nanoparticles (NPs) in bacterial biofilms. Monodisperse superparamagnetic iron oxide NPs

(SPIONs; black spheres) are coated with silver (gray shell), gold (yellow shell), and silver ring-coated, gold-coated SPIONs; silver ring-coated SPIONs and silver ring-coated,

gold-coated SPIONs have strong toxic effects on bacterial biofilms, by penetration into the biofilms. Both SPIONs cores and the intermediate gold shell have the capability

to induce heat by applying alternative magnetic and laser fields, respectively; the produced heat can be used as additional means to escalate bacterial death using these

NPs. The magnified section in the center illustrates the irreversible effects of NPs and their ions on the various parts of the bacteria (e.g., cell wall, DNA, and mitochondria).

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the polyunsaturated phospholipid component of the lipidmembrane and promote the disruption of cell respiration[35].

The toxicity of copper NPs depends on the combinationof several factors such as temperature, aeration, pH, con-centration of NPs, and concentration of bacteria (E. coli).The high temperature, high aeration, and low pH decreasethe agglomeration and increase the toxicity. In fact, thelower agglomeration provides more available surface areafor interaction with bacterial membranes and for solubili-zation of copper ions, which leads to more toxicity [36].Metallic and ionic forms of copper produce hydroxyl radi-cals that damage essential proteins and DNA [37].

Au NPs in solution, prepared by using the citrate re-duction method, are photomutagenic against Sal. typhi-murium () strainTA102. The photomutagenicity of AuNPs is dependent on coexisting Au3+ ions and citrateand it is not related to their intrinsic properties. Oxidationof Au3+ and decarboxylation of citrate in the presence oflight induce the generation of free radicals that damageessential proteins and DNA [38].

Among NPs such as CuO, NiO, ZnO, and Sb2O3 usedagainst E. coli, B. subtilis, and S. aureus, CuO NPs havethe highest toxicity, followed by ZnO (except for S. aureus),NiO and Sb2O3 NPs [39]. The toxicity of ions, which comeas a result of NPs, is not signicant and the toxicitystrength of metal oxide NPs depends on the natural toxicproperties of heavy metals. There appears to be a quanti-tative relation between colony size, colony number and theconcentration of metal oxide NPs [39]. Also, the toxicity ofoxide NPs (e.g., ZnO and CuO) does not always depend on

the bacteria internalizing the NPs; these NPs can locallychange microenvironments near the bacteria and produceROS or increase the NPs solubility, which can inducebacterial damage [40].

Biogenic Ag NPs, which are produced by living organ-isms or biological processes, have synergistic effects withantibiotics such as erythromycin, chloramphenicol, ampi-cillin, and kanamycin against Gram-negative and Gram-positive bacteria [41]. The combination of biogenic Ag NPswith antibiotics has efcient antibacterial activity. In fact,the ampicillin damages the cell wall and mediates theinternalization of Ag NPs into the bacteria. These NPsbind to DNA and inhibit DNA unwinding, which leads tocell death. Moreover, Ag NPs modied with titanium aretoxic to E. coli and S. aureus. Ag NPs naturally interactwith the membrane of bacteria and disrupt the membraneintegrity, and silver ions bind to sulfur, oxygen, and nitro-gen of essential biological molecules and inhibit bacterialgrowth [42]. The aforementioned studies show that suit-able NPs can be selected to ght against specic bacteria.

NPs against drug-resistant bacteriaThe emergence of antibiotic- and/or multidrug-resistantbacteria is recognized as a crucial challenge for publichealth. Killing of antibiotic-resistant bacteria requires mul-tiple expensive drugs that may have side effects. As a result,treatments are costly and require more time. NPs can offer anew strategy to tackle multidrug-resistant bacteria [43].Four types of silver carbon complexes (SCCs) with differentformulations including micelles and NPs have efcienttoxicity against medically important pathogens such as

Cell membrane

DNA damage

Release ofheavy metal

ions

Ag+

e

e

Zn2+

Mitochondriadamage

ROS producon

Reacve oxygenspecies (ROS)

Interrupted transmembraneelectron transport

Nucleus

Oxidized cellularcomponent

Protein

Cell membranedisrupon

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Figure 4. Mechanisms of toxicity of nanoparticles (NPs) against bacteria. NPs and their ions (e.g., silver and zinc) can produce free radicals, resulting in induction of

oxidative stress (i.e., reactive oxygen species; ROS). The produced ROS can irreversibly damage bacteria (e.g., their membrane, DNA, and mitochondria), resulting in

bacterial death.

