Investigation of Virulence Factors and Antibiotic Resistance of Pseudomonas aeruginosa Isolated from A Range of Clinical Samples

Abstract

Objective: Pseudomonas aeruginosa is an opportunistic pathogen, is one of the leading nosocomial infection-causing agents and over time has developed multidrug resistance. One of the most common patient groups affected by P. aeruginosa are on the intensive care unit (ICU), an optimal environment for the development of antibiotic resistance. The aim of this study was to investigate virulence factors and antibiotic resistance profiles of P. aeruginosa isolated from hospitalized patients in Turkey. Methods: Samples from the general wards and ICU-hospitalized patients were included. A nutrient agar-elastin method was used for the biochemical activity of elastase. For las B assessment PCR was used while special production medium was used to assay pyoverdine and pyocyanin. Isolate biofilm production was tested with the crystal violet method. Standard broth microdilution was used for antibiotic susceptibility. Results: A total of 208 samples were assessed. The virulence factor frequencies in ICU and ward isolates, were: pyocyanin 86.2% and 86.7%, pyoverdine 90.1%, and 89.6%, elastase 68.6% and 67.9%, las B 93.1% and 89.6%, and biofilm production 51.9% and 48.1%, respectively. Antibiotic resistance rates in ICU and ward were: meropenem 41.1% and 28.9%, colistin 11.7% and 13.2%, ceftazidime 43.1%,and 41.1%, and cefepime 52.9% and 48.5%. Conclusion: Virulence factors were present in most of the hospitalized patient samples. However, antibiotic resistance rates were below 50%, except for cefepime. In addition, low rates of colistin resistance suggest that colistin resistance is not yet widespread in our hospital.

Keywords

• Pseudomonas aeruginosa
• virulence factors
• biofilm
• antibiotic resistance

Project Number

TYL-2022-2829

References

1. Jurado-Martín I, Sainz-Mejías M, McClean S. Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors. Int J Mol Sci. 2021;22(6):3128. doi: 10.3390/ijms22063128.
2. Cigana C, Castandet J, Sprynski N, Melessike M, Beyria L, Ranucci S, Alcalá-Franco B, Rossi A, Bragonzi A, Zalacain M, Everett M. Pseudomonas aeruginosa Elastase Contributes to the Establishment of Chronic Lung Colonization and Modulates the Immune Response in a Murine Model. Front Microbiol. 2021;11:620819. doi: 10.3389/fmicb.2020.620819.
3. Coin D, Louis D, Bernillon J, Guinand M, Wallach J. LasA, alkaline protease and elastase in clinical strains of Pseudomonas aeruginosa: quantification by immunochemical methods. FEMS Immunol Med Microbiol. 1997;18(3):175-84. doi: 10.1111/j.1574-695X.
4. Kang D, Revtovich AV, Chen Q, Shah KN, Cannon CL, Kirienko NV. Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients. Front Microbiol. 2019;6;10:2048. doi: 10.3389/fmicb.2019.02048.
5. Hall-Stoodley L, McCoy KS. Biofilm aggregates and the host airway-microbial interface. Front Cell Infect Microbiol. 2022;12:969326. doi: 10.3389/fcimb.2022.969326.
6. Reyne N, McCarron A, Cmielewski P, Parsons D, Donnelley M. To bead or not to bead: A review of Pseudomonas aeruginosa lung infection models for cystic fibrosis. Front Physiol. 2023;14:1104856. doi: 10.3389/fphys.2023.1104856.
7. Guillaume O, Butnarasu C, Visentin S, Reimhult E. Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection. Bio. 2022;22;4:100089. doi: 10.1016/j.bioflm.2022.100089.
8. Diban F, Di Lodovico S, Di Fermo P, D'Ercole S, D'Arcangelo S, Di Giulio M, Cellini L. Biofilms in Chronic Wound Infections: Innovative Antimicrobial Approaches Using the In Vitro Lubbock Chronic Wound Biofilm Model. Int J Mol Sci. 2023; 5;24(2):1004. doi: 10.3390/ijms24021004.

