Genetic Diversity and Molecular Characterization of Clinically Isolated Pseudomonas aeruginosa
DOI:
https://doi.org/10.31530/cjnst.2026.2.1.5Keywords:
Pseudomonas aeruginosa, Biofilm, ERIC-PCR, Virulence geneAbstract
Background: Pseudomonas aeruginosa is a major opportunistic pathogen and a member of the ESKAPE group, known for causing serious nosocomial infections and exhibiting high levels of antimicrobial resistance. Its resistance arises from intrinsic, acquired, and adaptive mechanisms, complicating treatment, particularly in healthcare settings.
Aim: This study aimed to identify clinical P. aeruginosa isolates using molecular methods, assess their genetic diversity, detect key virulence genes, and evaluate biofilm formation and antibiotic resistance profiles.
Methodology: A total of 36 clinical isolates were collected from public and private hospitals in Sulaymaniyah City between September 2024 and July 2025. Samples included bronchoalveolar lavage, endotracheal aspiration, pleural fluid, wounds, blood, catheters, and ear swabs. Initial identification was based on morphological and biochemical characteristics, followed by confirmation using Vitek® 2 and BD Phoenix M50 systems. Molecular identification targeted the gyrB gene. Antimicrobial susceptibility was assessed using the Kirby–Bauer disk diffusion method and automated systems. Virulence genes (lasB, toxA, exoS, and algD) were detected by polymerase chain reaction (PCR). Biofilm formation was evaluated using a 96-well plate assay, and genetic relatedness was determined by Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR).
Results: All isolates (100%) carried the gyrB gene. High levels of antibiotic resistance were observed: 47.22% were multidrug-resistant (MDR), 36% extensively drug-resistant (XDR), and 8% pan-drug-resistant (PDR). Three isolates were resistant to colistin and all tested antibiotics. The lasB and algD genes were detected in all isolates, while toxA and exoS were present in 32 and 19 isolates, respectively. Biofilm analysis showed that 92% of isolates were strong producers. ERIC-PCR revealed clonal relatedness among isolates from the same hospitals and specimen types.
Conclusion: Wound samples were the most common source of P. aeruginosa. The high prevalence of drug-resistant, strong biofilm-forming strains highlights a significant clinical concern. ERIC-PCR proved effective for assessing clonal relationships, indicating persistence of identical genotypes within individual hospitals.
References
[1] Silverio MP, Schultz J, Parise MT, Parise D, Viana MVC, Nogueira W, et al. Genomic and phenotypic in-sight into antimicrobial resistance of Pseudomonas fluo-rescens from King George Island, Antarctica. Front Mi-crobiol. 2025;16:1535420. doi:10.3389/fmicb.2025.1535420 DOI: https://doi.org/10.3389/fmicb.2025.1535420
[2] Lewandowska W, Mahillon J, Drewnowska JM, Święcicka I. Insight into the phylogeny and antibiotic resistance of Pseudomonas spp. originating from soil of the Białowieża National Park in Northeastern Poland. Front Microbiol. 2025;16:1454510. doi:10.3389/fmicb.2025.1454510 DOI: https://doi.org/10.3389/fmicb.2025.1454510