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Table 1. Different nanostructured materials and their toxic effects in Gram-positive bacteria

Bacteria Bacterial property NP Composition Physicochemical

Properties of NPs

Applied

dosage

Mechanism of Toxicity Action Remarks Refs

S. aureus Biolm formation,normal ora of skin,production a matrixof exopolymericsubstances

Carboxyl-grafted SPIONs 1020 nm(size dened by TEM)ZP: 15.40.5 mV

0.35 mg/ml An external magnetic eld couldtarget carboxyl-grafted SPIONs into abiolm and increaseantibacterial efcacy

Carboxyl-grafted SPION, APTES-grafted and bare SPIONInternalized into the cell but doesnot affect mammalian celladhesion and spreading

[66]

APTES-grafted SPIONs 1020 nm(size dened by TEM)ZP: +32.60.3 mV

0.35 mg/ml ROS generation, electrostaticinteraction between NPs and bacteria

Bare SPIONs 1020 nm(size dened by TEM)ZP: +43.71.7 mV

0.35 mg/ml

PEGylated SPIONs 1020 nm(size dened by TEM)ZP: 7.710.9 mV

0.35 mg/ml No bacterial toxicity PEGylated SPION does notinternalize into the cell

Ag-coated SPIONs 1520 nm(size dened by TEM)

80 mg/ml Bacterial toxicity by penetration withinthe biolm and increase of the bacterialtoxicity in the presence of externalmagnetic eld,ROS generation, electrostatic interaction,and physical damage of bacteria

Ag-coated SPION andAgAu-coated SPION arefully compatible with the cell

[67]

AgAu-coated SPIONs 2030 nm(size dened by TEM)

80 mg/ml

Au-coated SPIONs 2540 nm(size dened by TEM)

80 mg/ml Spion-Au NPs have slight antibacterialactivity only in the presence of externalmagnetic eld due to penetrationwithin biolm of bacteria

S. epidermidis Biolm formation,normal ora of skin,production a matrixof exopolymericsubstances,gentamicin-resistant

NO-releasingMAP3(N-methyl aminopropyltrimethoxysilane)Si NPs

80100 nm(size dened by AFM)

8 mg/ml Biolm killing due to electrostaticproperties of NO-releasing NPs andincrease NO delivery to biolm-basedmicrobes

Rapid delivery of NO may bemore effective at biolmkilling than slow NO delivery

[68]

(Halophilic)bacterium sp.EMB4

Non-pathogenGram-positive halophilic,has a thicker PG layerwith higher percentageof neutralphosphatidylglycerol

ZnO

Table 1 (Continued )

Bacteria Bacterial property NP Composition Physicochemical

Properties of NPs

Applied

dosage

Mechanism of Toxicity Action Remarks Refs

B. subtilis Non pathogen, protectiveendospore forming

ZnO

Table 2. Different nanostructured materials and their toxic effects in Gram-negative bacteria

Bacteria Bacterial property NP composition Physicochemical

properties of NP

Applied

dosage

Mechanism of aoxicity action Remarks Refs

K. pneumoniae Medically importantpathogens, nitrogenxation, extended-spectrum b-lactamaseproduction Encapsulated

Ag Caron complex-L-tyrosine polyphosphateNP (SCC23-LTP NPs)

800 nm(size dened by DLS)

(MBC)NA(MIC)>10 mg/l

[43]

Ag NPs 43 nm(Hydrodynamic size in XRD)Surface area: 26 m2/g

30 mg/l Electrostatic interaction,adsorption, and penetrationof NPs and toxicity

The adsorption of Ag NPsincreases at 20 8Ccompared to 37 8C

[72]

NO NPs 1015 nm(size dened by TEM)

