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ISSN 2229 6824

IJPIs Journal of Biotechnology and Biotherapeutics

Effect of biologically synthesized nanoparticles with plant products and chemotherapeutics

against biofilm of clinical isolates of Staphylococcus aureus and Candida tropicalis

Karthick Raja Namasivayam Selvaraj1*, Brijesh Kumar Singh2 and Elamathi Krishnamoorthi3

Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu, India

Corresponding Author: K. R. N. Selvaraj Email address: [email protected]

ABSTRACT:

In the present study, the effect of synthesized silver nanoparticles together with plant

products and commercially available drugs were evaluated against biofilm inhibition and its

biochemical composition of clinical isolates of Candida tropicalisand Staphylococcus aureus.

Silver nanoparticles were synthesized from Lactobacillus acidophilus 01 strain and tested with

aqueous extracts of Aloe vera, ginger (Zingiberofficinale), garlic (Allium sativum), tulsi

(Ocimumtenuiflorum), oils such as coconut oil and antifungal drugs fluconazole and itraconazole

for C. tropicalis, and antibacterial antibiotics such as tetracycline, chloramphenicol for S. aureus.

The nanoparticles were synthesized by dried biomass washing of Lactobacillus acidophilus 01

strain adopting standard conditions and the synthesized purified particles were characterized by

UV-vis spectroscopy and scanning electron microscopy (SEM). The UV-vis spectroscopy

revealed the formation of silver nanoparticles by yielding the typical silver plasmon absorption

maxima at 430 nm and SEM micrograph demonstrated uniform spherical particles with the size

range of 4560 nm. The energy dispersive X-ray spectroscopy (EDX) of the nanoparticles confirmed the presence of elemental silver signal as a strong peak. Synthesized silver

nanoparticles, together with plant products and commercially available drugs, showed a

maximum inhibitory effect against both the tested clinical isolates. In the case of C. tropicalis,

themaximum inhibition was observed for silver nanoparticles with itraconazole, fluconazole and

garlic, followed by silver nanoparticles with itraconazole, and garlic. Total carbohydrates and

total proteins of the biofilm matrix were highly reduced in the respective treatments. In S.

aureus,silver nanoparticles with tetracycline, chloramphenicol and garlic recorded the maximum

inhibition, followed by silver nanoparticles with chloramphenicol, and garlic. Distinct reduction

in total carbohydrates and proteins of the biofilm matrix was also recorded in the respective

treatments.

Keywords: biofilm; silver nanoparticle; Staphylococcus aureus; Candida tropicalis

Vol 1: 3 (2011) IJPIS Journal of Biotechnology and Biotherapeutics

K. R. N. Selvaraj et al Page 2

1. INTRODUCTION

Biofilm, likely the predominant mode of device-related microbial infection exhibit resistance to antimicrobial

agents 1. They can serve as hides for disease and are often associated with high level antimicrobial resistance of the

associated organisms 2Candida tropicalisis a species of yeast in the genus Candida. It is easily recognized as common

medical yeast pathogen existing as a part of normal human flora and causes Septicemia and disseminated candidiasis

especially in patients with lymphoma, leukemia and diabetes.3Staphylococcus aureus is the major clinical pathogen

causing fatal pyogenic infection. Both the strains form biofilm which are now considered ubiquitous and observed to

be extensively heterogeneous in both structurally and with regard to physiology and provide resistance to antimicrobial

drugs 4. The inhibition of biofilm is now considered as drug target and the pharmacological inhibition of biofilm

development is now extensively studied for the treatment of various bacterial and fungal infections. The plants and

their metabolites, the metallic nanoparticle is now investigated in prevention of biofilm produced by various

microorganisms is suitable target as chemotherapeutic agents 5

The matrix is one of the most distinctive features of a microbial biofilm. It forms a three dimensional, gel-

like, highly hydrated and locally charged environment in which the microorganisms are largely immobilized 6. Matrix-

enclosed micro colonies, sometimes described as stacks or towers, are separated by water channels which provide a

mechanism for nutrient circulation within the biofilm 7.The composition of the matrix varies according to the nature of

the organisms present. In this point of view, the effect of sliver nanoparticle with aqueous extracts of Aloe vera, tulsi,

ginger, garlic, coconut oil, with fluconazole and itraconazole for C.tropicalis, silver nanoparticle with tetracycline and

chloramphenicol for S.aureus and was studied. Moreover the effect on biochemical composition of biofilm matrix

mainly total carbohydrate and total protein was also studied.

