Environment Health Techniques Antibiofilm activity of silver nanoparticles against P. aeruginosa
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Research Paper Gum arabic capped-silver nanoparticles inhibit biofilm formation by multi-drug resistant strains of Pseudomonas aeruginosa Mohammad Azam Ansari1, Haris Manzoor Khan1, Aijaz Ahmed Khan2, Swaranjit Singh Cameotra3, Quaiser Saquib4 and Javed Musarrat5 1
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Nanotechnology and Antimicrobial Drug Resistance Research Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, U.P., India Department of Anatomy, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, U.P., India Institute of Microbial Technology (IMTECH-CSIR), Chandigarh, India Quaiser Saquib, DNA Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Department of Ag. Microbiology, Aligarh Muslim University, Aligarh, U.P., India
Clinical isolates (n ¼ 55) of Pseudomonas aeruginosa were screened for the extended spectrum b-lactamases and metallo-b-lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped-silver nanoparticles (GA-AgNPs) against the multi-drug resistant (MDR) biofilm forming P. aeruginosa. The GA-AgNPs were characterized by UV-spectroscopy, X-ray diffraction, and high resolution-transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA-AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA-AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A-fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA-AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA-AgNPs at 50 mg ml1 has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA-AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA-AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings. Abbreviations: EPS – exopolysaccharides; GA-AgNPs – gum arabic-silver nanoparticles; CLSM – confocal laser scanning microscopy; SEM – scanning electron microscopy; HR-TEM – high resolution-transmission electron microscopy; ESBL – extended spectrum b-lactamases; MBL – metallo-b-lactamases; ConA-FITC – concanavalin A-f luorescein isothiocyanate; PI – propidium iodide; XRD – X-ray diffraction; MIC – minimum inhibitory concentration; MBC – minimum bactericidal concentration; CRA – Congo red agar; TM – tube method; TCP – tissue culture plate Keywords: GA-AgNPs / EPS / CLSM / SEM / ESBL / MBL / ConA-FITC Received: September 15, 2013; accepted: December 6, 2013 DOI 10.1002/jobm.201300748
Introduction Correspondence: Mohammad Azam Ansari, Nanotechnology and Antimicrobial Drug Resistance Research Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, U.P., India E-mail: [email protected] Phone: þ91 571 2720382 Fax: þ91 571 2720382 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Increased resistance of bacteria to antibiotic therapy is an emerging global health concern. In the last few decades, bacteria have shown to develop resistance to a majority of commercially available antibiotics, whereas the number of new antibiotics released or those expected
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to enter the market are limited. Therefore, infections resulting from microbial biofilm formation remain a perceptible threat, particularly in patients with chronic wound infections [1–3]. Most of the wound infections are polymicrobial in nature and caused by more than one species of bacteria or fungi [4], often including the Grampositive Staphylococcus aureus, S. epidermidis, and Gramnegative Pseudomonas aeruginosa [5]. These organisms are known to exhibit quorum sensing and produce strong biofilms [6]. Biofilms are the complex communities formed by a group of microorganisms, which secrete a polysaccharide matrix that retains nutrients for the constituent cells and protects them from the immune response and antimicrobial agents [7]. The biofilm matrix precludes the penetration of antibiotics and prevents them from reaching the embedded cells [8]. The protective action of b-lactamases in impairing the penetration of b-lactams in the biofilm has been demonstrated by Hoiby et al. [9]. The diffusion barrier in biofilms plays a significant role in b-lactamase producing P. aeruginosa, which hydrolyze the b-lactam antibiotics before it reaches to the bacterial cells in biofilm matrix [10, 11]. Thus, the biofilm producing bacteria, which also express higher levels of chromosomal b-lactamase would be exposed to a reduced concentration of b-lactam antibiotics, due to accumulation of the enzyme in the polysaccharide matrix. The extracellular b-lactamase would inactivate the antibiotic as it penetrates, thereby, protecting the deeper-lying cells [12]. Also, the sub-MIC concentrations of b-lactam antibiotics have been shown to induce increased levels of alginate synthesis in P. aeruginosa biofilms [13], and also enhance the biofilm matrix of some slime-producing coagulase-negative staphylococci [14]. Thus, killing the bacteria in a biofilm may require up to 1000 times greater antibiotic doses compared to a dose lethal to planktonic cells in suspension [15]. In order to choose appropriate antimicrobial agents and to optimize the dosing strategy on a case-by-case basis, it is necessary to improve our understanding of the interaction between various antimicrobial agents and biofilm forming bacteria. Conventional antimicrobial agents are known to develop multiple drug resistance and may also cause toxic side effects. Besides, the development of new antibiotics is cost, labor and time intensive. Therefore, lately the non-traditional antibiotic agents have stimulated tremendous interest in overcoming resistance that is developed by several pathogenic microorganisms against most of the commonly used antibiotics. Indeed, several classes of antimicrobial nanoparticles (NPs) and nanosized carriers for antibiotics delivery have proven their effectiveness for treating ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
infectious diseases caused by multi-drug resistant (MDR) bacteria [16]. Antimicrobial NPs offer many distinctive advantages in reducing acute toxicity, overcoming resistance, and lowering cost compared to conventional antibiotics [17, 18]. Moreover, the NPs are retained much longer in the body than small molecule antibiotics, which could be beneficial for achieving sustained therapeutic effects. High surface area to volume ratios and unique chemico-physical properties of various nanomaterials are suggested to contribute to effective antimicrobial activities [18]. It has also been demonstrated that naturally occurring bacteria do not develop antimicrobial resistance to metal NPs [19]. The inherent resistance of biofilms to antibacterial drugs and their pervasive involvement in implantrelated infections has prompted this study to investigate the role of gum arabic (GA) capped-silver nanoparticles (GA-AgNPs), as an effective surface coating antimicrobial agent, for inhibition of bacterial growth and colonization. Gum arabic coating of AgNPs stabilizes the NPs against aggregation, which impact the transport and toxicity of the NPs [20]. A correlation has already been established between stability of AgNPs against aggregation and the minimum concentration that inhibits the growth of a variety of bacteria [21]. Moreover, a strong association has been reported between nanoparticle aggregation and loss of an inhibitory effect on bacterial growth [22]. Yin et al. [20] demonstrated a much stronger inhibitory effect of GA-AgNPs on the growth of Lolium multiflorum, a common grass, compared to the equivalent dose of Ag ions added as AgNO3. To the best of our understanding, the impact of GA-AgNPs on multidrug resistant bacteria P. aeruginosa has not been thoroughly investigated. Therefore, the extended spectrum b-lactamase (ESBL) and metallo-b-lactamase (MBL) positive MDR isolates of P. aeruginosa have been used to assess the impact of GA-AgNPs, as nanoantibiotics on these isolates, with the aim to elucidate the (i) antibacterial potential of GA-AgNPs on the MDR biofilm producers, (ii) extent of structural and functional damage to the cells, and (iii) biofilm inhibition on the surface of plastic catheters.
Materials and methods Characterization GA-AgNPs A stock solution (200 mg ml1) of commercially available gum arabic stabilized silver nanoparticles (GA-AgNPs) (Cat No. A-03001) with particle size 5–10 nm were procured from the Nanoparticle Biochem, Inc. Columbia, USA.
