Enhanced antibacterial effects of green synthesized ZnO NPs using ...

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ARTICLE IN PRESS Journal of Infection and Public Health xxx (2017) xxx–xxx

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Enhanced antibacterial effects of green synthesized ZnO NPs using Aristolochia indica against Multi-drug resistant bacterial pathogens from Diabetic Foot Ulcer Katherin Steffy a,∗ , G. Shanthi a , Anson S. Maroky b , S. Selvakumar c a

Division of Microbiology, Rajah Muthiah Medical College, Annamalai University, Chidambaram 608002, Tamil Nadu, India Department of Pharmacy, Faculty of Engineering and Technology, Annamalai University, Chidambaram 608002, Tamil Nadu, India c Department of Zoology, Faculty of Science, Annamalai University, Chidambaram 608002, Tamil Nadu, India b

a r t i c l e

i n f o

Article history: Received 4 July 2017 Received in revised form 11 September 2017 Accepted 12 October 2017 Keywords: Aristolochia indica ZnO NPs Time-kill kinetics Protein leakage analysis Flow cytometry

a b s t r a c t Background: Increased incidence of Multi-drug resistance in microorganisms has become the greatest challenge in the treatment of Diabetic Foot Ulcer (DFU) and urges the need of a new antimicrobial agent. In this study, we determined the bactericidal effects of ZnO nanoparticles (ZnO NPs) green synthesized from Aristolochia indica against Multi-drug Resistant Organisms (MDROs) isolated from pus samples of DFU patients attending in a tertiary care hospital in South India. Methods: ZnO NPs were characterized by UV–vis-DRS spectroscopy, Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM) and for its zeta potential value. MIC/MBC assays were performed to determine bactericidal or bacteriostatic effects. Time-kill assays, Protein leakage and Flow cytometric analysis evaluated bacterial cell death at 1x MIC and 2x MIC concentrations of ZnO NPs. Results: ZnO NPs of size 22.5 nm with a zeta potential of −21.9 ± 1 mV exhibited remarkable bactericidal activity with MIC/MBC ranging from 25 to 400 ␮g/ml with a significant reduction in viable count from 2 h onwards. Protein leakage and Flow cytometric analysis confirmed bacterial cell death due to ZnO NPs. Conclusion: This study concluded that green synthesis protocol offers reliable, eco-friendly approach towards the development of antimicrobial ZnO NPs to combat antibiotic drug resistance. © 2017 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Diabetes mellitus (DM) is a chronic metabolic syndrome with a high risk of developing complications such as non-healing ulcers, retinopathy, neuropathy and macrovascular conditions [1]. Diabetic Foot Ulcers (DFU) have become an increasingly developed complication observed in persons with uncontrolled diabetes with increased morbidity and are more susceptible to polymicrobial infections spreading rapidly causing irreversible tissue damage. Diabetic foot infections (DFIs) lead not only to the morbidity but also associated with severe clinical dumps and increased death rates [2,3]. Antibiotic resistance is considered to be a major threat in the treatment of DFIs, mostly due to the chronic course of the wound, abuse of antibiotics, reduced effect of antibiotics in the wound environment and also acquired due to the exchange of resis-

∗ Corresponding author. E-mail address: [email protected] (K. Steffy).

tance genes between the bacterial chromosome and plasmids [4,5]. World Health Organizations (WHO) also emphasized the threatening level of antibiotic resistance and the urgent need for the development of new therapeutic drugs to overcome this distress situation [6]. Greener route synthesis of nanoparticles is getting more appreciation due to its eco-friendly approaches than chemical and physical means. Biological molecules within plants can undergo highly controlled assembly suitable for the metal nanoparticle synthesis [7]. ZnO nanostructures are gaining more importance for bio-applications due to its physiochemical properties, biocompatibility and biosafety approach [8]. For decades medicinal plants had been playing a key role in the development of new therapeutic agents. Aristolochia indica (family Aristolochiaceae) is a medicinal plant commonly known as Indian Birthwort. The plant has been used for treating leucoderma and eczema [9] rheumatism, inflammations, leprosy, dyspepsia, cough and dysmenorrhea [10,11] also applied externally as an antivenom [12]. Thus combining traditional medicine with nanotechnology can envisage

https://doi.org/10.1016/j.jiph.2017.10.006 1876-0341/© 2017 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Steffy K, et al. Enhanced antibacterial effects of green synthesized ZnO NPs using Aristolochia indica against Multi-drug resistant bacterial pathogens from Diabetic Foot Ulcer. J Infect Public Health (2017), https://doi.org/10.1016/j.jiph.2017.10.006


