Antimicrobial growth inhibition and mechanistic activities of synthesized Zinc oxide nanoparticles (ZnO NPs) were investigated in this study. Nanoparticle size 20-25 nm and concentrations of 0, 20, 40, 60, 80 and 100 µg/mL were used against pathogenic bacteria Staphylococcus aureus (Gram positive) and Salmonella typhimurium (Gram negative) and also first time against two plant fungi Aspergillus strain of flavus and fumigatus. The growth analysis data indicated that the ZnO NPs have significant bactericidal effect on both the bacteria. The quantity of dried fungal biomass was negligible when the fungal culture was grown in presence of 100 µg/mL NPs. These microbial analyses data indicates that the ZnO NPs (size ~20-25 nm) have shown potential activity against these tested bacteria and fungi. Further examine the antimicrobial mechanisms of the ZnO NPs by UV photocatlytic and GSH oxidation methods, which suggested that ZnO NPs could produce reactive oxygen species, and also the oxidation capacity of NPs towards the γ-L-Glutamyl-L-cysteinyl-glycine (GSH) oxidation stress, were responsible for antimicrobial behaviour of ZnO NPs.

Nanoparticles , Zinc oxide , Antibacterial, Antifungal, Reactive oxygen species

Navale GR, Thripuranthaka M, Late DJ, Shinde SS (2015) Antimicrobial Activity of ZnO Nanoparticles against Pathogenic Bacteria and Fungi. JSM Nanotechnol Nanomed 3(1): 1033.

Metal oxide nanoparticles and composite materials are widely applied in the field of research and development and diverse applications in industries including surface coatings, optoelectronics, bioengineering, biodignostics, and agriculture [1,2]. Their intrinsic properties are mainly determined by size, shape, composition, crystallinity and morphology. Highly ionic zinc oxide nanoparticles are unique in that they can be produced with high surface area, unusual crystal structures and size. The main advantages of using ZnO nanoparticles have excellent stability or long shelf life with organic antimicrobial agents [2]. In particular, the vigorous antimicrobial properties of nanoscale ZnO particles have been the focus of industrial applications in biocides coating in water treatment, paints and cosmetic products [3]. The scope of ZnO nanoparticles has been a keen area of interest for biologist due to their distinguished antimicrobial and distinct activity which has opened new frontiers to biological sciences [4]. Especially in its nanoscale form has a strong toxicity towards a wide range of micro-organisms including bacteria [5], fungi [6], fish [7], algae [8] and plants [9]. To find out the antibacterial activity of nanoparticles the pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus strains are being well know for initial investigation [10]. Growth of fungal pathogens in plants is the main cause of considerable economic loss during postharvest handling of grains and fruits. The fungal growth is difficult to control by organic substrate because fungi have developed resistance to many conventional fungicides such as benzimidazoles and dicarboximides [11]. Fungus Aspergillus species infection to plant will reduce the 20-30% total crop yield; this crop loss can be control by various metal nanoparticles because it has strong antifungal effect against specific fungi [12]. Infections of Aspergillus species have been identified and they are of major concern to the agriculture production losses.

We recently studied the ZnO NPs has different optical, field emission properties and applications in the field of nanotechnology [13-15]. Our continuous interest in ZnO nanoparticles, herein the structure and composition of freshly synthesized ZnO NPs 20-25 nm in size and spherical and hexagonal shaped were examined and evaluate influences against common pathogenic bacteria S. aureus (Gram-positive) and first time with S. typhimurium (Gramnegative) and two new agricultural important pathogenic fungus from Aspergillus family. As per our literature survey, this is first time to investigate the effect of ZnO nanoparticles with bacteria S. typhimurium, and fungus A. flavus and A. fumigatus. Also figure out the possible mechanism of antimicrobial effects by photo catalytic and GSH-Oxidation study.

