Main Article Content

Abstract

This study was aimed to adopt an ecofriendly method to synthesize nanoparticles with an effective antibacterial and anti-biofilm activity. A food origin hypermucoviscous Klebsiella pneumoniae was isolated from food samples and identified using biochemical tests and the Vitek system. A string test was depended on to identify hypermucoviscous isolates. Enterotoxicity of foodborne hypermucoviscous K. pneumoniae isolates was detected phenotypically using suckling mouse bioassay. Biofilm forming ability was tested for all the isolated bacteria using the Microtiter plate method and Congo Red Agar. A natural food additive Syzygium aromaticum (clove) aqueous extract was used for the biosynthesis of silver nanoparticles in optimized conditions. The biosynthesized clove-silver nanoparticles (Clove-AgNps) were characterized by several techniques, and their antimicrobial and antibiofilm activity was determined. The results of this study revealed that the isolation of 28 K. pneumoniae isolates were isolated from 200 food samples. String test results showed that 16/28 (57.14%) K. pneumoniae isolates were hypermucoviscous. Eleven of these isolates (68.75%) were enterotoxigenic. Using clove aqueous extract as a biological agent was successful in the biosynthesis of AgNPs with an average diameter of 14.12 nm as measured by AFM. The optimum biosynthesis conditions were: 1mM of AgNO3 concentration, pH 7, at 37 °C, and 24 hours.  The minimum inhibitory concentration of the clove-AgNPs was detected as 62.5 µg.mL-1. Sub-inhibitory concentration of 31.25 µg.mL-1 of Clove-AgNPs resulted in: 91% decrease in the formed biofilm. It can be concluded that using Syzygium aromaticum is an effective ecofriendly method for AgNPs biosynthesis with excellent antibacterial and anti-biofilm activity against enterotoxigenic hypermucoviscous K. pneumoniae.

Keywords

Antibacterial Agents Biofilm Clove Food Poisoning

Article Details

How to Cite
AlKhafaji, M. H. ., Mohsin, R. H. ., & Alshaikh Faqri, A. M. . (2024). Food Additive Mediated Biosynthesis of AgNPs with Antimicrobial Activity Against Hypermucoviscous Enterotoxigenic Foodborne Klebsiella pneumoniae. Basrah Journal of Agricultural Sciences, 37(1), 278–295. https://doi.org/10.37077/25200860.2024.37.1.21

References

  1. Abbas, H. H., & Faliyyah, M. T. (2019). Biosynthesis and optimization of silver nanoparticles from Pseudomonas aeruginosa. Biochemical & Cellular Archives, 19(1). https://www.connectjournals.com/pages/articledetails/toc029489
  2. Abdul-Karim, E. K., & Hussein, H. Z. (2022). The biosynthesis of nanoparticles by fungi and the role of nanoparticles in resisting of pathogenic fungi to plants: a review. Basrah Journal of Agricultural Sciences, 35(1), 243-256. https://doi.org/10.37077/25200860.2022.35.1.18
  3. Ahmad, S. A., Das, S. S., Khatoon, A., Ansari, M. T., Afzal, M., Hasnain, M. S., & Nayak, A. K. (2020). Bactericidal activity of silver nanoparticles: A mechanistic review. Materials Science for Energy Technologies, 3, 756-769. https://www.sciencedirect.com/science/article/pii/S2589299120300501
  4. Ahmed, S., Ahmad, M., Swami, B. L., & Ikram, S. (2016). A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise. Journal of advanced research, 7(1), pp.17-28. https://www.sciencedirect.com/science/article/pii/S2090123215000314
  5. Al-Khafaji, M. H. (2017). The Inhibition Activity of Silver Nanoparticles Compared with D-Glycin and Imipenem Effect on the Biofilm Formation by Food-origin Salmonella. Iraqi Journal of Science, 58(2B), 836-842. https://ijs.uobaghdad.edu.iq/index.php/eijs/article/view/6026