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successful alternate treatment procedures in this devastating situation of antibiotic drug resistance. The objective of the present study was to evaluate the antibacterial potential of eco-friendly A. indica mediated green synthesis of ZnO nanoparticles (ZnO NPs) against MDROs isolated from pus samples of Diabetic Foot Ulcer (DFU) patients attending in a tertiary care hospital in South India and also against ATCC bacterial strains through various microbiological assays.

firmed with VITEK 2 Compact automated system (Bio Mérieux, Marcy l’Etoile, France) using GN Test Kit VTK2/GP Test Kit VTK2. Standard bacterial strains Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, E. coli ATCC 25922 were obtained from CSIR-National Chemical Laboratory, Pune.

Materials and methods

Assays for MIC and MBC determination The MIC of the extracts was determined by Macro-broth dilution method [14] using Cation-adjusted Muller-Hinton broth (CAMHB) (Himedia Laboratories, Mumbai, India). Serially diluted ZnO NPs in 20% DMSO (Sigma-Aldrich, St. Louis, MO, USA) of 1 ml each was added to the set of tubes followed by 1 ml of bacterial suspensions whose colony count was adjusted to 5 × 105 cfu/ml. Thus final dilutions were adjusted to a concentration ranging from 400 ␮g/ml to 0.781 ␮g/ml. The tubes were incubated at 37 ◦ C for 24 h. The MIC values were considered as the lowest concentration of the sample, which showed clear fluid with no turbidity. MBC was determined by subculturing 0.01 ml aliquot from macroscopically clear tubes to sterile Nutrient agar (Himedia Laboratories, Mumbai, India) for 24 h at 37 ◦ C. Bactericidal end points (MBC) were calculated based on colony counts, correlating final inoculums with rejection values in accordance with CLSI guidelines [15]. MBC/MIC ratio was calculated to determine bactericidal or bacteriostatic properties of green synthesized ZnO NPs against bacterial pathogens [16].

Collection of plant material and preparation of A. indica leaf aqueous extract A. indica leaves were collected from forest areas of the Western Ghats, South India for the month of September 2016. The plants were identified and authenticated by the Botanical Survey of India (Southern Circle) Coimbatore (BSC/SRC/5/23/2016/Tech/1197). The leaves were cleaned with double distilled water and shade dried in room temperature for one week. Dried leaves were tattered and grounded in a blender to a coarse powder. 20 g of plant powder was heated with 100 ml of double distilled water for 20 min at 60 ◦ C. The light yellow coloured solution formed during the boiling was cooled to room temperature, filtered through Whatman no.1 and stored in the refrigerator until further use. Biosynthesis and characterization studies of ZnO NPs from A. indica leaf aqueous extract 40 ml of A. indica leaf Aq. extract was added with 2 g of Zinc nitrate hexahydrate crystals (Merck, Darmstadt, Germany) and allowed to dissolve using a magnetic stirrer. After complete dissolution of the mixture, the solution was kept for vigorous stirring at 100 ◦ C for 2 h until colour changes. After cooling to room temperature, the mixture was centrifuged twice at 5000 rpm for 10 min after thorough washing and heated at 60–80 ◦ C until the formation of deep yellow coloured paste and annealing was carried out in a muffle furnace at 400 ◦ C for 2 h and a fine off-white coloured material was obtained. Optical properties were analyzed using UV–vis DRS spectroscopy (Varian, Cary 5000, Palo Alto, California , U.S.). The surface topological studies were carried out using an Atomic Force Microscopy (AGILENT −5500, Santa Clara, CA, USA). The morphological features were characterized using Transmission Electron Microscopy (Jeol/JEM 2100, Tokyo, Japan). Zeta potential of the ZnO NPs was determined using Nano Particle Analyzer SZ-100 by Horiba Scientific (Kyoto, Japan). All the experiments were performed in triplicates and the data were analyzed using Origin Pro 7.5 SRO software (Origin Lab Corporation, USA). Isolation, identification and confirmation of MDROs from DFU Pus samples were collected from DFU patients admitted at Rajah Muthiah Medical College and Hospital (RMMCH), Annamalai University, Tamil Nadu, India with the approval of Institutional Human Ethical Committee (M18/RMMC/2015). DFU samples were taken with aseptic precautions after obtaining informed consents from the patient. The samples were further processed for Microscopy by Gram staining and bacterial culture on Columbia-based blood agar and Mac Conkey Agar (Himedia Laboratories, Mumbai, India) incubated overnight at 37 ◦ C. The microbial colonies were examined and identified using standard microbiological procedures. Antibiotic susceptibility testing was done by Kirby-Bauer method of disk diffusion on Muller Hinton Agar (Himedia Laboratories, Mumbai, India). The diameter of the zone of inhibition measured and interpreted according to guidelines of Clinical Laboratory Standard’s Institute [13]. The Multi-drug resistance of clinical isolates was con-

