Evaluation of Anti Carbapenem-Resistant Klebsiella Pneumonia of Zinc Oxide Nanoparticles Synthesized by Aspergillus Niger in Vitro and in Vivo

Currently, the mortality rate is increasing in Saudi Arabia's ICU, due to the spread of KPC. This project was carried out to evaluate the ability of the biological synthesized of zinc Oxide nanoparticles (ZnO-NPs) using Aspergillus niger to overcome Carbapenem-Resistant Klebsiella pneumonia (KPC) in vitro and in vivo. ZnO-NPs was synthesized via a biological method and characterized using UV-Vis spectroscopy, Zeta sizer and Zeta potential analyses, X-ray diffraction (XRD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). In vitro sensitivity of KPC to ZnO-NPs was identied using the well diffusion method, MIC and MBC was determined by macro dilution method. The morphological alteration of KPC cell after ZnO-NPs treatment was showed by SEM. In vivo susceptibility of KPC to ZnO-NPs ointment was evaluated using wound healing in experimental rats. The chemical characterization ndings showed the formation, stability, shape and size of the synthesized nanoparticles. The MIC and MBC results was found in 0.7mg\ml and 1.8mg\ml respectively. in vivo results displayed the inammation reduction and wound healing re-epithelialisation of kpc infected rats. these ndings demonstrated that ZnO-NPs has great potential to be developed as antibacterial agents and wound healing.


Introduction
Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile, rod-shaped opportunistic pathogen. It cause wide range of hospital acquired infections such as: wound infection, bacteremia, pneumonia, and urinary tract infections, particularly in immune compromised people 1 .
Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae cause infection may associated with the treatment failure and increased mortality 2 . It is increasingly recognized as a serious, worldwide public health concern. That is why is very important to nd out new strategies and ecofriendly drug to eradicate KPC and to control this emerging issue of multi drug resistant bacterial strains 3 . Among the genus Enterobacteriaceae, carbapenemases are more prevalent in K. pneumoniae isolates, which usually causes hospital acquired infections and outbreaks in Saudi Arabia. This is reported by Alotaibi 4 .
Also he found that both carbapenem producing E. coli and K. pneumoniae that isolated from a tertiary care hospital in Riyadh, are more frequently K. pneumoniae (63%) compared with E. coli (55%) 4 .
The international travelling is the primary rout of the spread of KPC spreading in Gulf Cooperation year, which further promotes the spread of KPC 5 .
In Saudi Arabia, KPC is high incidence this may due to the large number of Pilgrims, visitors and migrant workers receives from endemic countries such as Turkey, India, and Pakistan every year 6 Survey such as that conducted by Faiz and M 7 have shown that the incidence of KPC among Makkah hospitals is about 48.4% from other carbapenem producing organisms 7 .
Biogenic synthesis of nanoparticles, green synthesis of nanoparticles and biosynthesis of nanoparticles, all of these terms are called to synthesize Nano by using plants or microorganisms. Nanoparticles from such "green synthesis" have been used in the eld of drug, gene delivery and various medical treatments including antimicrobial, anticancer, anti-in ammatory, antiaging, antioxidant and anti-bio lm inhibition 8 . Synthesis of oxide nanoparticles by using eukaryotic organism like fungi is good for the synthesis of metal nanoparticles, because of their ability to produce large amount of enzymes 9 .
Several attempts have been done to evaluate and experiment the antimicrobial activity of Zinc oxide nanoparticles (Zn-ONPs), On October 2020a group of researcher were studied the antimicrobial activity of biosynthesis Zn-ONPs using aqueous extracts of pomegranate leaves and owers designated ZnO-NPs-PL, ZnO-NPs-PF. They found that ZnO-NPs were effective against all their selected pathogenic strains including Klebsiella pneumonia, then they reported that both ZnO-NPs can effectively be used as alternative antibacterial agents 10 . One study by 11 , showed that how ZnO NPs possess strong antimicrobial activity and can promote the antimicrobial activity of some beta-lactam antibiotics such as: Klebsiella pneumoniae and Escherichia coli 11 .
On the side of antimicrobial, ZnO appears to be strong potential to kill microorganisms whereas Zn-ONPs presented strong antibacterial activities on broad-spectrum pathogenic bacteria such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, E. coli O157:H7, Salmonella enteritidis, Salmonella typhimurium, Pseudomonas uorescens, and Listeria monocytogenes. ZnO nanoparticles's mode of action occur through the inhibition of the bacterial growth by fell a part of the cell membrane and increasing the membrane permeability, affect the synthesis of hydrogen peroxide and nal penetration of cell wall and disorganization of bacterial membrane 12,13 .

