Jülide Sedef GÖÇMEN, Şükran SABA ÇOPUR, Fikret ŞAHİN

Determination of virulence and multidrug resistance genes with polymerase chain reaction method in vancomycin-sensitive and -resistant enterococci isolated from clinical samples

Background/aim: Enterococci play an important role in nosocomial infections. Therefore, this study investigates multidrug resistance (MDR)1 gene areas in the pathogenicity of enterococci and virulence genes in both vancomycin-sensitive enterococci (VSE) and vancomycin-resistant enterococci (VRE) strains. Materials and methods: Virulence genes and MDR genes of enterococci were investigated by polymerase chain reaction (PCR). Results: We evaluated a total of 116 isolates, 93 being VRE and 23 being VSE. In this study, 95.6% of VRE (n = 93) were Enterococcus faecium (n = 89) and 4.3% were E. faecalis (n = 4), while 17.4% of VSE (n = 23) were E. faecium (n = 4) and 82.6% were E. faecalis (n = 19). The vanA MDR1 gene was detected in all VRE isolates. Among virulence genes, esp and hyl were detected in E. faecium, an enterococcus with the highest resistance to vancomycin, and gelE was detected in E. faecalis, an enterococcus with the highest sensitivity to vancomycin. Three or more virulence genes were identified only in VSE strains. We consider that it is a significant result that VSE had more virulence genes than VRE. Only esp was seen in VRE E. faecium strains. Conclusion: This study includes experimental results on the association of virulence characteristics in VRE and VSE strains.

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1. Gültekin M. Enterococci: microbiology, epidemiology and pathogenesis. In: Ulusoy S, Usluer G, Ünal S, editors. GramPositive Bacterial Infections. 2nd ed. Ankara, Turkey: Scientific Medical Publisher; 2012 (in Turkish).
2. Uttley AH, Collins CH, Naidoo J, George RC. Vancomycinresistant enterococci. Lancet 1988; 1: 57-58.
3. Vural T, Şekercioğlu AO, Ögünç D, Gültekin M, Çolak D, Yeşilipek A, Ünal S, Kocagöz S, Mutlu G. Vankomisine dirençli Enterococcus faecium suşu. ANKEM Derg 1999; 13: 1-4 (in Turkish).
4. Sava IG, Heikens E, Huebner J. Pathogenesis and immunity in enterococcal infections. Clin Microbiol Infect 2010; 16: 533- 540.
5. Kayaoğlu G, Orstavik D. Virulence factors of Enterococcus faecalis: relationship to endodontic disease. Crit Rev Oral Biol Med 2004; 15: 308-320.
6. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplements. CLSI Document M100- S19. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2012.
7. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995; 33: 24-27.
8. Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J Clin Microbiol 2004; 42: 4473-4479.
9. Panesso D, Reyes J, Rincon S, Diaz L, Galloway-Pena J, Zurita J, Carrillo C, Merentes A, Guzman M, Adachi JA et al. Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals. J Clin Microbiol 2010; 48: 1562-1569.
10. Turkish Ministry of Health. Antimicrobial Resistance Rates. National Nosocomial Infections Surveillance Study: 2011-2012 Statistics. Ankara, Turkey: Ministry of Health; 2013. Available online at http//www.uhes.saglik.gov.tr.
11. Turkish Ministry of Health. Antimicrobial Resistance Rates. National Nosocomial Infections Surveillance Study: 2008- 2009-2010 Statistics. Ankara, Turkey: Ministry of Health; 2013. Available online at http//www.uhes.saglik.gov.tr.
12. Aygün H, Memikoğlu OK, Tekeli A, Azap A, Yörük F. Hastanede yatan riskli hasta gruplarında vankomisin dirençli enterokok kolonizasyonunun surveyansı. Turk J Anaesth Reanim 2008; 36: 168-173 (in Turkish).
13. Aral M, Paköz NİE, Aral İ, Doğan S. Antibiotic resistance of Enterococcus faecalis and Enterococcus faecium strains isolated from various clinical samples. Turk Bull Hyg Exp Biol 2011; 68: 85-92 (in Turkish with English abstract).