Studies on antibacterial activity of green synthesized ZnO NPs

Time-kill kinetics Time-kill kinetics was performed as described by Messick et al. [17] with some modifications. Bacterial cultures whose colony count adjusted to 5 × 105 cfu/ml in CAMHB, was added against green synthesized ZnO NPs at concentrations equivalent to 1x MIC and 2x MIC of each particular strains at time intervals for 2, 4, 6 & 8 h. At each time interval, 0.5 ml of suspension was serially diluted (up to 10−5 ) and 100 ␮l from final dilution was spreaded on an agar plate. Growth control tubes was also included in the line of test tubes. After overnight incubation emergent bacterial colonies were counted, cfu/ml calculated and compared with the count of the growth control. Viable counts were made in triplicates for each organism. Deduction in the viable counts (3 log decrease in cfu/ml) for a specified time indicated bactericidal activity of green synthesized ZnO NPs. The bactericidal activity of the green synthesized ZnO NPs was determined by plotting log 10 colony counts (cfu/ml) against time. Protein leakage analysis Bacterial cells of MDROs and ATCC bacterial strains (washed in 0.9% w/v NaCl and colony count adjusted to 5 × 105 cfu/ml) were treated with different concentrations of (1x MIC and 2x MIC) biosynthesized ZnO NPs at various time intervals such as 2, 4, 6 and 8 h. Every tube was gently wobbled intermittently in a vortex mixer. At the end of each time period, each suspension was then centrifuged at Approx. 6000 × g for 15 min and supernatant obtained was evaluated for protein estimation using the assay as described by Bradford [18]. Optical density (O D) of the resulting solution was thereafter taken by UV–vis spectroscopy (Shimadzu, Japan) at 595 nm after 5 min. The O D of each of the samples was calculated from the equation of the best-fit linear regression line obtained from the graph of the Bovine Serum Albumin (BSA) standard curve. Cell viability count by fluorescence activated cell sorter (FACS) Flow cytometric analysis was performed to investigate the cell viability of MRSA (Gram-positive) and Acinetobacter baumannii (Gram-negative) when treated with 1x MIC and 2x MIC of green




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synthesized ZnO NPs at 2, 4, 6 and 8 h. At the end of each time period, treated bacterial cells were cold centrifuged at 300 × g for 5 min, washed twice with 1 ml of cold, filtered 1x PBS (pH 7.4). Bacterial cells were further resuspended in 2 ␮l of Propidium iodide (PI) incubated for 1 min in the dark. After incubation, cells were washed twice in PBS, filtered through 40 ␮m cell strainer (Himedia Laboratories, Mumbai, India) and analyzed by Flow cytometry (FACS MoFlo XDP Beckman Coulter). Untreated bacterial cells in PBS were used as the control. Statistical analysis All the experiments were performed in triplicates, with the results being expressed as Mean ± SEM of three independent experiments. The means were statistically compared using One-way ANOVA followed by post hoc Dunnett’s and Tukey’s Multiple Comparison Test using GraphPad Prism version 5. P < 0.05 was considered as statistically significant.

