The Role of Nanotechnology in Combating Multi-Drug Resistant Bacteria

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Nanoscience and NanotechnologyVol. 14, 4745–4756, 2014

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The Role of Nanotechnology in CombatingMulti-Drug Resistant Bacteria

Rajni Singh1�∗, M. S. Smitha1, and Surinder P. Singh2�∗1Amity Institute of Microbial Biotechnology, Amity University, Noida 201303, U.P., India2CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India

The development of antibiotics has played a significant role in combating the dreaded infectious dis-ease such as tuberculosis, pneumonia, typhoid fever and meningitis in 20th century. However, theimproper use of antibiotics led to the development of multidrug resistance (MDR) in microbial floraraising a global public health concern of 21st century. This unforeseen threat demands the devel-opment of new drugs and strategies for combating antibiotic resistance shown by many microbialspecies. Recent developments in nanotechnology to engineer nanoparticles with desired physico-chemical properties have been projected as a new line of defense against MDR micro-organism.In this review, we summarized and discussed the recent development demonstrating the potential ofnanomaterials to evade the MDR. Nanoparticles have shown effective antimicrobial activity againstMDR bacteria, such as Acinetobacter baumanii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Mycobacterium tuberculosis, vancomycin resistant enterococci, methicillin-resistant Staphylococ-cus aureus and others. Furthermore, new strategies like combination of radiation and drugs withnanoparticle that are being explored to potentiate the effectiveness against MDR bacteria have alsobeen summarized.

Keywords: Nanoparticles, Multidrug Resistance Bacteria, Antibiotics, Antibacterial,Nanomedicine.

CONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47452. Multidrug Resistance (MDR) and Nanoparticles . . . . . . . . . . . . 47463. Nano-Bactericidals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4748

3.1. Silver Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47483.2. NO Releasing Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . 47493.3. Metal Oxide Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . 4751

4. Nano-Photothermal Therapy of MDR Bacteria . . . . . . . . . . . . . 47525. Antibiotics Conjugated Nanoparticles . . . . . . . . . . . . . . . . . . . . 47536. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4754

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4754References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4754

1. INTRODUCTIONThe expansion of antibiotic resistance is a growing men-ace today due to the indiscriminate use of antibiotics.It has resulted in the proliferation of selective pathogenicbacteria which are resistant to multiple drugs.1 The eco-nomic impact of antimicrobial-drug resistance is esca-lating at an alarming rate due to the mounting medical

∗Authors to whom correspondence should be addressed.

and human cost.2 Antibiotic resistance currently spans allknown classes of natural and synthetic compounds leadingto an urgent requirement for new drugs and alternatives.But very few novel antibiotics have been discovered in thepast 40 years due to the cost and complexities associatedwith drug discovery and development.3

Bacteria develop resistance to antibacterial drugsthrough a variety of mechanisms4 that require a freshapproach to develop new bactericidal. The search for newantimicrobial agents or modifications in already exist-ing ones to improve their antimicrobial activity becomesindispensable. Nanotechnology provides a good platformto alter physico-chemical properties of different materi-als compared to their bulk counterpart that can be har-nessed for bioapplications.5–7 Nanomedicine an offshootof nanotechnology has taken a stride in diagnosis, mon-itoring, drug delivery and control of diseases. The appli-cation of nanotechnology to medicine appears relativelya recent trend and has been excellently summarized byDoane et al.8 in context to engineer nanoparticles (NPs)towards drug delivery imaging and therapy. Rai et al.9

have recently shown that silver nanoparticles could prove

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The Role of Nanotechnology in Combating Multi-Drug Resistant Bacteria Rajni Singh et al.

to be an alternative to antibiotics and to control microbialinfections caused by MDR organisms. Several NPs such assilver, iron oxide (Fe3O4), titanium di-oxide (TiO2), zincoxide (ZnO) and gold have been extensively studied fortheir antimicrobial activity.10–14

The Fe3O4 nanoparticles are the representative materialthat clearly shows the power of nano size (nanotechnol-ogy), as it is not antibacterial in its bulk form but exhibitantibacterial activity in its nanoparticulate form. Althoughthe mechanism responsible for antibacterial properties ofnanoparticles is not fully understood but it is clear that itvaries from particle to particle. Metal oxide nanoparticlessuch as ZnO and TiO2 are considered to exhibit bacterici-dal property through reactive oxygen species (ROS) gen-eration while others shows membrane damaging relatedto their physical structure and metal ion release.15–17 Thesize, shape and enhanced surface area of these nanopar-ticles play pivotal role through multiple mechanisms of

Rajni Singh is presently working as Additional Director and Head in Amity Institute ofMicrobial Biotechnology, Amity University Uttar Pradesh, Noida, India. At present she isworking on the formulation of microbial consortium, which can be used for biodegradationof the organic wastes (kitchen waste, feather waste, petroleum hydrocarbons) and produc-tion and purification of the proteases, keratinase, amylases and lipases and their molecularcharacterization. She received her B.Sc. in Microbiology (1992) from Delhi UniversityM.Sc. in Microbiology (1995) and Ph.D. Microbiology (1999) from G. B. Pant University,Pantnagar. During her doctorate, she worked on molecular ecology of Scytalidium ther-mophilum. She joined Post Doc fellowship in Centre for Biochemical Technology, CSIR,Indian Institute of Technology Delhi and University of Delhi South Campus, India. Shehas worked as Lecturer at Department of Microbiology, University of Delhi South Cam-

pus. Professor (Dr.) Rajni Singh has 13 patents in her credit, executed 5 different projects and authored, co-authoredor presented over 35 scientific papers, articles, book and chapters and received different grants from Govt. organizationto present paper at different international platforms.