Synthesis of ZnO Nanoparticles

To prepare of ZnO nanoparticles, precipitation method was applied according to modified procedure reported by Sourabh D et al., [16], The ZnO nanoparticles were successfully synthesised using zinc acetate di-hydrate (Zn (OAc)2 . 2H2 O, 0.3M) and xylene (C8 H10) in 100 mL of methanol (MeOH) under constant stirring. In a typical experiment: The zinc acetate dihydrate (Zn (OAc)2 . 2H2 O) and xylene (C8 H10) were diluted in equal (1:1) ratio in MeOH and stirred the solution for 30 min to complete dissolution of acetate salt. The pH of this solution was measured and it was reached around 6.7 to 6.8. The solution was transferred to the three necked refluxing flask and was refluxed at, 65?C for 6 h and then air cooled at room temperature. With rise in refluxing temperature, the transparent solution was changed in to white suspension and white precipitate was achieved in 6 h. No precipitate was observed before 6 h. The obtained precipitate has been washed several times by centrifuging it at 5000 rpm for 10 min with methanol, ethanol and acetone to remove the ionic impurities and dried it by keeping it in hot air oven at 100?C for 24 h in a glass petridish. The white powder sample then further crushed with a mortar and pestle to obtain fine nano sized ZnO NPs.

Characterization

The crystal structure, size, and shape of the synthesized ZnO nanoparticles were investigated using powder X-ray diffraction (XRD) and transmission electron microscopies (TEM). ZnO nanoparticles were suspended in de-ionized water and ultrasonicated for about 10 min and a clear dispersion of which is drop casted on to a carbon film coated Cu grids for collecting high resolution transmission electron microscopy images (HRTEM), For recording the X-ray diffraction (XRD) pattern ZnO nanoparticles were coated into a thin film on a cleaned glass substrate and for recording the Raman spectrum the sample was prepared in the similar way as for XRD.

TEM images were collected using FEI TECNAI TF-30 (FEG) instrument. XRD patterns were collected between the angles 20 to 80 degrees at a scan rate of 1 degree per minute using the XPERT PRO diffractometer. Raman spectra were collected with Lab RAM HR with a laser source of 632.8 nm at a power of ~200 µW at the sample in the back scattering geometry. UVVis spectrum was collected from JASCO v570 instrument. UV- Vis absorption spectrum of the ZnO nanoparticles was recorded in the solution form.

Microbial strains and culture conditions

In this experiment, four representative pathogenic microorganisms were selected, including two bacterial pathogens (Staphylococcus aureus ATCC29737/NCIM-2901 and Salmonella typhimurium ATCC 23564/NCIM 2501) and two fungal plant pathogens (Aspergillus flavus NCIM-524 and Aspergillus fumigatus NCIM-902). The four microbes were all taken from National Collection of Industrial Microorganisms (NCIM) Pune, India. S. aureus and S. typhimurium were grown in LB (LuriaBertani, Hi-Media, India) broth medium in a humidified incubator at 37ºC with constant agitation overnight whereas A. flavus and A. fumigatus were sub cultured and isolated on sabouraud dextrose agar and broth (Himedia Mumbai, India). Cultures were inoculated from isolated colony into Sabouraud Dextrose Broth and grown aerobically at 30°C.

Minimal Inhibitory Concentration (MIC) Determination

To investigate the minimum inhibitory concentration (MIC) of ZnO NPs against both S. aureus and S. typhimurium were determined by a method recommended in NCCLS [18]. Briefly, the sterile tubes were incubated aerobically at 37°C for 24 h with 180 rpm, which contained 5 mL Muller-Hinton (MH) broth (Himedia-India) with approximate 5×107 CFU/mL of Gram-positive S. aureus and Gram-negative S. typhimurium cells with various concentration of ZnO nanoparticles (20 µg/mL, 40 µg/mL, 60 µg/ mL, 80 µg/mL, 100 µg/mL). The concentration of tube without visible growth of both the bacterial cells was the MIC and which is measure by taking optical density at 600 nm by using UVspectrophotometer (JASCO-530) after 24 h incubation. All the measures were triplicate

