Ann Lab Med.  2014 Jul;34(4):286-292. 10.3343/alm.2014.34.4.286.

Correlation Between Virulence Genotype and Fluoroquinolone Resistance in Carbapenem-Resistant Pseudomonas aeruginosa

• Cho HH
• Kwon KC
• Kim S
• Koo SH

• KMID: 1791936
• DOI: http://doi.org/10.3343/alm.2014.34.4.286

Abstract

BACKGROUND
Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains.
METHODS
Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE.
RESULTS
A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P< or =0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased.
CONCLUSIONS
The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.

Keyword

TTSS effector genotype; exoS; exoU; Fluoroquinolone resistance

MeSH Terms

ADP Ribose Transferases/genetics
Anti-Bacterial Agents/*pharmacology
Bacterial Proteins/genetics
Bacterial Toxins/genetics
Carbapenems/pharmacology
Drug Resistance, Bacterial/*drug effects
Fluoroquinolones/*pharmacology
Genotype
Humans
Microbial Sensitivity Tests
Multilocus Sequence Typing
Mutation
Pseudomonas aeruginosa/*genetics/isolation & purification/pathogenicity
Sputum/microbiology
Virulence
ADP Ribose Transferases
Anti-Bacterial Agents
Bacterial Proteins
Bacterial Toxins
Carbapenems
Fluoroquinolones

Reference

1. Shaver CM, Hauser AR. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun. 2004; 72:6969–6977. PMID: 15557619.

2. Galán JE, Collmer A. Type III secretion machines: bacterial devices for protein delivery into host cells. Science. 1999; 284:1322–1328. PMID: 10334981.

3. Wong-Beringer A, Wiener-Kronish J, Lynch S, Flanagan J. Comparison of type III secretion system virulence among fluoroquinolone-susceptible and -resistant clinical isolates of Pseudomonas aeruginosa. Clin Microbiol Infect. 2008; 14:330–336. PMID: 18190571.

4. Veesenmeyer JL, Hauser AR, Lisboa T, Rello J. Pseudomonas aeruginosa virulence and therapy: evolving translational strategies. Crit Care Med. 2009; 37:1777–1786. PMID: 19325463.

5. Frank DW. The exoenzyme S regulon of Pseudomonas aeruginosa. Mol Microbiol. 1997; 26:621–629. PMID: 9427393.

6. Sato H, Frank DW. ExoU is a potent intracellular phospholipase. Mol Microbiol. 2004; 53:1279–1290. PMID: 15387809.

7. Feltman H, Schulert G, Khan S, Jain M, Peterson L, Hauser AR. Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology. 2001; 147:2659–2669. PMID: 11577145.

8. Fleiszig SM, Wiener-Kronish JP, Miyazaki H, Vallas V, Mostov KE, Kanada D, et al. Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun. 1997; 65:579–586. PMID: 9009316.

9. Roy-Burman A, Savel RH, Racine S, Swanson BL, Revadigar NS, Fujimoto J, et al. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis. 2001; 183:1767–1774. PMID: 11372029.

10. Schulert GS, Feltman H, Rabin SD, Martin CG, Battle SE, Rello J, et al. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J Infect Dis. 2003; 188:1695–1706. PMID: 14639541.

11. El Solh AA, Akinnusi ME, Wiener-Kronish JP, Lynch SV, Pineda LA, Szarpa K. Persistent infection with Pseudomonas aeruginosa in ventilator-associated pneumonia. Am J Respir Crit Care Med. 2008; 178:513–519. PMID: 18467510.

12. Thomson CJ. The global epidemiology of resistance to ciprofloxacin and the changing nature of antibiotic resistance: a 10 year perspective. J Antimicrob Chemother. 1999; 43(Suppl A):31–40. PMID: 10225569.

13. Karlowsky JA, Draghi DC, Jones ME, Thornsberry C, Friedland IR, Sahm DF. Surveillance for antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from hospitalized patients in the United States, 1998 to 2001. Antimicrob Agents Chemother. 2003; 47:1681–1688. PMID: 12709340.

14. Lee KW, Kim MY, Kang SH, Kang JO, Kim EC, Choi TY, et al. Korean nationwide surveillance of antimicrobial resistance in 2000 with special reference to vancomycin resistance in enterococci, and expanded-spectrum cephalosporin and imipenem resistance in gram-negative bacilli. Yonsei Med J. 2003; 44:571–578. PMID: 12950110.

15. Lee K, Jang SJ, Lee HJ, Ryoo N, Kim M, Hong SG, et al. Increasing prevalence of vancomycin-resistant Enterococcus faecium, expanded-spectrum cephalosporin-resistant Klebsiella pneumoniae, and imipenem-resistant Pseudomonas aeruginosa in Korea: KONSAR study in 2001. J Korean Med Sci. 2004; 19:8–14. PMID: 14966334.

16. Lee JY, Ko KS. OprD mutations and inactivation, expression of efflux pumps and AmpC, and metallo-β-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from South Korea. Int J Antimicrob Agents. 2012; 40:168–172. PMID: 22633564.

