•  
  •  
 

Abstract

Vaginitis is one of the most common women's health problems, and its severity increases in pregnant women with gestational diabetes due to higher susceptibility to infection and increased rates of antibiotic resistance. The research problem: The increasing prevalence of multidrug-resistant isolates of Klebsiella pneumoniae and its association with AcrAB-TolC ejaculation pumps limits treatment efficacy, while the effect of green-method nanoparticles on gene expression of these pumps remains unclear. This paper aimed to detect the acrA and acrB genes in K. pneumoniae isolates and evaluate the effect of zinc oxide nanoparticles prepared with garlic and rosemary extracts on their gene expression. Two hundred and three vaginal swabs were collected from pregnant women (aged 18–45) 72 hours after antibiotic discontinuation. The samples were cultured, and the isolates were identified using biochemical and Vitek assays. Susceptibility testing was then performed using the disk diffusion method according to CLSI 2025. Conventional PCR was used to detect acrA/acrB, and RT-qPCR was used to calculate expression folds using the 2^−ΔΔCt method with 16S as a reference gene. The results showed bacterial growth in 152 samples, and 44 isolates were from women with gestational diabetes, with K. pneumoniae being 15.9% of the isolates exhibiting MDR-like affinity with high resistance to some cephalosporins and piperacillin (up to 85.7%), and high sensitivity (up to 100%) to some fluoroquinolones. AcrA and acrB were present in all tested isolates. ZnO-NPs reduced acrA expression to 0.60 (garlic) and 0.43 (rosemary), while acrB remained close to unchanged with garlic (1.01) and increased with rosemary to 1.53, indicating a selective effect on ejaculation pump components and their clinical applications

References

[1] World Health Organization. WHO updates list of drugresistant bacteria most threatening to human health [Internet]. Geneva: WHO; 2024.

[2] Murray CJL, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022;399(10325):629—55 [Internet].

[3] World Health Organization. WHO bacterial priority pathogens list, 2024: bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance [Internet]. Geneva: WHO; 2024.

[4] Centers for Disease Control and Prevention. About Klebsiella [Internet]. Atlanta (GA): CDC; 2025.

[5] Maurya N, Jangra M, Tambat R, Nandanwar H. Alliance of efflux pumps with β-lactamases in multidrug-resistant Klebsiella pneumoniae isolates. Microb Drug Resist [Internet] 2019;25(8):1155—63.

[6] Vieira Da Cruz A, Jimenez-Castellanos JC, Bornsen C, Van Maele L, Compagne N, Pradel E, et al. Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae. EMBO Mol Med [Internet] 2024; 16(1):e17993.

[7] Zouine N, El Ghachtouli N, El Abed S, Ibnsouda Koraichi S. A comprehensive review on medicinal plant extracts as antibacterial agents: factors, mechanism insights and future prospects. Sci Afr [Internet] 2024;26:e02395.

[8] Vaou N, Stavropoulou E, Voidarou C, Tsimogiannis D, Rozos G. Towards advances in medicinal plant antimicrobial activity: a review study on challenges and future perspectives. Microorganisms 2021;9(10):2041 [Internet].

[9] Bhatwalkar SB, Mondal R, Krishna SBN, Adam JK, Govender P, Anupam RA. Antibacterial properties of organosulfur compounds of garlic (Allium sativum). Front Microbiol 2021;12:613077 [Internet].

[10] Mendes CR, Dilarri G, Forsan CF, Sapata VDMR, Lopes PRM, de Moraes PB, et al. Antibacterial action and target mechanisms of zinc oxide nanoparticles against bacterial pathogens. Sci Rep 2022;12(1):2658 [Internet]

[11] Al-darwesh MY, Ibrahim SS, Mohammed MA. A review on plant extract mediated green synthesis of zinc oxide nanoparticles and their biomedical applications. Results Chem [Internet] 2024;7:101368. https://doi.org/10.1016/j.rechem. 2024.101368.

[12] Gabriel T, Said-Canales C, Corzo-Rios L, et al. Antibacterial activity of nanoparticles of garlic (Allium sativum) extract and their application in edible coatings. Appl Sci [Internet] 2022;12(7):3491.

[13] Nieto G, Ros G, Castillo J. Antioxidant and antimicrobial properties of rosemary. Rosmarinus officinalis, L.): a review. Medicines [Internet] 2018;5(3):98.

[14] Cedeno-Pinos ~ C, Martínez-Tome � M, Mercatante D, Rodríguez-Estrada MT, Ban~on � S. Assessment of a diterpene-rich rosemary (Rosmarinus officinalis L.) extract as a natural antioxidant for salmon pat ^ e � formulated with linseed. Antioxidants [Internet] 2022;11(6):1057. https://doi.org/10.3390/ antiox11061057.

