Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Detection of Virulence Factors in Carbapenem-Resistant Pseudomonas aeruginosa Isolated from a Tertiary Hospital in Daejeon

대전지역의 3차 병원에서 분리된 Carbapenem 내성 Pseudomonas aeruginosa의 병독성 인자 검출

  • Cho, Hye Hyun (Department of Biomedical Laboratory Science, Daejeon Institute of Science and Technology)
  • 조혜현 (대전과학기술대학교 임상병리과)
  • Received : 2019.08.15
  • Accepted : 2019.08.27
  • Published : 2019.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The emergence and spread of multidrug resistant (MDR) Pseudomonas aeruginosa is a critical problem worldwide. The pathogenesis of P. aeruginosa is due partly to the production of several cell-associated and extracellular virulence factors. This study examined the distribution of virulence factors and antimicrobial resistance patterns of carbapenem-resistant P. aeruginosa (CRPA) isolated from a tertiary hospital in Daejeon, Korea. Antimicrobial susceptibility testing was performed using the disk diffusion method, and PCR and DNA sequencing were performed to determine for the presence of virulence genes. In addition, the sequence type (ST) of MDR P. aeruginosa was investigated by multilocus sequence typing (MLST). Among 32 CRPA isolates, 14 (43.8%) were MDR and the major ST was ST235 (10 isolates, 71.4%). All isolates were positive for the presence of virulence genes and the most prevalent virulence genes were toxA, plcN, and phzM (100%). All isolates carried at least eight or more different virulence genes and nine (28.1%) isolates had 15 virulence genes. The presence of the exoU gene was detected in 71.4% of the MDR P. aeruginosa isolates. These results indicate that the presence of the exoU gene can be a predictive marker for the persistence of MDR P. aeruginosa isolates.

다제내성 P. aeruginosa의 출현과 확산은 전 세계적으로 중요한 문제가 되고 있다. P. aeruginosa에 의한 발병은 일부 몇몇 세포 관련 및 세포외 병독성 인자의 생성에 기인한다. 본 연구에서는 대전지역의 3차 병원에서 분리된 carbapenem 내성 P. aeruginosa를 대상으로 병독성 인자의 분포와 항균제 내성 양상을 조사하였다. 항균제 감수성 시험은 디스크 확산법으로 확인하였고, 병독성 유전자의 분석을 위해 PCR과 염기서열분석을 수행하였다. 또한, 다제내성 P. aeruginosa의 sequence type (ST)은 multilocus sequence typing (MLST)을 통해 확인하였다. 32균주의 carbapenem 내성 P. aeruginosa 중, 14균주(43.8%)가 다제내성이었으며, 주요 ST는 ST235 (10균주, 71.4.%)임을 확인하였다. 병독성 유전자는 32균주 모두에서 확인되었고, 이 중 가장 높은 빈도로 확인된 병독성 유전자는 toxA, plcN, phzM (100.%)이었다. 또한, 32균주는 모두 8개 이상의 병독성 유전자를 가지고 있었으며, 9균주(28.1%)가 15개의 병독성 유전자를 가지고 있었다. exoU 유전자는 다제내성 P. aeruginosa 균주의 71.4%에서 확인되었다. 이러한 결과는 exoU 유전자가 다제내성 P. aeruginosa 균주의 지속성에 대한 예측 표지자가 될 수 있을 것으로 사료된다.