[3] Kharat AS, Makwana N, Nasser M, Gayen S, Yadav B, Kumar D, et al. Dramatic increase in antimicrobial re-sistance in ESKAPE clinical isolates over the 2010–2020 decade in India. J Infect Public Health. 2024;63(5):107125. doi:10.1016/j.jiph.2024.107125 DOI: https://doi.org/10.1016/j.ijantimicag.2024.107125
[4] Adenipekun EO, Akinleye EF, Tewogbade OA, Iwa-lokun BA. Detection of virulence genes and multidrug resistance in Pseudomonas aeruginosa clinical isolates from a public hospital in Lagos, Nigeria. New Microbes New Infect. 2023;22:e01950. doi:10.1016/j.nmni.2023.e01950 DOI: https://doi.org/10.1016/j.sciaf.2023.e01950
[5] Hematzadeh A, Haghkhah M. Biotyping of isolates of Pseudomonas aeruginosa isolated from human infections by RAPD and ERIC-PCR. Heliyon. 2021;7(9):e07967. doi:10.1016/j.heliyon.2021.e07967 DOI: https://doi.org/10.1016/j.heliyon.2021.e07967
[6] Cunrath O, Graulier G, Carballido-López A, Pérard J, Forster A, Geoffroy VA, et al. The pathogen Pseudomo-nas aeruginosa optimizes the production of the sidero-phore pyochelin upon environmental challenges. Metal-lomics. 2020;12(12):2108–2120. doi:10.1039/d0mt00029a DOI: https://doi.org/10.1039/d0mt00029a
[7] Gülhan B, Çıkman A, Aydın M, Hasbek M, Özekinci T, Akyüz S, et al. Rapid detection of pathogens and re-sistance genes grown in blood cultures with two multi-plex tandem real-time PCR kits. Infect Dis Clin Micro-biol. 2025;7(1):37–46. doi:10.36519/idcm.2025.465 DOI: https://doi.org/10.36519/idcm.2025.465
[8] Robert M, Ruffier d’Epenoux L, Paquin A, Boutoille D, Guillouzouic A, Corvec S, et al. Ciprofloxacin-susceptible but levofloxacin-resistant Pseudomonas ae-ruginosa clinical strains with Vitek® 2: mechanisms and clinical consequences. Eur J Clin Microbiol Infect Dis. 2025;44(3):549–558. doi:10.1007/s10096-024-05006-3 DOI: https://doi.org/10.1007/s10096-024-05006-3
[9] Tang KWK, Millar BC, Moore JE. Antimicrobial re-sistance (AMR). Br J Biomed Sci. 2023;80:11387. doi:10.3389/bjbs.2023.11387 DOI: https://doi.org/10.3389/bjbs.2023.11387
[10] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, exten-sively drug-resistant and pandrug-resistant bacteria: in-ternational expert proposal. Clin Microbiol Infect. 2012;18(3):268–281. doi:10.1111/j.1469-0691.2011.03570.x DOI: https://doi.org/10.1111/j.1469-0691.2011.03570.x
[11] Peña C, Suarez C, Gozalo M, Murillas J, Almirante B, Pomar V, et al. Impact of carbapenem resistance on mortality in Pseudomonas aeruginosa bloodstream infec-tions. Antimicrob Agents Chemother. 2012;56(3):1265–1272. doi:10.1128/AAC.05991-11 DOI: https://doi.org/10.1128/AAC.05991-11
[12] Jamaluddin IP, Musa SH, Ethica SN, Ansori AN, Yosephi V, Atmaja PY, et al. Detection of Pseudomonas aeruginosa using PCR targeting 16S rRNA and gyrB genes. Narra J. 2024;4(2):e774. doi:10.52225/narra.v4i2.774 DOI: https://doi.org/10.52225/narra.v4i2.774
[13] Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and virulence genes of Pseudomonas aeruginosa. Germs. 2018;8(1):31–40. doi:10.18683/germs.2018.1130 DOI: https://doi.org/10.18683/germs.2018.1130
[14] Cotar AI, Chifiriuc MC, Dinu S, Bucur M, Iordache C, Banu O, et al. Molecular virulence markers in Staphylo-coccus aureus and Pseudomonas aeruginosa. Int J Mol Sci. 2010;11(12):5273–5291. doi:10.3390/ijms11125273 DOI: https://doi.org/10.3390/ijms11125273
