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ORIGINAL RESEARCH ARTICLE

Antibacterial activity of green-synthesized silver nanoparticles against Gram-negative bacteria and insights into potential resistance mechanisms

Akamu J. Ewunkem1* ,  Bliss Daodu1 ,  Zahirah J. Williams2 ,  Lydia Merrills2 ,  Brittany L. Justice1 ,  Felicia Simpson3 ,  David Holland3 ,  Tatyana Bowers3 ,  Uchenna Iloghalu1
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1 Department of Biological Sciences, Faculty of Natural and Physical Sciences, Winston-Salem State University, Winston-Salem, North Carolina, United States of America
2 Department of Nursing, Faculty of Natural and Physical Sciences, Winston-Salem State University, Winston-Salem, North Carolina, United States of America
3 Department of Mathematics, Faculty of Natural and Physical Sciences, Winston-Salem State University, Winston-Salem, North Carolina, United States of America
INNOSC Theranostics and Pharmacological Sciences, 025080007 https://doi.org/10.36922/ITPS025080007
Received: 17 February 2025 | Revised: 3 June 2025 | Accepted: 11 June 2025 | Published online: 1 July 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Gram-negative bacterial infections pose a serious public health challenge due to their high global mortality rates and potential to cause severe complications. Antibiotics – one of the most impactful medical innovations of the 20th century – remain vital in treating life-threatening bacterial infections. However, the increasing prevalence of antibiotic resistance has made it progressively harder to treat Gram-negative bacterial infections effectively. Therefore, nanoparticles have gained attention as a promising alternative treatment owing to their targeted antibacterial properties. Among the various synthesis methods, green synthesis is considered one of the most effective approaches for nanoparticle production. In this study, silver nanoparticles were synthesized using a green approach that utilized silver nitrate salt and an extract derived from carpenter bee wings (CBWs). The synthesized nanoparticles were characterized using spectroscopic techniques and scanning electron microscopy. Their antibacterial activity was tested against two pathogenic Gram-negative bacteria using the broth dilution method. Furthermore, whole genome sequencing was conducted to assess the mutagenic effects of the biosynthesized silver nanoparticles on the two bacterial strains. The results demonstrated that the green-synthesized silver nanoparticles exhibit notable antibacterial activity, likely through electrostatic interactions that promote cell binding and induce significant morphological alterations. Genomic analysis revealed mutations associated with efflux pump regulation, neutralization, transport, energy metabolism, cell division, biosynthetic pathways, adaptation, and invasion in the tested strains. These findings demonstrate the potential of CBWs as a novel biological resource for the green synthesis of silver nanoparticles with antibacterial properties. However, the study also raises concerns regarding the potential for bacteria to develop resistance to nanoparticles over time.

Keywords
Carpenter bee wing extracts
Genomics
Gram-negative bacteria
Green synthesis
Nanoparticles
Funding
The study was supported by the Professional Development Committee Research Grant (grant number: 211444), Winston-Salem State University, and The Genomic Research and Data Science Center for Computation and Cloud Computing (grant number: 211512).
Conflict of interest
The authors declare that they have no competing interests.
References
  1. Peleg AY, Hooper DC. Hospital-acquired infections due to gram-negative bacteria. N Engl J Med. 2010;362(19): 1804-1813. doi: 10.1056/NEJMra0904124

 

  1. Santangelo L, Netti GS, Torres DD, et al. Peripheral nervous system manifestations of shiga toxin-producing E. Coli-induced haemolytic uremic syndrome in children. Ital J Pediatr. 2021;47:181. doi: 10.1186/s13052-021-01133-1

 

  1. Lung TWF, Charytonowicz D, Beaumont KG, et al. Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection. Cell Metab. 2022;34(5):761-774.e.9. doi: 10.1016/j.cmet.2022.03.009

 

