Aysegul GOZALAN, Özlem ÜNALDI, Fisun KIIRCA, Nilay ÇÖPLÜ, Tuba MUDERRİS, Ziya Cibali ACİKGOZ, Rıza DURMAZ

Yoğun bakım ünitelerinde kan dolaşımı enfeksiyonu etkeni karbapenem dirençli Acinetobacter baumannii izolatlarının moleküler yöntemlerle karakterizasyonu

Amaç: Bu çalışmada; yoğun bakım ünitelerinde takip edilen hastaların kan örneklerinden izole edilen karbapenem dirençli Acinetobacter baumannii izolatları arasındaki klonal ilişki ve karbapenem direnç genlerinin moleküler yöntemler ile araştırılması amaçlandı. Yöntem: Kan kültürü şişelerinden bakteri izolasyonu için Bactec 9240 sistemi Becton Dickinson, ABD kullanıldı. Çalışmaya; konvansiyonel testler, API 20NE bioMèrieux, Fransa ve Phoenix TM 100 sistemi Becton Dickinson, ABD ile tanımlanan ve blaOXA-51 gen varlığı gösterilerek doğrulanan 112 A. baumannii suşu dahil edildi. Antimikrobiyal duyarlılık testleri Kirby-Bauer disk difüzyon yöntemi ve Phoenix TM 100 sistemi ile gerçekleştirildi. Karbapenem direnç genleri; blaOXA-23, blaOXA-48, blaOXA-58, blaIMP, blaVIM ve blaNDM-1 Multipleks Polimeraz Zincir Reaksiyonu PZR yöntemi ile araştırıldı. Acinetobacter baumannii suşları arasındaki klonal ilişkinin belirlenmesi için Pulse Field Jel Elektroforezi PFGE yöntemi kullanıldı. Bulgular: Suşların antibiyotik direnç yüzdeleri gentamisin, amikasin, tobramisin, netilmisin, seftazidim, trimetoprim/sulfametoksazol, piperasilin, siprofloksasin, ampisilin/sulbaktam, piperasilin/ tazobaktam ve sefoperazon/sulbaktam için sırasıyla %88; %81; %78; %36; %98,5; %96; %89; %100; %100; %93; %91 olarak bulundu. İmipenem ve meropenem MİK değerleri tüm grupta ≥8 µg/mL’nin üzerinde saptandı. Çalışmaya alınan izolatların tümünde blaOXA-51 ve blaOXA-23 gen varlığı tespit edildi. PFGE yöntemi ile 62 farklı pulsotip saptandı. Pulsotiplerden 19 tanesi birbiriyle ayırt edilemez profil gösteren eşittir üzeri ≥2 suş içermekteydi. Toplam 108 %96,4 suşun benzerlik oranı pulsotipler için %85 ve üzeri kabul edildiği durumda klonal yönden ilişkili 11 grup içerisinde toplandıkları gözlendi. Sonuç: Bu çalışmada, karbapenem dirençli A. baumannii suşlarının netilmisin dışında çalışılan tüm antibiyotiklere çok yüksek yüzdelerle dirence sahip olduğu ve hastane içinde çapraz bulaş yoluyla yayıldığı gösterildi. Bu suşlar hastane enfeksiyonları açısından risk oluşturmaktadır, bunula birlikte klonal yönden ilişkili suşlar spesifik bir ünite ve zaman periyodu ile sınırlı değildir.

Anahtar Kelimeler:

Acinetobacter baumannii, antibiyotik direnci, yoğun bakım üniteleri, polimeraz zincir reaksiyonu, pulsed-field jel elektroforezi

Molecular characterisation of Carbapenem-resistant Acinetobacter baumannii bloodstream infections in intensive care units

Objective: In this study, the aim was to investigate the clonal relationship between carbapenem resistant Acinetobacter baumannii isolates and carbapenem resistance genes isolated from blood samples of patients followed in intensive care units by molecular methods. Methods: Bactec 9240 system Becton Dickinson, USA was used for the isolation of bacteria from blood culture flasks. Identification of 112 strains included in the study were performed by conventional tests, API 20NE bioMèrieux, France and Phoenix TM 100 system Becton Dickinson, USA and confirmed by the presence of blaOXA-51 gene. Antimicrobial susceptibility tests were performed by Kirby-Bauer disk diffusion method and Phoenix TM 100 system. Carbapenem resistance genes; blaOXA-23, blaOXA-48, blaOXA-58, blaIMP, blaVIM and blaNDM-1 were investigated by Multiplex Polymerase Chain Reaction PCR method. Pulsed Field Gel Electrophoresis PFGE was used to determine the clonal relationship between Acinetobacter baumannii strains. Results: The antibiotic resistance percentages of strains for gentamicin, amikacin, tobramycin, netil micin,ceftazidime,trimethoprim/sulfamethoxazole,piperacillin, ciprofloxacin, ampicillin/sulbactam, piperacillin/tazobactam and cefoperazone/ sulbactam, were 88%; 81%; 78%; 36%; 98.5%; 96%; 89%; 100%; 100%; 93%; 91% respectively. MIC values of imipenem and meropenem were determined above ≥8 µg/ml in the whole group. blaOXA-51 and blaOXA-23 genes were detected in all isolates included in the study. By PFGE method, 62 different pulsotypes were detected. Among the pulsotypes, 19 of them contained ≥2 strains. It was observed that 108 96.4% strains were clustered in 11 clonally related groups when the similarity between pulsotypes for grouping was limited to 85% or more. Conclusion: In this study, it was observed that carbapenem-resistant A. baumannii strains were resistant for all tested antibiotics at high levels except netilmicin and spread in the hospital via cross contamination. These strains posed a risk for hospital infections, however, clonal-related strains were not limited to a specific unit and time period.