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Antibiofilm activity of silver nanoparticles against P. aeruginosa
UV–visible spectral analysis The GA-AgNPs were characterized by UV–VIS spectroscopy, as described earlier by Musarrat et al. [23]. The suspension of GA-AgNPs in deionized Milli-Q water was subjected to sonication for 15 min. The spectrum of the surface plasmon resonance (SPR) of GA-AgNPs in aqueous suspension was recorded at wavelengths between 200 and 800 nm by use of a UV–Vis Cintra 10e GBC (Victoria, Australia). High-resolution transmission electron microscopy (HR-TEM) analysis of GA-AgNPs The size and morphology of the NPs were analyzed by high resolution-transmission electron microscopy (HRTEM). Samples were prepared by placing a drop of diluted suspension of NPs on carbon coated copper grid, and allowed to dry by evaporation at ambient temperature. The samples were kept in a desiccator until loaded on a specimen holder for analysis. The HR-TEM imaging and measurements were performed at an accelerating voltage of 200 kV on a transmission electron microscope (Technai G2, FEI, Electron Optics, USA). X-ray diffraction (XRD) analysis X-ray diffraction (XRD) analysis of GA-AgNPs was performed following the procedure described by Musarrat et al. [23]. The XRD pattern of GA-AgNPs was recorded by Bruker D8 diffractometer using Cu Ka radiation (l ¼ 1.54056 A) in the range of 20° 2u 80° at 40 keV. The lattice parameters were calculated by the PowderX software. The particle size (D) of the sample was calculated using the Scherrer’s relationship: D¼
0:9l Bcos u
where l is the wavelength of X-ray, B is the broadening of the diffraction line measured half of its maximum intensity in radians and u is the Bragg’s diffraction angle. The particle size of the sample was estimated from the line width of the (111) XRD peak. Bacterial strains A total of 55 isolates of P. aeruginosa were obtained from the frozen culture stocks of the Department of Microbiology, Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University (AMU), Aligarh, India. The isolates were originally isolated from the pus/wounds samples of the registered patients at JNMC, a tertiary care hospital at AMU, Aligarh. The identified and characterized isolates have been stored as frozen permanents (glycerol cultures) at 80 °C. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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In vitro antibiotic susceptibility test Antibiotic sensitivity test was performed on MullerHinton agar (MHA) based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [24], by the Kirby–Bauer disc diffusion method, with the following antibiotics: amikacin (AK, 30 mg), cefoperazone (Cs, 30 mg), cefoperazone-sulbactam (CFS, 75/10 mg), cefotaxime (Ce, 30 mg), ceftriaxone (Ci, 30 mg), ceftazidime (Ca, 30 mg), ceftazidime-clavulanate (Cac, 30/10 mg), chloramphenicol (C, 30 mg), gatifloxacin (Gf, 5 mg), gentamicin (G, 30 mg), tobramycin (Tb, 10 mg), piperacillin (Pi, 100 mg), piperacillin-tazobactam (Pt, 100/10 mg), ofloxacin (Of, 5mg), oxacillin (Ox, 5 mg), azithromycin (Az, 30 mg), amoxycillin (AMX, 10mg), co-trimoxazole (Co, 25 mg), levofloxacin (Le, 5 mg), ciprofloxacin (Cp, 5 mg), imipenem (I, 10 mg), cefepime (Cpm, 30 mg), cefepimetazobactam (CPT, 30/10 mg), ticarcillin (Ti, 75 mg), ticarcillin-clavulanate (Tc, 75/10 mg) obtained from Hi-Media, Mumbai, India. Screening of extended spectrum b-lactamases (ESBLs) The clinical isolates were thawed and sub-cultured. Freshly grown culture of each isolate was separately plated on MHA plates for screening of ESBL production. Antibiotic discs of cefotaxime (30 mg), ceftazidime (30 mg) and ceftriaxone (30 mg) were placed on the MHA plates. After 24 h of incubation, the growth inhibition was observed. Diameter of the zone of growth inhibition was measured and interpreted according to CLSI criteria [24]. The isolates with reduced sensitivity against two of four drugs mentioned above were interpreted as suspected ESBL producers. Double disk synergy test (DDST) Bacterial isolates that have exhibited resistance to the third generation cephalosporins were further verified for ESBL production. Phenotypic confirmation of these provisional ESBL producers was done by testing the sensitivity of ceftazidime and cefotaxime alone and in combination with clavulanic acid (10 mg), as recommended by CLSI [24]. The cefotaxime (30 mg) and cefotaxime-clavulanic acid (30 mg/10 mg) discs were placed almost 20 mm apart on a MHA plate. Similarly, the ceftazidime (30 mg) and ceftazidime-clavulanic acid (30 mg/10 mg) discs were also placed about 20 mm apart. The plates were incubated overnight at 37 °C. The increase in the zone diameter by >5 mm with the antibiotic in combination with clavulanic acid, as compared to the antibiotic alone was interpreted as positive for ESBL [24, 25]. Escherichia coli ATCC 35218 was used as reference control for b-lactamase detection.
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Detection of MBL by modified Hodge test (MHT) The MHT was performed following the method of Lee et al. [26]. In brief, 0.5 McFarland dilutions of the E. coli ATCC 25922 in 5 ml of broth or saline was prepared. A 1:10 dilution was streaked on to a MHA plate to form a lawn of bacterial growth. To this, imipenem (10 mg) susceptibility disk was placed in the center of the test area. Test organisms (all the ESBL, non-ESBL and MBL clinical isolates of P. aeruginosa) were individually streaked in a straight line from the edge of the disk to the edge of the plate. The plates were incubated overnight at 35 2 °C in ambient air for 16–24 h. Quality control of the carbapenem disks were performed according to CLSI guidelines. After 24 h, MHT positive tests showed a clover leaf-like indentation of the E. coli 25922, along the growth of test organisms, streaked within the disk diffusion zone. MHT negative test showed no growth of the E. coli 25922 along the test organisms (Supplementary Fig. S1).
wall and bottom of the tube. Simply the ring formation at the liquid interface was not indicative of biofilm formation. Tubes were examined and the biofilm formers were scored as () –negative, (þ) – weakly positive, (þþ) – moderately positive, and (þþþ) – strongly positive. Congo red agar method (CRA). Biofilm production by P. aeruginosa isolates was further established by CRA method following the method of Freeman et al. [29]. Freshly grown cultures were plated on BHI agar supplemented with 5% sucrose and Congo red (0.8 g L1). The plates were incubated aerobically for 24–48 h at 37 °C. Appearance of black colonies with a dry crystalline consistency indicated positive results. Weak slime producers usually remained pink, though occasional darkening at the centers of colonies was observed. Darkening of the colonies with the absence of dry crystalline colonial morphology indicated an indeterminate result.
Detection of biofilm formation
Evaluation of antibacterial activity of GA-AgNPs by MIC and MBC methods Minimal inhibitory concentrations (MICs). All clinical isolates of P. aeruginosa were grown overnight on MHA plates at 37 °C. The antimicrobial activity of GA-AgNPs was examined following the standard broth dilution method. The MIC was determined in Luria-Bertani (LB) broth (Hi-Media, Mumbai, India) using serial two-fold dilutions of GA-AgNPs in concentrations ranging from 180 to 1.40 mg ml1. Bacterial inoculums of 2 108 CFU ml1 were used in the experiments. The treated and untreated control samples were incubated for 24 h at 37 °C. The MIC was defined as the lowest concentration of antimicrobial agents that yielded no visible growth of the microorganisms. Minimal bactericidal concentrations (MBCs). For MBC determination, the aliquots of 50 ml from all tubes in which no visible bacterial growth was observed were seeded on the MHA plates not supplemented with GAAgNPs. The plates were then incubated for 24 h at 37 °C. The MBC endpoint is defined as the lowest concentration of antimicrobial agent that kills 100% of the initial bacterial population.
Tissue culture plate method (TCP). Biofilm formation by the isolates was screened by tissue culture plate (TCP) method [27]. Briefly, trypticase soy broth (TSB), 10 ml with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar. The broth was incubated at 37 °C for 24 h. The culture was further diluted 1:100 with a fresh medium and aliquots of 0.2 ml of culture were transferred to the wells of flat bottom microtitre plates. Steriled broth was used as a blank. The culture plates were incubated at 37 °C for 24 h. After incubation, the culture was removed and the wells were washed with 0.2 ml of phosphate buffer saline (PBS, pH 7.2) four times to remove free-floating bacteria. Biofilms, which remained adhered to the wells were fixed with 2% sodium acetate and stained with 0.1% crystal violet. Excess stain was washed with deionized water and plates were dried properly. Optical densities (OD) of the purple colored solution were recorded at 570 nm by use of an ELISA reader (Bio-Rad, Model 680, USA). Three independent experiments were performed in triplicate. Tube method (TM). A qualitative assessment of biofilm formation was performed following the method of Christensen et al. [28]. Briefly, the bacterial isolates were sub-cultured in 10 ml TSB with 1% glucose in 25 ml tubes for 24 h at 37 °C. The medium was discarded and the tubes were washed with 1 PBS (pH 7.3) and dried. The bacterial cells adhered to the tubes were then stained with crystal violet (0.1%) for 1 min. Excess stain was removed and tubes were again washed with deionized water. Tubes were then dried in inverted position and observed for biofilm formation. The isolates were considered as positive, when a visible biofilm lined the ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Scanning electron microscopy Biofilms were assessed as previously described with or without GA-AgNPs. Briefly, small pieces of plastic catheters were seeded in 5 ml of BHI (with 5% sucrose) in a 12-well microtiter plates. To this, 100 ml of exponentially grown cells of P. aeruginosa treated with 10, 20, 30, 40, and 50 mg ml1 of GA-AgNPs were added. The plates were incubated at 37 °C for 24 h. Cells without GA-AgNPs were used as control. The catheters were then
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removed from both the control and treated wells and gently washed with PBS, fixed with 2.5% glutaraldehyde. The fixed samples were again washed with PBS and dehydrated gently by passing through a series of ethanol solutions (30, 50, 70, 80, 95, and 100%), each for 10 min at room temperature. Cells were then mounted on the aluminum stubs and coated with gold. The topographic features of the biofilms were visualized by use of SEM (Carl Zeiss EVO 40, Germany) with accelerating voltage of 20 kV. Confocal laser scanning microscopy (CLSM) analysis Confocal laser scanning microscopy (CLSM) analysis of biofilms was performed following method as described by Banas et al. [30]. Uniform sized (2 1 mm2) blocks of plastic catheters were exposed to varying (10, 25, and 50 mg ml1) concentrations of GA-AgNPs for 12 h, by submerging in NPs suspension in a 12-well microtitre plate. The wells were washed and the untreated control and GA-AgNPs treated catheters were incubated with the cultures of ESBL and MBL positive biofilm producing representative isolates of P. aeruginosa for 24 h at 37 °C. The cells adhered and grown on the surface of catheters were then stained with 15 mM propidium iodide (PI; Product code 81845, Sigma–Aldrich) for 15 min at room temperature. After through washing with PBS, the cells were incubated with 50 mg ml1 of concanavalin Aconjugated fluorescein isothiocyanate (ConA-FITC, Product code 61761, Sigma–Aldrich) for 15 min at room temperature to stain the glycocalyx matrix green. The PI was excited at 540 nm and the emission was monitored at 608 nm. Similarly, the Con A-FITC was excited at 495 nm and monitored at an emission wave length of and 518 nm. Intact biofilms were examined non-destructively using a Fluoview FV1000 Espectral Olympus CSLM (Olympus Latin America, Miami, FL, USA) equipped with a UPlanSApo 100/1.40 oil UIS2 Olympus oil immersion lens. Statistical analysis MIC and MBC tests were performed in triplicate, and the results were expressed as the mean SD of the mean. Student’s t-test was used to compare these results. Values of p lower than 0.05 were considered significant.