  1. Hyun M, Lee JY, Lim KR, Kim HA. Clinical characteristics of uncomplicated acute pyelonephritis caused by Escherichia coli and Klebsiella pneumoniae. Infect Dis Ther. 2024;13(3):581-595. doi: 10.1007/s40121-024-00940-3

 

  1. Schwaderer AL, Rajadhyaksha E, Canas J, Saxena V, Hains DS. Intercalated cell function, kidney innate immunity, and urinary tract infections. Pflügers Arch. 2024;476(4):565-578. doi: 10.1007/s00424-024-02905-4

 

  1. Zykov IN. Old antibiotics as alternative treatment options for urinary tract infections caused by ESBL-, AmpC- and carbapenemase-producing Escherichia coli. Norway: The Arctic University of Norway. 2020.

 

  1. Kuo PY, Lin WH, Tang SF, et al. A longitudinal epidemiology study of fluoroquinolone-nonsusceptible Klebsiella pneumoniae reveals an increasing prevalence of qnrB and qnrS in Taiwan. J Infect Public Health. 2024;17(3):457-463. doi: 10.1016/j.jiph.2024.01.005

 

  1. Al-Khikani FH, Jasim SH, Abedulameeralhusayni A. Fosfomycin sensitivity among Escherichia coli and gram-positive cocci uropathogen. Indian J Med Specialities. 2024;15(2):143-144. doi: 10.4103/injms.injms_118_23

 

  1. Epand RM, Walker C, Epand RF, Magarvey NA. Molecular mechanisms of membrane targeting antibiotics. Biochim Biophys Acta. 2016;1858(5):980-987. doi: 10.1016/j.bbamem.2015.10.018

 

  1. Sabnis A, Edwards AM. Lipopolysaccharide as an antibiotic target. Biochim Biophys Acta Mol Cell Res. 2023;1870(7):119507. doi: 10.1016/j.bbamcr.2023.119507

 

  1. Fulke AB, Eranezhath S, Swain GK, Tiwari N, Dora GU. Land use land cover structural impact on abundance of antibiotic resistant Bacillus species in tropical estuary of India. Reg Stud Mar Sci. 2025;82:104005. doi: 10.1016/j.rsma.2024.104005

 

  1. Munita JM, Arias CA. Mechanisms of antibiotic resistance. In: Virulence Mechanisms of Bacterial Pathogens. United States: Wiley; 2016. p. 481-511. doi: 10.1128/9781555819286.ch17

 

  1. Breijyeh Z, Jubeh B, Karaman R. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules. 2020;25(6):1340. doi: 10.3390/molecules25061340

 

  1. Kitaba AA, Bonger ZT, Beyene D, et al. Antimicrobial resistance trends in clinical Escherichia coli and Klebsiella pneumoniae in Ethiopia. Afr J Lab Med. 2024;13(1):2268. doi: 10.4102/ajlm.v13i1.2268

 

  1. Pandey N, Cascella M. Beta-lactam antibiotics. In: StatPearls. Treasure Island, FL: StatPearls Publishing; 2023

 

  1. Tsai YK, Fung CP, Lin JC, et al. Klebsiella pneumoniae outer membrane porins ompK35 and ompK36 play roles in both antimicrobial resistance and virulence. Antimicrob Agents Chemother. 2011;55(4):1485-1493. doi: 10.1128/aac.01275-10

 

  1. Salam MA, Al-Amin MY, Salam MT, et al. Antimicrobial resistance: A growing serious threat for global public health. Healthcare (Basel). 2023;11(13):1946. doi: 10.3390/healthcare11131946

 

  1. Rudramurthy GR, Swamy MK, Sinniah UR, Ghasemzadeh A. Nanoparticles: Alternatives against drug-resistant pathogenic microbes. Molecules. 2016;21(7):836. doi: 10.3390/molecules21070836

 

  1. Hwang C, Choi MH, Kim HE, Jeong SH, Park JU. Reactive oxygen species-generating hydrogel platform for enhanced antibacterial therapy. NPG Asia Mater. 2022;14(1):72. doi: 10.1038/s41427-022-00420-5

