Effectiveness of Green Synthesized ZnO Nanoparticles Against MDR Resistant Klebsiella Pneumoniae Isolated From Diabetic Foot on Some Virulence Factors Genes Expression

• HaiderTurky Mousa Al-Mousawi

  Department of Applied Biotechnolog, College of Biotechnolog, Al-Qasim Green Universit

• Mustafa Muhammed Jadooa

  Department of Applied Biotechnolog, College of Biotechnolog, Al-Qasim Green Universit

• Nadhim Mushtaq Al-bderee

  Department of Applied Biotechnolog, College of Biotechnolog, Al-Qasim Green Universit

Diabetic foot ulcers are a major public health problem and are quite costly for diabetic patients. Besides, patients with diabetic foot ulcers often have a poor quality of life. Most of the main complications of diabetic foot ulcers are the bacterial infection that leads to gangrene when different types of antibiotics fail to prevent bacterial infections. The research proposed in this study aims to determine the most effective synthesis type of Zinc oxide nanoparticles (ZnO NPs) and study its effect on some virulence factor genes expressions. Research includes the isolation and identification of several bacterial species by biochemical tests, as well as confirmation by the Vitek system and molecular diagnostics. Out of 100 swabs, 26 samples were found to be contaminated with bacteria Klebsiella pneumoniae is one the main virulence factor that develops of diabetic foot ulcers. The biological production of Zinc Oxide was using bacteria Pseudomonas aeruginosa. The characterization of the Zinc Oxide products was using the Ultra-violet Visible spectrophotometer, Fourier transform infrared spectroscopy, Scanning electron microscopy, X-Ray diffraction spectrum very small crystals with a large spherical shape were formed, with a size of 13.5 nm. The genes were included as the bacterial virulence factors coding genes, MagA of gene Klebsiella pneumoniae, which are responsible for the production of exotoxins that cause sepsis, necrosis, tissue necrosis and non-healing.  The expression of MagA genes the different before and after treatment with ZnO NPs. The mRNA for both genes was extracted alongside the mRNA for 16SrRNA acting as housekeeping gene. All mRNAs were converted to cDNA through RT-PCR technique and then measure the expression by qPCR. The expressions of MagA genes for the biological product nanomaterial, bacterial nanomaterial, were significant for all the isolates with P<0.01. In conclusion from the foregoing the biological nanoparticles product was more sufficient for inhibition of most isolates.

Highlights:

1. Diabetic foot ulcers worsen by bacterial infections, causing severe complications.
2. ZnO nanoparticles synthesized biologically; bacterial virulence gene expression analyzed.
3. ZnO nanoparticles significantly inhibited K. pneumoniae virulence with P<0.01.

Keywords: K. pneumoniae, P. aeruginosa, ZnO NPs, Mucoviscosity associated a gene (MagA) and gene expression

M. Gadelkarim, A. I. Abushouk, E. Ghanem, A. M. Hamaad, A. M. Saad, and M. M. Abdel-Daim, “Adipose-derived stem cells: Effectiveness and advances in delivery in diabetic wound healing,” Biomedicine and Pharmacotherapy, vol. 107, pp. 625–633, 2018.

T. C. Quinn, D. A. Piggott, K. M. Erlandson, K. E. Yarasheki, D. Vancampfort, J. Mugisha, and B. Stubbs, “Global estimates of diabetes prevalence for 2013 and projections for 2035,” J. Clin. Exercise Physiol., vol. 8, no. 2, pp. 86–90, 2019.

P. G. Roy, K. Kumar, N. P. Singh, G. S. Ranga, and S. Giri, “Microbiological profile and antibiotic sensitivity pattern of bacteria in diabetic foot ulcers in a tertiary care hospital,” J. Adv. Res. Med., vol. 8, no. 1, pp. 2394–7047, 2021.

S. Wahjuni and N. W. Bogoriani, “Green Mustard Ethanol Extract (Brassica Rapa L.) Leaf Can Cell Damage (8-Hydroxy-2-Dioxiguanosine) in the Wistar Rat Hyperglycemic,” in IOP Conf. Ser.: Earth Environ. Sci., vol. 709, no. 1, 2021, p. 012046.

X. Li, H. Kou, C. Zhao, F. Zhu, Y. Yang, and Y. Lu, “Efficacy and safety of ALA-PDT in treatment of diabetic foot ulcer with infection,” Photodiagnosis and Photodynamic Therapy, vol. 38, p. 102822, 2022.

R. Ishwarya, B. Vaseeharan, S. Kalyani, B. Banumathi, M. Govindarajan, N. S. Alharbi, and G. Benelli, “Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm, and insecticidal activity,” J. Photochem. Photobiol. B: Biol., vol. 178, pp. 249–258, 2018.

