Materials Science and Engineering C 68 (2016) 939–947

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Review

Future prospects of antibacterial metal nanoparticles as enzyme inhibitor

Khan Behlol Ayaz Ahmed, Thiagarajan Raman ⁎, Anbazhagan Veerappan ⁎
School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Nanoparticles are being widely used as antibacterial agents with metal nanoparticles emerging as the most effi-
Received 29 March 2016 cient antibacterial agents. There have been many studies which have reported the mechanism of antibacterial ac-
Received in revised form 23 May 2016 tivity of nanoparticles on bacteria. In this review we aim to emphasize on all the possible mechanisms which are
Accepted 9 June 2016
involved in the antibacterial activity of nanoparticles and also to understand their mode of action and role as bac-
Available online 11 June 2016
terial enzyme inhibitor by comparing their antibacterial mechanism to that of antibiotics with enzyme inhibition
Keywords:
as a major mechanism. With the emergence of widespread antibiotic resistance, nanoparticles offer a better alter-
Nanoparticles native to our conventional arsenal of antibiotics. Once the biological safety of these nanoparticles is addressed,
Metal nanoparticles these nanoparticles can be of great medical importance in our fight against bacterial infections.
Antibacterial activity © 2016 Elsevier B.V. All rights reserved.
Enzyme inhibitors

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939
1.1. Enzyme inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939
1.1.1. Reversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940
1.1.2. Irreversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940
2. Synthetic drugs as enzyme inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940
3. Conventional antibiotics enzyme inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940
4. Bacteria resistance towards antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
5. Nanoparticles as new class of antibacterial agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
5.1. Antibacterial activity of nanoparticles (Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
5.2. Nanoparticles as efflux pump inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
5.3. Inhibition of enzyme by nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
5.4. Mechanism of nanoparticle toxicity on bacterial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
List of abbreviations used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 946

1. Introduction
Enzymes are biological agents which help in assisting a reaction
wherein a reaction starts with a particular substrate and then converted
to the desired products. Till date, enzymes are being known to assist in
nearly 5000 biochemical reactions [77]. Some enzymes contain metal
ions in their structure; these enzymes are called as metalloenzymes.
There are some chemical species which bind to the enzymes and
⁎ Corresponding authors.
E-mail addresses: raman@biotech.sastra.edu (T. Raman), anbazhagan@scbt.sastra.edu
(A. Veerappan).
make them active, these are called coenzymes. The complex of enzyme
and the coenzymes is called as a holoenzyme. The inactive or non-func-
tional enzymes are called as apoenzyme. Coenzymes bind to the apoen-
zymes by the means of electrostatic bonds or van der Waals forces and
produce several enzymatic actions [10].
1.1. Enzyme inhibition
In general, the blocking or stopping of the enzyme action is called as
enzyme inhibition. Indeed, both natural and synthetic inhibitors are
available that can be effectively used in therapeutics and as medicine
for controlling harmful diseases mediated by specific enzymes. Enzyme