Keywords:

Acinetobacter baumannii, antibiotic resistance, Intensive care units, Polymerase Chain Reaction, pulsed-field gel electrophoresis,

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1. Doi Y, Murray GL, Peleg AY. Acinetobacter baumannii: evolution of Antimicrobial resistancetreatment options. Semin Respir Crit Care Med, 2015; 36(1): 85-98.
2. Lee HY, Chen CL, Wu SR, Huang C .W, Chiu CH. Risk factors and outcome analysis of acinetobacter baumannii complex bacteremia in critical patients. Crit Care Med, 2014; 42(5): 1081-8.
3. Chusri S, Chongsuvivatwong V, Rivera JI, Silpapojakul K, Singkhamanan K, McNeil E et al. Clinical outcomes of hospital-acquired infection with Acinetobacter nosocomialis and Acinetobacter pittii. Antimicrob Agents Chemother, 2014; 58(7): 4172-9.
4. Wang J, Ruan Z, Feng Y, Fu Y, Jiang Y, Wang H et al. Species distribution of clinical Acinetobacter isolates revealed by different identification techniques. PLoS One, 2014; 13;9(8),e104882.
5. Gerner-Smidt P, Tjernberg I, Ursing. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol, 1991; 29(2): 277-82.
6. Nemec A, Krizova L, Maixnerova M, Sedo O, Brisse S, Higgins PG. Acinetobacter seifertii sp. nov., a member of the Acinetobacter calcoaceticusAcinetobacter baumannii complex isolated from human clinical specimens. Int J Syst Evol Microbiol, 2015; 65(Pt 3): 934-42.
7. Cosgaya, C, Marí-Almirall M, Van Assche A, Fernández-Orth D, Mosqueda N, Telli M et al. Acinetobacter dijkshoorniae sp. nov., a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries. Int J Syst Evol Microbiol, 2016; 66(10): 4105-11.
8. Wenzler E, Goff DA, Humphries R, Goldstein EJC. Anticipating the Unpredictable: A review of Antimicrobial Stewardship and Acinetobacter Infections. Infect Dis Ther, 2017; 6(2): 149-72.
9. Lin MF, Lan CY. Antimicrobial resistance in Acinetobacter baumannii: from bench to bedside. World J Clin Cases, 2014; 2(12): 787-814.
10. Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother, 2010; 65(2): 233-8.
11. Turton JF, Woodford N, Glover J, Yarde S, Kaufmann ME, Pitt TL. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. J Clin Microbiol, 2006; 44(8): 2974-6.
12. Anonymous. Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing. Twenty Fifth Informational Supplement, M100-S25. ISBN: 1-56238-989-0, Wayne, CLSI, 2015.
13. Gómez-Gil MR, Paño-Pardo JR, Romero-Gómez MP, Gasior M, Lorenzo M, Quiles I, Mingorance J. Detection of KPC-2-producing Citrobacter freundii isolates in Spain. J Antimicrob Chemother, 2010; 65(12): 2695-7.
14. Garza-Ramos U, Morfin-Otero R, Sader HS, Jones RN, Hernández E, Rodriguez-Noriega et al. Metallobeta-lactamase gene bla(IMP-15) in a class 1 integron, In95, from Pseudomonas aeruginosa clinical isolates from a hospital in Mexico. Antimicrob Agents Chemother, 2008; 52(8): 2943-6.
15. Poirel L, Bonnin RA, Nordmann P. Genetic features of the widespread plasmid coding for the carbapenemase OXA-48. Antimicrob Agents Chemother, 2012; 56(1): 559-62.
16. Lim J, Cho HH, Kim S, Kim J, Kwon KC, Park JW, Koo SH. The Genetic characteristics of Multidrugresistant Acinetobacter baumannii Coproducing 16S rRNA Methylase armA and Carbapenemase OXA-23. J Bacteriol Virol, 2013; (43)1: 27–36.
17. Anonymous. Center for disease control and Prevention. Multiplex Real-Time PCR Detection of Klebsiella pneumonia Carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM-1) genes, CDC . http://www.cdc. gov/HAI/settings/lab/kpcndm1-lab-protocol. (Erişim Tarihi: 2011).
18. Seifert H, Dolzani L, Bressan R, van der Reijden T, van Strijen B, Stefanik D et al. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. J Clin Microbiol, 2005; 43(9): 4328-35.
19. Tenover FC, Arbeit RD., Goering RV1. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epidemiology of America. Infect Control Hosp Epidemiol, 1997; 18(6): 426–39.
20. Vincent JL, Rello J, Marshall J, Silva E, Anzueto A, Martin CD et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA, 2009; 2: 302(21): 2323-9.
