Hastane kaynaklı enterokok izolatlarının pulsed-field jel elektroforezis yöntemiyle moleküler tiplendirilmesi

Yıl 2015, Cilt: 72 Sayı: 1, 1 - 10, 01.03.2015

Dilek Güldemir Alper Karagöz Tuba Dal Alicem Tekin Tuncer Özekinci Rıza Durmaz

Öz

Amaç: Enterokoklar hastaneden kaynaklanan üriner sistem ve yara enfeksiyonlarının ikinci, bakteriyemilerin ise üçüncü en sık nedenidir. Günümüzde, özellikle vankomisine dirençli enterokok VRE suşları nozokomiyal enfeksiyonlara sebep olarak morbidite, mortalite ve tedavi maliyetlerini artıran önemli patojenlerdir. Hastane kaynaklı VRE salgınlarının önlenmesi, kontrolü ve epidemiyolojik analizlerinin yapılması son derece önem taşımaktadır. Pulsed-field jel elektroforezis PFGE yöntemi enterokokal enfeksiyonların moleküler epidemiyolojik analizinde “altın standart” olarak kabul edilmektedir. Bu çalışmanın amacı, hastane kaynaklı enterokok izolatları arasındaki klonal ilişkiyi belirlemek ve olası çapraz bulaşı ortaya koymaktır. Yöntem: Kasım 2010 - Haziran 2012 tarihleri arasında Dicle Üniversitesi Tıp Fakültesi DÜTF Hastanesinin çeşitli kliniklerinde yatan ve hastane kaynaklı enfeksiyon tanısı alan hastalardan izole edilen 36 enterokok suş çalışıldı. 36 suşun 18’i idrar, altısı kan, beşi bronkoalveolar lavaj BAL , beşi yara, biri vajinal sürüntü ve biri ise kataterden izole edilmiş olup, dokuzu VRE olarak tanımlanmıştır. İzolasyon ve tanımlanan DÜTF Tıbbi Mikrobiyoloji Anabilim Dalı laboratuvarlarında gerçekleştirildi. Tanımlama için konvansiyonel yöntemler ve BD PhoenixTM 100 Becton Dickinson, MD, USA tam otomatik mikrobiyoloji sistemi kullanıldı. Enterococcus spp. suşlarının tam otomatik mikrobiyoloji sistemi ile Klinik ve Laboratuvar Standartlar Enstitüsü CLSI önerilerine uygun olarak antibiyotik duyarlılıkları belirlendi. İzolatların vankomisin duyarlılıkları E-test yöntemi ile de çalışıldı. Kalite kontrolü için Staphylococcus aureus ATCC 25923 suşu kullanıldı. Enterokok izolatları aralarındaki çapraz bulaş oranlarını belirlemek için PFGE çalışıldı. PFGE çalışması Türkiye Halk Sağlığı Kurumu, Ulusal Moleküler Mikrobiyoloji Referans Merkez Laboratuvarı’nda gerçekleştirildi. Kromozomal DNA; SmaI enzimi ile kesildi. DNA restriksiyon fragmanları CHEF DR II Bio-Rad Laboratories, Nazareth, Belgium sistemi kullanılarak gösterildi. İzolatlar arasındaki klonal ilişki, BioNumerics Software Program Applied Maths, Sinth-MartensLatem, Belgium kullanılarak oluşturulan dendogram ile değerlendirildi. Bulgular: PFGE yöntemiyle izolatların DNA fragment paternleri elde edildi ve bu paternlerin dendogramı yapıldı. DNA paternlerinin birebir karşılaştırılması sonucu 26 Enterococcus faecium suşunun dört küme ve bir ana grup, 10 Enterococcus faecalis suşunun ise üç küme ve bir majör grup oluşturduğu saptandı. 26 E. faecium izolatının, %97 benzerlikle ortak aynı grupta yer aldıkları görüldü. Kümeleşme oranı %77 20/26 olup, kümelerin 2-14 arasında suş içerdiği belirlendi. Sonuç: Bu çalışmada, Dicle Üniversitesi Tıp Fakültesi Hastanesinde “Kasım 2010 - Haziran 2012” tarihleri arasında hastane kaynaklı enfeksiyona neden olan enterokok suşları arasında çapraz bulaş oranının yüksek olduğu gösterilmiştir. Çalışmanın sonuçları, enfeksiyon kontrol programlarının daha etkin biçimde uygulanmasının önemini ortaya koymuştur.

