Microbial mediated synthesis, characterization, antibacterial and synergistic effect of gold nanoparticles using Klebsiella pneumoniae (MTCC-4030)

Abstract

Herein, the microbial mediated synthesis of gold nanoparticles (GNPs) was achieved via an easy biological protocol using Klebsiella pneumoniae (MTCC-4030). Gold ions in the reaction mixture were exposed to K. pneumoniae for the formation of colloidal GNPs. The characterization study indicated that the UV-vis spectral analysis of the GNPs showed a peak at 550 nm. XRD spectroscopy of the GNPs confirmed their crystalline nature. Scanning electron images of the GNPs showed that they were spherical in shape and were well dispersed. Atomic force microscopy revealed that the size range of the GNPs was between 10 and 15 nm. The FTIR study revealed the possible involvement of reductive groups on the surface of the nanoparticles. The antibacterial activity of the GNPs showed the highest inhibitory zone (25.60 mm) against Escherichia coli as an indicator strain. The synergistic effect of the GNPs obtained the highest fold increase (4.06) and activity index (3.210) against E. coli, followed by 2.610 activity index against Staphylococcus aureus using amoxycillin and streptomycin as standard antibiotics, respectively.

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Introduction

Nanotechnology is currently, one of the most rapidly advancing technologies. The role of microbial systems in the synthesis of nanometals is rapidly gaining importance due to their optical, chemical, photoelectrochemical and electronic properties. The thrust to develop reliable, ecofriendly procedures for the fabrication of nanoscale materials is an important aspect of nanotechnology study. Gold nanoparticles (GNPs) have gained increasing interest due to their specific features such as unusual optical and electronic properties, non cytotoxicity, high stability, biological compatibility, controllable morphology, size dispersion and easy surface functionalization.^1,2 The green biosynthesis of nanoparticles can be achieved via the selection of an environmentally acceptable solvent with eco-friendly reducing and stabilizing agents.³ Biologically synthesized nanoparticles are naturally protein-capped, which prevents aggregation and avoids the use of external toxic capping agents. Srinath and Ravishankar Rai⁴ stated that this green biosynthesis is the method used for the formation of monodispersed, small spherical GNPs of 4–10 nm in size by K. pneumoniae, and theirs was the first report of attempting to improve monodispersity and reduce size by varying the gold salt concentration. Therefore, biological approaches to nanoparticle synthesis have been suggested as valuable alternatives to physical and chemical methods.⁵

Biological synthesis of nanoparticles has emerged as a rapidly developing research area in nanotechnology across the globe with various biological entities being employed in the production of nanoparticles, which constantly form an attractive alternative for conventional methods. From simple prokaryotes to complex eukaryotic organisms, which include higher plants, are used for the fabrication of nanoparticles. The biosynthesis of GNPs has been reported in different prokaryotic organisms, including Bacillus subtilis,⁶ E. coli,⁷ Lactobacillus sp.,⁸ Pseudomonas aeruginosa,⁹ and Rhodopseudomonas capsulate;¹⁰ however, the molecular mechanisms involved in the metal ion reduction that occurs for the synthesis of nanoparticles has not yet been established.

Nanoparticles can act as antibacterial and antifungal agents due to their ability to interact with microorganisms. By exerting their antibacterial properties, nanoparticles can attach to the surface of the cell. This interaction causes structural changes and damage and markedly disturbs vital cell functions such as permeability causing pits and gaps, depressing the activity of respiratory chain enzymes and finally leading to cell death.¹¹ The demonstrated antibacterial activity of nanoparticles suggests its possible application in the food preservation field; however, it can be applied as a potent sanitizing agent for disinfecting and sterilizing food industry equipment and containers against attack and contamination from foodborne pathogenic bacteria.¹² In the present study, an attempt has been made on the microbial mediated fabrication of GNPs by the reduction of HAuCl₄ ions using K. pneumoniae (MTCC- 4030). The fabricated GNPs were characterized via UV, XRD, SEM, EDS, FTIR and AFM. Finally, the fabricated GNPs were applied in the field of antibacterial and synergistic studies in comparison with known antibiotics.