Bacterial cell growth and Viability Testing

To examine the bacterial growth rate or the bacterial growth behavior in the presence of 20-25 nm sized ZnO nanoparticles S. aureus and S. typhimurium, both the bacterial cultures were harvested in the mid exponential growth phase and the cells were collected by centrifugation at 6000 rpm for 10 min. Subsequently, the bacterial bread was washed three times with saline water to wipe off the medium constituents and other chemical macromolecules. Finally, the cells were re-suspended in saline water and the suspensions were progressively diluted to a desired concentration of 107 to 108 CFU/mL. 200µL of the diluted cell suspensions was mixed with 20 µL of five different concentrations of ZnO NPs (20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, 100 µg/mL) prepared in deionized water) along with a control and incubated at 37ºC for 2 h with gentle shaking. A control sample contained 200 µL of the cell suspensions and 20 mL of saline. The mixture was then transferred to 5 mL tubes, each containing 2 mL MH medium (Muller Hinton broth ,Himedia, India) and the tubes were incubated on a rotary shaker at 150 rpm and 37ºC. The value of optical density (OD) at a wavelength of 600 nm was measured on a UV-VIS spectrometer every 2 h. Bacterial growth curves were created by plotting OD values versus time. All treatments were prepared in triplicate.

For antibacterial activity (Viability) test, the bacteria (107 to 108 CFU/mL) were incubated with different concentrations of ZnO for 4 h, and then 20µL of a serial 106 -fold dilution of each bacterial suspension in sterile deionized water was spread onto LB plates and let to grow for 24 at 37ºC. Colonies were counted and the cell mortality (% of the control) was expressed as the percentage of [(counts of the control-counts of the treated samples)/counts of the control] X 100. All treatments were individually repeated at least three times.

Antifungal Activity

The antifungal activity of ZnO NPs towards, A. flavus and A. fumigatus was studied by using modified Broth dilution Method, recommended in NCCLS 2002 protocol [19]. Sterile tubes containing 5mL Sabouraud Dextrose broth (Himedia Mumbai, India) inoculated with A. flavus or A. fumigatus were incubated along with different concentrations of ZnO nanoparticles at 37°C and 225 rpm for 24 to 72 h. The OD was measured after every 24 h at 410 nm. The experiment was conducted in triplicate.

In order to measure the fungal biomass/dry weight we applied similar method as previously reported [20]. After 7 days incubated fungal samples were then filtered through Whatman No.1 filter paper. Then the paper with fungal filtrate was dried at 50°C for 24 h in hot air oven and was used to determine the fungal biomass. The net wet of mycelia was determined by eliminating the weight of filter paper.

Detection of superoxide radical anions (O2 ? ?)

To find out the reactive oxygen species antibacterial paths, the possibility of superoxide radical (O2 ? ?) anion production was evaluated by measuring the absorption at 470 nm after the exposure of ZnO NPs to UV radiation for 2 h to 6 h, as ZnO NPs has a photo catalytic activity previously reported [21]. The 1 mg/ mL ZnO disperse in DI water, After 10 min ultra-sonication, it was exposed to UV light ((365 nm, 66.2 mW/cm2 , Blak-Ray Longwave Ultraviolet Lamp B 100 AP) for different time interval. The produced superoxide radical was measured by taking out sample and then filtered it with whatmann filter paper. 250µL filtrate was taken and their absorbance at 470 nm was measured on a microplate spectrophotometer (Thermo Scientific VarioskanTM Flash Multimode Reader). TiO2 NPs used as a positive control.

In vitro GSH oxidation and quantification

For the quantification of GSH oxidation was examined by the Ellmans assay [22]. The dispersion of the ZnO (250 µl of 20 µg/mL and 100 µg/mL NPs) in 50 mM bicarbonate buffer (pH 8.6) was added into 250 µL of GSH (γ-L-Glutamyl-L-cysteinylglycine Reduced 98%, Sigma Aldrich) (0.8 mM in the bicarbonate buffer) to initiate oxidation. The mixtures were covered with alumina foil to prevent illumination, and then placed in a shaker with a speed of 160 rpm at room temperature for incubation of 2-6 h. After incubation, 785 µL of 0.05 M Tris–HCl and 15 µL of DNTB (Ellman’s reagent, 5, 50-dithio-bis-(2-nitrobenzoic acid), Sigma-Aldrich) were added into the mixtures to yield a yellow product. The ZnO NPs was removed from the mixtures by centrifuging it at 4000 RPM for 5 min. A 250 µL aliquot of supernatant from each sample was then placed in a 96-well plate. All tests were prepared in triplicate. Their absorbance at 412 nm was measured on a microplate spectrophotometer (Thermo Scientific VarioskanTM Flash Multimode Reader). GSH solution without ZnO Nps was used as a negative control. GSH (0.4 mM) oxidization by H2 O2 (1 mM) was used as a positive control. The loss of GSH was calculated by the following formula: loss of GSH  (%) = (absorbance of negative control -absorbance of sample)/ absorbance of negative control x 100.