M. S. Smitha received her M.Sc. Degree in Microbiology from H.N.B. Garhwal Uni-versity. Presently she is pursuing her PhD from Amity University in the field of Micro-bial nanotechnology. Her research interest is the metal nanomaterials for drug deliveryapplication.

Surinder P. Singh obtained his M.Sc. (1992) and Ph.D. (1998) degrees in Physics fromG.B. Pant University of Agriculture and Technology, Pantnagar, India. He is working asa Scientist at National Physical Laboratory, New Delhi, India. He served as AssistantProfessor, Engineering Science and Materials, department at University of Puerto Rico,Mayaguez, USA (2008–2011). His research is focused on nanomaterials, nanomedicine,nanobiointerface, bio-implants, biosensors for health care and gas sensors.

antibacterial activity. Recently, biodegradable nanoparti-cles has been used against MDR bacteria, capable ofdestroying bacterial membranes without targeting a veryspecific step in their metabolic pathways.18–20 Folic acidtagged chitosan nanoparticles have also been used todeliver vancomycin into bacterial cells.21 It has been foundthat vancomycin loaded into nanoconjugate is very effec-tive and has a strong bactericidal effect on vancomycin-resistant S. aureus (VRSA).In this review, we focus on the potential of NPs to fight

MDR infections as well as the synergistic activity betweennanoparticles, antibiotics and/or various irradiation basedstrategies against MDR bacteria.

2. MULTIDRUG RESISTANCE (MDR)AND NANOPARTICLES

Multidrug-resistant (MDR) bacteria pose a major threatto all fields of medical science as it can result in

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Rajni Singh et al. The Role of Nanotechnology in Combating Multi-Drug Resistant Bacteria

Figure 1. Mechanisms of antibiotic resistance.

treatment failure which can have severe consequences,especially in case of critical patients.22–24 At present someof the most challenging Multidrug-resistant (MDR) organ-isms are extensively drug-resistant (XDR) Mycobacteriumtuberculosis, Acinetobacter baumannii, Pseudomonasaeruginosa, methicillin-resistant Staphylococcus aureus(MRSA), Escherichia coli and Klebsiella pneumoniaebearing NDM-1 (New Delhi metallo-beta-lactamase-1),vancomycin resistant enterococci (VRE) and vancomycin-resistant MRSA.25

Bacterial antibiotic resistance exhibited by differentmicro-organisms could be attained through various intrin-sic or acquired mechanisms as summarized in Figure 1.Intrinsic or ‘natural’ resistance is inherent to a bacte-rial species and involves the absence of the target orthe presence of low-affinity targets, low cell permeabil-ity, inactivation of the antibiotics and the presence ofefflux mechanisms. On the other hand, acquired mecha-nisms include mutations in genes targeted by the antibi-otics, the transfer of resistance determinants borne onplasmids, bacteriophages, transposons, and other mobilegenetic materials.26 In general, this exchange is accom-plished through the processes of transduction (via bac-teriophages), conjugation (via plasmids and conjugativetransposons), and transformation (via incorporation intothe chromosome of chromosomal DNA, plasmids, andother DNAs from dying organisms).27

A very few novel antibiotics have been discovered totreat these MDR organisms in past decades.28�29 Famousmicrobiologist Alexander Fleming said that “There isprobably no chemotherapeutic drug to which in suitablecircumstances the bacteria can not react by in some wayacquiring fastness.” Therefore there is high probability thatthe organism may also become resistance to newly devel-oped drugs at later stage, further these drugs are highlyexpensive. To this end nanoparticles are considered to begood antibacterial agents and may overcome the barrier ofMDR owing to their multifunctional mechanisms to inter-vene normal cell functionality.Various theories and explanations have been proposed

for different nanoparticles for their microbicidal activity(Fig. 2) and have been studied on the basis of morpholog-ical and structural changes in the bacterial cells. Nanopar-ticles are shown to have the ability to anchor to thebacterial cell wall and subsequently penetrate it, therebycausing structural changes in the cell membrane perme-ability leading to cell death.30 Silver NPs (SNPs) showefficient antimicrobial property compared with other saltsdue to their extremely large surface area, which providesbetter contact with microorganisms.31 SNPs may target atthe bacterial membrane, leading to a dissipation of the pro-ton motive force which in turn cause blocking of oxidativephosphorylation.32 Another mechanism involved in micro-bicidal activity is the generation of free radicals by the

J. Nanosci. Nanotechnol. 14, 4745–4756, 2014 4747




Nanoparticle

Cell wall synthesisinhibition Oxidative stress

Inhibition of enzyme activity

Inactivation ofprotein synthesis

Penetration of cell membrane

Interference withcell signaling Hinder bio film formation

Incorporation intothe DNA bases

Modification of essentialproteins

OH.

OH.

OH. OH.