[15] Sabharwal N, Dhall S, Chhibber S, Harjai K. Molecular detection of virulence genes in Pseudomonas aeruginosa. Int J Appl Basic Med Res. 2014;4(2):103–107. doi:10.4103/2229-516X.136783 DOI: https://doi.org/10.4103/2229-516X.136783
[16] Ertugrul BM, Oryasin E, Lipsky BA, Willke A, Bozdogan B. Virulence genes in diabetic foot infections. Infect Dis (Lond). 2018;50(4):273–279. doi:10.1080/23744235.2017.1393839 DOI: https://doi.org/10.1080/23744235.2017.1393839
[17] Najem SA-A, Shubbar EE. Molecular analysis of Pseu-domonas aeruginosa by ERIC-PCR. Int J Health Sci. 2022;6(S1):10144–10151. doi:10.53730/ijhs.v6nS1.7203 DOI: https://doi.org/10.53730/ijhs.v6nS1.7203
[18] Cai Z, Zhang W, Liao G, Huang C, Wang J, Zhang J. Biofilm inhibition mechanisms. J Water Process Eng. 2025;69:106613. doi:10.1016/j.jwpe.2024.106613 DOI: https://doi.org/10.1016/j.jwpe.2024.106613
[19] Fuentes-González MF, Fernández-Rodríguez D, Colín-Castro CA, et al. Gram-negative bloodstream infections in burn patients. Int J Mol Sci. 2024;25(19):10458. doi:10.3390/ijms251910458 DOI: https://doi.org/10.3390/ijms251910458
[20] Akinsulie OC, Aliyu VA, Idris I, et al. The implications of handwashing and skin hygiene on infectious disease dynamics: the African scenario. Trop Med Infect Dis. 2024;9(4):82. doi:10.3390/tropicalmed9040082 DOI: https://doi.org/10.3390/hygiene4040036
[21] Qader AR, Muhamad JA. Nosocomial infection in Sulaimani burn hospital, Iraq. Ann Burns Fire Disasters. 2010;23(4):177–181.
[22] Chen Y, Xu M, Pan J, et al. Moxifloxacin-loaded nano-particles for overcoming multidrug resistance. ACS Appl Mater Interfaces. 2025;17(4):5695–5709. doi:10.1021/acsami.4c14991 DOI: https://doi.org/10.1021/acsami.4c14991
[23] Azemin A, Alias N, Kari A. Identification of Pseudo-monas aeruginosa from sheep milk. Malays J Fundam Appl Sci. 2022;18(1):30–42. doi:10.11113/mjfas.v18n1.2302 DOI: https://doi.org/10.11113/mjfas.v18n1.2302
[24] Hassan KI, Abdullah SR. Detection of Pseudomonas ae-ruginosa using PCR. Baghdad Sci J. 2018;15(4):401–405. doi:10.21123/bsj.2018.15.4.0401 DOI: https://doi.org/10.21123/bsj.15.4.401-405
[25] Aziz SH, Abdulrahman TT, Hamad A. Molecular char-acterization of virulence factors in Pseudomonas aeru-ginosa. J Adv Pharm Educ Res. 2024;14(S1):1–10. doi:10.51847/6zXyY9bJ8L
[26] Alkhulaifi ZM, Mohammed KA. Prevalence and molec-ular analysis of antibiotic resistance of Pseudomonas ae-ruginosa in Iraq. Iran J Microbiol. 2023;15(1):45–52. doi:10.18502/ijm.v15i1.11917 DOI: https://doi.org/10.18502/ijm.v15i1.11917
[27] Omer KA, Arif SK, Othman HE. Molecular detection of MBL genes in Pseudomonas aeruginosa. Bahrain Med Bull. 2024;46(2):2075-2079. doi:10.26715/bmb.46.2.2
[28] Alsaadi LA, Al-Dulaimi AAF, Rasheed HR. Car-bapenem resistance genes in Iraq. Indian J Public Health Res Dev. 2020;11(2):2550-2554.doi:10.37506/ijphrd.v11i2.195173
[29] Vaez H, Salehi-Abargouei A, Ghalehnoo ZR, Khademi F. Multidrug-resistant Pseudomonas aeruginosa in Iran. J Glob Infect Dis. 2018;10(4):212–217. doi:10.4103/jgid.jgid_113_17 DOI: https://doi.org/10.4103/jgid.jgid_113_17
[30] Mahfoud M, Al Najjar M, Hamzeh AR. Multidrug re-sistance in Pseudomonas aeruginosa in Syria. J Infect Dev Ctries. 2015;9(2):210–213. doi:10.3855/jidc.5643 DOI: https://doi.org/10.3855/jidc.5643