  6. Al-Khafaji, M. H. M., Majeed, S. M. A., & Basi, R. Q. (2017). Biosynthesis of silver nanoparticles by food-origin E. coli and Candida species and testing its antimicrobial activity against pathogenic bacteria and fungi. IOSR Journal of Pharmacy and Biological Sciences, 12, 29-34. https://doi.org/10.9790/3008-1203032934
  7. Almudhafar, S. M., & Al-Hamdani, M. A. (2022). Antibacterial and anticancer effects of silver nanoparticles synthesised using eragrostis tef and vitellaria paradoxa seeds extract. Basrah Journal of Agricultural Sciences, 35(2), 132-159. https://doi.org/10.37077/25200860.2022.35.2.10
  8. Al-Ogaidi, I. A. Z. (2017). Detecting the antibacterial activity of green synthesized silver (Ag) nanoparticles functionalized with ampicillin (Amp). Baghdad Science Journal, 14(1), 0117-0117. https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/view/2345
  9. Al-Shaabani, M. J. M., Turki, A. M., & Al-Mathkhury, H. J. F. (2020). The antibiofilm efficacy of gold nanoparticles against Acinetobacter baumannii. Iraqi Journal of Science, 61(4), 749-753. https://ijs.uobaghdad.edu.iq/index.php/eijs/article/view/1697
  10. Ansari, M. T., Sami, F., Majeed, S., Hasnain, M. S., & Badgujar, V. B., (2019). Design and evaluation of topical herbal antifungal stick containing extracts of Rhinacanthus nasutus. Journal of Herbal Medicine, 17, 100290. https://www.sciencedirect.com/science/article/abs/pii/S2210803319300375
  11. Alzubaidi, S. J., & Alkhafaji, M. H. (2023). Molecular Detection of bla TEM and bla CTX-M genes in clinical and food-borne Klebsiella pneumoniae isolates. Journal of Medicinal and Chemical Sciences, 6, 1706-1713. https://www.jmchemsci.com/article_163003.html
  12. Barapatre, A., Aadil, K. R., & Jha, H. (2016). Synergistic antibacterial and antibiofilm activity of silver nanoparticles biosynthesized by lignin-degrading fungus. Bioresources and Bioprocessing; 3(1), 1-3. https://bioresourcesbioprocessing.springeropen.com/articles/10.1186/s40643-016-0083-y
  13. Borges, A., Saavedra, M. J., & Simões, M. (2015). Insights on antimicrobial resistance, biofilms and the use of phytochemicals as new antimicrobial agents. Current medicinal chemistry, 22(21), 2590–2614. https://doi.org/10.2174/0929867322666150530210522
  14. Chang, D., Sharma, L., Dela Cruz, C. S., & Zhang, D. (2021). Clinical Epidemiology, Risk Factors, and Control Strategies of Klebsiella pneumoniae Infection. Frontiers in microbiology, 12, 750662. https://doi.org/10.3389/fmicb.2021.750662
  15. Choby, J. E., Howard-Anderson, J., & Weiss, D. S. (2020). Hypervirulent Klebsiella pneumoniae - clinical and molecular perspectives. Journal of internal medicine, 287(3), 283–300. https://doi.org/10.1111/joim.13007
  16. Coffey, B. M., & Anderson, G. G. (2014). Biofilm formation in the 96-well microtiter plate. Methods in molecular biology (Clifton, N.J.), 1149, 631–641. https://doi.org/10.1007/978-1-4939-0473-0_48
  17. Dauthal, P., & Mukhopadhyay, M. (2016). Noble metal nanoparticles: plant-mediated synthesis, mechanistic aspects of synthesis, and applications. Industrial & Engineering Chemistry Research, 55, 9557–9577. https://pubs.acs.org/doi/10.1021/acs.iecr.6b00861
  18. Decré, D., Verdet, C., Emirian, A., Le Gourrierec, T., Petit, J. C., Offenstadt, G., Maury, E., Brisse, S. & Arlet, G. (2011). Emerging severe and fatal infections due to Klebsiella pneumoniae in two university hospitals in France. Journal of clinical microbiology, 49(8), 3012-3014. https://journals.asm.org/doi/10.1128/jcm.00676-11
  19. Divya, M., Kiran, G.S., Hassan, S., & Selvin, J. (2019). Biogenic synthesis and effect of silver nanoparticles (AgNPs) to combat catheter-related urinary tract infections. Biocatalysis and agricultural biotechnology, 18, 101037. https://www.sciencedirect.com/science/article/abs/pii/S1878818118307321