Results Characterization of GA-AgNPs – UV–visible spectrophotometric analysis Fig. 1 shows the optical absorbance spectra of GA-AgNPs in aqueous medium. Exposure of GA-AgNPs to light ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Figure 1. UV–Vis spectra of GA-AgNPs.
leads to polarization of the free conduction electrons with respect to the much heavier ionic core of NPs, resulting in electron dipolar oscillation and appearance of a strong SPR band at 400 nm (Fig. 1). No absorption peak at the same wavelength range was observed in the AgNO3 solution in deionized Milli-Q water used as a control (data not shown). HR-TEM analysis of GA-AgNPs HR-TEM analysis revealed that the GA-AgNPs were predominantly spherical in shape. Significant variability in particle sizes was observed and the average size was estimated to be 7 nm (Fig. 2). The HR-TEM images also revealed that GA-AgNPs were not in aggregated state and appeared as monodispersed. XRD analysis of GA-AgNPs Fig. 3 shows the XRD pattern of GA-AgNPs. The XRD profile of GA-AgNPs exhibited four intense peaks in the spectrum of 2u values ranging from 20 to 80. The diffractions at 38°, 44.18°, 64.29°, and 77.19° can be indexed to the (111), (200), (220), and (311) planes of the face centered cubic (fcc) silver, respectively and suggest that the GA-AgNPs are crystalline in nature (JCPDS File No 03-0921). Antibiotic susceptibility testing The results representing the antibiotic resistance pattern of P. aeruginosa are shown in Fig. 4. The data revealed the multiple drug resistant P. aeruginosa, where the isolates exhibited the frequency of resistance against cephalosporins as: cefotaxime (99%), ceftazidime (95%), ceftazidime– clavulanate (88%), ceftriaxone (75%), and cefoperazone (60%), besides significantly greater resistance against the classical antibiotics like amoxicillin, oxacillin, co-trimoxazole, and chloramphenicol. The results of DDST and MHT
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Figure 2. HR-TEM micrographs of GA-AgNPs at (a) lower and (b) higher magnification.
assays indicating the ESBL and MBL production by the representative isolates are shown in the Supplementary Fig. S1. Out of a total 55 isolates of P. aeruginosa, 42 (76.3%) were ESBL producers, 9 (16.4%) were non-ESBL, and 4 (7.3%) were found to be MBL positive. Screening of biofilm formation The qualitative results obtained on Congo red agar (CRA) are shown in Table 1. Out of 55 isolates of P. aeruginosa tested for biofilm formation around 33 (60%) isolates produced black colonies. Amongst them, only 15 (27.2%) isolates formed black colonies with dry crystalline consistency, indicative of biofilm formation. About 18 (32.2%) isolates formed black colonies but were not dry and crystalline and were regarded as indeterminate for biofilm formation. These isolates were also taken as negative for biofilm formation along with other 22 (40%) isolates, which produced pink colonies on CRA plates. The detection of biofilm formation by tube method (TM) further reaffirmed that around 12 (21.82%) were strong
Figure 3. XRD analysis of GA-AgNPs. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
biofilm producers, whereas 20 (36.36%) were tagged as moderate (Table 1). Furthermore, the results of quantitative TCP assay categorized the isolates as highly proficient biofilm producers (strongly adherent), moderate biofilm producers (moderate adherent) and nonbiofilm producers (weak/non-adherent). The results demonstrated 10 (18.2%) isolates as strongly positive as compared to moderate adherent 28 (50.9%) and weak/ non-biofilm producers 17 (30.9%; Table 1). Evaluation of antibacterial activity of GA-AgNPs by MIC and MBC method Table 2a shows the MIC and MBC values of GA-AgNPs dispersion tested against ESBL positive and ESBL negative isolates of P. aeruginosa. About 23.81% ESBL positive isolates exhibited the MIC of 11.25 mg ml1; whereas 71.42% shows the MIC of 22.5 mg ml1. Also, the 33.33% non-ESBL isolates exhibited the MIC of 11.25 mg ml1 and 44.44% shows the MIC of 22.5 mg ml1. Similarly, the Table 2b shows the MIC and MBC of MBL producing P. aeruginosa. The MBC values for 76.89% ESBL positive and 77.78% negative was found to be 45 mg ml1. The lowest MIC for ESBL, non-ESBL, and MBL P. aeruginosa was determined to be 11.25 mg ml1, indicative of a very strong bacteriostatic activity. However, the MBC was found to be in the range of 11.25–45 mg ml1, which reflects an effective bactericidal activity of GA-AgNPs. Characterization of antibiofilm activity of GA-AgNPs on CRA plates GA-AgNPs induced inhibition of biofilm formation by P. aeruginosa was tested on brain heart infusion agar supplemented with Congo red (BHIC). The colonies in untreated control samples appeared as dry crystalline black colonies, indicating the production of exopolysaccharides (EPS), which is the prerequisite for the
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Figure 4. Antibiotic resistance patterns of clinical isolates of P. aeruginosa against different groups of antibiotics.
formation of biofilm. However, under treatment conditions in presence of increasing concentration of GAAgNPs on BHIC, the organisms did not survived. GAAgNPs at concentration of 20 mg ml1 inhibited the synthesis of EPSs, and precluded the formation of dry crystalline black colonies (Supplementary Fig. S2). At higher concentrations of GA-AgNPs, around 95% of bacterial growth was inhibited. As the EPSs synthesis is arrested, the cells do not form biofilm. The results
Table 1. Assessment of biofilm formation by P. aeruginosa isolates (n ¼ 55) by different methods. Methods
Biofilms formers isolates (%)
CRA Colony appearance Pink/red (negative) Black colonies without dry crystalline consistency (indeterminate) Black colonies with dry crystalline consistency (positive)
22 (40) 18 (32.73) 15 (27.27)
TM Biofilm production 0/1 (weak/non) 2 þ (moderate) 3 þ (strong)
23 (41.82) 20 (36.36) 12 (21.82)
Biofilm production Weak/non (0.24 OD)
17 (30.9) 28 (50.9) 10 (18.2)
TCP
CRA, Congo red agar; TM, tube method; TCP, tissue culture plate. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
demonstrated that GA-AgNPs at concentration of 30 mg ml1 significantly arrested the biofilm formation without affecting the cell viability, whereas at a concentration of 60 mg ml1, the biofilm formation was completely blocked and the bacterial growth was completely ceased (Supplementary Fig. S2). Characterization of antibiofilm activity of GA-AgNPs by CLSM CLSM analysis of biofilms formation was studied in the representative ESBL and MBL producing isolates of P. aeruginosa (Figs. 5 and 6). Bacterial cells and the surrounding glycocalyx matrix, which is indicative of bacterial biofilm formation, were observed by double staining using PI and ConA-FITC. Bacterial cells stained red with PI were easily identified by their size and morphologic features. ConA-FITC binds to mannose residues resulting in green staining and indicating the presence of a bacterial glycocalyx. The presence of dark areas within the biofilm is attributed to the water channels, the heterogeneity of matrix and the types of EPSs within the biofilm. Superimposed CLSM images with PI (red) and Con A-FITC (green) fluorescent intensities yielded yellow color, which reflects the extracellular polysaccharides being produced as a capsular component in biofilm. Also, the interconnected bacteria were found encased in a scaffolding network composed of extracellular matrix, suggesting a 3dimensional architecture of biofilm formations. The micrographs in Figs. 5 and 6(Panel a) exhibit the biofilm formation in absence of GA-AgNPs with a definite architecture. However, in the presence of GA-AgNPs
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Table 2. MIC and MBC determination of GA-AgNPs tested against ESBL, non-ESBL, and MBL P. aeruginosa (n ¼ 55) isolates. (a) ESBL positive P. aeruginosa 42 (76.34%) 1
Non-ESBL P. aeruginosa 9 (16.36%) 1
Isolates
MIC (mg ml )
Isolates
MBC (mg ml )
Isolates
MIC (mg ml1)
Isolates
MBC (mg ml1)
23.81% 71.42% 4.76%
11.25 1.01 22.50 2.02 45.00 4.04
9.52% 14.28% 76.19%
11.25 1.01 22.50 2.02 45.00 4.04
33.33% 44.44% 22.22%
11.25 1.01 22.50 2.02 45.00 4.04
22.22% 77.78% –
22.50 2.02 45.00 4.04 –
(b) MBL producing P. aeruginosa 4 (7.3%) Isolates
MIC (mg ml1)
Isolates
MBC (mg ml1)
25.00% 75.00%
11.25 1.01 22.50 2.02
25.00% 75.00%
22.50 2.02 45.00 4.04
(Figs. 5b–d and 6b–d), a scanty growth with few cells was observed with no distinct pattern of arrangement. The cells also exhibited morphological deformation upon exposure with increasing concentrations of GA-AgNPs.