Targetcell

Signalingcell

Peptidoglycan

Proteins

Phospholipid

Surface

Biofilm

Figure 2. Mechanisms for antibacterial activity of nanoparticles.

nanoparticles which have the ability to damage the cellmembrane and make it porous which can ultimately leadto cell death.33 Metal nanoparticles have the affinity tointeract with sulfur and phosphorus containing biomate-rials present in the bacterial cell e.g., DNA bases. Themetal nanoparticles can act on these soft bases and destroythe DNA which would lead to cell death.34 Nanoparticlesare known to modulate the bacterial signal transduction.The nanoparticles dephosphorylate the peptide substrateson tyrosine residues, which lead to signal transductioninhibition and inhibition of bacterial growth.35 It is alsoshown that there could be a release of silver ions from Agnanoparticles36 and these ions can interact with the thiolgroups of many vital enzymes and inactivate them, caus-ing disruption of cellular functions.37 NPs also exert theirantibacterial activities either by collapsing the membranepotential and inhibiting the ATPase activities to decreasethe ATP level and the other is by inhibiting the subunitof ribosome from binding to tRNA.38 Several findings onbactericidal effect of various NPs and nanoconjugate sys-tems are briefly reviewed in preceding sections.

3. NANO-BACTERICIDALS3.1. Silver NanoparticlesAmong metal nanoparticles silver nanoparticles havebeen extensively studied and used as effective antimicro-bial agents.39 The bactericidal effect of silver nanopar-ticles on micro-organisms is very well known; however,

the bactericidal mechanism is not completely understood.Studies showed that silver nanoparticles attacks Gram-negative bacteria by anchoring and penetrating the cellwall, and as a consequence, the leading structural changein the membrane morphology. This result in significantincrease in membrane permeability and alters transportthrough the plasma membrane resulting in cell death.40

It has also been proposed that the silver nanoparticlesstrongly interacts with thiol groups of vital enzymes andphosphorus-containing compounds such as DNA, prevent-ing cell division and DNA replication leading to celldeath.41�42 Kim et al.33 suggested that the antimicrobialmechanism of silver nanoparticles is associated to theformation of free radicals and subsequent free radical–induced membrane damage. Panacek et al.43 have studiedthe antimicrobial activity of silver nanoparticles againstGram +ve and Gram −ve bacteria, including MDR strainssuch as MRSA. The silver nanoparticles exhibited sizedependent bactericidal activity against MRSA and Gram+ve and Gram −ve bacteria. Marta et al.44 investi-gated the antibacterial efficiency of pluronic-coated sil-ver nanoprisms and found them to be effective againsttwo methicillin-resistant Staphylococcus aureus (MRSA)strains (UCLA 8076 and 1190R). The nano-prisms havedifferent crystalline facets with different surface energyi.e., with varying surface reactivity. The facets of higherreactivity and release of Ag ions from tips and edgesare considered a reason for efficient bactericidal activity.In a recent study, Jangra et al.45 have also showed the





importance of the role of crystalline structure, differentshapes and active facets in antibacterial activity of ZrO2

nanoparticles.It is seen that physico-chemical properties of nanopar-

ticles dictate the bactericidal activity.46 These propertiesare highly synthesis process and parameter dependent.Researchers have used various methods to synthesize metalnanoparticles e.g., the biosynthesis of metal nanopar-ticles has become popular for preparing biocompatiblenanoparticles. Duran et al.47 described the extracellularproduction of metal nanoparticles by several strains ofthe fungus Fusarium oxysporum. Vigneshwaran et al.48

have reported the extracellular synthesis of silver nanopar-ticles by a white rot fungus, Phaenerochaete chrysospo-rium. Biosynthetically produced silver nanoparticles usingfungus, yeast, bacteria and plant extracts were found tohave strong antibacterial efficacy against various MDRpathogens such as Mycobacterium tuberculosis, Pseu-domonas aeruginosa, S. pneumoniae, Methicillin-resistantS. aureus, Klebsiella pneumoniae, Methicillin-resistantStaph. Epidermidis (MRSE) and Strep. Pyogenes, Bacillussp., E. coli, S. typhi.49–61 The enhanced bioactivity of greennanoparticles with smaller particle size is attributed to thehigher surface area to volume ratio and surface reactivitywhen compared to the chemical nanoparticles.62 The coat-ing of phytochemicals on the surface of the NPs synthe-sized using plants has also resulted in elevated inhibitionof bacteria as compared to chemical nanoparticles.63

Agarwal et al.64 evaluated the biologically synthesizedsilver nanoparticles against the standard strain of M. tuber-culosis and 26 clinical isolates including drug sensitive(DS), multi-drug resistant (MDR), extensive-drug resis-tant (XDR) and Mycobacterium other than tuberculosis(MOTT) strains, exhibiting effective bactericidal activ-ity. Lara et al.65 have shown that silver nanoparticlesare effective bactericidal agents regardless of the drug-resistance mechanisms that exist in multidrug-resistantP. aeruginosa, ampicillin-resistant E. coli O157:H7 anderythromycin-resistant S. pyogenes. The bactericidal effectis attributed to the complexing of SNPs with electrondonor groups containing sulfur, oxygen or nitrogenatoms. Ninganagouda et al.66 developed a reliable,ecofriendly procedure for bioreduction of silver nanopar-ticles by Aspergillus flavus and evaluated its bactericidaleffects against MDR strains of Pseudomonas aeruginosa,Escherichia coli and Klebsiella pneumonia. Furthermore,researchers have investigated the composites of silvernanoparticles with other materials with or without antibac-terial activity to explore material synergistic to createapplication specific and better bactericidal agents. Leid etal.67 investigated four types of silver carbon complexes(SCCs) with different formulations including micelles andNPs and found them to be effective against pathogenicorganisms such as P. aeruginosa, methicillin-resistantS. aureus, multidrug-resistant Acinetobacter baumanniiand Klebsiella pneumoniae. L-tyrosine polyphosphate

(LTP) NPs have been formulated to encapsulate SCCsand provide a sustained delivery of the compounds.Kunkalekar et al.68 found the chemically synthesized sil-ver doped MnO2 nanoparticles have antibacterial activityagainst multidrug-resistant Staphylococcus aureus.

