Article Type: Research Article Article Citation: Tasha Anandya Tantyani, and Titik Taufikurohmah. (2020). ANTIBACTERIAL AND ANTIFUNGAL
ACTIVITY OF SILVER NANOPARTICLES AGAINST NEISSERIA GONORRHOEAE AND CANDIDA
ALBICANS. International Journal of Research -GRANTHAALAYAH, 8(6), 179-187. https://doi.org/10.29121/granthaalayah.v8.i6.2020.461 Received Date: 09 May 2020 Accepted Date: 29 June 2020 Keywords: Nanosilver Antibacterial Activity Antifungal Activity Disk Diffusion Neisseria Gonorrhoeae Candida Albicans This Research on the antibacterial and antifungal activity of nanosilver against Neisseria gonorrhoeae and Candida albincas fungi has been carried out. The purpose of this study was to determine antibacterial activity of nanosilver against Neisseria gonorrhoeae and antifungal activity against Candida albincas. Synthesis Nanosilver uses bottom up method and characterized using UV-Vis Spectrophotometer. Nanosliver concentrations used were 30, 40, 50, and 60 ppm. Antibacterial and antifungal activity tests using disk diffusion method. Observations obtained in form of the presence or absence of clear zones formed around paper discs indicate the inhibition of nanosilver on microbial growth. The results of testing the antifungal activity of Candida albicans on nanosilver with concentrations of 30, 40, 50 and 60 ppm resulted in clear zones of 9.73 nm, 11.46 nm, 11.93 nm, and 13 nm with fungal inhibition response categories is medium and strong. The results antibacterial activity test of Neisseria gonorrhoeae on nanosilver with concentrations of 30, 40, 50 and 60 ppm did not show any clear zone around the disc, it showed that nanosilver in this study did not have antibacterial activity against Neisseria gonorrhoeae.
1. INTRODUCTIONIn this era of
globalization there have been many advances in all fields and aspects of
science about the importance of hygiene and also the basic health of physical
and organ hygiene. One of the organs that really needs special care is the
reproductive organs. Reproductive organs are one of the important things in
every human life. Reproductive organs are the subject of several diseases.
Early knowledge and understanding of the health of reproductive organs can be
used as prevention for men and women so that they will be better able to
maintain the health of their reproductive organs.[1]. In women, the vaginal canal is very vulnerable to risk of infection from
the outside. In addition, the wrong way to clean the vagina and leave the
condition of the vagina moist also triggers the disease in female reproductive
organs [2]. In women, there is a term called Vaginal Infection. Vaginal infection is
one of the most frequent problems where women visit a doctor [3]. One example of vaginal infection is
vaginitis. Vaginitis is inflammation and vaginal infections commonly
encountered in clinical medicine [4]. Bacterial vaginosis, candidiasis
vaginalis (an infection caused by Candida
spesies) is responsible for the majority of vaginal infections in women,
especially in women of reproductive age [5]. An example of the most serious disease in
bacterial vaginosis is Gonorrhea disease caused by the bacterium Neisseria gonorrhoeae is Sexually
Transmitted Disease (STD). Gonorrhea in
Indonesia ranks highest of all STD. According to the World Health Organization
(WHO, 2011) as many as 70% of female patients and some male patients infected
with gonorrhoeae or chlamydia have asymptomatic symptoms. Sexually transmitted
diseases are also the most common cause of infertility, especially in women.
Several studies in Surabaya, Jakarta and Bandung in female sex workers show
that the prevalence of gonorrhoeae ranges from 7.4% - 50% [6]. Besides gonorrhea, there are also candidiasis vaginalis which is one of
the vaginal infections caused by fungus Candida species especially Candida albicans. Candida albicans is a normal flora that lives in oral mucrosa,
respiratory tract, digestive tract and in the vagina [7]. Candida albicans with excessive amounts
in the body can cause disease. One example is
Candidiasis vaginalis. Thin (1983) states that the most common cause of
vaginal candidiasis is candida albicans, which is 81%, the rest is 16% by
torulopsis glabarata, while the other 3% is caused by Candida tropicalis,
Candida pseudotropicalis, Candida krusei and Candida stellatoidea [8]. Candidiasis vaginalis can occur in women of
all ages, especially at puberty. The most prominent complaint in patients with
vaginal candidiasis is vaginal itching accompanied by discharge of the vaginal
body (fluor albus). Sometimes also found irritation, burning and dyspareunia.