Characterization of Zinc oxide nanoparticles synthesized by Aspergillus niger
The result of UV-Vis absorbance spectrophotometer (wave range 200-800 nm) of the synthesized Zn-ONPs using Aspergillus niger showed an absorbance peak at 304 nm (Fig. 1A). The X-ray diffraction pattern of ZnO-NPs found that the peaks of ZnO-NPs showed at 2θ of 31 (Fig. 1B). To determine the functional groups responsible for the synthesis of ZnO-NPs and to compare the functional group in Aspergillus niger which mediate the synthesized nanoparticles before and after calcination we used Furrier Transform Infra-Red Spectroscopy (FTIR) in the range of 400-4000 cm-1. FTIR of ZnO-NPs according to Fig. 1C, which presented the sharp peak at 3482cm-1 and 3401cm-1 corresponds to O-H strong, group which are found in ZnO-NPs before and after calcination and the aqueous extract of Aspergillus niger. ZO group and N-H medium, bend amins group were detected only in ZNO-NPs before calcination. After calcination showed S-H weak, stretching thiol, C = O stretch, α,β-unsaturated aldehyde keton, C-H medium, rock alkanes, C-N medium, stretching aliphatic amins, C-H meadium, wag alkyl halides and C-Br medium, stretching alkyl halides. The previous functional groups in ZnO-NPs after calcination makes it effective against tested bacteria. The aqueous extract of Aspergillus niger have several functional groups which responsible of the synthesized of zinc oxide nanoparticles such as: C = O, C-C, C-N, C-Cl) and C-Br groups.
The measured zeta potential value of biosynthesized zinc oxide nanoparticles showed in Fig.2A&B, were foundmean Z-average diameter (nm) of the ZnO-Nps and the size distribution were observed to be 176.5nm for zeta size and −0.734mV for zeta potential. The size distribution pro le was 100% and 0%. The results of SEM and EDX was conducted to con rm the formation of nanoparticles and their elemental composition, SEM is also analyze the structure of ZnO-NPs that were formed. SEM image of Fig. 3B, has shown an irregular, individual spherical shape, and most of them presented with aggregates hexagonal shape with a smooth surface and were apparently devoid of cracks. The elemental analysis of ZnO-NPs for Zinc and Oxygen components showed 61.63% zinc and 38.37% oxygen Fig. 3A.

Antibacterial activity:
Interestingly, the zone of inhibition (ZOI) results was observed promising outcoms of the synthesized ZnO-NPs against tested bacteria (KPC) using an agar well diffusion method. All tested KPC and Klebsiella pneumoniae (ATCC700603) as control were showed highly sensitivity to ZnO-NPs (20.8 ± 20.8 ± 2.7mm) at 7.5 mg\ml concentration, (Fig. 4A). The minimum inhibitory concentration (MIC) was determined by the macro dilution method in culture broth followed by the minimum bactericidal concentration by the agar dilution method. Figure 4B,C &D and Table 1, presented the experimental data of MIC and MBC for all tested KPC; the mean score for MIC was 0.7 mg/ml and for MBC was 1.8 mg/ml.

SEM for bacterial cell morphology
The bacterial cell morphology changes were studied by SEM. From the Fig. 3C, D&E, we can see that KPC cell treated with ZnO-NPs reported signi cantly changes in the bacterial cell included severe damage, change and decreased in size shape from rod-shaped to slightly coccus-shaped, also showed multiple dents on the cell surface these results. Whereas, the untreated KPC cells and those treated with impenem have no morphological change was reported. The ndings from SEM are matching with those 14-16 .