Anahtar Kelimeler

Enterokok, pulsed-field jel elektroforezis, hastane enfeksiyonları klonal ilişki

Molecular typing of nosocomial enterococci by pulsed-field gel electrophoresis

Öz

Objective: Enterococci are the second most common cause of nosocomial urinary tract and wound infections, also third most common cause of nosocomial bacteremia. Currently, especially vancomycin-resistant enterococci VRE are one of the significant pathogens that cause nosocomial infections and increase mortality, morbidity, and healthcare costs. Therefore prevention and control of the nosocomial VRE outbreaks and epidemiological analysis of the infection are important. Pulsed-Field Gel Electrophoresis PFGE is accepted as a “gold standard” method in the molecular epidemiological analysis of enterococcal infections. The aims of this study are to determine the clonal relationship among the nosocomial enterococcal isolates and the rate of cross-contamination between them. Method: Thirty-six Enterococcus strains isolated from hospitalized patients with nosocomial infection in different clinics of Dicle University Hospital between November 2010 and June 2012 were included in this study. A total of 36 isolates were obtained from various clinical samples including urine n=18 , blood n=6 , bronchoalveolar lavage BAL fluid n=5 , wound biopsy sample n=5 , vaginal smear n=1 and catheter tip n=1 . Nine of the thirty-six isolates were VRE. Isolation and identification of the isolates were conducted in the bacteriology laboratories of Dicle University Medical Faculty, Department of Medical Microbiology. The conventional methods and BD PhoenixTM 100 Becton Dickinson, MD, USA fully automated microbiology system were used for the identification. Antimicrobial susceptibilities of enterococcal strains were determined by a fully automated microbiology system according to the Clinical and Laboratory Standards Institute CLSI . In addition, vancomycin susceptibilities of the isolates were performed by E-test method. Staphylococcus aureus ATCC 25923 strain was used for quality control. PFGE was studied for determining of rate of cross-contamination. PFGE was performed in National Molecular Microbiology Referance Laboratory, Public Health Institution of Turkey PHIT . DNA restriction fragments were obtained by cutting bacterial DNA with the SmaI enzyme. DNA restriction fragments were used by CHEF DR II Bio-Rad Laboratories, Nazareth, Belgium system. PFGE results were evaluated by Bionumerics software system version 6.01; Applied Maths, Sint-Martens-Latem, Belgium . Results: DNA patterns of the isolates were obtained by PFGE, and dendrogram of DNA patterns were made. Comparison of DNA patterns obtained by PFGE showed that 26 E. faecium strains were divided into four different clusters and one major group, 10 E. faecalis strains were three clusters and one major group. Twenty-six Enterococcus faecium isolates were involved in a joint cluster 97% similarity . The cluster rate was found to be 77% 20/26 , number of E. faecium strains in each cluster ranged from 2 to 14 strains. Conclusion: In this study, cross-contamination was highlighted among enterococcal strains causing nosocomial infections in Dicle University Hospital between November 2010 and June 2012. Our data revealed that more effective infection control programs should be implemented to prevent cross-transmission.

Anahtar Kelimeler

Enterococcus, pulsed-field gel electrophoresis, nosocomial infections, clonal relationship