Results and discussion

In the present study, the Gram negative bacterium, K. pneumoniae, was found to be successful in the synthesis of GNPs which had a uniform distribution and were quite stable in solution. The colour of the reaction solution turned from pale yellow to deep red, which indicated the formation of gold nanoparticles (Fig. 1). The mechanism of the extracellular biosynthesis of the nanoparticles is proposed as a nitrate reductase-mediated synthesis that secretes the enzyme nitrate reductase, which then brings about the bioreduction of metal ions and synthesis of nanoparticles.¹³ Both extracellular and intracellular methods are used for the synthesis of biological nanoparticles.¹⁴ The exact mechanism for the synthesis of nanoparticles using biological agents has not yet been elucidated, but it has been suggested that various biomolecules are responsible for the synthesis of nanoparticles. It seems that the cell wall of microorganisms play a major role in the intracellular synthesis of nanoparticles. Mukherjee et al.¹⁴ postulated that the mechanism of the synthesis of nanoparticles occurs in three stages: trapping, bioreduction and synthesis. Honary et al.¹⁵ reported that the Enterobacteriaceae family biomass in 1 mM aqueous HAuCl₄ solution led to the development of a dark purple solution after 24 h of reaction.

Fig. 1 GNPs synthesized by K. pneumoniae.

The UV-visible spectroscopy result revealed that the reaction solution had an absorption maximum at 550 nm, which is attributed to the surface plasmon resonance band (SPR) of gold nanoparticles (Fig. 2). The optical absorption spectrum of the metal nanoparticles was dominated by SPR, which shifted to longer wavelengths with increasing particle size. The reduction of HAuCl₄ to GNPs can be identified from the peaks obtained at around 650 nm.¹⁶ Likewise, Skirtach et al.,¹⁷ reported that the absorption spectrum of GNPs was observed at 560 nm, which were synthesized using P. aeruginosa. It is well known that spherical nanoparticles of Au should exhibit single-surface plasmon bands, whereas anisotropic particles should exhibit two or three bands, which correspond to quadrupole and higher multipole plasmon excitations.¹⁸

Fig. 2 UV-vis Spectrum of GNPs synthesized by K. pneumoniae.

The FTIR spectrum of the GNPs, which is shown in Fig. 3, has absorption peaks at 1716, 1654, 1639, 1527, 1342, and 1249 cm⁻¹. The strong peak at 1654 cm⁻¹ was identified as C=O due to the carbonyl functional group. The peak at 1527 cm⁻¹ showed the characteristic of N–O which is an asymmetric nitro compound. The small band at 1639 cm⁻¹ arose from the –C=C stretching vibrations corresponding to the –C=O due to the carboxylic acid and carbonyl group. The small peak at 1249 cm⁻¹ can be assigned to the C–N of the aliphatic amine group. The presence of the intense peak of the C=O stretching mode indicated the presence of a carboxylic group in the material bound to the GNPs. The lower wavenumbers confirmed that the represented functional groups combined with the GNPs and reduction occurred on that particular surface. Similarly, Sandt et al.¹⁹ reported that the FTIR spectrum of another reference strain of K. pneumoniae displayed peaks within the ranges of 763–775, 1053–1085 and 1555–1595 cm⁻¹. Honary et al.¹⁵ pointed out that the FTIR spectrum of the nanoparticles indicates the presence of various chemical groups, one of which is an amide. The presence of –COO–, which is possibly due to amino acid residues, may indicate that protein co-exists with the GNPs. An amide I band was observed at 1630 to 1650 cm⁻¹. This was further confirmed by the band at 3406–3412 cm⁻¹. The band at 1626 cm⁻¹ corresponds to amide I due to the carbonyl stretch in proteins.

Fig. 3 FT-IR spectrum of GNPs synthesized by K. pneumoniae.