  20. Elken, E. M., Tan, Z. N., Wang, Q., Jiang, X. Y., Wang, Y., Wang, Y. M., & Ma, H. X. (2022). Impact of Sub-MIC Eugenol on Klebsiella pneumoniae Biofilm Formation via Upregulation of rcsB. Frontiers in Veterinary Science, 9, 945491. https://www.frontiersin.org/articles/10.3389/fvets.2022.945491/full
  21. Freeman, D. J., Falkiner, F. R., & Keane, C. T. (1989). New method for detecting slime production by coagulase negative staphylococci. Journal of clinical pathology, 42(8), 872-874.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142068/
  22. Gao, H., Yang, H., & Wang, C. (2017). Controllable preparation and mechanism of nano-silver mediated by the microemulsion system of the clove oil. Results in physics, 7, 3130-3136. https://www.sciencedirect.com/science/article/pii/S2211379717309257
  23. Guerra, M. E. S., Destro, G., Vieira, B., Lima, A. S., Ferraz, L. F. C., Hakansson, A. P., Darrieux, M., & Converso, T. R. (2022). Klebsiella pneumoniae biofilms and their role in disease pathogenesis. Frontiers in Cellular and Infection Microbiology, 12, 877995. https://doi.org/10.3389/fcimb.2022.877995
  24. Guo, Y., Zhou, H., Qin, L., Pang, Z., Qin, T., Ren, H., Pan, Z., & Zhou, J. (2016). Frequency, Antimicrobial Resistance and Genetic Diversity of Klebsiella pneumoniae in Food Samples. PloS one, 11(4), e0153561. https://doi.org/10.1371/journal.pone.0153561
  25. Gurunathan, S., Han, J. W., Kwon, D. N., & Kim, J. H. (2014). Enhanced antibacterial and anti-biofilm activities of silver nanoparticles against Gram-negative and Gram-positive bacteria. Nanoscale research letters, 9, 1-17. https://pubmed.ncbi.nlm.nih.gov/25136281/
  26. Hamida, R. S., Ali, M. A., Goda, D. A., Khalil, M. I.., & Redhwan, A. (2020). Cytotoxic effect of green silver nanoparticles against ampicillin-resistant Klebsiella pneumoniae. RSC advances, 10(36), 21136-21146. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9054378/
  27. Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., & Iqbal, M. (2011). Evaluation of different detection methods of biofilm formation in the clinical isolates. Brazilian journal of infectious diseases, 15, 305-311. https://pubmed.ncbi.nlm.nih.gov/21860999/
  28. Ibrhim, I. M. (2017). Synthesis and characteristics of Ag, Cu/Au core/shell nanoparticles produced by pulse laser ablation. Iraqi Journal of Science, 58(3C), 1651-1659. https://ijs.uobaghdad.edu.iq/index.php/eijs/article/view/5794
  29. Jay, J. M., Loessner, M. J., & Golden, D. A. (2005). Bioassay and related methods. Pp, 285-298. In Jay, J. M., Loessner, M. J., & Golden, D. A. (Editors). Modern food microbiology, Springer, Boston, M. A. https://link.springer.com/chapter/10.1007/0-387-23413-6_12
  30. Junaid, K., Ejaz, H., Younas, S., Alanazi, A., Yasmeen, H., & Rehman, A. (2022). Detection of Klebsiella pneumoniae antibiotic-resistant genes: An impending source of multidrug resistance dissemination through raw food. Saudi Journal of Biological Sciences, 29(5), 3347–3353. https://doi.org/10.1016/j.sjbs.2022.02.020
  31. Kaur, H., Kaur, S., & Singh, M. (2013). Biosynthesis of silver nanoparticles by natural precursor from clove and their antimicrobial activity. Biologia, 68(6), 1048-1053. https://doi.org/10.2478/s11756-013-0276-1
  32. Kaur, J., Kaul, M., & Chhibber, S. (1988). Enterotoxigenicity, klebocinogeny and antibiotic resistance pattern of food isolates of Klebsiella pneumoniae. Folia microbiologica, 33(6), 500–506. https://doi.org/10.1007/BF02925777
  33. Khalil, O., Enbaawy, M. I., Taher, S., & Mahmoud, H. & Ragab, E. (2020). In vitro investigation of the antibacterial effect of silver nanoparticles on ESBL-producing E. coli and Klebsiella spp. Isolated from Pet Animals. Worlds Veterinary Journal, 10(4), 514-524.