 

  1. Jiang Y, Zheng W, Tran K, et al. Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures. Nat Commun. 2022;13(1):197. doi: 10.1038/s41467-021- 27193-9

 

  1. Aflakian F, Mirzavi F, Aiyelabegan HT, et al. Nanoparticles-based therapeutics for the management of bacterial infections: A special emphasis on FDA approved products and clinical trials. Eur J Pharm Sci. 2023;188:106515. doi: 10.1016/j.ejps.2023.106515

 

  1. Urnukhsaikhan E, Bold BE, Gunbileg A, Sukhbaatar N, Mishig-Ochir T. Antibacterial activity and characteristics of silver nanoparticles biosynthesized from Carduus crispus. Sci Rep. 2021;11(1):21047. doi: 10.1038/s41598-021-00520-2

 

  1. Bruna T, Maldonado-Bravo F, Jara P, Caro N. Silver nanoparticles and their antibacterial applications. Int J Mol Sci. 2021;22(13):7202. doi: 10.3390/ijms22137202

 

  1. Ewunkem AJ, Williams ZJ, Johnson NS, Brittany JL, Maselugbo A, Nowlin K. Exploring the “carpenter” as a substrate for green synthesis: Biosynthesis and antimicrobial potential. Gene Protein Dis. 2023;2(4):2155. doi: 10.36922/gpd.2155

 

  1. Ewunkem AJ, Johnson N, Beard AF, Tshimanga I, Justice B, Meixner J. Synthesis of silver nanoparticles from honeybees and its antibacterial potential. Open J Med Microbiol. 2024;14(1):77-92. doi: 10.4236/ojmm.2024.141007

 

  1. Ewunkem AJ, Priester T, Williams D, et al. Rapid green synthesis of silver nanoparticles by Reishi and their antibacterial activity and mechanisms. J Biomater Nanobiotechnol. 2024;15(3):51-63. doi: 10.4236/jbnb.2024.153004

 

  1. Ameer MA, Wasey A, Salen P. Escherichia coli (e Coli 0157 H7). Treasure Island: StatPearls Publishing. 2013; pp. 1-11.

 

  1. Mouanga-Ndzime Y, Bisseye C, Longo-Pendy NM, Bignoumba M, Dikoumba AC, Onanga R. Trends in Escherichia coli and Klebsiella pneumoniae urinary tract infections and antibiotic resistance over a 5-year period in Southeastern Gabon. Antibiotics. 2024;14(1):14. doi: 10.3390/antibiotics14010014

 

  1. Mączyńska B, Frej-Mądrzak M, Sarowska J, Woronowicz, K, Choroszy-Król I, Jama-Kmiecik A. Evolution of antibiotic resistance in Escherichia coli and Klebsiella pneumoniae clinical isolates in a multi-profile hospital over 5 years (2017- 2021). J Clin Med. 2023;12(6):2414. doi: 10.3390/jcm12062414

 

  1. Gupta J, Fatima MT, Islam Z, Khan RH, Uversky VN, Salahuddin P. Nanoparticle formulations in the diagnosis and therapy of Alzheimer’s disease. Int J Biol Macromol. 2019;130:515-526. doi: 10.1016/j.ijbiomac.2019.02.156

 

  1. Ying S, Guan Z, Ofoegbu PC, et al. Green synthesis of nanoparticles: Current developments and limitations. Environ Technol Innov. 2022;26:102336. doi: 10.1016/j.eti.2022.102336

 

  1. Bhardwaj B, Singh P, Kumar A, Kumar S, Budhwar V. Eco-friendly greener synthesis of nanoparticles. Adv Pharm Bull. 2020;10(4):566-576. doi: 10.34172/apb.2020.067

 