XRD analysis is mainly undertaken to study the crystalline nature of nanoparticles. The intensive diffraction peak at the 2θ value of 38.20° from the (111) lattice plane of face centered cubic (fcc) gold unequivocally indicates that the particles were made of pure gold, which was broad, whereas the (200) plane was less distinct (2θ = 44.4°). Two additional broad bands were observed at 64.60° (2θ) and 77.60° (2θ) and they correspond to the (220) and (311) planes of gold, respectively (Fig. 4 and Table 1). In the obtained spectrum, the Bragg peak positions and their intensities were compared with the standard JCPDS files (file no. JCPDS 4-0783). The obtained GNPs were found to have an average size of 26 nm with a cubic structure. The fraction between the intensity of the (200), (220) and (311) diffraction peaks was much lower, which suggests that the (111) plane is the predominant orientation.²⁰ Likewise, Prema and Thangapandiyan²¹ reported that the intensive diffraction peaks at 2θ value of 38.38°, 44.7°, 64.5° and 77.3° corresponding to (111), (200), (220) and (311) lattice plane of face centered cubic (fcc) for unstabilized and stabilized AuNPs respectively. Senapati²² reported that the GNPs produced by Fusarium oxysporum had intense peaks at 38° (111), 45° (200), 67° (220) and 78° (311).

Fig. 4 X-ray diffraction pattern of GNPs synthesized by K. pneumoniae.

Table 1 X-ray diffraction peak list of the GNPs synthesized by K. pneumoniae

Pos. [° 2Th.]	Height [cts]	FWHM left [° 2Th.]	d-Spacing [Å]	Rel. int. [%]
38.20(1)	83(24)	0.33(3)	2.35380	100.00
40.93(7)	9(9)	0.3(2)	2.20314	11.39
44.4(1)	34(30)	0.4(1)	2.03912	40.54
64.60(9)	38(34)	1(1)	1.44149	45.41
77.6(2)	24(80)	1(1)	1.22933	29.50

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The morphology of the synthesized GNPs was observed under a scanning electron microscopy (Fig. 5). The scanning electron micrograph indicated that the synthesized particles were small in size and almost spherical in shape and some of them were aggregated. Similarly, Biradar and Lingappa²³ revealed that the surface morphology of GNPs adhere to the surface in a scaly pattern. They also observed that smaller sized particles were almost spherical in shape and some of them were aggregated.

Fig. 5 Scanning electron micrograph of the synthesized GNPs.

Two and three dimensional images of the GNPs were visualized via AFM (Fig. 6 and 7). The images revealed that the synthesized nanoparticles were in the form of spheres. The GNPs were formed in several different sizes, ranging from small to large nanoparticles (10–40 nm). AFM data obtained in the present study revealed that the 3D profile of the GNPs showed strong shape control with a size of around 10–15 nm. Similarly, Srivastava et al.²⁴ reported that the 2D profile obtained by AFM for their GNPs suggested strong shape control with a size of around 50 nm. This strong shape control indicated that apart from the reducing proteins present in the membrane bound fraction (MBF), certain organic groups must be acting as stabilizing agents.

Fig. 6 Two dimensional AFM image of the GNPs synthesized by K. pneumoniae.

Fig. 7 Three dimensional AFM image of the GNPs synthesized by K. pneumoniae.

The antibacterial activity of the GNPs was investigated against human bacterial pathogens, such as E. coli, S. epidermidis, S. aureus, P. aeruginosa, and B. subtilis, and the inhibitory zone (mm) result is presented in Table 2. The GNPs gave the highest zone of inhibition (25.68 mm) against E. coli, whereas the lowest zone of inhibition (18.70 mm) was recorded against S. aureus as an indicator strain. Similarly, effective antimicrobial activity against E. coli and S. aureus using GNPs was reported earlier by Kim et al.²⁵

Table 2 Zone of inhibition (mm) of the GNPs synthesized by K. pneumoniae against selected bacterial pathogens^a

Bacterial pathogens	Zone of inhibition (mean ± SD)
a Each value is the mean ± SD of five individual replicates.
E. coli	25.68 ± 1.50
S. epidermidis	20.70 ± 1.25
S. aureus	18.70 ± 1.22
P. aeruginosa	20.90 ± 0.92
B. subtilis	20.08 ± 1.01