17. Ruiz J. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother. 2003; 51:1109–1117. PMID: 12697644.

18. Valdezate S, Vindel A, Echeita A, Baquero F, Cantó R. Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility. Antimicrob Agents Chemother. 2002; 46:665–671. PMID: 11850246.

19. Mouneimné H, Robert J, Jarlier V, Cambau E. Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1999; 43:62–66. PMID: 9869566.

20. Vila J, Ruiz J, Goñi P, Jimenez de Anta T. Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. J Antimicrob Chemother. 1997; 39:757–762. PMID: 9222045.

21. Sáenz Y, Zarazaga M, Briñas L, Ruiz-Larrea F, Torres C. Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans and animals. J Antimicrob Chemother. 2003; 51:1001–1005. PMID: 12654733.

22. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: Twentieth Informational supplement, M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute;2010.

23. Agnello M, Wong-Beringer A. Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa. PLoS One. 2012; 7:e42973. PMID: 22905192.

24. Lee JK, Lee YS, Park YK, Kim BS. Alterations in the GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subunits of topoisomerase IV in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents. 2005; 25:290–295. PMID: 15784307.

25. Gasink LB, Fishman NO, Weiner MG, Nachamkin I, Bilker WB, Lautenbach E. Fluoroquinolone-resistant Pseudomonas aeruginosa: assessment of risk factors and clinical impact. Am J Med. 2006; 119:526.e19–526.e25. PMID: 16750968.

26. Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother. 2000; 44:710–712. PMID: 10681343.

27. Holder IA, Neely AN, Frank DW. Type III secretion/intoxication system important in virulence of Pseudomonas aeruginosa infections in burns. Burns. 2001; 27:129–130. PMID: 11226648.

28. Kulasekara BR, Kulasekara HD, Wolfgang MC, Stevens L, Frank DW, Lory S. Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa. J Bacteriol. 2006; 188:4037–4050. PMID: 16707695.

29. Hauser AR, Cobb E, Bodi M, Mariscal D, Vallés J, Engel JN, et al. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit Care Med. 2002; 30:521–528. PMID: 11990909.

30. Hsu DI, Okamoto MP, Murthy R, Wong-Beringer A. Fluoroquinolone-resistant Pseudomonas aeruginosa: risk factors for acquisition and impact on outcomes. J Antimicrob Chemother. 2005; 55:535–541. PMID: 15728150.

31. Cho HH, Kwon KC, Sung JY, Koo SH. Prevalence and genetic analysis of multidrug-resistant Pseudomonas aeruginosa ST235 isolated from a hospital in Korea, 2008-2012. Ann Clin Lab Sci. 2013; 43:414–419. PMID: 24247798.

32. Lee JY, Peck KR, Ko KS. Selective advantages of two major clones of carbapenem-resistant Pseudomonas aeruginosa isolates (CC235 and CC641) from Korea: antimicrobial resistance, virulence and biofilm-forming activity. J Med Microbiol. 2013; 62:1015–1024. PMID: 23558139.

33. Matsumoto M, Shigemura K, Shirakawa T, Nakano Y, Miyake H, Tanaka K, et al. Mutations in the gyrA and parC genes and in vitro activities of fluoroquinolones in 114 clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections and their rapid detection by denaturing high-performance liquid chromatography. Int J Antimicrob Agents. 2012; 40:440–444. PMID: 22884856.

34. Hrabák J, Cervená D, Izdebski R, Duljasz W, Gniadkowski M, Fridrichová M, et al. Regional spread of Pseudomonas aeruginosa ST357 producing IMP-7 metallo-β-lactamase in Central Europe. J Clin Microbiol. 2011; 49:474–475. PMID: 20980582.

35. Nakano M, Deguchi T, Kawamura T, Yasuda M, Kimura M, Okano Y, et al. Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1997; 41:2289–2291. PMID: 9333065.

36. Higgins PG, Fluit AC, Milatovic D, Verhoef J, Schmitz FJ. Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents. 2003; 21:409–413. PMID: 12727072.

37. Akasaka T, Tanaka M, Yamaguchi A, Sato K. Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob Agents Chemother. 2001; 45:2263–2268. PMID: 11451683.

38. Oh H, Stenhoff J, Jalal S, Wretlind B. Role of efflux pumps and mutations in genes for topoisomerase II and IV in fluoroquiniolone-resistant Pseudomonas aeruginosa strains. Microb Drug Resist. 2003; 9:323–328. PMID: 15000738.

39. Shultz TR, Tapsall JW, White PA. Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Antimicrob Agents Chemother. 2001; 45:734–738. PMID: 11181352.

40. Parry CM, Thuy CT, Dongol S, Karkey A, Vinh H, Chinh NT, et al. Suitable disk antimicrobial susceptibility breakpoints defining Salmonella enterica serovar Typhi isolates with reduced susceptibility to fluoroquinolones. Antimicrob Agents Chemother. 2010; 54:5201–5208. PMID: 20837759.