In vitro antibacterial activity of ZnO and Nd doped ZnO nanoparticles against ESBL producing Escherichia coli and Klebsiella pneumoniae

Pure ZnO and Neodymium (Nd) doped ZnO nanoparticles (NPs) were synthesized by the co-precipitation method. The synthesized nanoparticles retained the wurtzite hexagonal structure. From FESEM studies, ZnO and Nd doped ZnO NPs showed nanorod and nanoflower like morphology respectively. The FT-IR spectra confirmed the Zn-O stretching bands at 422 and 451 cm−1 for ZnO and Nd doped ZnO NPs respectively. From the UV-VIS spectroscopic measurement, the excitonic peaks were found around 373 nm and 380 nm for the respective samples. The photoluminescence measurements revealed that the broad emission was composed of ten different bands due to zinc vacancies, oxygen vacancies and surface defects. The antibacterial studies performed against extended spectrum β-lactamases (ESBLs) producing strains of Escherichia coli and Klebsiella pneumoniae showed that the Nd doped ZnO NPs possessed a greater antibacterial effect than the pure ZnO NPs. From confocal laser scanning microscopic (CLSM) analysis, the apoptotic nature of the cells was confirmed by the cell shrinkage, disorganization of cell wall and cell membrane and dead cell of the bacteria. SEM analysis revealed the existence of bacterial loss of viability due to an impairment of cell membrane integrity, which was highly consistent with the damage of cell walls.


Antibacterial assay
The antibacterial activity was determined by the disc diffusion method against the test bacteria on Muller-Hinton agar, according to the Clinical and Laboratory Standards Institute (CLSI) 1 . The media plates (MHA) were swabbed with bacteria 2-3 times by rotating the plate at 60˚ angles for each streak to ensure the homogeneous distribution of the inocula. After inoculation, discs (6 mm Hi-Media) loaded with 2 mg of the test samples were placed on the bacteria-seeded plates using sterile forceps. The plates were then incubated at 37 ˚C for 24 h. The inhibition zone around the discs was measured and recorded. DMSO served as the control, and assays were carried out in duplicates. We have simultaneously studied whether or not the DMSO without the ZnO NPs plays any active role as a biocide. We found that DMSO did not show any biocidal properties.

Determination of MIC and MBC
In the present study, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for ZnO and Nd-doped ZnO NPs by the agar dilution method. 2 mL of the test sample was added to 19 mL of molten nutrient agar and mixed adequately through mixing of the sample into the medium. The final concentrations of the sample in each plate were 150, 250, 350, 500, 650, 800 and 1000 µg/mL. Each plate was inoculated with test bacteria and the growth was determined by colony formation. The minimal quantity of the undoped ZnO and Nd doped ZnO NPs required for the bacterial activity was studied by adding 150, 250, 350, 500, 650, 800 and 1000 µg/mL concentrations of the nanoparticles into the bacterial culture and after 24 h, the Optical density values (600 nm) were measured. The lowest concentration, at which there was no regrowth of the bacteria, when 3 transferred from the test plate into new media, is called the MBC, and the MIC is the lowest concentration at which 99% of the bacterial growth was inhibited. The experiment was carried out in triplicates.

Confocal laser scanning microscopic studies
Apoptotic cell Acridine Orange/Ethidium Bromide (AO/EB) dual staining was used in our experiment. The collected bacteria were resuspended in 1 mL LB medium and then incubated with 10 μL AO/EB at 37 °C for 10 min (450-490 nm, and the final concentration was 5 μg/mL). Subsequently, the bacterial suspension was centrifuged at 5000 g for 5 min at 4 °C, and the supernatant was discarded. The unincorporated dyes were removed by washing with phosphate-buffered saline (PBS). One droplet of cell suspension (5 μL) was dropped on the freshly treated glass slide, and then it was covered with the coverslip without bubbles. The cells were microphotographed with the magnification of 60X using a laser scanning confocal microscope (Carl Zeiss 710, Zen Software 2011).

Characterization techniques
The ZnO and Nd doped ZnO NPs were characterized by X-ray diffractometer (model: paramagnetic resonance (EPR) measurement was conducted with a Bruker EMX Plus spectrometer using an X band (9.78 GHz) at room temperature.

Morphology and Chemical Composition
The morphology and composition of as prepared nanoparticles are further investigated by FESEM and EDAX analyses. From Fig. S1(a-b), the pure ZnO NPs look as a hexagonal nanorod like morphology with uniform grain boundaries and Nd doped ZnO NPs exhibit flower like morphology. The size reduction is due to the distortion in the host material incorporated with Nd 3+ . The Nd 3+ ions decrease the nucleation and subsequent growth rate of ZnO NPs.
The chemical composition of the pure ZnO and Nd doped ZnO NPs are found out using EDAX analysis. The typical EDAX spectra of pure ZnO and Nd doped ZnO NPs are shown in Fig. S1(c-d)

FT-IR spectroscopic studies
The FT-IR spectra of the prepared ZnO and Nd doped ZnO NPs are shown in Fig. S2.
The FT-IR measurements are performed for the samples using the KBr pallet method in the wave number range 400-4000 cm -1 . The broad absorption in the frequency band 3750-3000 cm -1 is assigned to O-H stretching from residual alcohols, water and Zn-OH 2 . The absorption peak is observed at 3436 cm -1 for the ZnO NPs sample. The CO 2 peak is observed at 2369 cm -1 for the Nd doped ZnO samples. These CO 2 bands may arise due to some trapped CO 2 in air ambience 3 .  6 . A good fit with three peaks labeled as A1, A2 and A3 is obtained using a Gaussian function for the FT-IR spectra of undoped and Nd doped ZnO NPs. The fitting was carried out in the wave number range 400-600 cm -1 and is shown in Fig. S3 (a-b).
The three peaks are observed at 428, 474 and 534 cm -1 for pure ZnO NPs sample. The peak A1 at 428 cm -1 can be assigned to [E 1 (TO)]. The interaction between electromagnetic (EM) 6 radiations and the particles depends on the size, shape and state of aggregation of the crystals 7 .
Since the particle size in this study is much lower than the EM radiation, surface phonon modes (SPM) can be observed in the IR spectra. The SPM [A 1 (TO)] band (A2) around 473 cm -1 and [E 1 (TO)] band (A3) around 534 cm -1 can be identified as the SPM absorption in the IR spectra.
As compared to undoped ZnO NPs, the entire IR modes shift to the blue side in the case of Nd doped ZnO NPs. The shift in the position of the bands can be correlated with their respective ionic radii as well as the structural changes due to the Nd doping in ZnO NPs. The values are given in Table S1.

UV-Vis-NIR spectroscopic studies
The absorbance of the samples depends on several factors such as band gap, oxygen deficiency, surface roughness and impurity centers 8  The value of n = 1/2, 3/2, 2, or 3 depends on the nature of the electronic transition (1/2 for allowed direct transition, 2 for allowed in-direct transition, 3/2 and 3 for forbidden direct and forbidden indirect transition, respectively). Considering a direct band transition in ZnO, a plot between (αhυ) 2 versus photon energy (hυ) is drawn for ZnO and Nd doped ZnO NPs and is shown in Fig. S4 (a-b). Extrapolation of the linear region of these plots to (αhυ) 2 Figure S1 FESEM images of (a) pure ZnO NPs, (b) Nd doped ZnO NPs and EDAX spectra of (c) pure ZnO NPs (d) Nd doped ZnO NPs.