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The result on the synergistic effect of the GNPs synthesized by K. pneumoniae is given in Table 3. It revealed that a distinct difference is observed between the inhibitory zones by antibiotics with and without GNPs. An enhanced zone of inhibition was observed and it was increased from 9 to 20 mm when the GNPs were incorporated with streptomycin antibiotics against S. aureus. However, the synergistic activity between the GNPs and ampicillin, gentamycin, kanamycin, streptomycin and vancomycin was subsequently shown to be greater against E. coli and P. aeruginosa than against S. aureus. The highest fold increase (4.06) was observed against P. aeruginosa, followed by a 3.52 fold increase, which was observed while using the combination of GNPs and streptomycin against B. subtilis. Two way ANOVA for the data on the fold increase of the GNPs against the selected bacterial pathogens revealed that the obtained data are statistically significant between antibiotics with GNPs and bacterial pathogens (F = 13.24 and 4.47; P < 0.05). The zone of inhibition showed an increase in activity index for all the cases with a range between 1.10 and 2.25. The highest activity index (3.21) was observed against E. coli using amoxicillin as the standard antibiotic, followed by 2.61 activity index, which was observed against S. aureus compared with streptomycin (Table 4). Two way ANOVA for the data on the activity index of the GNPs against selected bacterial pathogens revealed that the obtained data are statistically significant between antibiotics with GNPs and bacterial pathogens (F = 11.39 and 3.45; P < 0.05). The present findings corroborate the report of Shahverdi et al.,²⁶ who stated that there was an increase in the synergistic effects of penicillin G, amoxycillin, erythromycin, clindamycin and vancomycin in combination with mycosynthesized GNPs against E. coli, P. aeruginosa, and S. aureus. Fayaz et al.²⁷ also reported an increase in the antibacterial activity of ampicillin, kanamycin, erythromycin and chloramphenicol in combination with AuNPs against S. typhi, E. coli, S. aureus and Micrococcus luteus. Similarly, Agnihotri et al.²⁸ suggested that combined antibiotic therapy produces synergistic effects in the treatment of bacterial infections and has been shown to delay the emergence of antimicrobial resistance.

Table 3 Synergistic effect of antibiotics in combination with or without GNPs against selected human bacterial pathogens^a

Pathogens	Antibiotics (μg per disc)	Zone of inhibition (mm)		Increased zone size (mm)
		Antibiotics alone	Antibiotics + GNPs
a Each value is the mean ± SD of five individual replicates.b Values in parentheses indicate the fold increase of the zone of inhibition.
E. coli	Amoxycillin (30 μg)	8.00 ± 0.63	13.00 ± 0.71	5.00 ± 1.41 (1.640 ± 0.013)^b
	Amikacin (30 μg)	11.00 ± 1.41	17.00 ± 1.41	6.00 ± 1.79 (1.390 ± 0.007)
	Streptomycin (10 μg)	10.00 ± 0.63	16.00 ± 1.27	6.00 ± 1.41 (1.560 ± 0.012)
	Vancomycin (30 μg)	16.00 ± 1.27	19.00 ± 0.89	3.00 ± 1.26 (0.410 ± 0.010)
S. epidermidis	Amoxycillin (30 μg)	9.00 ± 1.41	14.00 ± 1.41	5.00 ± 1.79 (1.420 ± 0.007)
	Amikacin (30 μg)	12.00 ± 1.26	16.00 ± 1.27	4.00 ± 1.41 (0.790 ± 0.006)
	Streptomycin (10 μg)	10.00 ± 0.63	16.00 ± 1.41	6.00 ± 1.79 (1.560 ± 0.013)
	Vancomycin (30 μg)	18.00 ± 1.60	22.00 ± 1.79	4.00 ± 1.41 (0.490 ± 0.005)
S. aureus	Amoxycillin (30 μg)	9.00 ± 1.41	18.00 ± 1.60	9.00 ± 0.63 (3.000 ± 1.337)
	Amikacin (30 μg)	10.00 ± 0.63	18.00 ± 1.79	8.00 ± 0.89 (2.240 ± 0.008)
	Streptomycin (10 μg)	9.00 ± 0.63	20.00 ± 2.83	11.00 ± 1.41 (3.040 ± 0.009)
	Vancomycin (30 μg)	17.00 ± 1.41	23.00 ± 1.26	6.00 ± 1.79 (0.830 ± 0.004)
P. aeruginosa	Amoxycillin (30 μg)	8.00 ± 0.63	18.00 ± 1.79	10.00 ± 0.63 (4.060 ± 0.008)
	Amikacin (30 μg)	11.00 ± 1.41	17.00 ± 1.41	6.00 ± 1.41 (1.390 ± 0.007)
	Streptomycin (10 μg)	8.00 ± 0.89	18.00 ± 1.60	10.00 ± 0.89 (4.060 ± 0.005)
	Vancomycin (30 μg)	14.00 ± 1.41	16.00 ± 1.27	2.00 ± 1.26 (0.310 ± 0.007)
B. cereus	Amoxycillin (30 μg)	10.00 ± 1.79	19.00 ± 0.89	9.00 ± 0.63 (2.610 ± 0.014)
	Amikacin (30 μg)	12.00 ± 1.26	19.00 ± 0.63	7.00 ± 0.89 (1.510 ± 0.007)
	Streptomycin (10 μg)	8.00 ± 0.89	17.00 ± 1.41	9.00 ± 1.41 (3.520 ± 0.014)
	Vancomycin (30 μg)	14.00 ± 1.41	20.00 ± 2.83	6.00 ± 1.79 (1.040 ± 0.006)