14. Protonotariou E, Dimitroulia E, Pournaras S, Pitiriga V, Sofianou D, Tsakris A. Trends in antimicrobial resistance of clinical isolates of Enterococcus faecalis and Enterococcus faecium in Greece between 2002 and 2007. J Hosp Infect 2010; 75: 225-227.
15. Mendes RE, Woosley LN, Farrell DJ, Sader HS, Jones RN. Oritavancin activity against vancomycin-susceptible and vancomycin-resistant enterococci with molecularly characterized glycopeptide resistance genes recovered from bacteremic patient, 2009-2010. Antimicrob Agents Chemother 2012; 56: 1639-1642.
16. Wang JT, Chang SC, Wang HY, Chen PC, Shiau YR, Lauderdale TL. High rates of multidrug resistance in Enterococcus faecalis and E. faecium isolated from inpatient and outpatient in Taiwan. Diagn Microbiol Infect Dis 2013; 75: 406-411.
17. Altun B, Cengiz AB, Kara A, Ceyhan M, Ünal S, Seçmeer G, Gür D. First vancomycin-resistant blood isolate of Enterococcus faecium in a childrens hospital and molecular analysis of the mechanism of resistance. Turk J Pediatr 2008; 50: 554-558.
18. Yiş R, Aslan S, Çıtak Ç, Değirmenci S. Evaluation of vancomycin-resistant Enterococcus colonization at Gaziantep Childrens Hospital, Turkey. Mikrobiyol Bul 2011; 45: 646-654 (in Turkish with English abstract).
19. McCracken M, Wong A, Mitchell R, Gravel D, Conly J, Embil J, Johnston L, Matlow A, Ormiston D, Simor AE et al. Molecular epidemiology of vancomycin-resistant enterococcal bacteraemia: results from the Canadian Nosocomial Infection Surveillance Program 1999-2009. J Antimicrob Chemother 2013; 68: 1505-1509.
20. Cakirlar FK, Samasti M, Baris I, Kavakli H, Karakullukcu A, Sirekbasan S, Bagdatli Y. The epidemiological and molecular characterization of vancomycin-resistant enterococci isolated from rectal swab samples of hospitalized patient in Turkey. Clin Lab 2014, 60: 1807-1812.
21. Hasani A, Sharifi Y, Ghotaslou R, Naghili B, Hasani A, Aghazadeh M, Milani M, Bazmai A. Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran. Indian J Med Microbiol 2012; 30: 175-181.
22. Sharifi Y, Hasani A, Ghotaslou R, Varshochi M, Hasani A, Aghazadeh M, Milani M. Survey of virulence determinants among vancomycin resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens of hospitalized patients of North west of Iran. Open Microbiol J 2012; 6: 34-39.
23. Ira P, Sujatha S, Chandra PS. Virulence factors in clinical and commensal isolates of Enterococcus species. Indian J Pathol Microbiol 2013; 56: 24-30.
24. Billström H, Lund B, Sullivan A, Nord CE. Virulence and antimicrobial resistance in clinical Enterococcus faecium. Int J Antimicrob Agents 2008; 32: 374-377.
25. Woodford N, Soltani M, Hardy KJ. Frequency of esp in Enterococcus faecium isolates. Lancet 2001; 358: 584.
26. Lopes-Salas P, Llaca-Diaz J, Morfin-Otero R, Tinoco JC, Rodriguez-Noriega E, Salcido-Gutierres L, Gonzales GM, Mendoza-Olazaran S, Garza-Gonzales E. Virulence and antibiotic resistance of Enterococcus faecalis clinical isolates recovered from three states of Mexico. Detection of linezolid resistance. Arch Med Res 2013; 44: 422-428.
27. Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, Klare I, Nallapareddy SR, Huang W, Murray BE. A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. J Infect Dis 2003; 187: 508-512.
28. Comerlato CB, Resende MC, Caierao J, dAzevedo PA. Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin. Mem Inst Oswaldo Cruz 2013; 108: 590-595.