Characterization of ZnO NPs

The synthesized ZnO NPs were characterized based on data as shown in Figure 1.

Figure 1: As synthesized ZnO nanoparticle (a, b) TEM images, (c) SAED pattern, (d) UV-Visible spectrum, (e) Raman spectrum, and (f) XRD pattern and also corresponding JCPDS Card No 36-1451 for hexagonal Wurtzite-typed ZnO were shown in inset down.

The TEM images of the ZnO NPs from which the average of nanoparticle were calculated to be ~20 nm with spherical and hexagonal shaped was observed (Figure 1a and 1b). Figure c of 1 shows the typical selected area of electron diffraction pattern (SAED) of the ZnO NPs which confirms the highly crystalline nature of ZnO NPs [23]. The Raman spectrum of ZnO NPs was excited with 632.8 nm laser source with a power of 200 µW recorded in the standard back scattering geometry (Figure 1e). From the spectrum we observed two significant peaks 436.5 cm-1 corresponding to the Raman active E2 mode and the 607.9 cm-1. These observed peaks consistent with the wurtzite structure of ZnO NPs reported earlier [24]. The powder XRD pattern of the ZnO NPs is shown in Figure f of 1. The typical peaks observed in the pattern correspond to the planes (100), (002), (101), (102), (110), (103), (112) and (201) and are in good agreement with the spherical and hexagonal wurtzite structure of bulk ZnO as confirmed by comparison with the JCPDS card (#36-1451). This powder XRD data indicate the ZnO NPs are in the wurtzite structure. The UV-Vis absorption spectra of the obtained ZnO NPs are in the range of 200 nm to 800 nm. We observe the a typical absorption peak at ? 310 nm as shown in Figure 1 (d), which corresponds to a band gap of 3.9 eV, was consistent to the previous report [25].

Antibacterial Activity

The minimal inhibitory concentration (MIC) of ZnO NPs towards the both the pathogenic bacteria Gram positive S. aureus and Gram negative S. typhimurium were 40 µg/mL, which were determined by taking absorbance at 600 nm after 24 h of incubation of bacterial cells. Chemically synthesized nanoparticles were unable to diffuse through agar medium. We observed the MIC value of ZnO NPs against S. aureus and S. typhimurium was almost similar (40 µg/mL). The minimum bactericidal concentration (MBC), after plating of 100 µg/mL ZnO NPs treated bacterial medium it showed the no growth on the LB plates (<5 colonies). In bacterial growth curve experiment, ZnO NPs had slightly more activity against S. aureus than S. typhimurium till 8 h. Concentration of 60 µg/mL ZnO NPs showed the bacteriostatic affect against both the pathogens but 80 µg/ mL ZnO NPs had shown the completely inhibitory effect against S. typhimurium (Figure 2).

Figure 2: The effect of ZnO NPs on the growth of S. aureus (a) and S. typhimurium (b) culture in MH broth containing various concentrations of ZnO NPs (0 µg/mL control, 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, 100 µg/mL) was grown at 37ºC with shaking at 225 rpm. The growth curve was determined by measuring the OD600nm of the sample versus time (h).

In case of S. aureus, 80 µg/mL and 100 µg/mL ZnO showed the completely antibacterial activity, ZnO NPs retards the growth of both the pathogens when they are present in the bacterial suspension. Since the gram positive bacteria more susceptible towards metal nanoparticles as previously reported [5], surprisingly we found this ZnO NPs has shown quite similar activity against both bacteria.

In the viability test, the viability of the S. aureus and S. typhimurium decreases with increase in the concentration of ZnO NPs (Figure 3).

Figure 3: The viability of S.aureus and S. typhimurium cells after incubating with ZnO suspensions (20μg/mL, 40μg/mL, 60μg/mL, 80μg/mL and 100μg/mL) for 4 h with 220 rpm shaking speed at 37°C. Loss of viability was calculated by the following formula: loss of viability % = {(counts of control − counts of samples after incubation with suspensions)/counts of control}X 100.