R. V. Kumar, S. Vinoth, V. Baskar, M. Arun, and P. Gurusaravanan, “Synthesis of zinc oxide nanoparticles mediated by Dictyota dichotoma endophytic fungi and its photocatalytic degradation of fast green dye and antibacterial applications,” South Afr. J. Bot., vol. 151, pp. 337–344, 2022.

E. C. Constance, C. Perpetua, and S. A. Steve, “Media and Air Sterilization Required for Microbial Fermentation,” Constance Chinyere Ezemba, 2020.

K. Locher, C. R. Belanger, E. Eckbo, M. Caza, B. Velapatino, and M. K. Charles, “Automated 16S sequencing using an r-based analysis module for bacterial identification,” Microbiol. Spectrum, vol. 10, no. 2, pp. e00408-22, 2022.

A. Bazzi, B. M. Masini, A. Zanella, and I. Thibault, “On the performance of IEEE 802.11 p and LTE-V2V for the cooperative awareness of connected vehicles,” IEEE Trans. Veh. Technol., vol. 66, no. 11, pp. 10419–10432, 2017.

A. M. Abdo, A. Fouda, A. M. Eid, N. M. Fahmy, A. M. Elsayed, A. M. A. Khalil, and A. M. Soliman, “Green Synthesis of Zinc Oxide Nanoparticles (ZnO-NPs) by Pseudomonas aeruginosa and Their Activity Against Pathogenic Microbes and Common House Mosquito, Culex pipiens,” Materials, vol. 14, no. 22, p. 6983, 2021.

M. Barsainya and D. P. Singh, “Green Synthesis of Zinc Oxide Nanoparticles by Pseudomonas aeruginosa and Their Broad-Spectrum Antimicrobial Effects,” J. Pure Appl. Microbiol., vol. 12, no. 4, pp. 2123–2134, 2018.

I. Y. Tafinta, K. Shehu, H. Abdulganiyyu, A. M. Rabe, and A. Usman, “Isolation and Identification of Fungi Associated With the Spoilage of Sweet Orange (Citrus sinensis) Fruits in Sokoto State,” Nigerian J. Basic Appl. Sci., vol. 21, no. 3, pp. 193–196, 2013.

H. Anton, H. Al-Ghoshae, M. Chandra, M. F. Baobaid, T. Aung, H. Hazman, and A. Al-Kabsi, “Formalin-Resistant Fungi Isolated From Cadavers at a Medical School’s Dissection Hall in Malaysia,” Asian J. Med. Health Sci., vol. 5, no. 1, p. 56, 2022.

A. Es-Haghi, M. E. Taghavizadeh, M. Sharifalhoseini, M. Baghani, E. Yousefi, A. Rahdar, and F. Baino, “Application of Response Surface Methodology for Optimizing the Therapeutic Activity of ZnO Nanoparticles Biosynthesized From Aspergillus niger,” Biomimetics, vol. 6, no. 2, p. 34, 2021.

A. Shamim, M. B. Abid, and T. Mahmood, “Biogenic Synthesis of Zinc Oxide (ZnO) Nanoparticles Using a Fungus (Aspergillus niger) and Their Characterization,” Int. J. Chem., vol. 11, no. 2, pp. 119–126, 2019.

A. E. Mekky, A. A. Farrag, A. A. Hmed, and A. R. Sofy, “Preparation of Zinc Oxide Nanoparticles Using Aspergillus niger as Antimicrobial and Anticancer Agents,” J. Pure Appl. Microbiol., vol. 15, pp. 1547–1566, 2021.

S. Jacob, R. Bharathkumar, and G. Ashwathram, “Aspergillus niger Mediated Synthesis of ZnO Nanoparticles and Their Antimicrobial and In Vitro Anticancerous Activity,” World J. Pharm. Res., vol. 3, no. 2, pp. 3044–3054, 2014.

G. Baskar, J. Chandhuru, K. S. Fahad, and P. A. S. Praveen, “Mycological Synthesis, Characterization and Antifungal Activity of Zinc Oxide Nanoparticles,” Asian J. Pharm. Technol., vol. 3, no. 4, pp. 142–146, 2013.

P. P. George, “Value-Addition of Pomegranate Fruit Peel Biowaste: Microwave-Mediated Green Synthesis, Characterization and Antimicrobial Activity of Zinc Oxide Nanoparticles,” Asian Basic Appl. Res. J., pp. 17–24, 2022.

S. R. Senthilkumar and T. Sivakumar, “Green Tea (Camellia sinensis) Mediated Synthesis of Zinc Oxide (ZnO) Nanoparticles and Studies on Their Antimicrobial Activities,” Int. J. Pharm. Pharm. Sci., vol. 6, no. 6, pp. 461–465, 2014.

D. B. Stensen, R. A. N. Cñadas, L. Småbrekke, K. Olsen, C. S. Nielsen, K. Svendsen, and A. S. Furberg, “Social Network Analysis of Staphylococcus aureus Carriage in a General Youth Population,” Int. J. Infect. Dis., vol. 123, pp. 200–209, 2022.