ZnO-NPs improves KPC-infected wound healing in rats
Wound healing evaluation was based on the change of fresh wounds of rats and the degree of closure on days 3, 7, 11, and 14 after wounding (Fig. 5). Group-1 (G-1), the infected and untreated control group showed severe tissue in ammation with purulence on the wound surface on all days. Group-2 (G-2), the infected and untreated control group showed tissue in ammation on day-3,7 and 11, with slight bleeding on day-7. Group-3 (G-3), the infected and treated with imipenem ointment group showed sever in ammation on day-3, 7 and 11 with slight bleeding thick mixture of layers of organisms and purulence, while on day-14 the tissue recovered the damage and in ammation. Group-4 (G-4), the infected and treated with ZnO-NPs ointment, showed severe tissue in ammation with slight bleeding on day-3 and 7, which disappeared on day-11 with signi cant improvement of healing on day-14. Statistical analyses of the mean percentage of wound healing on day 14 after wounding showed that G-1 displayed comparable wound heal rate (63%) healing with G-2 (64%) healing, while, G-3 exhibited moderate result (70%) healing and G-4 presented promising result (96%) ( Table 2. A histogram of Table 2 is shown in Fig. 4E).

General histopathology of wound healing
Hematoxylin and eosin (H&E) used to stained the stages of wound healing from the four groups of rats under investigation on days 3, 7, and 14. Unwounded control skin posted normal skin feature with epidermis layer of strati ed squamous epithelia thickened (348 µm) sheathed with keratinized layer and underneath with dermis layer of connective tissue (Fig. 6A). Untreated wounded skin after 3 days of post operation day (POD) altered hemorrhage and granulomatous reaction of in ammatory cells (Fig. 6B). Whereas, untreated infected wounded skin revealed more granulomatous reaction 3 days of POD (Fig.  6C).
Untreated wounded skin after 7 days of POD represented a primary formation of scab underneath with in ammatory granulomatous reaction (Fig. 7A). While, infected untreated wounded skin showed heavy incidence of hemorrhage foci and in ammation (Fig. 7B). Moreover, infected wound skin treated with Imipenem antibiotic exhibited formation of scab and in ammation (Fig. 7C). Furthermore, infected wound and treated with ZnO-NPs also showed more scab formation and granulomatous reaction (Fig.   7D).
Untreated wounded skin 14 days post to wound cut displayed a super cial scab to in ammatory granulomatous reaction (Fig. 8A). Whereas, infected untreated wounded skin showed more concentrated in ammation beneath the scab (Fig. 8B). Additionally, infected wound treated with Imipenem antibiotic showed thickened regenerated epidermis (710µm) sheathed with keratinized layer (Fig. 8C). Furthermore, infected wound and treated with ZnO-NPs posted marked improvement of wound with regenerated differentiate epidermis (391µm) and more improved dermis (Fig. 8D).

Discussion
An initial objective of this project was to nd out and develop new antibacterial agent to manage bacterial infections which occur due to KPC. The current study found that the Aspergillus niger mediate the biological synthesis of ZnO-NPs was appeared promising activity against KPC, also the most interesting nding was that shows in the recovery of wounds, this may due to the stability of the synthesized nanoparticles for long time. In reviewing the literature, no more in vivo studies was found.
Optical properties of ZnO-NPs were characterized using UV-Vis spectrophotometer. Zinc oxide formation was con rmed as the absorption peak (lambda max) was found near 304 nm. This result correlate with the result which reported by Aldalbahi 17 , who was observed the maximum peaks of ZnO NPs at 300 and 359 nm. The results of UV-Vis were con rmed the present of ZnO-NPs. The measured zeta potential value of ZnO-NPs possesses a negative zeta potential value of -0.734 mv, which indicated that these nanoparticles have a high stability due to the electrostatic repulsive force 18 . For zeta sizer, ZnO-NPs showed (176nm) in size. The XRD results are consistent with those of 15,19 , Although, these results differ from some published studies 17 . On the side of nanoparticles structure, ZnO-NPs showed a crystalline hexagonal structure similar to the synthesized nanoparticles structure reported by 15,20 . XRD indicates that the synthesized nanoparticles are in their purest form and have a crystalline structure. A possible explanation for these results due to their tight and strong diffraction peaks. To identi ed the various functional groups found in synthesized nanoparticles and the aqueous extract of Aspergillus niger as control were characterized under FTIR. The functional group O-H was presented in the aqueous extract of Aspergillus niger and ZnO-NPs before and after calcination, this result explain that O-H group is the reducing agent which responsible of ZnO-NPs formation. While most of the functional groups detected as shown in Fig. 1C, such as C-H, C = O, C-C, N-H, N-O, C-N, OH, and ZnO, were similar to those obtained previous [21][22][23] . Size and the morphology of the ZnO-NPs were identi ed using the SEM. The average particle size was around 48-88 nm, with a hexagonal shape, this study produced results which corroborate the ndings of a great deal of the previous work in SEM image [24][25][26] . Interestingly, ZnO-NPs was found to have a promising antibacterial activity, ZOI mean for the tested KPC was 20.8 mm vis 22.9 for the ATCC 27 . This nding corroborates the results of many researchers who obtained 16-27 mm of ZOI 11,20,28,29 .
According to Van vuuren 30 , and others natural products with MIC values below 1.0 mg/ml are considered noteworthy. The mean MIC and MBC of ZnO-NPs was 0.7mg\ml, 1.8 mg\ml respectively, It is encouraging to compare this result with that found by Yousef and others 20 , Their MIC result was 500 mg/mL against Bacillus subtilis, Bacillus megaterium, Sarcina lutea, Klebsiella pneumoniae, and Proteus vulgaris. This result is con rmed that the biosynthesis of nanoparticles is better than other method of synthesis. Nonetheless, ZnO-NPs achieved high stability and potency as antibacterial, according to the MIC and MBC result's. SEM analysis assists in the prediction that ZnO-NPs that interact with KPC surface may lead to transformations in cell size and shape these changes resulted to the disruption of the cell membrane and the death of the cell 23 . Surprisingly, in the side of in vivo experiments, the fresh wound healing area was found to have a promising result in G-4, which treated with ZnO-NPs ointment, while G-3 showed moderate healing it seems possible that these results are due to the resistance of the tested bacteria to Imipenem and G-1 and G-2 were presented the lowest wound healing, this may due to their dependent to the recovery of immune response only.