After 4 h of incubation nearly 50 ± 1.9 % of S. aureus cells lost their viability in the concentration at 60 and 100 µg/mL of NPs, as result the almost 95 ± 1.5 % of lost viability as compare to the control (Figure 4 compare a-c).

Figure 4 compare a-c)The viability of bacterial cells with ZnO NPs after incubating for 4 h with 220 rpm shaking speed at 37°C. S.aureus with a) control 0 μg/mL, b) 80 μg/mL and c) 100 μg/mL; S. typhimurium cells

On the other hand after 4 h of incubation S. typhimurium lost 75 ± 1.8 % viability in presence of 80µg/mL and more than 90 ± 1.4 % in 100 µg/ mL ZnO NPs concentration compared to the control (Figure 4 compare d-f).

Figure 4 compare d-f)The viability of bacterial cells with ZnO NPs after incubating for 4 h with 220 rpm shaking speed at 37°C. S.aureusd) control 0 μg/mL, e) 80 μg/mL and f) 100 μg/mL concentration.

This excellent antibacterial activity of ZnO NPs is due to the high surface area of smaller in size nanoparticles and ROS production.

Antifungal Activity

As per the our literature survey there is few studies over the antifungal activity of ZnO NPs. Fungi static activity of ZnO nanoparticles were investigated by the broth dilution method according to protocol in NCCLS [19]. Antifungal activity of the ZnO NPs against two major plant and animal pathogenic fungi viz. A. fumigatus and A. flavus is first time reported in this study. From the concentration of 20 µg/mL ZnO NPs started to elicit inhibitory activity against plant pathogenic fungi A. flavus and A. fumigatus (graph not shown). However after 3 days of incubation by comparing optical density at 410 nm, 60 µg/mL and 80 µg/ mL ZnO NPs showed the excellent activity against A. fumigatus and A. flavus respectively. After 7 days of incubation of both the fungi with different concentration of ZnO NPs, the fungal biomass was significantly decreased as the concentration of ZnO NPs was increases greater than 40 µg/mL. Figure 5

Figure 5: Effect of different concentrations of ZnO NPs on direct biomass of A. flavus (blue) and A.  fumigatus (orange) after 7 day of incubation (values ± 0.02 g).

, clearly reveals that the up to 75% of fungal biomass was reduced in presence of ZnO NPs 80 µg/mL concentrations. After a 7 days of incubation, the all the fungal biomasses were collected on filter paper, which showed significant antifungal role of ZnO NPs (Figure 6).

Figure 6: Fungal biomass harvested on filter paper treated with ZnO NPs, A. flavus with (a) control, (b) 80µg/mL. A. fumigatus with (c) control, (d) 80µg/mL concentration.

However, the both fungi treated with ZnO NPs showed nearly similar inhibitory effects but A. fumigatus slightly was more susceptible as compared to A. flavus (compare Figure 5 and 6). These results indicated that as the concentration of ZnO increases, the dry biomass of fungi decreases, up to the concentration 100 µg/mL. The analysis of the data can be applied to the analysis of protein quantity from total fungal mass [20].

These results clearly demonstrate that the zinc oxide could be the good antifungal agent against pathogenic fungi that can help to avoid crop infection of not only A. flavus and A. fumigatus but also the other species of Aspergillus as well as other similar fungi.

Antimicrobial Mechanism

Previous studies on the antibacterial Behavior of Zinc NPs have shown that the morphology and oxidative stress play important roles in the antibacterial activity [16,17]. Due to the quite similar nano sized structure of our ZnO NPs to other oxide nano particles, we examine oxidative stress in presence of NPs in order to explore how ZnO NPs kills the microorganisms including bacteria and fungi. To demonstrate the antibacterial mechanism of the ZnO NPs, we applied two methods, photo catalytic and GSH oxidation protocol to measure the superoxide anion production. Normally, oxidative stress may come from nanomaterials itself and other one it may came through disturbing microbial process or oxidizing a vital cellular structure or components without ROS production, i.e ROS independent oxidative stress [26,27]

TiO2 radiated with UV light was used as a positive control to validate our tests and along ZnO NPs (without radiating with UV light) as a negative control. As shown in Figure 7,

Figure 7: Time dependent photo catalytic activity of ZnO NPs. ZnO NPs was incubated in the presence of UV radiation, 2h, 4 h and 6 h.  TiO2 was used as a positive control and ZnO Nps without UV-radiation was used as a negative control.

we found that ZnO NPs has the property to generate ROS in the presence of UV light; it could show significant antimicrobial activity in the presence of UV light because of ROS generation in the medium. After the 2, 4 and 6h incubation in UV radiation ZnO NPs have shown ascending level of absorbance that confirmed the ZnO NPs could be produce super oxide anion.