21. Aydın M, Ergönül Ö, Azap A, Bilgin H, Aydın G, Çavuş SA, et al. Rapid emergence of colistin resistance and its impact on fatality among healthcareassociated infections. J Hosp Infect, 2018; 98(3): 260-3.
22. Zander E, Higgins PG, Fernández-González A, Seifert H. Detection of intrinsic blaOXA-51-like by multiplex PCR on its own is not reliable for the identification of Acinetobacter baumannii. Int J Med Microbiol, 2013; 303(2): 88-9.
23. Lee YT, Kuo SC, Chiang MC, Yang SP, Chen CP, Chen TL, et al. Emergence of carbapenem-resistant non-baumannii species of Acinetobacter harboring a blaOXA-51-like gene that is intrinsic to A. baumannii. Antimicrob Agents Chemother, 2012; 56(2): 1124-7.
24. Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of multidrugresistant Acinetobacter baumannii. Antimicrob Agents Chemother, 2007; 51(10): 3471-84.
25. Sarı AN, Biçmen M, Gülay Z. The first report on the outbreak of OXA-24/40-like carbapenemaseproducing Acinetobacter baumannii in Turkey. Jpn J Infect Dis, 2013; 66(5): 439-42.
26. Mugnier PD, Poirel L, Naas T, Nordmann P. Worldwide dissemination of the blaOXA-23 carbapenemase gene of Acinetobacter baumannii. Emerg Infect Dis, 2010; 16(1): 35-40.
27. Ozen N, Ergani A, Naas T, Ogunc D, Gultekin M, Colak D, et al. Outbreak of CarbapenemResistant Acinetobacter baumannii producing the carbapenemase OXA-58in Turkey. The Open Antimicrob Agents J, 2009; 1: 1-8.
28. Vahaboglu H, Budak F, Kasap M, Gacar G, Torol S, Karadenizli A, et al. High prevalence of OXA-51- type class D beta-lactamases among ceftazidimeresistant clinical isolates of Acinetobacter spp.: co-existence with OXA-58 in multiple centres. J Antimicrob Chemother, 2006; 58(3): 537-42.
29. Kulah C, Mooij MJ, Comert F, Aktas E, Celebi G, Ozlu N, et al. Characterisation of carbapenemresistant Acinetobacter baumannii outbreak strains producing OXA-58 in Turkey. Int J Antimicrob Agents, 2010; 36(2): 114-8.
30. Gur D, Korten V, Unal S, Deshpande LM, Castanheira M. Increasing carbapenem resistance due to the clonal dissemination of oxacillinase (OXA-23 and OXA-58)-producing Acinetobacter baumannii: report from the Turkish SENTRY Program sites. J Med Microbiol, 2008; 57(Pt 12): 1529-32.
31. Ergin A, Hascelik G, Eser OK Molecular characterization of oxacillinases and genotyping of invasive Acinetobacter baumannii isolates using repetitive extragenic palindromic sequence-based polymerase chain reaction in Ankara between 2004 and 2010. Scand J Infect Dis, 2013; 45(1): 26-31.
32. Ahmed SS, Alp E, Ulu-Kilic A, Dinc G, Aktas Z, Ada B, et al. Spread of carbapenem-resistant international clones of Acinetobacter baumannii in Turkey and Azerbaijan: a collaborative study. Eur J Clin Microbiol Infect Dis, 2016; 35(9): 1463-8.
33. Ciftci IH, Aşık G, Karakeçe E, Oksüz L, Yağcı S, Sesli Çetin E, et al. Distribution of blaOXA genes in Acinetobacter baumannii strains: a multicenter study]. Mikrobiyol Bul, 2013; 47(4): 592-602.
34. Keskin H, Tekeli A, Dolapci İ, Öcal D. Molecular characterization of beta-lactamase-associated resistance in Acinetobacter baumannii strains isolated from clinical samples. Mikrobiyol Bul, 2014; 48(3): 365-76.
35. Castanheira M, Costello SE, Woosley LN, Deshpande LM., Davies TA, Jones RN. Evaluation of clonality and carbapenem resistance mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus complex and Enterobacteriaceae isolates collected in European and Mediterranean countries and detection of two novel β-lactamases, GES-22 and VIM-35. Antimicrob Agents Chemother, 2014; 58(12): 7358-66.
36. Anonymous. Surveillance of antimicrobial resistance in Europe, Antimicrobial Resistance Surveillance Network) 2016, Stockholm: ECDC; 2017. https:// ecdc.europa.eu/sites/portal/files/documents/ AMR-surveillance-Europe-2016.pdf (Erişim tarihi: 2017). doi 10.2900/296939.
37. Lob SH, Hoban DJ, Sahm DF, Badal E. Regional differences and trends in antimicrobial susceptibility of Acinetobacter baumannii. Int J Antimicrob Agents, 2016; 47(4): 317-23.