J. G. Collee, “A Dreadful Challenge,” The Lancet, vol. 347, no. 9006, pp. 917–918, 1996.

J. MacFaddin, “Deoxyribonuclease (DNase) and Thermos Nuclease (Tnase) Tests,” Biochemical Tests for Identification of Medical Bacteria, 3rd ed., Philadelphia, PA: Lippincott Williams and Wilkins, 2000, pp. 136–159.

C. Brennan, H. Momota, D. Hambardzumyan, T. Ozawa, A. Tandon, A. Pedraza, and E. Holland, “Glioblastoma Subclasses Can Be Defined by Activity Among Signal Transduction Pathways and Associated Genomic Alterations,” PLoS One, vol. 4, no. 11, p. e7752, 2009.

P. Lakshmi, A. Bharadwaj, and R. K. Srivastava, “Molecular Detection and Identification of Bacteria in Urine Samples of Asymptomatic and Symptomatic Pregnant Women by 16S rRNA Gene Sequencing,” Archives of Clin. Infect. Dis., vol. 15, no. 3, 2020.

R. Srinivasan, U. Karaoz, M. Volegova, J. MacKichan, M. Kato-Maeda, S. Miller, and S. V. Lynch, “Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens,” PLoS One, vol. 10, no. 2, p. e0117617, 2015.

H. T. M. Al-Mousawi, M. I. AL-Taee, and A. T. Al-Musawi, “Evaluation of the Effects of Fe3O4 Nanoparticles on Biofilm Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Assay,” J. Global Pharma Technol., vol. 11, no. 3, pp. 579–589, 2019.

R. H. A. Al-Saeedi and R. H. Raheema, “Molecular Diagnosis of Some Virulence Genes in Pseudomonas aeruginosa Clinical Isolates in Wasit Province,” Indian J. Public Health, vol. 10, no. 4, 2019.

P. Basnet, T. I. Chanu, D. Samanta, and S. Chatterjee, “A Review on Bio-Synthesized Zinc Oxide Nanoparticles Using Plant Extracts as Reductants and Stabilizing Agents,” J. Photochem. Photobiol. B: Biol., vol. 183, pp. 201–221, 2018.

Q. N. Al-Hajjar and H. T. M. Al-Mousawi, “Sero-Prevalence of Helicobacter pylori Among Staff and Students in Faculty of Pharmacy,” Indian J. Forensic Med. Toxicol., vol. 13, no. 4, p. 2, 2019.

A. K. Mandal, S. Katuwal, F. Tettey, A. Gupta, S. Bhattarai, S. Jaisi, and N. Parajuli, “Current Research on Zinc Oxide Nanoparticles: Synthesis, Characterization, and Biomedical Applications,” Nanomaterials, vol. 12, no. 17, p. 3066, 2022.

K. Koupadi, S. C. Boyatzis, M. Roumpou, N. Kalogeropoulos, and D. Kotzamani, “Organic Remains in Early Christian Egyptian Metal Vessels: Investigation With Fourier Transform Infrared Spectroscopy and Gas Chromatography–Mass Spectrometry,” Heritage, vol. 4, no. 4, pp. 3611–3629, 2021.

M. J. Mustafa, M. Al-Bderee, and H. T. M. Al-Mousawi, “Biosynthesis Nanoparticles and Molecular Study to Detect Bacterial Isolates From Patients With Diabetic Foot Ulcers in Babylon Province,” J. Pharm. Negative Results, vol. 13, no. 5, pp. 391–395, 2022.

M. F. Hamza, K. A. Salih, A. H. Adel, Y. E. Zayed, Y. Wei, J. Liang, and E. Guibal, “Sulfonic-Functionalized Algal/PEI Beads for Scandium, Cerium and Holmium Sorption From Aqueous Solutions (Synthetic and Industrial Samples),” Chem. Eng. J., vol. 403, p. 126399, 2021.

M. Ebadi, M. R. Zolfaghari, S. S. Aghaei, M. Zargar, M. Shafiei, H. S. Zahiri, and K. A. Noghabi, “A Bio-Inspired Strategy for the Synthesis of Zinc Oxide Nanoparticles (ZnO NPs) Using the Cell Extract of Cyanobacterium Nostoc sp. EA03: From Biological Function to Toxicity Evaluation,” RSC Adv., vol. 9, no. 41, pp. 23508–23525, 2019.

S. Sarwar, S. Chakraborti, S. Bera, I. A. Sheikh, K. M. Hoque, and P. Chakrabarti, “The Antimicrobial Activity of ZnO Nanoparticles Against Vibrio cholerae: Variation in Response Depends on Biotype,” Nanomedicine: Nanotechnol., Biol. Med., vol. 12, no. 6, pp. 1499–1509, 2016.