Kaynakça

• 1. Moellering RC. Enterococcus species, Streptococcus bovis and Leuconostoc species. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases. Fifth edition. New York. Churchill Livingstone, 2005: 2411-21.
• 2. Eliopoulos GM. Vancomycin-resistant enterococci. Mechanism and clinical relevance. Infect Dis Clin North Am, 1997; 11 (4): 851-65.
• 3. Papanicolaou GA, Meyers BR, Meyers J, Mendelson MH, Lou W, Emre S, et al. Nosocomial infections with vancomycin-resistant Enterococcus faeciumin liver transplant recipients: risk factors for acquisition and mortality. Clin Infect Dis, 1996; 23 (4): 760-6.
• 4. Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbiol etiology of nosocomial infections. Am J Med, 1991; 91(Suppl B): 72-5.
• 5. Yüce A, Karaman M, Gülay Z, Yulug N. Yeni doğanlarda vankomisin dirençli enterokokların fekal taşıyıcılığı. ANKEM, 1999; 13: 7-11.
• 6. Linden PK, Pasculle AW, Manez R, Kramer DJ, Fung JJ, Pinna AD, et al. Differences in outcomes for patients with bacteremia due to vancomycinresistant Enterococcus faecium or vancomycinsusceptible E. faecium. Clin Infect Dis, 1996; 22 (4): 663-70.
• 7. Woodford N, Johnson AP, Morrison D, Speller DC. Current perspectives on glycopeptide resistance. Clin Microbiol Rev, 1995; 8 (4): 585-615.
• 8. Murray BE. Vancomycin-resistant enterococcal infections. N Eng J Med, 2000; 342: 710-21.
• 9. English BK, Shenep JL. Enterococcal and viridans streptococcal infections. In: Feigin RD, Cherry JD, eds. Textbook of Pediatric Infectious Diseases. 5th edition. Philedelphia: WB Saunders Company, 2004: 1175-92.
• 10. Uttley AH, Collins CH, Naidoo J, George RC. Vancomycin-resistant enterococci. Lancet, 1988; 1 (8575-6): 57-8.
• 11. Leclercq R, Derlot E, Duval J, Courvalin P. Plasmidmediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med, 1988; 319 (3): 157-61.
• 12. Woodford N. Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci. Microb Drug Resist, 2001; 7: 229-34.
• 13. Anonymous. Centers for Disease Control and Prevention. Nosocomial enterococci resistant to vancomycin in the United States, 1989-1993. MMWR 1993;42:597-9.
• 14. Vural T, Şekercioğlu AO, Öğünç D, Gültekin M, Çolak D, Yeşilipek A, ve ark. Vankomisine dirençli Enterococcus casseliflavus suşu. ANKEM, 1998; 12 (2): 113.
• 15. Basustaoglu A, Aydogan H, Beyan C, Yalcin A, Unal SFirst glycopeptide-resistant Enterococcus faecium isolate from blood culture in Ankara, Turkey. Emerg Infect Dis, 2001; 7: 160-1.
• 16. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989.
• 17. Top J, Schouls LM, Bonten MJM, Willems RJ. Multiple-locus variable-number tandem repeat analysis, a novel typing scheme to study the genetic relatedness and epidemiology of Enterococcus faecium isolates. J Clin Microbiol, 2004; 42 (10): 4503-11.
• 18. Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, et al. Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol, 2002; 40: 1963-71.
• 19. Kühn I, Burman LG, Haeggman S, Tullus K, Murray BE. Biochemical fingerprinting compared with ribotyping and pulsed-field gel electrophoresis of DNA for epidemiological typing of enterococci. J Clin Microbiol, 1995; 33: 2812-7.
• 20. 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: 24-7.
• 21. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol, 1995; 33 (9): 2233-9.
• 22. Cetinkaya Y, Falk P, Mayhall CG. Vancomycinresistant enterococci. Clin Microbiol Rev, 2000; 13: 686-707.
• 23. Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC definitions for nosocomial infections, 1988. Am J Infect Control, 1988; 16(3): 128-40.