In the acute situation, the body of the vagina is runny while the chronic is
thicker. Candidiasis that has entered the bloodstream can spread to various
organs such as the kidneys, spleen, heart, brain, and cause various diseases
such as endocarditis, meningitis, endophthalmitis and pyelonephritis [9]. Various attempts
have been made to reduce various infectious diseases of the vagina that require
antimicrobial agents to inhibit or kill bacteria and fungi that cause vaginal
infections. Now many new technologies have been developed that are used to
overcome problems for microbes such as bacteria and fungi. One of them is
nanotechnology. Nanotechnology is the science of technology that uses the
properties of molecular structures or atomic structures or materials in
nanometer sizes. Nanometer-sized materials have better properties than
large-sized materials. A nanomaterial or nanoparticle is a particle with the
size of a nanometer (1-100 nm) [10]. One of the nanoparticles that has been
developed is silver or nanosilver. Nanosilver can be obtained by synthesis
using the bottom-up method, which is a synthesis process that involves chemical
reactions by growing nanoparticles from a number of starting materials so that
other nanometer-sized materials are produced [11]. Nanosilver has a
very broad antimicrobial spectrum including antiviral activity such as HIV-AID
and HSV herpes virus [12]. Nanosilver is proven to have the
ability, among others, as an antibacterial and antifungal [13]. Silver nanoparticles (AgNP) also have
antifungal, anti-inflammatory, antiviral, and antiplatelet activity [14]. Nanosilver as a strong antibacterial
because of its chemically reactive and easily ionized nature and antimicrobial
ability of nanosilver can kill all pathogenic microorganisms and there have not
been reported any silver-resistant microbes [15]. The antibacterial activity of silver
nanoparticles is influenced by several things, such as the concentration of
silver nanoparticles, the shape of silver nanoparticles, the size of silver
nanoparticles, the type of bacteria, the number of bacterial colonies and the
contact time of silver nanoparticles with bacteria [16].The antibacterial ability of silver
nanoparticles include damaging bacterial cell walls, disrupting cell
metabolism, and inhibiting bacterial cell synthesis. Nanosilver can approach
the microbial cell membrane and penetrate into the microbe. Furthermore, the
nanosilver diffuses and attacks the respiratory chain of the microbes, so it
can kill the microbes [17]. Nanosilver is a natural mineral that is
non-toxic and safe for daily use. The combined minerals are also found in
groundwater or natural water sources [18]. In this research,
basic ingredients of nanosilver in the form of
AgNO3 by reducing sodium citrate in aquo media [19]. Synthesis of nanosilver
was carried out by bottom-up method using sodium citrate as a reducing agent.
Several variations of nanosilver concentrations were performed by 30, 40, 50,
60 ppm. Nanosilver with several variations of concentration will be analyzed by
UV-Vis spectrophotometer at a wavelength of 400-513 nm to determine the peak
absorbance. In the antibacterial assay of Neisseria gonorrhoeae and antifungal
assay of Candida albicans used disc diffusion method. The observation results
obtained are the presence or absence of clear areas formed around the disc
paper which shows inhibition zones on microbial growth. 2. MATERIALS AND METHODS2.1.
MATERIALS
This research was carried out using several materials including 250 ml beaker glass, 10 ml
beaker glass, 50 ml beaker glass, hot plate and stirrer, Ohauss analytical
balance, spatula, 100 ml measuring flask, Shimadzu UV-Vis Spectrophotometer
1800, micropipette, autoclave, incubator, calipers, ose needles, tweezers,
vortex, petri dishes, AgNO3 (≥99%, Merck), sodium citrate (99%, Merck), aquades
(PT Bratachem), aquabides
(PT Bratachem), liquid media Potato Dextrosa Agar (DPA), ketocenazole, dimethyl sufoxide (DMSO), Neisseria gonnorhoeae
stock colony, Candida
albicans stock colony, filter
paper, paper discs, Ciprofloxacin. 2.2.