3.2. NO Releasing NanoparticlesBesides silver nanoparticles, nitric-oxide-releasing NPs(NO NPs) also possess broad spectrum antibacterial activ-ity which can inhibit the growth of many antibiotic-resistant and sensitive bacteria such as K. pneumoniae,E. faecalis, S. pyogenes, E. coli, and P. aeruginosa. NOis a lipophilic and hydrophilic natural gas, and is unstablein an oxygen environment.69 Reactions of NO with oxy-gen or superoxide spontaneously produce reactive nitro-gen and oxygen intermediates that are toxic to the celland act as antimicrobial species. The formation of theseintermediates becomes biologically significant once theconcentration of NO is greater than 1 �M. At these con-centrations, reactive nitrogen oxide species (RNOS) likeperoxynitrite (OONO−), S-nitrosothiols (RSNO), nitro-gen dioxide (NO•

2), dinitrogen trioxide and dinitrosyl-ironcomplexes are generated.70 NO-associated lipid damagehas been demonstrated with peroxynitrite and nitrogendioxide.71 Peroxynitrite mediate lipid peroxidation of lipo-somes which contribute to the antimicrobial activitiesof NO.72 NO interactions with proteins involve reactivethiols, heme groups, iron-sulfur clusters, phenolic or aro-matic amino acid residues, tyrosyl radicals, or amines. Per-oxynitrite and NO•

2 also nonspecifically oxidize proteinsat a variety of sites.73 Studies of NO-related cytotoxic-ity have demonstrated inactivation of enzymes containingFe-S clusters (e.g., aconitase, NADH dehydrogenase, suc-cinate dehydrogenase) suggesting that NO• (NO radical)might directly release iron from metalloenzymes and causeiron depletion.74

RNOS also causes DNA damage through autoxidationof NO where N-nitrosating intermediates deaminate cyto-sine, adenine and guanine.75 In this category of RNOS,peroxynitrite and NO•

2 in particular induce DNA strandbreaks, abasic sites and other DNA alterations.76 On otherhand DNA alkyl transferases have cysteine residues whose-SH group reacts with NO to form S-NO adducts. Theseadducts inhibit the transfer of the alkyl group from gua-nine to the protein. Thus NO inhibits DNA repair enzymesassociated with the repair of alkylation to DNA.77�78

Prokaryotes are more sensitive to NO treatment becausebacteria depend on iron sulfur clusters to a greater extentthan mammalian cells.79 Therefore it seems plausible thatan efficient NO releasing NPs could prove to be aneffective solution against MDR exhibited by microbialspecies.The antimicrobial and healing efficacy of sustained

release nitric oxide nanoparticles have been investigatedagainst methicillin-resistant SA (MRSA) and Acineto-bacter baumannii using a murine wound and soft





Table I. Nanoparticles exhibiting antimicrobial activity.

ParticleNanoparticles size (nm) Mode of action Target MDR organisms Refs.

Silver 5–100 • AgNPs could damage the structure ofbacteria cell membrane and resulted in theleakage of reducing sugars, leading to thedeath of bacteria.

Mycobacterium tuberculosis [64, 54]

Multidrug-resistant S. pneumoniae [49]

• It affects the integrity of the surface of thebacterial cell wall. The cytoplasmic materialis extruded from the cell leading to thecollapse of the cell.

Multidrug-resistant Pseudomonasaeruginosa

[50, 65, 57, 66]

Methicillin-resistant S. aureus [47, 53, 56, 58–60]

• Increase in membrane permeability bypenetrating cell wall.

Multidrug-resistant K. pneumoniae [56, 66, 60]

Methicillin-resistant Staph. Epidermidis(MRSE)

[60]

• AgNP forms complex with electron donorgroups containing sulfur, oxygen or nitrogenatoms that are normally present as thiols orphosphates on amino acids and nucleic acids.

Methicillin-resistant Strep. Pyogenes [60]

Multidrug-resistant E. coli [55, 58, 59, 65, 66]

• Prevent cell division and DNA replication Multidrug-resistant S. typhi [49, 58, 60]

• Nanosilver cause damage by interacting withphosphorus and sulfur-containing compoundssuch as DNA.

Erythromycin-resistant S. pyogenes [65]

Nitric oxidereleasingnanoparticles

20–100 (NOdonorparticle)

• RNOS irreversibly binds to heme proteins,resulting in heme removal from the protein.

Acinetobacter baumannii [80]

Methicillin-resistant S. aureus [81, 82]

• Lipid peroxidation of liposomes by theReactive nitrogen oxide species (RNOS)formed.

K.pneumoniae, E. coli E. faecalis,S. pyogenes P.aeruginosa.

