VITEK-MS Sistemi ile Karbapenemaz ID Üretiminin Hızlı Tespiti

Son yıllarda karbapenemaz üreten Enterobacteriaceae kaynaklı enfeksiyonların dünya çapında artması önemli halk sağlığı sorunudur. Araştırıcılar, mikroorganizmanın kısa sürede tanımlanmasına olanak sağlayan MALDI-TOF sisteminin antibiyotik duyarlılık testlerinde de kullanılabileceğini gösteren çalışmalar bildirilmektedir. Çalışmamızda karbapenemaz üretiminin hızlı saptanmasına yönelik olarak ertapenemin karbapenemazlar tarafından hidrolizinin VITEK-MS (bioMérieux, Fransa) sistemi tarafından tespit edilebilirliği değerlendirilmiştir. Çalışmamıza çeşitli klinik örneklerden izole edilen karbapenem dirençli (n=86) ve duyarlı (n=20) toplam 106 Klebsiella pneumoniae kökeni dahil edilmiştir. KPC, NDM, IMP, VIM ve OXA-48 genlerinin varlığını PCR ile tespit edilmiştir. Bakteri süspansiyonları 4 McFarland bulanıklığında hazırlanmış, 0.5 μg/ml’lik ertapenem ile muamele edildikten sonra iki ve dört saat inkübe edilip analiz edilmek üzere VITEK-MS RUO (bioMérieux, Fransa) sistemine aktarılmıştır. Karbapenem dirençli 86 K.pneumoniae kökeninin blaOXA-48 (n=39), blaNDM (n= 37), blaIMP (n=8) ve blaKPC (n=2) karbapenemaz genlerini taşıdığı tespit edilmiştir. Tüm antibiyotiklere duyarlı olan kökenlerde hiçbir karbapenemaz genine rastlanmamıştır. Ertapenemin tek başına sahip olduğu spektrumlar çalışma kökenlerinin ertapenem ile muamele edilip inkübe edilmesi sonucu elde edilen spektrumlar ile karşılaştırıldığında; iki saatlik inkübasyon sonrasında NDM, IMP ve KPC geni pozitif kökenlerin tamamında ertapenem hidrolizi gözlenirken, OXA-48 geni pozitif olan 14 kökende ertapenem hidrolizinin zayıf olduğu tespit edilmiştir. Dört saatlik inkübasyon sonrası OXA-48 geni pozitif olan kökenlerin sadece % 64.1’inde ertapenem hidrolizi gözlemlenmiştir. İki ve dört saat inkübasyon sonrası karbapenemaz geni içermeyen kökenlerin hiçbirinde ertapenem hidrolizi gözlemlenmemiştir. Rutin laboratuvarda mikroorganizmaların hızlı tespitinde önemli başarı sağlayan MALDI-TOF sistemlerinin antibiyotik duyarlılık testlerinde de benzer başarı göstermesi oldukça ümit vericidir. Ancak OXA-48 pozitif kökenlerin saptanmasını kolaylaştıracak farklı protokollerin geliştirilmesi gereklidir. MALDI-TOF sistemleri ile kısa sürede, düşük maliyetle karbapenemaz üretiminin tespit edilmesi özellikle salgınların çok daha kısa sürede kontrol altına alınmasında önemli katkı sağlayacaktır.