[15] Uysal Y, et al. Rosemary-mediated green synthesis of ZnO nanoparticles and evaluation of antibacterial activity. Glob Chall 2024;8:e2400120 [Internet].

[16] Struelens MJ, Ludden C, Werner G, Sintchenko V, Jokelainen P, Ip M. Real-time genomic surveillance for enhanced control of infectious diseases and antimicrobial resistance. Front Sci [Internet] 2024;2:1298248.

[17] Russell AD, Hammond SA, Morgan JR. Bacterial resistance to antiseptics and disinfectants. J Hosp Infect 1986;7(3): 213—25 [Internet].

[18] Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol [Internet] 1966;45:493—6.

[19] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008;3(6):1101—8 [Internet].

[20] Hameed SJ. Evaluating the antimicrobial efficacy of apple cider vinegar on bacteria and fungi isolated from vaginitis. Al-Kitab J Pure Sci [Internet] 2024;8(1):29—39.

[21] Abdulla SR, Kareem SR, Hasan AH. Vaginal microbiota profile in first-trimester miscarriages cases. Cell Mol Biol [Internet] 2023;69(8):9—17.

[22] Abid FAS. Detection of AcrA and AcrB efflux pumps in multidrug-resistant Klebsiella pneumoniae isolated from wound infection patients in Al-Diwaniyah province [Internet]. 2023.

[23] Nasir AA, Al-Mousawi AA, Abdul-Hussein ZR. Detection of AcrAB efflux pumps gene, biofilm formation in Escherichia coli and Klebsiella pneumoniae isolated from pregnant women suffered of urinary tract infections and diabetes mellitus in Basrah City, Iraq. Rev Diabet Stud [Internet] 2025:297—312.

[24] Hussein ZR, Ghareeb AM. Enhancing the antibacterial activity of ciprofloxacin against Klebsiella pneumoniae by inhibiting the AcrAB-TolC efflux pump system using phenylalanine-arginine β-naphthylamide. Al-Rafidain J Med Sci [Internet] 2025;8(1):56—61.

[25] Razavi S, Mirnejad R, Babapour E. Involvement of AcrAB and OqxAB efflux pumps in antimicrobial resistance of clinical isolates of Klebsiella pneumoniae. J Appl Biotechnol Rep [Internet] 2020;7(4):251—7

[26] Li Y, Cross TS, Dorr T. Analysis of AcrB in Klebsiella pneumoniae reveals natural variants promoting enhanced multidrug resistance. Res Microbiol [Internet] 2022;173(3): 103901.

[27] Shahbazi S, Shivaee A, Nasiri M, Mirshekar M, Sabzi S, Sariani OK. Zinc oxide nanoparticles impact the expression of the genes involved in toxin—antitoxin systems in multidrug-resistant Acinetobacter baumannii. J Basic Microbiol [Internet] 2023;63(9):1007—15.

[28] Venter H, Mowla R, Ohene-Agyei T, Ma S. RND-type drug efflux pumps from Gram-negative bacteria: Molecular mechanism and inhibition. Front Microbiol [Internet] 2015;6: 377.

[29] Khames SASK, Ahmed ST. Effect of chemically synthesis compared to biosynthesized zinc oxide nanoparticles using extract of Vitex agnus on the expression of MexAB-OprM efflux pump genes of multi-drug resistance Pseudomonas aeruginosa. Baghdad Sci J [Internet] 2024;21(12 Suppl): 4050—66.

[30] Modi S, Fulekar MH. Green synthesis of zinc oxide nanoparticles using garlic skin extract and its characterization. J Nanostruct [Internet] 2020;10(1):20—7.

[31] Saleh F, Hosseini F, Kheirandish F, Yazdian F. Evaluation of the inhibitory effect of biosynthetic zinc oxide nanoparticles on the expression of efflux pumps genes (AdeA, AdeC) in clinical strains of Acinetobacter baumannii. Genet Eng Biosaf J [Internet] 2021;10(1):1—10.

[32] Kudaer NB, Risan MH, Yousif E, Kadhom M, Raheem R, Salman I. Effect of zinc oxide nanoparticles on capsular gene expression in Klebsiella pneumoniae isolated from clinical samples. Biomimetics 2022;7(4):180 [Internet].

[33] Guo XP, Sha RR, Wang YC, Yan J, Peng B, Niu ZS, et al. ZnO nanorod coatings attenuate antibiotic resistance genes (ARGs) on their surface biofilms in estuarine environment. J Environ Chem Eng [Internet] 2024;12(3):112877.

Included in

Biology Commons

Share

COinS