Nilgün ÖZBEY, Müşerref TATMAN-OTKUN

Molecular typing and investigation of carbapenemases in carbapenem resistant Acinetobacter baumannii isolates

Amaç: Bu çalışmada karbapenem dirençli A. baumannii'lerde karbapenemazların varlığı ve bunların kromozomal veya plazmid kaynaklı olduğunun araştırılması amaçlanmıştır. Yöntemler: Çalışılan 65 A. baumannii'nin %66'sı yoğun bakım ünitesi (YBÜ)'ndeki hastalardan, en çok kandan (%37) ve alt solunum yolu örneklerinden (%32) izole edilmiştir. Antibiyotik duyarlılıklarını değerlendirmede VITEK 2 otomatize sistemi ve disk difüzyon testi kullanılmıştır. Ayrıca, karbapenemler için MİK değerleri, MİK değerlendirici stripler kullanılarak belirlenmiştir. İzolatlar arasındaki klonal ilişki M13 ve DAF4 primerleri kullanılarak AP-PZR ile değerlendirilmiştir. Karbapenemaz genleri multipleksPZR ile taranmış ve plazmit kaynaklı karbapenemaz araştırılmıştır.Bulgular: İzolatlar tobramisin, netilmisin ve kolistine sırasıyla %98.5, %98.5 ve %96.9 oranında duyarlı bulunmuştur. Diğer antibiyotiklere direnç oranları oldukça yüksek olup %78.5-100.0 arasında saptanmıştır. Tikarsilin, piperasilin, piperasilin+tazobaktam, seftazidim, seftriakson, sefepim, imipenem, meropenem ve siprofloksasin için %100.0; tikarsilin+klavulanik asit, gentamisin, ciprofloxacin, 98.5% to ticarcillin+clavulanic acid, gentamicin, levofloxacin, tetracycline, doxycycline, and trimethoprim-sulfamethoxazole, 93.8% to minocycline, 92.3% to amikacin, and 78.5% to ampicillin+sulbactam. In the study, two different patterns were observed by AP-PCR. Chromosomal OXA-23 and OXA-51 carbapenemase enzyme genes positively were found in all isolates. Plasmids were isolated neither of isolates.Conclusion: Isolates were detected more frequently from hemocultures in ICU. Isolates showed clonal similarity in AP-PCR where M13 and DAF4 primers were used. The presence of the single Acinetobacter clone in our hospital suggests that they all originated from the same source. For this reason, it is concluded that universal infection control measures should be taken in consideration to prevent cross-infection by carbapenem resistant isolates.Key Words: Acinetobacter baumannii, levofloksasin, tetrasiklin, doksisiklin ve trimethoprim+sulfametoksazol için %98.5; minosiklin için %93.8; amikasin için %92.3 ve ampisilin+sulbaktam için %78.5 oranında direnç saptanmıştır. AP-PZR ile araştırmada iki farklı patern gözlenmiştir. Bütün izolatlarda kromozomal kaynaklı OXA-23 ve OXA51 karbapenemaz enzim geni pozitif bulunmuştur. İzolatların hiçbirinde plazmit izole edilmemiştir.Sonuç: İzolatlar en sık YBÜ'de yatmakta olan hastaların kan örneklerinde saptanmıştır. İzolatlar M13 ve DAF4 primerlerin kullanıldığı AP-PZR'de klonal benzerlik göstermiştir. Hastanemizde tek Acinetobacter kolonu bulunması, hepsinin aynı kaynaktan köken aldığını düşündürmektedir. Bu nedenle karbapeneme dirençli izolatların çapraz enfeksiyonlarını önlemek için evrensel enfeksiyon kontrol önlemlerine dikkat etmek gerektiği sonucuna varılmıştır

Karbapenemlere dirençli Acinetobacter baumannii izolatlarında moleküler tiplendirme ve karbapenemazların araştırılması