Rapid Detection of Carbapenemase Detection by VITEK-MS

In recent years, the increasing of infections caused by carbapenemase-producing Enterobacteriaceae (CPE) is an important public health problem in the worldwide. Some researchers have been reported studies showing that MALDI-TOF MS system can also be used in antibiotic susceptibility testing. The aim of this study was to evaluate the performance of VITEK-MS (bioMérieux, France) system based on ertapenem hidrolysis for the detection of carbapenemase activity. One hundered-six Klebsiella pneumoniae strains isolated from various clinical samples were tested in this study, which included 86 carbapenem resistant and 20 carbapenem susceptible isolates. The presence of KPC, NDM, IMP, VIM ve OXA-48 genes were detected by PCR. After the 4 McFarland bacterial suspensions was treated with 0.5 mg/ml of ertapenem, the suspension was incubated for 2h and 4h, and transferred to VITEK-MS RUO (bioMérieux, France) system for analysis. Eighty-six carbapenem resistant K.pneumoniae strains had blaOXA-48 (n=39), blaNDM (n= 37), blaIMP (n=8) ve blaKPC (n=2) genes. No carbapenemase gene was found in the carbapenem susceptible K.pneumoniae strains. According to results of VITEK MS, ertapenem hydrolysis was observed in all of the NDM, IMP, and KPC after 2h of incubation. Poor hydrolysis was observed in 14 OXA-48 positive strains within 2h. After 4h of incubation strong ertapenem hydrolysis was observed in 64.1 % of the OXA-48 positive strains. All non-carbapenemase producing strains were detected as negative. According to our study results, the detection of NDM, IMP and KPC using VITEK-MS seems to be a rapid, reliable and cost-effective method for routine laboratory. Since similar results were not observed in OXA-48, different protocols should be developed for the detection of these enzymes. The detection of carbapenemases by MALDI-TOF-MS systems in a short time will make a significant contribution especially in controlling the outbreaks.

Kaynakça

1. Aktaş Z, Kayacan CB, Schneider I, et al. Carbapenemhydrolyzing oxacillinase, OXA-48, persists in Klebsiella pneumoniae in Istanbul, Turkey. Chemotherapy. 2008;54(2):101-6. doi: 10.1159/000118661. Epub 2008 Feb 25. PubMed PMID: 18303258. https://doi.org/10.1159/000118661

2. Aktaş Z, Kayacan CB. Investigation of metallo-betalactamase producing strains of Pseudomonas aeruginosa and Acinetobacter baumannii by E-test, disk synergy and PCR. Scand J Infect Dis. 2008;40(4):320-5. https://doi.org/10.1080/00365540701704698

3. Burckhardt I, Zimmermann S. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 hours. J Clin Microbiol. 2011;49(9):3321-4. https://doi.org/10.1128/JCM.00287-11

4. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother. 2010;54(3):969-76. doi: 10.1128/AAC.01009-09. Epub 2009 Dec 7. Review. PubMed PMID: 19995920 https://doi.org/10.1128/AAC.01009-09

4. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother. 2010;54(3):969-76. doi: 10.1128/AAC.01009-09. Epub 2009 Dec 7. Review. PubMed PMID: 19995920; PubMed Central PMCID: PMC2825993. https://doi.org/10.1128/AAC.01009-09

5. Carvalhaes CG, Cayô R, Visconde MF, et al. Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision. J Antimicrob Chemother. 2014;69(8):2132-6. https://doi.org/10.1093/jac/dku094

6. Carvalhaes CG, da Silva AC, Streling AP, et al. Detection of c carbapenemase activity using VITEK MS: interplay of carbapenemase type and period of incubation. J Med Microbiol. 2015;64(8):946-7. https://doi.org/10.1099/jmm.0.000102

7. Chong PM, McCorrister SJ, Unger MS, et al. MALDITOF MS detection of carbapenemase activity in clinical isolates of Enterobacteriaceae spp., Pseudomonas aeruginosa, and Acinetobacter baumannii compared against the Carba-NP assay. J Microbiol Methods. 2015;111:21-3. https://doi.org/10.1016/j.mimet.2015.01.024

8. Cohen Stuart J, Leverstein-Van Hall MA; Dutch working party on the detection of highly resistant microorganisms. Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents. 2010;36(3):205-10. https://doi.org/10.1016/j.ijantimicag.2010.05.014

9. Cole JM, Schuetz AN, Hill CE, et al. Development and evaluation of a real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase genes. J Clin Microbiol. 2009;47(2):322-6. https://doi.org/10.1128/JCM.01550-08