Keywords

References

  1. Kumari H, Balasubramanian D, Zincke D, Mathee K. Role of Pseudomonas aeruginosa AmpR on ${\beta}$-lactam and non-${\beta}$-lactam transient cross-resistance upon pre-exposure to subinhibitory concentrations of antibiotics. J Med Microbiol. 2014;63:544-555. https://doi.org/10.1099/jmm.0.070185-0.
  2. Fazeli N, Momtaz H. Virulence gene profiles of multidrug-resistant Pseudomonas aeruginosa isolated from Iranian hospital infections. Iran Red Crescent Med J. 2014;16:E15722. http://dx.doi.org/10.5812/ircmj.15722.
  3. Strateva T, Yordanov D. Pseudomonas aeruginosa - a phenomenon of bacterial resistance. J Med Microbiol. 2009;58:1133-1148. https://doi.org/10.1099/jmm.0.009142-0.
  4. Lanotte P, Watt S, Mereghetti L, Dartiguelongue N, Rastegar-Lari A, Goudeau A, et al. Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins. J Med Microbiol. 2004;53:73-81.https://doi.org/10.1099/jmm.0.05324-0.
  5. Choy MH, Stapleton F, Willcox MD, Zhu H. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens- and non-contact lens-related keratitis. J Med Microbiol. 2008;57:1539-1546. https://doi.org/10.1099/jmm.0.2008/003723-0.
  6. Goldufsky J, Wood S, Hajihossainlou B, Rehman T, Majdobeh O, Kaufman HL, et al. Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer celllines. J Med Microbiol. 2015;64:164-173. https://doi.org/10.1099/jmm.0.000003.
  7. Wolska K, Szweda P. Genetic features of clinical Pseudomonas aeruginosa strains. Pol J Microbiol. 2009;58:255-260.
  8. Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect. 2006;36:78-91. https://doi.org/10.1016/j.medmal.2005.10.007.
  9. Haghi F, Zeighami H, Monazami A, Toutouchi F, Nazaralian S, Naderi G. Diversity of virulenc egenes in multidrug resistant Pseudomonas aeruginosa isolated from burn wound infections. Microb Pathog. 2018;115:251-256. https://doi.org/10.1016/j.micpath.2017.12.052.
  10. Makedou KG, Tsiakiri EP, Bisiklis AG, Chatzidimitriou M, Halvantzis AA, Ntoutsou K, et al. Changes in antibiotic resistance of the most common Gram-negative bacteria isolated in intensive care units. J Hosp Infect. 2005;60:245-248. https://doi.org/10.1016/j.jhin.2005.01.013.
  11. Tsukayama DT, van Loon HJ, Cartwright C, Chmielewski B, Fluit AC, van der Werken C, et al. The evolution of Pseudomonas aeruginosa during antibiotic rotation in a medical intensive care unit: the RADAR-trial. Int J Antimicrob Agents. 2004;24:339-345. https://doi.org/10.1016/j.ijantimicag.2004.04.011.
  12. Rossi Goncalves I, Dantas RCC, Ferreira ML, Batistao DWDF, Gontijo-Filho PP, Ribas RM. Carbapenem-resistant Pseudomonas aeruginosa: association with virulence genes and biofilm formation. Braz J Microbiol. 2017;48:211-217. https://doi.org/10.1016/j.bjm.2016.11.004.
  13. Khosravi Y, Tay ST, Vadivelu J. Analysis of integrons and associated gene cassettes of metallo-${\beta}$-lactamase-positive Pseudomonas aeruginosa in Malaysia. Med Microbiol. 2011;60:988-994. https://doi.org/10.1099/jmm.0.029868-0.
  14. Cho HH. Molecular analysis of carbapenem-resistant Pseudomonas aeruginosa isolated from patients hospitalized in Daejeon between 2008 and 2014 year. Korean J Clin Lab Sci. 2018;50:406-413. https://doi.org/10.15324/kjcls.2018.50.4.406.
  15. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement, M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.
  16. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18:268-281. https://doi.org/10.1111/j.1469-0691.2011.03570.x.
  17. Finnan S, Morrissey JP, O'Gara F, Boyd EF. Genome diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients and the hospital environment. J Clin Microbiol. 2004;42:5783-5792. https://doi.org/10.1128/JCM.42.12.5783-5792.2004.
  18. Khodayary R, Nikokar I, Mobayen MR, Afrasiabi F, Araghian A, Elmi A, et al. High incidence of type III secretion system associated virulence factors (exoenzymes) in Pseudomonas aeruginosa isolated from Iranian burn patients. BMC Res Notes. 2019;12:28. https://doi.org/ 10.1186/s13104-019-4071-0.
  19. Samuelsen O, Toleman MA, Sundsfjord A, Rydberg J, Leegaard TM, Walder M, et al. Molecular epidemiology of metallo-beta-lactamase-producing Pseudomonas aeruginosa isolates from Norway and Sweden shows import of international clones and local clonal expansion. Antimicrob Agents Chemother. 2010;54:346-352. http://doi.org/10.1128/AAC.00824-09.