Objective: In this study, it was aimed to investigate the presence of carbapenemases in carbapenem-resistant A. baumannii strains and their chromosomal or plasmid origin.Methods: Of the total 65 A. baumannii studied, 66% were isolated from patients in intensive care unit (ICU); most of strains were blood (37%) and lower respiratory tract samples (32%). VITEK2 automated system and disk diffusion tests were used to evaluate antibiotic susceptibilities. In addition, MIC values for carbapenems were determined using M.I.C. evaluator strips. Clonal relationship between the isolates was assessed by AP-PCR, using M13 and DAF4 primers. Carbapenemase genes were screened by multiplexPCR, and presence of plasmid-borne carbapenemase was investigated.Results: The The isolates were found to be susceptible to tobramycin, netilmicin, and colistin by 98.5%, 98.5% and 96.9%, respectively. The rates of resistance to other antibiotics were quite high, and resistance was found between 78.5% - 100%. The resistance rates were detected as 100% to ticarcillin, piperacillin, piperacillin+tazobactam, ceftazidime, ceftriaxone, sefepime, imipenem, meropenem, and iprofloxacin, 98.5% to ticarcillin+clavulanic acid, gentamicin, levofloxacin, tetracycline, doxycycline, and trimethoprim-sulfamethoxazole, 93.8% to minocycline, 92.3% to amikacin, and 78.5% to ampicillin+sulbactam. In the study, two different patterns were observed by AP-PCR. Chromosomal OXA-23 and OXA-51 carbapenemase enzyme genes positively were found in all isolates. Plasmids were isolated neither of isolates. Conclusion: Isolates were detected more frequently from hemocultures in ICU. Isolates showed clonal similarity in AP-PCR where M13 and DAF4 primers were used. The presence of the single Acinetobacter clone in our hospital suggests that they all originated from the same source. For this reason, it is concluded that universal infection control measures should be taken in consideration to prevent cross-infection by carbapenem resistant isolates

