Identification and antimicrobial susceptibility testing of microorganisms from positive blood cultures by a combined lysis-centrifugation method with MALDI-TOF MS and VITEK2 System

  • Donatella Maria Rodio Dep. Public Health and Infectious Diseases, Sapienza University, Rome
  • Filomena Febbraro Department of Pediatrics, “Sapienza” University Rome
  • Gianluca Puggioni Department of Clinical Medicine, “Sapienza” University Rome
  • Camilla Paradisi Department of Public Health and Infectious Diseases, “Sapienza” University Rome
  • Flavia Stangherlin Department of Public Health and Infectious Diseases, “Sapienza” University Rome
  • Carla Prezioso Department of Public Health and Infectious Diseases, “Sapienza” University Rome
  • Guido Antonelli Department of Molecular Medicine and Pasteur Institute-Cenci Bolognetti Foundation, “Sapienza” University Rome
  • Maria Trancassini Department of Public Health and Infectious Diseases, “Sapienza” University Rome
  • Valeria Pietropaolo Dep. Public Health and Infectious Diseases, Sapienza University, Rome
Keywords: Bacteremia, LCM, MALDI-TOF MS, VITEK®2, AST


Background: Rapid identification and the application of antimicrobial susceptibility testing (AST) to microorganisms causing bloodstream infections is pivotal to guide antimicrobial therapy. This study aims to: 1) utilize the Lysis-Centrifugation Method (LCM) not only for identification of microorganisms from positive blood culture bottles, but also for direct AST full panel by Vitek®2 system (bioMérieux, Inc. France) and by disc diffusion plate (Kirby Bauer Method) and 2) analyze the accuracy of these combined methods.Methods: 124 mono-microbial positive blood culture bottles were included in this study. An aliquot was subjected to LCM and used for the identification by the MALDI-TOF System. Moreover the microbial pellet was used for direct AST testing full panel by VITEK®2  system and by Kirby Bauer Method.Results: 123 isolates were correctly identified to the species level and 1 isolate was identified to the genus level. Comparing the two utilized AST methods, it was observed that Gram-positive isolates showed an agreement rate of 96.6% (58/60). Enterococcus faecalis was the only microorganism with a major error rate of 0.6% (2/324) related to erythromycin. Among the Gram-negative, the overall agreement rate was 93.3 (56/60). Klebsiella pneumoniae, Escherichia coli and Enterobacter spp. were the major cause of minor error rates (0.6%, 4/709) and major error rates (1.1%, 8/709). Among the yeasts, results showed an agreement rate of 100% (4/4).Conclusions: Our simple and cost-effective sample preparation method is very useful for rapid identification as well as AST of microorganisms directly from positive blood culture bottles in a clinical setting. DOI: 10.21276/APALM.1212