[83]

Fe3O4@TiO2

core/shellmagneticnanoparticles

• Damage cell components through theproduction of ROS.

Multiantibiotic-resistant S. pyogenesand Methicillin-resistant S. aureus

[100]

Iron oxide 9 nm±4 nm • ROS (Reactive oxygen species) generateoxidative stress which cause damage toproteins and DNA.

Staphylococcus aureus [89]

Copper oxide 20–95 • The Cu2+ ions released by the CuOnanoparticles are able to disrupt cellmembrane and enzyme function.

Meticillin-resistant Staphylococcusaureus (MRSA)

[87]

E. coli [88]

Ferrocene-carboranederivativeswith nanoTiO2

• Damage cell components through theproduction of reactive oxygen species

MDR-resistant A. baumannii [90]

Titaniumoxide

20 • Cell components are damaged through theproduction of reactive oxygen species

E coli, B subtilis [126]

S. aureus, S. typhimurium, E. coliK. pneumoniae

[128]

MDR S. Aureus [129, 130]

Zinc oxide 12–60 • Nanoparticles get internalized in the cell andincrease the membrane permeability bydisorganizing and damaging the cellmembrane.

Methicillin resistant Streptococcusagalactiae and S. aureus

[92]

Methicillin-resistant S. aureus [93, 95]

• Generation of oxidative stress inside thebacterial cell

Methicillin-resistant S. epidermidis(MRSE)

[95]

Extended spectrum �

lactmases-producing E.coli andK pneumoniae.

[96]





Table I. Continued.

ParticleNanoparticles size (nm) Mode of action Target MDR organisms Refs.

Nanocomposites(a) TiO2/Agnanocomposite

20–50 nm TiO2/Ag nanocomposite shows antibacterialcapability because the electrical attractionbetween negative E. coli and thenanocomposite with its high positive chargepromotes the interaction between bacterialcells and silver nanoparticles

S. mutans and A.actinomycetemcomitans

[127]

(b) Chitosan/silvernanocomposite

(c) Chitosan–hyaluronicacid/nanosilvercomposite

43–55 nm Chitosan either interact with anionic groups onthe cell surface leading to increasedpermeability or chelate trace elements ornutrients essential for enzyme activity. Theantimicrobial activity of silver nanoparticleshas been related to the action of the ionsreleased from them.

Methicillin-resistant S. aureus [102, 104, 106]

(d) ZnTe/dendrimernanocomposites

3 nm Amine terminated ZnTe/dendrimernanocomposites were positively charged andthey had preferential recognition for thenegatively charged outer surfaces of bacterialmembranes and this give high penetratingpower which make them effective to bind tothe substrates on the outer membrane andcell membranes of organisms.

Multi-drug resistant V. cholerae andE. coli

[105]

(e) Coppernanoparticle–cottoncomposites

5 nm The mechanism of biocidal action of coppernanoparticles is based on the fact that Cunanoparicles release Cu(II) ions on contactwith moisture. These copper ions bind withthe –SH and –COOH groups of proteinmolecules of bacterial cell wall.

A.baumannii [103]

tissue models.80–82 It was observed that NO-NP treat-ment resulted in enhanced healing by induction of col-lagen deposition. Furthermore, they showed that NO-NPcan alter the immune responses to aid in the reduc-tion of bacterial load. Recently Friedman et al.83 haveused a number of clinically important Gram-positive(S. pyogenes and E. faecalis) and negative (E. coli,K. pneumoniae and P. aeruginosa) isolates to checkthe antimicrobial efficiency of NO-NP in-vitro. Forboth Gram-negative and Gram positive bacteria, reduc-tion in bacterial growth was NO-NP dose dependent.NO-NP significantly reduced all Gram-negative bacte-rial growth within 24 h, while in case of Gram posi-tive bacteria lower NO-NP concentrations were able toachieve 90–100% growth inhibition within 8–16 h ofexposure.

3.3. Metal Oxide NanoparticlesMetal oxides nanoparticles (NPs) such as TiO2, CuO andZnO are known to exhibit good antibacterial properties.84

One of the common properties they share is theirphotocatalytic activity due to wide band gap. Thephotocatalytic activity is attributed to generation of reac-tive oxygen species (ROS).85�86 Copper (CuO) nanopar-ticles have shown activity against a range of bacterialpathogens, including methicillin-resistant Staphylococcus

aureus (MRSA) and Escherichia coli,87�88 Furthermore,the ability of CuO nanoparticles to reduce bacterial popu-lations to zero was found to be enhanced in the presence ofsilver nanoparticles. The bactericidal effect of iron oxidenanoparticles on Staphylococcus aureus was evaluated byTran et al.89 Li et al.90 has explored the antibacterialeffect of a pair of geometrical isomers ferrocene-carboranederivatives (designated as FcSB1 and FcSB2) combiningwith nano-scaled titania (nano TiO2) against five MDRA. baumannii strains. The studies indicated that the syner-gistic antibacterial activity of FcSB1/or FcSB2 with nanoTiO2 have a deadly effect on the target bacteria. Neculaet al.91 studied the synthesis of a porous TiO2-Ag com-posite coating and assessed its in vitro bactericidal activityagainst methicillin-resistant Staphylococcus aureus. Thecomposite TiO2-Ag coating showed complete killing ofmethicillin-resistant S. aureus within 24 h in all cultureconditions.ZnO NPs have shown acute toxicity to antibiotic