  34. https://doi.org/10.54203/scil.2020.wvj62
  35. Kienesberger, S., Cosic, A., Kitsera, M., Raffl, S., Hiesinger, M., Leitner, E., Halwachs B., Gorkiewicz G., Glabonjat R. A., Raber G., Lembacher-Fadum C., Breinbauer R., Schild, S., & Zechner, E. L. (2022). Enterotoxin tilimycin from gut-resident Klebsiella promotes mutational evolution and antibiotic resistance in mice. Nature Microbiology, 7(11), 1834-1848. https://doi.org/10.1038/s41564-022-01260-3
  36. Kulshrestha, S., Khan, S., Hasan, S., Khan, M. E., Misba, L., & Khan, A. U. (2016). Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach. Applied microbiology and biotechnology, 100, 1901-1914. https://doi.org/10.1007/s00253-015-7154-4
  37. Lee, C. R., Lee, J. H., Park, K. S., Jeon, J. H., Kim, Y. B., Cha, C. J., Jeong, B. C., & Lee, S. H. (2017). Antimicrobial Resistance of Hypervirulent Klebsiella pneumoniae: Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms. Frontiers in cellular and infection microbiology, 7, 483. https://doi.org/10.3389/fcimb.2017.00483
  38. Lee, H. C., Chuang, Y. C., Yu, W.L., Lee, N. Y., Chang, C. M., Ko, N.Y., Wang, L. R., & Ko, W.C. (2006). Clinical implications of hypermucoviscosity phenotype in Klebsiella pneumoniae isolates: association with invasive syndrome in patients with community‐acquired bacteraemia. Journal of internal medicine, 259(6), 606-614. https://doi.org/10.1111/j.1365-2796.2006.01641.x
  39. Lee, I. R., Molton, J. S., Wyres, K. L., Gorrie, C., Wong, J., Hoh, C. H., Teo, J., Kalimuddin, S., Lye, D. C., Archuleta, S., & Gan, Y. H. (2016). Differential host susceptibility and bacterial virulence factors driving Klebsiella liver abscess in an ethnically diverse population. Scientific reports, 6(1), 29316. https://doi.org/10.1038/srep29316
  40. Li, W., Sun, G., Yu, Y., Li, N., Chen, M., Jin, R., Jiao, Y. & Wu, H. (2014). Increasing occurrence of antimicrobial-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in China. Clinical infectious diseases, 58(2), 225-232. https://doi.org/10.1093/cid/cit675
  41. Logeswari, P., Silambarasan, S., & Abraham, J. (2015). Synthesis of silver nanoparticles using plants extract and analysis of their antimicrobial property. Journal of Saudi Chemical Society, 19(3), 311-317. https://doi.org/10.1016/j.jscs.2012.04.007
  42. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2014). Textbook of diagnostic microbiology, 5th edition. Saunders. 1104pp. https://hsrc.himmelfarb.gwu.edu/smhs_crl_facpubs/37/
  43. Mazhir, S. N., Ali, I. A. M., & Al-Ahmed, H. I. (2019). Utilizing cold plasma in preparing silver nanoparticle from Origanum vulgare. Iraqi Journal of Science, 2433-2442. https://doi.org/10.24996/ijs.2019.60.11.14
  44. Mittal, A. K., Chisti, Y., & Banerjee, U. C. (2013). Synthesis of metallic nanoparticles using plant extracts. Biotechnology advances, 31(2), 346–356. https://doi.org/10.1016/j.biotechadv.2013.01.003
  45. Mohanta, Y. K., Biswas, K., Jena, S.K., Hashem, A., Abd_Allah, E. F., & Mohanta, T. K. (2020) Anti-biofilm and antibacterial activities of silver nanoparticles synthesized by the reducing activity of phytoconstituents present in the Indian medicinal plants. Frontiers in Microbiology, 11, 1143. https://doi.org/10.3389/fmicb.2020.01143