Maximum biofilm inhibition was noticed at concentration of 50 mg ml1 GA-AgNPs, and only few scattered cells were noticed on the surface of the solid matrix (Figs. 5c and d and 6c and d). Thus, the treatment of plastic
Figure 5. CLSM micrographs of ESBL P. aeruginosa biofilm. The panel (a) represents CLSM images of untreated control P. aeruginosa biofilm. Red color depicts the PI staining of bacterial nucleic acids and green fluorescent staining with ConA-FITC indicates the presence of EPSs. The panels (b–d) represent CLSM images of P. aeruginosa biofilm treated with 10 (b), 25 (c), and 50 (c) mg ml1 of GA-AgNPs. Magnification at 400. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 6. CLSM micrographs of MBL P. aeruginosa biofilm. The panel (a) represents CLSM images of untreated control P. aeruginosa biofilm. Red color depicts the PI staining of bacterial nucleic acids and green fluorescent staining with ConA-FITC indicates the presence of EPSs. The panels (b–d) represent CLSM images of P. aeruginosa biofilm treated with 10 mg ml1 (b), 25 mg ml1 (c), and 50 mg ml1 (c) of GA-AgNPs. Magnification at 400.
catheters with GA-AgNPs significantly restricted the colonization of ESBL and MBL bacterial cells, compared to the massive growth and biofilm formation in untreated control.
surface, resulting in inhibition of bacterial colonization on the surface of plastic catheters (Fig. 7b–f).
Discussion Characterization of antibiofilm activity of GA-AgNPs by SEM The effect of GA-AgNPs on biofilm formation by the ESBL and MBL positive isolates of P. aeruginosa on catheter was further confirmed by SEM. Figure 7a shows the untreated control cells of P. aeruginosa with normal cellular morphology, exhibiting a smooth cell surface. Under identical growth conditions, the cells treated with increasing concentrations of GA-AgNPs exhibited perceptible changes in cell morphology. The SEM images explicitly revealed GA-AgNPs induced damage with prominent indentations and depression on the cell ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Silver has long been used for its antimicrobial properties as its toxicity to microorganisms is greater than many other metals, while maintaining low toxicity to mammalian cells [31]. It has been shown that AgNPs are more efficient in mediating antimicrobial activity [32–36] than silver ions and as a result have been incorporated into wound dressings, medical devices, water purification systems, linings of washing machines, dish washers, refrigerators, toilet seats, and clothing [37]. In this study, we have assessed the antibacterial activity of highly stable and water soluble suspension of GA-AgNPs against
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Figure 7. SEM images of P. aeruginosa biofilms formed on the surface of catheters after 24 h of incubation. Panels are represented as: (a) Untreated, (b) treated with 10 mg ml1, (c) 20 mg ml1, (d) 30 mg ml1, (e) 40 mg ml1, and (f) 50 mg ml1 of GA-AgNPs.
the ESBL and MBL positive clinical isolates of P. aeruginosa, and determined their potential for biofilm inhibition by using sophisticated techniques, such as SEM and CLSM. In order to restrict the growth of biofilms, the antimicrobials must penetrate through the polysaccharide matrix to gain access to the embedded population of microbial cells. Our results demonstrated that the GAAgNPs can easily penetrate the biofilms and reduce its formation. The results of CRA assay revealed the antibiofilm efficacy of GA-AgNPs against the ESBL and MBL producers. At higher concentrations, the GA-AgNPs completely inhibited the bacterial growth and arrested the exopolysachharide synthesis and biofilm formation. Similar results have been reported by Kalishwaralal et al. [6] against the P. aeruginosa and S. epidermidis biofilms, where 100 nM of AgNPs resulted in 95–98% reduction in biofilm formation and Ansari et al. [38] against the clinical isolates of E. coli and Klebsiella spp. biofilms, where 50 mg ml1 of AgNPs resulted about 95% reduction in biofilm formation. Our SEM results also revealed that GA-AgNPs reduce the surface coverage and bacterial colonization, which ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
supports the observations of Ansari et al. [38] and Kostenko et al. [39]. However, the major limitation of SEM analysis is that the EPS within P. aeruginosa biofilms cannot be detected. The demonstration of bacterial biofilms is often challenging because of the problem in concurrent staining of both the bacterial cells and EPS. Also, the electron microscopy techniques require a dehydration process that reduces the total volume of the matrix and alters its architecture [40]. Thus, to demonstrate the presence of bacterial biofilms, it is important to detect both the bacteria and the EPS matrix simultaneously. For such an investigation, confocal scanning laser microscopy (CLSM) is an ideal tool for spatial resolution and non-destructive examination of the layers of biofilm at different depths, and helps in reconstructing a three-dimensional structure [41, 42]. The results obtained with the double-staining technique using CLSM, revealed GA-AgNPs induced cell death with no EPS matrix (green fluorescent cells) around P. aeruginosa cells and a disrupted 3-dimensional structure of the biofilm. This inhibitory effect of GA-AgNPs on the biofilm has been attributed to the malfunction of the
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water channels though out the biofilm. Water channels (pores) are present in all biofilms for nutrient transportation. GA-AgNPs may directly diffuse through the EPSs layer and impart antimicrobial action. Masurkar et al. [43] have reported the antibiofilm activity of AgNPs (32 nm) synthesized from Cymbopogan citratus. Similarly, Kostenko et al. [39] have also reported that acticoat nanocrystalline silver exhibit antibiofilm efficacy compared to aquacel silver and silverlon, and suggested that the silver concentration alone cannot account for the antibiofilm efficacy of the silver dressings. The reduction of the silver particle to the nanoscale level increases the relative surface area, which provides higher Agþ release rates than for elemental silver particles. It has been suggested that the silver ions bound to bacterial cells and EPS, interfere with intermolecular forces in biofilms and facilitate biofilm dispersion [44]. However, the exact mechanism of action of AgNPs on biofilms is yet to be elucidated. Overall, our results suggested that the GAAgNPs possesses an inherent capacity to attach and penetrate the bacterial membranes. The trans-membrane movement and intracellular NPs accumulation may trigger the release of silver ions, which supposedly causes oxidative stress and eventually leads to cell death.
Conclusion The effective antiseptic treatment of medical implants and surgical tools demands new approaches that target the biofilm forming MDR bacteria for control of infections. This study explicitly demonstrated that the potential of GA-AgNPs to inhibit the growth and colonization of MDR P. aeruginosa on plastic catheters. The GA-AgNPs are suggested as effective antimicrobial agent for protecting the surfaces of medical implants against pathogen colonization.
Acknowledgments The authors would like to acknowledge AIRF, Jawaharlal Nehru University, New Delhi, India, for SEM and CLSM and SAIF-DST, Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi, India, for HR-TEM observation. JM is grateful to the visiting professor program (VVP) of the King Saud University, Riyadh, SA for all the help and support in conducting this collaborative research work. Authors also would like to thank the Indian Council of Medical Research (ICMR) New Delhi-India, grant number 35/15/BMS-11 for their partial support and funding of this project. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Conflict of interest There is no conflict of interest between the authors.