2. MATERIALS AND METHODS

2.1 Synthesis of silver nanoparticles by Lactobacillus acidophilus SK 01 strain:

Lactobacillus acidophilus 01 strain was isolated by serial dilution technique using Modified Lactobacillus

Agar (Hi-media, India). The bacterium was identified based on cultural and biochemical characteristics. For inoculum

preparation, a loopful of bacterial culture was inoculated in a 250 ml of Erenlymer flask containing 100 ml sterile MRS

broth. The flask was incubated at 300 C, for 32hr on a rotary shaker at 200 rpm, and the cells were harvested by

centrifugation at 3000 rpm for 15 min. Thus obtained biomass was dried in an oven at 600C for 24hrs.

In a typical procedure of nanoparticle synthesis, the dried biomass was washed thrice with Milli-Q-deinosied

water to remove any further medium components. The dried biomass was brought in contact with 200 ml of milli-Q-

deinosied water for 72 hr and agitated under same conditions as described earlier. After the incubation the cell filtrate

was obtained by passing it through Whatmann filter paper no.1.Silver nitrate AgNO3, 10-3

M (1mM) final

concentration was mixed with 50 ml of cell filtrate in a 250 ml of Erlenmeyer flask and agitated at 300C under dark

conditions. Control was run along with experimental flask. Change in color was observed to brown in the silver nitrate

solution incubated with Lactobacillus acidophilus and the nanoparticles were separated from the reaction mixture by

centrifugation at 10,000 rpm for 10 minutes at 4o C. The synthesized silver nanoparticles were analyzed periodically

using UV-Vis spectrophotometer .The absorbance of the nanoparticles was measured in the range 400-800nm, which

includes the plasmon absorbance peak of the silver nanoparticles centered at 430 nm. Further the samples were

characterized by SEM. It was observed that the nanoparticle solution was extremely stable for more than six months

and no signs of aggregation even at the end of this period.

2.2 Inhibition of Biofilm formation:

Clinical strains:Candida tropicalisand Staphylococcus aureus MTCC 29213.



C.tropicalis was obtained from disseminated candidiasis patient who was admitted in Sri Ramachandra

Medical College, Porur, Chennai, Tamilnadu, India. The clinical isolate was identified based on the cultural and

biochemical characteristics described by standard methods. Pure culture was maintained on Sabouraud maltose Yeast

Extract Agar slant.

S.aureus was obtained from Microbial Type Culture Collection (MTCC), Chandigarh, Punjab, India. Pure culture was

maintained on Trypticase Soy Agar.

2.3 Test Materials:

Silver nanoparticle (AgNp) synthesized with Lactobacillus acidophilus , aqueous extract of Aloe

vera(Av),ginger(G) (Zingiberofficinale), garlic(Gc) (Alliumsativum),tulsi(T)(Ocimumtenuiflorum), coconut oil(CN) and

antifungal drugs-fluconazole(F)and itraconazole(I)final concentration 100mg/ml for C.tropicalis, antibacterial drugs-

tetracycline(Tc), chloramphenicol(C) final concentration 100mg/ml for S.aureus. The biofilm inhibition assay was

performed by the tested materials as single and combination. The tested materials as single and combination with silver

nanoparticle for the biofilm inhibition assay was performed with following treatments.