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Table 4 Activity index of GNPs synthesized by K. pneumoniae against selected human bacterial pathogens in comparison with selected standard antibiotics^a

Antibiotics	Activity index/pathogens
	E. coli	S. epidermidis	S. aureus	P. aeruginosa	B. cereus
a Each value is the mean ± SD of five individual replicates.
Amoxycillin (30 μg)	3.210 ± 0.010	2.300 ± 0.126	2.080 ± 0.009	2.610 ± 0.010	2.010 ± 0.013
Amikacin (30 μg)	2.330 ± 0.006	1.730 ± 0.006	1.870 ± 0.014	1.900 ± 0.048	1.670 ± 0.017
Streptomycin (10 μg)	2.570 ± 0.015	2.070 ± 0.015	2.080 ± 0.029	2.610 ± 0.006	2.510 ± 0.010
Vancomycin (30 μg)	1.610 ± 0.007	1.150 ± 0.006	1.100 ± 0.049	1.490 ± 0.007	1.430 ± 0.011

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Conclusions

The overall results clearly emphasize that the biobased approach towards the synthesis of GNPs has many advantages such as ease with which the process can be scaled up and economic viability. The applications of nanoparticles in the medical and other fields make this method potentially useful for the large-scale synthesis of other inorganic nanomaterials. Narrow size distribution and small nanosized GNPs also offer advantages for self-assembled monolayer formation and enhanced surface areas. Gold colloidal solutions are biologically well suited and have the potential to be used in medical and pharmaceutical applications due to their homologous size distribution.

Experimental section

Chemicals and cultures

Gold chloride (chloroauric acid; HAuCl₄), nutrient broth and nutrient agar were purchased from Himedia (P) Ltd., Mumbai, as starting materials without further purification. Sterile MilliQ water was used throughout the experiment. The microorganisms used in the experiment are K. pneumoniae (MTCC-4030), E. coli (MTCC-4296), S. epidermidis (MTCC-435), S. aureus (MTCC-3160), P. aeruginosa (MTCC-424) and B. cereus (MTCC-619) were procured from the Microbial Type Culture Collection (MTCC), Chandigarh, India.

Fabrication of GNPs

A loopful of culture of freshly grown K. pneumoniae was inoculated in a 250 mL conical flask containing 100 mL sterile nutrient broth. The inoculated medium was incubated at 37 °C for 24 h in a rotary shaker at 120 rpm. The overnight culture broth was centrifuged at 6000 rpm for 10 minutes and the supernatant was used for the synthesis of the GNPs. To this cell-free supernatant, 1 mM gold chloride was added, mixed well, and the solution was incubated at 37 °C for 24 h.

Characterization of GNPs

Visual inspection. After 24 h of incubation, the preliminary detection of gold nanoparticles was done by visual observation of the colour change in the culture filtrate.