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[38] Ansari, M.A., Khan, H.M., Khan, A.A., Cameotra, S.S. et al., 2013. Antibiofilm efficacy of silver nanoparticles against biofilm of extended spectrum b-lactamase isolates of Escherichia coli and Klebsiella pneumoniae. Appl. Nanosci., DOI: 10.1007/s13204-013-0266-1 [39] Kostenko, V., Lyczak, J., Turner, K., Martinuzzi, T.J., 2010. Impact of silver-containing wound dressings on bacterial biofilm viability and susceptibility to antibiotics during prolonged treatment. Antimicrob. Agents Chemother., 54, 5120–5131. [40] Akiyama, H., Hamada, T., Huh, W.K., Yamasaki, O. et al., 2003. Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of Bullous impetigo, Atopic dermatitis and Pemphigus foliaceus. Br. J. Dermatol., 148, 526–532. [41] Lawrence, J.R., Neu, T.R., 1999. Confocal laser scanning microscopy for analysis of microbial biofilms. Meth. Enzymol., 310, 131–144.
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Research Paper Gum arabic capped-silver nanoparticles inhibit biofilm formation by multi-drug resistant strains of Pseudomonas aeruginosa Mohammad Azam Ansari1, Haris Manzoor Khan1, Aijaz Ahmed Khan2, Swaranjit Singh Cameotra3, Quaiser Saquib4 and Javed Musarrat5 1
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Nanotechnology and Antimicrobial Drug Resistance Research Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, U.P., India Department of Anatomy, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, U.P., India Institute of Microbial Technology (IMTECH-CSIR), Chandigarh, India Quaiser Saquib, DNA Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Department of Ag. Microbiology, Aligarh Muslim University, Aligarh, U.P., India
Clinical isolates (n ¼ 55) of Pseudomonas aeruginosa were screened for the extended spectrum b-lactamases and metallo-b-lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped-silver nanoparticles (GA-AgNPs) against the multi-drug resistant (MDR) biofilm forming P. aeruginosa. The GA-AgNPs were characterized by UV-spectroscopy, X-ray diffraction, and high resolution-transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA-AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA-AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A-fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA-AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA-AgNPs at 50 mg ml1 has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA-AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA-AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings. Abbreviations: EPS – exopolysaccharides; GA-AgNPs – gum arabic-silver nanoparticles; CLSM – confocal laser scanning microscopy; SEM – scanning electron microscopy; HR-TEM – high resolution-transmission electron microscopy; ESBL – extended spectrum b-lactamases; MBL – metallo-b-lactamases; ConA-FITC – concanavalin A-f luorescein isothiocyanate; PI – propidium iodide; XRD – X-ray diffraction; MIC – minimum inhibitory concentration; MBC – minimum bactericidal concentration; CRA – Congo red agar; TM – tube method; TCP – tissue culture plate Keywords: GA-AgNPs / EPS / CLSM / SEM / ESBL / MBL / ConA-FITC Received: September 15, 2013; accepted: December 6, 2013 DOI 10.1002/jobm.201300748
Introduction Correspondence: Mohammad Azam Ansari, Nanotechnology and Antimicrobial Drug Resistance Research Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, U.P., India E-mail: [email protected] Phone: þ91 571 2720382 Fax: þ91 571 2720382 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Increased resistance of bacteria to antibiotic therapy is an emerging global health concern. In the last few decades, bacteria have shown to develop resistance to a majority of commercially available antibiotics, whereas the number of new antibiotics released or those expected
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to enter the market are limited. Therefore, infections resulting from microbial biofilm formation remain a perceptible threat, particularly in patients with chronic wound infections [1–3]. Most of the wound infections are polymicrobial in nature and caused by more than one species of bacteria or fungi [4], often including the Grampositive Staphylococcus aureus, S. epidermidis, and Gramnegative Pseudomonas aeruginosa [5]. These organisms are known to exhibit quorum sensing and produce strong biofilms [6]. Biofilms are the complex communities formed by a group of microorganisms, which secrete a polysaccharide matrix that retains nutrients for the constituent cells and protects them from the immune response and antimicrobial agents [7]. The biofilm matrix precludes the penetration of antibiotics and prevents them from reaching the embedded cells [8]. The protective action of b-lactamases in impairing the penetration of b-lactams in the biofilm has been demonstrated by Hoiby et al. [9]. The diffusion barrier in biofilms plays a significant role in b-lactamase producing P. aeruginosa, which hydrolyze the b-lactam antibiotics before it reaches to the bacterial cells in biofilm matrix [10, 11]. Thus, the biofilm producing bacteria, which also express higher levels of chromosomal b-lactamase would be exposed to a reduced concentration of b-lactam antibiotics, due to accumulation of the enzyme in the polysaccharide matrix. The extracellular b-lactamase would inactivate the antibiotic as it penetrates, thereby, protecting the deeper-lying cells [12]. Also, the sub-MIC concentrations of b-lactam antibiotics have been shown to induce increased levels of alginate synthesis in P. aeruginosa biofilms [13], and also enhance the biofilm matrix of some slime-producing coagulase-negative staphylococci [14]. Thus, killing the bacteria in a biofilm may require up to 1000 times greater antibiotic doses compared to a dose lethal to planktonic cells in suspension [15]. In order to choose appropriate antimicrobial agents and to optimize the dosing strategy on a case-by-case basis, it is necessary to improve our understanding of the interaction between various antimicrobial agents and biofilm forming bacteria. Conventional antimicrobial agents are known to develop multiple drug resistance and may also cause toxic side effects. Besides, the development of new antibiotics is cost, labor and time intensive. Therefore, lately the non-traditional antibiotic agents have stimulated tremendous interest in overcoming resistance that is developed by several pathogenic microorganisms against most of the commonly used antibiotics. Indeed, several classes of antimicrobial nanoparticles (NPs) and nanosized carriers for antibiotics delivery have proven their effectiveness for treating ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
infectious diseases caused by multi-drug resistant (MDR) bacteria [16]. Antimicrobial NPs offer many distinctive advantages in reducing acute toxicity, overcoming resistance, and lowering cost compared to conventional antibiotics [17, 18]. Moreover, the NPs are retained much longer in the body than small molecule antibiotics, which could be beneficial for achieving sustained therapeutic effects. High surface area to volume ratios and unique chemico-physical properties of various nanomaterials are suggested to contribute to effective antimicrobial activities [18]. It has also been demonstrated that naturally occurring bacteria do not develop antimicrobial resistance to metal NPs [19]. The inherent resistance of biofilms to antibacterial drugs and their pervasive involvement in implantrelated infections has prompted this study to investigate the role of gum arabic (GA) capped-silver nanoparticles (GA-AgNPs), as an effective surface coating antimicrobial agent, for inhibition of bacterial growth and colonization. Gum arabic coating of AgNPs stabilizes the NPs against aggregation, which impact the transport and toxicity of the NPs [20]. A correlation has already been established between stability of AgNPs against aggregation and the minimum concentration that inhibits the growth of a variety of bacteria [21]. Moreover, a strong association has been reported between nanoparticle aggregation and loss of an inhibitory effect on bacterial growth [22]. Yin et al. [20] demonstrated a much stronger inhibitory effect of GA-AgNPs on the growth of Lolium multiflorum, a common grass, compared to the equivalent dose of Ag ions added as AgNO3. To the best of our understanding, the impact of GA-AgNPs on multidrug resistant bacteria P. aeruginosa has not been thoroughly investigated. Therefore, the extended spectrum b-lactamase (ESBL) and metallo-b-lactamase (MBL) positive MDR isolates of P. aeruginosa have been used to assess the impact of GA-AgNPs, as nanoantibiotics on these isolates, with the aim to elucidate the (i) antibacterial potential of GA-AgNPs on the MDR biofilm producers, (ii) extent of structural and functional damage to the cells, and (iii) biofilm inhibition on the surface of plastic catheters.
Materials and methods Characterization GA-AgNPs A stock solution (200 mg ml1) of commercially available gum arabic stabilized silver nanoparticles (GA-AgNPs) (Cat No. A-03001) with particle size 5–10 nm were procured from the Nanoparticle Biochem, Inc. Columbia, USA.