http://dx.doi.org/10.1016/j.msec.2016.06.034
0928-4931/© 2016 Elsevier B.V. All rights reserved.
940 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947
inhibition is mainly of two types: reversible and irreversible. In both re-
versible and irreversible inhibition, the inhibitor need not cover the
complete binding (active) site of the enzyme. Binding of the inhibitor
to any part of the enzyme or the active site results in bringing a confor-
mational change in the enzyme resulting in distortion of the active site
of the enzyme due to which the substrate does not fit in the active site
and the enzyme action is inhibited.
1.1.1. Reversible inhibition
Reversible inhibition is facilitated by enzyme inhibitors which bind
to the enzyme for a specific period of time and then releases itself
from the active site of the enzyme. The binding causes a specific change
in the conformation of the enzyme. Reversible enzyme inhibition is
mainly of two types, namely competitive and non-competitive inhibi-
tion. Competitive inhibition involves the reversible binding of the inhib-
itor to the active site of the enzyme. In this case, the inhibitor has the
same shape as that of the substrate and thus it competes with the sub-
strate to bind at the active site of the enzyme. As a result, enzymes bind
to the inhibitors and minimize the overall enzyme action of the meta-
bolic process. In the case of non-competitive inhibition, the inhibitor
does not resemble the substrate hence it does not compete with the
substrate for binding to the active site of the enzyme. In this method,
the inhibitor reversibly binds to any part of the enzyme. As a result, en-
zyme undergoes a conformational change and this results in the change
in the active site of the enzyme and thus, blocks the substrate binding
and inhibits the enzyme action [40].
1.1.2. Irreversible inhibition
Irreversible inhibition occurs through the binding of the enzymes ei-
ther by strong covalent or non-covalent bond. In the case of a non-cova-
lent bond, the inhibitors detach from the active site of the enzyme for a
short time after the attachment and make the enzyme-free to carry out
the normal reaction [28]. Irreversible inhibition is mainly caused by the
help of active site-directed inhibitors. These inhibitors bind with the
functional groups present at the active site or near the active site of
the enzyme. As a result, the enzyme becomes unavailable for the reac-
tion, which results in a reduction in the formation of essential products
[28].
Enzyme inhibition has been regarded as an important process for
regulating metabolic activities. In this review, we provide an overview
of different commercially available drugs which act as an enzyme inhib-
itor and also provide information about how the antibacterial activity of
the metal nanoparticles resembles the natural and conventional
antibiotics.
2. Synthetic drugs as enzyme inhibitors
A drug called difluoro methyl ornithine, which is used to treat Afri-
can trypanosomiasis (sleeping sickness) works on the principle of irre-
versible inhibition of enzyme action. A compound called as
diisopropylfluorophosphate (DFFP) is a potent inhibitor of acetylcholin-
esterase (ACE). It follows irreversible inhibition mechanism and binds
at the active site of ACE, thereby blocking the enzyme activity to break
down acetylcholine [78]. This results in accumulation of the excess
amount of acetylcholine in the body and improper function of respirato-
ry muscles leading to death due to suffocation. Similar effect is also ob-
served by the compound malaoxon which is a toxic derivative from
malathion which binds reversibly and then irreversibly to the active
site serine and inactivates ACE leading to death due to suffocation [78]
DFFP and malathion are the prime components of the current organo-
phosphorus nerve gases like sarin and other organophosphorus toxins.
Gout is a disease caused due to over production of uric acid in the
body [23]. The enzyme xanthine oxidase helps in the production of
uric acid crystals in the body by its oxidase property [11]. Allopurinol
is an anti-gout drug which acts on the enzyme xanthine oxidase by
the mechanism of suicidal irreversible inhibition [78]. The enzyme
xanthine oxidase first converts allopurinol into its active form called
oxypurinol, which thereby binds to the molybdenum sulfide complex
present at the active site of the enzyme xanthine oxidase rendering
the enzyme inactive. This results in less production of uric acid in the
body and thus a cure for gout disease.
Methotrexate is a commonly used antimetabolite which is usually
used as an anticancer drug and also for many autoimmune diseases.
Methotrexate acts as a competitive inhibitor of the enzyme
dihydrofolate reductase (DHFR) [78]. DHFR converts dihydrofolate to
tetrahydrofolate. Being similar to dihydrofolate, methotrexate blocks
the active site of DHFR thereby reducing the availability of tetrahydrofo-
late which acts as a carrier molecule in for carbon moieties important for
anabolic pathways. Blocking the anabolic pathways results in blockage
of synthesis of purine nucleotides for DNA replication [71].
Sulfanilamides are another class of drugs which belong to the sulfa
drugs group. They are mainly used as antibacterial and anticancer
drugs. The principle behind their activity lies in competitive inhibition.
They block the folic acid synthesizing enzyme [78]. Folic acid is vitamin
B9 which is required for important functions in the body. Humans and
bacteria cannot synthesize folic acid in the body but it is an essential vi-
tamin for synthesizing important compounds like methionine, DNA,
RNA in the body. Hence, folic acid is supplied externally in the form of
folate which is converted to folic acid by the folic acid synthesizing en-
zyme. Hence, sulfanilamides act as a competitive inhibitor for the sub-
strate p-aminobenzoic acid and prevent it from binding to folic acid
synthesizing enzyme thereby blocking the synthesis of folic acid. Un-
availability of folic acid kills the bacteria as important constituents re-
quired for the growth cannot be synthesized [50].
Aciclovir, a commonly used antiviral drug, is a guanosine analog
called as acycloguanosine and is used mainly for treatment of herpes
simplex virus [13]. Acyclovir is converted to acycle-guanosine
monophosphate by the enzyme viral thymidine kinase. This
acycloguanosine monophosphate can be easily phosphorylated to
acycloguanosine triphosphate (acyclo–GTP) by cellular kinases. The
formed acylo GTP is a potent inhibitor of viral DNA polymerase leading
to termination of DNA replication and virus inactivation [3].
Apart from the examples of the drugs mentioned above, there exist
many other synthetic drugs which are being used as an enzyme inhibi-
tor. A list of the drugs and that acts as enzyme inhibitors are provided in
Table 1.
3. Conventional antibiotics enzyme inhibitors
Many newly developed antibiotics for various diseases cure by act-
ing on the essential enzymes which are required by the bacteria or the
virus to develop infection. Some of the common antibiotics which use
enzyme inhibition mechanism as their tool to kill the infectious bacteria
and virus are being discussed below.
The most common and widely used antibiotic Penicillin discovered
in 1928 in England is a very promising antibiotic against the bacterial in-
fection arising from Streptococci and Staphylococci. Penicillin is known to
act as a suicidal inhibitor following the irreversible inhibition mecha-
nism towards the enzyme glycopeptide transpeptidase belonging to
the family of serine protease. Enzyme glycopeptide transpeptidase is re-
quired for the synthesis of the cell wall of bacteria which is essential for
the growth and survival of the bacteria [78]. It cleaves the peptide link-
age between the alanine residues in the polypeptide. Penicillin structure
possesses a β-lactam ring that is similar to the transition state of the
product formed in the cleavage reaction [53]. This helps penicillin to ac-
tively bind at the active site of the enzyme thus rendering it inactive due
to which the bacterial cell wall is compromised and resulting in cell wall
rupture and bacterial death. Other antibiotics like ofloxacin, ciprofloxa-
cin, kanamycin are also expected to follow the same mechanism for kill-
ing the bacteria.
Acetylsalicylic acid commonly known as aspirin is a well-recognized
anti-inflammatory and analgesic drug [47]. The mechanism of action of
 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947 941