(methicillin)-resistant bacteria such as S. aureus andStreptococcus agalactiae.92 It has been observed thatnanoparticles get internalized in the cell resulting cell walldisorganization and damage the cell membrane. The cellu-lar internalization of ZnO NPs are also known to increasethe oxidative stress inside the bacterial cell and cause dam-age to all components of the cell including proteins, lipid





and DNA.92 The generation of ROS from ZnO NPs isattributed to the direct wide band gap of this well knownsemiconductor. The toxicity of ZnO NPs is concentration-dependent and they are mildly toxic at low concentration.The antibacterial properties of zinc oxide nanoparticlesagainst both gram-positive and gram-negative microor-ganisms have been investigated by Raghupathi et al.93

They found that the ZnO colloidal suspension inhibited95% of MRSA, Enterococcus faecalis, a high-biofilm-producing strain Staphylococcus epidermidis, and variousother clinically relevant pathogen growths. Joshi et al.94

have shown that the unintentional surface adsorbed specieson ZnO nanoparticles plays a vital role in deciding theantibacterial activity against E-coli with a conclusion thatmodifying surface with electron donar groups may leadto the development of new class of nanobactericidal.Ansari et al.95 shown that the ZnO–NPs inhibited bacterialgrowth of methicillin-sensitive S. aureus (MSSA), MRSAand methicillin-resistant S. epidermidis (MRSE) strainsindependent of drug-resistant mechanisms of MRSA andMRSE. These nanoparticles were also found to be effectiveagainst extended-spectrum �-lactamases-producing E coliand K pneumoniae.96

The band gap engineering (BGE) of these metal oxidenanoparticles leads to manipulate the photocatalytic activ-ity that in turn affect the antibacterial property. BGEcould be done by surface modification, size variationand doping by appropriate cation or anion.97�98 The sur-face modification either by organic or inorganic materi-als creates multifunctionality in the nanoparticles.99 Thesemultifunctional NPs can be used for targeting specificbacteria or infected tissue to overcome the side effectsinvolved and for enhancing the antibacterial activity.For instance, a magnetic nanoprobe comprising of ironoxide/titania Fe3O4@TiO2 core/shell magnetic nanoparti-cles have been developed by Chen et al.100 which canbe used as photokilling agents for MDR bacteria suchas multiantibiotic-resistant S. pyogenes M9022434 andM9141204 and Methicillin-resistant S. aureus, (MRSA).They demonstrated that the IgG-Fe3O4@TiO2 magneticnanoparticles can target several pathogenic bacteria andeffectively inhibit the cell growth of the bacteria under UVirradiation. The eradication of MDR bacteria by a novelZn-doped CuO nanocomposite was reported by Malkaet al.101 The antibacterial activity of the Zn-doped CuOdeposited on the fabric was tested against E. coli andS. aureus and it was found to be 10,000 times betteras compared to pure CuO and ZnO nanoparticles. Simi-lar results were observed against multidrug-resistant bac-teria (MDR) example Methicillin-resistant S. aureus andMDR E. coli which further emphasized the efficacy ofthis composite. Chitosan and cellulose based nanocompos-ites have also been found to be effective against MDRpathogens.102–106 Table I summarizes the various nanopar-ticles with antimicrobial activities.

4. NANO-PHOTOTHERMAL THERAPY OFMDR BACTERIA

Photothermal therapy using inorganic nanoparticles is anexcellent technique which has been utilized for killingof biological cells. Nanoparticles convert the absorbedelectromagnetic radiation into heat and transfer heat tothe cells or bacteria in their immediate vicinity by ther-mal conduction or processes related to strong heatingsuch as the generation of vapor bubbles.107 Gold NPshave been extensively studied for photothermal therapyof cancer.108�109 Huang et al.110 have demonstrated thatfunctional gold nanoparticles can be used as photothermalagents for selective-killing of pathogenic bacteria. Goldnanoparticles with polygonal shapes, capable of absorb-ing near infrared (NIR) light was used in conjugationwith vancomycin. The results indicated that large portion(>99%) of bacteria targeted by the gold nanoparticleswere destroyed under illumination. The rapid photother-mal lysis of the pathogenic bacteria E coli using goldnanorods has been reported by Kim et al.111 The goldnanorods covalently linked to primary antibodies havebeen used to selectively destroy the pathogenic gramnegative bacterium, P. aeruginosa.112 It was found thatfollowing nanorod attachment to bacterial cell surface,exposure to near-infrared radiation results in a signifi-cant reduction in bacterial cell viability. A multifunctionalpopcorn-shaped iron magnetic core–shell gold nanoparti-cle has also been reportedly used for photothermal destruc-tion of multidrug-resistant bacteria. Their experimentalresults shows that when the M3038 antibody-conjugatedhybrid nanoconjugate are attached to MDR SalmonellaDT104 bacterial cells, the localized heating that occurson 670 nm light irradiation is able to cause irreparablecellular-damage and kill MDR Salmonella bacteria within10 min of exposure.113

Perni et al.114 reported the formation of polysiloxanepolymers containing embedded methylene blue and goldnanoparticles. These polymers show significant antimi-crobial activity against methicillin-resistant Staphylococ-cus aureus and Escherichia coli with up to a 3.5 log(10)reduction in the viable count when exposed for 5 min tolight from a low power 660 nm laser. The bacterial deathoccurred due to the light-induced production of singletoxygen and other reactive oxygen species by the methy-lene blue. Luo et al.115 have investigated a X-ray irra-diation based strategy using bismuth nanoparticles thatcan be used to kill multidrug resistant (MDR) P. aerugi-nosa. Polyclonal antibody modified bismuth nanoparticleswere introduced into bacterial culture to specifically targetP. aeruginosa. The bacteria are irradiated with X-rays ina deep wound model. Results showed that the killing ofMDR P. aeruginosa was 15 times better in the presence ofbismuth nanoparticles compared to X-rays alone. In addi-tion, this technique had no detrimental effect on humancell lines which make it a valuable contender for futureapplications.