Fig. 1. UV-DRS spectrum of green synthesized ZnO NPs.

Results Characterization studies of green synthesized ZnO NPs Green synthesized ZnO NPs exhibited clear and strongly observed reflectance below at 367 nm indicating blue-shift (Fig. 1) [19]. The optical direct band-gap energy (Eg) estimated from Tauc’s plot was found to be 3.37 eV. AFM images of green synthesized ZnO NPs (Fig. 2A and B) from 3 × 3 ␮m scan showed spherical, homogenous, uneven surface morphology and porous nature of nanoparticles at a size range of 22.5 nm. The TEM image of green synthesized ZnO NPs shown in Fig. 2C, showing agglomerated quasi-spherical shaped nanoparticles. The surface zeta potential of

a colloidal suspension of ZnO NPs was −21.9 ± 1 mV (Fig. 3) indicating stable nature of nanoparticles [20]. FTIR spectra of A. indica plant extract has shown broad IR absorption bands indicating the presence phytochemical compounds having functional groups of amines, alcohols, ketones and carboxylic acid. FTIR spectra of ZnO NPs exhibited IR absorption bands highly shifted proving the presence of phytochemical components of plant in the biosynthesis process and EDAX analysis showed only the presence of Zinc and Oxygen elements (Steffy et al., Unpublished data).

Fig. 2. Morphological features of green synthesized ZnO NPs (A) AFM 2Dimensional image, (B) AFM 3Dimensional image and (C) TEM image with inset picture of higher magnification (HRTEM).



≥32 (R) ≥32 (R) A. baumannii E. coli

CX—Cefoxitin Screening, BP—Benzyl pencillin, Ox—Oxacillin, Gen—Gentamicin, Cip—Ciproflixacin, Lev—Levofloxacin, iCd—Inducible Clindamycin Resistance, Er—Erythromycin, Cd—Clindamycin, Lz—Linezolid, Dap—Daptomycin, Teico—Teicoplanin, V—Vancomycin, T—Tetracyclin, Tig—Tigecycline, Nt—Nitrofurantine, Rif—Rifampicin, Cotri—Cotrimoxazole. TC—Ticarcillin/Clavulanicacid, PT—Piperacillin/Tazobactum, Cz—Ceftazidime, CS—Cefaperazone/Sulbactum, Cf—Cefepime, D—Doripenem, I—Imipenem, Mero—Meropenem, Ak—Amikacin, G—Gentamicin, Cip—Ciprofloxacin, Lev—Levofloxacin, M—Minocycline, Col—Colistin, Cotri—Co-trimoxazole, A—Ampicillin, AC—Amoxicillin/Clavulanic acid, Cx—Cefuroxime, Cu—Cefuroxime Axetil, Ctr—Ceftaxone, Na—Nalidixic acid. a Cefoxitin MIC >4 ␮g/ml (mecA positive), Cefoxitin is used as a surrogate marker for mecA-mediated oxacillin resistance. b Indicates growth of organism (inducible clindamycin resistant). c Antibiotic panel for Gram Positive cocci. d Antibiotic panel for Non-fermenting Gram Negative bacteria. e Antibiotic panel for Fermenting Gram Negative bacteria.

Nil Nil

Nil Cotri

≥320 (R) ≥320 (R) ≤0.5 (S) ≤0.5 (S)

Col Nt

≥512 (R) ≤16 (S) ≤0.5 (S) ≤0.5 (S)

Tig Cip

≥4 (R) ≥4 (R) ≥32 (R) ≥32 (R)

Na G

≥16 (R) ≥16 (R) ≥16 (R) ≤0.25 (S)

Mer I

≥16 (R) ≤0.5 (S) ≥64 (R) ≥64 (R)

Cf CS

32 (I) 16 (S) ≥64 (R) ≥64 (R)

Ctr Cu

≥64 (R) ≥64 (R) ≥64 (R) ≥64 (R)

Cx AC

≥32 (R) ≥32 (R)

A Antibiotice

≥128 (R) 8 (S)

≥128 (R) ≥128 (R) P. aeruginosa

PT

Nil

Nil Nil

Nil Cotri

≥320 (R) ≤0.5 (S)