Table 1 cassette ([87] resistance nodulation cell division (RND) [73], major facil-
Common enzyme inhibiting drugs. itator superfamily (MFS) [59], small multidrug resistance family [60],
Sr. no. Drug name Enzyme inhibited References multidrug and toxic compound extrusion family (MATE) [9]). These
1 Metyrosine Tyrosine hydroxylase [2]
pumps have the ability to uptake or efflux drugs, sugars, ions and pro-
2 MAO inhibitors Monoamine oxidase [52] teins [45]. For example, NorA belongs to MFS provide resistance to-
3 Physostigmine Choline esterase [29] wards tetracycline drugs [17].
4 Methyldopa Aromatic L-amino acid decarboxylase [19] It has been found that infectious bacteria becomes smarter by
5 Captopril Angiotensin converting enzyme [80]
attaching themselves to damaged tissues or implanted medical devices
6 Lovastatin HMG–CoA reductase [31]
8 Trimethoprim Dihydrofolate reductase [20] and protect themselves by forming a layer of polysaccharide, generally
9 Cephalosporins Membrane transpeptidase [76] termed as a biofilm. These biofilms prevent the antibiotics entry, more-
10 Rifampin DNA dependent RNA polymerase [44] over, the different chemical microenvironment of the biofilm precludes
11 Acyclovir DNA polymerase [85] the action of antibiotics. For example, aminoglycoside has reduced tox-
12 Zidovudine Reverse transcriptase [22]
13 Methotrexate Dihydrofolate reductase [38]
icity towards the same bacteria when there is a change in the condition
14 Fluorouracil Thymidylate synthase [1] from anaerobic to aerobic condition [81]. Other than efflux pumps and
biofilm, bacteria also develop resistance through mutation. As a result,
these bacteria produce drug-degrading enzymes or mutate antibiotic-
specific binding sites to resist the effects of antibiotics [51]. For example,
aspirin is by covalently attaching to the enzyme prostaglandin endoper- the enzyme New Delhi metallo-β-lactamase-1 (NMD-1) was identified
oxide synthase. Aspirin binds to serine present in the active site of the in multidrug resistance tuberculosis and it seems to be responsible for
enzyme, thereby inhibiting the enzyme action. A part of the enzyme re- bacterial resistance to a broad range of β-lactam antibiotics [56]. Most
sembles the prostaglandin structure which in turn helps aspirin in ac- bacterial isolates with NMD-1 enzyme showed resistance to all standard
tive site binding and blocking the enzyme. Ibuprofen is an example of intravenous antibiotics [61]. The treatment of drug-resistant bacteria re-
drug resembling the aspirin in its function as an enzyme inhibitor [35, quires high-dose administration of multiple expensive drugs, which
72]. often develops undesirable side effects. Therefore, there is a growing de-
Kanamycin chemically an aminoglycoside is a common antibiotic mand for an alternative strategy to treat bacterial infections with no or
which has been isolated from Streptomyces kanamyceticus. This antibiot- minimal side effects along with the lack of resistance development on
ic is used for treatment against gram-negative bacteria, by inhibiting the the part of the pathogen.
enzymes involved in translocation during protein synthesis [54].
Ciprofloxacin is another commonly used and broad spectrum antibi-
otic against bacterial infections (both gram-positive and -negative or- 5. Nanoparticles as new class of antibacterial agents
ganisms) like respiratory tract infection, urinary tract infection, skin
infections and diarrhea. It is known to inhibit enzymes like DNA gyrase, Particles ranging from 10 to 100 nm in size are termed as nanoparti-
topoisomerase 2 and 4, and the enzymes which are responsible for bac- cles (NPs). Nanoparticles are being widely used in biological applica-
terial DNA replication [62]. tions including diagnosis, cancer therapeutics, biosensing and
phototherapy [43]. Nevertheless, one of the major applications pro-
4. Bacteria resistance towards antibiotics posed for the metal nanoparticles is the antibacterial activity. For exam-
ple, the antibacterial properties of silver nanoparticle are well studied
Besides the benefits, the repeated use of the antibiotics over the and it has found application in wound dressings [93]. NPs either
years generated drug resistance in the bacteria. These bacteria develop decrease the bacterial growth or kill the bacteria through disrupting
antibiotic resistance majorly through efflux pumps and biofilm. Bacteri- the cell wall or by the unavailability of the food source [93]. The antibi-
al efflux pumps are classified into five families namely, ATP-binding otics in clinical use today are known to exert their bactericidal or