5. ANTIBIOTICS CONJUGATEDNANOPARTICLES

The combination of nanoparticles with existing antibioticsseems to be very enthralling option by combining thetwo treatment modalities. Availability of different types ofnanoparticles has opened up the avenue for probing theimpact of this association on various pathogens. Variousresearch groups have adopted this approach using differentnanoparticles and drugs.Vancomycin has been used for the treatment of MRSA

infections since 1980, however, reports suggests thatS. aureus has developed the resistant against vancomycinand this new strain is named as vancomycin-resistantS. aureus (VRSA).116 Several therapies for low-levelvancomycin-resistant (vancomycin-intermediate S. aureus)and VRSA have been recommended but still they are notperfect. The recent studies are focused on the combinationof vancomycin and other antibiotics with nanoparticles toenhance the antibacterial efficacy. In 2003, Gu et al.117

have conjugated vancomycin to gold nanoparticles andtested it against VRE (vancomycin resistant enterococci).Increased activity of vancomycin was observed in the formof Au@Van nanoparticles against VRE. Fayaz et al.118

coated gold nanoparticles with vancomycin through ionicinteractions between positively charged amine groupsof vancomycin and negatively charged surface of goldnanoparticles to form vancomycin bound gold nanoparti-cles (VBGNPs) and tested it against E. coli, S. aureus andvancomycin resistant S. aureus. The antibacterial mech-anism of VBGNP is attributed to non-specific bindingof VBGNPs to transpeptidases, instead of terminal pepti-dases of the glycopeptidyl precursors on the cell surfaceof test strain. These nanoparticles demonstrated activityagainst VRSA, E. coli and S. aureus. The VBGNP exhib-ited notable antibacterial activity against E. coli which isnormally resistant to vancomycin owing to its inabilityto penetrate the outer membrane of Gram-negative bacte-ria. This implies that gold nanoparticles assist in bindingof vancomycin to the bacterial cell surface independentof its structure in both Gram-positive and Gram-negativebacteria.Chamundeeswari et al.119 have described antimicrobial

activity of C-AuNp-Amp (chitosan-capped gold nanopar-ticles coupled with ampicillin). The results revealed thatthere was a two fold increase in antimicrobial effi-cacy of C-AuNp-Amp when compared with that of freeampicillin. Amino-substituted pyrimidines which them-selves do not possess any antibiotic action, but whenattached to gold nanoparticles (NPs), show antibac-terial activities against multidrug-resistant clinical iso-lates of E. coli and P. aeruginosa.120 Among thevarious amino-substituted pyrimidine-capped gold NPsused Au_DAPT(4,6-Diamino-2-pyrimidinethiol) showedthe best antibacterial activities and efficient inhibitionof proliferation of MDR bacteria. It has been arguedthat pyrimidine-capped gold NPs exert their antibiotic

actions via sequestration of magnesium or calcium ionsto disrupt the bacterial cell membrane, resulting in leak-age of cytoplasmic contents including nucleic acids fromcompromised cell membranes. The other possibility isinteraction with DNA and inhibition of protein synthesisby internalized NPs. The non-toxic property of Au_DAPTto primary human cells make it valuable for clinicalapplication.120

Duran et al.121 have carried out a study withclindamycin and silver nanoparticles produced chemi-cally and/or biosynthetically. The silver nanoparticleswere chemically associated with clindamycin and puri-fied before being used as an antibacterial compound.These nanoparticles showed significant bactericidal activ-ity towards methicillin-resistant S. aureus strains (MRSA)and Staphylococcus epidemidis. The biosynthetically pro-duced nanoparticles were found to be more stable than thechemical ones. However, the activity exhibited was depen-dent upon the bacterial strain analyzed and also on thesize of the nanoparticles. Shahverdi et al.122 have evaluatedthe antibacterial activity of silver nanoparticles and antibi-otics nanoconjugates against S. aureus and E coli. Thesilver nanoparticles conjugated with penicillin G, amox-icillin, erythromycin, clindamycin, and vancomycin andwere evaluated for antimicrobial activities against S aureusand E coli. The antibacterial activities of these antibioticswere increased in the presence of Ag-NPs against both teststrains. However, their results were in contrast with Fayazet al.123 and Birla et al.124 in case of S aureus.The combined antibacterial effects of silver nanopar-