Col Tig

≥8 (R) ≥8 (R)

M Lev

≥8 (R) ≥4 (R)

Cip G

≥16 (R) 32 (I)

Ak Mero

≥16 (R) ≥16 (R)

I D

≥8 (R) ≥64 (R)

Cf CS PT TC Antibioticd

Cz

POS 4 (R)

≥8 (R) ≥16 (R) ≥4 (R) POS MRSA

≥0.5 (R)

≥64 (R)

≥8 (R)

Ery

b

iCd Lev Cip Gen Ox BP

Determination of bactericidal activity of green synthesized ZnO NPs by MIC/MBC assays MIC-MBC values of biosynthesized ZnO NPs against ATCC bacterial strains and MDROs summarized in Table 2. The minimum concentration of green synthesized ZnO NPs which inhibited the growth of bacteria was 25 ␮g/ml for MDR-A. baumannii, 100 ␮g/ml for E. faecalis ATCC 29212, MDR and ATCC bacterial strains of P. aeruginosa and E. coli. MIC values were 200 ␮g/ml for MDR and ATCC bacterial strains of S. aureus. Whereas MBC values ranged from 100 ␮g/ml (MDR-A. baumannii, P. aeruginosa ATCC 27853 and E. coli ATCC 25922), 200 ␮g/ml (MDR-P. aeruginosa, MDR- E. coli, S. aureus ATCC 29213 and E. faecalis ATCC 29212) and 400 ␮g/ml (MDR-MRSA). An antimicrobial agent can be considered bacterici-

a

Antibacterial assays against green synthesized ZnO NPs

CX

A total of 180 bacterial strains was isolated from the 80 ulcer specimens, averaging 2 species per patient. Among bacterial pathogens, E. coli (45%) were common and prominent followed by P. aeruginosa (20%), S. aureus (15%), A. baumannii (10%), K. pneumonia (8.4%) E. faecalis (1.16%), Coagulase-negative staphylococci (CNS) (0.44%) (Fig. 4). The majority of bacterial isolates exhibited reduced susceptibility towards antibiotics. Four selected bacterial pathogens that showed Multi-drug resistance pattern (MDR-MRSA, MDR-E. coli, MDR-P. aeruginosa, MDR-A. baumannii) in disc diffusion assay and automated antibiotic susceptibility system (Table 1) were selected for further bactericidal experiments against A. indica mediated green synthesized ZnO NPs.

Antibioticc

Isolation, identification and confirmation of MDROs from DFU

Table 1 ® Susceptibility testing pattern of clinical bacterial isolates done with VITEK 2 compact automated system.

Fig. 4. Prevalence of bacterial isolates from Diabetic Foot Ulcer (DFU).

≥64 (R)

2 (I) ≥128 (R) ≥16 (R) ≥16 (R) 1 (S) ≤0.5 (S) 1 (S) 2 (S) 1–2 (I)

Rif Nt Tig Tet Cd

Lz

Dap

Teic

V

Fig. 3. Surface zeta potential of green synthesized ZnO NPs.

≥320 (R)

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Table 2 The MIC, MBC (99.9%kill) and MIC-MBC ratio of Green synthesized ZnO NPs against ATCC bacterial strains and MDR bacterial isolates from DFU. Standard bacterial strains

MIC (␮g/ml)

MBC (␮g/ml)

MBC/MIC ratio

Control Antibiotica (␮g/ml)

S. aureus ATCC 29213 P. aeruginosa ATCC 27853 E. faecalis ATCC 29212 E. coli ATCC 25922 MDR-MRSA MDR-P. aeruginosa MDR-A. baumannii MDR-E. coli

200 100 100 100 200 100 25 100

200 100 200 100 400 200 100 200

1 1 2 1 2 2 4 2

0.5 1 1 1 1 0.5 0.5 0.5

a

Control Antibiotic Vancomycin for Gram Positive organisms and Colistin for Gram Negative organisms.