The treatment with tested materials for C. tropicalis are T1-silver nanoparticles(AgNp), T2-Aloe vera(Av),

T3-tulsi(T), T4-ginger(G),T5-garlic(Gc), T6-coconutoil(Cn), T7-itraconazole(I), T8- fluconazole (F),T9-silver

nanoparticle and Aloe vera(Av), T10-silver nanoparticle(AgNp) and tulsi(T), T11-silver nanoparticle(AgNp) and

ginger(G), T12-silver nanoparticle (AgNp) and garlic(Gc),T13-silver nanoparticles (AgNp) and coconut oil(Cn), T14-

silver nanoparticle (AgNp) and itraconazole(I), T15-silver nanoparticle (AgNp) and fluconazole(F), T16-silver

nanoparticle(AgNp) ,Aloe vera (Av)itraconazole(I), T17-silver nanoparticle(AgNp),tulsi(T) itraconazole(I), T18-silver

nanoparticle(AgNp) ginger(G) itraconazole(I),T19-silver nanoparticle(AgNp),garlic(Gc), itraconazole(I), T20-silver

nanoparticle(AgNp), coconutoil(Cn), itraconazole(I), T21-silver nano particle (AgNp), Aloevera(Av), fluconazole(F),

T22 silver nanoparticle(AgNp), tulsi(T) and fluconazole(F), T23- silver nanoparticle(AgNp), ginger(G) and

fluconazole(F),T24- silver nanoparticle(AgNp),garlic(Gc) and fluconazole(F),T25-silver nanoparticle (AgNp)

coconutoil(Cn), fluconazole(F), T26-silver nanoparticle (AgNp), Aloevera(Av) itraconazole(I), fluconazole(F), T27-

silver nanoparticle (AgNp) tulsi(T) itraconazole(I), fluconazole(F), T28- (AgNp),ginner(G),

itraconazole(I),fluconazole(F),T29-silver nanoparticle (AgNp), garlic(Gc), itraconazole(I), fluconazole(F) ,T30-silver

nanoparticle (AgNp), coconut oil(Cn) itraconazole(I) and fluconazole(F). For S. aureus, the inhibitory assay was

performed with the above mentioned treatments except fluconazolitraconazol and coconut oil..chloramphenicol(C) and

tetracycline (Tc) was used.

2.4 Biofilm inhibition assay:

The Biofilm inhibition assay with the tested substance was done with modification of Toole and Kolter8. An

overnight culture of C.tropicalis in Sabouraud maltose Yeast Extract broth and S.aureus in Trypticase Soy broth was

diluted 1:100 ratio in respective fresh medium and grown in for another hour. 100l of diluted respective strains was

added into 96 well titre plate, 200 l of respective test materials as single and combination(1:1 ratio) as described

earlier was added and incubated 37 C for 3 days. After the incubation the medium was removed and 100 l of 1% w/v

aqueous solution of crystal violet was added. Following staining at room temperature for 30 minutes the dye was

removed and the wells were washed thoroughly, 95% ethanol was added and incubated for 15 minutes. The reaction

mixture was read spectrophotometrically at 570nm. Inhibition mediated reduction of biofilm formation was correlated

to the color obtained without addition of test materials.

2.5 Effect of Test Materials on Biochemical Composition of Biofilm Matrix:

The carbohydrate and the total protein of the extracted biofilm matrix of both tested strain was performed by

modified method of Mohammad et al 9.