29. Baylan O, Nazik H, Bektöre B, Çitil BE, Turan D, Öngen B, Özyurt M, Açıkel CH, Haznedaroğlu T. The relationship between antibiotic resistance and virulence factors in urinary Enterococcus isolates. Mikrobiyol Bul 2011; 45: 430-445 (in Turkish with English abstract).
30. Sun H, Wang H, Xu Y, Jones RN, Costello AJ, Liu Y, Li G, Chen M, Mendes RE. Molecular characterization of vancomycinresistant Enterococcus spp. clinical isolates recovered from hospitalized patients among several medical institutions in China. Diagn Microbiol Infect Dis 2012; 74: 399-403.
31. Chang CM, Wang LR, Lee HC, Lee NY, Wu CJ, Ko WC. Characterisation of vancomycin-resistant enterococci from hospitalised patients at a tertiary centre over a seven-year period. J Hosp Infect 2010; 74: 377-384.
32. Al-Talib H, Zuraina N, Kamarudin B, Yean CY. Genotypic variations of virulent genes in Enterococcus faecium and Enterococcus faecalis isolated from three hospitals in Malaysia. Adv Clin Exp Med 2015; 24: 121-127.
33. Kang M, Xie Y, He C, Chen ZX, Guo L, Yang Q, Liu JY, Du Y, Ou QS, Wang LL. Molecular characteristics of vancomycinresistant Enterococcus faecium from a tertiary care hospital in Chengdu, China: molecular characteristics of VRE in China. Eur J Clin Microbiol Infect Dis 2014; 33: 933-939.
34. Bourdon N, Fines-Guyon M, Thiolet JM, Maugat S, Coignard B, Leclercq R, Cattoir V. Changing trends in vancomycinresistant enterococci in French hospitals, 2001-08. J Antimicrob Chemother 2011; 66: 713-721.
35. Thierfelder C, Keller PM, Kocher C, Gaudenz R, Hombach M, Bloemberg GV, Ruef C. Vancomycin-resistant Enterococcus. Swiss Med Wkly 2012; 142: 13540.
36. Camargo IL, Gilmore MS, Darini AL. Multilocus sequence typing and analysis of putative virulence factors in vancomycinresistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil. Clin Microbiol Infect 2006; 12: 1123-1130.
37. Palazzo IC, Pitondo-Silva A, Levy CE, da Costa Darini AL. Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil. J Hosp Infect 2011; 79: 70-74.
38. Khan MA, van der Wal M, Farrell DJ, Cossins L, van Belkum A, Alaidan A, Hays JP. Analysis of vanA vancomycin-resistant Enterococcus faecium isolates from Saudi Arabian hospitals reveals the presence of clonal cluster 17 and two new Tn1546 lineage types. J Antimicrob Chemother 2008; 62: 279-283.
39. Udo EE, Al-Sweih N. Frequency of virulence-associated genes in Enterococcus faecalis isolated in Kuwait hospitals. Med Princ Pract 2011; 20: 259-264.
40. Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, Baldassarri L. Survey for virulence determinants among Enterococcus faecalis isolated from different sources. J Med Microbiol 2004; 53: 13-20.
41. Gülhan T, Aksakal A, Ekin İH, Savaşan S, Boynukara B. Virulence factors of Enterococcus faecium and Enterococcus faecalis strains isolated from humans and pets. Turk J Vet Anim Sci 2006; 30: 477-482.
42. Song JY, Cheong HJ, Seo YB, Kim IS, Heo JY, Noh JY, Choi WS, Kim WJ. Clinical and microbiological characteristics of vancomycin-resistant enterococci with the vanD phenotype and vanA genotype. Jnp J Infect Dis 2013; 66: 1-5.
43. Brilliantova AN, Kliasova GA, Mironova AV, Tishkov VI, Novichkova GA, Bobrynina VO, Sidorenko SV. Spread of vancomycin-resistant Enterococcus faecium in two haematological centres in Russia. Int J Antimicrob Agents 2010; 35: 177-181.