UV-visible spectroscopy analysis. UV measurements were carried out on a Shimadzu dual beam spectrophotometer (model UV – 1650 PC) operated at a resolution of 1 nm using deionized water as the reference. The colloidal gold solution was transferred into a quartz cuvette cell followed by immediate spectral measurements.

Fourier transform infrared (FTIR) spectroscopy analysis. The synthesized colloidal gold solution was measured using a Nicolet Impact 400FT-IR spectrophotometer in the spectral range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹. Powder samples for FTIR were prepared similar to the samples for powder diffraction measurements.¹⁵

X-ray diffraction analysis. Crystallographic information about the colloidal gold solution was obtained from its X-ray diffraction pattern. The XRD pattern was measured using the scanning mode of an X'pert PRO PANanalytical instrument operated at 40 kV and a current of 30 mA with Cu Kα radiation (λ = 1.5404 Å) and the 2θ scanning range of 30–80 °C at 2° min⁻¹.

The average size of the particles was estimated using the following Debye Scherrer equation:

D = Kλ/β cos θ

where D = thickness of the nanoparticles, K = constant, λ = wavelength of X-rays, β = width of half maxima of reflection and θ = Bragg angle.

Scanning electron microscopy (SEM) study. The determination of the nanoparticle morphology by high resolution analytical scanning electron microscopy (SEM) was performed using a JEOL-6390 SEM instrument. A thin film of the sample was prepared by placing a drop of the suspension of nanoparticles on a carbon coated copper grid, extra solution was removed using blotting paper and then the film on the SEM grid was allowed to dry by putting it under a mercury lamp for 5 min. Sample surface images were taken at different magnifications.

Atomic force microscopy (AFM) study. To determine their exact particle size the GNPs were characterized using Atomic Force Microscopy (Nanonics Imaging MN1000), which measures the atomic range of particles, in tapping mode.²⁹

Antibacterial activity of GNPs against human bacterial pathogens

Antibacterial assay. The antibacterial activity of the synthesized GNPs was evaluated using the agar well diffusion method proposed by Nithya et al.³⁰ Pure cultures of selected human pathogenic bacteria were subcultured individually in nutrient broth for 12 h at 37 °C. A 20 mL volume of sterile Mueller Hinton Agar medium was poured into each petriplate and each bacterial strain was swabbed uniformly into plates using sterile cotton swabs. Wells of 6 mm diameter were made onto each bacterium inoculated agar plate using a sterile gel puncher. 100 μL of GNPs suspension was introduced into the corresponding wells. Bactericidal activity was determined by the clear inhibition zone around the sample loaded well after incubation of the plates overnight at 37 °C.

Synergistic effect of GNPs. The synergistic effect of the GNPs was determined using the disc diffusion method by Vadivel and Suja.³¹ To determine the synergistic effect, four standard antibiotic discs, vancomycin, streptomycin, amikacin and amoxicillin, were each impregnated individually with 100 μL of freshly prepared GNPs and were placed onto Mueller Hinton Agar medium inoculated with individual test organisms. Standard antibiotic discs alone were used as positive controls. The plates were incubated overnight at 37 °C. After incubation, the result was recorded by measuring the inhibitory zone diameter (mm).

Assessment of increase in fold area of zone of inhibition. The increase in fold area was assessed using the method by Birla et al.³² It was assessed by calculating the mean surface area of the inhibition zone generated by an antibiotic alone and in combination with the GNPs using the following equation:

Fold increase = b² − a²/a²

where a & b are the zone of inhibition for the antibiotic alone and antibiotic with gold nanoparticles, respectively.

Activity index. The Activity index of the synthesized GNPs was calculated according to the method of Singariya et al.³³ The inhibition zones were measured and compared with the standard reference antibiotics. The activity index for each sample was calculated by using the formula:

Statistical analysis

The data obtained in the present study are expressed as mean ± SD and were analysed using two-way ANOVA at 5% level of significance using the computer software STATISTICA 06 (Statosoft, Bedford, UK).

Acknowledgements

The authors thank V. H. N. S. N. College Managing Board, Virudhunagar for providing facilities to complete the experiment in a successful manner.

Notes and references

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