Fig. 5. Time-kill curve determination at different time intervals of (A) Untreated and Treated ATCC bacterial strains at 1x MIC of green synthesized ZnO NPs (B) Untreated and Treated ATCC bacterial strains at 2x MIC of green synthesized ZnO NPs, (C) Untreated and Treated MDROs at 1x MIC of green synthesized ZnO NPs and (D) Untreated and Treated MDROs at 2x MIC of green synthesized ZnO NPs. Each point represents the relative viable count of bacterial cells at a particular time intervals (n = 3); dashed line indicates 99.9% kill.

dal against tested bacterial strains if the MBC/MIC ratio exhibited is within ≤8 [16]. The results of this study support this suggestion since MBC/MIC ratio of all test bacterial strains were within ≤4.

Thus green synthesized ZnO NPs from A. indica exhibited strong bactericidal properties against all test bacterial strains.


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Fig. 6. Protein leakage analysis at different time intervals of (A) Untreated and Treated ATCC bacterial strains at 1x MIC and 2x MIC of green synthesized ZnO NPs and (B) Untreated and Treated MDROs at 1x MIC and 2x MIC of green synthesized ZnO NPs. Each point represents the amount of protein leaked (␮g/ml) from the cells at a particular time interval in the presence of the green synthesized ZnO NPs (n = 3).

The extent and rate of killing of green synthesized ZnO NPs against bacterial isolates A bactericidal effect of an antimicrobial agent is explained in terms of 3 log decrease in cfu/ml or 99.9% kill over a specified time [15]. A 90% kill of an antimicrobial agent at 6 h was corresponding to a 99.9% kill at 24 h [21]. In agreement with this statement, at a time period of 6 h green synthesized ZnO NPs at 1x MIC and 2x MIC respective to S. aureus ATCC 29213 showed 99.52 and 99.91% kill, P. aeruginosa ATCC 27853 showed 98.14 and 99.10% kill, E. faecalis ATCC 29212 showed 99.67 and 99.92% kill and E. coli ATCC 25922 showed 99.94 and 99.99% kill (Fig. 5A and B). Among clinically isolated MDROs, within 6 h of contact with 1x MIC and 2x MIC of green synthesized ZnO NPs, MDR-MRSA exhibited 96.47 and 99.01% kill, MDR-P. aeruginosa exhibited 89.50 and 97.97% kill, MDR-A. baumannii exhibited 99.11 and 99.83% kill and MDR-E. coli exhibited 98.99 and 99.7% kill (Fig. 5C and D). This proves remarkable bactericidal effects of green synthesized ZnO NPs against MDROs from DFU and ATCC bacterial strains. Protein leakage analysis The amount of protein leaked from the bacterial cells was proportionally in accordance with increasing concentration and contact period of biosynthesized ZnO NPs. The study clearly represents that at the end of 8 h amount of protein leaked due to antibacterial effects of A. indica formed green synthesized ZnO NPs

was severe in E. coli ATCC 25922 (267.19 ␮g/ml at 1x MIC and 296.19 ␮g/ml at 2x MIC) and S. aureus ATCC 29213 (250.22 ␮g/ml at 1x MIC and 278.18 ␮g/ml at 2x MIC) than P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212 (Fig. 6A). Whereas among clinical MDROs, MDR-MRSA (269.44 ␮g/ml at 1x MIC and 289.70 ␮g/ml at 2x MIC) and MDR-A. baumannii (249.96 ␮g/ml at 1x MIC and 288.05 ␮g/ml at 2x MIC) showed more obvious protein leakage when compared to MDR-P. aeruginosa and MDR-E. coli (Fig. 6B). Interestingly reports of protein leakage analysis pointed out the bactericidal effects of green synthesized ZnO NPs due to cell membrane damage resulting outflow of protoplasmic inclusions. Analysis of dead bacteria by Flow cytometry Fig. 7A illustrated the number of dead bacterial cells representing a shift of peak from R5 (live cells) to R6 (dead cells) on treatment with biosynthesized ZnO NPs, relative to the untreated control group. Dead cell percentage of MDR-A. baumannii representing Gram-negative bacteria when treated with 1x MIC of green synthesized ZnO NPs were 30, 69.14, 94.24 and 99.54% and at 2x MIC dead cell percentage of MDR-A. baumannii were 63, 96.7, 97.17 and 99.54% at 2, 4, 6 and 8 h. Dead bacterial cell percentage using PI staining for MDR-MRSA representing Gram-positive bacteria when treated with 1x MIC of green synthesized ZnO NPs were 34.68, 64.05, 83.64, 99.85% whereas at 2x MIC, dead cell percentage was 52.65, 84.09, 94.24, 99.90% at 2, 4, 6 and 8 h. Biosynthesized ZnO NPs