[31] Abulhasan YB, Abdullah AA, Shetty SA, et al. Healthcare-associated infections in neurocritical care. Neurocrit Care. 2020;32(3):836–846. doi:10.1007/s12028-019-00856-8 DOI: https://doi.org/10.1007/s12028-019-00856-8
[32] Al-Tawfiq JA, Rabaan AA, Saunar JV, Bazzi AM. An-timicrobial resistance in Saudi Arabia. J Infect Public Health. 2020;13(5):737–745. doi:10.1016/j.jiph.2020.01.004 DOI: https://doi.org/10.1016/j.jiph.2020.01.004
[33] Ghanem SM, Abd El-Baky RM, Abourehab MA, et al. Virulence and resistance genes. Infect Drug Resist. 2023;16:2371–2385. doi:10.2147/IDR.S403441 DOI: https://doi.org/10.2147/IDR.S403441
[34] Bogiel T, Depka D, Rzepka M, et al. Biofilm and toxin genes. Antibiotics (Basel). 2021;10(3):241. doi:10.3390/antibiotics10030241 DOI: https://doi.org/10.3390/antibiotics10030241
[35] Hibbert TM, Whiteley M, Renshaw SA, et al. Emerging strategies to target virulence in Pseudomonas aeruginosa respiratory infections. Crit Rev Microbiol. 2024;50(6):1037–1052. doi:10.1080/1040841X.2023.2285995 DOI: https://doi.org/10.1080/1040841X.2023.2285995
[36] Ali AM, Al-Kenanei KA, Hussein SA, Bdaiwi QO. Mo-lecular study of virulence genes of Pseudomonas aeru-ginosa. Med J Babylon. 2020;31(1):26–41. doi:10.4103/MJOB.MJOB_35_19 DOI: https://doi.org/10.1097/MRM.0000000000000194
[37] Ghasemian S, Karami-Zarandi M, Heidari H, et al. Mo-lecular characterization of Pseudomonas aeruginosa. J Clin Lab Anal. 2023;37(4):e24850. doi:10.1002/jcla.24850 DOI: https://doi.org/10.1002/jcla.24850
[38] Shariat A, Alizadeh S, Hoseinabadi MJ, Movagharnejad M. Molecular detection of virulence genes. Avicenna J Clin Microbiol Infect. 2018;5(1):28–34. doi:10.5812/ajcmi.64332 DOI: https://doi.org/10.21859/ajcm.25.1.28
[39] Berber I, Avsar C, Yegin Z, et al. Molecular epidemiol-ogy of Pseudomonas aeruginosa. Braz J Infect Dis. 2016;20(2):224–225. doi:10.1016/j.bjid.2015.12.002 DOI: https://doi.org/10.1016/j.bjid.2015.12.002
[40] Ali SA, Ghamry AA, Soliman AM, et al. Virulence and resistance in Egypt. J Infect Dev Ctries. 2025;19(5):712–722. doi:10.3855/jidc.20957 DOI: https://doi.org/10.3855/jidc.20957
[41] Mitov I, Strateva T, Markova B. Virulence genes in Bulgaria. Braz J Microbiol. 2010;41(3):588–595. doi:10.1590/S1517-83822010000300008 DOI: https://doi.org/10.1590/S1517-83822010000300008
[42] Qin S, Xiao W, Zhou C, et al. Pseudomonas aeruginosa: pathogenesis and resistance. Signal Transduct Target Ther. 2022;7(1):199. doi:10.1038/s41392-022-01056-1 DOI: https://doi.org/10.1038/s41392-022-01056-1
[43] Fazeli H, Akbari R, Moghim S, et al. Pseudomonas ae-ruginosa infections in hospital settings. J Res Med Sci. 2012;17(4):332–338.
[44] Naji H, Saleh MJ. Genetic diversity and resistance in Iraq. J Life Sci Appl Res. 2024;5(1):14–27. doi:10.59807/jlsar.v5i1.93
[45] Elahi G, Goli HR, Shafiei M, Nikbin VS, Gholami M. Antimicrobial resistance and genetic diversity of Pseu-domonas aeruginosa. BMC Microbiol. 2024;24(1):546. doi:10.1186/s12866-024-03707-5 DOI: https://doi.org/10.1186/s12866-024-03707-5
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Charmo Journal of Natural Sciences and Technologies
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