10. Hammoudi D, Moubareck CA, Sarkis DK. How to detect carbapenemase producers? A literature review of phenotypic and molecular methods. J Microbiol Methods. 2014;107:106-18. https://doi.org/10.1016/j.mimet.2014.09.009

11. Hrabák J, Chudáčková E, Papagiannitsis CC. Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories. Clin Microbiol Infect. 2014;20(9):839-53. https://doi.org/10.1111/1469-0691.12678

12. Hrabak J, Walkova R, Studentova V, et al. Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2011;49(9):3222-7. https://doi.org/10.1128/JCM.00984-11

13. Hrabák J, Chudácková E, Walková R. Matrix-assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis. Clin Microbiol Rev. 2013;26(1):103-14. https://doi.org/10.1128/CMR.00058-12

14. Hoyos-Mallecot Y, Cabrera-Alvargonzalez JJ, Miranda- Casas C, et al. MALDI-TOF MS, a useful instrument for differentiating metallo-β-lactamases in Enterobacteriaceae and Pseudomonas spp. Lett Appl Microbiol. 2014;58(4):325-9. https://doi.org/10.1111/lam.12203

15. Johansson A, Ekelöf J, Giske CG, et al. The detection and verification of carbapenemases using ertapenem and matrix assisted laser desorption Ionizationtime of flight. BMC Microbiol. 2014;14:89. https://doi.org/10.1186/1471-2180-14-89

16. Knox J, Jadhav S, Sevior D, et al. Phenotypic detection of carbapenemase-producing Enterobacteriaceae by use of matrix-assisted laser desorption ionizationtime of flight mass spectrometry and the Carba NP test. J Clin Microbiol. 2014;52(11):4075-7. https://doi.org/10.1128/JCM.02121-14

17. Lee CR, Lee JH, Park KS, et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: Epidemiology, genetic context, treatment options, and detection methods. Front Microbiol. 2016;7:1- 30. https://doi.org/10.3389/fmicb.2016.00895

18. Mirande C, Canard I, Croix Blanche SB, et al. Rapid detection of carbapenemase activity: benefits and weaknesses of MALDI-TOF MS. Eur J Microbiol Infect Dis. 2015;34(11):2225-34. https://doi.org/10.1007/s10096-015-2473-z

19. Morency-Potvin P, Schwartz DN, Weinstein RA. Antimicrobial stewardship: How the microbiology laboratory can right the ship. Clin Microbiol Rev. 2016;30(1):381-407. https://doi.org/10.1128/CMR.00066-16

20. Osei Sekyere J, Govinden U, Essack SY. Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria. J Appl Microbiol. 2015;119(5):1219-33 https://doi.org/10.1111/jam.12918

21. Ovia-o M, Barba MJ, Fernández B, et al. Rapid detection of OXA-48-Producing Enterobacteriaceae by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2016;54(3):754-9. https://doi.org/10.1128/JCM.02496-15

22. Perry JD, Naqvi SH, Mirza IA, et al. Prevalence of faecal carriage of Enterobacteriaceae with NDM-1 carbapenemase at military hospitals in Pakistan, and evaluation of two chromogenic media. J Antimicrob Chemother. 2011;66(10):2288-94. https://doi.org/10.1093/jac/dkr299

23. Pitout JD, Gregson DB, Poirel L, et al. Detection of Pseudomonas aeruginosa producing metallo-betalactamases in a large centralized laboratory. J Clin Microbiol. 2005;43(7):3129-35. https://doi.org/10.1128/JCM.43.7.3129-3135.2005

24. Tijet N, Boyd D, Patel SN, et al. Evaluation of the Carba NP test for rapid detection of carbapenemaseproducing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013;57(9):4578-580. https://doi.org/10.1128/AAC.00878-13

25. Vogne C, Prod’hom G, Jaton K, et al. A simple, robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry, microarray and PCR. Clin Microbiol Infect. 2014;20(12): O1106-12. https://doi.org/10.1111/1469-0691.12715

26. Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 2009;53(12):5046-54. https://doi.org/10.1128/AAC.00774-09

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