Table 2
Antibacterial nanoparticles and their mechanism of action.

Sr. no. NPs Bacteria Mechanism of action Ref

1 Ag-SPION S. aureus Penetration through biofilm [37]

2 ZnO NPs Halophilic bacterium spp. Increased membrane permeability [7]
3 ZnO NPs B. subtilis Decreased growth rate and viable count [41]
4 CuO NPs B. subtilis Cell wall damage, disruption of biochemical process [75]
5 Al2O3 NPs B. subtilis Cell wall damage and increased cell permeability [69]
6 TiO2 NPs M. segmatis Release of Cu, decreased enzymatic activity, NADPH production [91]
7 AgNPs K. pneumoniae Penetration of NPs [42]
8 NO NPs K. pneumoniae Bacterial membrane damage [34]
9 TiO2 NPs P. aeruginosa Peroxidation of membrane lipids. Loss of respiratory activity [82]
10 AgNPs P. aeruginosa Disrupts membrane permeability, cellular respiration and cell division [83]
11 ZnO NPs P. aeruginosa ROS generation, membrane damage [25]
12 AgNPs E. coli Cell wall lysis, prevention of DNA unwinding [26]
13 NiO NPs E. coli Growth inhibition, cellular damage [27]
14 CuNPs E. coli Hydroxyl radicals generation [70]
15 ZnO NPs S. typhimurium ROS generation, frameshift mutation [5]
16 AgNPs A. baumannii Cell wall alteration [49]
17 AgNPs P. aeruginosa Membrane permeability, cell wall damage [24]
18 AgNPs S. aureus Inhibition of bacterial DNA replication [24]
19 Bismuth NPs S. mutans Cell growth inhibition [33]
20 AgNPs E. coli, BCG Cell wall disruption [95]
21 AuNPs E. coli, BCG Cell wall disruption [95]
22 AuNPs E. coli, S. aureus, P. aeruginosa, S. typhi Cell wall damage [6]
23 ZnO NPs C. jejuni Loss of membrane integrity [94]
24 Ch-AgNPs E. coli, S. aureus Inhibiting cell growth and membrane damage [92]
942 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947
bacteriostatic action by causing damage to the bacterial cell wall or
blocking food source. The antibiotic uses the principle of enzyme inhibi-
tion by acting as enzyme inhibitors to stop a vital metabolic process in
bacteria and thus leads to bacterial death. In this part of the review,
we will discuss the different types of nanoparticles reported as antibac-
terial agents, their mechanism of action and the future prospects of the
potential and use of nanoparticles as bacterial enzyme inhibitors.
5.1. Antibacterial activity of nanoparticles (Table 2)
Nanoparticles are efficiently being used as antibacterial agents. A lot
of studies have been reported which displays the antibacterial nature of
nanoparticles. Chakrapani et al. have reported the synthesis of casein
stabilized copper nanoparticles (CuNPs) which displayed efficient anti-
bacterial activity against pathogenic bacteria like Klebsiella pneumoniae
MTCC 109, Pseudomonas aeruginosa MTCC 1688, Salmonella
typhimurium MTCC 98, Shigella exneri MTCC 1457, Bacillus thuringinesis
MTCC 869, and Staphylococcus aureus MTCC 3160. The antibacterial ac-
tivity of CuNPs was determined using zone of inhibition assay using
ofloxacin and kanamycin antibiotics as standards. CuNPs displayed
good zone of inhibition which was nearly equal to the zone of inhibition
obtained from the standard antibiotics, which implies that CuNPs have
an effect similar to the effect displayed by the standard antibiotics
(Fig. 1). As stated above the mechanism by which kanamycin displays
its antibacterial activity is by inhibiting the enzymes involved in protein
synthesis, on similar lines it can be suggested that CuNPs also display
antibacterial activity by enzyme inhibition [14].
Veerappan et al. have reported the synthesis of silver nanoparti-
cles using N-stearoyl ethanolamine (NSEA) as a capping agent. The
antibacterial activity of N-stearoyl ethanolamine silver nanoparticles
(NSEA-AgNPs) was tested against pathogenic bacteria like
Escherichia coli (MTTC 1655), Salmonella typhi (MTTC 98), Shigella
(MTTC1457), Klebsiella pneumoniae (MTTC 109), Staphylococcus au-
reus (MTTC3160), and Bacillus thuringinesis (MTTC 869). The MIC
value of NSEA-AgNPs estimated using resazurin assay was found to
be 6–12 μg/ml. The NSEA-AgNPs displayed good zone of inhibition
against the pathogenic bacteria tested [86]. Pramanik et al. [63]
have reported the antibacterial activity of copper iodide (CuI) nano-
particle towards the pathogenic bacteria including Bacillus subtilis
(ATCC 6633), Staphylococcus aureus (ATCC 29737), Escherichia coli
(ATCC 10536), Shigella dysenteriae (ATCC 12039). It was observed
that CuI nanoparticles were able to display antibacterial toxicity to-
wards the bacteria at a minimum concentration of 15–66 μg/ml.
Other than the presented data, there is a wealth of literature that de-
scribes nanoparticles as effective antibacterial agents with their
mode of action similar to that of antibiotics.