  46. Osama, D. M., Zaki, B. M., Khalaf, W. S., Mohamed, M. Y. A., Tawfick, M. M., & Amin, H. M., (2023). Occurrence and Molecular Study of Hypermucoviscous/Hypervirulence Trait in Gut Commensal K. pneumoniae from Healthy Subjects. Microorganisms, 11(3), 704. https://www.mdpi.com/2076-2607/11/3/704
  47. Rahal, B. S., Salman, A. A. A. N. Y., & Mohamed, K. K. G. M. M. (2021). The Role of EDTA in biofilm eradication of Klebsiella pneumoniae isolated from wound infections. Iraqi Journal of Biotechnology, 1(20), 96-102. https://jige.uobaghdad.edu.iq/index.php/IJB/article/view/422
  48. Ramos, A. P., Cruz, M. A. E., Tovani, C. B., & Ciancaglini, P. (2017). Biomedical applications of nanotechnology. Biophysical reviews, 9(2), 79–89. https://doi.org/10.1007%2Fs12551-016-0246-2
  49. Rodríguez-Félix, F., Graciano-Verdugo, A. Z., Moreno-Vásquez, M. J., Lagarda-Díaz, I., Barreras-Urbina, C. G., Armenta-Villegas, L., Olguín-Moreno, A., & Tapia-Hernández, J. A. (2022). Trends in sustainable green synthesis of silver nanoparticles using agri-food waste extracts and their applications in health. Journal of Nanomaterials, 2022, 1-37. https://doi.org/10.1155/2022/8874003
  50. Saeed, E. A., Bnyan, I. A., & Saadi, M. (2013). Quorum sensing and biofilm formation by bacterial isolates from hemodialysis patients. Research in Pharmacy, 3(2), 33-40. https://updatepublishing.com/journal/index.php/rip/article/view/230
  51. Saleh, G. M. (2020). Green synthesis concept of nanoparticles from environmental bacteria and their effects on pathogenic bacteria. Iraqi Journal of Science, 61(6), 1289-1297. https://doi.org/10.24996/ijs.2020.61.6.6
  52. Seifi, K., Kazemian, H., Heidari, H., Rezagholizadeh, F., Saee, Y., Shirvani, F., & Houri, H. (2016). Evaluation of biofilm formation among Klebsiella pneumoniae isolates and molecular characterization by ERIC-PCR. Jundishapur journal of microbiology, 9(1), e30682. https://doi.org/10.5812/jjm.30682
  53. Shareef, A. A., Farhan, F. J., & Alriyahee, F. A. (2023). Antibacterial activity of silver nanoparticles composed by fruit aqueous extract of Abelmoschus esculentus (L.) Moench alone or in combination with Antibiotics. Basrah Journal of Agricultural Sciences, 36(2), 144-174. https://doi.org/10.37077/25200860.2023.36.2.12
  54. Shon, A. S., Bajwa, R. P., & Russo, T. A. (2013). Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence, 4, 107–18. https://doi.org10.4161/viru.22718
  55. Singh, A. K., Talat, M., Singh, D. P., & Srivastava, O. N. (2010). Biosynthesis of gold and silver nanoparticles by natural precursor clove and their functionalization with amine group. Journal of Nanoparticle Research, 12(5), 1667-1675. https://doi.org/10.1007/s11051-009-9835-3
  56. Singh, B. R., & Kulshreshtha, S. B. (1992). Preliminary examinations on the enterotoxigenicity of isolates of Klebsiella pneumoniae from seafoods. International journal of food microbiology, 16(4), 349–352. https://doi.org/10.1016/0168-1605(92)90037-4
  57. Some, S., Mondal, R., Dam, P., & Mandal, A. K. (2022). Synthesis of biogenic silver nanoparticles using medicinal plant extract: A new age in nanomedicine to combat multidrug-resistant pathogens. Pp. 359-387. In Abd-El-Salam, K. A. (Editor). Green Synthesis of Silver Nanomaterials Elsevier, 765pp.
