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High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS

  • Clinical Microbiology - Research Paper
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Abstract

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)–producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as “KPC-positive.” The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

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References

  1. Centers for Disease Control (2013) Antibiotic resistance threats in the United States. https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf Acessed 30 August 2019

  2. Jacob JT, Klein E, Laxminarayan R, Beldavs Z, Lynfield R, Kallen AJ, Ricks P, Edwards J, Srinivasan A, Fridkin S, Rasheed JK, Lonsway D, Bulens S, Herrera R, McDonald C, Patel J, Bell M, Cardo D (2013) Vital signs: carbapenem-resistant Enterobacteriaceae. Morb Mortal Wkly Rep 62(9):165–170

    Google Scholar 

  3. World Health Organization (2014) Antimicrobial resistance: global report on surveillance. http://wwwwhoint/drugresistance/documents/surveillancereport/en/ Acessed 30 August 2019

  4. Campos AC, Albiero J, Ecker AB, Kuroda CM, Meirelles LE, Polato A, Tognim MC, Wingeter MA, Teixeira JJ (2016) Outbreak of Klebsiella pneumoniae carbapenemase–producing K. pneumoniae: a systematic review. Am J Infect Control 44(11):1374–1380

    Article  Google Scholar 

  5. White House National Action Plan for Combating Antibiotic-Resistant Bacteria (2015) https://www.whitehouse.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic-resistant_bacteria.pdf. Acessed 30 August 2019

  6. Hrabák J, Studentová V, Walková R, Zemlicková H, Jakubu V, Chudácková E, Gniadkowski M, Pfeifer Y, Perry JD, Wilkinson K, Bergerová T (2012) Detection of NDM-1, VIM-1, KPC, OXA-48, and OXA-162 carbapenemases by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 50:2441–2443

    Article  Google Scholar 

  7. Peaper DR, Kulkarni MV, Tichy AN, Jarvis M, Murray TS, Hodsdon ME (2013) Rapid detection of carbapenemase activity through monitoring ertapenem hydrolysis in Enterobacteriaceae with LC-MS/MS. Bioanalysis 5:147–157

    Article  CAS  Google Scholar 

  8. Kulkarni MV, Zurita AN, Pyka JS, Murray TS, Hodsdon ME, Peaper DR (2014) Use of imipenem to detect KPC, NDM, OXA, IMP, and VIM carbapenemase activity from gram-negative rods in 75 minutes using liquid chromatography-tandem mass spectrometry. J Clin Microbiol 52:2500–2505

    Article  CAS  Google Scholar 

  9. Sauget M, Cabrolier N, Manzoni M, Bertrand X, Hocquet D (2014) Rapid, sensitive and specific detection of OXA-48-like producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Microbiol Methods 105:88–91

    Article  CAS  Google Scholar 

  10. Lasserre C, De Saint ML, Cuzon G, Bogaerts P, Lamar E, Glupczynski Y, Naas T, Tandé D (2015) Efficient detection of carbapenemase activity in Enterobacteriaceae by matrix-assisted laser desorption ionization-time of flight mass spectrometry in less than 30 minutes. J Clin Microbiol 53:2163–2171

    Article  CAS  Google Scholar 

  11. Foschi C, Franza V, Conti M, Tamburini MV, Roncarati G, Cordovana M, Smirnova V, Patrono D, Mancini R, Landini MP, Ambretti S (2015) Use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect carbapenemase production in Enterobacteriaceae by a rapid meropenem degradation assay. New Microbiol 38:571–576

    CAS  Google Scholar 

  12. Mirande C, Canard I, Buffet Croix Blanche S, Charrier JP, van Belkum A, Welker M, Chatellier S (2015) Rapid detection of carbapenemase activity: benefits and weaknesses of MALDI-TOF MS. Eur J Clin Microbiol Infect Dis 34:2225–2234

    Article  CAS  Google Scholar 

  13. Ghebremedhin B, Halstenbach A, Smiljanic M, Kaase M, Ahmad-Nejad P (2016) MALDI-TOF MS based carbapenemase detection from culture isolates and from positive blood culture vials. Ann Clin Microbiol Antimicrob 15:5

    Article  CAS  Google Scholar 

  14. Huber CA, Sidjabat HE, Zowawi HM, Kvaskoff D, Reed S, McNamara JF, McCarthy KL, Harris P, Toh B, Wailan AM, Paterson DL (2016) Detection of carbapenemase activity in Enterobacteriaceae using LC-MS/MS in comparison with the neo-rapid CARB kit using direct visual assessment and colorimetry. J Microbiol Methods 131:68–72

    Article  CAS  Google Scholar 

  15. Monteferrante CG, Sultan S, Ten Kate MT, Dekker LJ, Sparbier K, Peer M, Kostzrewa M, Luider TM, Goessens WH, Burgers PC (2016) Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF. J Antimicrob Chemother 71:2856–2867

    Article  CAS  Google Scholar 

  16. Ramos AC, Carvalhaes CG, Cordeiro-Moura JR, Rockstroh AC, Machado AMO, Gales AC (2016) Influence of culture media on detection of carbapenem hydrolysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 54:1896–1898