Fig. 1. (A) ZOI against methicillin resistant S. aureus strain, (i) casein CuNPs, (ii) ampicillin,
(iii) kanamycin and (iv) ofloxacin. About 7.2 mg of materials was added to the respective
disk. (B) ZOI against MR-SA at different concentration of casein CuNPs, (i) 7.2 mg, (ii)
14.4 mg, (iii) 21.6 mg and (iv) 27.8 mg.
Reprinted with permission from RSC.
5.2. Nanoparticles as efflux pump inhibitors
Efflux pumps are the proteinaceous transporters which are present
in the plasma membrane. They help in transporting ions and toxic
chemicals out of the bacterial cell. Efflux pumps are also known to
flush out antibiotics from the bacteria cell [79]thus making them resis-
tant. This problem can be overcome by designing efflux pump inhibi-
tors, which might increase bacterial kill by antibiotics. Christena et al.,
have displayed the use of casein stabilized CuNPs as a potent efflux
pump inhibitor against P. aeruginosa and S. aureus (Fig. 2). To evaluate
the efflux pump activity of CuNPs, a cartwheel assay was performed in
which ethidium bromide (EtBr) acts as a substrate for the efflux
pump. Efflux pump inhibitors are known to accumulate EtBr in the
cells which in turn gives fluorescence. In the cartwheel assay, the TSA
agar plates were mixed with EtBr and CuNPs (0.25× and 0.5× of MIC)
and then the culture of P. aeruginosa and S. aureus was swabbed and in-
cubated for 24 h. It was observed that 0.25× concentration of CuNPs it-
self was able to inhibit the efflux pump which was observed by the
fluorescence emitted by the bacterial cells. This implies that CuNP
acted as effective efflux pump inhibitor. To evaluate the effect of CuNP
on membrane permeability of P. aeruginosa and S. aureus two specific
dyes 1-napthylamine (NPN) (for gram-negative P. aeruginosa) and
propidium iodide (for gram-positive S. aureus) were used. Outer mem-
brane of gram-negative bacteria prevents the access of hydrophobic
molecules into the bacterial cells and hence the accumulation of NPN
on the cell wall may result in increased fluorescence. It was observed
that as the concentration of CuNPs was increased the fluorescence in-
tensity was increased which implies that the outer membrane of the
bacterial cell has been made permeable by the CuNPs which leads to ac-
cumulation of NPN inside the cell. In the case of gram-positive organism
the membrane permeability was evaluated by propidium iodide using
permeability index. PI cannot penetrate the cells. Hence a detergent
CTAB was used as a positive control which leads to membrane breakage
of the bacterial cell and accumulation of PI inside the bacterial cell that
was evaluated as permeability index. It was observed that CuNPs treat-
ed cells exhibited more permeability index (approx. 38%) as compared
to the CTAB treated cells which displayed a permeability index of 33%.
These findings suggest that CuNPs effectively caused damage to the
cell wall of both gram-positive and gram-negative bacteria. To screen
for live and dead bacterial cells the culture was treated with CuNP and
then mixed with a mixture of acridine orange and propidium iodide. Ac-
ridine orange will stain the nucleic acid in live cell as it can penetrate
through the cell membrane and gives green fluorescence on excitation,
whereas propidium iodide stains the DNA of dead cells red. Treatment
of P. aeruginosa with CuNPs displayed cell death while S. aureus showed
loss of colony formation which may be the cause of cell death (Fig. 3).
Thus, it clear that CuNPs act as potent efflux pump inhibitors and also
caused cell death by damaging the cell membrane which is similar to
the mode of action of antibiotics [15].
5.3. Inhibition of enzyme by nanoparticles
Enzymes are one of the major bacterial virulence factors during in-
fection caused by the bacteria. For example, urease produced by
uropathogenic, Ureaplasma urealyticum, Klebsiella spp., Pseudomonas
spp., Corynebacterium sp. D2, Proteus penneri, Providencia stuartii and
Morganella morganii, is one of the major bacterial virulence factors dur-
ing urinary tract infections caused by these bacteria [39]. The inhibition
of the virulent enzymes is the simple approach to tackling the infection.
Inhibition of Urease by conventional antibiotics ciprofloxacin increases
significantly when used as ciprofloxacin capped AgNPs/AuNPs [57].
Cha et al., demonstrated small zinc oxide nanoparticles possess the abil-
ity to inhibit the activity of a typical enzyme β-galactosidase (GAL) in a
biomimetic fashion and showed strong antibacterial activity against
methicillin-resistant Staphylococcus aureus [12]. Silver nanoparticles
are proposed to exhibit antibacterial activity by releasing silver ions
 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947 943