3. RESULTS AND DISCUSSION

Several groups have demonstrated that Candida biofilm lifestyle lead to dramatically increased level of

resistance to the most commonly used antifungal agents. However the determination of effectiveness of different

antifungal agents against biofilms in this setting has important clinical implications in that it may guide therapeutic

decisions that potentially affect the outcomes of patients suffering from these difficult-to-treat infections. A recently

developed microtitre-plate based biofilm model that is compatible with the 96 well platform technology has proven

valuable for determination and standardization of antimicrobial susceptibility testing in Candida and S.aureus..In the

present study, the various plant products, antimicrobial drugs, Silver Nanoparticle, as single and combination was

evaluated against biofilm development. Various bioactive compounds in the plant such as polyphenols, alkaloids,

flavonoids, antimicrobial peptides known to inhibit bacterial biofilm formation and quorum sensing The effect of

polyphenols of various medicinal plants on E.coli, Pseudomonas putida bacterial biofilm formation. Both the tested

strains, biofilm formation was highly affected by tested polyphenols. Inhibiton of quoram sensing controlled virulent

factor mediate biofilm formation in Pseudomonas aeuroginosa by South Florida plant extracts was studied by

Adonizioet al 1 Similarly efficacy of antifungal drug amphotericin B lipid formulations and Echinocandins on the

biofilm of Candida albicans reported by Kuhn et al5. Candida biofilm showed high susceptibility to both the tested

drugs. Most of the secondary metabolites of plants serve as plant defense mechanisms against predation by

microorganisms insects and herbivores. It was reported that 60% of essential oil derivatives examined data were

inhibitory to fungi while 30% inhibited in bacteria 10

. Less report is available regarding combined effect of silver

nanoparticle with plant products and antimicrobial drugs. Though all the tested plant products commercially available

antimicrobial drugs and silver nanoparticles inhibited biofilm development in both tested strains, significant reduction

could be observed in combination of silver nanoparticle with plant products and commercially available antimicrobial

agents. (P>0.05) (Table.1 and 2)

Synthesis of silver nanoparticles

The conical flask with Lactobacillus acidophilus 01 strain biomass were a pale yellow color before the

addition of Ag ions and this change to a brown color clearly indicators the formation of silver nanoparticles in the

reaction mixture (Figure 1), the characteristic brown color due to the excitation of Plasmon vibrations in the

nanoparticles and provides a convenient signature of their formation The formation of brown color suggests the

presence of silver nanoparticles. The conical flask with biomass is mixed with 10-3

M silver nitrate of final

concentration and stored at dark conditions. Synthesized silver nanoparticles are characterized by UV-Vis

spectroscope, a strong broad surface Plasmon peak located at 430 nm on 14 and 21 day (Figure 2), The surface

plasmon band remains in the range of 420 -440nm throughout the reaction period that suggesting that the particles are

dispersed in the aqueous solution with no evidence for aggregation after complete of the reaction .The solution was

extremely stable even for several weeks after reaction, it is known that silver cations area higly reactive and tend binds

strongly to electron donar groups containgsulphur, oxygen or nitrogen5. But Mineianet al

6 did not observed any extra

cellular bio synthesis activity from Lactobacillus acidophilus, when we challenged the cell biomass of Lactobacillus

acidophilus it was observed that the silver was reduced by Lactobacillus acidophilus. The primordial assay of silver

nanoparticles was performed by EDX on the SEM. (Figure 3) shows the EDX spectrum of the silver nanoparticles.

Strong signals from the silver particles were observed (42.44% in mass), while weaker signal from C, O, Al and S

atoms are also recorded. The SEM micrograph at 30000 times magnification was shown in (Figure 4). In this

micrograph observed spherical nanopartilces in the size range of 45-60nm.



Figure 1: Photograph shows biosynthesized Silver Nanopartilces

Figure 2: UV-Vis absorption of Silver Nanoparticles



Figure 3: Energy dispersive spectroscopy spectrum of Silver Nanoparticles

Figure 4: SEM micrograph of Silver Nanoparticles



Inhibition of Biofilm

In C.tropicalis the maximum effect on biofilm was recorded in silver nanoparticle, itraconazole, fluconazole

and garlic (45.92%) followed by silver nanoparticle, itraconazole and garlic (40.82%).But 13.26%, 14.28%, 16.33%,

12.24% of inhibition was recorded in silver anoparticle, fluconazole, itraconazole and garlic as single treatment.