PREPARATION OF AgNO3 SOLUTION
1000 ppm AgNO3 solution was obtained by dissolving
1.57 g of AgNO3 crystal (colored white) which was put into a 1000 ml
volumetric flask, then diluted with aquabides to mark
limits.Then the mixture is
homogeneous and a 1000 ppm AgNO3 solution is formed which is ready
to be used as material in nanosilver synthesis. 1000
ppm AgNO3 is used for synthesis. 2.3.
SYNTHESIS OF NANOSILVER
Synthesis of
nanisilver was carried out by the bottom up method by heating 200 ml of
distilled water to boiling, then adding sodium gratrate
0.4 g and 1000 ppm 4 ml AgNO3 solution for a concentration of 20
ppm. Warming is continued until the solution turns from colorless to yellow
then grayish yellow for about 15-20 minutes. Furthermore, the colloid is cooled
at room temperature. Synthesis was
continued with variations of concentrations of 30, 40, 50, 60 ppm with a volume
of 1000 ppm AgNO3 solution of 6, 8, 10, 12 ml. The synthesized nanosilver measured its maximum wavelength on a UV-Vis
spectrophotometer in the wavelength range of 400-513 nm. 2.4.
ANTIBACTERIAL ASSAY OF NEISSERIA GONORRHOEAE USING DISC
DIFFUSION METHOD
The steps taken
in this antibacterial assay are make a
suspension of the bacteria Neisseria gonorrhoeae with a turbidity of 1mc farland then
insert a sterile swab into the suspension of the Neisseria gonorrhoeae and
rubbed evenly into the MH plate media then saturate the disc paper in
nanosilver for 15 minutes and insert the saturated disc paper into the MH plate
media. The positive control used 5 mcg ciprofloxacin antibiotic disk and
negative control used aquadest. Incubation at 35°-37°C for 24 hours. The
results of observations can be measured use calipers. 2.5.
ANTIFUNGAL ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION
METHOD
The steps of make
a specific culture media for Candida albicans are embed Candida albicans stock
on SDB (Sabour Dextrosa Broth) media by applying 1 ose needle pure
culture Candida albicans then incubated for 24 hours at 37°C. Antifungal assay
use sterile paper discs dropped with 10 microliter nanosilver, then the paper
discs containing nanosilver are placed on the surface of SDB media and
incubated for 48 hours. The positive control used ketokenazol and negative
control used aquadest. The results of observations can be measured use
calipers. 3.
RESULTS AND DISCUSSION
3.1.
SYNTHESIS AND CHARACTERIZAION OF NANOSILVER
Synthesis of
Nanosilver use bottom up method which is reducing chemicals by using sodium
citrate. In synthesis of nanosilver with this method produces a change in the
color of solution from initially colorless to stable yellow, so that heating
can be stopped. The colorless solution indicates that there is no interaction
with each other between Ag atoms, while the stable yellow color indicates that
Ag particles enter nano size and the growth of particle size (cluster) is
getting bigger. The results synthesis are brownish-yellow colloids as shown in Figure 1. Figure 1: Nanosilver colloids with concentrations of
30, 40, 50, 60 ppm There is a difference in the intensity of the brownish yellow color that occurs in the results of nanosilver synthesis, the greater the concentration of nanosilver shows the more concentrated brownish yellow color. This happens because there are differences in the size of the cluster produced. If the concentration of nanosilver is greater then more clusters will form so that the color intensity results will be stronger. Silver atoms will interact with their fellow metal bonds and produce large numbers of nano clusters. Reaction that occurs in synthesis of nanosilver is written in equation (1). 4Ag+ + C6H5O7Na3
+ 2H2O → 4Ag0 +C6H5O7H3
+ 3Na+ + H+ + O2
(1) Nanosilver was
then characterized by a UV-Vis Spectrophotometer in the wavelength range of
300-450 nm to determine the maximum wavelength of nanosilver used to measure
the cluster diameter. Cluster diameter of nanosilver was calculated
using the Brush equation. The results of the characterization are shown in Table 1. Table 1: Maximum wavelength and diameter of
nanosilver cluster
3.2.