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UV–visible spectral analysis The GA-AgNPs were characterized by UV–VIS spectroscopy, as described earlier by Musarrat et al. [23]. The suspension of GA-AgNPs in deionized Milli-Q water was subjected to sonication for 15 min. The spectrum of the surface plasmon resonance (SPR) of GA-AgNPs in aqueous suspension was recorded at wavelengths between 200 and 800 nm by use of a UV–Vis Cintra 10e GBC (Victoria, Australia). High-resolution transmission electron microscopy (HR-TEM) analysis of GA-AgNPs The size and morphology of the NPs were analyzed by high resolution-transmission electron microscopy (HRTEM). Samples were prepared by placing a drop of diluted suspension of NPs on carbon coated copper grid, and allowed to dry by evaporation at ambient temperature. The samples were kept in a desiccator until loaded on a specimen holder for analysis. The HR-TEM imaging and measurements were performed at an accelerating voltage of 200 kV on a transmission electron microscope (Technai G2, FEI, Electron Optics, USA). X-ray diffraction (XRD) analysis X-ray diffraction (XRD) analysis of GA-AgNPs was performed following the procedure described by Musarrat et al. [23]. The XRD pattern of GA-AgNPs was recorded by Bruker D8 diffractometer using Cu Ka radiation (l ¼ 1.54056 A) in the range of 20° 2u 80° at 40 keV. The lattice parameters were calculated by the PowderX software. The particle size (D) of the sample was calculated using the Scherrer’s relationship: D¼
0:9l Bcos u
where l is the wavelength of X-ray, B is the broadening of the diffraction line measured half of its maximum intensity in radians and u is the Bragg’s diffraction angle. The particle size of the sample was estimated from the line width of the (111) XRD peak. Bacterial strains A total of 55 isolates of P. aeruginosa were obtained from the frozen culture stocks of the Department of Microbiology, Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University (AMU), Aligarh, India. The isolates were originally isolated from the pus/wounds samples of the registered patients at JNMC, a tertiary care hospital at AMU, Aligarh. The identified and characterized isolates have been stored as frozen permanents (glycerol cultures) at 80 °C. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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In vitro antibiotic susceptibility test Antibiotic sensitivity test was performed on MullerHinton agar (MHA) based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [24], by the Kirby–Bauer disc diffusion method, with the following antibiotics: amikacin (AK, 30 mg), cefoperazone (Cs, 30 mg), cefoperazone-sulbactam (CFS, 75/10 mg), cefotaxime (Ce, 30 mg), ceftriaxone (Ci, 30 mg), ceftazidime (Ca, 30 mg), ceftazidime-clavulanate (Cac, 30/10 mg), chloramphenicol (C, 30 mg), gatifloxacin (Gf, 5 mg), gentamicin (G, 30 mg), tobramycin (Tb, 10 mg), piperacillin (Pi, 100 mg), piperacillin-tazobactam (Pt, 100/10 mg), ofloxacin (Of, 5mg), oxacillin (Ox, 5 mg), azithromycin (Az, 30 mg), amoxycillin (AMX, 10mg), co-trimoxazole (Co, 25 mg), levofloxacin (Le, 5 mg), ciprofloxacin (Cp, 5 mg), imipenem (I, 10 mg), cefepime (Cpm, 30 mg), cefepimetazobactam (CPT, 30/10 mg), ticarcillin (Ti, 75 mg), ticarcillin-clavulanate (Tc, 75/10 mg) obtained from Hi-Media, Mumbai, India. Screening of extended spectrum b-lactamases (ESBLs) The clinical isolates were thawed and sub-cultured. Freshly grown culture of each isolate was separately plated on MHA plates for screening of ESBL production. Antibiotic discs of cefotaxime (30 mg), ceftazidime (30 mg) and ceftriaxone (30 mg) were placed on the MHA plates. After 24 h of incubation, the growth inhibition was observed. Diameter of the zone of growth inhibition was measured and interpreted according to CLSI criteria [24]. The isolates with reduced sensitivity against two of four drugs mentioned above were interpreted as suspected ESBL producers. Double disk synergy test (DDST) Bacterial isolates that have exhibited resistance to the third generation cephalosporins were further verified for ESBL production. Phenotypic confirmation of these provisional ESBL producers was done by testing the sensitivity of ceftazidime and cefotaxime alone and in combination with clavulanic acid (10 mg), as recommended by CLSI [24]. The cefotaxime (30 mg) and cefotaxime-clavulanic acid (30 mg/10 mg) discs were placed almost 20 mm apart on a MHA plate. Similarly, the ceftazidime (30 mg) and ceftazidime-clavulanic acid (30 mg/10 mg) discs were also placed about 20 mm apart. The plates were incubated overnight at 37 °C. The increase in the zone diameter by >5 mm with the antibiotic in combination with clavulanic acid, as compared to the antibiotic alone was interpreted as positive for ESBL [24, 25]. Escherichia coli ATCC 35218 was used as reference control for b-lactamase detection.
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Detection of MBL by modified Hodge test (MHT) The MHT was performed following the method of Lee et al. [26]. In brief, 0.5 McFarland dilutions of the E. coli ATCC 25922 in 5 ml of broth or saline was prepared. A 1:10 dilution was streaked on to a MHA plate to form a lawn of bacterial growth. To this, imipenem (10 mg) susceptibility disk was placed in the center of the test area. Test organisms (all the ESBL, non-ESBL and MBL clinical isolates of P. aeruginosa) were individually streaked in a straight line from the edge of the disk to the edge of the plate. The plates were incubated overnight at 35 2 °C in ambient air for 16–24 h. Quality control of the carbapenem disks were performed according to CLSI guidelines. After 24 h, MHT positive tests showed a clover leaf-like indentation of the E. coli 25922, along the growth of test organisms, streaked within the disk diffusion zone. MHT negative test showed no growth of the E. coli 25922 along the test organisms (Supplementary Fig. S1).
wall and bottom of the tube. Simply the ring formation at the liquid interface was not indicative of biofilm formation. Tubes were examined and the biofilm formers were scored as () –negative, (þ) – weakly positive, (þþ) – moderately positive, and (þþþ) – strongly positive. Congo red agar method (CRA). Biofilm production by P. aeruginosa isolates was further established by CRA method following the method of Freeman et al. [29]. Freshly grown cultures were plated on BHI agar supplemented with 5% sucrose and Congo red (0.8 g L1). The plates were incubated aerobically for 24–48 h at 37 °C. Appearance of black colonies with a dry crystalline consistency indicated positive results. Weak slime producers usually remained pink, though occasional darkening at the centers of colonies was observed. Darkening of the colonies with the absence of dry crystalline colonial morphology indicated an indeterminate result.
Detection of biofilm formation
Evaluation of antibacterial activity of GA-AgNPs by MIC and MBC methods Minimal inhibitory concentrations (MICs). All clinical isolates of P. aeruginosa were grown overnight on MHA plates at 37 °C. The antimicrobial activity of GA-AgNPs was examined following the standard broth dilution method. The MIC was determined in Luria-Bertani (LB) broth (Hi-Media, Mumbai, India) using serial two-fold dilutions of GA-AgNPs in concentrations ranging from 180 to 1.40 mg ml1. Bacterial inoculums of 2 108 CFU ml1 were used in the experiments. The treated and untreated control samples were incubated for 24 h at 37 °C. The MIC was defined as the lowest concentration of antimicrobial agents that yielded no visible growth of the microorganisms. Minimal bactericidal concentrations (MBCs). For MBC determination, the aliquots of 50 ml from all tubes in which no visible bacterial growth was observed were seeded on the MHA plates not supplemented with GAAgNPs. The plates were then incubated for 24 h at 37 °C. The MBC endpoint is defined as the lowest concentration of antimicrobial agent that kills 100% of the initial bacterial population.
Tissue culture plate method (TCP). Biofilm formation by the isolates was screened by tissue culture plate (TCP) method [27]. Briefly, trypticase soy broth (TSB), 10 ml with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar. The broth was incubated at 37 °C for 24 h. The culture was further diluted 1:100 with a fresh medium and aliquots of 0.2 ml of culture were transferred to the wells of flat bottom microtitre plates. Steriled broth was used as a blank. The culture plates were incubated at 37 °C for 24 h. After incubation, the culture was removed and the wells were washed with 0.2 ml of phosphate buffer saline (PBS, pH 7.2) four times to remove free-floating bacteria. Biofilms, which remained adhered to the wells were fixed with 2% sodium acetate and stained with 0.1% crystal violet. Excess stain was washed with deionized water and plates were dried properly. Optical densities (OD) of the purple colored solution were recorded at 570 nm by use of an ELISA reader (Bio-Rad, Model 680, USA). Three independent experiments were performed in triplicate. Tube method (TM). A qualitative assessment of biofilm formation was performed following the method of Christensen et al. [28]. Briefly, the bacterial isolates were sub-cultured in 10 ml TSB with 1% glucose in 25 ml tubes for 24 h at 37 °C. The medium was discarded and the tubes were washed with 1 PBS (pH 7.3) and dried. The bacterial cells adhered to the tubes were then stained with crystal violet (0.1%) for 1 min. Excess stain was removed and tubes were again washed with deionized water. Tubes were then dried in inverted position and observed for biofilm formation. The isolates were considered as positive, when a visible biofilm lined the ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Scanning electron microscopy Biofilms were assessed as previously described with or without GA-AgNPs. Briefly, small pieces of plastic catheters were seeded in 5 ml of BHI (with 5% sucrose) in a 12-well microtiter plates. To this, 100 ml of exponentially grown cells of P. aeruginosa treated with 10, 20, 30, 40, and 50 mg ml1 of GA-AgNPs were added. The plates were incubated at 37 °C for 24 h. Cells without GA-AgNPs were used as control. The catheters were then
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removed from both the control and treated wells and gently washed with PBS, fixed with 2.5% glutaraldehyde. The fixed samples were again washed with PBS and dehydrated gently by passing through a series of ethanol solutions (30, 50, 70, 80, 95, and 100%), each for 10 min at room temperature. Cells were then mounted on the aluminum stubs and coated with gold. The topographic features of the biofilms were visualized by use of SEM (Carl Zeiss EVO 40, Germany) with accelerating voltage of 20 kV. Confocal laser scanning microscopy (CLSM) analysis Confocal laser scanning microscopy (CLSM) analysis of biofilms was performed following method as described by Banas et al. [30]. Uniform sized (2 1 mm2) blocks of plastic catheters were exposed to varying (10, 25, and 50 mg ml1) concentrations of GA-AgNPs for 12 h, by submerging in NPs suspension in a 12-well microtitre plate. The wells were washed and the untreated control and GA-AgNPs treated catheters were incubated with the cultures of ESBL and MBL positive biofilm producing representative isolates of P. aeruginosa for 24 h at 37 °C. The cells adhered and grown on the surface of catheters were then stained with 15 mM propidium iodide (PI; Product code 81845, Sigma–Aldrich) for 15 min at room temperature. After through washing with PBS, the cells were incubated with 50 mg ml1 of concanavalin Aconjugated fluorescein isothiocyanate (ConA-FITC, Product code 61761, Sigma–Aldrich) for 15 min at room temperature to stain the glycocalyx matrix green. The PI was excited at 540 nm and the emission was monitored at 608 nm. Similarly, the Con A-FITC was excited at 495 nm and monitored at an emission wave length of and 518 nm. Intact biofilms were examined non-destructively using a Fluoview FV1000 Espectral Olympus CSLM (Olympus Latin America, Miami, FL, USA) equipped with a UPlanSApo 100/1.40 oil UIS2 Olympus oil immersion lens. Statistical analysis MIC and MBC tests were performed in triplicate, and the results were expressed as the mean SD of the mean. Student’s t-test was used to compare these results. Values of p lower than 0.05 were considered significant.