Moreover 37.76% of inhibition was recorded in silver nanoparticle,itraconazole, fluconazole and coconut oil

combination.. Coconut oil alone inhibited 10.2% of biofilm development. Tulsi alone inhibited 5.1% of biofilm

development 15.31%.of inhibition was observed intulsi with silver nanoparticle The inhibition recorded in

combination of silver nanoparticle, tulsi and itraconazole was 18.37%.. 22.45%. of inhibition was reported in silver

nanoparticle with tulsi, fluconazole 31.63% of inhibition was observed in silver nanoparticle, tulsi, itraconazole,

fluconazole

Ginger alone inhibited 10.2%.13.35% of inhibition was noticed in ginger combined with silver nanoparticle.

The inhibitory activity was increased to 30.61% in silver nanoparticle combined with itraconazole and ginger.32.65%

inhibition was in silver nanoparticle with ginger and fluconazole. 34.69% of inhibition was in silver nanoparticles with

ginger, itraconazole and fluconazole. Aloe vera combined with silver nanoparticle and antifungal drugs shows distinct

biofilmcidal effect. 27.55%,26.53% and 32.65% of inhibition was recorded in silver nanoparticle with Aloe

veraanditraconazole, silver nanoparticle with Aloe veraand fluconazole, and silver nanoparticles with Aloe vera,

itraconazole and fluconazole respectively(Table 1)

In S.aureus distinct effect on biofilmcidal activity was observed in combination of silver nanoparticles with

plant products and anti bacterial agents. Nanoparticle with tetracycline, chloramphenicol and garlic recorded maximum

inhibition (44.68%) followed by 41.48% in silver nanoparticle with chloramphenicol and garlic. 30.85% of inhibition

was in silver nanoparticle with tetracycline and ginger (30.85%). 12.76% of inhibition recorded in silver nanoparticle

with ginger. Ginger combined with silver nanoparticle and tetracycline recorded 14.89% of inhibition. 27.65% of

inhibition was reported in silver nanoparticles with ginger and chloramphenicol.(Table 2) Silver nanoparticle with Aloe

vera and tetracycline inhibited 23.40%; Silver nanoparticle with Aloe vera and chloramphenicol inhibited 26.95%;

Silver nanoparticles combined with Aloe vera tetracycline and chloramphenicol reveals 25.53% inhibition.. 11.70% of

inhibition was recorded in treatment of silver nanoparticles with tulsi.13.82%, 19.14% and 21.27% of inhibition was

observed in silver nanoparticles with tulsi and tetracycline, silver nanoparticles with tulsi and chloramphenicol and

silver nanoparticles with tulsi, tetracycline and chloramphenicol respectively.

The biochemical composition of biofilm matrix mainly total carbohydrate and total protein of both tested

strains was highly reduced in combinations of silver nanoparticle with plant products and antifungal and antibacterial

drugs. (Table 1 and 2). Mohammed et al 9 extensively studied biofilm matrix of Candida albicans,Candida tropicalis.

The chemical composition and their role drug resistance was extensively studied by them. Their results demonstrate

that the matrix can make a significant contribution to drug resistance to Candida biofilm under adverse conditions.

In C.tropicalis, the protein content was highly reduced in silver nanoparticle with itraconazole, fluconazole and

garlic (10g/mg) followed by silver nanoparticle with itraconazole, and garlic(30g/mg).The protein content of biofilm

matrix of S.aureus was also reduced in silver nanoparticle with tetracycline, chloramphenicol and garlic (16g/mg)

followed by silver nanoparticle with choramphenicol and garlic(33g/mg) .The carbohydrate content was highly

reduced in silver nanoparticle with itraconazole, fluconazole and garlic in C.tropicalis (57g/mg) followed by silver

nanoparticle with itraconazole, and garlic(67g/mg).