(MIC)10 mg/mlin 24 h

Alteration of the bacterialmembrane, antimicrobialeffects via nitrosation of proteinthiols and the nitrosylation ofmetal centers

NO oxidized to RNS, whichexert antimicrobial effects

[47]

P. aeruginosa Opportunistic, normalora of skin andintestine, biolmformation

NO NPs 815 nm(size dened by TEM)

(MIC)10 mg/mlin 16h

Alteration of the bacterialmembrane, antimicrobialeffects via nitrosation ofprotein thiols and thenitrosylation of metalcenters

NO oxidizes to RNS whichexert antimicrobial effects

[47]

NO-releasing MAP3(N-methyl aminopropyltrimethoxysilane)Si NPs

80100 nm(size dened by AFM)

8 mg/ml Biolm killing due toelectrostatic properties ofNO-releasing NPs andincreased NO deliveryto biolm-based microbes

Rapid delivery of NOmay be more effective atbiolm killing thanslow NO delivery

[68]

TiO2 1025 nm(ST-01, IshiharaSangyo Kaisha Ltd.,Osaka, Japan)

10 mg/l Photoactivation of TiO2promotes bactericidal effectPeroxidation of thepolyunsaturated phospholipidof membrane, loss ofrespiratory activity

[73]

Ag 110 nm(size dened by TEM)

25100 mg/l Disturbs permeability,respiration, and cell division,interacts with cell membraneand sulfur- and phosphorus-containing compounds

[74]

ZnO 1020 nm(size dened by TEM)

14.25 mM in100 `L of LB

Bacterial attachment byElectrostatic interaction,ROS generation, membranedisruption, and disturbanceof permeability

[75]

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Table 2 (Continued )

Bacteria Bacterial property NP composition Physicochemical

properties of NP

Applied

dosage

Mechanism of aoxicity action Remarks Refs

E. coli Extracellular biogenicsynthetic Ag NPs byTrichoderma viride fungi

540 nm(size dened by TEM)

(MIC)30 mg/ml

Ag NPampicillin leads to cellwall lysis, penetration ofAg NPs, and prevents DNAunwinding

Biogenic Ag NPs havesynergistic effectswith antibiotics

[76]

NO-releasing MAP3(N-methyl aminopropyltrimethoxysilane)Si NPs

80100 nm(size dened by AFM)

8 mg/ml Biolm killing due to electrostaticproperties of NO-releasing NPsand increased NO delivery tobiolm-based microbes

Rapid delivery of NOmay be more effectiveat biolm killing thanslow NO delivery

[68]

Al2O3 5070 nm(Zhejiang HongshengMaterial Technology Co.)+30 mV

20 mg/l Bacterial attachment(electrostatic interaction)Damage to the bacterial cellwall and increased permeability

Toxicity of NPs is fromtheir high tendency tobind to the cell walls

[71]

ZnO 20 nm(Zhejiang HongshengMaterial Technology Co.)5 mV

20 mg/l

TiO2 50 nm(Zhejiang HongshengMaterial Technology Co.)21 mV

20 mg/l No toxicity in dark condition

NiO 2030 nm(NanoAmor, Houston, USA)

20 mg/l Growth inhibition (in aqueousmedium). Signicant damagedcellular functions, physical/mechanical stresses on cellularstructure integrity (in aerosolexposure)

Synergistic effectbetween the solubleion stress and thenano-related stress(in aerosol exposureof NiO, ZnO, CuO)

[77]

zero valent Cu NPs (ZVCN) 25 nm(Sun Innovations, USA)

Release of Cu ions andgeneration of hydroxyl radicalin the cytoplasm

[78]

TiO2 20 nmST-01 (Ishihara SangyoKaisha Ltd.)

10mg/l Photoactivation of TiO2 promotesbactericidal effect. Peroxidationof the polyunsaturatedphospholipid of membrane,loss of respiratory activity

[73]

Ag 110 nm(size dened by TEM)

25100 mg/l Disturbed permeability,respiration, and cell divisionInteracts with cell membraneand sulfur- and phosphorus-containing compounds

[74]

Sal. typhimurium ZnO 2540 nm(core size in TEM)

8 and 80 ng/ml ZnO: cellular uptake, ROSgeneration and has nosignicant toxicity. Frameshiftmutation in the presenceof metabolic activationsystem (S9).