ticles with four different antibiotics (ampicillin, ery-thromycin, kanamycin and chloramphenicol) against twoGram-positive (S. aureus and Micrococcus luteus) and twoGram-negative (E. coli and Salmonella typhi) microorgan-isms was investigated by Fayaz et al.123 The antibacterialactivity was found to be higher against Gram-negative bac-teria as compared to Gram-positive bacteria. The highestenhancing effect was observed for ampicillin against teststrains and this has been attributed to the differences incell wall structures of Gram-positive and Gram-negativebacteria. The crosslinking and rigidity of paptidoglycan ofgram positive cell wall make it difficult for silver nanopar-ticles to penetrate. However, in case of Gram negativebacteria there is a thin layer of peptidoglycan that can beeasily attacked by Ag NP-Ampicillin conjugate leading tothe internalization of these particles and in turn the celllysis.In another study Birla et al.124 studied the com-

bined effect of silver nanoparticles with five antibiotics(ampicillin, gentamicin, kanamycin, streptomycin and van-comycin) on the three most common human pathogens–S. aureus, E. coli and Pseudomonas aeruginosa. Theantibacterial activities of ampicillin, gentamycin, strep-tomycin and vancomycin were found to be increasedin combination with Ag-NPs against the Gram-negative





micro-organisms, i.e., E� coli and Pseudomonas aerugi-nosa as compared with S� aureus which is a gram positivebacterium. Brown et al.125 showed that silver nanoparticles(AgNP) were intrinsically antibacterial, whereas goldnanoparticles (AuNP) were antimicrobial only when ampi-cillin was bound to their surfaces. AuNP functionalizedwith ampicillin (AuNP-AMP) were bactericidal. UnlikeAuNP, silver nanoparticles (AgNP) without ampicillinexhibited antimicrobial activity against both Gram-positiveand Gram-negative bacteria. Silver nanoparticles function-alized with ampicillin (AgNP-AMP) were effective againstall E. coli strains and the ampicillin-resistant P. aeruginosaisolate.Instead of metal nanoparticles, metal oxide nanopat-

icles has also been enormously used as antimicrobialagents. One of the commonly used non-silver nanoparti-cles is titanium dioxide (TiO2).

126–129 Antibacterial actionof TiO2 nanoparticles is photodependent and it generatesfree radicals during photocatalytic reactions. These freeradicals degrade lipopolysaccharide (LPS), peptidoglycanand phospholipids bilayer owing to peroxidation in thebacterial cell. The efficacy of 22 different antibiotics withTiO2 nanoparticles has been studied against MRSA by Royet al.130 They found that the antibacterial activities of allantibiotics have been increased in the presence of nanosizetitanium dioxide against test strains and optimum resultswere observed with penicillin and amikacin.Banoee et al.131 observed the effect of ZnO nanopar-

ticles on the antibacterial activity of different antibioticsagainst Staphylococcus aureus and Escherichia coli. Theantibacterial activity of ciprofloxacin increased in the pres-ence of ZnO nanoparticles in both test strains. The authorsput forward the theory that ZnO nanoparticles may inter-fere with the pumping activity of NorA protein whichis involved in the active efflux of fluoroquinolones fromthe bacterial cell to make them resistant to the antibac-terial activity of ciprofloxacin. They induce faster elec-tron transfer kinetics in its active site which hinderswith the activity of NorA protein and helps restoringciprofloxacin action. Another mechanism includes inter-fering with the functioning of membrane Omf protein,which is associated with infiltration of the cell membraneby quinolones. In this way, ZnO nanoparticles increaseciprofloxacin absorption into the cell. Thati et al.132 andLuo et al.133 have investigated the synergistic role of zincoxide (ZnO) nanoparticles with more than 25 differentantibiotics against S� aureus and E. coli. The findingsdemonstrated that ZnO nanoparticles can enhance antibac-terial activities of penicillins, cephalosporins, aminoglyco-sides, glycopeptides, macrolides, lincosamides, gentamicin,clarithromycin, ofloxacin and ceftriaxone and tetracycline.The studies summarized in this review clearly displays

that nanoparticles/and or their coupled derivatives arepotential candidate for enhancing or restoring the alreadyexisting antibiotics or new substances to combat the MDRproblem.

6. CONCLUSIONSThe wide spread antibiotic resistance has put immensepressure on pharmaceutical industries to search newantimicrobial agents or modification of the existing drugs.In an area such as MDR, nanotechnology has poten-tial to change the scenario and prevent the spread ofdrug resistance. Metallic nanoparticles of both biologi-cal and chemical origin are shown to be potential agentsin antibacterial treatment. Nanoparticles not only demon-strated activity against MDR bacteria themselves, but alsoshowed potential for the development of synergistic com-binations that increases the antibacterial effect of existingantibiotics. They helped to revive the antibacterial activityof old generation antibiotics against which microbes havedeveloped resistance. The review showcased the effect ofvarious forms of radiations in combination with nanopat-icles enhancing the antibacterial efficacy. Further workis still required in order to elucidate the entire mecha-nism of action of nanoparticles as bactericidal, toxicity ofnanoparticles in human and better delivery of drug insidehuman system using nanodrug carriers. Nanoparticles cou-pled with either antibiotics and/or irradiation may providea potential strategic remedy to combat MDR.

Acknowledgments: Rajni Singh gratefully acknowl-edge the financial support from Ministry of Environ-ment and Forest (19-118/2008-RE) and Surinder P. Singhacknowledges the support from CSIR network grant Nano-SHE (BSC0112).

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Received: 23 September 2013. Accepted: 14 January 2014.


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