Fig. 2. CuNPs inhibit efflux activity in Staphylococcus aureus, Pseudomonas aeruginosa, MRSA and mutant strains of Staphylococcus aureus. TSA agar plates containing EtBr (0.05 mg ml−1)
was swabbed with either Staphylococcus aureus and Pseudomonas aeruginosa or with MRSA/mutant strains of Staphylococcus and efflux inhibitory activity of 0.25× MIC concentrations of
(a) CuNPs, (b) CuCl2 and (c) CuNPs + EDTA, and EPI activity of 0.5× MIC concentrations of (d) CuNPs, (e) CuCl2 and (f) CuNPs + EDTA and (g) untreated control. Treatments (a) to (g)
were against wild type strains of both Staphylococcus and Pseudomonas swabbed in a cartwheel pattern on the same plate (h) MRSA with 0.5× MIC of CuNPs and (i) mutant strains of
Staphylococcus aureus with 0.5 × MIC of CuNPs. All treatments were evaluated after overnight incubation for growth followed by determining residual fluorescence in a UV
transilluminator.
Reprinted with permission from RSC.

and inhibit the respiratory enzymes, as a result, excess reactive oxygen
species is generated which damage the cell membrane and inactivates
cellular enzymes [58]. Raffi et al., showed that the post treatment of E.
coli cells with AgNPs affects the DNA replication, suggesting that the
nanoparticles have an effect on the DNA polymerase enzyme [66].
These studies suggest the NPs either directly or indirectly inhibit the cel-
lular enzyme and cause antibacterial action.
5.4. Mechanism of nanoparticle toxicity on bacterial cells
The specific mechanism of nanoparticle toxicity towards bacteria
still needs to be addressed. It is believed that nanoparticle can bind to
the bacterial cell membrane by means of electrostatic forces and result
in the disintegration of the bacterial cell membrane [84]. Parashar et
al. have reported a comparative study between the green synthesized
silver nanoparticles from guava plant leaves and chemically synthesized
silver nanoparticles. It was observed that the green synthesized silver
nanoparticles showed a bigger zone of inhibition as compared to the
nanoparticles synthesized using chemical capping agents. It was also
observed by TEM images (Fig. 4) that the nanoparticles disrupted the
cell wall of the bacteria and the silver nanoparticles took 12 h to
completely disrupt the cell wall of the bacteria. After 1 h incubation of
the bacteria cell with the nanoparticles, it was observed that nanoparti-
cles had accumulated near the bacteria. It was only after the end of 8 h
nanoparticle internalization was observed. These studies suggest that
the bactericidal activity of nanoparticle is due to disruption of the cell
wall of the bacteria similar to the action carried by antibiotics [64].
Chatterjee et al. reported that copper nanoparticles (CuNPs) act as
efficient antibacterial agents. The authors have reported the bactericidal
mechanism to be related to bacterial filament formation and cell death.
Filament formation in a bacterial cell is induced due to the presence of
external agent through SOS like stress response. In this study, the au-
thors report that the bacterial cell filamentation after exposure to
CuNPs was due to the generation of cellular ROS. It was found that
ROS level had increased in the nanoparticle treated sample as compared
to the untreated sample. It was also observed that the overproduction of
ROS resulted in considerable increase in lipid peroxidation, and protein
oxidation [16]. Lipid peroxidation leads to bacterial cell toxicity as
higher lipid peroxidation will result in oxidative degradation of polyun-
saturated lipids, resulting in plasma membrane damage, decreases in
membrane fluidity and increase in membrane leakage [96]. It was ob-
served that bacterial sample treated with CuNPs showed increased
lipid peroxidation values which in turn resulted in cell death. In addi-
tion, membrane potential changes were also observed with the poten-
tial of normal E. coli cell found to be around -185 mV and this was
drastically reduced to − 105 and − 75 at different concentrations of
CuNPs exposure. The decrease in membrane potential signifies the
damage to the cell membrane, which is the main reason for bacterial
cell death. In addition, CuNPs also facilitated DNA damage resulting in
the death of bacterial cells.
944 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947