Synthesis of ZnO-NPs by using Aspergillus species:
The biosynthesis of nanoparticles by Aspergillus niger was modi ed from two references to develop the stability of the synthesized nanoparticles. Aspergillus niger (ATCC 16404 NA) was obtained from mycology department, King Khaled Hospital and inoculated aerobically in Czepak Dox agar plate for 96 hours at room temperature in orbital shaker at 160 rpm. The fungal biomass was harvested by Whatman no.1 lter paper to collect free fungal ltrate. Then the ltrate was washed by centrifuge at 10.000 rpm for 20 min, after that was transferred to 250 ml sterile ask and mix with 200ml of deionized water and incubated at room temperature in shaker incubator for 72 h at 200 rpm. Then, ltrated by Whatman no.1 lter paper and collected the ltrate of Aspergillus niger. To prepared zinc oxide nanoparticles we prepared 100 ml of 0.25 mM solution of zinc nitrate and added to 10 ml of Aspergillus niger ltrate and incubated in shaker incubator at 180 rpm for one week at 37 o c. Finally, white to yellow paste of ZnO-Nps were formed as showed in Fig. 9, then, packed it until used 31,32 .

The synthesized ZnO-NPs Characterization
The formation of ZnO-NPs was con rmed by UV-vis spectroscopy (UV-1800; Shimadzu UV Spectrophotometer, Kayoto, Japan), with the wave length range of 200-800 nm at resolution of 10nm. The zeta sizer and potential measurement was performed using dynamic light scattering and Malvern Zetasizer nanoseries compact scattering spectrometer (Malvern Instruments Ltd. Malvern, UK) respectively, the histogram was developed by Zetasizer software (version 7.11) (Malveren Panalytical, Malveren, UK). Zeta potential was measured by using the folded capillary cell of Malvern. Fourier Transform Infra-Red Spectroscopy (FTIR) was recorded in the range of 400-4000 cm-1 Nicolet (Parkin Elmer, Spectrum BX, Waltham, UK). To identify and determine the different functional groups present in the Aspergillus niger ltrate before and after mixed with Zinc nitrate, and the synthesized ZnO-NPs. The structure and crystalline nature and composition of synthesized ZnO NPs was analyzed by X-Ray diffraction (XRD). The formation, symmetry, size and shape of nanoparticles were analyzed in the form of powdered by an X-Ray diffractometer (Brucker-Discover D8, CUK-alpha, Sangamon Ave, Gibson, USA) of 2 /min scan speed the range 10-100•.Scanning electron microscopy (SEM) analysis was done to measure the size and the composition of synthesized nanoparticles using SEM (JEOL model, JSM-761OF, Tokyo, Japan) operated at an accelerating voltage of 10 kV with an EDX detector SEM machine. small amount of dry ZnO-NPs was prepared as thin lm on a carbon coated copper grid, then dried the lm by putting it on the SEM grid under a mercury lamp for ve minutes.