PDF

___

1. Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger PC, Woods GL. The Nonfermentative Gram-Negative Bacilli. In: Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger PC, Woods GL eds. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams-Wilkins, 2006: 303-391.
2. Lolans K, Rice TW, Munoz-Price S L, Quinn JP. Multicity outbreak of carbapenem- resistant Acinetobacter baumannii isolates producing the carbapenemase OXA-40. Antimicrob Agents Chemother, 2006; 50 (9): 2941-5.
3. Higgins PG, Perez-Llarena FJ, Zander E, Fernandez A, Bou G, Seifert H. OXA-235, a novel class D β-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob Agents Chemother, 2013; 57(5): 2121-6.
4. Turton JF, Ward ME, Wood N, Kaufmann MEE, Pike R, Livermore DM, et al. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett, 2006; 258: 72-7.
5. Poriel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: Mechanisms and epidemiology. Clin Microbiol Infect, 2006; 12: 826– 36.
6. Clinical And Laboratory Standarts Institute. Performance standards for antimicrobial susceptibility testing: Twenty-Second Informational Supplement. CLSI document M100-S22. Clinical and Laboratory Standards Institute, USA. 2012.
7. European Committe On Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. v2.0. 2012.
8. Grundmann HJ, Towner KJ, Dijkshoorn L, GernerSmidt P, Maher M, Seifert H, et al. Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp. J Clin Microbiol, 1997; 35: 3071–7.
9. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents, 2006; 27 (4): 351-3.
10. Bier KES, Luiz SO, Scheffer MC, Gales AC, Paganini MC, Nascimento AJ, et al. Temporal evolution of carbapenem resistant Acinetobacter baumannii in Curitiba, southern Brazil. American J Infect Control, 2010; 38: 308-14.
11. Turton JF, Gabriel SN, Valderrey C, Kaufmann ME, Pitt TL. Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin Microbiol Infect, 2007; 13: 807-15.
12. Higgins PG, Lehmann M, Wisplinghoff H, Seifert H. gryB multiplex PCR to differentiate between Acinetobacter calcoaceticus and Acinetobacter genomic species 3. J Clin Microbiol, 2010; 48 (12): 4592-4.
13. 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. Journal Plos One, 2014 Aug 14; 9(8): e104882. DOI; 10.1371/journal.pone.0104882.
14. Nowak P, Paluchowska P, Budak A. Co-occurrence of carbapenem and aminoglycoside resistance genes among multidrug-resistant clinical isolates of Acinetobacter baumannii from Cracow, Poland. Med Sci Monit Basic Res, 2014; 20: 9-14.
15. Schreckenberger PC, Daneshvar MI, Hollis DG. Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and other nonfermantative Gramnegative rods. In: Murray PR, Baron EJ, Pfaller MA, Jorgensen JH, Landry M.L, eds. Manual of Clinical Microbiology. 9th ed. Washington DC: ASM Press, 2007; 770-802.
16. Zarakolu P, Hasçelik G, Ünal S. Antimicrobial susceptibility pattern of nosocomial Gram negative pathogens: Results from MYSTIC study in Hacettepe University adult Hospital (2000-2004). Mikrobiyol Bul, 2006; 40: 147-54.
17. Iraz M, Ceylan A, Akkoyunlu Y. Çeşitli klinik örneklerden izole edilen Acinetobacter türlerinde antibiyotik direnç oranlarının incelenmesi. ANKEM Dergisi, 2012; 26(2): 80-5.
18. Gordon NC, Wareham DW. Multidrug-resistant Acinetobacter baumannii; mechanism of virulence and resistance. Int J Antimicrob Agents, 2010; 35: 219-26.
19. Nigro SJ, Post V, Hall RM. Aminoglycoside resistance in multiply antibiotic-resistant Acinetobacter baumannii belonging to global clone 2 from Australian hospitals. J Antimirob Chemother, 2011; DOİ; 10.1093/jac/dkr163.
20. Levin AS, Barone AA, Penco J, Santos MV, Marinho IS, Arruda EAG, et al. Intravenous colistin therapy for nosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Clin Infect Dis, 1999; 28: 1008-11.
21. Hawley JS, Murray CK, Jorgensen JH. Colistin heteroresistance in Acinetobacter and its association with previous colistin therapy. Antimicrob Agents Chemother, 2008; 52 (1): 351-2.
22. Olive DM, Principles PB. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol, 1999; 37(6): 1661-9.
23. Schuetz AN, Huard RC, Eshoo MW, Massire C, Della-Latta P, Wu F, et al. Identification of a novel Acinetobacter baumannii clone in a US hospital outbreak by multilocus polymerase chain reaction/ electrospray-ionization mass spectrometry. Diagnostic Microbiol Infect Dis, 2012; 72: 14-9.
24. Aygün G. Yoğun bakım birimi infeksiyonlarında çevre şartlarının önemi. Klimik Dergisi, 2003; 16(3): 106- 7.
25. Gür D, Korten V, Ünal 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: 1529–32.
26. Çiftçi İH, Aşık G, Karakeçe E, Öksüz L, Yağcı S, Sesli-Çetin E, ve ark. Acinetobacter baumannii izolatlarında blaOXA genlerinin dağılımı: Çok merkezli bir çalışma. Mikrobiyol Bul, 2013; 47(4): 592-602.
27. Çiçek AC, Saral A, Iraz M, Ceylan A, Düzgün AO, Peleg AY, et al. OXA- and GES-type b-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital. Clin Microbiol Infect, 2014; 20(5): 410-5.
28. Pournaras S, Markogiannakis A, Ikonomidis A, Kondyli L, Bethimouti K, Maniatis AN, et al. Outbreak of multiple clones of imipenem-resistant Acinetobacter baumannii isolates expressing OXA58 carbapenemase in an intensive care unit. J Antimicrob Chemother, 2006; 57: 557–61.