  1. Jakinala P, Lingampally N, Hameeda B, et al. Silver nanoparticles from insect wing extract: Biosynthesis and evaluation for antioxidant and antimicrobial potential. PLoS One. 2021;16(3):e0241729. doi: 10.1371/journal.pone.0241729

 

  1. Luceri A, Francese R, Lembo D, Ferraris M, Balagna C. Silver nanoparticles: Review of antiviral properties, mechanism of action and applications. Microorganisms. 2023;11(3):629. doi: 10.3390/microorganisms11030629

 

  1. More PR, Pandit S, Filippis AD, Franci G, Mijakovic I, Galdiero M. Silver nanoparticles: Bactericidal and mechanistic approach against drug resistant pathogens. Microorganisms. 2023;11(2):369. doi: 10.3390/microorganisms11020369

 

  1. Yin IX, Zhang J, Zhao IS, Mei ML, Li Q, Chu CH. The antibacterial mechanism of silver nanoparticles and its application in dentistry. Int J Nanomedicine. 2020;15: 2555-2562. doi: 10.2147/IJN.S246764

 

  1. Tymoszuk A, Kulus D. Silver nanoparticles induce genetic, biochemical, and phenotype variation in chrysanthemum. Plant Cell Tissue Organ Cult. 2020;143(2):331-344. doi: 10.1007/s11240-020-01920-4

 

  1. Pan X, Yang Y, Zhang JR. Molecular basis of host specificity in human pathogenic bacteria. Emerg Microbes Infect. 2014;3(1):e23. doi: 10.1038/emi.2014.23

 

  1. Garmory HS, Titball RW. ATP-binding cassette transporters are targets for the development of antibacterial vaccines and therapies. Infect Immun. 2004;72(12):6757-6763. doi: 10.1128/iai.72.12.6757-6763.2004

 

  1. Almeida S, Sousa C, Abreu V, et al. Exploration of nitrate reductase metabolic pathway in Corynebacterium pseudotuberculosis. Int J Genomics. 2017;(1):9481756. doi: 10.1155/2017/9481756

 

  1. Martinez JL, Sánchez MB, Martínez-Solano L, et al. Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. FEMS Microbiol Rev. 2009;33(2):430-449. doi: 10.1111/j.1574-6976.2008.00157.x

 

  1. Baghal SM, Gargari SL, Rasooli I. Production and immunogenicity of recombinant ferric enterobactin protein (FepA). Int J Infect Dis. 2010;14:e166-e170. doi: 10.1016/j.ijid.2009.12.009

 

  1. Casanova-Hampton K, Carey A, Kassam S, et al. A genome-wide screen reveals the involvement of enterobactin-mediated iron acquisition in Escherichia coli survival during copper stress. Metallomics. 2021;13(9):mfab052. doi: 10.1093/mtomcs/mfab052

 

  1. Huang SW, Lim SK, Yu YA, et al. Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy. Elife. 2024;12:RP90798. doi: 10.7554/eLife.90798.3

 

  1. Moynié L, Milenkovic S, Mislin GL, et al. The complex of ferric-enterobactin with its transporter from Pseudomonas aeruginosa suggests a two-site model. Nat Commun. 2019;10(1):3673. doi: 10.1038/s41467-019-11508-y

 

  1. Lau CK, Krewulak KD, Vogel HJ. Bacterial ferrous iron transport: The Feo system. FEMS Microbiol Rev. 2016;40(2):273-298. doi: 10.1093/femsre/fuv049

 

  1. Ghssein G, Ezzeddine Z. The key element role of metallophores in the pathogenicity and virulence of Staphylococcus aureus: A review. Biology (Basel). 2022;11(10):1525. doi: 10.3390/biology11101525

 

  1. Bhagavan NV, Ha CE. Essentials of Medical Biochemistry. With Clinical Cases. 2nd ed. London, UK. Academic Press; 2015.