Results Characterization of GA-AgNPs – UV–visible spectrophotometric analysis Fig. 1 shows the optical absorbance spectra of GA-AgNPs in aqueous medium. Exposure of GA-AgNPs to light ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Figure 1. UV–Vis spectra of GA-AgNPs.
leads to polarization of the free conduction electrons with respect to the much heavier ionic core of NPs, resulting in electron dipolar oscillation and appearance of a strong SPR band at 400 nm (Fig. 1). No absorption peak at the same wavelength range was observed in the AgNO3 solution in deionized Milli-Q water used as a control (data not shown). HR-TEM analysis of GA-AgNPs HR-TEM analysis revealed that the GA-AgNPs were predominantly spherical in shape. Significant variability in particle sizes was observed and the average size was estimated to be 7 nm (Fig. 2). The HR-TEM images also revealed that GA-AgNPs were not in aggregated state and appeared as monodispersed. XRD analysis of GA-AgNPs Fig. 3 shows the XRD pattern of GA-AgNPs. The XRD profile of GA-AgNPs exhibited four intense peaks in the spectrum of 2u values ranging from 20 to 80. The diffractions at 38°, 44.18°, 64.29°, and 77.19° can be indexed to the (111), (200), (220), and (311) planes of the face centered cubic (fcc) silver, respectively and suggest that the GA-AgNPs are crystalline in nature (JCPDS File No 03-0921). Antibiotic susceptibility testing The results representing the antibiotic resistance pattern of P. aeruginosa are shown in Fig. 4. The data revealed the multiple drug resistant P. aeruginosa, where the isolates exhibited the frequency of resistance against cephalosporins as: cefotaxime (99%), ceftazidime (95%), ceftazidime– clavulanate (88%), ceftriaxone (75%), and cefoperazone (60%), besides significantly greater resistance against the classical antibiotics like amoxicillin, oxacillin, co-trimoxazole, and chloramphenicol. The results of DDST and MHT
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Figure 2. HR-TEM micrographs of GA-AgNPs at (a) lower and (b) higher magnification.
assays indicating the ESBL and MBL production by the representative isolates are shown in the Supplementary Fig. S1. Out of a total 55 isolates of P. aeruginosa, 42 (76.3%) were ESBL producers, 9 (16.4%) were non-ESBL, and 4 (7.3%) were found to be MBL positive. Screening of biofilm formation The qualitative results obtained on Congo red agar (CRA) are shown in Table 1. Out of 55 isolates of P. aeruginosa tested for biofilm formation around 33 (60%) isolates produced black colonies. Amongst them, only 15 (27.2%) isolates formed black colonies with dry crystalline consistency, indicative of biofilm formation. About 18 (32.2%) isolates formed black colonies but were not dry and crystalline and were regarded as indeterminate for biofilm formation. These isolates were also taken as negative for biofilm formation along with other 22 (40%) isolates, which produced pink colonies on CRA plates. The detection of biofilm formation by tube method (TM) further reaffirmed that around 12 (21.82%) were strong
Figure 3. XRD analysis of GA-AgNPs. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
biofilm producers, whereas 20 (36.36%) were tagged as moderate (Table 1). Furthermore, the results of quantitative TCP assay categorized the isolates as highly proficient biofilm producers (strongly adherent), moderate biofilm producers (moderate adherent) and nonbiofilm producers (weak/non-adherent). The results demonstrated 10 (18.2%) isolates as strongly positive as compared to moderate adherent 28 (50.9%) and weak/ non-biofilm producers 17 (30.9%; Table 1). Evaluation of antibacterial activity of GA-AgNPs by MIC and MBC method Table 2a shows the MIC and MBC values of GA-AgNPs dispersion tested against ESBL positive and ESBL negative isolates of P. aeruginosa. About 23.81% ESBL positive isolates exhibited the MIC of 11.25 mg ml1; whereas 71.42% shows the MIC of 22.5 mg ml1. Also, the 33.33% non-ESBL isolates exhibited the MIC of 11.25 mg ml1 and 44.44% shows the MIC of 22.5 mg ml1. Similarly, the Table 2b shows the MIC and MBC of MBL producing P. aeruginosa. The MBC values for 76.89% ESBL positive and 77.78% negative was found to be 45 mg ml1. The lowest MIC for ESBL, non-ESBL, and MBL P. aeruginosa was determined to be 11.25 mg ml1, indicative of a very strong bacteriostatic activity. However, the MBC was found to be in the range of 11.25–45 mg ml1, which reflects an effective bactericidal activity of GA-AgNPs. Characterization of antibiofilm activity of GA-AgNPs on CRA plates GA-AgNPs induced inhibition of biofilm formation by P. aeruginosa was tested on brain heart infusion agar supplemented with Congo red (BHIC). The colonies in untreated control samples appeared as dry crystalline black colonies, indicating the production of exopolysaccharides (EPS), which is the prerequisite for the
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Figure 4. Antibiotic resistance patterns of clinical isolates of P. aeruginosa against different groups of antibiotics.
formation of biofilm. However, under treatment conditions in presence of increasing concentration of GAAgNPs on BHIC, the organisms did not survived. GAAgNPs at concentration of 20 mg ml1 inhibited the synthesis of EPSs, and precluded the formation of dry crystalline black colonies (Supplementary Fig. S2). At higher concentrations of GA-AgNPs, around 95% of bacterial growth was inhibited. As the EPSs synthesis is arrested, the cells do not form biofilm. The results
Table 1. Assessment of biofilm formation by P. aeruginosa isolates (n ¼ 55) by different methods. Methods
Biofilms formers isolates (%)
CRA Colony appearance Pink/red (negative) Black colonies without dry crystalline consistency (indeterminate) Black colonies with dry crystalline consistency (positive)
22 (40) 18 (32.73) 15 (27.27)
TM Biofilm production 0/1 (weak/non) 2 þ (moderate) 3 þ (strong)
23 (41.82) 20 (36.36) 12 (21.82)
Biofilm production Weak/non (0.24 OD)
17 (30.9) 28 (50.9) 10 (18.2)
TCP
CRA, Congo red agar; TM, tube method; TCP, tissue culture plate. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
demonstrated that GA-AgNPs at concentration of 30 mg ml1 significantly arrested the biofilm formation without affecting the cell viability, whereas at a concentration of 60 mg ml1, the biofilm formation was completely blocked and the bacterial growth was completely ceased (Supplementary Fig. S2). Characterization of antibiofilm activity of GA-AgNPs by CLSM CLSM analysis of biofilms formation was studied in the representative ESBL and MBL producing isolates of P. aeruginosa (Figs. 5 and 6). Bacterial cells and the surrounding glycocalyx matrix, which is indicative of bacterial biofilm formation, were observed by double staining using PI and ConA-FITC. Bacterial cells stained red with PI were easily identified by their size and morphologic features. ConA-FITC binds to mannose residues resulting in green staining and indicating the presence of a bacterial glycocalyx. The presence of dark areas within the biofilm is attributed to the water channels, the heterogeneity of matrix and the types of EPSs within the biofilm. Superimposed CLSM images with PI (red) and Con A-FITC (green) fluorescent intensities yielded yellow color, which reflects the extracellular polysaccharides being produced as a capsular component in biofilm. Also, the interconnected bacteria were found encased in a scaffolding network composed of extracellular matrix, suggesting a 3dimensional architecture of biofilm formations. The micrographs in Figs. 5 and 6(Panel a) exhibit the biofilm formation in absence of GA-AgNPs with a definite architecture. However, in the presence of GA-AgNPs
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Table 2. MIC and MBC determination of GA-AgNPs tested against ESBL, non-ESBL, and MBL P. aeruginosa (n ¼ 55) isolates. (a) ESBL positive P. aeruginosa 42 (76.34%) 1
Non-ESBL P. aeruginosa 9 (16.36%) 1
Isolates
MIC (mg ml )
Isolates
MBC (mg ml )
Isolates
MIC (mg ml1)
Isolates
MBC (mg ml1)
23.81% 71.42% 4.76%
11.25 1.01 22.50 2.02 45.00 4.04
9.52% 14.28% 76.19%
11.25 1.01 22.50 2.02 45.00 4.04
33.33% 44.44% 22.22%
11.25 1.01 22.50 2.02 45.00 4.04
22.22% 77.78% –
22.50 2.02 45.00 4.04 –
(b) MBL producing P. aeruginosa 4 (7.3%) Isolates
MIC (mg ml1)
Isolates
MBC (mg ml1)
25.00% 75.00%
11.25 1.01 22.50 2.02
25.00% 75.00%
22.50 2.02 45.00 4.04
(Figs. 5b–d and 6b–d), a scanty growth with few cells was observed with no distinct pattern of arrangement. The cells also exhibited morphological deformation upon exposure with increasing concentrations of GA-AgNPs.