[79]

TiO2 4060 nm(core size in TEM)

8 and 80 ng/ml TiO2: cellular uptake, ROSgeneration and has nosignicant toxicity. Frameshiftmutation independent ofmetabolic activation system(s9)

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Table 2 (Continued )

Bacteria Bacterial property NP composition Physicochemical

properties of NP

Applied

dosage

Mechanism of aoxicity action Remarks Refs

Waste waterbiolm bacteria

Biolm formation Ag 5100 nm(Sky-Spring Nanomaterials,Houston, USA)

1200 mg/l Wastewater biolms withoriginal EPS are highly tolerantto the Ag-NP. After removingof EPS, the bacteria arevulnerable to Ag NPs

EPS and microbialcommunity interactionsin the biolms playimportant roles ininhibition of AgNPs toxicity

[80]

Sh. oneidensis MR-1 Reduce poisonousheavy metals, resistantto heavy metals suchas iron and uranium

Cu-doped TiO2 NPs 20 nm(size dened by TEM)

20 mg/l Cu-doped TiO2 NPs do not affectSh. oneidensis MR-1 growth

Sh. oneidensis MR-1tolerate against NPsdue to production ofa large amount ofEPS and reductionof ionic Cu

[51]

P. putida KT2442 Non-pathogen,biolm formation,benecial soil bacterium

Ag 10 nmSigmaAldrich,St. Louis, MO, USA

1 mg/l Cell membrane damage andbactericidal effect

Nano-Ag andnano-CuO NPs havedifferent targets forkilling bacteria

[58]

CuO 2540 nmSigmaAldrich

10 mg/l

ZnO 5070 nmSigmaAldrich

10 mg/l Bacteriostatic effect

C. metalliduransCH34

Non-pathogen,resistant in thepresence of severalforms of heavy metal

TiO2

P. aeruginosa (), Burkholderia cepacia (), methicillin-re-sistant S. aureus, multidrug-resistant Acinetobacter bau-mannii (), and Klebsiella pneumoniae () in the range of0.590 mg/l [43]. The SCCs are able to inhibit the growth ofbio-defense bacteria such B. subtilis and Yersinia pestis ()[43].

Targeting bactericidal NPs to specic bacteria or specif-ic infected tissue is an efcient prospect in treating infec-tion because this phenomenon minimizes side effects andenhances antibacterial activity [44,45]. In this case, mul-tifunctional NPs can be very useful; for instance, multi-functional IgGFe3O4@TiO2 magnetic NPs are able totarget several pathogenic bacteria and have efcient anti-bacterial activity under UV irradiation. The IgG and TiO2play a critical role in the targeting and killing properties ofthese NPs respectively. These NPs are toxic to Streptococ-cus pyogenes M9022434 and M9141204 [46].

Nitric-oxide-releasing NPs (NO NPs) are broad spec-trum antibacterial agents that are able to inhibit thegrowth of many antibiotic-resistant and sensitive clinicallyisolated bacteria such as K. pneumoniae, Enterococcusfaecalis (+), Str. pyogenes, E. coli, and P. aeruginosa ().The toxicity of these NPs depends on the delivery of NO tothe target. These NPs are able to change the structure ofthe bacterial membrane and produce reactive nitrogenspecies (RNS), which lead to modication of essentialproteins of bacteria [47]. Beside NO NPs, ZnO NPs aretoxic to antibiotic (methicillin)-resistant bacteria such asStreptococcus agalactiae (+) and S. aureus. These NPs areable to disorganize and damage the cell membrane andincrease the permeability, which leads to cell death. Thepolyvinyl alcohol (PVA)-coated ZnO NPs are able to inter-nalize the bacteria and induce oxidative stress [48]. Thetoxicity of ZnO NPs is concentration-dependent and theseNPs are mildly toxic at low concentration [49].