ANTIBACTERIAL
ASSAY OF NEISSERIA GONORRHOEAE USING DISC DIFFUSION METHOD
Nanosilver with
concentration of 30,40,50 and 60 ppm then tested for antibacterial activity
against Neisseria gonorrhoeae. In this study, antibacterial assay use disc
diffusion method that’s make a suspension of Neisseria
gonorrhoeae with a turbidity of 1mc farland then insert a sterile swab into the
suspension of the Neisseria gonorrhoeae and rubbed evenly into the MH plate
media then saturate the disc paper in nanosilver for 15 minutes and insert the
saturated disc paper into the MH plate media. The positive control used 5 mcg
ciprofloxacin antibiotic disk and negative control used aquadest. Incubation at
35°-37°C for 24 hours. The results of research using this method are the clear
zone diameters that occur around the paper disk. The results of the qualitative test
antibacterial activity of nanosilver against Neisseria gonorrhoeae as shown in Figure 2. and the results of the qualitative test antibacterial activity of
positive control (Ciprofloxacin) against Neisseria gonorrhoeae as shown in Figure 3.
Figure 2: Result of antibacterial assay of
nanosilver with concentration 30,40,50, and 60 ppm against Neisseria
gonorrhoeae. (a) Replication I (b) Replication II Figure 3: Results of the qualitative test
antibacterial activity of positive control (Ciprofloxacin) against Neisseria
gonorrhoeae The quantitative
test results of the antibacterial assay of nanosilver which is diameter of
clear zone can be measured using the calipers shown in Table 2. Table 2: Clear zone diameter of the antibacterial
test on nanosilver against Neisseria gonorrhoeae
In this study,
four replications were carried out and from the results of the clear zone
diameter it can be stated that the nanosilver in this study does not have
antibacterial activity against Neisseria gonorrhoeae, it can be known because
there is no clear zone diameter that occurs around the disc paper that has been
saturated on the nanosilver. The criteria for antibacterial activity according
to Muharni (2016) [20] were clear zone diameters <10 nm
included in the weak category, 10-16 nm included in the moderate category and
>16 nm included in the strong category. Nanosilver has good antibacterial
activity but there is no research that states that nanosilver can inhibit the
growth of the bacteria Neisseria gonorrhoeae. In this study, nanosilver used
concentrations of 30, 40, 50 and 60 ppm and nanosilver was allowed to stand for
2 weeks after the synthesis process, after that testing was carried out on the
Neisseria gonorrhoeae bacteria, it also could reduce the antibacterial
properties of the nanosilver because it was not directly tested after the
process synthesis is done. The absence of antibacterial activity in this study
can also occur because the concentration of nanosilver used is too small or not
optimal to inhibit the growth of Neisseria gonorrhoeae. N. gonorrheae is a
gram-negative bacterium that has a selection system for certain substances. According to Muharini (2017) [21], the structure of gram negative bacteria
also affects the results of bacterial inhibition zones because it has a more
complex structure with three layers, namely the outer layer in the form of
lipoprotein, the middle layer in the form of lipopolysaccharide and the inner
layer in the form of peptidoglycan, which causes Gram-negative bacterial cell
walls are more difficult to penetrate by antibacterial compounds and
gram-negative bacteria have the property of less susceptible to some
antibacterial compounds. In negative
control area (aquades) no clear zone appears. The absence of this clear zone
indicates that aquades do not have antibacterial properties and can not inhibit
the growth of Neisseria gonorrhoeae bacteria while in the positive control area
(ciprofloxacin) shows the presence of a clear zone diameter of 48 mm which
indicates that the antibiotic ciprofloxacin has a strong antibacterial ability
against Neisseria bacteria gonorrhoeae. 3.3.
ANTIFUNGAL
ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION METHOD
Nanosilver with
concentration of 30,40,50 and 60 ppm then tested for antifungal activity
against Candida albicans. In this study, antifungal assay use disc diffusion
method that’s make a specific culture media for Candida albicans are embed
Candida albicans stock on SDB (Sabour Dextrosa
Broth) media by applying 1 ose needle pure culture
Candida albicans then incubated for 24 hours at 37°C. Antifungal assay use
sterile paper discs dropped with 10 microliter nanosilver, then the paper discs
containing nanosilver are placed on the surface of SDB media and incubated for
48 hours. The positive control used ketokenazol and negative control used
aquadest. The results of research using this method are the clear zone
diameters that occur around the paper disk. The results of the qualitative test
antifungal activity of nanosilver against Candida albicans as shown in Figure 4. and the results of the
qualitative test antifungal activity of positive control (ketokenazol) against
Candida albicans as shown in Figure 5.