    Article  CAS  Google Scholar 

  17. Lau AF, Wang H, Weingarten RA, Drake SK, Suffredini AF, Garfield MK, Chen Y, Gucek M, Youn JH, Stock F, Tso H, DeLeo J, Cimino JJ, Frank KM, Dekker JP (2014) A rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for single plasmid tracking in an outbreak of carbapenem-resistant Enterobacteriaceae. J Clin Microbiol 52:2804–2812

    Article  CAS  Google Scholar 

  18. Youn JH, Drake SK, Weingarten RA, Frank KM, Dekker JP, Lau AF (2016) Clinical performance of a matrix-assisted laser desorption ionization-time of flight mass spectrometry method for detection of certain blaKPC-containing plasmids. J Clin Microbiol 54:35–42

    Article  CAS  Google Scholar 

  19. Charretier Y, Charrier JP, Franceschi C, Zambardi G, Cecchini T, Degout-Charmette E (2012) Method of detecting at least one mechanism of resistance to carbapenems by mass spectrometry. Patent US 20150031063

  20. Charretier Y, Schrenzel J (2016) Mass spectrometry methods for predicting antibiotic resistance. Proteomics Clin Appl 10:964–981

    Article  CAS  Google Scholar 

  21. Charretier Y, Dauwalder O, Franceschi C, Degout-Charmette E, Zambardi G, Cecchini T, Bardet C, Lacoux X, Dufour P, Veron L, Rostaing H, Lanet V, Fortin T, Beaulieu C, Perrot N, Dechaume D, Pons S, Girard V, Salvador A, Durand G, Mallard F, Theretz A, Broyer P, Chatellier S, Gervasi G, Van Nuenen M, Roitsch CA, Van Belkum A, Lemoine J, Vandenesch F, Charrier JP (2015) Rapid bacterial identification, resistance, virulence and type profiling using selected reaction monitoring mass spectrometry. Sci Rep 5:13944

    Article  Google Scholar 

  22. Wang H, Drake SK, Youn JH, Rosenberg AZ, Chen Y, Gucek M, Suffredini AF, Dekker JP (2017) Peptide markers for rapid detection of KPC carbapenemase by LC-MS/MS. Sci Rep 7(1):2531

    Article  Google Scholar 

  23. Magagnin CM, Rozales FP, Antochevis L, Nunes LS, Martins AS, Barth AL, Sampaio JM, Zavascki AP (2017) Dissemination of blaOXA-370 gene among several Enterobacteriaceae species in Brazil. Eur J Clin Microbiol Infect Dis 36:1907

    Article  CAS  Google Scholar 

  24. Monteiro J, Widen RH, Pignatari AC, Kubasek C, Silbert S (2012) Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother 67(4):906–909

    Article  CAS  Google Scholar 

  25. Pires J, Novais A, Peixe L (2013) Blue-carba, an easy biochemical test for detection of diverse carbapenemase producers directly from bacterial cultures. J Clin Microbiol 51:4281–4283

    Article  CAS  Google Scholar 

  26. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV (2012) Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67(11):2640–2644

    Article  CAS  Google Scholar 

  27. Geneious Prime 2019.1.3. (https://www.geneious.com)

  28. Naas T, Cuzon G, Truong H, Nordmann P (2012) Role of ISKpn7 and deletions in blaKPC gene expression. Antimicrob Agents Chemother 56(9):4753–4759

    Article  CAS  Google Scholar 

  29. Kitchel B, Rasheed JK, Endimiani A, Hujer AM, Anderson KF, Bonomo RA, Patel JB (2010) Genetic factors associated with elevated carbapenem resistance in KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 54(10):4201–4207

    Article  CAS  Google Scholar 

  30. Jousset AB, Rosinski-Chupin I, Takissian J, Glaser P, Bonnin RA, Naas T (2018) Transcriptional landscape of a blaKPC-2 plasmid and response to Imipenem exposure in Escherichia coli TOP10. Front Microbiol 9(2929)

  31. Roth AL, Kurpiel PM, Lister PD, Hanson ND (2011) blaKPC RNA expression correlates with two transcriptional start sites but not always with gene copy number in four genera of gram-negative pathogens. Antimicrob Agents Chemother 55(8):3936–3938

    Article  CAS  Google Scholar 

  32. Roth AL, Lister PD, Hanson ND (2013) Effect of drug treatment options on the mobility and expression of blaKPC. J Antimicrob Chemother 68:2779–2785

    Article  CAS  Google Scholar 

  33. Daikos GL, Markogiannakis A (2011) Carbapenemase-producing Klebsiella pneumoniae: (when) might we still consider treating with carbapenems? Clin Microbiol Infect 17:1135–1141

    Article  CAS  Google Scholar 

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Funding

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministry of Science, Technology, Inovation and Comunications, Brasília, Brazil; FIPE/HCPA (Research and Events Support Fund at Hospital de Clínicas de Porto Alegre); MAPA (Ministry of Agriculture, Livestock and Food Supply of Brazil). A.L.B. and A.F.M are research fellows from the CNPq, Ministry of Science, Technology, Inovation and Comunications, Brazil. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Correspondence to Andreza F. Martins.

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Lovison, O.A., Rau, R.B., Lima-Morales, D. et al. High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS. Braz J Microbiol 51, 1029–1035 (2020). https://doi.org/10.1007/s42770-019-00222-y

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