NPs in water can signicantly promote the horizontalconjugative transfer of multidrug-resistance genes medi-ated by the RP4, RK2, and pCF10 plasmids [50]. Here,nanoalumina can promote the conjugative transfer of theRP4 plasmid from E. coli to Salmonella spp. by up to200-fold compared with untreated cells. The nanoaluminais able to induce oxidative stress, damage bacterial cellmembranes, enhance the expression of mating pair forma-tion genes and DNA transfer and replication genes, anddepress the expression of global regulatory genes thatregulate the conjugative transfer of RP4 [50].

Defense mechanisms of tolerant bacteria against NPsSeveral naturally adapted bacteria are tolerant to specictoxins or NPs that are present in the environment.Cu-doped TiO2 NPs are able to inhibit the growth ofMycobacterium smegmatis (+), but have no effect againstShewanella oneidensis MR-1() [51]. These NPs releaseCu2+ ions, which might be the main cause of toxicity,because the antibacterial activity of Cu-doped TiO2 NPswas decreased in the presence of chelating agents such asEDTA. Sh. oneidensis MR-1 has excellent resistant againstseveral concentrations of Cu2+ and Cu-doped TiO2 NPsbecause of the production of extracellular polymeric sub-stances (EPSs) under NP stress. This bacterium is able toabsorb NPs on the cell surface and to decrease the amount

of ionic Cu in the culture medium. Therefore this bacteri-um can be regarded as a promising candidate for cleaningof metal oxide NPs from the environment.

B. subtilis and Pseudomonas putida () can physicallyadapt to nC60 [buckminsterfullerene (C60) introduced ascolloidal aggregates in water] [52]. P. putida increasescyclopropan fatty acids and decreases unsaturated fattyacid levels, but B. subtilis increases the transition temper-ature and membrane uidity in the presence of nC60. Thesephysiological adaptation responses of bacteria help to pro-tect the bacterial membrane against oxidative stress. TiO2and Al2O3 NPs are able to be internalized by E. coli andCupriavidus metallidurans CH34, but these NPs are toxiconly against E. coli [53]. The resistance mechanism of C.metallidurans CH34 is not yet understood completely. Thetolerance mechanism of this bacterium may be related tophysical properties of their PG layer and/or products ofgenes that are located in the plasmids and are able tostabilize the plasma membrane or efux of NPs.

Many bacteria are able to tolerate NO NPs using vari-ous mechanisms. For example P. aeruginosa, E. coli, andSal. typhimurium induce the expression of genes that areresponsible for repairing of DNA and altering the metalhomeostasis in the presence of NO NPs [5456]. In thiscondition, K. pneumoniae produces the enzyme avohemo-globin, which neutralizes nitrosative stress [57].

NPs against environment and ecosystemsExtensive use of NPs in biological science, medical science,and commercial products leads to leakage and accumula-tion of NPs in the environment (e.g., soil and water).Protection of the environment and benecial bacteria fromNPs is very important because, for example, the indiscrim-inate use of nanosized Ag materials leads to release of Aginto the environment. The leakage of NPs into the envi-ronment is one of the most serious threats to benecialmicrobes, microbial communities in ecosystems, and publichealth [58]. Many microbes benet the environment andthe ecosystem, because they play an important role inbioremediation, element cycling, and nitrogen xationfor plant growth [5961]. For instance, in the nitricationprocess, ammonium nitrogen is converted to nitrite andthen to nitrate by ammonia- and nitrite-oxidizing bacteria,respectively; the nitrifying bacteria are spread in theregions that have a high amount of ammonia; Ag NPs(

environment and human health, in spite of their benecialcommercial use.

Concluding remarksAntibacterial activities of NPs depend on two main factors:(i) physicochemical properties of NPs and (ii) type of bac-teria. Although there are good trends of correlation in a fewaspects of antibacterial activity of NPs (e.g., for biolms),individual studies are difcult to generalize. This is mainlydue to the fact that the majority of researchers performexperiments based on available NPs and bacteria, ratherthan targeting specic, desired NPs or bacteria. In partic-ular, often poorly dened and characterized NPs are usedand thus correlation with basic physicochemical propertiesis not possible. Without agreement on standard NPs andbacteria as reference systems, which should be included infuture studies, there is still a long way to go in order tounravel systematically the antibacterial properties of NPs.

AcknowledgmentsThis work was funded in part by BMBF Germany (project UMSICHT).

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