Figure 4: Result of antifungal assay of nanosilver with
concentration 30,40,50,60 ppm against Candida albicans Figure 5: Result of antifungal assay of Ketokenazol
against Candida albicans The quantitative
test results of the antifungal assay of nanosilver which is diameter of clear
zone can be measured using the calipers shown in Table 3. and graphs of nano
silver concentrations with clear zone diameters are shown in Figure 6. Table 3: Clear zone diameter of the antifungal test
on nanosilver against Candida albicans
Figure 6: Graphs of nano
silver concentrations with clear zone diameters In this study,
four replications were carried out and it can be seen from the results of the
diameter of the clear zone that occurs around the disc that the nanosilver in
this study has inhibitory properties as an antifungal against Candida albicans.
The greater the concentration of nanosilver, the greater the diameter of the
clear zone that occurs. In the nanosilver with a concentration of 30 ppm the
clear zone diameter that occurred was 9.73 mm, the nanosilver with a concentration
of 40 ppm the clear zone diameter that occurred was 11.46 nm, the nanosilver
with a concentration of 50 ppm the clear zone diameter that occurred was 11.93
nm, and nanosilver with a concentration of 60 ppm diameter of a clear zone that
occurs by 13 mm. According to Mandey and Londok (2014) [22], there are categories of antifungal
activity based on inhibition zone diameter that occurs namely <5 mm is weak
category, 5-10 mm is moderate category, 10-20 mm is strong category, and >20
is very strong category. Nanosilver in this study included in medium and strong
antifungal because at the concentration of 30 ppm the diameter of the
inhibitory zone produced was in the range <10 mm or in the category of moderate
antifungal and at concentrations of 40, 50, and 60 ppm the diameter of the
inhibitory zone produced was included in the range of 10-20 mm or a strong
antifungal category against Candida albicans. In the research
of Keuk-jun et al (2008) [23], states that the mechanism by which
nano-Ag breaks down the membrane permeability barrier, it is possible that
nano-Ag perturbs the membrane lipid bilayers, causing the leakage of ions and
other materials as well as forming pores and dissipating the electrical
potential of the membrane and also the interaction between nano-Ag and the
membrane structure. C. albicans cells, during nano-Ag exposure, show
significant changes to their membranes, which are recognized by the formation
of 'pits' on their surfaces, and finally, the result in the formation of pores
and cell death. So nanosilver can inhibit the growth of Candida albicans
through destruction of membrane integrity; therefore, it was concluded that
nano-Ag has strong antifungal activity against Candida albicans. The positive
control used in this study was ketocenazole which produced clear zone diameter
at 16.16 mm. Ketocenazole is also included in a powerful antifungal to inhibit
the growth of Candida albicans. While the negative control used in this study
is aquadest, in negative control (aquades) no clear zone appears around the
disc. The absence of this clear zone indicates that aquades has no antifungal
activity and can not inhibit the growth of Candida albicans. 4.
CONCLUSION
Based on the
results of the study it can be seen that nanosilver in this study does not have
antibacterial activity against Neisseria gonorrhoeae and can not inhibit the
growth of Neisseria gonorrhoeae as indicated by the absence of clear zone that
occur around the discs, but nanosilver in this study has a strong antifungal
activity in inhibiting growth Candida albicans, with an increase in
concentration on nanosilver can increase the growth inhibition of Candida
albicans shown by a wider diameter of the clear zone that occurs around the
disk. The clear zone diameters that occur in Candida albicans were at a
conmcentration of 30 ppm at 9.73 mm, 40 ppm at 11.46 mm, 50 ppm at 11.93 nm,
and 60 ppm at 13 mm. SOURCES OF FUNDINGNone. CONFLICT OF INTERESTNone. ACKNOWLEDGMENTThank you is conveyed to
Prof. Dr. Titik Taufikurohmah,
M.Si., who has funded all of activities in this study
including nanosilver, antifungal and antibacterial
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