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Fig. 7. Flowcytometric analysis of bacterial cell death at different time intervals of (A) MDR-A. baumannii and MDR-MRSA at 1x MIC and 2x MIC of green synthesized ZnO NPs using PI staining. (B) Analysis for a dead bacterial percentage of MDR-A. baumannii at 1x MIC and 2x MIC (C) analysis of dead bacterial percentage of MDR-MRSA at 1x MIC and 2x MIC. Statistic analysis by One-way ANOVA test, followed by Tukey’s multiple comparison tests (n = 3).

inhibited the growth of MDR-A. baumannii and MDR-MRSA cells with a statistically significant reduction in viable cells compared to control (P < 0.001) (Fig. 7B and C). Discussion Management of DFU has to be followed by systematic evaluation towards the assessment of microbial etiology and antimicrobial

susceptibility. Bacterial etiology of DFU in the present study was polymicrobial in nature (Fig. 4). Previous studies showed that commonly isolated Gram-negative bacteria were P. aeruginosa followed by E. coli, Acinetobacter spp., Proteus spp. and Klebsiella spp. and Gram-positive organisms were S. aureus, E. faecalis, CNS [22,23]. Increased Multi-drug resistance pattern was observed in bacteria strains isolated from DFU (Table 1). Antibiotic resistance in


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Gram-negative bacteria has become a terrorizing due to the fast development of drug resistance even to the higher level of antibiotics such as carbapenems and third generation cephalosporins [24]. Among Gram-positive bacteria, OS-MRSA strains have been increasingly reported. Failure of detection of these strains and further treatment with ␤-lactam antibiotics causes, the emergence of highly resistant MRSA causing failure of therapy [25]. Alternate therapeutic methods can overcome antibiotic resistance, morbidity and mortality occurred due to treatment failure. Applications of nanotechnology in human health research have resulted in immense development in drug and gene delivery and diagnostics [26]. The present study has performed biosynthesis of ZnO NPs from A. indica and the UV–vis spectroscopy analysis of ZnO NPs (Fig. 1) confirmed the presence of nanoparticles by indicating a shift of Surface Plasmon resonance (SPR) to higher energies resulting absorption and scattering of peaks to shorter wavelengths below 400 nm [27,28]. TEM and AFM analysis identified quasispherical shape of ZnO NPs with an average size of 22.5 nm (Fig. 2) The magnitude of the zeta potential of green synthesized ZnO NPs (Fig. 3) within the range of −30 mV to +30 mV indicated potential steadiness with a mutual repulsion and minimal chances of aggregation of nanoparticles [29]. MIC and MBC values of green synthesized ZnO NPs were in the range of 25–200 ␮g/ml and 100–200 ␮g/ml, respectively against all bacterial strains. The MBC/MIC ratio is a parameter that reflects the bactericidal capacity of the analyzed compound and all test strains showed ≤4 confirms its significant bactericidal properties (Table 2). Both MDROs and ATCC bacterial strains seem to be significantly inhibited by green synthesized ZnO NPs at lower concentrations (25–200 ␮g/ml) in agreement with earlier studies of green synthesized ZnO NPs [30]. Whereas chemically synthesized ZnO nanoparticles in earlier studies showed bactericidal activity only at a higher concentration of 400–8000 ␮g/ml [31]. Time-kill curves that monitor bacterial growth and death have been often used to estimate the effect of antimicrobial agents over time [32]. Time-kill kinetics demonstrated bactericidal properties of green synthesized ZnO NPs at 25–200 ␮g/ml with a significant decline of viable bacterial cells from 2 h onwards against MDROs as well as ATCC bacterial strains (Fig. 5). This is in coherence with earlier reports on time-kill kinetics of chemically synthesized nanoparticles of ZnO and Ag+ ZnO composite against dental pathogens at a concentration of 100–2500 ␮g/ml with significant decrease in growth within 4 h, reaching 100% within 2 h for Porphyromonas gingivalis and within 3 h for Fusobacterium nucleatum and Prevotella intermedia [33]. Protein leakage analysis revealed significant release of proteins from the cell membranes of MDROs and ATCC bacterial strains on contact with biosynthesized ZnO NPs. The amount of protein released was directly proportional to the concentration and contact time (Fig. 6) and well explains the antibacterial mechanism of green synthesized ZnO NPs by damaging the cell membrane leading to the leakage of minerals, proteins and genetic materials leading to cell death. Earlier investigations demonstrated that interaction of ZnO NPs with outer membrane proteins (OMP) present in the cell membrane of Gram-negative bacteria resulted, decreased membrane permeability finally causing cell death. They also demonstrated the leakage of protoplasmic inclusions proportional to the interaction with increased amount of ZnO NPs [34]. Analysis of bacterial cell death percentage by Flow cytometry for MDR-MRSA and MDR-A. baumannii were statistically significant (P < 0.001) from 2 h onwards (Fig. 7) in agreement with previous reports [35]. The highest positive ratio of the bacterial cells was observed in the 2x MIC of green synthesized ZnO NPs. Thus the present study confirms the remarkable bactericidal potential of A. indica mediated green synthesis of ZnO NPs against ATCC bacterial strains as well as MDROs in a concentration-dependent manner.