 

  1. Rahman MM, Hunter HN, Prova S, Verma V, Qamar A, Golemi-Kotra D. The Staphylococcus aureus methicillin resistance factor FmtA is a d-amino esterase that acts on teichoic acids. MBio. 2016;7(1):e02070-15. doi: 10.1128/mbio.02070-15

 

  1. Holmquist M. Alpha/beta-hydrolase fold enzymes: Structures, functions and mechanisms. Curr Protein Pept Sci. 2000;1(2):209-235. doi: 10.2174/1389203003381405

 

  1. Singleton C, White GF, Todd JD, et al. Heme-responsive DNA binding by the global iron regulator Irr from Rhizobium leguminosarum. J Biol Chem. 2010;285(21):16023-16031. doi: 10.1074/jbc.M109.067215

 

  1. Mehrez M, Lecampion C, Ke H, Gorsane F, Field B. Insights into the function of the chloroplastic ribosome-associated GTPase high frequency of lysogenization X in Arabidopsis thaliana. Plant Direct. 2024;8(1):e559. doi: 0.1002/pld3.559

 

  1. Neuhaus FC, Baddiley J. A continuum of anionic charge: Structures and functions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev. 2003;67(4):686-723. doi: 10.1128/mmbr.67.4.686-723.2003

 

  1. Razew A, Schwarz JN, Mitkowski P, Sabala I, Kaus-Drobek M. One fold, many functions-M23 family of peptidoglycan hydrolases. Front Microbiol. 2022;13:1036964. doi: 10.3389/fmicb.2022.1036964

 

  1. Hecker M, Völker U. General stress response of Bacillus subtilis and other bacteria. Adv Microb Physiol. 2001;44:35-91. doi: 10.1016/S0065-2911(01)44011-2

 

  1. Karzai AW, Susskind MM, Sauer RT. SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA). EMBO J. 1999;18:3793-3799. doi: 10.1093/emboj/18.13.3793

 

  1. White HE, Sherman MB, Brasilès S, et al. Capsid structure and its stability at the late stages of bacteriophage SPP1 assembly. J Virol. 2012;86(12):6768-6777. doi: 10.1128/jvi.00412-12

 

  1. Lu A, Disoma C, Zhou Y, et al. Protein interactome of the deamidase phosphoribosylformylglycinamidine synthetase (PFAS) by LC-MS/MS. Biochem Biophys Res Commun. 2019;513(3):746-752. doi: 10.1016/j.bbrc.2019.04.039

 

  1. Bommisetti P, Bandarian V. Insights into the mechanism of installation of 5-carboxymethylaminomethyl uridine hypermodification by tRNA-modifying enzymes MnmE and MnmG. J Am Chem Soc. 2023;145(49):26947-26961. doi: 10.1021/jacs.3c10182

 

  1. Dym O, Pratt EA, Ho C, Eisenberg D. The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme. Proc Natl Acad Sci. 2000;97(17):9413-9418. doi: 10.1073/pnas.97.17.9413

 

  1. Dubey S, Majumder P, Penmatsa A, Sardesai AA. Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues. J Biol Chem. 2021;297(4):101168. doi: 10.1016/j.jbc.2021.101168

 

  1. Jorgenson MA, Young KD. YtfB, an OapA domain-containing protein, is a new cell division protein in Escherichia coli. J Bacteriol. 2018;200(13):e00046-48. doi: 10.1128/jb.00046-18

 

  1. Lancaster MS, Graham BH. Succinyl-CoA synthetase dysfunction as a mechanism of mitochondrial encephalomyopathy: More than just an oxidative energy deficit. Int J Mol Sci. 2023;24(13):10725. doi: 10.3390/ijms241310725

 

  1. Monti SM, De Simone G, D’Ambrosio K. L-Histidinol dehydrogenase as a new target for old diseases. Curr Top Med Chem. 2016;16(21):2369-2378. doi: 10.2174/1568026616666160413140000

 