• 24. Mulvey MR, Chui L, Ismail J, Louie L, Murphy C, Chang N, et al. Development of a Canadian standardized protocol for subtyping methicillinresistant Staphylococcus aureus using pulsed-field gel electrophoresis. J Clin Microbiol, 2001; 39 (10): 3481-5.
• 25. Esel D, Doganay M, Alp E, Sumerkan B. Prospective evaluation of blood cultures in a Turkish university hospital: epidemiology, microbiology and patient outcome. Clin Microbiol Infect, 2003; 9(10): 1038-44.
• 26. Leblebicioglu H, Esen S. Turkish nosocomial urinary tract infection study group. hospital-acquired urinary tract infections in Turkey: a nationwide multicenter point prevalence study. J Hosp Infect, 2003; 53 (3): 207-10.
• 27. Arslan U, Demir E, Oryaşin E, Türk Dağı H, Tuncer I, Fındık D, et al. MLST types of vancomycin-resistant Enterococcus faecium strains isolated from blood cultures. Mikrobiyol Bul, 2013; 47 (3): 432-41.
• 28. Akan O, Kanra G, Ecevit Z, Ceyhan M, Seçmeer G, Berkman E. Antibiotic susceptibilities of enterococci isolated from Turkish children. Turk J Pediatr, 1997; 39 (1): 13-7.
• 29. Siegel JD, Rhinehart E, Jackson M, Chiarello L, Healthcare infection control practices advisory committee. management of multidrug-resistant organisms in health care settings, 2006. Am J Infect Control, 2007; 35 (Suppl 2): S165-93.
• 30. Hayden MK. Insights into the epidemiology and control of infection with vancomycin-resistant enterococci. Clin Infect Dis, 2000; 31: 1058-65.
• 31. Mato R, de Lencastre H, Roberts RB, Tomasz A. Multiplicity of genetic back grounds among vancomycin-resistant Enterococcus faecium isolates recovered from an outbreak in a New York City Hospital. Microbiol Drug Res, 1996; 2: 309-17.
• 32. Goossens H, Jabes D, Rossi R, Lammens C, Privitera G, Courvalin P. European survey of vancomycinresistant enterococci in at-risk hospital wards and in vitro susceptibility testing of ramoplanin against these isolates. J Antimicrob Chemother, 2003; 51 Suppl 3: iii5-12.
• 33. Çolak D, Naas T, Gunseren F, Fortineau N, Ogunc D, Gultekin M, et al. First outbreak of vancomycinresistant enterococci in a tertiary hospital in Turkey. J Antimicrob Chemother, 2002; 50: 397- 401.
• 34. Comert FB, Kulah C, Aktas E, Ozlu N, Celebi G. First isolation of vancomycin-resistant enterococci and spread of a single clone in a university hospital in northwestern Turkey. Eur J Clin Microbiol Infect Dis, 2007; 26: 57-61.
• 35. Rudy M, Zientara M, Bek T, Martirosian G. Occurrence of antibiotic resistant enterococci in clinical specimens from a pediatric hospital. Pol J Microbiol, 2005; 54 (1): 77-80.
• 36. Kuzucu C, Cizmeci Z, Durmaz R, Durmaz B, Ozerol IH. The prevalence of fecal colonization of enterococci, the resistance of the isolates to ampicillin, vancomycin, and high-level aminoglycosides, and the clonal relationship among isolates. Microb Drug Resist, 2005; 11 (2): 159-64.
• 37. Abele-Horn M, Vogel U, Klare I, Konstabel C, Trabold R, Kurihara R, et al. Molecular epidemiology of hospital-acquired vancomycinresistant enterococci. J Clin Microbiol, 2006; 44 (11): 4009-13.
• 38. Top J, Banga NM, Hayes R, Willems RJ, Bonten MJ, Hayden MK. Comparison of multiple-locus variablenumber tandem repeat analysis and pulsed-field gel electrophoresis in a setting of polyclonal endemicity of vancomycin-resistant Enterococcus faecium. Clin Microbiol Infect, 2008; 14 (4): 363-9.
• 39. Bourdon N, Fines-Guyon M, Thiolet JM, Maugat S, Coignard B, Leclercq R, et al. Changing trends in vancomycin-resistant enterococci in French hospitals, 2001-08. J Antimicrob Chemother, 2011; 66 (4): 713-21.
• 40. Böhme H, Königsmark C, Klare I, Zischka M, Werner G. Cross-transmission rates of enterococcal isolates among newborns in a neonatal intensive care unit. Pediatr Rep, 2012; 4(1): e15.