ZnO nanostructures due to its large surface area can generate more H2 O2 which is potentially lethal towards bacterial cells [36,37]. Increased production of reactive oxygen species by ZnO NPs causes increased oxidative stress within bacterial cells that involves the destruction of cellular components as a result of their internalization into their cell membrane. ZnO NPs with negligible particles size exhibited increased efficiency of biological activity due to its increased specific surface area to volume ratio and surface reaction of ZnO [38,39]. Conclusions Our study demonstrated a reliable, eco-friendly, environmentally safe approach on the plant-mediated biosynthesis of ZnO NPs from a well known medicinal plant A. indica. Biosynthesized ZnO NPs was notably remarkable for its optical, morphological and colloidal properties. Green synthesized ZnO NPs exhibited strong bactericidal properties against clinically isolated Multi-drug resistant strains isolated from Diabetic Foot Ulcer (MDR-MRSA, MDR-P. aeruginosa, MDR-A. baumannii and MDR-Escherichia coli) and also against ATCC bacterial strains (S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212 and E. coli ATCC 25922). Thus the present study provides a new insight towards the development of green synthesized ZnO NPs as an alternate therapeutic agent, due to its enhanced bactericidal effects even against the clinical bacterial pathogens that are almost resistant to existing higher antibiotics. But further studies have to be done to elucidate the mechanism of antibacterial activity and it is suggested that this synthesis process can be used for other biological application such as antifungal and larvicidal agents. Funding No funding sources. Competing interests None declared. Ethical approval Not required. Acknowledgments The authors wish to acknowledge the financial support from the Department of Science and Technology, New Delhi for providing under DST-INSPIRE fellowship scheme received by Ms Katherin Steffy (IF140576). Special thanks to Professor and Head of Division of Microbiology, RMMC and Professor and Head of Department of Pharmacy, Annamalai University for providing necessary facilities to carry out this work. The authors cordially thank Dr. K. Chitra, Research Scientist, TRPVB-TANUVAS, Chennai for Flow cytometry study, Sophisticated Analytical Instrument Facility, STIC-CUSAT, Cochin for recording HR-TEM and VIT University, Vellore for Spectroscopy analysis. References [1] Unnikrishnan R, Anjana RM, Mohan V. Diabetes mellitus and complications its in India. Nat Rev Endocrinol 2016:1–14, http://dx.doi.org/10.1038/nrendo.2016.53. [2] Spichler A, Hurwitz BL, Armstrong DG, Lipsky BA. Microbiology of diabetic foot infections: from Louis Pasteur to “crime scene investigation”. BMC Med 2015;13:2–13, http://dx.doi.org/10.1186/s12916-014-0232-0. [3] Game FL, Apelqvist J, Attinger C, Hartemann A, Hinchliffe RJ, Londahl M, et al. Effectiveness of interventions to enhance healing of chronic ulcers of the foot




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