1. Centers for Diseases Control and prevention (CDC), CDC/NHSN surveillance definitions for specific types of infections., 2014.
2. Ferrer R, Martin-Loeches I, Phillips G, Osborn TM, Townsend S, Dellinger RP, et al. Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program. Crit Care Med 2014; 42(8): 1749–55.
3. Bearman GML, Wenzel RP. Bacteremias: a leading cause of death Arch Med Res 2005; 36: 646–59.
4. Seifert H. The clinical importance of microbiological findings in the diagnosis and management of bloodstream infections. Clin Infect Dis 2009; 48: S238–45.
5. Mayr FB, Yende S, Angus DC. Epidemiology of severe sepsis. Virulence 2014;5:4–11.
6. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect 2015; 21(4): 313–22.
7. Buehler SS, Madison B, Snyder SR, Derzon JH, Cornish NE, Saubolle MA, et al. Effectiveness of practices to increase timeliness of providing targeted therapy for inpatients with bloodstream infections:a laboratory medicine best practices systematic review and meta-analysis. Clin Microbiol Rev 2016; 29(1): 59–103.
8. Hall M, Levant S, DeFrances C. Trends in inpatient hospital deaths: National Hospital Discharge Survey, 2000–2010. NCHS Data Brief 118. National Center for Health Statistics, Centers for Disease Control and Prevention, 2013; Atlanta, GA.
9. Liu V, Escobar GJ, Greene JD, Soule J, Whippy A, Angus DC, Iwashyna TJ. Hospital deaths in patients with sepsis from 2 independent cohorts. JAMA 2014; 312: 90–2.
10. Beekmann SE, Diekema DJ, Doern GV. Determining the clinical significance of coagulase- negative staphylococci isolated from blood cultures. Infect Control Hosp Epidemiol 2005; 26(6): 559–66.
11. Micklos L. Do Needle-Free Connectors Prevent Catheter-Related Bloodstream Infections in Patients Receiving Hemodialysis Treatments Using Central Venous Catheters. Nephrol Nurs J 2015; 42(4): 383–6.
12. Machen A, Drake T, Wang YF. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system. PLoS One 2014; 9(2): e87870.
13. Suarez S, Nassif X, Ferroni A. Applications of MALDI-TOF technology in clinical microbiology. Pathol Biol 2015; 63: 43–52.
14. Bizzini A, Greub G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin Microbiol Infect 2010; 16: 1614–9.
15. Bizzini A, Durussel C, Bille J, Greub G, Prod’hom G. Performance of Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 2010; 48: 1549–54.
16. Bizzini A, Jaton K, Romo D, Bille J, Prod’hom G, Greub G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial stains. J Clin Microbiol 2011; 49: 693–6.
17. Jo SJ, Park KG, Han K, Park DJ, Park YJ. Direct Identification and Antimicrobial Susceptibility Testing of Bacteria From Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and the Vitek 2 System. Ann Lab Med 2016; 36: 117–23.
18. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2009; 48: 444–7.
19. Kok J, Thomas LC, Olma T, Chen SC, Iredell JR. Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper™ and time of flight mass spectrometry. PLoS One 2011; 6: e23285.
20. Schneiderhan W, Grundt A, Wörner S, Findeisen P, Neumaier M. Work flow analysis of around-the-clock processing of blood culture samples and integrated MALDI-TOF mass spectrometry analysis for the diagnosis of bloodstream infections. Clin Chem 2013; 59: 1649–56.
21. Lavergne RA, Chauvin P, Valentin A, Fillaux J, Roques-Malecaze C, Arnaud S, et al. An extraction method of positive blood cultures for direct identification of Candida species by Vitek MS matrix-assisted laser desorption ionization time of flight mass spectrometry. Med Mycol 2013; 51: 652–6.
22. Prod'hom G, Bizzini A, Durussel C, Bille J, Greub G. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. J Clin Microbiol 2010; 48: 1481–3.
23. Monteiro J, Inoue FM, Lobo AP, Sugawara EK, Boaretti FM, Tufik S. Fast and reliable bacterial identification direct from positive blood culture using a new TFA sample preparation protocol and the Vitek®MS system. J Microbiol Methods 2015; 109: 157–9.
24. Febbraro F, Rodio DM, Puggioni G, Antonelli G, Pietropaolo V, Trancassini M. MALDI- TOF MS versus VITEK®2: Comparison of Systems for the Identification of Microorganisms Responsible for Bacteremia. Curr Microbiol 2016; 73(6): 843–50.
25. Harris P, Winney I, Ashhurst-Smith C, O'Brien M, Graves S. Comparison of Vitek MS (MALDI-TOF) to standard routine identification methods: an advance but no panacea. Pathology 2012; 4(6): 583–5.
26. Pavlovic M, Konrad R, Iwobi AN, Sing A, Busch U, Huber I. A dual approach employing MALDI-TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex. FEMS Microbiol Lett 2012; 328(1): 46–53.
27. Patel R. Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology. Clin Infect Dis. 2013; 57(4): 564–72.
28. Romero-Gómez MP, Gómez-Gil R, Paño-Pardo JR, Mingorance J. Identification and susceptibility testing of microorganism by direct inoculation from positive blood culture bottles by combining MALDI-TOF and Vitek-2 Compact is rapid and effective. J Infect 2012; 65: 513-20.
29. Wimmer JL, Long SW, Cernoch P, Land GA, Davis JR, Musser JM, et al. Strategy for rapid identification and antibiotic susceptibility testing of gram-negative bacteria directly recovered from positive blood cultures using the Bruker MALDI Biotyper and the BD Phoenix system. J Clin Microbiol 2012; 50(7): 2452–4.
30. Hayek SS, Abd TT, Cribbs SK, Anderson AM, Melendez A, Kobayashi M, et al. Rare Elizabethkingia meningosepticum meningitis case in an immunocompetent adult. Emerg Microbes Infec 2013; 2: e17.
Original Article