9. Bakthavatchalam YD, Pragasam AK, Biswas I, Veeraraghavan B. Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update. J Glob Antimicrob Resist. 2018;12:124-136. doi: 10.1016/j.jgar.2017.09.011.
10. Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev. 2002;15(2):194-222. doi: 10.1128/CMR.15.2.194-222.2002.
11. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv. 2019;37(1):177-192. doi: 10.1016/j.biotechadv.2018.11.013.
12. Coleman SR, Blimkie T, Falsafi R, Hancock REW. Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells. Antimicrob Agents Chemother. 2020;21;64(3):e01999-19. doi: 10.1128/AAC.01999-19.
13. Huszczynski SM, Lam JS, Khursigara CM. The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology. J Pathogens. 2019; 19;9(1):6. doi: 10.3390/pathogens9010006.
14. Chung ES, Lee JY, Rhee JY, Ko KS. Colistin resistance in Pseudomonas aeruginosa that is not linked to arnB. J Med Microbiol. 2017;66(6):833-841. doi: 10.1099/jmm.0.000456.
15. Mapipa, Q., Digban, T.O., Nnolim, N.E. et al. Antibiogram profile and virulence signatures of Pseudomonas aeruginosa isolates recovered from selected agrestic hospital effluents. Sci Rep 2021;11,11800. doi:10.1038/s41598-021-91280-6
16. Kafil HS, Mobarez AM. Assessment of biofilm formation by enterococci isolates from urinary tract infections with different virulence profiles. J King Saud University-Sci. 2015;27(4):312-317. doi:10.1016/j.jksus.2014.12.007
17. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0; 2023.
18. Hall S, McDermott C, Anoopkumar-Dukie S, McFarland AJ, Forbes A, Perkins AV, Davey AK, Chess-Williams R, Kiefel MJ, Arora D, Grant GD. Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa. Toxins. 2016;9;8(8):236. doi: 10.3390/toxins8080236.
19. El-Mahdy R, El-Kannishy G. Virulence Factors Of Carbapenem-Resistant Pseudomonas aeruginosa In Hospital-Acquired Infections In Mansoura, Egypt. Infect Drug Resist. 2019;12:3455-3461. doi: 10.2147/IDR.S222329
20. Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs. 2018;3;8(1):31-40. doi: 10.18683/germs.2018.1130
21. Hamza EH, El-Shawadfy AM, Allam AA, Hassanein WA. Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa. Saudi J Biol Sci. 2023;30(1):103492. doi: 10.1016/j.sjbs.2022.103492.
22. Uzunbayir-Akel N, Tekintaş Y, Yılmaz FF, et al. Klinik Pseudomonas aeruginosa izolatlarının virülans özellikleri ve epidemiyolojik ilişkisi. Turk Hij Den Biyol Derg. 2019; 76(4): 395-404 doi: 10.5505/TurkHijyen.2019.68235
23. Zupetic J, Peñaloza HF, Bain W, Hulver M, Mettus R, Jorth P, Doi Y, Bomberger J, Pilewski J, Nouraie M, Lee JS. Elastase Activity From Pseudomonas aeruginosa Respiratory Isolates and ICU Mortality. Chest. 2021;160(5):1624-1633. doi: 10.1016/j.chest.2021.04.015.
24. Çoban AY, Çiftci A, Onuk EE, Erturan Z, Çaycı Y, Durupınar B. Investigation of Plasmid-Mediated Quinolone Resistance in Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients. Mikrobiyol Bul. 2009; 43:563-57
25. Ghadaksaz A, Fooladi AAI, Hosseini HMH, Amin M. The prevalence of some Pseudomonas virulence genes related to biofilm formation and alginate production among clinical isolates. J Appl Biomed. 2015;13:61-68, doi: 10.1016/j.jab.2014.05.002
26. Hirsch EB, Tam VH. Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes. Expert Rev Pharmacoecon Outcomes Res. 2010;10(4):441-51. doi: 10.1586/erp.10.49.
27. Babaeekhou L, Karshenasan H, Pishkar L. Antibiotic Resistance in Clinical Isolates of Pseudomonas aeruginosa: A New Viewpoint for Antibiotic Prescription. Avicenna J Clin Microbiol Infect. 2018; 5(3), 55–60. doi: 10.34172/ajcmi.2018.11
28. Abd El-Baky RM, Masoud SM, Mohamed DS, Waly NG, Shafik EA, Mohareb DA, Elkady A, Elbadr MM, Hetta HF. Prevalence and Some Possible Mechanisms of Colistin Resistance Among Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa. Infect Drug Resist. 2020;3;13:323-332. doi: 10.2147/IDR.S238811.
29. Sonmezer MC, Ertem G, Erdinc FS, Kaya Kilic E, Tulek N, Adiloglu A, Hatipoglu C. Evaluation of Risk Factors for Antibiotic Resistance in Patients with Nosocomial Infections Caused by Pseudomonas aeruginosa. Can J Infect Dis Med Microbiol. 2016;1321487. doi: 10.1155/2016/1321487.
30. Kal Çakmaklıoğulları E, Kuru C. Pseudomonas aeruginosa Suşlarının Antibiyotik Duyarlılıkları: Farklı Örnek Türlerinde Değerlendirme. Ank. Derg. 2019;33(2):37-42 doi: 10.5222/ankem.2019.197
31. Dursun A, Özsoylu S, Kılıç H, Ulu Kılıç A, Akyıldız BN. Antibiotic Susceptibilities of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii Strains Isolated from Patients in the Pediatric Intensive Care Unit. J Turk Soc Intense Care. 2018; 16(3), 109–114. doi:10.4274/TYBD.63825
32. Ceken, N. , Duran, H. & Atik, B. Yoğun bakım ünitelerinden izole edilen Pseudomonas aeruginosa suşlarının 4 yıllık direnç profili. Pam Tıp Derg. 2021; 14 (2) , 306-311 doi: 10.31362/patd.789332
33. Uğur M, Genç S. A 3-year Resistance Profile of Acinetobacter baumannii and Pseudomonas aeruginosa Strains Isolated from Intensive Care Units. J Turk Soc Intense Care. 2019;17:130-7 doi: 10.4274/tybd.galenos.2018.94103
34. Şafak B, Kilinç O, Tunç N, Topçu B. Türkiye’de Bir Devlet Hastanesinde 2010-2016 Yılları Arasında Pseudomonas aeruginosa Antimikrobiyal Duyarlılık Sonuçları . Ank. Derg. 2018;32(1):31-36 doi: 10.5222/ankem.2018.031
35. Santos SO, Rocca SM, Hörner R. Colistin resistance in non-fermenting Gram-negative bacilli in a university hospital. Braz J Infect Dis. 2016 Nov-Dec;20(6):649-650. doi: 10.1016/j.bjid.2016.08.009.
36. Sader HS, Castanheira M, Arends SJR, Goossens H, Flamm RK. Geographical and temporal variation in the frequency and antimicrobial susceptibility of bacteria isolated from patients hospitalized with bacterial pneumonia: results from 20 years of the SENTRY Antimicrobial Surveillance Program (1997-2016). J Antimicrob Chemother. 2019 Jun 1;74(6):1595-1606. doi: 10.1093/jac/dkz074.