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.
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- 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