  20. Cholley P, Thouverez M, Hocquet D, van der Mee-Marquet N, Talon D, Bertrand X. Most multidrug-resistant Pseudomonas aeruginosa isolates from hospitals in eastern France belong to a few clonal types. J Clin Microbiol. 2011;49:2578-2583. http://doi.org/10.1128/JCM.00102-11.
  21. Hong JS, Yoon EJ, Lee H, Jeong SH, Lee K. Clonal dissemination of Pseudomonas aeruginosa sequence type 235 isolates carrying $bla_{IMP-6}$ and emergence of $bla_{IMP-10}$ and blaIMP-10 on novel genomic islands PAGI-15 and -16 in South Korea. Antimicrob Agents Chemother. 2016;60:7216-7223. http://doi.org/10.1128/AAC.01601-16.
  22. Wi YM, Choi JY, Lee JY, Kang CI, Chung DR, Peck KR, et al. Emergence of colistin resistance in Pseudomonas aeruginosa ST235 clone in South Korea. Int J Antimicrob Agents. 2017;49:767-769. http://doi.org/10.1016/j.ijantimicag.2017.01.023.
  23. Kim D, Ahn JY, Lee CH, Jang SJ, Lee H, Yong D, et al. Increasing resistance to extended-spectrum cephalosporins, fluoroquinolone, and carbapenem in Gram-negative bacilli and the emergence of carbapenem non-susceptibility in Klebsiella pneumoniae: Analysis of Korean Antimicrobial Resistance Monitoring System (KARMS) data from 2013 to 2015. Ann Lab Med. 2017;37:231-239. https://doi.org/10.3343/alm.2017.37.3.231.
  24. Breidenstein EB, de la Fuente-Nunez C, Hancock RE. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 2011;19:419-426. http://doi.org/10.1016/j.tim. 2011. 04.005.
  25. Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever-present adversary. J Hosp Infect. 2009;73:338-344. http://doi.org/10.1016/j.jhin.2009.04.020.
  26. Badr RI, Nagdy M, Sabagh A, Din AB. Pseudomonas aeruginosa exotoxin A as a virulence factor in burn wound infections. Egypt J Med Microbiol. 2008;17:125-132.
  27. Cotar AI, Chifiriuc MC, Banu O, Lazar V. Molecular characterization of virulence patterns in Pseudomonas aeruginosa strains isolated from respiratory and wound samples. Biointerface Res Appl Chem. 2013;3:551-558.
  28. Georgescu M, Gheorghe I, Curutiu C, Lazar V, Bleotu C, Chifiriuc MC. Virulence and resistance features of Pseudomonas aeruginosa strains isolated from chronic leg ulcers. BMC Infect Dis. 2016;16:92. http://doi.org/10.1186/s12879-016-1396-3.
  29. Faraji F, Mahzounieh M, Ebrahimi A, Fallah F, Teymournejad O, Lajevardi B. Molecular detection of virulence genes in Pseudomonas aeruginosa isolated from children with cystic fibrosis and burn wounds in Iran. Microb Pathog. 2016;99:1-4. http://doi.org/10.1016/j.micpath.2016.07.013.
  30. Jamunadevi S, Balashanmugam P, Muralitharan G, Kalaichelvan PT. Molecular characterization of pathogenic and non-pathogenic Pseudomonas aeruginosa with special reference to phenazine gene. J Mod Biotechnol. 2012;1:70-74.
  31. Galle M, Carpentier I, Beyaert R. Structure and function of the type III secretion system of Pseudomonas aeruginosa. Curr Protein Pept Sci. 2012;13:831-842. https://doi.org/10.2174/138920312804871210
  32. Hauser AR. The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat Rev Microbiol. 2009;7:654-665. http://doi.org/10.1038/nrmicro2199.
  33. Allydice-Francis K, Brown PD. Diversity of antimicrobial resistance and virulence determinants in Pseudomonas aeruginosa associated with fresh vegetables. Int J Microbiol. 2012;2012:426241. http://doi.org/10.1155/2012/426241.
  34. Kruczek C, Kottapalli KR, Dissanaike S, Dzvova N, Griswold JA, Colmer-Hamood JA, et al. Major transcriptome changes accompany the growth of Pseudomonas aeruginosa in blood from patients with severe thermal injuries. PLoS One. 2016;11:E0149229. http://doi.org/10.1371/journal.pone.0149229.
  35. Islamieh DI, Afshar D, Esmaeili D. Effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in Pseudomonas aeruginosa. Iran J Microbiol. 2019;11:55-59.
  36. Andrejko M, Sieminska-Kuczer A, Janczarek M, Janik E, Bednarczyk M, Gagos M, et al. Pseudomonas aeruginosa alkaline protease exhibits a high renaturation capability. Acta Biochim Pol. 2019;66:91-100. http://doi.org/10.18388/abp.2018_2741.