The carbohydrate content of biofilm matrix of S.aureus was also reduced in silver nanoparticle with

tetracycline, chloramphenicol and garlic (43g/mg) followed by silver nanoparticle with chloramphenicol and garlic

(63g/mg).In the present study, both the tested strain biofilm formation and development was highly inhibited or

reduced by the combined effect of Silver Nanoparticles with plant products and commercially available antimicrobial

drugs.



Table 1: Table 1: Inhibition of biofilm formation by C. tropicalis and changes in total protein and

carbohydrate concentration of C.tropicalis

Treatment Biofilm OD 570

as a % of

control value

Total

protein(g/mg)

Total

Carbohydrate(g/mg)

Control 0.98 240 197

SilverNanoparticle(AgNp)T1 13.26 183 130

Aloe vera(Av) T2 7.14 190 186

Tulsi(T) T3 5.1 200 170

Ginger(G) T4 10.2 197 146

Garlic(Gc) T5 12.24 186 131

Coconut oil(Cn) T6 10.2 198 160

Itraconazole(I) T7 16.33 173 114

Fluconazole(F) T8 14.28 180 123

AgNp+Av T9 24.48 170 117

AgNp+T 10 15.31 186 120

AgNp+G T11 13.35 177 97

AgNp+Gc T12 19.36 156 87

AgNp+Cn T13 16.32 183 114

AgNp+I T14 24.48 70 90

AgNp+F T15 20.41 123 103

AgNp+I+Av T16 27.55 53 87

AgNp+I+T T17 18.37 67 83

AgNp+I+G T18 30.61 60 77

AgNp+I+Gc T19 40.82* 30* 67*

AgNp+I+Cn T20 20.41 156 93

AgNp+F+Av T21 26.53 97 97

AgNp+F+T T22 22.45 111 90

AgNp+F+G T23 32.65 70 82

AgNp+F+Gc T24 36.73 47 77

AgNp+F+Cn T25 34.7 164 83

AgNp+I+F+Av T26 32.65 70 73

AgNp+I+F+T T27 31.63 90 77

AgNp+I+F+G T28 34.69 36 70

AgNp+I+F+Gc T29 45.92* 10* 57*

AgNp+I+F+Cn T30 37.76 117 70

* Statistically significant at p>0.05 level by DMRT



Table 2: Inhibition of biofilm formation by S.aureus and changes in total protein and

carbohydrate concentration of S.aureus

Treatment Biofilm

OD 570

as a % of

control

value

Total

protein(g/mg)

Total

Carbohydrate(g/mg)

Control 0.94 273 183

Silvernanoparticle(AgNp)T1 9.57 146 143

Aloe vera(Av) T2 5.3 170 177

Tulsi(T) 3 4.25 170 166

Ginger(G) T4 4.25 130 164

Garlic(Gc) T5 7.44 153 156

Tetracycline(Tc) T6 11.70 140 131

Chloramphenicol(C) T7 9.57 183 143

AgNp+Av T8 12.76 143 110

AgNp+T 9 11.70 146 131

AgNp+G T10 12.76 130 126

AgNp+Gc 11 14.89 120 120

AgNp+Tc T12 13.82 131 117

AgNp+C T13 15.95 110 107

AgNp+Tc+Av T14 23.40 111 100

AgNp+Tc+T T15 13.82 107 97

AgNp+Tc+G T16 14.89 97 90

AgNp+Tc+Gc T17 32.97 36 90

AgNp+C+Av T18 26.59 100 107

AgNp+C+T T19 19.14 107 100

AgNp+C+G T20 27.65 47 97

AgNp+C+Gc 21 41.48* 33* 63*

AgNp+Tc+C+Av T22 25.53 90 90

AgNp+Tc+C+T T23 21.27 70 83

AgNp+Tc+C+G T24 30.85 47 70

AgNp+Tc+C+Gc T25 44.68* 16* 43*

* Statistically significant at p>0.05 level by DMRT

4. ACKNOWLEDGEMENTS

Thanks due to Anna University, Chennai, Tamil Nadu for SEM and EDX analysis of silver nanoparticles.

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