  58. Tawfeeq, S. M. T., Maaroof, M. N., & Al-Ogaidi, I. (2017). Synergistic effect of biosynthesized silver nanoparticles with antibiotics against multi-drug resistance bacteria isolated from children with diarrhoea under five years. Iraqi Journal of Science, 58(1), 14-52. https://ijs.uobaghdad.edu.iq/index.php/eijs/article/view/6189
  59. Trower, C. J., Abo, S., Majeed, K. N., & Itzstein, M. V. (2000). Production of an enterotoxin by a gastro-enteritis-associated Aeromonas strain. Journal of Medical Microbiology, 49(2), 121-126. https://doi.org/10.1099/0022-1317-49-2-121
  60. Venugopal, K., Rather, H. A., Rajagopal, K., Shanthi, M. P., Sheriff, K., Illiyas, M., Rather, R.A., Manikandan, E., Uvarajan, S., Bhaskar, M. & Maaza, M. (2017). Synthesis of silver nanoparticles (Ag NPs) for anticancer activities (MCF 7 breast and A549 lung cell lines) of the crude extract of Syzygium aromaticum. Journal of Photochemistry and Photobiology B: Biology, 167, 282-289. https://doi.org/10.1016/j.jphotobiol.2016.12.013
  61. Wang, G., Zhao, G., Chao, X., Xie, L., & Wang, H. (2020). The Characteristic of Virulence, Biofilm and Antibiotic Resistance of Klebsiella pneumoniae. International journal of environmental research and public health, 17(17), 6278. https://doi.org/10.3390/ijerph17176278
  62. Xu, Q., Yang, X., Chan, E. W. C., & Chen, S. (2021). The hypermucoviscosity of hypervirulent K. pneumoniae confers the ability to evade neutrophil-mediated phagocytosis. Virulence, 12, 2050–2059. https://pubmed.ncbi.nlm.nih.gov/34339346/
  63. Yang, D., & Zhang, Z. (2008). Biofilm-forming Klebsiella pneumoniae strains have greater likelihood of producing extended-spectrum beta-lactamases. The Journal of hospital infection, 68(4), 369–371. https://pubmed.ncbi.nlm.nih.gov/18353499/
  64. Zhang, Y., Zhao, C., Wang, Q., Wang, X., Chen, H., Li, H., Zhang F., Li S., Wang R., & Wang, H. (2016). High prevalence of hypervirulent Klebsiella pneumoniae infection in China: geographic distribution, clinical characteristics, and antimicrobial resistance. Antimicrobial agents and chemotherapy, 60(10), 6115-6120. https://pubmed.ncbi.nlm.nih.gov/27480857/
  65. Zhao, X., Xu, X., Ai, C., Yan, L., Jiang, C., & Shi, J. (2022). Advantages of silver nanoparticles synthesized by microorganisms in antibacterial activity. Pp. 571-586. In Abd-Elsalam, K. A. (Editor). Green Synthesis of Silver Nanomaterials Elsevier. 765pp. https://doi.org/10.1016/B978-0-12-824508-8.00005-8
  66. Zhu, J., Wang, T., Chen, L., & Du, H. (2021). Virulence factors in hypervirulent Klebsiella pneumoniae. Frontiers in microbiology, 12, 642484. https://doi.org/10.3389/fmicb.2021.642484