Fig. 3. Live dead imaging shows that CuNPs decreases viability in Pseudomonas aeruginosa and retards colonization in Staphylococcus aureus. Confocal imaging of acridine orange and
propidium iodide stained Pseudomonas aeruginosa (left panel) and Staphylococcus aureus (right panel) with and without CuNPs treatment. Control – represents bacterial biomass
grown for 3 days on glass surface in media without copper nanoparticles. Treated represent bacterial biomass grown for 3 days on glass surface in media containing (1× MIC) of
CuNPs. (Reprinted with permission from RSC).

The nanoparticle toxicity is mainly due to the formation of free rad-
icals resulting in oxidative stress [74]. The antibacterial mechanism of
nanoparticle also depends on the composition of bacterial cell wall. In
recent years, many studies have been reported for the effect of nanopar-
ticle on the bacterial cell wall [8], which suggests that the exact mecha-
nism of nanoparticle toxicity towards bacteria is quite varied and really
complicated. It has been reported that zinc oxide (ZnO) and titanium di-
oxide (TiO2) show antibacterial activity by inducing frameshift muta-
tions [55]. It was observed that the internalization of ZnO by the
bacterial cell resulted in frameshift mutations leading to bacterial
death. TiO2 nanoparticles are known to assist in photocatalysis which
synergistically increases the peroxidation of lipids in the cell membrane
leading to membrane rupture and inhibition of cellular respiration and
death [4]. The toxicity of certain nanoparticles like copper nanoparticles
is dependent on factors such as pH, temperature etc. It was observed
that at high temperature the toxicity of CuNPs is increased. Also low
pH results in decreasing the colony formation of bacteria resulting in
bacterial cell death and enhancing the toxicity. Due to less colony for-
mation, the copper ions are able to dissolve efficiently in the bacterial
membrane which translates to the enhanced toxicity of the nanoparti-
cles [30]. It was also reported that metallic forms of copper induce over-
production of hydroxyl radicals resulting in DNA damage [65]. Gold
nanoparticles synthesized using citrate display mutagenicity against
Salmonella typhi implicating the DNA damage potential of such nano-
particles. The mutagenic effect is termed photomutagenicity as the
gold and citrate induce the generation of free radicals in the presence
of light. The generated free radicals in turn induce DNA damage
resulting in bacterial cell death [88]. It has been reported that silver

Fig. 4. (A) The typical TEM micrograph of E. coli without Ag-NP dose. The cell looks healthy with proper shape and size without any surface or internal damage or defects. (B1) and (B2) the
typical TEM micrograph of E. coli with Gr-Ag-NPs and Ch-Ag-NPs at an early stage of interaction (after 1 h incubation). (C1) and (C2) show a low magnification TEM micrograph after 5 h
incubation with the Gr-Ag-NPs and Ch-Ag-NPs. (D1) and (D2) show the TEM micrograph of the bacterial cell after 8 h incubation with Gr-Ag-NPs and Ch-Ag-NPs. (E1) shows disintegrated
bacteria after overnight incubation (12 h) with Gr-Ag-NPs, black arrows indicate the disintegrated bacterial parts and red arrows the Gr-Ag-NPs. (E2) shows the bacterial cell after
overnight incubation (12 h) with Ch-Ag-NPs. The cell is still not disintegrated and is visible even at lower magnification. (F) Shows the magnified view of a selected port of (C1)
indicating the partial insertion of nanoparticles in bacterial cell membrane.
Reprinted with permission from [44].
 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947 945