We further carried in vitro GSH oxidation protocol to figure out the second possible mechanism, as GSH is an antioxidant to bacteria and fungi of plants, to examine the possibility of ROS independent oxidative stress because this path play a significant role in the antimicrobial as well as toxic activity of nanoparticles [28,29]. Mechanism of GSH is a tripeptide with thiol groups (–SH) can be oxidized to form disulphide (–S–S–), converting GSH to glutathione disulphide. In bacteria GSH found of a concentration in the range from 0.1 to 10.0 mM preventing damages to cellular components. the employed the assay content, bicarbonate buffer (50.0 mM at pH 8.6) was used as negative control and H2 O2 (1.0 mM) without ZnO NPs was used as a positive control in GSH oxidation experiments. As shown in Figure 8,

Figure 8: Time and concentration dependent GSH oxidation of ZnO Nps. 20 µg/mL and 100 µg/mL ZnO NPs were incubated with GSH (0.4 mM) for 3 h and 6 h.  H2O2 (1 mM) was used as a positive control.

the ZnO NPs had a capacity to oxidise GSH. The oxidation capacity of the ZnO NPs toward GSH was above 91.5 ± 1.4 % and above 55.6 ± 1.5 % after the incubation of 6 h with 100 µg/mL and 20 µg/mL ZnO NPs concentrations. The oxidation capacity of the ZnO NPs toward GSH was examine by taking absorbance at 412 nm. This incubation time and concentration dependent oxidation capacity is well consistent with the antibacterial activity as observed in Figure 8. These experiments proved that the antimicrobial activity of ZnO nanoparticles is dependent on the size of the nanoparticles and is mainly due to the particulate ZnO as the release of free Zn2+ ions and Reactive Oxygen Species (ROS) from ZnO colloidal solution is low in the experiment, these results are consistent with earlier report of ZnO NPs with spherical shapes and 15-20 nm size [17]. We realize the potential applications of ZnO NPs in medical fields so, to get a better understanding of the antimicrobial toxicity mechanism, the possibility of superoxide anion induced by ROS production was evaluated by UV-mediated photo catalytic experiment, and in vitro GSH (g-L-glutamyl-Lcysteinylglycine) oxidation methods. Our study suggests that the ZnO NPs and their photo catalytic properties contribute greatly to their antimicrobial activity with membrane and oxidation stress.

In conclusion, the ZnO NPs sizes of 20-25 nm possess significant antibacterial properties against S. aureus and S. typhimurium. Our results suggested that the ZnO NPs have greater efficacy in inhibiting the growth of S. aureus compared to the S. typhimurium and it’s inhibitory effects increases as the concentrations of ZnO NPs increased. The concluded MIC for S. aureus and S. typhimurium was 40 µg/mL, In case of both fungi the concentration of ZnO NPs 80 to 100 µg/mL were more enough to significantly control their growth, which was demonstrated by weighing the fungal biomass. The antimicrobial mechanisms of the ZnO NPs, suggested that oxidation capacity of NPs toward GSH oxidation stress were responsible for antimicrobial behaviour of ZnO NPs. The results of S. typhimurium, and fungi A. flavus and A. fumigates suggest that ZnO nanoparticles are not only useful as an effective fungicide in agricultural and food packaging applications but also to control the growth of pathogenic bacteria.

Authors, S.S.S. and D.J.L., would like to thank DST (Government of India) for the Ramanujan fellowship (Grant No. SR/S2/RJN-111/2012 and SR/S2/RJN-130/2012). The research was primarily supported by DST Fast Track scheme for Young scientist project is gratefully acknowledged (Grant No. SB/FT/CS042/2013 and SB/FT/CS-116/2013), and NCL-MLP project grant 028626, and the partial support by INUP IITB project sponsored by DeitY, MCIT, Government of India. Thanks to Director CSIRNCL for constant support and encouragement.

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