Maximum biofilm inhibition was noticed at concentration of 50 mg ml1 GA-AgNPs, and only few scattered cells were noticed on the surface of the solid matrix (Figs. 5c and d and 6c and d). Thus, the treatment of plastic
Figure 5. CLSM micrographs of ESBL P. aeruginosa biofilm. The panel (a) represents CLSM images of untreated control P. aeruginosa biofilm. Red color depicts the PI staining of bacterial nucleic acids and green fluorescent staining with ConA-FITC indicates the presence of EPSs. The panels (b–d) represent CLSM images of P. aeruginosa biofilm treated with 10 (b), 25 (c), and 50 (c) mg ml1 of GA-AgNPs. Magnification at 400. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 6. CLSM micrographs of MBL P. aeruginosa biofilm. The panel (a) represents CLSM images of untreated control P. aeruginosa biofilm. Red color depicts the PI staining of bacterial nucleic acids and green fluorescent staining with ConA-FITC indicates the presence of EPSs. The panels (b–d) represent CLSM images of P. aeruginosa biofilm treated with 10 mg ml1 (b), 25 mg ml1 (c), and 50 mg ml1 (c) of GA-AgNPs. Magnification at 400.
catheters with GA-AgNPs significantly restricted the colonization of ESBL and MBL bacterial cells, compared to the massive growth and biofilm formation in untreated control.
surface, resulting in inhibition of bacterial colonization on the surface of plastic catheters (Fig. 7b–f).
Discussion Characterization of antibiofilm activity of GA-AgNPs by SEM The effect of GA-AgNPs on biofilm formation by the ESBL and MBL positive isolates of P. aeruginosa on catheter was further confirmed by SEM. Figure 7a shows the untreated control cells of P. aeruginosa with normal cellular morphology, exhibiting a smooth cell surface. Under identical growth conditions, the cells treated with increasing concentrations of GA-AgNPs exhibited perceptible changes in cell morphology. The SEM images explicitly revealed GA-AgNPs induced damage with prominent indentations and depression on the cell ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Silver has long been used for its antimicrobial properties as its toxicity to microorganisms is greater than many other metals, while maintaining low toxicity to mammalian cells [31]. It has been shown that AgNPs are more efficient in mediating antimicrobial activity [32–36] than silver ions and as a result have been incorporated into wound dressings, medical devices, water purification systems, linings of washing machines, dish washers, refrigerators, toilet seats, and clothing [37]. In this study, we have assessed the antibacterial activity of highly stable and water soluble suspension of GA-AgNPs against
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Figure 7. SEM images of P. aeruginosa biofilms formed on the surface of catheters after 24 h of incubation. Panels are represented as: (a) Untreated, (b) treated with 10 mg ml1, (c) 20 mg ml1, (d) 30 mg ml1, (e) 40 mg ml1, and (f) 50 mg ml1 of GA-AgNPs.
the ESBL and MBL positive clinical isolates of P. aeruginosa, and determined their potential for biofilm inhibition by using sophisticated techniques, such as SEM and CLSM. In order to restrict the growth of biofilms, the antimicrobials must penetrate through the polysaccharide matrix to gain access to the embedded population of microbial cells. Our results demonstrated that the GAAgNPs can easily penetrate the biofilms and reduce its formation. The results of CRA assay revealed the antibiofilm efficacy of GA-AgNPs against the ESBL and MBL producers. At higher concentrations, the GA-AgNPs completely inhibited the bacterial growth and arrested the exopolysachharide synthesis and biofilm formation. Similar results have been reported by Kalishwaralal et al. [6] against the P. aeruginosa and S. epidermidis biofilms, where 100 nM of AgNPs resulted in 95–98% reduction in biofilm formation and Ansari et al. [38] against the clinical isolates of E. coli and Klebsiella spp. biofilms, where 50 mg ml1 of AgNPs resulted about 95% reduction in biofilm formation. Our SEM results also revealed that GA-AgNPs reduce the surface coverage and bacterial colonization, which ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
supports the observations of Ansari et al. [38] and Kostenko et al. [39]. However, the major limitation of SEM analysis is that the EPS within P. aeruginosa biofilms cannot be detected. The demonstration of bacterial biofilms is often challenging because of the problem in concurrent staining of both the bacterial cells and EPS. Also, the electron microscopy techniques require a dehydration process that reduces the total volume of the matrix and alters its architecture [40]. Thus, to demonstrate the presence of bacterial biofilms, it is important to detect both the bacteria and the EPS matrix simultaneously. For such an investigation, confocal scanning laser microscopy (CLSM) is an ideal tool for spatial resolution and non-destructive examination of the layers of biofilm at different depths, and helps in reconstructing a three-dimensional structure [41, 42]. The results obtained with the double-staining technique using CLSM, revealed GA-AgNPs induced cell death with no EPS matrix (green fluorescent cells) around P. aeruginosa cells and a disrupted 3-dimensional structure of the biofilm. This inhibitory effect of GA-AgNPs on the biofilm has been attributed to the malfunction of the
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water channels though out the biofilm. Water channels (pores) are present in all biofilms for nutrient transportation. GA-AgNPs may directly diffuse through the EPSs layer and impart antimicrobial action. Masurkar et al. [43] have reported the antibiofilm activity of AgNPs (32 nm) synthesized from Cymbopogan citratus. Similarly, Kostenko et al. [39] have also reported that acticoat nanocrystalline silver exhibit antibiofilm efficacy compared to aquacel silver and silverlon, and suggested that the silver concentration alone cannot account for the antibiofilm efficacy of the silver dressings. The reduction of the silver particle to the nanoscale level increases the relative surface area, which provides higher Agþ release rates than for elemental silver particles. It has been suggested that the silver ions bound to bacterial cells and EPS, interfere with intermolecular forces in biofilms and facilitate biofilm dispersion [44]. However, the exact mechanism of action of AgNPs on biofilms is yet to be elucidated. Overall, our results suggested that the GAAgNPs possesses an inherent capacity to attach and penetrate the bacterial membranes. The trans-membrane movement and intracellular NPs accumulation may trigger the release of silver ions, which supposedly causes oxidative stress and eventually leads to cell death.
Conclusion The effective antiseptic treatment of medical implants and surgical tools demands new approaches that target the biofilm forming MDR bacteria for control of infections. This study explicitly demonstrated that the potential of GA-AgNPs to inhibit the growth and colonization of MDR P. aeruginosa on plastic catheters. The GA-AgNPs are suggested as effective antimicrobial agent for protecting the surfaces of medical implants against pathogen colonization.
Acknowledgments The authors would like to acknowledge AIRF, Jawaharlal Nehru University, New Delhi, India, for SEM and CLSM and SAIF-DST, Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi, India, for HR-TEM observation. JM is grateful to the visiting professor program (VVP) of the King Saud University, Riyadh, SA for all the help and support in conducting this collaborative research work. Authors also would like to thank the Indian Council of Medical Research (ICMR) New Delhi-India, grant number 35/15/BMS-11 for their partial support and funding of this project. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Conflict of interest There is no conflict of interest between the authors.
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