nanoparticles synthesized using bacteria or other living organism are List of abbreviations used
known to display symbiotic effect with common antibiotics like ampi-
cillin, penicillin, and kanamycin [89]. These nanoparticles are known
to readily pass through bacterial cell membrane and cause bacterial
cell death. DFFP diisopropylfluorophosphate
It has been reported that silver nanoparticles (AgNPs) can bind to ACE acetylcholinesterase
and accumulate in the cell membrane of bacteria leading to membrane DHFR dihydrofolate reductase
damage and increasing membrane permeability [21]. In addition, AgNPs DNA deoxyribose nucleic acid
are known to trigger ROS generation resulting in cell death [48]. The ex- RNA ribose nucleic acid
tent of permeability of the bacterial cell membrane is dependent on the GTP guanosine Triphosphate
size and concentration of nanoparticles [67]. If the size of the nanopar- MAO monoamine oxidase
ticles is less than it can easily settle on the bacterial surface and cause HMG CoA 3-hydroxyl-3-methyl-glutaryl-CoA
the rupturing of the cell wall of the bacteria thereby making it perme- CuNPs copper nanoparticles
able. It has been reported that accumulation of silver nanoparticles on ZOI zone of inhibition
the surface of E. coli results in the formation of pits on the bacterial MRSA methicillin resistant Staphylococcus aureus
cell membrane leading to increased cell permeability and cell death NSEA N stearoyl ethanolamine
[32]. Silver nanoparticles of size less than 30 nm were found to be AgNPs silver nanoparticles
toxic against S. aureus and K. pneumoniae [68,90]. This is because small CuI copper iodide
particle size facilitates easy penetration into the cell membrane and MIC minimum inhibitory concentration
also helps in good interaction with cell organelles, such as ribosomes EtBr ethidium bromide
[83], which may eventually lead to cell death. Based on the available lit- TSA Tryptone Soya agar
erature, the probable mechanisms of bactericidal action of NPs are pre- NPN 1-napthylamine
sented in Fig. 5. CTAB cetyl trimethyl ammonium bromide
PI propidium iodide
6. Conclusion TEM transmission electron microscope
Gr-AgNPs green synthesized silver nanoparticles from guava leaves
In this mini-review, we have discussed the similarities in the mech- Ch-AgNPS chemically synthesized silver nanoparticles
anism of action of metal nanoparticles with commonly used antibiotics ROS reactive oxygen species
and other molecules on the bacterial cell. The bactericidal mechanism of ZnO zinc oxide
action of NPs is mainly attributed to cell wall damage, altered mem- TiO2 titanium oxide
brane permeability, and generation of ROS, which is in accordance NPs nanoparticles
with the enzyme inhibitors. In addition to accumulation in the cell NO NP nitric oxide nanoparticles
membrane, one possible reason for membrane damage exhibited by Ni NP nickel nanoparticles
metal nanoparticles could be their ability to inhibit enzymes involved Si NP silica nanoparticles
in bacterial cell membrane synthesis. Recently, it was shown that the Al2O3 aluminium oxide nanoparticles
urease activity was significantly inhibited with metal nanoparticles. SPION super magnetic iron oxide nanoparticles
Thus, nanoparticles can be efficiently used as antibacterial agents due S. aureus Staphylococcus aureus
to their ability to inhibit membrane synthesis enzymes in place of B. subtilis Bacillus subtilis
more expensive enzyme inhibitors or antibiotics, and this warrants ex- M. smegmatis Mycobacterium smegmatis
tensive studies. Moreover, with the advent of antibiotic resistance, K. pneumoniae Klebsiella pneumoniae
nanoparticle-based antibacterial strategies could open up a new tool P. aeruginosa Pseudomonas aeruginosa
in our arsenal of antibiotics. E. coli Escherichia coli

Fig 5. Schematic representation of nanoparticle toxicity on bacteria.

946 K.B.A. Ahmed et al. / Materials Science and Engineering C 68 (2016) 939–947
S. typhimurium Salmonella typhimurium
A. baumannii Acinetobacter baumanniii
E. faecalis Enterococcus faecalis
S. typhi Salmonella typhi
B. thuringiensis Bacillus thuringiensis
C. jejuni Campylobacter jejuni
BCG Bacillus Calmette guerin
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