In vitro microbial susceptibility testing of the synthesized ZnO-NPs
Microbial susceptibility testing was performed by agar well diffusion technique. Tested bacteria (carbapenemase-producing Klebsiella pneumoniae (KPC)) were isolated as a retrospective sample from wounds of ICU patients at Prince Mohammed Bin Abdul Aziz Hospital -Al Madinah. Then, all samples were identi ed in microbiology department in the hospital by using VITEK 2 systems version: 08.01. Klebsiella pneumoniae (ATCC 700603) was tested as control and was obtained from the College of Applied Medical Science, King Saud University. All bacterial samples were kept in Nutrient Agar slant (NA) and stored at 4 0 c until used 33 . well plate agar diffusion method, was carried out by inoculated the tested bacteria on nutrient broth overnight, and adjusted to 0.5 McFarland turbidity standards, then each bacteria streaked on a Mueller-Hinton agar (MHA) plate by swab. A sterile cork borer was used to form wells (6 mm in diameter) on the agar plates. Then, we added 7.5 mg of ZnO-NPs dissolved in 1 mL of deionized water to obtain a concentration of 7.5 mg/mL, and add 0.

Scanning Electron Microscopy technique for Bacterial Samples images
Scanning electron microscopy (SEM) was carried out for KPC cells untreated (negative control), treated with Imipenem (Positive control) and treated with ZnO-NPs (tested sample) to study the morphological cell alterations. The treated cells of KPC were xed with 2.5% glutaraldehyde in a phosphate buffer having pH 7.2. The samples were post-xed in 1% osmium tetroxide, followed by dehydration through an ascending ethanol series, critical point dried and coated with Au-Pd (80:20) using a Polaron E5000 sputter coater, Quorum Technologies, Laughton, UK. The samples were checked at an accelerating voltage of 25 kV in FEI Quanta 250 using an SE detector 34,35 . 4.5 In vivo KPC susceptibility for ZnO-NPs Huawei mate-9 Came. At day 14, the rats were sacri ced by physical method (carbon dioxide inhalation) after lled the chamber with carbon dioxide for several minutes, and the skin tissues of the wound site were collected at day 3, 7 and 11 by chosen one rat from each group and under weak anesthesia we collected the skin samples to avoid any pain during the collection. While, at day 14 the samples collected after sacri ced all rat's groups. Hematoxylin and eosin (H&E) staining were performed for the histological analysis 38 (Fig. 10). Tissue sections were checked under a light microscope (Nikon, Eclipse i80), and images were taken at different magni cations using a Nikon mounted digital camera (OXM 1200C; Nikon, Japan).

Preparation of gel based ointments
Equal volumes of polyethylene glycol (PEG) 400-2000 was added to ZnO-NPs and imipenem separately, followed by boiling them at 65 _C for 5 min Ointment formulations to prepared 5 mg/mL of the oinment 39 .

Statistical analysis
Statistical analysis of the rate of wound recovery was performed using multivariate analysis of variance, and the statistical analysis of MIC, MBC, and ZOI was performed using paired samples t-test and ANOVA, with the results presented as mean ± SD with SPSS statistical software version 22 (SPSS Inc., Chicago, IL, USA).

Conclusion
This project was undertaken to design new way to treat the Carbapenem-Resistant Klebsiella pneumonia and evaluate the activity of it in vitro and in vivo. In this study we showed promising results in both in vivo and in vitro and we found that there was a correspondence between in vitro and in vivo results. The chemical characterization results of this study indicate that, the biological synthesis of nanoparticles provided nanoparticles with small size, suitable shape, stable for long time and have antibacterial activity. UV-Vis showed 304 nm which con rmed the formation of ZnO-NPs. XRD and zeta potential results presented the purity and the crystalline form of ZnO-NPs. O-H functional group was playing signi cant role in the formation of ZnO-NPs, according to the FTIR ndings. Zeta sizer found that the size of ZnO-NPs was 176nm and EDX result was detected the components of the synthesized ZnO-NPs which appeared 61.63% for Zinc and 38.37% for the Oxygen. The MIC of ZnO-NPs was 0.7 mg\ml and MBC was 1.8 mg\ml with 20.8mm of ZOI. ZnO-NPs ointment presented interesting results in wound healing, these results support the aim of this study of using ZnO-NPs as antibacterial agent.

Competing interest
The authors declare no competing interests.