  1. Aravind L, Anantharaman V, Balaji S, Babu MM, Iyer LM. The many faces of the helix-turn-helix domain: Transcription regulation and beyond. FEMS Microbiol Rev. 2005;29(2):231-262. doi: 10.1016/j.fmrre.2004.12.008

 

  1. Jeckelmann JM, Erni B. Transporters of glucose and other carbohydrates in bacteria. Pflügers Arch. 2020;472(9):1129-1153. doi: 10.1007/s00424-020-02379-0

 

  1. Pan B, Kaldhone PR, Alund AW, et al. Mutagenicity of silver nanoparticles evaluated using whole-genome sequencing in mouse lymphoma cells. Nanotoxicology. 2021;15(3):418-432. doi: 10.1080/17435390.2021.1894614

 

  1. Pasqua M, Grossi M, Zennaro A, et al. The varied role of efflux pumps of the MFS family in the interplay of bacteria with animal and plant cells. Microorganisms. 2019;7(9):285. doi: 10.3390/microorganisms7090285

 

  1. Facey SJ, Kuhn A. Biogenesis of bacterial inner-membrane proteins. Cell Mol Life Sci. 2010;67:2343-2362. doi: 10.1007/s00018-010-0303-0

 

  1. Motouchi S, Kobayashi K, Nakai H, Nakajima M. Identification of enzymatic functions of osmo-regulated periplasmic glucan biosynthesis proteins from Escherichia coli reveals a novel glycoside hydrolase family. Commun Biol. 2023;6(1):961. doi: 10.1038/s42003-023-05336-6

 

  1. Roncarati D, Vannini A, Scarlato V. Temperature sensing and virulence regulation in pathogenic bacteria. Trends Microbiol. 2024;33:66-79. doi: 10.1016/j.tim.2024.07.009

 

  1. McCoy JG, Levin EJ, Zhou M. Structural insight into the PTS sugar transporter EIIC. Biochim Biophys Acta. 2015;1850(3):577-585. doi: 10.1016/j.bbagen.2014.03.013

 

  1. Fath MK, Garousi S, Mottahedi M, et al. The role of hypoxia-inducible factors in breast cancer stem cell specification. Pathol Res Pract. 2023;243:154349. doi: 10.1016/j.prp.2023.154349

 

  1. Rathore AS, Gupta RD. Chitinases from bacteria to human: Properties, applications, and future perspectives. Enzyme Res. 2015;(1):791907. doi: 10.1155/2015/791907

 

  1. Modrzejewska M, Kawalek A, Bartosik AA. The LysR-type transcriptional regulator BsrA (PA2121) controls vital metabolic pathways in Pseudomonas aeruginosa. Msystems. 2021;6(4):e0001521. doi: 10.1128/msystems.00015-21

 

  1. Zawilak-Pawlik A, Nowaczyk M, Zakrzewska-Czerwińska J. The role of the N-terminal domains of bacterial initiator DnaA in the assembly and regulation of the bacterial replication initiation complex. Genes (Basel). 2017;8(5):136. doi: 10.3390/genes8050136

 

  1. Dakal TC, Kumar A, Majumdar RS, Yadav V. Mechanistic basis of antimicrobial actions of silver nanoparticles. Front Microbiol. 2016;7:1831. doi: 10.3389/fmicb.2016.01831

 

  1. Li Y, Shi Z, Radauer-Preiml I, et al. Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation. Nanotoxicology. 2017;11(9-10):1157-1175. doi: 10.1080/17435390.2017.1401142

 

  1. Whitfield C, Williams DM, Kelly SD. Lipopolysaccharide O-antigens-bacterial glycans made to measure. J Biol Chem. 2020;295(31):10593-10609. doi: 10.1074/jbc.REV120.009402

 

  1. Clements A, Tull D, Jenney AW, et al. Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides. J Biol Chem. 2007;282(21):15569-15577. doi: 10.1074/jbc.M701454200

 

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